CN118556123A - HBB modulating compositions and methods - Google Patents

Abstract

The present disclosure provides compositions, systems, and methods, e.g., for targeting, editing, modifying, or manipulating a host cell genome at one or more locations of a DNA sequence in a cell, tissue, or subject. A genetic modification system for treating Sickle Cell Disease (SCD) is described.

Description

HBB modulating compositions and methods

Sequence Listing

This application contains a sequence listing, which has been submitted electronically in XML format in accordance with WIPO Standard ST.26 and is hereby incorporated by reference in its entirety. The XLM copy was created on September 1, 2022, named V2065-7027WO_SL.XML, and is 15,727,019 kb in size.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/241,994, filed on September 8, 2021, U.S. Provisional Application No. 63/250,143, filed on September 29, 2021, and U.S. Provisional Application No. 63/303,900, filed on January 27, 2022. The contents of the foregoing applications are hereby incorporated by reference in their entirety.

Background Art

In the absence of specialized proteins to promote insertion events, the frequency of integration of the target nucleic acid into the genome is low and the site specificity is extremely low. Some existing methods, such as CRISPR/Cas9, are more suitable for small edits that rely on host repair pathways and are less efficient when integrating longer sequences. Other existing methods such as Cre/loxP require the first step of inserting the loxP site into the genome, and then the second step of inserting the target sequence into the loxP site. Improved compositions (e.g., proteins and nucleic acids) and methods are needed in the art to insert, change, or delete the target sequence in the genome.

Sickle cell disease is an inherited blood disorder that affects red blood cells. There are several types of sickle cell disease (e.g., hemoglobin SS disease, hemoglobin SC disease; sickle beta plus thalassemia; sickle beta-zero thalassemia). People with sickle cell disease have red blood cells that contain mostly hemoglobin S, an abnormal type of hemoglobin. The sickle-shaped cells die prematurely, which can lead to a shortage of red blood cells (anemia). The sickle-shaped cells are rigid and can block small blood vessels, causing severe pain and organ damage. Tissues that do not receive normal blood flow eventually become damaged. This is what causes the complications of sickle cell disease.

The HBB gene provides instructions for making the protein beta-globulin. Beta-globulin is a component (subunit) of a larger protein called hemoglobin, which is located inside red blood cells. In adults, hemoglobin is usually composed of four protein subunits: two subunits of beta-globulin and two subunits of another protein called alpha-globulin, which is produced by another gene called HBA. Each of these protein subunits is bound to an iron-containing molecule called heme; each heme contains an iron molecule in its center that can bind to one oxygen molecule. The hemoglobin in the red blood cells binds to oxygen molecules in the lungs. These cells then travel through the bloodstream and deliver oxygen to tissues throughout the body.

Sickle cell anemia, the common form of sickle cell disease, is caused by a specific mutation in the HBB gene. The mutation results in the production of an abnormal form of β-globulin (called hemoglobin S or HbS). In this case, hemoglobin S replaces the β-globin subunit in hemoglobin. The mutation changes a single amino acid in the β-globulin. In particular, the amino acid glutamic acid is replaced by the amino acid valine at position 6 in β-globulin (written as Glu6Val or E6V). Replacing glutamic acid with valine causes the abnormal hemoglobin S subunits to stick together and form a long rigid molecule that bends the red blood cells into a sickle or crescent shape. Mutations in the HBB gene can also cause other abnormalities in β-globulin, leading to other types of sickle cell disease. In these other types of sickle cell disease, only one β-globulin subunit is replaced with hemoglobin S. The other β-globulin subunits are replaced by different abnormal variants (such as hemoglobin C or hemoglobin E).

There is currently no universal cure for sickle cell disease. Available options for treating sickle cell disease are limited to bone marrow or stem cell transplants. Therefore, new and more effective treatments for sickle cell disease that exploit the HBB E6V mutation are needed.

Summary of the invention

The present disclosure relates to novel compositions, systems and methods for altering the genome at one or more locations in a host cell, tissue or subject in vivo or in vitro. In particular, the present invention features compositions, systems and methods for inserting, altering or deleting a sequence of interest in a host genome. For example, the present disclosure provides systems capable of modulating (e.g., inserting, altering or deleting a sequence of interest) the activity of the HBB gene and methods for treating sickle cell disease (SCD) by administering one or more such systems to alter the genomic sequence at the HBB nucleotide to correct a pathogenic mutation that causes SCD.

In one aspect, the present disclosure relates to a system for modifying DNA to correct a human HBB gene mutation that causes SCD, the system comprising (a) a nucleic acid encoding a gene-modifying polypeptide capable of targeted initiation of reverse transcription, the polypeptide comprising (i) a reverse transcriptase domain and (ii) a Cas9 nickase that binds DNA and has endonuclease activity; and (b) a template RNA comprising (i) a gRNA spacer complementary to a first portion of a human HBB gene, (ii) a gRNA scaffold that binds to the polypeptide, (iii) a heterologous subject sequence comprising a mutation region to correct the mutation, and (iv) a primer binding site (PBS) sequence comprising at least 3, 4, 5, 6, 7 or 8 bases having 100% homology to the target DNA strand at the 3′ end of the template RNA. The HBB gene may comprise an E6V mutation. The template RNA sequence may comprise a sequence described herein (e.g., in Tables 1, 3, 4, A, AA, B, B1, 5A-5D, X4 or X4A).

The gRNA spacer may comprise at least 15 bases with 100% identity to the target DNA at the 5' end of the template RNA. The template RNA may further comprise a PBS sequence comprising at least 5 bases with at least 80% homology to the target DNA strand. The template RNA may comprise one or more chemical modifications.

The domains of the genetically modified polypeptide can be connected by peptide linkers. The polypeptide may comprise one or more peptide linkers. The genetically modified polypeptide may further comprise a nuclear localization signal. The polypeptide may comprise more than one nuclear localization signal, for example, multiple adjacent nuclear localization signals or one or more nuclear localization signals in different regions of the polypeptide, for example, one or more nuclear localization signals in the N-terminus of the polypeptide and one or more nuclear localization signals in the C-terminus of the polypeptide. The nucleic acid encoding the genetically modified polypeptide may encode one or more intein domains.

Introducing the system into a target cell can result in the insertion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, or 1000 base pairs of exogenous DNA. Introducing the system into a target cell can result in a deletion, wherein the deletion is less than 2, 3, 4, 5, 10, 50, or 100 base pairs of genomic DNA upstream or downstream of the insertion. Introducing the system into a target cell can result in a substitution, such as a substitution of 1, 2, or 3 nucleotides (e.g., consecutive nucleotides).

The heterologous subject sequence can be at least 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, or 700 base pairs.

On the one hand, the present disclosure relates to a pharmaceutical composition comprising the above-mentioned system and a pharmaceutically acceptable excipient or carrier, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of: a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle. On the one hand, the present disclosure relates to a pharmaceutical composition comprising the above-mentioned system and a plurality of pharmaceutically acceptable excipients or carriers, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of: a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle, for example, wherein the above-mentioned system is delivered by two different excipients or carriers, such as two lipid nanoparticles, two viral vectors, or a lipid nanoparticle and a viral vector. The viral vector can be an adeno-associated virus (AAV).

In one aspect, the disclosure relates to a host cell (eg, a mammalian cell, eg, a human cell) comprising the above-described system.

In one aspect, the present disclosure relates to a method for correcting a mutation in a human HBB gene in a cell, tissue or subject, the method comprising administering the above system to the cell, tissue or subject, wherein optionally, the correction of the mutant HBB gene comprises an amino acid substitution of V6E (reversing the pathogenic substitution E6V). The system can be introduced in vivo, in vitro, ex vivo or in situ. The nucleic acid of (a) can be integrated into the genome of the host cell. In some embodiments, the nucleic acid of (a) is not integrated into the genome of the host cell. In some embodiments, the heterologous subject sequence is inserted into only one target site in the genome of the host cell. The heterologous subject sequence can be inserted into two or more target sites in the genome of the host cell, for example, into the same corresponding sites in two homologous chromosomes, or two different sites on the same or different chromosomes. The heterologous subject sequence can encode a mammalian polypeptide or a fragment or variant thereof. The components of the system can be delivered on 1, 2, 3, 4 or more different nucleic acid molecules. The system can be introduced into the host cell by electroporation or by using at least one vector selected from plasmid vectors, viral vectors, vesicles and lipid nanoparticles.

Features of these compositions or methods may include one or more of the embodiments listed below.

Examples of Examples

1. A template RNA, which comprises, for example, from 5' to 3':

(i) a gRNA spacer complementary to a first portion of a human HBB gene, wherein the gRNA spacer has a sequence comprising core nucleotides of a gRNA spacer sequence of Table 1, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the gRNA spacer (e.g., comprising one or more flanking nucleotides adjacent to the core nucleotides), or wherein the gRNA spacer has the sequence of a spacer selected from Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4, or Table X4A;

(ii) a gRNA scaffold that binds to a gene modifying polypeptide (e.g., a Cas domain that binds to the gene modifying polypeptide),

(iii) a heterologous subject sequence comprising a mutation region, for introducing a mutation into the second portion of the human HBB gene (e.g., correcting a mutation therein) (wherein, optionally, the heterologous subject sequence comprises, from 5' to 3', a post-editing homology region, a mutation region, and a pre-editing homology region), and

(iv) a primer binding site (PBS) sequence comprising at least 3, 4, 5, 6, 7 or 8 bases having 100% identity with the third part of the human HBB gene.

2. The template RNA of embodiment 1, wherein the heterologous object sequence comprises the following core nucleotides: an RT template sequence in Table 3, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence, or wherein the heterologous object sequence comprises the sequence of the RT template sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4, or Table X4A.

3. The template RNA of Example 1, wherein the heterologous object sequence comprises the following core nucleotides: an RT template sequence in Table 3 corresponding to the gRNA spacer sequence, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence (e.g., comprising one or more flanking nucleotides adjacent to the core nucleotides), or wherein the heterologous object sequence comprises a sequence of an RT template sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A corresponding to the gRNA spacer sequence.

4. The template RNA of any one of embodiments 1-3, wherein the PBS sequence has a sequence comprising core nucleotides from a PBS sequence in the same row as the RT template sequence in Table 3, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence (e.g., comprising one or more flanking nucleotides adjacent to the core nucleotides).

5. The template RNA of any one of embodiments 1-3, wherein the PBS sequence has a sequence comprising core nucleotides of a PBS sequence in Table 3 corresponding to the RT template sequence, or a sequence having 1, 2 or 3 substitutions therewith, the gRNA spacer sequence, or both, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence has a sequence comprising a PBS sequence from Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A corresponding to the RT template sequence, or a sequence having 1, 2 or 3 substitutions therewith, the gRNA spacer sequence, or both.

6. The template RNA of any one of embodiments 1-5, wherein the gRNA scaffold comprises the sequence of the gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

7. The template RNA of any one of embodiments 1-5, wherein the gRNA scaffold comprises a sequence of the gRNA scaffold in Table 12 corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

8. A template RNA, which comprises, for example, from 5' to 3':

(i) a gRNA spacer complementary to the first part of the human HBB gene,

(ii) a gRNA scaffold that binds to a gene modifying polypeptide (e.g., a Cas domain that binds to the gene modifying polypeptide),

(iii) a heterologous subject sequence comprising a mutation region for introducing a mutation into the second portion of the human HBB gene (e.g., correcting a mutation therein), wherein the heterologous subject sequence comprises the core nucleotides of the RT template sequence in Table 3 or a sequence having 1, 2 or 3 substitutions therewith, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence, or wherein the heterologous subject sequence comprises the RT template sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A; and

(iv) a PBS sequence comprising at least 3, 4, 5, 6, 7 or 8 bases that are 100% identical to the third part of the human HBB gene.

9. The template RNA of Example 8, wherein the gRNA spacer comprises the following core nucleotides: a gRNA spacer sequence in Table 1, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprising one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the gRNA spacer sequence, or wherein the gRNA spacer comprises a gRNA spacer sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A.

10. The template RNA of any one of embodiments 1-9, wherein the gRNA spacer comprises CATGGTGCATCTGACTCCTG (SEQ ID NO: 21668) or CATGGTGCACCTGACTCCTG (SEQ ID NO: 19249) or a sequence having 1, 2 or 3 substitutions therefrom.

11. The template RNA of any one of embodiments 1-9, wherein the gRNA spacer comprises GTAACGGCAGACTTCTCCAC (SEQ ID NO: 19971) or a sequence having 1, 2 or 3 substitutions thereto.

12. The template RNA of embodiment 8, wherein the heterologous subject sequence comprises core nucleotides of a gRNA spacer sequence in Table 1 corresponding to the RT template sequence or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the gRNA spacer sequence, or wherein the heterologous subject sequence comprises nucleotides of a gRNA spacer sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4, or Table X4A corresponding to the RT template sequence or a sequence having 1, 2, or 3 substitutions thereto.

13. The template RNA of any one of embodiments 8-12, wherein the PBS sequence has a sequence comprising core nucleotides from a PBS sequence in the same row as the RT template sequence in Table 3, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence.

14. The template RNA of any one of embodiments 8-12, wherein the PBS sequence has a sequence comprising core nucleotides of a PBS sequence in Table 3 corresponding to the RT template sequence, or a sequence having 1, 2 or 3 substitutions relative thereto, the gRNA spacer sequence, or both, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence has a sequence comprising core nucleotides of a PBS sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A corresponding to the RT template sequence, the gRNA spacer sequence, or both.

15. The template RNA of any one of embodiments 8-14, wherein the gRNA scaffold comprises the sequence of the gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

16. The template RNA of any one of embodiments 8-14, wherein the gRNA scaffold comprises a sequence of the gRNA scaffold in Table 12 corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

17. The template RNA of any one of the preceding embodiments, wherein the gRNA spacer has a sequence of a gRNA spacer sequence of Table A or Table B or a sequence having 1, 2 or 3 substitutions thereto.

18. The template RNA of Example 17, wherein the gRNA spacer has a sequence of SEQ ID NO: 21668.

19. The template RNA of embodiment 17 or 18, wherein the PBS sequence has a sequence from a PBS sequence in the same row as the gRNA spacer sequence in Table A or B or a sequence having 1, 2 or 3 substitutions relative thereto.

20. The template RNA of any one of embodiments 17-19, wherein the PBS sequence has a sequence of core nucleotides of the PBS sequence of SEQ ID NO: 21669, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence.

21. A template RNA as described in any of embodiments 17-19, wherein the gRNA scaffold has a sequence from a gRNA scaffold in the same row as the gRNA spacer sequence in Table A or B or a sequence having 1, 2 or 3 substitutions relative thereto.

22. The template RNA of any one of embodiments 17-20, wherein the heterologous subject sequence has a sequence from the RT template sequence in the same row as the gRNA spacer sequence in Table A or B, or a sequence having 1, 2, or 3 substitutions relative thereto, wherein optionally, the bold T shown in the RT template sequence in Table A is replaced by G (e.g., a sequence without a PAM-killing mutation), or wherein further optionally, the bold C shown in the RT template in Table B is replaced by T or U (e.g., a sequence without the following SNP, which is present in HEK293T cells but not in the hg38 human reference genome).

23. The template RNA of any one of embodiments 17-22, wherein the heterologous subject sequence has a sequence comprising core nucleotides of the RT template sequence of SEQ ID NO: 21670, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence.

24. The template RNA of any one of embodiments 17-23, wherein the heterologous subject sequence has a sequence comprising core nucleotides of the RT template sequence of SEQ ID NO: 21671, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence.

25. The template RNA of any one of embodiments 17-24, wherein the template RNA has a template RNA sequence of Table A or Table B, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, wherein optionally the template RNA comprises one or more (e.g., all) chemical modifications shown in the sequence of Table A or Table B.

26. A gene modification system for modifying DNA, comprising:

(a) a first RNA comprising, from 5' to 3', (i) a guide RNA sequence complementary to a first portion of a human HBB gene, wherein the guide RNA sequence has a sequence of core nucleotides comprising a spacer sequence of Table 1, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the guide RNA sequence, or wherein the guide RNA sequence has a sequence comprising a spacer from Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A; and (ii) a sequence (e.g., a scaffold region) that binds to a gene modifying polypeptide (e.g., a Cas domain that binds to the gene modifying polypeptide), and

(b) a second RNA comprising: (iii) a heterologous subject sequence comprising nucleotide substitutions to introduce mutations into the second portion of the human HBB gene (optionally, the heterologous subject sequence comprises a post-editing homology region, a mutation region, and a pre-editing homology region from 5' to 3'); (iv) a primer region comprising at least 5, 6, 7 or 8 bases having 100% identity with the third portion of the human HBB gene; and (v) an RRS (RNA binding protein recognition sequence) that binds to a gene modification protein.

27. The gene modification system of embodiment 26, wherein the heterologous subject sequence comprises the following core nucleotides: an RT template sequence in Table 3, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence, or wherein the heterologous subject sequence comprises a sequence of an RT template sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4, or Table X4A, or a sequence having 1, 2 or 3 substitutions relative thereto.

28. The gene modification system of embodiment 26, wherein the heterologous subject sequence comprises the following core nucleotides: an RT template sequence in Table 3 corresponding to the gRNA spacer sequence, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence, or wherein the heterologous subject sequence comprises a sequence of an RT template sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A corresponding to the gRNA spacer sequence, or a sequence having 1, 2 or 3 substitutions relative thereto.

29. A gene modification system as described in any of embodiments 26-28, wherein the PBS sequence has a sequence comprising core nucleotides from a PBS sequence in Table 3 in the same row as the RT template sequence, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence.

30. A gene modification system as described in any of embodiments 26-28, wherein the PBS sequence has a sequence comprising core nucleotides of a PBS sequence in Table 3 corresponding to the RT template sequence, or a sequence having 1, 2 or 3 substitutions therewith, the gRNA spacer sequence, or both, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence comprises a PBS sequence in Table A, Table AA, Table B, Table Bl, Table 5A-5D, Table X4 or Table X4A corresponding to the RT template sequence, or a sequence having 1, 2 or 3 substitutions therewith, the gRNA spacer sequence, or both.

31. A gene modification system as described in any of Examples 26-30, wherein the gRNA scaffold comprises the sequence of the gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

32. A gene modification system as described in any of Examples 26-30, wherein the gRNA scaffold comprises a sequence of the gRNA scaffold in Table 12 corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

33. A gene modification system for modifying DNA, comprising:

(a) a first RNA comprising, from 5' to 3', (i) a guide RNA sequence complementary to a first portion of a human HBB gene, and (ii) a sequence (e.g., a scaffold region) that binds to a gene modifying polypeptide (e.g., a Cas domain that binds to the gene modifying polypeptide), and

(b) a second RNA comprising: (iii) a heterologous subject sequence comprising nucleotide substitutions for introducing mutations into the second portion of the human HBB gene, wherein the heterologous subject sequence comprises the following core nucleotides: the RT template sequence in Table 3, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence, or wherein the heterologous subject sequence comprises the RT sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A, or a sequence having 1, 2 or 3 substitutions relative thereto, and (iv) a primer region comprising at least 5, 6, 7 or 8 bases having 100% homology to the third portion of the human HBB gene, and (v) an RRS (RNA binding protein recognition sequence) that binds to a gene modifying protein.

34. A gene modification system as described in Example 33, wherein the gRNA spacer comprises the following core nucleotides: a gRNA spacer sequence in Table 1, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprising one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the gRNA spacer sequence, or wherein the gRNA spacer comprises a gRNA spacer sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A.

35. The gene modification system of embodiment 33, wherein the heterologous subject sequence comprises core nucleotides of a gRNA spacer sequence in Table 1 corresponding to the RT template sequence or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the gRNA spacer sequence, or wherein the gRNA spacer comprises a gRNA spacer sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4, or Table X4A corresponding to the RT template sequence or a sequence having 1, 2, or 3 substitutions thereto.

36. A gene modification system as described in any of embodiments 33-35, wherein the PBS sequence has a sequence comprising core nucleotides from a PBS sequence in Table 3 in the same row as the RT template sequence, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence.

37. A gene modification system as described in any of embodiments 33-35, wherein the PBS sequence has a sequence comprising core nucleotides of a PBS sequence in Table 3 corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having 1, 2, or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence comprises a PBS sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4, or Table X4A corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having 1, 2, or 3 substitutions relative thereto.

38. A gene modification system as described in any of Examples 33-37, wherein the gRNA scaffold comprises the sequence of the gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

39. A gene modification system as described in any of Examples 33-37, wherein the gRNA scaffold comprises a sequence of the gRNA scaffold in Table 12 corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

40. A gRNA comprising (i) a gRNA spacer sequence complementary to a first portion of a human HBB gene, wherein the gRNA spacer has a sequence comprising core nucleotides of a gRNA spacer sequence of Table 1, Table 2 or Table 4, or a sequence having 1, 2 or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the gRNA spacer sequence; and (ii) a gRNA scaffold, or wherein the gRNA spacer has a sequence of a gRNA spacer sequence from Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A, or a sequence having 1, 2 or 3 substitutions thereto.

41. The gRNA of embodiment 40, wherein the gRNA scaffold comprises the sequence of the gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

42. The gRNA of embodiment 40, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12 corresponding to the gRNA spacer sequence, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

43. A template RNA comprising: (iii) a heterologous subject sequence comprising a mutation region for introducing a mutation into a second portion of a human HBB gene, wherein the heterologous subject sequence comprises the following core nucleotides: an RT template sequence in Table 3, or a sequence having 1, 2 or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence, or wherein the heterologous subject sequence comprises an RT sequence in Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A, or a sequence having 1, 2 or 3 substitutions thereto, and (iv) a PBS sequence comprising at least 5, 6, 7 or 8 bases having 100% homology to a third portion of the human HBB gene.

44. The template RNA of embodiment 43, wherein the PBS sequence has a sequence comprising core nucleotides from a PBS sequence in the same row as the RT template sequence in Table 3, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence.

45. The template RNA of embodiment 43, wherein the PBS sequence has a sequence comprising core nucleotides of a PBS sequence in Table 3 corresponding to the RT template sequence, or a sequence having 1, 2 or 3 substitutions relative thereto, and optionally comprises one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence has a sequence comprising a PBS sequence from Table A, Table AA, Table B, Table B1, Table 5A-5D, Table X4 or Table X4A, or a sequence having 1, 2 or 3 substitutions relative thereto.

46. The template RNA of any one of embodiments 1-16 or 43-45, the gene modification system of any one of embodiments 26-39, or the gRNA of any one of embodiments 31-33, wherein the mutation introduced by the system is a V6E mutation of the HBB gene (e.g., to correct a pathogenic E6V mutation).

47. The template RNA of any one of embodiments 1-16 or 43-46, or the gene modification system of any one of embodiments 36-39 or 46, wherein the pre-editing sequence length comprises about 1 nucleotide to about 35 nucleotides (e.g., comprises about 1-5, 5-10, 10-15, 15-20, 20-25, 25-30 or 30-35 nucleotides).

48. The template RNA of any one of embodiments 1-16 or 43-47, or the gene modification system of any one of embodiments 36-39, 46 or 47, wherein the mutation region comprises a single nucleotide.

49. The template RNA of any one of embodiments 1-16 or 43-47, or the gene modification system of any one of embodiments 26-39, 46 or 47, wherein the mutation region is at least two nucleotides in length.

50. The template RNA of any one of embodiments 1-14, 41-45 or 47, or the gene modification system of any one of embodiments 24-37, 44-45 or 47, wherein the mutation region is up to 32 (e.g., up to 5, 10, 15, 20, 25, 30 or 32) nucleotides in length and comprises one, two or three sequence differences relative to the second portion of the human HBB gene.

51. The template RNA of any one of embodiments 1-16, 43-47, 49 or 50, or the gene modification system of any one of embodiments 26-39, 46, 47, 49 or 50, wherein the mutation region comprises two sequence differences relative to the second portion of the human HBB gene.

52. A template RNA as described in any one of embodiments 1-16, 43-47 or 49-51, or a gene modification system as described in any one of embodiments 26-39, 46, 47 or 49-51, wherein the mutation region comprises a first region (e.g., a first nucleotide) designed to correct a pathogenic mutation in the HBB gene and a second region (e.g., a second nucleotide) designed to inactivate a PAM sequence (e.g., a "PAM-killing" mutation exemplified in Table A, AA, B or B1).

53. The template RNA of any one of embodiments 1-16, 43-51, or the gene modification system of any one of embodiments 26-39 or 46-51, wherein the mutation region comprises less than 80%, 70%, 60%, 50%, 40% or 30% identity with the corresponding portion of the human HBB gene.

54. The template RNA of any one of the preceding embodiments, wherein the template RNA comprises one or more silent mutations (e.g., silent substitutions), e.g., as exemplified in Table 7A, X4 or X4A.

55. The template RNA of embodiment 54, wherein the one or more silent mutations comprise a silent substitution at the codon encoding the 6th amino acid (proline) of the HBB gene counting the initial methionine, for example, a substitution to CCC or CCG.

56. The template RNA of any one of the preceding embodiments, wherein the mutation region comprises a first region designed to correct a pathogenic mutation in the HBB gene and a second region designed to introduce a silent substitution.

57. The template RNA of any one of the preceding embodiments, comprising one or more chemically modified nucleotides.

58. A gene modification system comprising:

A template RNA as described in any one of embodiments 1-16, 43-57, or a system as described in any one of embodiments 26-39 or 46-57, and

A gene-modifying polypeptide or a nucleic acid (eg, RNA) encoding the gene-modifying polypeptide.

59. The gene modification system of embodiment 58, wherein the gene modification polypeptide comprises:

a reverse transcriptase (RT) domain (e.g., an RT domain from a retrovirus, or a polypeptide domain having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto); and

a Cas domain (e.g., a Cas9 domain) that binds to the target DNA molecule and is heterologous to the RT domain; and

Optionally, a linker is arranged between the RT domain and the Cas domain.

60. The gene modification system of embodiment 59, wherein:

(a) The RT domain comprises:

(i) the RT domain of Table 6, or

(ii) an RT domain from murine leukemia virus (MMLV), porcine endogenous retrovirus (PERV), avian reticuloendotheliosis virus (AVIRE), feline leukemia virus (FLV), simian foamy virus (SFV) (e.g., SFV3L), bovine leukemia virus (BLV), Mason-Fischer monkey virus (MPMV), human foamy virus (HFV), or bovine foamy/syncytial virus (BFV/BSV); or

(b) the genetically modified polypeptide comprises an amino acid sequence according to Table C, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

61. The gene modification system of Embodiment 59 or 60, wherein the Cas domain comprises the Cas domain of Table 7 or Table 8.

62. The gene modification system of any one of embodiments 59-61, wherein the Cas domain:

(a) is the Cas9 domain;

(b) is a SpCas9 domain, a B1atCas9 domain, a Nme2Cas9 domain, a PnpCas9 domain, a SauCas9 domain, a SauCas9-KKH domain, a SauriCas9 domain, a SauriCas9-KKH domain, a ScaCas9-Sc++ domain, a SpyCas9 domain, a SpyCas9-NG domain, a SpyCas9-SpRY domain or a St1Cas9 domain; and/or

(c) is a Cas9 domain comprising an N670A mutation, an N611A mutation, an N605A mutation, an N580A mutation, an N588A mutation, an N872A mutation, an N863 mutation, an N622A mutation or an H840A mutation.

63. The gene modification system of embodiment 62, wherein the Cas9 domain binds to a PAM sequence listed in Table 7 or Table 12.

64. The gene modification system of embodiment 63, wherein the second portion of the human HBB gene overlaps with the PAM recognized by the Cas domain, for example, wherein the second portion of the human HBB gene is within the PAM or wherein the PAM is within the second portion of the human HBB gene.

65. The gene modification system of any one of embodiments 58-64, wherein the gRNA spacer is a gRNA spacer according to Table 1, and the Cas domain comprises a Cas domain listed in the same row of Table 1, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

66. The gene modification system of any one of embodiments 58-64, wherein the template RNA comprises a sequence of a template RNA sequence in Table 3, Table A, Table AA, Table B, Table Bl, Table 5A-5D, Table X4, or Table X4A, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

67. The gene modification system of any one of embodiments 58-66, wherein:

(a) the template RNA comprises a sequence of a template RNA sequence in Table 3, Table A, Table AA, Table B, Table Bl, Table 5A-5D, Table X4, or Table X4A;

(b) the Cas domain comprises the Cas domain of Table 7 or Table 8;

(c) the linker comprises a linker sequence of Table 10 (e.g., a linker sequence of any one of SEQ ID NOs: 5217, 5106, 5190, and 5218); and

(d) the genetically modified polypeptide comprises one or two NLS sequences from Table 11 (e.g., an NLS sequence of any one of SEQ ID NOs: 5245, 5290, 5323, 5330, 5349, 5350, 5351 and 4001).

68. The gene modification system of any one of embodiments 58-67, which produces a first nick in the first strand of the human HBB gene.

69. The gene modification system of embodiment 68, further comprising a second-strand targeting gRNA that directs a second cut to the second strand of the human HBB gene.

70. The gene modification system of embodiment 69, wherein the second strand targeting gRNA comprises:

(i) a sequence comprising the core nucleotides of a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2, and optionally comprising one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the left gRNA spacer sequence or the right gRNA spacer sequence; or

(ii) a second-strand targeting gRNA comprising a spacer sequence of Table 6A or a spacer sequence having 1, 2 or 3 substitutions therewith.

71. A gene modification system as described in Example 69, wherein the second-strand targeting gRNA comprises a sequence of core nucleotides of a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2 containing the gRNA spacer sequence corresponding to (i), and optionally comprises one or more consecutive nucleotides starting from the 3’ end of the flanking nucleotides of the left gRNA spacer sequence or the right gRNA spacer sequence.

72. The gene modification system of embodiment 69, wherein the second strand targeting gRNA comprises:

(i) a sequence containing the core nucleotides of the second nicking gRNA sequence from Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and optionally comprising one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the second nicking gRNA sequence; or

(ii) a second strand targeting gRNA comprising a spacer sequence from Table 6A, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

73. A gene modification system as described in Example 69, wherein the second strand targeting gRNA comprises a sequence of core nucleotides of a second nick gRNA sequence from Table 4 containing a gRNA spacer sequence corresponding to (i), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the second nick gRNA sequence.

74. A gene modification system as described in any of Examples 58-73, wherein the second-strand targeting gRNA and the template RNA of the gene modification system have a "PAM-inward orientation", for example, as exemplified in Table 4, 6A, X4 or X4A.

75. A gene modification system as described in any of Examples 58-63, wherein the second-strand targeting gRNA targets a sequence overlapping with the target mutation of the template RNA.

76. The gene modification system of embodiment 75, wherein the second strand targeting gRNA comprises:

(i) a sequence complementary to a sickle cell mutation (e.g., a spacer sequence);

(ii) a sequence that is complementary to the wild-type sequence at the sickle cell locus (e.g., a spacer sequence);

(iii) a sequence complementary to the Makassar sequence at the sickle cell locus (e.g., a spacer sequence);

(iv) a sequence (e.g., a spacer sequence) that is complementary to a SNP proximal to the sickle cell locus, e.g., a SNP contained in genomic DNA of a subject (e.g., a patient);

(v) a sequence (e.g., a spacer sequence) that is complementary to or comprises one or more silent substitutions proximal to the sickle cell locus.

77. The template RNA, gene modification system or gRNA of any of the preceding embodiments, wherein the gRNA spacer comprises approximately 1, 2, 3 or more flanking nucleotides of the gRNA spacer.

78. The template RNA or gene modification system of any of the preceding embodiments, wherein the heterologous subject sequence comprises about 2, 3, 4, 5, 10, 20, 30, 40 or more flanking nucleotides of the RT template sequence.

79. The template RNA or gene modification system of any of the preceding embodiments, wherein the heterologous subject sequence comprises about 8-30, 9-25, 10-20, 11-16, or 12-15 (e.g., about 11-16) nucleotides.

80. The template RNA or gene modification system of any one of the preceding embodiments, wherein the mutation region comprises a sequence difference of 1, 2 or 3 nucleotide positions relative to the corresponding portion of the human HBB gene.

81. The template RNA or gene modification system of any one of the preceding embodiments, wherein the mutation region comprises a sequence difference of at least 2 nucleotide positions relative to the corresponding portion of the human HBB gene.

82. The template RNA or gene modification system of any one of the preceding embodiments, wherein the post-editing homology region and/or the pre-editing homology region comprises 100% identity with the HBB gene.

83. The template RNA or gene modification system of any one of the preceding embodiments, wherein the PBS sequence further comprises about 1, 2, 3, 4, 5, 6, 7 or more flanking nucleotides.

84. The template RNA or gene modification system of any of the preceding embodiments, wherein the PBS sequence comprises about 5-20, 8-16, 8-14, 8-13, 9-13, 9-12 or 10-12 (e.g., about 9-12) nucleotides.

85. The template RNA or gene modification system of any of the preceding embodiments, wherein the PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of the HBB gene nicking site.

86. The gene modification system of any one of the preceding embodiments, wherein the domains of the gene modifying polypeptide are connected by a peptide linker.

87. The gene modification system of embodiment 86, wherein the linker comprises a linker sequence of Table 10 (eg, a linker sequence of any one of SEQ ID NOs: 5217, 5106, 5190, and 5218).

88. The gene modification system of any one of the preceding embodiments, wherein the gene modifying polypeptide further comprises one or more nuclear localization sequences (NLS).

89. The gene modification system of embodiment 88, wherein the gene modification polypeptide comprises a first NLS and a second NLS.

90. The gene modification system of embodiment 88 or 89, wherein the NLS comprises the NLS sequence of Table 11 (e.g., any one of SEQ ID NOs: 5245, 5290, 5323, 5330, 5349, 5350, 5351 and 4001).

91. A template RNA comprising the sequence of a template RNA in Table A, Table AA, Table B, Table Bl, Tables 5A-5D, Table X4, or Table X4A, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

92. A template RNA comprising the sequence of a template RNA in Table A, Table AA, Table B, Table Bl, Tables 5A-5D, Table X4, or Table X4A.

93. A gene modification system comprising:

(i) a template RNA comprising the sequence of a template RNA in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto; and

(ii) a second nicking gRNA sequence from the same row as (i) in Table 4, a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

94. A gene modification system comprising:

(i) a template RNA comprising the sequence of the template RNA of Table 4; and

(ii) The second cut gRNA sequence from the same row as (i) in Table 4.

95. A DNA encoding the template RNA of any one of embodiments 1-16, 43-53, 77-85, 91 or 92, or the gRNA of any one of embodiments 40-42.

96. A pharmaceutical composition comprising the system of any one of embodiments 58-90, 93 or 94, or one or more nucleic acids encoding the system, and a pharmaceutically acceptable excipient or carrier.

97. The pharmaceutical composition of embodiment 96, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle.

98. The pharmaceutical composition of Example 97, wherein the viral vector is an adeno-associated virus.

99. A host cell (eg, a mammalian cell, eg, a human cell) comprising the template RNA or gene modification system as described in any one of the preceding embodiments.

100. A method for preparing a template RNA as described in any one of embodiments 1-16, 43-53, 77-85, 91 or 92, the method comprising synthesizing the template RNA by in vitro transcription (e.g., solid-state synthesis) or by introducing DNA encoding the template RNA into a host cell under conditions that allow production of the template RNA.

101. A method for modifying a target site in a human HBB gene in a cell, the method comprising contacting the cell with a gene modification system as described in any one of embodiments 58-90, 93 or 94, or a DNA encoding the gene modification system, thereby modifying the target site in the human HBB gene in the cell.

102. A method for modifying a target site in a human HBB gene in a cell, the method comprising contacting the cell with: (i) a template RNA as described in any one of embodiments 58-90, 93 or 94, or a DNA encoding the same; and (ii) a gene modifying polypeptide or a nucleic acid encoding a gene modifying polypeptide, thereby modifying the target site in the human HBB gene in the cell.

103. A method for treating a subject suffering from a disease or condition associated with a human HBB gene mutation, the method comprising administering to the subject a gene modification system as described in any one of Examples 58-90, 93 or 94, or a DNA encoding the gene modification system, thereby treating the subject suffering from the disease or condition associated with the human HBB gene mutation.

104. A method for treating a subject suffering from a disease or condition associated with a human HBB gene mutation, the method comprising administering to the subject a template RNA or a DNA encoding the same as described in any one of embodiments 58-90, 93 or 94; and (ii) a gene-modified polypeptide or a nucleic acid encoding a gene-modified polypeptide, thereby treating the subject suffering from the disease or condition associated with the human HBB gene mutation.

105. The method of embodiment 103 or 104, wherein the disease or condition is sickle cell disease (SCD) (eg, sickle cell anemia).

106. A method as described in any of Examples 103-105, wherein the subject has a pathogenic EV6 mutation.

107. A method for treating a subject having SCD, the method comprising administering to the subject a gene modification system as described in any one of Examples 58-90, 93 or 94, or a DNA encoding the gene modification system, thereby treating the subject having SCD.

108. A method for treating a subject with SCD, the method comprising administering to the subject (i) a template RNA or a DNA encoding the same as described in any one of embodiments 58-90, 93 or 94, and (ii) a gene-modified polypeptide or a nucleic acid encoding a gene-modified polypeptide, thereby treating the subject with SCD.

109. The gene modification system or method of any preceding embodiment, wherein introducing the system into a target cell can correct a pathogenic mutation in the HBB gene.

110. The gene modification system or method of any of the preceding embodiments, wherein the pathogenic mutation is an E6V mutation, and wherein the correction comprises an amino acid substitution of V6E.

111. The gene modification system or method of any of the preceding embodiments, wherein correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70% or more) of the target nucleic acid.

112. The gene modification system or method of any of the preceding embodiments, wherein correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70% or more) of the target cells.

113. A gene modification system or method as described in any of the preceding embodiments, wherein the gene modification system comprises a second strand targeting gRNA, and wherein correction of mutations in the target cell population is increased relative to a target cell population treated with a gene modification system comprising a template RNA but without a second strand targeting gRNA.

114. The gene modification system or method of any of the preceding embodiments, wherein the template RNA comprises one or more silent substitutions (e.g., as exemplified in Tables 7A, X4, and X4A), and wherein correction of mutations in the target cell population is increased relative to a target cell population treated with a gene modification system comprising a template RNA that does not contain the one or more silent substitutions.

115. The method of any one of the preceding embodiments, wherein the cell is a mammalian cell, eg, a human cell.

116. The method of any one of the preceding embodiments, wherein the subject is a human.

117. The method of any one of the preceding embodiments, wherein the contacting occurs ex vivo, e.g., wherein the DNA of the cell or subject is modified ex vivo.

118. The method of any preceding embodiment, wherein the contacting occurs in vivo, e.g., wherein the DNA of the cell or subject is modified in vivo.

119. The method of any of the preceding embodiments, wherein contacting the cell or the subject with the system comprises contacting the cell or a cell in the subject with a nucleic acid (e.g., DNA or RNA) encoding the genetically modified polypeptide under conditions that allow production of the genetically modified polypeptide.

120. A method as described in any of the preceding embodiments, wherein the gRNA spacer is fully complementary to the first portion of the human HBB gene in the cell at all nucleotide positions, wherein the first portion is located on the second strand of the HBB gene.

121. The method of any one of the preceding embodiments, wherein the heterologous subject sequence is fully complementary to a second portion of the human HBB gene in the cell at all nucleotide positions except the mutation region, wherein the second portion is located on the first strand of the HBB gene.

122. The method of any one of the preceding embodiments, wherein the PBS sequence is fully complementary to a third portion of the human HBB gene, wherein the third portion is located on the first strand of the HBB gene.

Further Examples

A1. A template RNA comprising from 5' to 3':

(i) a gRNA spacer complementary to the first part of the human HBB gene, wherein the gRNA spacer has a nucleotide sequence comprising CATGGTGCATCTGACTCCTG (SEQ ID NO: 21668), or a nucleotide sequence having 1 substitution relative thereto;

(ii) a gRNA scaffold that binds to the Cas domain of the gene modifying polypeptide;

(iii) a heterologous subject sequence comprising a mutation region for correcting a mutation in the second portion of the human HBB gene, and

(iv) a primer binding site (PBS) sequence comprising at least 5 bases having 100% identity with the third portion of the human HBB gene.

A2. The template RNA as described in Example A1, wherein the gRNA spacer has a nucleotide sequence comprising CATGGTGCATCTGACTCCTG (SEQ ID NO: 21668) or CATGGTGCACCTGACTCCTG (SEQ ID NO: 19249).

A3. The template RNA as described in Example A1 or A2, wherein the gRNA spacer has a nucleotide sequence consisting of CATGGTGCATCTGACTCCTG (SEQ ID NO: 21668) or CATGGTGCACCTGACTCCTG (SEQ ID NO: 19249).

A4. The template RNA as described in any of the preceding embodiments, wherein the gRNA spacer has a length of 20 nucleotides.

A5. The template RNA as described in Example A1, wherein the gRNA scaffold has a sequence according to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 11,012), or a sequence with at least 90% identity thereto.

A6. The template RNA as described in Example A1, wherein the gRNA scaffold has a sequence according to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 11,012).

A7. The template RNA as described in Example A1, wherein the heterologous object sequence comprises a sequence of at least 8 nucleotides from the 3' end of the sequence according to AGTAACGGCAGACTTCTCTTCAG (SEQ ID NO: 20954), or a sequence having 1, 2 or 3 substitutions therefrom.

A8. The template RNA of embodiment A1, wherein the heterologous object sequence comprises a sequence of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides from the 3' end of the sequence according to AGTAACGGCAGACTTCTCTTCAG (SEQ ID NO: 20954), or a sequence having 1, 2 or 3 substitutions therefrom.

A9. The template RNA as described in Example A1, wherein the heterologous object sequence comprises a sequence of at least 8 nucleotides from the 3' end of the sequence according to AGTAACGGCAGACTTCTCTTCAG (SEQ ID NO: 20954).

A10. The template RNA as described in embodiment A1, wherein the heterologous object sequence comprises a sequence of at least 8 nucleotides from the 3' end of the sequence according to AGTAACGGCAGACTTCTCTGCAG (SEQ ID NO: 20955).

A11. The template RNA as described in Example A1, wherein the PBS sequence comprises a sequence of at least 8 nucleotides from the 5' end of the sequence according to GAGTCAGGTGCACCATG (SEQ ID NO: 19431), or a sequence having 1 substitution relative thereto.

A12. The template RNA as described in Example A1, wherein the PBS sequence comprises a sequence of at least 8 nucleotides from the 5' end of the sequence according to GAGTCAGGTGCACCATG (SEQ ID NO: 19431).

A13. The template RNA as described in Example A1, wherein the PBS sequence comprises a sequence of 9, 10, 11, 12, 13, 14, 15, 16 or 17 nucleotides from the 5' end of the sequence according to GAGTCAGGTGCACCATG (SEQ ID NO: 19431), or a sequence having 1 substitution relative thereto.

A14. The template RNA as described in Example A1, wherein

the gRNA scaffold having a sequence according to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 11,012), or a sequence at least 90% identical thereto;

The heterologous subject sequence comprises a sequence of at least 8 nucleotides from the 3' end of the sequence according to AGTAACGGCAGACTTCTCTTCAG (SEQ ID NO: 20954), or a sequence having 1, 2 or 3 substitutions therewith; and

The PBS sequence comprises a sequence of at least 8 nucleotides from the 5' end of the sequence according to GAGTCAGGTGCACCATG (SEQ ID NO: 19431), or a sequence having 1 substitution therewith.

A15. The template RNA as described in Example A1, wherein

The gRNA scaffold had a sequence according to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 11,012).

wherein the heterologous subject sequence comprises a sequence of at least 8 nucleotides from the 3' end of the sequence according to AGTAACGGCAGACTTCTCTTCAG (SEQ ID NO: 20954); and

The PBS sequence comprises a sequence of at least 8 nucleotides from the 5' end of the sequence according to GAGTCAGGTGCACCATG (SEQ ID NO: 19431).

A16. The template RNA as described in any of the preceding embodiments, which does not comprise a sequence according to GCATGGTGCACCTGACTCCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAGACTTCTCCACAGGAGTCAGGTGCAC (SEQ ID NO: XXX).

A17. A template RNA comprising from 5' to 3':

(i) a gRNA spacer complementary to the first part of the human HBB gene, wherein the gRNA spacer has a nucleotide sequence comprising GTAACGGCAGACTTCTCCAC (SEQ ID NO: 19971), or a nucleotide sequence having 1 substitution relative thereto;

(ii) a gRNA scaffold that binds to the Cas domain of a gene modifying polypeptide;

(iii) a heterologous subject sequence comprising a mutation region for introducing a mutation into the second portion of the human HBB gene, and

(iv) a primer binding site (PBS) sequence comprising at least 5 bases having 100% identity with the third portion of the human HBB gene.

A18. The template RNA as described in Example A17, wherein the gRNA spacer has a nucleotide sequence comprising GTAACGGCAGACTTCTCCAC (SEQ ID NO: 19971).

A19. The template RNA of embodiment A17 or A18, wherein the gRNA scaffold has a sequence according to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 11,012), or a sequence having at least 90% identity thereto.

A20. The template RNA of any one of embodiments A17-19, wherein the gRNA scaffold has a sequence according to GTTTTAGGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 11,012).

A21. The template RNA of any one of embodiments A17-20, wherein the heterologous object sequence comprises a sequence of at least 8 nucleotides from the 3' end of the sequence according to CCATGGTGCACCTGACTCCTGAG (SEQ ID NO: 20956) or CCATGGTGCACCTGACTCCTGCG (SEQ ID NO: 21906), or a sequence having 1, 2 or 3 substitutions therefrom.

A22. The template RNA of any one of embodiments A17-21, wherein the heterologous subject sequence comprises a sequence of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides from the 3' end of the sequence according to CCATGGTGCACCTGACTCCTGAG (SEQ ID NO: 20956) or CCATGGTGCACCTGACTCCTGCG (SEQ ID NO: 21906), or a sequence having 1, 2 or 3 substitutions therefrom.

A23. The template RNA of any one of embodiments A17-22, wherein the heterologous object sequence comprises a sequence of at least 8 nucleotides from the 3' end of the sequence according to CCATGGTGCACCTGACTCCTGAG (SEQ ID NO: 20956) or CCATGGTGCACCTGACTCCTGCG (SEQ ID NO: 21906).

A24. The template RNA of any one of embodiments A17-23, wherein the heterologous object sequence comprises a sequence of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides from the 3' end of the sequence according to CCATGGTGCACCTGACTCCTGAG (SEQ ID NO: 20956) or CCATGGTGCACCTGACTCCTGCG (SEQ ID NO: 21906).

A25. The template RNA of any one of embodiments A17-24, wherein the PBS sequence comprises a sequence of at least 8 nucleotides from the 5' end of the sequence according to GAGAAGTCTGCCGTTAC (SEQ ID NO: 20957), or a sequence having 1 substitution relative thereto.

A26. The template RNA of any one of embodiments A17-25, wherein the PBS sequence comprises a sequence of at least 8 nucleotides from the 5' end of the sequence according to GAGAAGTCTGCCGTTAC (SEQ ID NO: 20957).

A27. The template RNA of any one of embodiments A17-26, wherein the PBS sequence comprises a sequence of 9, 10, 11, 12, 13, 14, 15, 16 or 17 nucleotides from the 5' end of the sequence according to GAGAAGTCTGCCGTTAC (SEQ ID NO: 20957), or a sequence having 1 substitution relative thereto.

A28. The template RNA of any one of embodiments A17-27, wherein the PBS sequence comprises a sequence of 9, 10, 11, 12, 13, 14, 15, 16 or 17 nucleotides from the 5' end of the sequence according to GAGAAGTCTGCCGTTAC (SEQ ID NO: 20957).

A29. The template RNA of any one of embodiments A17-28, which does not comprise a sequence according to GTAACGGCAGACTTCTCCACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCGACTCCTGaGGAGAAGTCTGCC (SEQ ID NO: YYY).

A30. The template RNA of any one of the preceding embodiments, wherein the mutation region comprises a single nucleotide.

A31. The template RNA of any one of the preceding embodiments, wherein the mutation region is at least two nucleotides in length.

A32. The template RNA of any of the preceding embodiments, wherein the mutation region is up to 20 nucleotides in length and comprises one, two or three sequence differences relative to the second part of the human HBB gene.

A33. The template RNA as described in any of the preceding embodiments, wherein the mutation region comprises a first region designed to correct the pathogenic mutation in the HBB gene and a second region designed to inactivate the PAM sequence.

A34. The template RNA as described in any of the preceding embodiments, wherein the mutation region comprises a first region designed to correct a pathogenic mutation in the HBB gene and a second region designed to introduce a silent substitution.

A35. The template RNA of any of the preceding embodiments, which is configured to edit the E6V mutation in the human HBB gene.

A36. The template RNA of embodiment A35, which is configured to convert the E6V mutation to glutamine or alanine.

A37. The template RNA of any one of the preceding embodiments, comprising one or more chemically modified nucleotides.

A38. A gene modification system comprising:

The template RNA as described in any one of the preceding embodiments, and

A gene-modified polypeptide or a nucleic acid encoding the gene-modified polypeptide.

A39. A gene modification system as described in embodiment A38, wherein the gene modification polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,003, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity thereto.

A40. The gene modification system of embodiment A38, wherein the gene modification polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,020, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity thereto.

A41. A gene modification system as described in embodiment A38, wherein the gene modification polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,074, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity thereto.

A42. A gene modification system as described in embodiment A38, wherein the gene modification polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,113, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity thereto.

A43. A gene modification system as described in embodiment A38, wherein the gene modification polypeptide comprises a DNA binding domain having the sequence of a Cas9 nickase comprising an N863A mutation, such as a sequence according to SEQ ID NO: 11,096, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity thereto.

A44. A gene modification system as described in Example A38, which produces a first nick in the first strand of the human HBB gene.

A45. The gene modification system of embodiment A44, further comprising a second strand targeting gRNA, which directs the second cut to the second strand of the human HBB gene.

A46. The gene modification system of embodiment A45, wherein the first nick and the second nick are separated by 80-120 nucleotides.

A47. A gene modification system as described in Example A45, wherein the template RNA and the second-strand targeting gRNA are configured to produce an outward incision orientation.

A48. A gene modification system as described in embodiment A45, wherein the second strand targeting gRNA comprises a spacer sequence complementary to a human HBB gene having a sickle cell disease mutation, a wild-type sequence, or a Makassar variant.

A49. A method for modifying a target site in a human HBB gene in a cell, the method comprising contacting the cell with a gene modification system as described in Example 38, thereby modifying the target site in the human HBB gene in the cell.

A50. A method as described in embodiment A49, wherein correction of the mutation occurs in at least 30% of the target nucleic acid.

A51. A method for treating a subject having a disease or condition associated with a human HBB gene mutation, wherein the disease or condition is sickle cell disease (SCD), the method comprising administering to the subject a gene modification system as described in Example 38, thereby treating the subject having the disease or condition associated with the human HBB gene mutation.

A52. A template RNA comprising from 5' to 3':

(i) a gRNA spacer complementary to the first part of the human HBB gene, wherein the gRNA spacer has a nucleotide sequence comprising the core nucleotides of the gRNA spacer sequence of Table 1, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the gRNA spacer, or a nucleotide sequence having 1, 2 or 3 substitutions relative to the nucleotide sequence;

(ii) a gRNA scaffold that binds to the Cas domain of the gene modifying polypeptide;

(iii) a heterologous subject sequence comprising a mutation region for correcting a mutation in the second portion of the human HBB gene, and

(iv) a primer binding site (PBS) sequence comprising at least 5 bases having 100% identity with the third portion of the human HBB gene.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 depicts a gene modification system as described herein. The left-hand figure shows a gene modification polypeptide comprising a Cas nickase domain (e.g., spCas9 N863A) and a reverse transcriptase domain (RT domain) connected by a linker. The right-hand figure shows a template RNA, which comprises a gRNA spacer, a gRNA scaffold, a heterologous object sequence, and a primer binding site sequence (PBS sequence) from 5′ to 3′. The heterologous object sequence may comprise a mutation region comprising one or more sequence differences relative to the target site. The heterologous object sequence may also comprise a pre-editing homology region and a post-editing homology region flanking the mutation region. Without wishing to be bound by theory, it is believed that the gRNA spacer of the template RNA binds to the second strand of the target site in the genome, and the gRNA scaffold of the template RNA binds to the gene modification polypeptide, for example, positioning the gene modification polypeptide at the target site in the genome. It is believed that the Cas domain of the gene modification polypeptide produces a nick at the target site (e.g., the first strand of the target site), for example, allowing the PBS sequence to bind to a sequence adjacent to the site to be changed on the first strand of the target site. It is believed that the RT domain of the gene modifying polypeptide uses the first strand of the target site that binds to the complementary sequence (comprising the PBS sequence of the template RNA) as a primer and the heterologous subject sequence of the template RNA as a template, for example, polymerizing a sequence complementary to the heterologous subject sequence. Without wishing to be bound by theory, it is believed that reverse transcription can then proceed through the editing pre-homologous region, then through the mutation region, and then through the editing post-homologous region, thereby generating a DNA strand comprising the mutation specified by the heterologous subject sequence.

FIG2 is a pair of graphs showing the rewriting levels in 293T cells (left panel) and CD34+ primary human HSCs after transfection with a gene modification system comprising a gene modification polypeptide and various template RNAs.

FIG3 is a pair of graphs showing the rewriting levels in 293T cells (left panel) and CD34+ primary human HSCs after transfection with a gene modification system comprising a gene modification polypeptide and various template RNAs.

FIG. 4 shows graphs showing the editing percentage in primary human fibroblasts after electroporation with a gene modification system comprising tgRNA14 with or without a second nick.

5 shows the percentage of editing in wild-type human primary fibroblasts (for installation of the Makassar mutation) and sickle human primary fibroblasts (for installation of the wild-type sequence) after electroporation with a gene modification system comprising tgRNA14 with or without a second nick.

The graph in Figure 6 shows the percentage of rewriting achieved using RNAV209-013 or RNAV214-040 gene modifying polypeptides with the indicated template RNAs.

The graph in FIG7 shows the amount of Fah mRNA relative to the wild type when the template RNA is used with the RNAV209-013 or RNAV214-040 gene modifying polypeptide.

The middle graph of FIG8 shows the percentage of Cas9-positive hepatocytes 6 hours after administration of LNPs containing various gene-modifying polypeptides and template RNA.

FIG. 9 shows the graph showing the rewriting levels in liver samples 6 days after administration of LNPs containing various gene-modifying polypeptides and template RNA.

The middle graph of FIG. 10 shows that after administration of LNPs containing various gene-modified polypeptides and template RNA, wild-type Fah mRNA was restored in liver samples compared to heterozygous littermates.

The graph in Figure 11 shows the distribution of Fah protein in liver samples after administration of LNPs containing various gene-modified polypeptides and template RNA.

Figure 12 is a series of Western blots showing Cas9-RT expression 6 hours after infusion of Cas9-RT mRNA + TTR guide LNP. Each lane represents an individual animal, with 20 μg of tissue homogenate added to each lane. The positive control is from an in vitro cell experiment expressing Cas9-RT (previously described). GAPDH was used as a loading control for each sample. n = 4 per group, vehicle or treated.

Figure 13 shows gene editing of the TTR locus after treatment with Cas9-RT mRNA+TTR-guided LNPs. The level of indels detected at the TTR locus was measured by TIDE analysis of Sanger sequencing of the protospacer-targeted TTR locus.

The graph in Figure 14 shows that TTR serum levels decreased after treatment with Cas9-RT mRNA + TTR guide LNPs. Circulating TTR levels were measured 5 days after treatment of mice with LNPs encapsulating Cas9-RT + TTR guide RNA.

Figure 15, middle panel shows Cas9-RT expression after infusion of Cas9-RT mRNA + TTR guide LNPs. Relative expression was quantified by ProteinSimple Jess capillary electrophoresis western blot. Numbers in symbols are the number of animals in the group. Vehicle n=2, Cas9-RT + TTR guide n=3.

Figure 16 shows gene editing of the TTR locus after infusion of Cas9-RT mRNA + TTR guide LNP. The level of indels detected at the TTR locus was measured by amplicon sequencing of the protospacer-targeted TTR locus. The liver of each animal was biopsied 8 different times, where amplicon sequencing measured the percentage of reads showing indels.

FIG. 17 , in which a graph is shown, shows the average perfect rewriting levels in primary human HSCs after transfection with various gene modifying polypeptides and template RNA.

The graphs in Figures 18A and 18B show the average perfect rewriting levels in primary human HSCs after transfection with various gene modifying polypeptides and template RNA comprising an HBB5 spacer (Figure 18A) or an HBB8 spacer (Figure 18B).

Figures 19A and 19B are heat maps (Figure 19A) and graphs (Figure 19B) showing average perfect rewriting levels in primary human HSCs following transfection with various gene modifying polypeptides and template RNA comprising either the HBB5 spacer (Figure 19A) or the HBB8 spacer (Figure 19B).

Figures 20A-20C show the average perfect rewriting levels in primary human HSCs after transfection with various gene modifying polypeptides and template RNA comprising an HBB5 spacer (Figures 20A and 20C) or an HBB8 spacer (Figure 20B).

Figures 21A and 21B are a pair of graphs showing the perfect rewriting level in primary human HSCs (Figure 21A) and the percentage of HSC subpopulations (Figure 21B) after transfection with various gene modifying polypeptides and template RNA.

Figures 22A and 22B show perfect reprogramming levels in primary human HSC subsets following transfection with various gene modifying polypeptides and template RNA.

The graphs in Figures 23A-23C show the total colony number (Figure 23A), colony number (Figure 23B), and percentage of enucleated CD235+ cells (Figure 23C) after transfection with various gene-modifying polypeptides and template RNA.

DETAILED DESCRIPTION

definition

As used herein, the term "expression cassette" refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a nucleic acid molecule of the present invention.

As used herein, "gRNA spacer" refers to a nucleic acid portion that has complementarity with a target nucleic acid and that, together with a gRNA scaffold, can target a Cas protein to a target nucleic acid.

As used herein, "gRNA scaffold" refers to a nucleic acid portion that can bind to a Cas protein and can target the Cas protein to a target nucleic acid together with a gRNA spacer. In some embodiments, the gRNA scaffold comprises a crRNA sequence, a tetraloop, and a tracrRNA sequence.

As used herein, "genetically modified polypeptide" refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in a host cell). In some embodiments, the gene-modified polypeptide is capable of integrating a sequence substantially independent of the host machinery. In some embodiments, the gene-modified polypeptide integrates the sequence into a random position in the genome, and in some embodiments, the gene-modified polypeptide integrates the sequence into a specific target site. In some embodiments, the gene-modified polypeptide comprises one or more domains that together promote 1) binding to the template nucleic acid, 2) binding to the target DNA molecule, and 3) promoting the integration of at least a portion of the template nucleic acid into the target DNA. Genetically modified polypeptides include naturally occurring polypeptides and engineered variants of the aforementioned polypeptides, for example, these variants have one or more amino acid substitutions relative to the naturally occurring sequence. Genetically modified polypeptides also include heterologous constructs, for example, wherein one or more of the above-mentioned domains are heterologous to each other, whether by heterologous fusions (or other conjugates) of domains that are otherwise wild-type, and fusions of modified domains, for example, by replacement or fusion of heterologous subdomains or other substituted domains. Exemplary genetically modified polypeptides that can be used for the methods provided herein, systems comprising them, and methods using them are described, for example, in PCT/US2021/020948, which are incorporated herein by reference with respect to genetically modified polypeptides comprising retroviral reverse transcriptase domains. In some embodiments, the genetically modified polypeptide integrates a sequence into a gene. In some embodiments, the genetically modified polypeptide integrates a sequence into a sequence outside a gene. As used herein, "genetically modified system" refers to a system comprising a genetically modified polypeptide and a template nucleic acid.

As used herein, the term "domain" refers to the structure of a biomolecule that contributes to a specific function of the biomolecule. A domain may comprise a continuous region (e.g., a continuous sequence) or different non-continuous regions (e.g., a non-continuous sequence) of a biomolecule. Examples of protein domains include, but are not limited to, endonuclease domains, DNA binding domains, reverse transcription domains; examples of domains of nucleic acids are regulatory domains, such as transcription factor binding domains. In some embodiments, a domain (e.g., a Cas domain) may comprise two or more smaller domains (e.g., a DNA binding domain and an endonuclease domain).

As used herein, the term "exogenous" when used with respect to a biomolecule (e.g., a nucleic acid sequence or a polypeptide) means that the biomolecule is artificially introduced into a host genome, cell, or organism. For example, a nucleic acid added to an existing genome, cell, tissue, or subject using recombinant DNA technology or other methods is exogenous to the existing nucleic acid sequence, cell, tissue, or subject.

As used herein, "first strand" and "second strand" used to describe a single DNA strand of a target DNA distinguish the two DNA strands based on the strand on which the reverse transcriptase domain initiates polymerization, e.g., based on where target-triggered synthesis is initiated. The first strand refers to the strand of the target DNA on which the reverse transcriptase domain initiates polymerization, e.g., where target-triggered synthesis is initiated. The second strand refers to the other strand of the target DNA. The first and second strand designations are otherwise not descriptive of the target site DNA strands; for example, in some embodiments, the first and second strands are nicked by a polypeptide described herein, but the designations of the "first" and "second" chains are not related to the order in which such nicking occurs.

When used herein to describe a first element with reference to a second element, the term "heterologous" means that the first element and the second element do not exist in the arrangement as described in nature. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to a polypeptide, nucleic acid molecule or a part of a polypeptide or nucleic acid molecule sequence that is not natural (a) for a cell expressing it, (b) a polypeptide or nucleic acid molecule or a part of a polypeptide or nucleic acid molecule that has been changed or mutated relative to its natural state, or (c) a polypeptide or nucleic acid molecule with expression that is changed compared to the natural expression level under similar conditions. For example, a heterologous regulatory sequence (e.g., a promoter, an enhancer) can be used to regulate the expression of a gene or nucleic acid molecule in a manner different from that in which a gene or nucleic acid molecule is usually expressed in nature. In another example, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or a nucleic acid encoding a DNA binding domain of a polypeptide) can be arranged relative to other domains, or can be a different sequence or relative to other domains or parts of a polypeptide or its encoding nucleic acid from different sources. In certain embodiments, the heterologous nucleic acid molecule may be present in the native host cell genome, but may have an altered expression level or have a different sequence or both. In other embodiments, the heterologous nucleic acid molecule may not be endogenous to the host cell or host genome, but is introduced into the host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may be integrated into the host genome, or may exist transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vectors, plasmids or other self-replicating vectors) as extrachromosomal genetic material.

As used herein, "insertion" of a sequence into a target site refers to a net addition of a DNA sequence at the target site, e.g., where there are new nucleotides in the heterologous subject sequence that have no homologous position in the unedited target site. In some embodiments, nucleotide alignment of the PBS sequence and the heterologous subject sequence with the target nucleic acid sequence will result in alignment gaps in the target nucleic acid sequence.

As used herein, a "deletion" of a heterologous subject sequence at a target site refers to a net loss of a DNA sequence at the target site, e.g., a nucleotide present in the unedited target site that has no homologous position in the heterologous subject sequence. In some embodiments, nucleotide alignment of a PBS sequence and a heterologous subject sequence with a target nucleic acid sequence will result in alignment gaps in the molecule comprising the PBS sequence and the heterologous subject sequence.

As used herein, the term "inverted terminal repeat" or "ITR" refers to the cis-elements of the AAV virus, so named because of its symmetry. These elements promote efficient multiplication of the AAV genome. The minimal elements hypothesized to function as ITRs are the Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3', for AAV2; SEQ ID NO: 4601) and the terminal dissociation site (TRS; 5'-AGTTGG-3', for AAV2; SEQ ID NO: 4602) plus a variable palindromic sequence that allows hairpin formation. According to the present invention, ITRs contain at least these three elements (RBS, TRS, and a sequence that allows hairpin formation). In addition, in the present invention, the term "ITR" refers to ITRs of known natural AAV serotypes (e.g., ITRs of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV), chimeric ITRs formed by fusion of ITR elements derived from different serotypes, and functional variants thereof. "Functional variant" refers to a sequence having at least 80%, 85%, 90%, preferably at least 95% sequence identity with a known ITR, allowing the sequence comprising said ITR to multiply in the presence of Rep protein.

As used herein, the term "mutation region" refers to a region in a template RNA that has one or more sequence differences relative to a corresponding sequence in a target nucleic acid. The sequence difference may include, for example, a substitution, insertion, frameshift, or deletion.

When applied to nucleic acid sequences, the term "mutated" means that the nucleotides in the nucleic acid sequence are inserted, deleted or changed compared to a reference (e.g., natural) nucleic acid sequence. A single change (point mutation) can be made at the locus, or multiple nucleotides can be inserted, deleted or changed at a single locus. In addition, one or more changes can be made at any number of loci within the nucleic acid sequence. Nucleotide sequences can be mutated by any method known in the art.

Nucleic acid molecules refer to both RNA and DNA molecules, including but not limited to complementary DNA ("cDNA"), genomic DNA ("gDNA"), and messenger RNA ("mRNA"), and also include synthetic nucleic acid molecules, such as chemically synthesized or recombinantly produced nucleic acid molecules, such as RNA templates as described herein. Nucleic acid molecules can be double-stranded or single-stranded, circular or linear. If single-stranded, the nucleic acid molecule can be a sense strand or an antisense strand. Unless otherwise indicated, and as an example of all sequences described herein in the general format "SEQ ID NO:", "a nucleic acid comprising SEQ ID NO: 1" refers to a nucleic acid, at least a portion thereof, having (i) a sequence of SEQ ID NO: 1 or (ii) a sequence complementary to SEQ ID NO: 1. The choice between the two depends on the context in which SEQ ID NO: 1 is used. For example, if the nucleic acid is used as a probe, the choice between the two depends on the requirement that the probe is complementary to the desired target. As will be readily appreciated by those skilled in the art, the nucleic acid sequences disclosed herein may be chemically or biochemically modified or may contain non-natural or derived nucleotide bases. Such modifications include, for example, tags, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as uncharged connections (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged connections (e.g., phosphorothioates, phosphorodithioates, etc.), side chain moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified connections (e.g., α-anomeric nucleic acids, etc.). Chemically modified bases (see, e.g., Table 13), backbones (see, e.g., Table 14), and modified caps (see, e.g., Table 15) are also included. Also included are synthetic molecules that simulate the ability of polynucleotides to bind to a specified sequence via hydrogen bonds and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide connections replace phosphate connections in the backbone of the molecule, such as peptide nucleic acids (PNAs). Other modifications may include, for example, analogs in which the ribose ring contains a bridging moiety or other structure (e.g., modifications found in "locked" nucleic acids (LNAs)). In various embodiments, the nucleic acid is operably associated with additional genetic elements, such as one or more tissue-specific expression control sequences (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences) and additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, such as AAV ITRs) and tandem repeats, inverted repeats/direct repeats, homology regions (segments with varying degrees of homology to the target DNA), untranslated regions (UTRs) (5′, 3′, or 5′ and 3′ UTRs), and various combinations of the foregoing. The nucleic acid elements of the systems provided by the present invention can be provided in a variety of topological structures, including single-stranded, double-stranded, circular, linear, linear with open ends, linear with closed ends, and special versions of these, such as doggybone DNA (dbDNA), closed end DNA (ceDNA).

As used herein, a "gene expression unit" is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably linked to at least one effector sequence. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, if a promoter or enhancer affects the transcription or expression of a coding sequence, the promoter or enhancer is operably linked to the coding sequence. Operably linked DNA sequences can be continuous or non-continuous. In the case where it is necessary to connect two protein coding regions, the operably linked sequences can be in the same reading frame.

As used herein, the term "host genome" or "host cell" refers to a cell and/or its genome into which proteins and/or genetic materials have been introduced. It should be understood that such terms are intended not only to refer to specific subject cells and/or genomes, but also to the progeny of such cells and/or the genomes of the progeny of such cells. Because some modifications may occur in offspring due to mutations or environmental influences, such progeny may actually be different from parental cells, but are still included in the scope of the term "host cell" as used herein. The host genome or host cell can be an isolated cell or cell line grown in culture, or a genomic material isolated from such a cell or cell line, or can be a host cell or host genome constituting a living tissue or organism. In some cases, the host cell can be an animal cell or a plant cell, for example, as described herein. In some cases, the host cell can be a mammalian cell, a human cell, a bird cell, a reptile cell, a cattle cell, a horse cell, a pig cell, a goat cell, a sheep cell, a chicken cell or a turkey cell. In some cases, the host cell can be a corn cell, a soybean cell, a wheat cell or a rice cell.

As used herein, "operable association" describes a functional relationship between two nucleic acid sequences (e.g., 1) a promoter and 2) a heterologous subject sequence), and in such instances means that the orientation of the promoter and the heterologous subject sequence (e.g., a target gene) is such that under appropriate conditions, the promoter drives the expression of the heterologous subject sequence. For example, the template nucleic acid carrying the promoter and the heterologous subject sequence can be single-stranded, such as in (+) or (-) orientation. The "operable association" between the promoter and the heterologous subject sequence in the template means that, regardless of whether the template nucleic acid is transcribed in a specific state, when it is in the appropriate state (e.g., in the (+) orientation, in the presence of the required catalytic factors and NTPs, etc.), it is accurately transcribed. Operable association is similarly applicable to other nucleic acid pairs, including other tissue-specific expression control sequences (e.g., enhancers, repressors, and microRNA recognition sequences), IR/DR, ITR, UTR, or homology regions and heterologous subject sequences or sequences encoding retroviral RT domains.

As used herein, the term "primer binding site sequence" or "PBS sequence" refers to a portion of a template RNA that is capable of binding to a region contained in a target nucleic acid sequence. In some cases, the PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases that are 100% identical to a region contained in a target nucleic acid sequence. In some embodiments, the primer region comprises at least 5, 6, 7, or 8 bases that are 100% identical to a region contained in a target nucleic acid sequence. Without wishing to be bound by theory, in some embodiments, when the template RNA comprises a PBS sequence and a heterologous object sequence, the PBS sequence binds to a region contained in a target nucleic acid sequence, thereby allowing the reverse transcriptase domain to use the region as a primer for reverse transcription and use the heterologous object sequence as a template for reverse transcription.

As used herein, "stem-loop sequence" refers to a nucleic acid sequence (e.g., RNA sequence) having sufficient self-complementarity to form a stem-loop, e.g., having a stem comprising at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs, and having a loop having at least three (e.g., four) base pairs. The stem may contain mismatches or bulges.

As used herein, "tissue-specific expression control sequence" means a nucleic acid element that increases or decreases the transcript level of a heterologous object sequence in a tissue-specific manner in a target tissue, for example, in one or more target tissues, relative to one or more off-target tissues. In some embodiments, a tissue-specific expression control sequence preferentially drives or inhibits the transcription, activity or half-life of a transcript containing a heterologous object sequence in a tissue-specific manner in a target tissue, for example, in one or more target tissues, relative to one or more off-target tissues. Exemplary tissue-specific expression control sequences include tissue-specific promoters, repressors, enhancers, or combinations thereof, and tissue-specific microRNA recognition sequences. Tissue-specificity refers to on-target (one or more tissues that expect or tolerate the expression or activity of a template nucleic acid) and off-target (one or more tissues that do not expect or tolerate the expression or activity of a template nucleic acid). For example, relative to off-target tissues, a tissue-specific promoter preferentially drives expression in the target tissue. On the contrary, relative to the target tissue, a microRNA that binds to a tissue-specific microRNA recognition sequence preferentially expresses in off-target tissues, thereby reducing the expression of template nucleic acids in off-target tissues. Therefore, promoters and microRNA recognition sequences specific to the same tissue (e.g., target tissue) have different functions with respect to the transcription, activity, or half-life of the associated sequences in the tissue (promotion and inhibition, respectively, with consistent expression levels, i.e., high levels of microRNA in off-target tissues and low levels in on-target tissues, while the promoter drives high expression in on-target tissues and low expression in off-target tissues).

Table of contents

1) Introduction

2) Gene modification system

a) Polypeptide components of the gene modification system

i) Writing domain

ii) Endonuclease domain and DNA binding domain

(1) Gene-modifying polypeptides containing Cas domains

(2) TAL effectors and zinc finger nucleases

iii) Connectors

iv) Localization sequence of gene modification system

v) Evolutionary variants of gene-modified polypeptides and systems

vi) Inteins

vii) Additional domains

b) Template nucleic acid

i) gRNA spacer and gRNA scaffold

ii) Heterogeneous object sequence

iii) PBS sequence

iv) Exemplary template sequences

c) gRNA with inducible activity

d) Circular RNA and ribozymes in gene modification systems

e) Target nucleic acid site

f) Second chain cut

3) Generation of compositions and systems

4) Therapeutic applications

5) Administration and delivery

a) Tissue-specific activity/administration

i) Promoter

ii) MicroRNA

b) Viral vectors and their components

c) AAV administration

d) Lipid Nanoparticles

6) Kits, products and pharmaceutical compositions

7) Chemistry, Manufacturing and Controls (CMC)

introduction

The present disclosure relates to methods for treating sickle cell disease (SCD) and compositions for targeting, editing, modifying or manipulating a DNA sequence at one or more positions in a DNA sequence in a cell, tissue or subject, for example, in vivo or in vitro (e.g., inserting a heterologous subject sequence into a target site in a mammalian genome). The heterologous subject DNA sequence can include, for example, a substitution.

More particularly, the present disclosure provides methods for treating SCD that use a reverse transcriptase-based system to alter a genomic DNA sequence of interest, e.g., by inserting one or more nucleotides into, deleting one or more nucleotides from, or substituting one or more nucleotides in a sequence of interest.

The present disclosure provides, in part, methods for treating SCD, which use a gene modification system comprising a gene modification polypeptide component and a template nucleic acid (e.g., template RNA) component. In some embodiments, the gene modification system can be used to introduce changes to a target site in a genome. In some embodiments, the gene modification polypeptide component comprises a writing domain (e.g., a reverse transcriptase domain), a DNA binding domain, and an endonuclease domain (e.g., a nickase domain). In some embodiments, the template nucleic acid (e.g., template RNA) comprises a sequence that binds to a target site in a genome (e.g., binding to the second strand of the target site) (e.g., a gRNA spacer), a sequence that binds to the gene modification polypeptide component (e.g., a gRNA scaffold), a heterologous object sequence, and a PBS sequence. Without wishing to be bound by theory, it is believed that the template nucleic acid (e.g., template RNA) binds to the second strand of the target site in the genome and binds to the gene modification polypeptide component (e.g., locating the polypeptide component to the target site in the genome). It is believed that the endonuclease (e.g., nickase) of the gene modification polypeptide component cuts the target site (e.g., the first strand of the target site), for example, allowing the PBS sequence to bind to a sequence adjacent to the site to be changed on the first strand of the target site. It is believed that the writing domain (e.g., reverse transcriptase domain) of the polypeptide component uses the PBS sequence comprising the template nucleic acid as a primer and the heterologous subject sequence of the template nucleic acid as a template to, for example, polymerize the first strand of the target site that binds to the complementary sequence of the sequence complementary to the heterologous subject sequence. Without wishing to be bound by theory, it is believed that selecting an appropriate heterologous subject sequence can result in substitution, deletion, and/or insertion of one or more nucleotides at the target site.

Gene modification system

In some embodiments, the gene modification system described herein comprises: (A) a gene modification polypeptide or a nucleic acid encoding the gene modification polypeptide, wherein the gene modification polypeptide comprises (i) a reverse transcriptase domain and (x) a nuclease domain containing a DNA binding function or (y) an nuclease domain and a separate DNA binding domain; and (B) a template RNA. In some embodiments, the gene modification polypeptide is a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in a host cell), substantially independent of the host machine. For example, a gene modification protein may comprise a DNA binding domain, a reverse transcriptase domain, and an endonuclease domain. In some embodiments, the DNA binding function may involve an RNA component that guides the protein to a DNA sequence (e.g., a gRNA spacer). In other embodiments, the gene modification polypeptide may comprise a reverse transcriptase domain and an endonuclease domain. The RNA template element of the gene modification system is typically heterologous to the gene modification polypeptide element and provides a target sequence to be inserted (reverse transcribed) into the host genome. In some embodiments, the gene modification polypeptide is capable of targeted reverse transcription. In some embodiments, the genetically modified polypeptide is capable of second strand synthesis.

In some embodiments, the gene modification system is combined with a second polypeptide. In some embodiments, the second polypeptide may include a nucleic acid endonuclease domain. In some embodiments, the second polypeptide may include a polymerase domain, such as a reverse transcriptase domain. In some embodiments, the second polypeptide may include a DNA-dependent DNA polymerase domain. In some embodiments, the second polypeptide helps to complete genome editing, for example, by helping second strand synthesis or DNA repair dissociation.

Functional gene modification polypeptides can be composed of unrelated DNA binding domains, reverse transcription domains, and endonuclease domains. This modular structure allows the combination of functional domains, such as dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), FokI (endonuclease). In some embodiments, multiple functional domains can be from a single protein, for example, Cas9 or Cas9 nickase (DNA binding, endonuclease).

In some embodiments, the genetically modified polypeptide comprises one or more domains that collectively promote 1) binding to the template nucleic acid, 2) binding to the target DNA molecule, and 3) promoting the integration of at least a portion of the template nucleic acid into the target DNA. In some embodiments, the genetically modified polypeptide is an engineered polypeptide, for example, having one or more amino acid substitutions relative to a naturally occurring sequence. In some embodiments, the genetically modified polypeptide comprises two or more domains that are heterologous to each other, for example, by heterologous fusion (or other conjugates) of domains that are otherwise wild-type, or fusions of modified domains, for example, by replacement or fusion of heterologous subdomains or other substituted domains. For example, in some embodiments, one or more of the following: the RT domain is heterologous to the DBD; the DBD is heterologous to the endonuclease domain; or the RT domain is heterologous to the endonuclease domain.

In some embodiments, the template RNA molecule used in the system comprises from 5′ to 3′ (1) a gRNA spacer; (2) a gRNA scaffold; (3) a heterologous target sequence; and (4) a primer binding site (PBS) sequence. In some embodiments:

(1) is a gRNA spacer of about 18-22 nt (e.g., 20 nt)

(2) is a gRNA scaffold comprising one or more hairpin loops (e.g., 1, 2, or 3 loops) for associating the template with a Cas domain, such as a nickase Cas9 domain. In some embodiments, the gRNA scaffold comprises the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC (SEQ ID NO: 5008) from 5′ to 3′.

(3) In some embodiments, the heterologous subject sequence length is, for example, 7-74, such as 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nt or 80-90 nt. In some embodiments, the first (most 5′) base of the sequence is not C.

(4) In some embodiments, the PBS sequence that binds to the target priming sequence after nicking is, for example, 3-20 nt, for example, 7-15 nt, for example, 12-14 nt. In some embodiments, the PBS sequence has a GC content of 40%-60%.

In some embodiments, a second gRNA associated with the system may help drive full integration. In some embodiments, the second gRNA can target a position 0-200 nt from the first strand nick, such as 0-50, 50-100, 100-200 nt from the first strand nick. In some embodiments, the second gRNA can only bind to its target sequence after editing, for example, the gRNA binds to a sequence that is present in the heterologous subject sequence but not in the initial target sequence.

In some embodiments, the gene modification system described herein is used to edit in HEK293, K562, U2OS, or HeLa cells. In some embodiments, the gene modification system is used to edit in primary cells (e.g., primary cortical neurons from E18.5 mice).

In some embodiments, a genetically modified polypeptide as described herein comprises a reverse transcriptase or RT domain comprising a MoMLV RT sequence or a variant thereof (e.g., as described herein). In embodiments, the MoMLV RT sequence comprises one or more mutations selected from the group consisting of D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, and K103L. In embodiments, the MoMLV RT sequence comprises a combination of mutations (e.g., D200N, L603W, and T330P), optionally further comprising T306K and/or W313F.

In some embodiments, the endonuclease domain (e.g., as described herein) nCas9, e.g., comprises a N863A mutation (e.g., in spCas9) or a H840A mutation.

In some embodiments, the heterologous subject sequence (e.g., of a system as described herein) is about 1-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, or more nucleotides in length.

In some embodiments, the RT and the endonuclease domain are connected by a flexible linker, for example, comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 5006).

In some embodiments, the endonuclease domain is at the N-terminus relative to the RT domain. In some embodiments, the endonuclease domain is at the C-terminus relative to the RT domain.

In some embodiments, the system incorporates the heterologous subject sequence into the target site via TPRT, eg, as described herein.

In some embodiments, the gene-modified polypeptide comprises a DNA binding domain. In some embodiments, the gene-modified polypeptide comprises an RNA binding domain. In some embodiments, the RNA binding domain comprises an RNA binding domain of an element of a B-box protein, MS2 coat protein, dCas, or a sequence in the table herein. In some embodiments, the RNA binding domain can bind to the template RNA with greater affinity than the reference RNA binding domain.

In some embodiments, the genetic modification system is capable of producing at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides) insertions in the target site. In some embodiments, the genetic modification system is capable of producing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides) insertions in the target site. In some embodiments, the gene modification system is capable of producing at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases) insertion in the target site. In some embodiments, the gene modification system is capable of producing at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides) deletion. In some embodiments, the gene modification system is capable of producing at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides) deletion. In some embodiments, the gene modification system is capable of producing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides) deletion. In some embodiments, the gene modification system is capable of producing at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases) deletion. In some embodiments, the gene modification system is capable of producing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotide substitutions in the target site. In some embodiments, the gene modification system is capable of producing 1-2, 2-3, 3-4, 4-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 nucleotide substitutions in the target site.

In some embodiments, the substitution is a transposition mutation. In some embodiments, the substitution is a transversion mutation. In some embodiments, the substitution converts adenine to thymine, adenine to guanine, adenine to cytosine, guanine to thymine, guanine to cytosine, guanine to adenine, thymine to cytosine, thymine to adenine, thymine to guanine, cytosine to adenine, cytosine to guanine, or cytosine to thymine.

In some embodiments, insertion, deletion, substitution, or a combination thereof increases or decreases the expression (e.g., transcription or translation) of a gene. In some embodiments, insertion, deletion, substitution, or a combination thereof increases or decreases the expression (e.g., transcription or translation) of a gene by changing, adding, or deleting a sequence in a promoter or enhancer (e.g., a sequence that binds a transcription factor). In some embodiments, insertion, deletion, substitution, or a combination thereof changes the translation of a gene (e.g., changes the amino acid sequence), inserts or deletes a start or stop codon, changes or fixes the translation frame of a gene. In some embodiments, insertion, deletion, substitution, or a combination thereof changes the splicing of a gene, for example, by inserting, deleting, or changing a splicing acceptor or donor site. In some embodiments, insertion, deletion, substitution, or a combination thereof changes transcript or protein half-life. In some embodiments, insertion, deletion, substitution, or a combination thereof changes the localization of a protein in a cell (e.g., from the cytoplasm to mitochondria, from the cytoplasm to the extracellular space (e.g., adding a secretion tag)). In some embodiments, insertion, deletion, substitution, or a combination thereof changes (e.g., improves) protein folding (e.g., to prevent the accumulation of misfolded proteins). In some embodiments, insertion, deletion, substitution, or a combination thereof changes, increases, or decreases the activity of a gene, such as the activity of a protein encoded by a gene.

Exemplary gene-modifying polypeptides, systems comprising them, and methods of using them are described, for example, in PCT/US2021/020948, which is incorporated herein by reference with respect to retroviral RT domains, including the amino acid and nucleic acid sequences therein.

Exemplary gene-modified polypeptides and retroviral RT domain sequences are also described in, for example, International Application No. PCT/US21/20948 filed on March 4, 2021, such as Tables 30, 31, and 44 therein; the entire application is incorporated herein by reference with respect to retroviral RT, such as in the sequences and tables. Thus, the gene-modified polypeptides described herein may comprise an amino acid sequence or a domain thereof (e.g., a retroviral RT domain) according to any table mentioned in this paragraph, or a functional fragment or variant of any of the foregoing, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto.

In some embodiments, the polypeptide used in any system described herein can be a molecular reconstruction or genetic reconstruction of the aligned polypeptide sequence based on multiple homologous proteins. In some embodiments, the reverse transcriptase domain used in any system described herein can be a molecular reconstruction or genetic reconstruction, or can be modified at a specific residue based on the comparison of the reverse transcriptase domain from the same or different sources. Based on the accession number provided herein, the technician can, for example, compare polypeptides or nucleic acid sequences by using conventional sequence analysis tools such as basic local alignment search tools (BLAST) or CD-search (for conservative domain analysis). Molecular reconstruction can be created based on a consensus sequence, for example, using Ivics et al., Cell [cell] 1997, 501-510; Wagstaff et al., Molecular Biology and Evolution [molecular biology and evolution] 2013, 88-99 described in the method.

Peptide components of gene modification systems

In some embodiments, the gene modification polypeptide has the functions of DNA target site binding, template nucleic acid (e.g., RNA) binding, DNA target site cutting, and template nucleic acid (e.g., RNA) writing (e.g., reverse transcription). In some embodiments, each function is contained in different domains. In some embodiments, the function can be attributed to two or more domains (e.g., two or more domains display the function together). In some embodiments, two or more domains can have the same or similar functions (e.g., two or more domains each independently have a DNA binding function, such as for two different DNA sequences). In other embodiments, one or more domains may be able to achieve one or more functions, for example, the Cas9 domain can simultaneously achieve DNA binding and target site cutting. In some embodiments, these domains are all located in a single polypeptide. In some embodiments, the first domain is in a polypeptide and the second domain is in a second polypeptide. For example, in some embodiments, the sequence can be broken between the first polypeptide and the second polypeptide, for example, wherein the first polypeptide comprises a reverse transcriptase (RT) domain and wherein the second polypeptide comprises a DNA binding domain and an endonuclease domain, such as a nickase domain. As another example, in some embodiments, the first polypeptide and the second polypeptide each comprise a DNA binding domain (e.g., a first DNA binding domain and a second DNA binding domain). In some embodiments, the first and second polypeptides can be post-translationally bound together by splitting the intein to form a single gene-modified polypeptide.

In some aspects, the gene modifying polypeptide described herein comprises (e.g., the system described herein comprises a gene modifying polypeptide comprising): 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain of Table D, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical thereto, wherein the RT domain is located at the C-terminus of the Cas domain; and a linker located between the RT domain and the Cas domain, wherein the linker has a sequence from the same row as the RT domain in Table D, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical thereto.

In some embodiments, the RT domain has a sequence that is 100% identical to the RT domain of Table D, and the linker has a sequence that is 100% identical to the linker sequence from the same row as the RT domain in Table D. In some embodiments, the Cas domain comprises a sequence of Table 8, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical thereto. In some embodiments, the gene modifying polypeptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-3332 in the sequence listing, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical thereto.

In some embodiments, the gene-modified polypeptide comprises a GG amino acid sequence between the Cas domain and the linker, an AG amino acid sequence between the RT domain and the second NLS, and/or a GG amino acid sequence between the linker and the RT domain. In some embodiments, the gene-modified polypeptide comprises a sequence of SEQ ID NO: 4000 containing a first NLS and a Cas domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity thereto. In some embodiments, the gene-modified polypeptide comprises a sequence of SEQ ID NO: 4001 containing a second NLS, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity thereto.

Exemplary N-terminal NLS-Cas9 domains

Exemplary C-terminal sequences containing NLS

Writing domain (RT domain)

In certain aspects of the invention, the writing domain of the gene modification system has reverse transcriptase activity, also referred to as a reverse transcriptase domain (RT domain). In some embodiments, the RT domain comprises a RT catalytic portion and an RNA binding region (e.g., a region that binds to template RNA).

In some embodiments, the nucleic acid encoding the reverse transcriptase is changed from its native sequence to have a changed codon usage, for example, for human cells to improve. In some embodiments, the reverse transcriptase domain is a heterologous reverse transcriptase from a retrovirus. In some embodiments, the RT domain comprising a genetically modified polypeptide has been mutated from its original amino acid sequence, for example, with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 substitutions. In some embodiments, the RT domain is derived from the RT of a retrovirus, such as HIV-1 RT, Moloney murine leukemia virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous sarcoma virus (RSV) RT.

In some embodiments, the retroviral reverse transcriptase (RT) domain shows the enhanced stringency of the reverse transcription (TPRT) initiated by the target, for example, relative to the endogenous RT domain. In some embodiments, when the 3nt immediately upstream of the first strand nick in the target site, such as the genomic DNA of the triggering RNA template, has at least 66% or 100% complementarity with the homologous 3nt in the RNA template, the RT domain starts TPRT. In some embodiments, when there is less than 5nt mismatch (for example, less than 1,2,3,4 or 5nt mismatch) between the template RNA homology and the target DNA triggering reverse transcription, the RT domain starts TPRT. In some embodiments, the modification of the RT domain increases the stringency of the mismatch in the TPRT reaction initiation, for example, wherein relative to the wild-type (for example, unmodified) RT domain, the RT domain does not allow any mismatch or allows less mismatch in the triggering region. In some embodiments, the RT domain includes the HIV-1RT domain. In embodiments, the HIV-1 RT domain initiates lower levels of synthesis, even with three nucleotide mismatches relative to an alternative RT domain (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol [Journal of Molecular Biology] 407(5): 661-672 (2011); which is incorporated herein by reference in its entirety). In some embodiments, the RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, the RT domain is monomeric. In some embodiments, the RT domain naturally functions as a monomer or dimer (e.g., a heterodimer or homodimer). In some embodiments, the RT domain naturally functions as a monomer, e.g., derived from a virus in which it functions as a monomer. In embodiments, the RT domain is selected from the RT domains from murine leukemia virus (MLV; sometimes referred to as MoMLV) (e.g., P03355), porcine endogenous retrovirus (PERV) (e.g., UniProt Q4VFZ2), mouse mammary tumor virus (MMTV) (e.g., UniProt P03365), avian reticuloendotheliosis virus (AVIRE) (UniProtKB Accession No.: P03360), feline leukemia virus (FLV or FeLV) (e.g., UniProtKB Accession No.: P10273), Mason-Fischer monkey virus (MPMV) (e.g., UniProt P07572), bovine leukemia virus (BLV) (e.g., UniProt P03361), human T-cell leukemia virus-1 (HTLV-1) (e.g., UniProt P03362), human foamy virus (HFV) (e.g., UniProt P14350), simian foamy virus (SFV) (e.g., SFV3L) (e.g., UniProt P23074 or P27401), or bovine foamy/syncytial virus (BFV/BSV) (e.g., UniProt O41894), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto). In some embodiments, the RT domain is a dimer in its native function. In some embodiments, the RT domain is derived from a virus, wherein it functions as a dimer. In an embodiment, the RT domain is selected from an RT domain from avian sarcoma/leukemia virus (ASLV) (e.g., UniProt A0A142BKH1), Rous sarcoma virus (RSV) (e.g., UniProt P03354), avian myeloblastosis virus (AMV) (e.g., UniProt Q83133), human immunodeficiency virus type I (HIV-1) (e.g., UniProt P03369), human immunodeficiency virus type II (HIV-2) (e.g., UniProt P15833), simian immunodeficiency virus (SIV) (e.g., UniProt P05896), bovine immunodeficiency virus (BIV) (e.g., UniProt P19560), equine infectious anemia virus (EIAV) (e.g., UniProt P03371), or feline immunodeficiency virus (FIV) (e.g., UniProt P16088) (Herschhorn and HiziCell Mol Life Sci [Cellular and Molecular Life Sciences] 67(16): 2717-2747 (2010)), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, 80%, 90%, 95% or 99% identity thereto). In some embodiments, the native heterodimeric RT domain can also function as a homodimer. In some embodiments, the dimeric RT domain is expressed as a fusion protein, e.g., a homodimeric fusion protein or a heterodimeric fusion protein. In some embodiments, the RT function of the system is achieved by multiple RT domains (e.g., as described herein). In other embodiments, the multiple RT domains are fused or separated, e.g., on the same polypeptide or on different polypeptides.

In some embodiments, the gene modification system described herein includes an integrase domain, for example, wherein the integrase domain can be a part of the RT domain. In some embodiments, the RT domain (for example, as described herein) includes an integrase domain. In some embodiments, the RT domain (for example, as described herein) lacks an integrase domain, or includes an integrase domain inactivated by mutation or deletion. In some embodiments, the gene modification system described herein includes an RNase H domain, for example, wherein the RNase H domain can be a part of the RT domain. In some embodiments, the RNase H domain is not a part of the RT domain and is covalently connected by a flexible joint. In some embodiments, the RT domain (for example, as described herein) includes an RNase H domain, for example, an endogenous RNase H domain or a heterologous RNase H domain. In some embodiments, the RT domain (for example, as described herein) lacks an RNase H domain. In some embodiments, the RT domain (for example, as described herein) includes an RNase H domain of addition, deletion, mutation or exchange of a heterologous RNase H domain. In some embodiments, the polypeptide includes an inactivated endogenous RNase H domain. In some embodiments, the endogenous RNase H domain is genetically removed from one of the other domains of the polypeptide so that it is not included in the polypeptide, e.g., the endogenous RNase H domain is partially or completely truncated from the included domain. In some embodiments, mutations in the RNase H domain produce a polypeptide that exhibits lower RNase activity, e.g., as determined by the methods described in Kotewicz et al. Nucleic Acids Res 16(1):265-277 (1988), which is incorporated herein by reference in its entirety, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to an otherwise similar domain without the mutation. In some embodiments, RNase H activity is eliminated.

In some embodiments, the RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation. For example, in some embodiments, a YADD or YMDD motif in a RT domain (e.g., in a reverse transcriptase) is replaced with a YVDD. In embodiments, replacing the YADD or YMDD or YVDD results in higher fidelity of retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol [Molecular Biology] 2011; which is incorporated herein by reference in its entirety).

In some embodiments, the genetically modified polypeptides described herein comprise an RT domain having an amino acid sequence according to Table 6, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. In some embodiments, the nucleic acids described herein encode an RT domain having an amino acid sequence according to Table 6, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

Table 6: Exemplary reverse transcriptase domains from retroviruses

In certain embodiments, the reverse transcriptase domain is modified, for example, by site-specific mutation. In certain embodiments, the reverse transcriptase domain is engineered to have improved characteristics, for example, SuperScript IV (SSIV) reverse transcriptase derived from MMLV RT. In certain embodiments, the reverse transcriptase domain can be engineered to have a lower error rate, for example, as described in WO 2001068895 (incorporated herein by reference). In certain embodiments, the reverse transcriptase domain can be engineered to be more heat-resistant. In certain embodiments, the reverse transcriptase domain can be engineered to have more continuous synthesis ability. In certain embodiments, the reverse transcriptase domain can be engineered to have tolerance to inhibitors. In certain embodiments, the reverse transcriptase domain can be engineered to be faster. In certain embodiments, the reverse transcriptase domain can be engineered to better tolerate the modified nucleotides in the RNA template. In certain embodiments, the reverse transcriptase domain can be engineered to insert modified DNA nucleotides. In certain embodiments, the reverse transcriptase domain is engineered to bind template RNA. In some embodiments, the one or more mutations are selected from D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, H8Y, T306K or D653N in the RT domain of murine leukemia virus reverse transcriptase, or corresponding mutations in corresponding positions of another RT domain.

In some embodiments, the genetically modified polypeptide comprises an RT domain from a retroviral reverse transcriptase, such as a wild-type M-MLV RT, for example comprising the sequence:

M-MLV(WT):

In some embodiments, the gene modifying polypeptide comprises an RT domain from a retroviral reverse transcriptase, such as M-MLV RT, for example comprising the sequence:

In some embodiments, the genetically modified polypeptide comprises an RT domain from a retroviral reverse transcriptase comprising a sequence of amino acids 659-1329 of NP_057933. In embodiments, the genetically modified polypeptide further comprises an additional amino acid at the N-terminus of the sequence of amino acids 659-1329 of NP_057933, for example as shown below:

Core RT (bold), as noted above

RNase H (underlined), as noted above

In embodiments, the genetically modified polypeptide further comprises an additional amino acid at the C-terminus of the sequence of amino acids 659-1329 of NP_057933. In embodiments, the genetically modified polypeptide comprises an RNase H1 domain (eg, amino acids 1178-1318 of NP_057933).

In some embodiments, a retroviral reverse transcriptase domain, such as M-MLV RT, can comprise one or more mutations of a wild-type sequence that can improve characteristics of RT, such as thermostability, processivity, and/or template binding. In some embodiments, the M-MLV RT domain comprises one or more mutations relative to the above-mentioned M-MLV (WT) sequence, such as selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, such as a combination of mutations, such as D200N, L603W, and T330P, optionally further comprising T306K and W313F. In some embodiments, the M-MLV RT used herein comprises mutations D200N, L603W, T330P, T306K and W313F. In an embodiment, the mutant M-MLV RT comprises the following amino acid sequence:

M-MLV(PE2):

In some embodiments, the writing domain (e.g., RT domain) comprises an RNA binding domain, e.g., which specifically binds to an RNA sequence. In some embodiments, the template RNA comprises an RNA sequence that is specifically bound by the RNA binding domain of the writing domain.

In certain embodiments, the reverse transcription domain only recognizes and reverse transcribes a specific template, such as the template RNA of the system. In certain embodiments, the template comprises a sequence or structure that can be recognized and reverse transcribed by the reverse transcription domain. In certain embodiments, the template comprises a sequence or structure that can be associated with the RNA binding domain of the polypeptide component of the genome engineering system described herein. In certain embodiments, the genome engineering system preferably reverse transcribes a template that comprises an associated sequence, rather than a template that lacks an associated sequence.

The writing domain may also include DNA-dependent DNA polymerase activity, for example, including an enzyme activity capable of writing DNA from a template DNA sequence into a genome. In some embodiments, DNA-dependent DNA polymerization is used to complete the second strand synthesis of target site editing. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in a polypeptide. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain, which is also capable of DNA-dependent DNA polymerization, such as second strand synthesis. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a second polypeptide in the system. In some embodiments, the DNA-dependent DNA polymerase activity is provided by an endogenous host cell polymerase, which is optionally recruited to the target site by components of a genome engineering system.

In some embodiments, the reverse transcriptase domain has a lower probability of premature termination (P _off ) in vitro relative to a reference reverse transcriptase domain. In some embodiments, the reference reverse transcriptase domain is a viral reverse transcriptase domain, such as an RT domain from M-MLV.

In some embodiments, the reverse transcriptase domain has a lower probability of an in vitro premature termination rate ( _Poff ) of less than about ^5x10-3 /nt, ^5x10-4 /nt, or ^5x10-6 /nt, e.g., as measured on a 1094nt RNA. In embodiments, the in vitro premature termination rate is determined as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (which is incorporated herein by reference in its entirety).

In some embodiments, the reverse transcriptase domain is capable of completing at least about 30% or 50% integration in the cell. The percentage of complete integration can be measured by dividing the number of substantially full-length integration events (e.g., genomic sites containing at least 98% of the expected integration sequence) by the number of total (including substantially full-length and partial) integration events in the cell population. In an embodiment, integration in a cell (e.g., across integration sites) is determined using long read amplicon sequencing, for example, as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (which is incorporated herein by reference in its entirety).

In embodiments, quantifying integration in a cell comprises counting the integrated portion comprising at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a DNA sequence corresponding to a template RNA (e.g., a template RNA of at least 0.05, 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, or 5 kb in length, such as 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 1.0-1.2, 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2-3, 3-4, or 4-5 kb in length).

In some embodiments, the reverse transcriptase domain is capable of polymerizing dNTPs in vitro. In embodiments, the reverse transcriptase domain is capable of polymerizing dNTPs in vitro at a rate of 0.1-50 nt/sec (e.g., 0.1-1, 1-10, or 10-50 nt/sec). In embodiments, polymerization of dNTPs by the reverse transcriptase domain is measured by a single molecule assay, e.g., as described in Schwartz and Quake (2009) PNAS [Proceedings of the National Academy of Sciences of the United States of America] 106(48): 20294-20299 (which is incorporated by reference in its entirety).

In some embodiments, the reverse transcriptase domain has an in vitro error rate (e.g., misincorporation of nucleotides) of ^{1x10-3 -1x10-4} or ^{1x10-4 -1x10-5} substitutions/nt, e.g., as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2):147-153 (which is incorporated herein by reference in its entirety). In some embodiments, the reverse transcriptase domain has an error rate (e.g., misincorporation of nucleotides) of ^{1x10-3 -1x10-4} or ^{1x10-4 -1x10-5} substitutions/nt in cells (e.g., HEK293T cells), e.g., by long read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (which is incorporated herein by reference in its entirety).

In some embodiments, the reverse transcriptase domain is capable of reverse transcription of a target RNA in vitro. In some embodiments, the reverse transcriptase requires a primer of at least 3 nucleotides to initiate reverse transcription of a template. In some embodiments, reverse transcription of a target RNA is determined by detecting cDNA from the target RNA (e.g., when a ssDNA primer is provided, for example, it anneals to the target at the 3′ end by at least 3, 4, 5, 6, 7, 8, 9 or 10 nt), for example, as described in Bibillo and Eickbush (2002) J Biol Chem [Journal of Biological Chemistry] 277 (38): 34836-34845 (which is incorporated herein by reference in its entirety).

In some embodiments, the reverse transcriptase domain performs reverse transcription at least 5 or 10 times more efficiently than an RNA template lacking a protein binding motif (e.g., 3'UTR), e.g., when its RNA template is converted into cDNA (e.g., produced by cDNA). In embodiments, reverse transcription efficiency is measured as described in Yasukawa et al. (2017) Biochem Biophys Res Commun [Biochemistry and Biophysics Research Communications] 492(2): 147-153 (which is incorporated herein by reference in its entirety).

In some embodiments, the reverse transcriptase domain specifically binds to a particular RNA template at a higher frequency (e.g., about 5 or 10 times higher frequency) than any endogenous cellular RNA (e.g., when expressed in a cell (e.g., HEK293T cell). In an embodiment, the specific binding frequency between the reverse transcriptase domain and the template RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11): 5490-5501 (which is incorporated herein by reference in its entirety).

In some embodiments, the RT domain (e.g., as listed in Table 6) comprises one or more mutations as listed in Table 2A below. In some embodiments, the RT domain as listed in Table 6 comprises one, two, three, four, five or six mutations listed in the corresponding row of Table 2A below.

Table 2A. Exemplary RT domain mutations (relative to the corresponding wild-type sequence listed in the corresponding row of Table 6)

Template nucleic acid binding domain

Genetically modified polypeptides typically include a region that can be associated with a template nucleic acid (e.g., a template RNA). In some embodiments, the template nucleic acid binding domain is an RNA binding domain. In some embodiments, the RNA binding domain is a modular domain that can be associated with an RNA molecule containing a specific feature (e.g., a structural motif). In other embodiments, the template nucleic acid binding domain (e.g., an RNA binding domain) is contained within a reverse transcription domain, for example, a component derived from a reverse transcriptase has a known RNA preference feature.

In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) is included in the target DNA binding domain. For example, in some embodiments, the DNA binding domain is a CRISPR-associated protein that recognizes the structure of the template nucleic acid (e.g., template RNA) containing gRNA. In some embodiments, the gene-modified polypeptide comprises a DNA binding domain, which comprises a CRISPR-associated protein associated with a gRNA scaffold, which allows the DNA binding domain to bind to a target genomic DNA sequence. In some embodiments, the gRNA scaffold and the gRNA spacer are included in the template nucleic acid (e.g., template RNA), so the DNA binding domain is also a template nucleic acid binding domain. In some embodiments, the polypeptide has an RNA binding function in multiple domains, for example, it can be combined with the gRNA structure in the CRISPR-associated DNA binding domain and another sequence or structure in the reverse transcriptase domain.

In some embodiments, the RNA binding domain is capable of binding to the template RNA with a greater affinity than the reference RNA binding domain. In some embodiments, the reference RNA binding domain is an RNA binding domain of Cas9 from Streptococcus pyogenes (S. pyogenes). In some embodiments, the RNA binding domain is capable of binding to the template RNA with an affinity of 100pM-10nM (e.g., 100pM-1nM or 1nM-10nM). In some embodiments, the affinity of the RNA binding domain to its template RNA is measured in vitro, for example by thermophoresis, for example, as described in Asmari et al. Methods [method] 146: 107-119 (2018) (which is incorporated herein by reference in its entirety). In some embodiments, the affinity of the RNA binding domain to its template RNA is measured in cells (e.g., by FRET or CLIP-Seq).

In some embodiments, the RNA binding domain is associated with the template RNA in vitro at a frequency at least about 5 times or 10 times higher than the scrambled RNA. In some embodiments, the binding frequency between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, for example, as described in Lin and Miles (2019) Nucleic Acids Res [Nucleic Acids Research] 47 (11): 5490-5501 (which is incorporated herein by reference in its entirety). In some embodiments, the RNA binding domain is associated with the template RNA in a cell (e.g., HEK293T cell) at a frequency at least about 5 times or 10 times higher than the scrambled RNA. In some embodiments, the association frequency between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, for example, as described in Lin and Miles (2019) supra.

Endonuclease domain and DNA binding domain

In some embodiments, the gene-modified polypeptide has the function of cutting the DNA target site by the endonuclease domain. In some embodiments, the gene-modified polypeptide comprises a DNA binding domain, for example, for binding to a target nucleic acid. In some embodiments, the domain (e.g., Cas domain) of the gene-modified polypeptide comprises two or more smaller domains (e.g., DNA binding domain and endonuclease domain). It should be understood that when the DNA binding domain (e.g., Cas domain) is described as being bound to a target nucleic acid sequence, in some embodiments, the binding is mediated by gRNA.

In some embodiments, the domain has two functions. For example, in some embodiments, the endonuclease domain is also a DNA binding domain. In some embodiments, the endonuclease domain is also a template nucleic acid (e.g., template RNA) binding domain. For example, in some embodiments, the polypeptide comprises an endonuclease domain associated with CRISPR, which binds to a template RNA comprising a gRNA, binds to a target DNA sequence (e.g., complementary to a portion of the gRNA), and cuts the target DNA sequence. In some embodiments, an endonuclease domain or an endonuclease/DNA binding domain from a heterologous source may be used or may be modified in a gene modification system as described herein (e.g., by inserting, deleting, or replacing one or more residues).

In some embodiments, the nucleic acid encoding the endonuclease domain or endonuclease/DNA binding domain is changed from its native sequence to have an altered codon usage, e.g., to improve for human cells. In some embodiments, the endonuclease element is a heterologous endonuclease element, e.g., a Cas endonuclease (e.g., Cas9), a type II restriction endonuclease (e.g., Fok1), a meganuclease (e.g., I-SceI), or other endonuclease domains.

In certain aspects, the DNA binding domain of the gene-modified polypeptide described herein is selected, designed or constructed to bind to the desired host DNA target sequence. In certain embodiments, the DNA binding domain of the polypeptide is a heterologous DNA binding element. In some embodiments, the heterologous DNA binding element is a zinc finger element or a TAL effector element, such as a zinc finger or TAL polypeptide or a functional fragment thereof. In some embodiments, the heterologous DNA binding element is a sequence-guided DNA binding element, such as Cas9, Cpf1 or other CRISPR-associated proteins that have been altered to have no endonuclease activity. In some embodiments, the heterologous DNA binding element retains endonuclease activity. In some embodiments, the heterologous DNA binding element retains partial endonuclease activity to cut ssDNA, for example, with nickase activity. In specific embodiments, the heterologous DNA binding domain can be any one or more of Cas9, TAL domains, ZF domains, Myb domains, combinations thereof, or multiples thereof.

In some embodiments, the DNA binding domain is modified, for example, by site-specific mutations, increasing or decreasing the number and/or specificity of DNA binding elements (e.g., zinc fingers), etc., to change DNA binding specificity and affinity. In some embodiments, the nucleic acid sequence encoding the DNA binding domain is changed from its native sequence to have altered codon usage, for example, to improve for human cells. In an embodiment, the DNA binding domain comprises one or more modifications relative to the wild-type DNA binding domain, for example, modifications via directed evolution (e.g., phage-assisted continuous evolution (PACE)).

In some embodiments, the DNA binding domain comprises a meganuclease domain (e.g., as described herein, e.g., in an endonuclease domain portion), or a functional fragment thereof. In some embodiments, the meganuclease domain has endonuclease activity, e.g., double-strand cleavage and/or nickase activity. In other embodiments, the meganuclease domain has reduced activity, e.g., lacks endonuclease activity, e.g., the meganuclease is catalytically inactive. In some embodiments, a catalytically inactive meganuclease is used as a DNA binding domain, e.g., as described in Fonfara et al. Nucleic Acids Res [Nucleic Acids Research] 40(2): 847-860 (2012), which is incorporated herein by reference in its entirety.

In some embodiments, the genetically modified polypeptide comprises a modification to a DNA binding domain, for example, relative to a wild-type polypeptide. In some embodiments, the DNA binding domain comprises an addition, deletion, replacement or modification to the amino acid sequence of the original DNA binding domain. In some embodiments, the DNA binding domain is modified to include a heterologous functional domain that specifically binds to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the functional domain replaces at least a portion (e.g., all) of a previous DNA binding domain of a polypeptide. In some embodiments, the functional domain comprises a zinc finger (e.g., a zinc finger that specifically binds to a target nucleic acid (e.g., DNA) sequence of interest). In some embodiments, the functional domain comprises a Cas domain (e.g., a Cas domain that specifically binds to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the Cas domain comprises Cas9 or a mutant or variant thereof (e.g., as described herein). In embodiments, the Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In embodiments, the Cas domain is guided to a target nucleic acid (e.g., DNA) sequence of interest by a gRNA. In embodiments, the Cas domain and the gRNA are encoded in the same nucleic acid (e.g., RNA) molecule. In embodiments, the Cas domain and the gRNA are encoded in different nucleic acid (e.g., RNA) molecules.

In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with a greater affinity than a reference DNA binding domain. In some embodiments, the reference DNA binding domain is a DNA binding domain of Cas9 from Streptococcus pyogenes. In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with an affinity between 100 pM-10 nM (e.g., between 100 pM-1 nM or 1 nM-10 nM).

In some embodiments, the affinity of a DNA binding domain for its target sequence (e.g., a dsDNA target sequence) is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018), incorporated herein by reference in its entirety.

In embodiments, the DNA binding domain is capable of binding its target sequence (e.g., a dsDNA target sequence) with an affinity of, e.g., between 100 pM-10 nM (e.g., between 100 pM-1 nM or 1 nM-10 nM) in the presence of a molar excess of, e.g., about a 100-fold molar excess of a scrambled sequence competitor dsDNA.

In some embodiments, a DNA binding domain is found to be associated with its target sequence (e.g., a dsDNA target sequence) more frequently than any other sequence in the genome of a target cell (e.g., a human target cell), e.g., as measured by ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol [Current Protocols in Molecular Biology] Chapter 21 (which is incorporated herein by reference in its entirety). In some embodiments, a DNA binding domain is found to be associated with its target sequence (e.g., a dsDNA target sequence) at least about 5-fold or 10-fold more frequently than any other sequence in the genome of a target cell, e.g., as measured by ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010), supra.

In some embodiments, the endonuclease domain has nickase activity and cuts a strand of the target DNA. In some embodiments, the nickase activity reduces the formation of double-strand breaks at the target site. In some embodiments, the endonuclease domain produces a staggered nick structure in the first and second strands of the target DNA. In some embodiments, the staggered nick structure produces a free 3' overhang at the target site. In some embodiments, the free 3' overhang at the target site improves editing efficiency, for example, by enhancing access and annealing of the 3' homologous region of the template nucleic acid. In some embodiments, the staggered nick structure reduces the formation of double-strand breaks at the target site.

In some embodiments, the endonuclease domain cuts both strands of the target DNA, for example, resulting in blunt-end cleavage of the target, and there are no ssDNA overhangs on either side of the cleavage site. The amino acid sequence of the endonuclease domain of the gene modification system described herein can be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of the endonuclease domain described herein (e.g., the endonuclease domain of Table 8).

In certain embodiments, the heterologous endonuclease is Fok1 or a functional fragment thereof. In certain embodiments, the heterologous endonuclease is a Holliday ligation resolvase or a homolog thereof, such as a Holliday ligation resolvase from Sulfolobus solfataricus-Ssol Hje (Govindaraju et al., Nucleic Acids Research 44:7, 2016). In certain embodiments, the heterologous endonuclease is an endonuclease of a large fragment of a spliceosomal protein such as Prp8 (Mahbub et al., Mobile DNA 8:16, 2017). In certain embodiments, the heterologous endonuclease is derived from a CRISPR-associated protein, such as Cas9. In certain embodiments, the heterologous endonuclease is engineered to have only ssDNA cleavage activity, such as only nickase activity, such as a Cas9 nickase, such as SpCas9 with a D10A, H840A or N863A mutation. Table 8 provides exemplary Cas proteins and mutations associated with nickase activity. In yet other embodiments, the homologous endonuclease domain is modified, for example, by site-specific mutations, to change the DNA endonuclease activity. In yet other embodiments, the endonuclease domain is modified to reduce DNA sequence specificity, for example, by truncating to remove the domain that confers DNA sequence specificity or by mutating to inactivate the region that confers DNA sequence specificity.

In some embodiments, the endonuclease domain has nickase activity and does not form double-strand breaks. In some embodiments, the endonuclease domain forms single-strand breaks at a higher frequency than double-strand breaks, for example, at least 90%, 95%, 96%, 97%, 98%, or 99% of the breaks are single-strand breaks, or less than 10%, 5%, 4%, 3%, 2%, or 1% of the breaks are double-strand breaks. In some embodiments, the endonuclease does not form double-strand breaks substantially. In some embodiments, the endonuclease does not form double-strand breaks of detectable levels.

In some embodiments, the endonuclease domain has a nickase activity that cuts the target site DNA of the first chain; for example, in some embodiments, the endonuclease cuts the target site of the genomic DNA, which is near the change site on the chain to be extended by the writing domain. In some embodiments, the endonuclease domain has a nickase activity that cuts the target site DNA of the first chain and does not cut the target site DNA of the second chain. For example, when the polypeptide comprises a CRISPR-related endonuclease domain with nickase activity, in some embodiments, the CRISPR-related endonuclease domain cuts the target site DNA chain containing the PAM site (for example, and does not cut the target site DNA chain that does not contain the PAM site). As another example, when the polypeptide comprises a CRISPR-related endonuclease domain with nickase activity, in some embodiments, the CRISPR-related endonuclease domain cuts the target site DNA chain that does not contain the PAM site (for example, and does not cut the target site DNA chain that contains the PAM site).

In some other embodiments, the endonuclease domain has a nickase activity that cuts the target site DNA of the first and second strands. Without wishing to be bound by theory, after the writing domain (e.g., RT domain) of the polypeptide described herein is polymerized (e.g., reverse transcribed) from a heterologous subject sequence of a template nucleic acid (e.g., template RNA), the cellular DNA repair mechanism must repair the nick on the first DNA strand. The target site DNA now contains two different first DNA strand sequences: one corresponding to the original genomic DNA (e.g., with a free 5' end), and the second corresponding to the one polymerized from the heterologous subject sequence (e.g., with a free 3' end). It is believed that the two different sequences are balanced with each other, the first hybridizing with the second strand, then the other, and the sequence that the cellular DNA repair apparatus incorporates into the target site it repairs can be a random process. Without wishing to be bound by theory, it is believed that the introduction of additional nicks into the second strand may bias the cellular DNA repair mechanism to adopt sequences based on heterologous subject sequences more frequently than the original genomic sequence (Anzalone et al. Nature [Nature] 576: 149-157 (2019)). In some embodiments, the additional nicks are located at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides 5′ or 3′ of the target site modification (e.g., insertion, deletion, or substitution) or the nick on the first strand.

Alternatively or additionally, without wishing to be bound by theory, it is believed that additional nicking of the second strand may facilitate second strand synthesis. In some embodiments, when the gene modification system has inserted or replaced a portion of the first strand, a new sequence corresponding to the insertion/replacement in the second strand needs to be synthesized.

In some embodiments, the polypeptide comprises a single domain (e.g., a single endonuclease domain) with endonuclease activity and the domain cuts the first and second chains. For example, in such an embodiment, the endonuclease domain can be a CRISPR-related endonuclease domain, and the template nucleic acid (e.g., template RNA) comprises a gRNA spacer for guiding the cutting of the first chain and another gRNA spacer for guiding the cutting of the second chain. In some embodiments, the polypeptide comprises a plurality of domains with endonuclease activity, and the first endonuclease domain cuts the first chain and the second endonuclease domain cuts the second chain (optionally, the first endonuclease domain does not (e.g., cannot) cut the second chain, and the second endonuclease domain does not (e.g., cannot) cut the first chain).

In some embodiments, the endonuclease domain is capable of nicking the first and second chains. In some embodiments, the first and second chain nicks occur at the same position in the target site but on the opposite chain. In some embodiments, the second chain nick occurs at the staggered position of the first nick, such as upstream or downstream. In some embodiments, if the second chain nick is upstream of the first chain nick, the endonuclease domain produces a target site deletion. In some embodiments, if the second chain nick is downstream of the first chain nick, the endonuclease domain produces a target site repetition. In some embodiments, if the nicks of the first and second chains occur at the same position of the target site, the endonuclease domain does not produce repetitions and/or deletions. In some embodiments, the endonuclease domain has a changed activity, which depends on the protein conformation or RNA binding state, for example, this promotes the nicking of the first or second chain (for example, as described in Christensen et al. PNAS [Proceedings of the National Academy of Sciences of the United States] 2006; it is incorporated herein by reference in its entirety).

In some embodiments, the endonuclease domain comprises a meganuclease or a functional fragment thereof. In some embodiments, the endonuclease domain comprises a homing endonuclease or a functional fragment thereof. In some embodiments, the endonuclease domain comprises a meganuclease from the LAGLIDADG, GIY-YIG, HNH, His-Cys box or PD-(D/E)XK family, or a functional fragment or variant thereof, for example, having a conserved amino acid motif, for example, as indicated by the family name. In some embodiments, the endonuclease domain comprises a meganuclease or a fragment thereof selected from, for example, I-SmaMI (Uniprot F7WD42), I-SceI (Uniprot P03882), I-AniI (Uniprot P03880), I-Dmol (Uniprot P21505), I-Crel (Uniprot P05725), I-TevI (Uniprot P13299), I-OnuI (Uniprot Q4VWW5), or I-Bmol (Uniprot Q9ANR6). In some embodiments, the meganuclease is a natural monomer, such as I-SceI, I-TevI, or a dimer, such as I-Crel, in its functional form. For example, LAGLIDADG meganucleases with a single copy of the LAGLIDADG motif typically form homodimers, while members with two copies of the LAGLIDADG motif are typically found as monomers. In some embodiments, meganucleases that are normally formed as dimers are expressed as fusions, e.g., two subunits are expressed as a single ORF and optionally linked by a linker, e.g., an I-Crel dimer fusion (Rodriguez-Fornes et al. Gene Therapy 2020; incorporated herein by reference in its entirety). In some embodiments, meganucleases or functional fragments thereof are altered to favor nickase activity of one strand of a double-stranded DNA molecule, e.g., I-Scel (K122I and/or K223I) (Niu et al. J Mol Biol 2008), I-AniI (K227M) (McConnell Smith et al. PNAS 2009), I-Dmol (Q42A and/or K120M) (Molina et al. J Biol Chem 2015). In some embodiments, meganucleases or functional fragments thereof having such a preference for single-stranded cleavage are used as endonuclease domains, e.g., having nickase activity. In some embodiments, the endonuclease domain comprises a meganuclease or a functional fragment thereof that is naturally targeted or engineered to target a safe harbor site, such as I-Crel (Rodriguez-Fornes et al., supra) that targets the SH6 site. In some embodiments, the endonuclease domain comprises a meganuclease or a functional fragment thereof that has a sequence-tolerant catalytic domain, such as I-TevI (Kleinstiver et al. PNAS [Proceedings of the National Academy of Sciences of the United States of America] 2012) that recognizes the minimal motif CNNNG. In some embodiments, a target sequence-tolerant catalytic domain is fused to a DNA binding domain, e.g., to direct activity, e.g., by fusing I-TevI to: (i) a zinc finger to produce Tev-ZFE (Kleinstiver et al. PNAS [Proceedings of the National Academy of Sciences of the United States of America] 2012), (ii) other meganucleases to produce MegaTevs (Wolfs et al. Nucleic Acids Res [Nucleic Acids Research] 2014), and/or (iii) Cas9 to produce TevCas9 (Wolfs et al. PNAS [Proceedings of the National Academy of Sciences of the United States of America] 2016).

In some embodiments, the endonuclease domain comprises a restriction enzyme, e.g., a type IIS or type IIP restriction enzyme. In some embodiments, the endonuclease domain comprises a type IIS restriction enzyme, e.g., FokI, or a fragment or variant thereof. In some embodiments, the endonuclease domain comprises a type IIP restriction enzyme, e.g., PvuII, or a fragment or variant thereof. In some embodiments, a dimeric restriction enzyme is expressed as a fusion so that it functions as a single chain, e.g., a FokI dimer fusion (Minczuk et al. Nucleic Acids Res 36(12):3926-3938 (2008)).

For example, the use of additional endonuclease domains is described in Guha and Edgell Int J Mol Sci 18(22):2565 (2017), which is incorporated herein by reference in its entirety.

In some embodiments, the gene modification polypeptide comprises a modification to an endonuclease domain, for example, relative to a wild-type Cas protein. In some embodiments, the endonuclease domain comprises an addition, deletion, replacement or modification to the amino acid sequence of a wild-type Cas protein. In some embodiments, the endonuclease domain is modified to include a heterologous functional domain that specifically binds and/or induces endonuclease cleavage of a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the endonuclease domain comprises a zinc finger. In an embodiment, the endonuclease domain comprising a Cas domain is associated with, for example, a guide RNA (gRNA) as described herein. In some embodiments, the endonuclease domain is modified to include a functional domain that does not target a specific target nucleic acid (e.g., DNA) sequence. In an embodiment, the endonuclease domain comprises a Fok1 domain.

In some embodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than the scrambled dsDNA. In some embodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than the scrambled dsDNA, for example in a cell (e.g., HEK293T cell). In some embodiments, the association frequency between the endonuclease domain and the target DNA or scrambled DNA is measured by ChIP-seq, for example, as described in He and Pu (2010) Curr. Protoc Mol Biol [Current Protocols in Molecular Biology] Chapter 21 (which is incorporated herein by reference in its entirety).

In some embodiments, the endonuclease domain can catalyze the formation of a nick at the target sequence, such as an increase of at least about 5-fold or 10-fold relative to a non-target sequence (e.g., relative to any other genomic sequence in the target cell genome). In some embodiments, the level of nick formation is determined using NickSeq, for example, as described in Elacqua et al. (2019) bioRxivdoi.org/10.1101/867937 (which is incorporated herein by reference in its entirety).

In some embodiments, the endonuclease domain is capable of nicking DNA in vitro. In an embodiment, the nicking results in exposed bases. In an embodiment, the exposed bases can be detected using a nuclease sensitivity assay, for example, as described in Chaudhry and Weinfeld (1995) Nucleic Acids Res [Nucleic Acids Research] 23 (19): 3805-3809 (which is incorporated herein by reference in its entirety). In an embodiment, the level of exposed bases (e.g., detected by a nuclease sensitivity assay) is increased by at least 10%, 50% or more relative to a reference endonuclease domain. In some embodiments, the reference endonuclease domain is an endonuclease domain of Cas9 from Streptococcus pyogenes.

In some embodiments, the endonuclease domain is capable of nicking DNA in a cell. In an embodiment, the endonuclease domain is capable of nicking DNA in a HEK293T cell. In an embodiment, an unrepaired nick that undergoes replication in the absence of Rad51 results in an increase in the NHEJ rate at the nick site, which can be detected, for example, by using a Rad51 inhibition assay, for example, as described in Bothmer et al. (2017) Nat Commun [Natural Communications] 8: 13905 (which is incorporated herein by reference in its entirety). In an embodiment, the NHEJ rate is increased to more than 0-5%. In an embodiment, for example, after Rad51 inhibition, the NHEJ rate is increased to 20%-70% (e.g., 30%-60% or 40%-50%).

In some embodiments, the endonuclease domain releases the target after cleavage. In some embodiments, the release of the target is indirectly indicated by evaluating multiple turnovers of the enzyme, for example, as described in Yourik et al. RNA 25(1): 35-44 (2019) (which is incorporated herein by reference in its entirety) and shown in Figure 2. In some embodiments, the _kexp of the endonuclease domain is ^{1x10-3-1x10-5min} -1 as measured by such methods.

In some embodiments, the endonuclease domain has a catalytic efficiency (k _cat / K _m ) greater than about 1x10 ⁸ s ^-1 M ^-1 in vitro. In embodiments, the endonuclease domain has a catalytic efficiency greater than about 1x10 ⁵ , 1x10 ⁶ , 1x10 ⁷ or 1x10 ⁸ s ^-1 M ^-1 in vitro. In embodiments, the catalytic efficiency is determined as described in Chen et al ^. (2018) Science 360(6387): 436-439 (which is incorporated herein by reference in its entirety). In some embodiments, the endonuclease domain has a catalytic efficiency (k _cat / K _m ) greater than about 1x10 ⁸ s ^-1 M ^-1 in cells. In embodiments, the catalytic efficiency of the endonuclease domain is greater than about 1x10 ⁵ , 1x10 ⁶ , 1x10 ⁷ or 1x10 ⁸ s ^-1 M ^-1 in cells.

Gene modifying polypeptides containing Cas domains

In some embodiments, the gene modification polypeptide described herein comprises a Cas domain. In some embodiments, the Cas domain can guide the gene modification polypeptide to the target site specified by the gRNA spacer, thereby "cis" modifying the target nucleic acid sequence. In some embodiments, the gene modification polypeptide is fused to the Cas domain. In some embodiments, the gene modification polypeptide comprises a CRISPR/Cas domain (also referred to herein as a CRISPR-associated protein). In some embodiments, the CRISPR/Cas domain comprises proteins (e.g., Cas proteins) involved in a clustered regulatory interspaced short palindromic repeat (CRISPR) system, and is optionally combined with a guide RNA, such as a single guide RNA (sgRNA).

The CRISPR system is an adaptive defense system originally found in bacteria and archaea. The CRISPR system uses RNA-guided nucleases (e.g., Cas9 or Cpf1) called CRISPR-related or "Cas" endonucleases to cut foreign DNA. For example, in a typical CRISPR-Cas system, the endonuclease is directed to a target nucleotide sequence (e.g., a site to be sequenced in a genome) by targeting a sequence-specific non-coding "guide RNA" of a single-stranded or double-stranded DNA sequence. Three types (I-III) of CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). A Class II CRISPR system includes type II Cas endonucleases, such as Cas9, CRISPR RNA ("crRNA"), and trans-activating crRNA ("tracrRNA"). CrRNA contains a "spacer" sequence, i.e., an RNA sequence ("protospacer") of about 20 nucleotides that generally corresponds to a target DNA sequence. In wild-type systems and some engineered systems, crRNA also includes a region bound to tracrRNA to form a partial double-stranded structure cut by RNA enzyme III, producing crRNA/tracrRNA hybrid molecules. Then, crRNA/tracrRNA hybrids guide Cas endonucleases to recognize and cut target DNA sequences. The target DNA sequence is usually adjacent to a "protospacer adjacent motif" ("PAM"), which is specific for a given Cas endonuclease and is required for the cutting activity at the target site matched to the crRNA spacer. CRISPR endonucleases identified from different prokaryotic species have unique PAM sequence requirements, e.g., as listed in Table 7 for exemplary Cas enzymes; examples of PAM sequences include 5′-NGG (Streptococcus pyogenes; SEQ ID NO: 11,019), 5′-NNAGAA (Streptococcus thermophilus CRISPR1; SEQ ID NO: 11,020), 5′-NGGNG (Streptococcus thermophilus CRISPR3; SEQ ID NO: 11,021), and 5′-NNNGATT (Neisseria meningiditis; SEQ ID NO: 11,022). Some endonucleases, such as the Cas9 endonuclease, are associated with G-rich PAM sites (e.g., 5'-NGG (SEQ ID NO: 11,023)) and perform blunt-end cleavage of the target DNA at a position 3 nucleotides upstream (5' of the site) of the PAM site. Another class II CRISPR system includes a type V endonuclease, Cpf1, which is smaller than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.). The Cpf1-associated CRISPR array is processed into mature crRNA without the need for tracrRNA; in other words, in some embodiments, the Cpf1 system comprises only the Cpf1 nuclease and crRNA to cleave the target DNA sequence. The Cpf1 endonuclease is typically associated with T-rich PAM sites, such as 5'-TTN. Cpf1 can also recognize a 5'-CTA PAM motif. Cpf1 typically cuts the target DNA by introducing a misplaced or staggered double-strand break with a 5' overhang of 4 or 5 nucleotides, for example, cutting the target DNA in which the misplaced or staggered cut of 5 nucleotides is located at a position 18 nucleotides downstream (3') from the PAM site on the coding strand and at a position 23 nucleotides downstream from the PAM site on the complementary strand; the 5-nucleotide overhang generated by such misplaced cuts allows DNA insertion by homologous recombination to be more accurately edited than the insertion of DNA cut at the blunt end. See, for example, Zetsche et al. (2015) Cell, 163: 759-771.

A variety of CRISPR-related (Cas) genes or proteins can be used in the technology provided by the present disclosure, and the selection of Cas proteins will depend on the specific conditions of the method. Specific examples of Cas proteins include Class II systems, including Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1 or C2C3. In some embodiments, Cas proteins (e.g., Cas9 proteins) can be from any of a variety of prokaryotic species. In some embodiments, specific Cas proteins (e.g., specific Cas9 proteins) are selected to recognize specific protospacer adjacent motif (PAM) sequences. In some embodiments, DNA binding domains or endonuclease domains include sequences of targeting polypeptides (e.g., Cas proteins, such as Cas9). In certain embodiments, Cas proteins (e.g., Cas9 proteins) can be obtained from bacteria or archaea or synthesized using known methods. In certain embodiments, Cas proteins can be from Gram-positive bacteria or Gram-negative bacteria. In certain embodiments, the Cas protein can be from Streptococcus (e.g., Streptococcus pyogenes or Streptococcus thermophilus), Francisella (e.g., Francisella novicida), Staphylococcus (e.g., Staphylococcus aureus), Acidaminococcus (e.g., Acidaminococcus species BV3L6), Neisseria (e.g., Neisseria meningitidis), Cryptococcus, Corynebacterium, Haemophilus, Eubacterium, Pasteurella, Prevotella, Veillonella, or Oceanobacter.

In some embodiments, the gene-modified polypeptide may comprise the following amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereof. In embodiments, the following amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereof is located at the N-terminus of the gene-modified polypeptide. In embodiments, the following amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereof is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 amino acids of the N-terminus of the gene-modified polypeptide.

Exemplary N-terminal NLS-Cas9 domains

In some embodiments, the gene-modified polypeptide may comprise the following amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the following amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto is located at the C-terminus of the gene-modified polypeptide. In embodiments, the following amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 amino acids of the C-terminus of the gene-modified polypeptide.

Exemplary C-terminal sequences containing NLS

Exemplary Benchmark Sequences

In some embodiments, the gene-modified polypeptide may comprise a Cas domain as listed in Table 7 or 8, or a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

Table 7. CRISPR/Cas proteins, species and mutations

Table 8 Amino acid sequences, species and mutations of CRISPR/Cas proteins

In some embodiments, the Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to the target DNA sequence in order for the Cas protein to bind and/or function. In some embodiments, the PAM from 5′ to 3′ is or comprises NGG (SEQ ID NO: 11,024), YG (SEQ ID NO: 11,025), NNGRRT (SEQ ID NO: 11,026), NNNRRT (SEQ ID NO: 11,027), NGA (SEQ ID NO: 11,029), TYCV (SEQ ID NO: 11,030), TATV (SEQ ID NO: 11,031), NTTN (SEQ ID NO: 11,032) or NNNGATT (SEQ ID NO: 11,033), wherein N represents any nucleotide, Y represents C or T, R represents A or G, and V represents A or C or G. In some embodiments, the Cas protein is a protein listed in Table 7 or 8. In some embodiments, the Cas protein comprises one or more mutations that change its PAM. In some embodiments, the Cas protein comprises E1369R, E1449H and R1556A mutations or similar substitutions of amino acids corresponding to the positions. In some embodiments, the Cas protein comprises E782K, N968K and R1015H mutations or similar substitutions of amino acids corresponding to the positions. In some embodiments, the Cas protein comprises D1135V, R1335Q and T1337R mutations or similar substitutions of amino acids corresponding to the positions. In some embodiments, the Cas protein comprises S542R and K607R mutations or similar substitutions of amino acids corresponding to the positions. In some embodiments, the Cas protein comprises S542R, K548V and N552R mutations or similar substitutions of amino acids corresponding to the positions. Exemplary progress in engineering Cas enzymes to recognize altered PAM sequences is reviewed in Collias et al. Nature Communications [Natural Communications] 12: 555 (2021), which is incorporated herein by reference in its entirety.

In some embodiments, the Cas protein has catalytic activity and cuts one or both strands of the target DNA site. In some embodiments, the target DNA site is cut and then an alteration is formed, such as an insertion or deletion, such as by a cellular repair mechanism.

In some embodiments, the Cas protein is modified to inactivate or partially inactivate the nuclease, for example, a nuclease-deficient Cas9. While on a specific DNA sequence targeted by a gRNA, the wild-type Cas9 produces a double-strand break (DSB), and many CRISPR endonucleases with modified functionality are available, for example: a partially inactivated version of the Cas9 "nickase" produces only single-strand breaks; a catalytically inactive Cas9 ("dCas9") does not cut the target DNA. In some embodiments, the binding of dCas9 to a DNA sequence can interfere with transcription at the site by steric hindrance. In some embodiments, the binding of dCas9 to an anchor sequence can interfere with (e.g., reduce or prevent) the formation and/or maintenance of a genomic complex (e.g., ASMC). In some embodiments, the DNA binding domain comprises a catalytically inactive Cas9, such as dCas9. Many catalytically inactive Cas9 proteins are known in the art. In some embodiments, dCas9 comprises mutations in each endonuclease domain of the Cas protein, such as D10A and H840A or N863A mutations. In some embodiments, the catalytically inactive or partially inactive CRISPR/Cas domain comprises a Cas protein comprising one or more mutations, such as one or more mutations listed in Table 7. In some embodiments, the Cas protein described in a given row of Table 7 comprises one, two, three, or all of the mutations listed in the same row of Table 7. In some embodiments, the Cas protein, e.g., not described in Table 7, comprises one, two, three, or all of the mutations listed in a row of Table 7 or corresponding mutations at corresponding sites in the Cas protein.

In some embodiments, catalytically inactive, for example, dCas9 or partially inactivated Cas9 proteins include D11 mutations (e.g., D11A mutations) or similar substitutions corresponding to the amino acids at the positions. In some embodiments, catalytically inactive Cas9 proteins, such as dCas9 or partially inactivated Cas9 proteins, include H969 mutations (e.g., H969A mutations) or similar substitutions corresponding to the amino acids at the positions. In some embodiments, catalytically inactive Cas9 proteins, such as dCas9 or partially inactivated Cas9 proteins, include N995 mutations (e.g., N995A mutations) or similar substitutions corresponding to the amino acids at the positions. In some embodiments, catalytically inactive Cas9 proteins, such as dCas9, include mutations (e.g., D11A, H969A, and N995A mutations) at one, two, or three positions in positions D11, H969, and N995, or similar substitutions corresponding to the amino acids at the positions.

In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9 or partially inactivated Cas9 protein, comprises a D10 mutation (e.g., a D10A mutation) or a similar substitution of an amino acid corresponding to the position. In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9 or partially inactivated Cas9 protein, comprises a H557 mutation (e.g., an H557A mutation) or a similar substitution of an amino acid corresponding to the position. In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9, comprises a D10 mutation (e.g., a D10A mutation) and a H557 mutation (e.g., an H557A mutation) or a similar substitution of an amino acid corresponding to the position.

In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9 or partially inactivated Cas9 protein, comprises a D839 mutation (e.g., a D839A mutation) or a similar substitution corresponding to the amino acid at the position. In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9 or partially inactivated Cas9 protein, comprises a H840 mutation (e.g., an H840A mutation) or a similar substitution corresponding to the amino acid at the position. In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9 or partially inactivated Cas9 protein, comprises a N863 mutation (e.g., an N863A mutation) or a similar substitution corresponding to the amino acid at the position. In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9, comprises a D10 mutation (e.g., D10A), a D839 mutation (e.g., D839A), an H840 mutation (e.g., H840A), and an N863 mutation (e.g., N863A) or a similar substitution corresponding to the amino acid at the position.

In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9 or a partially inactive Cas9 protein, comprises an E993 mutation (eg, an E993A mutation) or a similar substitution of an amino acid corresponding to that position.

In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9 or partially inactivated Cas9 protein, comprises a D917 mutation (e.g., a D917A mutation) or a similar substitution corresponding to the amino acid at the position. In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9 or partially inactivated Cas9 protein, comprises an E1006 mutation (e.g., an E1006A mutation) or a similar substitution corresponding to the amino acid at the position. In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9 or partially inactivated Cas9 protein, comprises a D1255 mutation (e.g., a D1255A mutation) or a similar substitution corresponding to the amino acid at the position. In some embodiments, a catalytically inactive Cas9 protein, such as a dCas9, comprises a D917 mutation (e.g., D917A), an E1006 mutation (e.g., E1006A), and a D1255 mutation (e.g., D1255A) or a similar substitution corresponding to the amino acid at the position.

In some embodiments, Cas9 proteins without catalytic activity, such as dCas9 or partially inactivated Cas9 proteins, include D16 mutations (e.g., D16A mutations) or similar substitutions corresponding to the amino acids at the positions. In some embodiments, Cas9 proteins without catalytic activity, such as dCas9 or partially inactivated Cas9 proteins, include D587 mutations (e.g., D587A mutations) or similar substitutions corresponding to the amino acids at the positions. In some embodiments, the partially inactivated Cas domain has nickase activity. In some embodiments, the partially inactivated Cas9 domain is a Cas9 nickase domain. In some embodiments, Cas domains without catalytic activity or inactivated Cas domains do not produce detectable double-strand break formation. In some embodiments, Cas9 proteins without catalytic activity, such as dCas9 or partially inactivated Cas9 proteins, include H588 mutations (e.g., H588A mutations) or similar substitutions corresponding to the amino acids at the positions. In some embodiments, Cas9 proteins without catalytic activity, such as dCas9 or partially inactivated Cas9 proteins, include N611 mutations (e.g., N611A mutations) or similar substitutions corresponding to the amino acids at the positions. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16 mutation (e.g., D16A), a D587 mutation (e.g., D587A), a H588 mutation (e.g., H588A), and a N611 mutation (e.g., N611A), or similar substitutions of the amino acids corresponding to said positions.

In some embodiments, the DNA binding domain or the endonuclease domain can comprise a Cas molecule that comprises or is linked (e.g., covalently) to a gRNA (e.g., a template nucleic acid, e.g., a template RNA comprising a gRNA).

In some embodiments, the endonuclease domain or DNA binding domain comprises Streptococcus pyogenes Cas9 (SpCas9) or a functional fragment or variant thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises a modified SpCas9. In an embodiment, the modified SpCas9 comprises a modification that changes the specificity of the protospacer adjacent motif (PAM). In an embodiment, the PAM is specific for the nucleic acid sequence 5′-NGT-3′. In an embodiment, the modified SpCas9 comprises one or more amino acid substitutions, for example, at one or more of positions L1111, D1135, G1218, E1219, A1322, or R1335, for example, the one or more amino acid substitutions are selected from L1111R, D1135V, G1218R, E1219F, A1322R, R1335V. In an embodiment, the modified SpCas9 comprises the amino acid substitution T1337R and one or more additional amino acid substitutions, for example, the one or more additional amino acid substitutions are selected from L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or their corresponding amino acid substitutions. In an embodiment, the modified SpCas9 comprises: (i) one or more amino acid substitutions selected from D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337; and (ii) one or more amino acid substitutions selected from L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or their corresponding amino acid substitutions.

In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas domain, such as a Cas9 domain. In an embodiment, the endonuclease domain or DNA binding domain comprises a nuclease active Cas domain, a Cas nickase (nCas) domain, or a nuclease-free Cas (dCas) domain. In an embodiment, the endonuclease domain or DNA binding domain comprises a nuclease active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease-free Cas9 (dCas9) domain. In some embodiments, the endonuclease domain or DNA binding domain comprises a domain Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i of Cas9. In some embodiments, the endonuclease domain or DNA binding domain comprises Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA binding domain comprises Streptococcus pyogenes or Streptococcus thermophilus Cas9, or a functional fragment thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 sequence, for example, as described in Chylinski, Rhun, and Charpentier (2013) RNA Biology 10: 5, 726-737; the document is incorporated herein by reference. In some embodiments, the endonuclease domain or DNA binding domain comprises the HNH nuclease subdomain and/or RuvC1 subdomain of Cas, for example, Cas9 as described herein, or a variant thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas polypeptide (e.g., an enzyme) or a functional fragment thereof. In an embodiment, the Cas polypeptide (e.g., an enzyme) is selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2c1, Cas1 2c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5 , Csm6, Cmr1, Cmr3, Cmr4 、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx1S、Csx11、Csf1、Csf2、CsO、Csf4、Csd1、Csd2、Cst1、Cst2、Csh1、Csh2、Csa1、Csa2、Csa3、Csa4、Csa5、Type II Cas effector protein、Type V Cas Effector protein, type VI Cas effector protein, CARF, DinG, Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12b/C2c1, Cas12c/C2c3, SpCas9 (K855A), eSpCas9 (1.1), SpCas9-HF1, ultra-precise Cas9 variant (HypaCas9), homologs thereof, modified or engineered versions thereof, and/or functional fragments thereof. In an embodiment, Cas9 comprises one or more substitutions, for example, selected from H840A, D10A, P475A, W476A, N477A, D1125A, W1126A, and D1127A. In an embodiment, Cas9 comprises one or more mutations at positions selected from: D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987, for example, one or more substitutions selected from D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas (e.g., Cas9) sequence or a fragment or variant thereof from Corynebacterium ulcerans, Corynebacterium diphtheriae, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria meningitidis, Streptococcus pyogenes, or Staphylococcus aureus.

In some embodiments, the endonuclease domain or DNA binding domain comprises a Cpf1 domain, e.g., comprising one or more substitutions (e.g., at positions D917, E1006A, D1255), or any combination thereof, the one or more substitutions, e.g., selected from D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, and D917A/E1006A/D1255A.

In some embodiments, the nuclease domain or DNA binding domain comprises spCas9, spCas9-VRQR (SEQ ID NO: 5019), spCas9-VRER (SEQ ID NO: 5020), xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER (SEQ ID NO: 5021), spCas9-LRKIQK (SEQ ID NO: 5022), or spCas9-LRVSQL (SEQ ID NO: 5023).

In some embodiments, the gene modifying polypeptide has an endonuclease domain comprising a Cas9 nickase, such as Cas9 H840A. In an embodiment, Cas9 H840A has the following amino acid sequence: Cas9 nickase (H840A):

In some embodiments, the gene modifying polypeptide comprises a dCas9 sequence comprising a D10A and/or H840A mutation, for example, the following sequence:

TAL effectors and zinc finger nucleases

In some embodiments, the endonuclease domain or DNA binding domain comprises a TAL effector molecule. TAL effector molecules, such as TAL effector molecules that specifically bind to a DNA sequence, typically comprise multiple TAL effector domains or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effectors (e.g., N and/or C termini of multiple TAL effector domains). Many TAL effectors are known to those skilled in the art and are commercially available, for example, from Thermo Fisher Scientific.

Naturally occurring TALEs are natural effector proteins secreted by a variety of bacterial pathogens, including the plant pathogen Xanthomonas, that regulate gene expression in host plants and promote bacterial colonization and survival. The specific binding of TAL effectors is based on a central repeat domain (repeat variable diresidue, RVD domain) of nearly identical, typically 33 or 34 amino acid repeats arranged in tandem.

Members of the TAL effector family differ primarily in the number and order of their repeat sequences. The number of repeat sequences typically ranges from 1.5 to 33.5 repeats, and the C-terminal repeat is typically shorter in length (e.g., about 20 amino acids) and is often referred to as a "half repeat". Each repeat sequence of a TAL effector typically has a one repeat sequence to one base pair correlation, with different repeat sequence types exhibiting different base pair specificities (one repeat sequence recognizes one base pair on the target gene sequence). Generally, the fewer the number of repeat sequences, the weaker the protein-DNA interaction. It has been shown that a number of 6.5 repeat sequences is sufficient to activate transcription of a reporter gene (Scholze et al., 2010).

Repeat-to-repeat variation occurs primarily at amino acid positions 12 and 13, which are therefore referred to as "hypervariable," and are responsible for the specificity of interaction with the target DNA promoter sequence, as shown in Table 9, which lists exemplary repeat variable diresidues (RVDs) and their correspondence to nucleic acid base targets.

Table 9 - RVD and nucleic acid base specificity

Therefore, it is possible to modify the repeat sequence of TAL effectors to target specific DNA sequences. Further studies have shown that RVD NK can target G. The target sites of TAL effectors also tend to include T flanking the 5′ base targeted by the first repeat sequence, but the exact mechanism of this recognition is unclear. More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include Hax2, Hax3, Hax4, AvrXa7, AvrXa10, and AvrBs3.

Accordingly, the TAL effector domain of the TAL effector molecules described herein can be derived from TAL effectors from any bacterial species (e.g., Xanthomonas species, such as African strains of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C, and Xanthomonas oryzae pv. oryzicola strain BLS256 (Bogdanove et al. 2011)). In some embodiments, the TAL effector domain comprises an RVD domain and one or more flanking sequences (sequences on the N-terminal and/or C-terminal side of the RVD domain) that are also from a naturally occurring TAL effector. It may contain more or fewer repeat sequences than the RVD of a naturally occurring TAL effector. TAL effector molecules can be designed to target a given DNA sequence based on the above-mentioned encodings and other encodings known in the art. The number of TAL effector domains (e.g., repeat sequences (monomers or modules)) and their specific sequences can be selected based on the desired DNA target sequence. For example, in order to adapt to a specific target sequence, TAL effector domains, such as repeat sequences, can be removed or added. In one embodiment, the TAL effector molecules of the present invention contain 6.5 to 33.5 TAL effector domains, such as repeat sequences. In one embodiment, the TAL effector molecules of the present invention contain 8 to 33.5 TAL effector domains, such as repeat sequences, such as 10 to 25 TAL effector domains, such as repeat sequences, such as 10 to 14 TAL effector domains, such as repeat sequences.

In some embodiments, the TAL effector molecule comprises a TAL effector domain corresponding to a complete match with a DNA target sequence. In some embodiments, mismatches between the repeat sequence and the target base pair on the DNA target sequence are allowed as long as it allows the function of the polypeptide containing the TAL effector molecule. Generally, TALE binding is negatively correlated with the number of mismatches. In some embodiments, the TAL effector molecule of the polypeptide of the present invention contains no more than 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, 2 mismatches or 1 mismatch with the target DNA sequence, and optionally no mismatches. Without wishing to be bound by theory, in general, the fewer the number of TAL effector domains in the TAL effector molecule, the fewer the number of mismatches that will be tolerated, and the function of the polypeptide containing the TAL effector molecule is still allowed. Binding affinity is believed to depend on the sum of the matched repeat-DNA combinations. For example, a TAL effector molecule with 25 or more TAL effector domains may be able to tolerate up to 7 mismatches.

In addition to the TAL effector domain, the TAL effector molecules of the present invention may also include additional sequences derived from naturally occurring TAL effectors. The length of one or more C-terminal and/or N-terminal sequences contained on each side of the TAL effector domain portion of the TAL effector molecule can vary and be selected by a person skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al. have characterized many C-terminal and N-terminal truncation mutants in TAL effector-based proteins derived from Hax3, and have identified key elements that contribute to optimal binding to target sequences and thereby activate transcription. In general, transcriptional activity was found to be negatively correlated with the length of the N-terminus. With respect to the C-terminus, an important element of DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Therefore, in some embodiments, the first 68 amino acids on the C-terminal side of the TAL effector domain of a naturally occurring TAL effector are included in the TAL effector molecule. Thus, in an embodiment, the TAL effector molecule comprises 1) one or more TAL effector domains derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids from a naturally occurring TAL effector on the N-terminal side of the TAL effector domain; and/or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids from a naturally occurring TAL effector on the C-terminal side of the TAL effector domain.

In some embodiments, the endonuclease domain or the DNA binding domain is or comprises a Zn finger molecule. The Zn finger molecule comprises a Zn finger protein, such as a naturally occurring Zn finger protein or an engineered Zn finger protein, or a fragment thereof. Many Zn finger proteins are known to those skilled in the art and are commercially available, for example, from Sigma-Aldrich.

In some embodiments, the Zn finger molecule comprises a non-naturally occurring Zn finger protein that is engineered to bind to a selected target DNA sequence. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242, 6,534,264. 1, 6,599,692, 6,503,717, 6,689,558, 7,030,215, 6,794,136, 7,067,317, 7,262,054, 7,070,934, 7,361,635, 7,253,273; and U.S. Patent Publication Nos. 2005/0064474, 2007/0218528, and 2005/0267061, all of which are incorporated herein by reference in their entirety.

Compared to naturally occurring Zn finger proteins, engineered Zn finger proteins may have novel binding specificities. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using a database comprising triplet (or quadruple) nucleotide sequences and single Zn finger amino acid sequences, wherein each triplet or quadruple nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplet or quadruple sequence. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261, which are incorporated herein by reference in their entirety.

Exemplary selection methods (including phage display and two-hybrid systems) are disclosed in the following: U.S. Patent Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,410,248, 6,140,466, 6,200,759, and 6,242,568; and International Patent Publication Nos. WO 98/37186, WO 98/53057, WO 00/27878, and WO 01/88197 and GB 2,338,237. In addition, the binding specificity of zinc finger proteins has been enhanced, for example, described in International Patent Publication No. WO 02/077227.

In addition, as disclosed in these and other references, zinc finger domains and/or multi-finger zinc finger proteins can be linked together using any suitable linker sequence (including, for example, a linker of 5 or more amino acids in length). See also U.S. Patent Nos. 6,479,626, 6,903,185, and 7,153,949 for exemplary linker sequences of 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancing the binding specificity of zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227.

Zn finger proteins and methods for designing and constructing fusion proteins (and polynucleotides encoding the same) are known to those skilled in the art and are described in detail in the following: U.S. Patent Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; International Patent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536, and WO 03/016496.

In addition, as disclosed in these and other references, Zn finger proteins and/or multi-finger Zn finger proteins can be linked together, for example as fusion proteins, using any suitable linker sequence (including, for example, a linker of 5 or more amino acids in length). See also U.S. Pat. Nos. 6,479,626; 6,903,185, and 7,153,949 for exemplary linker sequences of 6 or more amino acids in length. The Zn finger molecules described herein may include any combination of suitable linkers between the individual zinc finger proteins and/or multi-finger Zn finger proteins of the Zn finger molecule.

In certain embodiments, the DNA binding domain or the endonuclease domain comprises a Zn finger molecule comprising an engineered zinc finger protein that binds to a target DNA sequence (in a sequence-specific manner). In some embodiments, the Zn finger molecule comprises a Zn finger protein or a fragment thereof. In other embodiments, the Zn finger molecule comprises a plurality of Zn finger proteins (or fragments thereof), such as 2, 3, 4, 5, 6 or more Zn finger proteins (and optionally, no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 Zn finger proteins). In some embodiments, the Zn finger molecule comprises at least three Zn finger proteins. In some embodiments, the Zn finger molecule comprises four, five or six fingers. In some embodiments, the Zn finger molecule comprises 8, 9, 10, 11 or 12 fingers. In some embodiments, a Zn finger molecule comprising three Zn finger proteins recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger molecule comprising four Zn finger proteins recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger molecule comprising six Zn finger proteins recognizes a target DNA sequence comprising 18 to 21 nucleotides.

In some embodiments, the Zn finger molecule comprises a two-handed Zn finger protein. A two-handed zinc finger protein is a protein in which two clusters of zinc finger proteins are separated by intervening amino acids, so that the two zinc finger domains bind to two non-contiguous target DNA sequences. An example of a two-handed zinc finger binding protein is SIP1, in which a cluster of four zinc finger proteins is located at the amino terminus of the protein and a cluster of three Zn finger proteins is located at the carboxyl terminus (see Remade et al. (1999) EMBO Journal [European Molecular Biology Journal] 18 (18): 5073-5084). Each cluster of zinc fingers in these proteins is capable of binding to a unique target sequence, and the interval between the two target sequences can contain many nucleotides.

Connectors

In some embodiments, the gene-modified polypeptide may comprise a linker, such as a peptide linker, such as a linker described in Table 10. In some embodiments, the gene-modified polypeptide comprises a Cas domain (e.g., a Cas domain of Table 8), a linker of Table 10 (or a sequence having at least 70%, 80%, 85%, 90%, 95% or 99% identity thereto), and a RT domain (e.g., an RT domain of Table 6) in the N-terminal to C-terminal direction. In some embodiments, the gene-modified polypeptide comprises a flexible linker between the endonuclease and the RT domain, for example, a linker comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 11,002). In some embodiments, the RT domain of the gene-modified polypeptide may be located at the C-terminus of the endonuclease domain. In some embodiments, the RT domain of the gene-modified polypeptide may be located at the N-terminus of the endonuclease domain.

Table 10 Exemplary linker sequences

In some embodiments, the linker of the gene modifying polypeptide comprises a motif selected from the group consisting of: (SGGS) _n (SEQ ID NO: 5025), (GGGS) _n (SEQ ID NO: 5026), (GGGGS) _n (SEQ ID NO: 5027), (G) _n , (EAAAK) _n (SEQ ID NO: 5028), (GGS) _n , or (XP) _n .

Selection of gene-modifying peptides by pooled screening

Candidate gene modification polypeptides can be screened to evaluate the gene editing ability of the candidate. For example, an RNA gene modification system designed for targeted editing of coding sequences in the human genome can be used. In certain embodiments, such a gene modification system can be used in combination with a pooled screening method.

For example, a gene-modified polypeptide candidate library and a template guide RNA (tgRNA) can be introduced into mammalian cells to test the gene editing ability of the candidate by a pooled screening method. In a particular embodiment, a gene-modified polypeptide candidate library is introduced into mammalian cells, and then tgRNA is introduced into the cells.

Representative, non-limiting examples of mammalian cells that can be used for screening include HEK293T cells, U2OS cells, HeLa cells, HepG2 cells, Huh7 cells, K562 cells, or iPS cells.

The gene-modified polypeptide candidate may comprise 1) a Cas nuclease, such as a wild-type Cas nuclease (e.g., a wild-type Cas9 nuclease), a mutant Cas nuclease (e.g., a Cas nickase, such as a Cas9 nickase, such as a Cas9 N863A nickase), or a Cas nuclease selected from Table 7 or Table 8, 2) a peptide linker, such as a sequence from Table D or Table 10, which may exhibit varying degrees of length, flexibility, hydrophobicity, and/or secondary structure; and 3) a reverse transcriptase (RT), such as an RT domain from Table D or Table 6. The gene-modified polypeptide candidate library comprises: a plurality of different gene-modified polypeptide candidates, which differ from each other in one, two, or all three aspects of the Cas nuclease, peptide linker, or RT domain components; or a plurality of nucleic acid expression vectors encoding such gene-modified polypeptide candidates.

In order to screen gene modification polypeptide candidates, a two-component system comprising a gene modification polypeptide component and a tgRNA component can be used. The gene modification component may include, for example, an expression vector, such as an expression plasmid or a lentiviral vector, encoding a gene modification polypeptide candidate, such as a nucleic acid comprising human codon optimization, encoding a gene modification polypeptide candidate, such as a Cas-linker-RT fusion as described above. In a particular embodiment, a lentiviral cassette is utilized, comprising: (i) a promoter for expression in mammalian cells, such as a CMV promoter; (ii) a gene modification library candidate, such as a Cas nuclease comprising Table 7 or Table 8, a peptide linker of Table 10, and a Cas-linker-RT fusion of RT of Table 6, such as a Cas-linker-RT fusion of Table D; (iii) a self-cleaving polypeptide, such as a T2A peptide; (iv) a marker capable of selection in mammalian cells, such as a puromycin resistance gene; and (v) a termination signal, such as a poly A tail.

The tgRNA component can include a tgRNA or an expression vector, such as an expression plasmid, which produces the tgRNA, for example using a U6 promoter to drive the expression of the tgRNA, wherein the tgRNA is a non-coding RNA sequence that is recognized by Cas and localized to the genomic locus of interest, and also serves as a template for reverse transcription of the desired edits into the genome through the RT domain.

In order to prepare a cell pool expressing a candidate for a gene-modified polypeptide library, a mammalian cell (e.g., HEK293T or U2OS cell) can be transduced with a pooled gene-modified polypeptide candidate expression vector preparation (e.g., a lentiviral preparation) of a gene-modified candidate polypeptide library. In a specific embodiment, a lentiviral plasmid is used, and HEK293 Lenti-X cells are inoculated in a 15 cm plate (about 12x10 ⁶ cells) before lentiviral plasmid transfection. In such an embodiment, a lentiviral packaging mixture (Biosettia) can be used for lentiviral plasmid transfection, and the plasmid DNA of the gene-modified candidate library can be transfected using Lipofectamine 2000 and Opti-MEM medium according to the manufacturer's protocol on the second day. In such an embodiment, extracellular DNA can be removed by replacing the complete medium on the second day, and the culture medium containing the virus can be harvested after 48 hours. The lentiviral culture medium can be concentrated using Lenti-X concentrate (TaKaRa Biosciences), and then 5 mL lentiviral aliquots can be prepared and stored at -80 ° C. Lentivirus titer determination is performed by counting colony forming units after selection (eg, after puromycin selection).

In order to monitor the gene editing of the target DNA, mammalian cells carrying the target DNA, such as HEK293T or U2OS cells, can be used. In other embodiments of monitoring the gene editing of the target DNA, mammalian cells carrying the target DNA genome landing pad, such as HEK293T or U2OS cells, can be used. In a specific embodiment, the target DNA genome landing pad may include a gene to be edited to treat a disease or condition of interest. In other specific embodiments, the target DNA is a gene sequence expressing a protein that exhibits detectable characteristics, and these characteristics can be monitored to determine whether gene editing has occurred. For example, in certain embodiments, a genome landing pad expressing blue fluorescent protein (BFP) or green fluorescent protein (GFP) is utilized. In certain embodiments, mammalian cells (e.g., HEK293T or U2OS cells) containing the target DNA (e.g., the target DNA genome landing pad) are inoculated in a culture plate with 500x-3000x cells per gene modification library candidate and transduced at a multiplicity of infection (MOI) of 0.2-0.3 to minimize multiple infections of each cell. Puromycin (2.5 ug/mL) can be added 48 hours after infection to select infected cells. In such embodiments, cells can be maintained under puromycin selection for at least 7 days and then expanded to introduce tgRNA, for example, tgRNA electroporation.

To determine whether gene editing occurs, mammalian cells containing the target DNA to be edited can be infected with a gene modifying polypeptide library candidate and then transfected with a tgRNA designed to edit the target DNA. Subsequently, the cells can be analyzed to determine whether editing of the target locus occurs according to the designed results, or whether no editing occurs or imperfect editing occurs, for example, by using cell sorting and sequence analysis.

In a specific embodiment, in order to determine whether genome editing occurs, mammalian cells (such as HEK293T or U2OS cells) expressing BFP or GFP can be infected with gene modification library candidates, and then transfected or electroporated with tgRNA plasmids or RNA, such as by using 200ng tgRNA plasmids to electroporate 250,000 cells/well, the plasmid is designed to convert BFP into GFP or GFP into BFP, wherein the cell count ensures that the coverage of each library candidate is >250x-1000x. In such an embodiment, the genome editing ability of various constructs in the assay can be evaluated by sorting cells with fluorescence activated cell sorting (FACS) for the expression of color-converted fluorescent protein (FP) 4-10 days after electroporation. Cells are sorted and harvested, divided into different groups of unedited cells (displaying original fluorescent protein signals), edited cells (displaying converted fluorescent protein signals) and imperfectly edited cells (not showing fluorescent protein signals). Unsorted cell samples can also be harvested as input populations to determine candidate enrichment during analysis.

In order to determine which gene modification library candidates show genome editing ability in the assay, genomic DNA (gDNA) is harvested from the sorted cell population, and the gene modification library candidates in each population are sequenced for analysis. In short, the gene modification candidates can be amplified from the genome using primers specific for a gene modification polypeptide expression vector (such as a lentiviral cassette), amplified in the second round of PCR to dilute the genomic DNA, and then sequenced, such as by a next-generation sequencing platform. After quality control of sequencing reads, at least about 1500 nucleotides and generally no more than about 3200 nucleotides are mapped to the gene modification polypeptide library sequence, and those reads that match at least about 80% of the library sequence are considered to have been successfully compared with a given candidate, for this pooling screening. In order to identify candidates that can be gene edited (for example, BFP to GFP or GFP to BFP editing) in the assay, the read counts of each library candidate in the editing population are compared with the read counts in the initial unsorted population.

In order to perform pool screening, gene modification candidates with genome editing capabilities are identified based on the enrichment of the edited (converted FP) population relative to unsorted (input) cells. In some embodiments, an enrichment of at least 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or at least 100 times relative to the input indicates potentially useful gene editing activity, such as at least 2 times enrichment. In some embodiments, the enrichment is converted to a logarithm with a base of 2 for the enrichment rate. In some embodiments, a log2 enrichment score of at least 0, 1, 2, 3, 4, 5, 5.5, 6.0, 6.2, 6.3, 6.4, 6.5 or at least 6.6 indicates potentially useful gene editing activity, such as a log2 enrichment score of at least 1.0. In certain embodiments, a reference (eg, element ID number: 17380) can be used to compare the observed enrichment values of a gene modification candidate to the enrichment values observed under similar conditions.

In some embodiments, multiple tgRNAs can be used to screen a library of candidate genes for gene modification. In certain embodiments, multiple tgRNAs can be used to optimize template/Cas-linker-RT fusion pairs, such as for gene editing of specific target genes, such as gene targets for treating diseases. In particular embodiments, a pooling method for screening candidate genes for gene modification can be performed using a variety of different tgRNAs in array form.

In some embodiments, multiple types of edits, such as insertions, substitutions, and/or deletions of varying lengths, can be used to screen a library of gene modification candidates.

In some embodiments, multiple target sequences (e.g., different fluorescent proteins) can be used to screen the gene modification candidate library. In some embodiments, multiple target sequences (e.g., different fluorescent proteins) can be used to screen the gene modification candidate library. In some embodiments, multiple cell types, such as HEK293T or U2OS, can be used to screen the gene modification candidate library. It will be understood by those of ordinary skill in the art that a given candidate may exhibit a changed editing ability, even obtaining or losing any observable or useful activity under different conditions, including tgRNA sequence (e.g., nucleotide modification, PBS length, RT template length), target sequence, target position, editing type, mutation position relative to the first chain nick of the gene modification polypeptide, or cell type. Therefore, in some embodiments, the gene modification library candidate is screened across multiple parameters, for example, using at least two different tgRNAs in at least two cell types, and identifying gene editing activity by enrichment under any single condition. In other embodiments, candidates with stronger activity in different tgRNAs and cell types are identified by enrichment under at least two conditions (e.g., under all screening conditions). For clarity, candidates that show little or no enrichment under any given condition are not considered inactive under all conditions and can be screened with different parameters or reconfigured at the polypeptide level, for example by swapping, shuffling or evolving domains (e.g., RT domains), linkers or other signals (e.g., NLS).

Exemplary Cas9-Linker-RT Fusion Sequences

In some embodiments, the genetically modified polypeptide comprises a linker sequence and an RT sequence. In some embodiments, the genetically modified polypeptide comprises a linker sequence as listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises an amino acid sequence of an RT domain as listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the gene modifying polypeptide comprises a linker sequence as listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and an amino acid sequence of an RT domain as listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the gene modifying polypeptide comprises: (i) a linker sequence as listed in a row of Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and (ii) an amino acid sequence of an RT domain as listed in the same row of Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

Exemplary Gene Modifying Polypeptides

In some embodiments, a genetically modified polypeptide (e.g., a genetically modified polypeptide as part of a system described herein) comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, a genetically modified polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence that is at least 80% identical thereto. In some embodiments, a genetically modified polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence that is at least 90% identical thereto. In some embodiments, a genetically modified polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence that is at least 95% identical thereto. In some embodiments, a genetically modified polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence that is at least 99% identical thereto. In some embodiments, a genetically modified polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743. In some embodiments, the gene-modified polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gene-modified polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

In some embodiments, the genetically modified polypeptide comprises an amino acid sequence as listed in Table A1, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

In some embodiments, the genetically modified polypeptide comprises an amino acid sequence as listed in Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises a linker comprising a linker sequence as listed in Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the gene-modified polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table T1, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table T1, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto.

Table T1. Selection of Exemplary Gene Modifying Polypeptides

In some embodiments, the genetically modified polypeptide comprises an amino acid sequence as listed in Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises a linker comprising a linker sequence as listed in Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the gene-modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table T2, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table T2, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto.

Table T2. Selection of Exemplary Gene Modifying Polypeptides

Subsequences of Exemplary Gene Modifying Polypeptides

In some embodiments, the gene-modified polypeptide comprises one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS), a DNA binding domain, a linker, a RT domain, and/or a second NLS in order from N-terminus to C-terminus. In some embodiments, the gene-modified polypeptide comprises an NLS (e.g., a first NLS), a DNA binding domain, a linker, and a RT domain in order from N-terminus to C-terminus, wherein the linker and RT domain are the linker and RT domain of the gene-modified polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the linker and RT domain. In some embodiments, the gene-modified polypeptide comprises a DNA binding domain, a linker, a RT domain, and an NLS (e.g., a second NLS) in N-terminal to C-terminal order, wherein the linker and RT domain are the linker and RT domain of the gene-modified polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the linker and RT domain. In some embodiments, the gene-modified polypeptide comprises a first NLS, a DNA binding domain, a linker, a RT domain, and a second NLS in N-terminal to C-terminal order, wherein the linker and RT domain are the linker and RT domain of the gene-modified polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the linker and RT domain. In some embodiments, the gene-modified polypeptide further comprises an N-terminal methionine residue.

In some embodiments, the gene modifying polypeptide comprises one or more (e.g., 1, 2, 3, 4, 5, or all 6) of the following in order from N-terminus to C-terminus: an N-terminal methionine residue, a first nuclear localization signal (NLS) (e.g., a gene modifying polypeptide belonging to any one of SEQ ID NOs: 1-7743 and/or as listed in any one of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto), a DNA binding domain (e.g., a Cas domain, e.g., a SpyCas9 domain, e.g., as listed in Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; or a DNA binding domain of the following gene modifying polypeptide: SEQ ID NOs: NO: 1-7743 and/or as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto), a linker (e.g., a gene modifying polypeptide belonging to any one of SEQ ID NO: 1-7743 and/or as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto), an RT domain (e.g., a gene modifying polypeptide belonging to any one of SEQ ID NO: 1-7743 and/or as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto), and a second NLS (e.g., a gene modifying polypeptide belonging to SEQ ID NO: 1-7743 and/or as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto). In some embodiments, the gene-modified polypeptide further comprises (e.g., C-terminal to the second NLS) a T2A sequence and/or a puromycin sequence (e.g., a gene-modified polypeptide belonging to any one of SEQ ID NOs: 1-7743 and/or an amino acid sequence listed in any one of Tables A1, T1 or T2, or at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto). In some embodiments, the nucleic acid encoding the gene-modified polypeptide (e.g., as described herein) encodes a T2A sequence, e.g., wherein the T2A sequence is located between the region encoding the gene-modified polypeptide and the second region, wherein the second region optionally encodes a selectable marker, e.g., puromycin.

In certain embodiments, the first NLS comprises a first NLS sequence of a gene-modifying polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the first NLS comprises a first NLS sequence of a gene-modifying polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the first NLS sequence comprises C-myc NLS. In certain embodiments, the first NLS comprises the amino acid sequence PAAKRVKLD (SEQ ID NO: 11,095), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In certain embodiments, the genetically modified polypeptide further comprises a spacer sequence between the first NLS and the DNA binding domain. In certain embodiments, the spacer sequence between the first NLS and the DNA binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In certain embodiments, the spacer sequence between the first NLS and the DNA binding domain comprises the amino acid sequence GG.

In certain embodiments, the DNA binding domain comprises the DNA binding domain of the following gene modification polypeptide: any one of SEQ ID NO: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the DNA binding domain comprises the DNA binding domain of a gene modification polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the DNA binding domain comprises a Cas domain (e.g., as listed in Table 8). In certain embodiments, the DNA binding domain comprises the amino acid sequence of a SpyCas9 polypeptide (e.g., as listed in Table 8, e.g., Cas9 N863A polypeptide), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the DNA binding domain comprises the amino acid sequence:

or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

In certain embodiments, the genetically modified polypeptide further comprises a spacer sequence between the DNA binding domain and the joint. In certain embodiments, the spacer sequence between the DNA binding domain and the joint comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In certain embodiments, the spacer sequence between the DNA binding domain and the joint comprises the amino acid sequence GG.

In certain embodiments, the linker comprises a linker sequence of a gene-modifying polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of a gene-modifying polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the linker comprises an amino acid sequence as listed in Table D or 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In certain embodiments, the gene modification polypeptide further comprises a spacer sequence between a joint and a RT domain. In certain embodiments, the spacer sequence between a joint and a RT domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In certain embodiments, the spacer sequence between a joint and a RT domain comprises an amino acid sequence GG.

In certain embodiments, the RT domain comprises the RT domain sequence of the following gene-modifying polypeptide: any one of SEQ ID NO: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the RT domain comprises the RT domain sequence of the gene-modifying polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the RT domain comprises an amino acid sequence as listed in Table D or 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the RT domain is about 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids in length.

In certain embodiments, the genetically modified polypeptide further comprises a spacer sequence between the RT domain and the second NLS. In certain embodiments, the spacer sequence between the RT domain and the second NLS comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In certain embodiments, the spacer sequence between the RT domain and the second NLS comprises the amino acid sequence AG.

In certain embodiments, the second NLS comprises a second NLS sequence of a gene-modifying polypeptide of any one of SEQ ID NOs: 1-7743. In certain embodiments, the second NLS comprises a second NLS sequence of a gene-modifying polypeptide as listed in any one of Tables A1, T1 or T2. In certain embodiments, the second NLS sequence comprises a plurality of partial NLS sequences. In an embodiment, an NLS sequence (e.g., a second NLS sequence) comprises a first partial NLS sequence, for example, comprising the amino acid sequence KRTADGSEFE (SEQ ID NO: 11,097), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In an embodiment, an NLS sequence (e.g., a second NLS sequence) comprises a second partial NLS sequence. In embodiments, the NLS sequence (e.g., the second NLS sequence) comprises an SV40A5 NLS, e.g., a two-component SV40A5 NLS, e.g., comprising the amino acid sequence KRTADGSEFESPKKKAKVE (SEQ ID NO: 11,098), or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In certain embodiments, the NLS sequence (e.g., the second NLS sequence) comprises the amino acid sequence KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 11,099), or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto.

In certain embodiments, the genetically modified polypeptide further comprises a spacer sequence between a second NLS and a T2A sequence and/or a puromycin sequence. In certain embodiments, the spacer sequence between a second NLS and a T2A sequence and/or a puromycin sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In certain embodiments, the spacer sequence between a second NLS and a T2A sequence and/or a puromycin sequence comprises the amino acid sequence GSG.

Linker and RT domains

In some embodiments, the gene-modified polypeptide comprises a linker (e.g., as described herein) and a RT domain (e.g., as described herein). In certain embodiments, the gene-modified polypeptide comprises a linker (e.g., as described herein) and a RT domain (e.g., as described herein) in the order from N-terminus to C-terminus.

In certain embodiments, the linker comprises a linker sequence as listed in Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the linker comprises the following linker sequence: any one of SEQ ID NO: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the linker comprises the following linker sequence: any one of SEQ ID NO: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the linker comprises the following linker sequence: any one of SEQ ID NO: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of any one of the exemplary gene-modifying polypeptides listed in Table A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the RT domain comprises an RT domain sequence as listed in Table 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the RT domain comprises an RT domain sequence of any one of the exemplary gene-modifying polypeptides listed in Table A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In some embodiments, the genetically modified polypeptide comprises a portion of the genetically modified polypeptide of any one of SEQ ID NOs: 1-7743 (wherein the portion comprises a linker and an RT domain), or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the portion.

In some embodiments, the gene-modified polypeptide comprises a linker of a gene-modified polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gene-modified polypeptide comprises a linker of a gene-modified polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gene-modified polypeptide comprises a linker of a gene-modified polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gene-modified polypeptide comprises a linker of a gene-modified polypeptide as listed in any one of Tables A1, T1 or T2, or a linker comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In some embodiments, the gene-modified polypeptide comprises the RT domain of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the RT domain. In some embodiments, the gene-modified polypeptide comprises the RT domain of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the RT domain. In some embodiments, the gene-modified polypeptide comprises the RT domain of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the RT domain. In some embodiments, the gene-modified polypeptide comprises an RT domain of a gene-modified polypeptide as listed in any one of Tables A1, T1 or T2, or an RT domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprises an amino acid sequence of a linker and RT domain of a gene modifying polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-7743 (or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto). In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprises an amino acid sequence of a linker and RT domain having at least 80% identity with a linker and RT domain of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprises an amino acid sequence of a linker and RT domain having at least 90% identity with a linker and RT domain of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprises an amino acid sequence of a linker and RT domain having at least 95% identity with a linker and RT domain of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprises an amino acid sequence of a linker and RT domain having at least 99% identity to the linker and RT domain of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprises an amino acid sequence of a linker and RT domain of a gene modifying polypeptide having an amino acid sequence of any one of SEQ ID NOs: 6001-7743 (or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereof). In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprises an amino acid sequence of a linker and RT domain of a gene modifying polypeptide having an amino acid sequence of any one of SEQ ID NOs: 4501-4541 (or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereof). In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprises an amino acid sequence of a linker and RT domain from a single row of any one of Tables A1, T1 or T2 (e.g., from a single exemplary gene modifying polypeptide listed in any one of Tables A1, T1 or T2) (or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto).

In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprise an amino acid sequence of a linker and RT domain from two different amino acid sequences selected from SEQ ID NOs: 1-7743 (or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereof). In certain embodiments, the linker and RT domain of the gene modifying polypeptide comprise an amino acid sequence of a linker and RT domain from different rows of any one of Tables A1, T1 or T2 (or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereof).

In certain embodiments, the genetically modified polypeptide further comprises a first NLS (e.g., a 5' NLS), e.g., as described herein. In certain embodiments, the genetically modified polypeptide further comprises a second NLS (e.g., a 3' NLS), e.g., as described herein. In certain embodiments, the genetically modified polypeptide further comprises an N-terminal methionine residue.

RT families and mutants

In certain embodiments, the gene modifying polypeptide comprises an amino acid sequence from an RT domain sequence selected from the following families: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, XMRV6, BLVAU, BLVJ, HTL1A, HTL1C, HTL1L, HTL32, HTL3P, HTLV2, JSRV, MLVF5, MLVRD, MMTVB, MPMV, SFVCP, SMRVH, SRV1, SRV2, and WDSV. In certain embodiments, the gene modifying polypeptide comprises an amino acid sequence from an RT domain sequence selected from the following families: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, and XMRV6.

In certain embodiments, the genetically modified polypeptide comprises an amino acid sequence of an RT domain sequence from an MLVMS RT domain. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations listed in column 1 of Table M1, or point mutations corresponding thereto. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations listed in column 3 of Table M1 (Gen1MLVMS), or point mutations corresponding thereto. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations at the amino acid positions of the RT domain listed in columns 1 and 2 of Table M2, or one or more point mutations at amino acid positions corresponding thereto.

In certain embodiments, the genetically modified polypeptide comprises an amino acid sequence of an RT domain sequence from an AVIRE RT domain. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations listed in column 2 of Table M1, or point mutations corresponding thereto. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations listed in column 4 of Table M1 (Gen2AVIRE), or point mutations corresponding thereto. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations at the amino acid positions of the RT domain listed in columns 3 and 4 of Table M2, or one or more point mutations at amino acid positions corresponding thereto. In certain embodiments, the RT domain comprises IENSSP (e.g., at the C-terminus).

Table M1. Exemplary point mutations in the MLVMS and AVIRE RT domains

Table M2. Exemplary positions that can be mutated in MLVMS and AVIRE RT domains

In certain embodiments, the genetically modified polypeptide comprises a RT domain of gamma retrovirus origin. In certain embodiments, the RT domain of gamma retrovirus origin of the genetically modified polypeptide comprises an amino acid sequence from an RT domain sequence selected from the following families: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV and XMRV6. In some embodiments, the RT domain of gamma retrovirus origin of the genetically modified polypeptide is not derived from PERV. In some embodiments, the RT comprises one, two, three, four, five, six or more mutations shown in Table 2A, corresponding to mutations D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K or D653N in the RT domain of murine leukemia virus reverse transcriptase. In some embodiments, the genetically modified polypeptide further comprises a linker having at least 99% identity to the linker domain of any one of SEQ ID NOs: 1-7743. In some embodiments, the genetically modified polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO: 11,041.

In embodiments, the RT domain comprises an amino acid sequence of the RT domain of AVIRE RT (e.g., an AVIRE_P03360 sequence, e.g., SEQ ID NO: 8001), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of AVIRE RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, G330P, L605W, T306K, and W313F, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of AVIRE RT further comprising one, two, or three mutations selected from the group consisting of D200N, G330P, and L605W, or corresponding positions in homologous RT domains.

In an embodiment, the RT domain comprises an amino acid sequence of an RT domain of BAEVM RT (e.g., a BAEVM_P10272 sequence, e.g., SEQ ID NO: 8004), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of BAEVM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L602W, T304K, and W311F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of BAEVM RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L602W, or corresponding positions in a homologous RT domain.

In an embodiment, the RT domain comprises an amino acid sequence of the RT domain of FFV RT (e.g., FFV_093209 sequence, e.g., SEQ ID NO: 8012), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of FFV RT further comprising one, two, three, or four mutations selected from the group consisting of D21N, T293N, T419P and L393K, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of FFV RT further comprising one, two, or three mutations selected from the group consisting of D21N, T293N and T419P, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of FFV RT further comprising mutation D21N. In some embodiments, the RT domain comprises an amino acid sequence of FFV RT further comprising one, two, or three mutations selected from the group consisting of T207N, T333P, and L307K, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of FFV RT further comprising one or two mutations selected from the group consisting of T207N and T333P, or corresponding positions in a homologous RT domain.

In an embodiment, the RT domain comprises an amino acid sequence of the RT domain of FLV RT (e.g., FLV_P10273 sequence, e.g., SEQ ID NO: 8019), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of FLV RT further comprising one, two, three, or four mutations selected from the group consisting of D199N, L602W, T305K and W312F, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of FLV RT further comprising one or two mutations selected from the group consisting of D199N and L602W, or corresponding positions in homologous RT domains.

In an embodiment, the RT domain comprises an amino acid sequence of the RT domain of FOAMV RT (e.g., a FOAMV_P14350 sequence, e.g., SEQ ID NO: 8021), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of FOAMV RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, S420P, and L396K, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of FOAMV RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and S420P, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of FOAMV RT further comprising mutation D24N, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of FOAMV RT further comprising one, two, or three mutations selected from the group consisting of T207N, S331P, and L307K, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of FOAMV RT further comprising one or two mutations selected from the group consisting of T207N and S331P, or corresponding positions in a homologous RT domain.

In embodiments, the RT domain comprises an amino acid sequence of an RT domain of GALV RT (e.g., a GALV_P21414 sequence, e.g., SEQ ID NO: 8027), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of GALV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W311F, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of GALV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or corresponding positions in homologous RT domains.

In embodiments, the RT domain comprises an amino acid sequence of the RT domain of KORV RT (e.g., a KORV_Q9TTC1 sequence, e.g., SEQ ID NO: 8047), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of GALV RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D32N, D322N, E452P, L274W, T428K, and W435F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of GALV RT further comprising one, two, three, or four mutations selected from the group consisting of D32N, D322N, E452P, and L274W, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of GALV RT further comprising mutation D32N. In some embodiments, the RT domain comprises an amino acid sequence of KORV RT further comprising one, two, three, four or five mutations selected from the group consisting of D231N, E361P, L633W, T337K and W344F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of KORV RT further comprising one, two, or three mutations selected from the group consisting of D231N, E361P and L633W, or corresponding positions in a homologous RT domain.

In embodiments, the RT domain comprises an amino acid sequence of the RT domain of MLVAV RT (e.g., MLVAV_P03356 sequence, e.g., SEQ ID NO: 8053), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of MLVAV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of MLVAV RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or corresponding positions in homologous RT domains.

In an embodiment, the RT domain comprises an amino acid sequence of the RT domain of MLVBM RT (e.g., MLVBM_Q7SVK7 sequence, e.g., SEQ ID NO: 8056), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of MLVBM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D199N, T329P, L602W, T305K, and W312F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of MLVBM RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or corresponding positions in a homologous RT domain.

In an embodiment, the RT domain comprises an amino acid sequence of the RT domain of MLVCB RT (e.g., MLVCB_P08361 sequence, e.g., SEQ ID NO: 8062), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of MLVCB RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of MLVCB RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or corresponding positions in homologous RT domains.

In embodiments, the RT domain comprises the amino acid sequence of the RT domain of MLVFF RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of MLVFF RT further comprising one, two, three, four or five mutations selected from the group consisting of D200N, T330P, L603W, T306K and W313F, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises the amino acid sequence of MLVFF RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P and L603W, or corresponding positions in homologous RT domains.

In embodiments, the RT domain comprises an amino acid sequence of an RT domain of MLVMS RT (e.g., a MLVMS_reference sequence, e.g., SEQ ID NO: 8137; or a MLVMS_P03355 sequence, e.g., SEQ ID NO: 8070), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of MLVMS RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D200N, T330P, L603W, T306K, W313F, and H8Y, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of MLVMS RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises the amino acid sequence of MLVMSRT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or corresponding positions in a homologous RT domain.

In an embodiment, the RT domain comprises an amino acid sequence of the RT domain of PERV RT (e.g., a PERV_Q4VFZ2 sequence, e.g., SEQ ID NO: 8099), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of PERV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D196N, E326P, L599W, T302K, and W309F, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of PERV RT further comprising one, two, or three mutations selected from the group consisting of D196N, E326P, and L599W, or corresponding positions in homologous RT domains.

In embodiments, the RT domain comprises an amino acid sequence of the RT domain of SFV1 RT (e.g., SFV1_P23074 sequence, e.g., SEQ ID NO: 8105), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of SFV1 RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N420P, and L396K, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of SFV1 RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N420P, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of SFV1 RT further comprising D24N, or corresponding positions in homologous RT domains.

In embodiments, the RT domain comprises an amino acid sequence of the RT domain of SFV3L RT (e.g., SFV3L_P27401 sequence, e.g., SEQ ID NO: 8111), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of SFV3L RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N422P, and L396K, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of SFV3L RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N422P, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of SFV3L RT further comprising mutation D24N, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of SFV3L RT further comprising one, two, or three mutations selected from the group consisting of T307N, N333P, and L307K, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of SFV3L RT further comprising one or two mutations selected from the group consisting of T307N and N333P, or corresponding positions in a homologous RT domain.

In embodiments, the RT domain comprises an amino acid sequence of the RT domain of WMSV RT (e.g., a WMSV_P03359 sequence, e.g., SEQ ID NO: 8131), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of WMSV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W311F, or corresponding positions in homologous RT domains. In some embodiments, the RT domain comprises an amino acid sequence of WMSV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or corresponding positions in homologous RT domains.

In embodiments, the RT domain comprises an amino acid sequence of an RT domain of XMRV6 RT (e.g., an XMRV6_A1Z651 sequence, e.g., SEQ ID NO: 8134), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises an amino acid sequence of an XMRV6 RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises an amino acid sequence of an XMRV6 RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or corresponding positions in a homologous RT domain.

In certain embodiments, the RT domain of the genetically modified polypeptide comprises the amino acid sequence of the RT domain of AVIRE RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In embodiments, the RT domain comprises the amino acid sequence of the RT domain contained in the sequence listed in column 1 of Table A5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the genetically modified polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO: 11,041.

In certain embodiments, the RT domain of the genetically modified polypeptide comprises the amino acid sequence of the RT domain of MLVMS RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In embodiments, the RT domain comprises the amino acid sequence of the RT domain contained in any one of the sequences listed in columns 2-6 of Table A5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In some embodiments, the genetically modified polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO: 11,041.

Table A5. Exemplary gene-modifying polypeptides comprising an AVIRE RT domain or an MLVMS RT domain.

system

On the one hand, the present disclosure relates to a system comprising a nucleic acid molecule encoding a gene-modified polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein). In certain embodiments, the nucleic acid molecule encoding the gene-modified polypeptide comprises one or more silent mutations in the coding region (e.g., in the sequence encoding the RT domain) relative to the nucleic acid molecule described herein. In certain embodiments, the system further comprises a gRNA (e.g., a gRNA bound to a polypeptide that induces a nick, e.g., in the opposite strand of the target DNA to which the gene-modified polypeptide binds).

In certain embodiments, the nucleic acid molecule encoding a genetically modified polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding a genetically modified polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NO: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding a genetically modified polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NO: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the genetically modified polypeptide encodes a polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In certain embodiments, the nucleic acid molecule encoding a gene-modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and an RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the portion. In certain embodiments, the nucleic acid molecule encoding a gene-modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and an RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the portion. In certain embodiments, the nucleic acid molecule encoding a gene-modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541, wherein the portion comprises a linker and an RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the portion. In certain embodiments, the nucleic acid molecule encoding the genetically modified polypeptide comprises a sequence encoding a portion of a polypeptide listed in any one of Tables A1, T1 or T2 (wherein the portion comprises a linker and an RT domain), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In certain embodiments, the nucleic acid molecule encoding a genetically modified polypeptide comprises a sequence encoding a linker of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding a genetically modified polypeptide comprises a sequence encoding a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding a genetically modified polypeptide comprises a sequence encoding a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene-modified polypeptide comprises a sequence encoding a linker of a polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In certain embodiments, the nucleic acid molecule encoding a genetically modified polypeptide comprises a sequence encoding the RT domain of an amino acid sequence selected from SEQ ID NO: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereof. In certain embodiments, the nucleic acid molecule encoding a genetically modified polypeptide comprises a sequence encoding the RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NO: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereof. In certain embodiments, the nucleic acid molecule encoding a genetically modified polypeptide comprises a sequence encoding the RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NO: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereof. In certain embodiments, the nucleic acid molecule encoding the genetically modified polypeptide comprises a sequence encoding the RT domain of a polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In one aspect, the disclosure relates to a system comprising a gene modifying polypeptide (eg, as described herein) and a template nucleic acid (eg, a template RNA, eg, as described herein).

In certain embodiments, the genetically modified polypeptide comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises a polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In certain embodiments, the gene-modified polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and an RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the gene-modified polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and an RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the gene-modified polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541, wherein the portion comprises a linker and an RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises a portion of a polypeptide listed in any one of Tables A1, T1 or T2 (wherein the portion comprises a linker and an RT domain), or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the portion.

In certain embodiments, the genetically modified polypeptide comprises a linker comprising an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereof. In certain embodiments, the genetically modified polypeptide comprises a sequence encoding a linker for a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereof. In certain embodiments, the genetically modified polypeptide comprises a sequence encoding a linker for a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereof. In certain embodiments, the gene-modified polypeptide comprises a linker of a polypeptide as listed in any one of Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

In certain embodiments, the genetically modified polypeptide comprises an RT domain of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises the RT domain of any of the polypeptides listed in Tables A1, T1 or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

Targeting sequences for gene modification systems

In certain embodiments, the gene editor system RNA further comprises an intracellular localization sequence, e.g., a nuclear localization sequence (NLS). In some embodiments, the gene modifying polypeptide comprises an NLS as contained in SEQ ID NO: 4000 and/or SEQ ID NO: 4001, or an NLS having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

The nuclear localization sequence can be an RNA sequence that promotes RNA to be imported into the nucleus. In certain embodiments, the nuclear localization signal is located on the template RNA. In certain embodiments, the gene modification polypeptide is encoded on the first RNA, and the template RNA is the second separate RNA, and the nuclear localization signal is located on the template RNA instead of on the RNA encoding the gene modification polypeptide. Although it is not desired to be bound by theory, in some embodiments, the RNA encoding the gene modification polypeptide mainly targets the cytoplasm to promote its translation, and the template RNA mainly targets the nucleus to promote its insertion into the genome. In some embodiments, the nuclear localization signal is at the 3' end, 5' end or internal region of the template RNA. In some embodiments, the nuclear localization signal is at the 3' end of the heterologous sequence (for example, directly at the 3' of the heterologous sequence) or at the 5' end of the heterologous sequence (for example, directly at the 5' of the heterologous sequence). In some embodiments, the nuclear localization signal is placed outside the 5'UTR of the template RNA or outside the 3'UTR. In some embodiments, the nuclear localization signal is placed between the 5'UTR and the 3'UTR, wherein optionally, the nuclear localization signal is not transcribed with the transgene (for example, the nuclear localization signal is antisense oriented or downstream of the transcription termination signal or polyadenylation signal). In some embodiments, the nuclear localization sequence is located inside an intron. In some embodiments, a plurality of identical or different nuclear localization signals are in RNA, for example, in a template RNA. In some embodiments, the length of the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 bp. Various RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature [Nature] 555 (107-111), 2018 describes an RNA sequence that drives RNA localization into the nucleus. In some embodiments, the nuclear localization signal is a nuclear RNA localization (SIRLOIN) signal derived from SINE. In some embodiments, the nuclear localization signal binds to a nuclear enriched protein. In some embodiments, the nuclear localization signal binds to an HNRNPK protein. In some embodiments, the nuclear localization signal is rich in pyrimidines, for example, a region rich in C/T, rich in C/U, rich in C, rich in T or rich in U. In some embodiments, the nuclear localization signal is derived from long non-coding RNA. In some embodiments, the nuclear localization signal is derived from MALAT1 long non-coding RNA or the M region of 600 nucleotides of MALAT1 (described in Miyagawa et al., RNA 18, (738-751), 2012). In some embodiments, the nuclear localization signal is derived from BORG long non-coding RNA or is an AGCCC motif (described in Zhang et al., Molecular and Cellular Biology [Molecular and Cellular Biology] 34, 2318-2329 (2014)). In some embodiments, the nuclear localization sequence is described in Shukla et al., The EMBO Journal [EMBO Magazine] e98452 (2018). In some embodiments, the nuclear localization signal is derived from a retrovirus.

In some embodiments, the polypeptides described herein comprise one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, such as nuclear localization sequences (NLS). In some embodiments, the NLS is a two-component NLS. In some embodiments, the NLS promotes the import of proteins comprising the NLS into the nucleus. In some embodiments, the NLS is fused to the N-terminus of a genetically modified polypeptide described herein. In some embodiments, the NLS is fused to the C-terminus of a genetically modified polypeptide. In some embodiments, the NLS is fused to the N-terminus or C-terminus of a Cas domain. In some embodiments, a linker sequence is arranged between the NLS and the adjacent domains of the genetically modified polypeptide.

In some embodiments, the NLS comprises the amino acid sequences MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 5009), PKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5010), RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 5011), KRTADGSEFESPKKKRKV (SEQ ID NO: 5012), KKTELQTT NAENKTKKL(SEQ IDNO： 5013), or KRGINDRNFWRGENGRKTR (SEQ ID NO: 5014), KRPAATKKAGQAKKKK (SEQ ID NO: 5015), PAAKRVKLD (SEQ ID NO: 4644), KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4649), KRTADGSEFE (SEQ ID NO: 4649), NO: 4650), KRTADGSEFESPKKKAKVE (SEQ ID NO: 4651), AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4001), or a functional fragment or variant thereof. Exemplary NLS sequences are also described in PCT/EP2000/011690, the contents of which are directed to Its disclosure of exemplary nuclear localization sequences is incorporated herein by reference. In some embodiments, the NLS comprises an amino acid sequence as disclosed in Table 11. The NLS of this table can be present in one or more copies of a polypeptide in a polypeptide. Multiple locations are used, such as 1, 2, 3 or more copies of the NLS in the N-terminal domain, between peptide domains, in the C-terminal domain, or a combination of multiple locations to improve subcellular localization in the nucleus Multiple unique sequences may be used in a single polypeptide. The sequences may be mono- or bi-component in nature, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bi-component sequences. Sequence references correspond to UniProt accession numbers unless indicated as SeqNLS for sequences mined using the subcellular localization prediction algorithm (Lin et al. BMC Bioinformat [BMC Bioinformatics] 13: 157 (2012), incorporated herein by reference in its entirety).

Table 11 Exemplary nuclear localization signals for gene modification systems

In some embodiments, the NLS is a two-component NLS. A two-component NLS typically comprises two basic amino acid clusters separated by a spacer sequence (whose length can be, for example, about 10 amino acids). A single-component NLS typically lacks a spacer. An example of a two-component NLS is a nucleoplasmin NLS, having the sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 5015), wherein the spacer is placed in brackets. Another exemplary two-component NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5016). Exemplary NLSs are described in International Application WO 2020051561, which is incorporated herein by reference in its entirety, including its disclosure about nuclear localization sequences.

In certain embodiments, the gene editor system polypeptide (e.g., a gene modification polypeptide as described herein) further comprises an intracellular localization sequence, for example, a nuclear localization sequence and/or a nucleolar localization sequence. The nuclear localization sequence and/or the nucleolar localization sequence can be an amino acid sequence that promotes protein import into the nucleus and/or the nucleolus, wherein it can promote the integration of heterologous sequences into the genome. In certain embodiments, the gene editor system polypeptide (e.g., a gene modification polypeptide as described herein) further comprises a nucleolar localization sequence. In certain embodiments, the gene modification polypeptide is encoded on a first RNA, the template RNA is a second separate RNA, and the nucleolar localization signal is encoded on the RNA encoding the gene modification polypeptide, and not on the template RNA. In some embodiments, the nucleolar localization signal is located at the N-terminus, C-terminus, or internal region of the polypeptide. In some embodiments, a plurality of identical or different nucleolar localization signals are used. In some embodiments, the length of the nuclear localization signal is less than 5, 10, 25, 50, 75, or 100 amino acids. Various polypeptide nucleolar localization signals can be used. For example, Yang et al., Journal of Biomedical Science 22, 33 (2015) describes a nuclear localization signal that also functions as a nucleolar localization signal. In some embodiments, a nucleolar localization signal may also be a nuclear localization signal. In some embodiments, a nucleolar localization signal may overlap with a nuclear localization signal. In some embodiments, a nucleolar localization signal may comprise a segment of basic residues. In some embodiments, a nucleolar localization signal may be rich in arginine and lysine residues. In some embodiments, a nucleolar localization signal may be derived from a protein that is enriched in the nucleolus. In some embodiments, a nucleolar localization signal may be derived from a protein that is enriched at a ribosomal RNA locus. In some embodiments, a nucleolar localization signal may be derived from a protein that binds rRNA. In some embodiments, a nucleolar localization signal may be derived from MSP58. In some embodiments, a nucleolar localization signal may be a single component motif. In some embodiments, a nucleolar localization signal may be a two-component motif. In some embodiments, a nucleolar localization signal may be composed of a plurality of single component or two-component motifs. In some embodiments, a nucleolar localization signal may be composed of a mixture of single component and two-component motifs. In some embodiments, a nucleolar localization signal may be a dual two-component motif. In some embodiments, the nucleolar localization motif may be KRASSQALGTIPKRRSSSRFIKRKK (SEQ ID NO: 5017). In some embodiments, the nucleolar localization signal may be derived from nuclear factor-κB-inducing kinase. In some embodiments, the nucleolar localization signal may be a RKKRKKK motif (SEQ ID NO: 5018) (described in Birbach et al., Journal of Cell Science, 117 (3615-3624), 2004).

Genetically modified peptides and evolutionary variants of the system

In some embodiments, the invention provides an evolutionary variant of a gene modified polypeptide as described herein. In some embodiments, an evolutionary variant can be produced by mutagenizing a reference gene modified polypeptide or one of the fragments or domains contained therein. In some embodiments, one or more domains (e.g., reverse transcriptase domains) evolve. In some embodiments, one or more such evolutionary variant domains can be made to evolve alone or with other domains. In some embodiments, one or more evolutionary variant domains can be combined with one or more unevolved homologous components or the evolutionary variant of one or more homologous components, for example, the evolutionary variant of the one or more homologous components can evolve in parallel or in a continuous manner.

In some embodiments, the process of mutagenizing a reference gene modified polypeptide or its fragment or domain includes mutagenizing a reference gene modified polypeptide or its fragment or domain. In an embodiment, mutagenesis includes a continuous evolution method (e.g., PACE) or a discontinuous evolution method (e.g., PANCE), for example, as described herein. In some embodiments, the evolved gene modified polypeptide or its fragment or domain comprises one or more amino acid variations introduced into its amino acid sequence relative to the amino acid sequence of the reference gene modified polypeptide or its fragment or domain. In an embodiment, amino acid sequence variation may include one or more mutated residues (e.g., conservative substitutions, non-conservative substitutions, or a combination thereof) within the amino acid sequence of the reference gene modified polypeptide, for example, the one or more mutated residues are due to changes in the nucleotide sequence encoding the gene modified polypeptide (e.g., changes in codons at any particular position in the coding sequence), which causes one or more amino acids (e.g., truncated proteins) to be deleted, one or more amino acids to be inserted, or any combination of the foregoing. Evolution variant gene modified polypeptides may include variants in one or more components or domains of a gene modified polypeptide (e.g., variants introduced into a reverse transcriptase domain).

In some aspects, the disclosure provides genetically modified polypeptides, systems, kits and methods using or comprising evolutionary variants of genetically modified polypeptides, e.g., genetically modified polypeptides produced or producible by PACE or PANCE are employed. In embodiments, the unevolved reference genetically modified polypeptide is a genetically modified polypeptide as disclosed herein.

As used herein, the term "phage-assisted continuous evolution (PACE)" generally refers to continuous evolution using bacteriophage as viral vectors. Examples of PACE technology have been described, for example, in International PCT Application No. PCT/US 2009/056194, filed September 8, 2009, published as WO 2010/028347 on March 11, 2010; International PCT Application No. PCT/US 2011/066747, filed December 22, 2011, published as WO 2012/088381 on June 28, 2012; U.S. Patent No. 9,023,594, issued May 5, 2015; U.S. Patent No. 9,771,574, issued September 26, 2017; U.S. Patent No. 9,394,537, issued July 19, 2016; International PCT Application No. PCT/US 2015/066747, filed December 22, 2011, published as WO 2012/088381 on June 28, 2012 2015/012022, which was published on September 11, 2015 as WO 2015/134121; U.S. Patent No. 10,179,911, issued on January 15, 2019; and International PCT Application PCT/US 2016/027795, filed on April 15, 2016, which was published on October 20, 2016 as WO 2016/168631, the entire contents of each of which are incorporated herein by reference.

As used herein, the term "phage-assisted discontinuous evolution (PANCE)" generally refers to discontinuous evolution using phage as a viral vector. Examples of PANCE technology have been described, for example, in Suzuki T. et al., Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase, Nat Chem Biol. 13 (12): 1261-1266 (2017), which is incorporated herein by reference in its entirety. In brief, PANCE is a technique for rapid in vivo directed evolution using continuous flask transfers of evolving selected phage (SP), which contain target genes to be evolved in fresh host cells (e.g., E. coli cells). Genes within the host cell may remain unchanged, while genes contained in the SP evolve continuously. After phage growth, aliquots of infected cells can be used to transfect subsequent flasks containing host E. coli. This process can be repeated and/or continued until the desired phenotype has evolved, e.g., for a desired number of transfers.

The skilled person can easily understand the methods of applying PACE and PANCE to gene-modified polypeptides by referring to, inter alia, the aforementioned documents. Additional exemplary methods for directing the continuous evolution of genome-modified proteins or systems, such as using phage particles, for example, in a host cell population, can be used to generate evolutionary variants of gene-modified polypeptides or fragments or subdomains thereof. Non-limiting examples of such methods are described in the following: International PCT Application No. PCT/US2009/056194, filed September 8, 2009, published as WO 2010/028347 on March 11, 2010; International PCT Application No. PCT/US 2011/066747, filed December 22, 2011, published as WO 2012/088381 on June 28, 2012; U.S. Patent No. 9,023,594, issued May 5, 2015; U.S. Patent No. 9,771,574, issued September 26, 2017; U.S. Patent No. 9,394,537, issued July 19, 2016; International PCT Application No. PCT/US 2011/066747, filed December 22, 2011, published as WO 2012/088381 on June 28, 2012; U.S. Patent No. 9,023,594, issued May 5, 2015; U.S. Patent No. 9,771,574, issued September 26, 2017; U.S. Patent No. 9,394,537, issued July 19, 2016; International PCT Application No. PCT/US 2015/012022, which was published as WO 2015/134121 on September 11, 2015; U.S. Patent No. 10,179,911, issued on January 15, 2019; International Application No. PCT/US2019/37216, filed on June 14, 2019; International Patent Publication WO 2019/023680, published on January 31, 2019; International PCT Application PCT/US2016/027795, filed on April 15, 2016, which was published as WO 2016/168631 on October 20, 2016; and International Patent Publication No. PCT/US2019/47996, filed on August 23, 2019; each of which is incorporated herein by reference in its entirety.

In some non-limiting illustrative embodiments, a method for evolving an evolutionary variant genetically modified polypeptide, or a fragment or domain thereof, comprises: (a) contacting a population of host cells with a population of viral vectors comprising a gene of interest (the starting genetically modified polypeptide or a fragment or domain thereof), wherein: (1) the host cells are susceptible to infection by the viral vector; (2) the host cells express viral genes required for the production of viral particles; (3) the expression of at least one viral gene required for the production of infectious viral particles depends on the function of the gene of interest; and/or (4) the viral vector allows the protein to be expressed in the host cells and can be replicated and packaged into viral particles by the host cells. In some embodiments, the method comprises (b) contacting the host cells with a mutagen, using a host cell with a mutation that increases the mutation rate (e.g., by carrying a mutant plasmid or some genomic modification - for example, a proofreading-impaired DNA polymerase, an SOS gene, such as UmuC, UmuD', and/or RecA, which, if combined with a plasmid, may be under the control of an inducible promoter) or a combination thereof. In some embodiments, the method includes (c) incubating a host cell colony under conditions that allow viral replication and production of viral particles, wherein host cells are removed from the host cell colony, and fresh, uninfected host cells are introduced into the host cell colony, thereby supplementing the host cell colony and producing a host cell flow. In some embodiments, the cells are incubated under conditions that allow the target gene to acquire mutations. In some embodiments, the method further includes (d) isolating a mutant version of the viral vector from the host cell colony, the mutant version encoding an evolved gene product (e.g., an evolutionary variant gene modified polypeptide or a fragment or domain thereof).

Technicians will appreciate the various features that can be adopted within the above framework.For example, in some embodiments, viral vector or phage are filamentous phages, such as M13 phages, such as M13 selection phages.In certain embodiments, the gene required for producing infectious viral particles is M13 gene III (gIII).In an embodiment, phage may lack functional gIII, but different is to comprise gI, gII, gIV, gV, gVI, gVII, gVIII, gIX and gX.In certain embodiments, the generation of infectious VSV particles relates to envelope protein VSV-G.Various embodiments can use different retroviral vectors, such as murine leukemia virus vectors or slow virus vectors.In an embodiment, utilizing VSV-G envelope protein (for example, as a substitute for the natural envelope protein of virus) can effectively package retroviral vectors.

In some embodiments, the host cells are incubated for a suitable number of viral life cycles, e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000 or more consecutive viral life cycles, each of which is 10-20 minutes in the illustrative and non-limiting example of M13 bacteriophage. Similarly, conditions can be adjusted to adjust the time that a host cell remains in a host cell population, e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 150, or about 180 minutes. The host cell population can be controlled in part by the density of the host cells, or in some embodiments, the host cell density in the influent is, for example, 10 ³ cells/ml, about 10 ⁴ cells/ml, about 10 ⁵ cells/ml, about 5-10 ⁵ cells/ml, about 10 ⁶ cells/ml, about 5-10 ⁶ cells/ml, about 10 ⁷ cells/ml, about 5-10 ⁷ cells/ml, about 10 ⁸ cells/ml, about 5-10 ⁸ cells/ml, about 10 ⁹ cells/ml, about 5.10 ⁹ cells/ml, about 10 ¹⁰ cells/ml, or about 5.10 ¹⁰ cells/ml.

Intein

In some embodiments, as described in more detail below, the intein-N (intN) domain can be fused to the N-terminal portion of the first domain of a genetically modified polypeptide described herein, and the intein-C (intC) domain can be fused to the C-terminal portion of the second domain of a genetically modified polypeptide described herein for connecting the N-terminal portion to the C-terminal portion, thereby connecting the first and second domains. In some embodiments, the first and second domains are each independently selected from a DNA binding domain, an RNA binding domain, an RT domain, and an endonuclease domain.

Inteins can occur as self-splicing protein introns (e.g., peptides), for example, which connect flanking N-terminal and C-terminal exteins (e.g., fragments to be connected). In some cases, inteins can comprise fragments of proteins that are able to self-excise and connect the remaining fragments (exteins) with peptide bonds in a process called protein splicing. Inteins are also called "protein introns." The process of intein self-excision and connecting the remaining parts of the protein is referred to herein as "protein splicing" or "intein-mediated protein splicing."

In certain embodiments, the intein of the precursor protein (the protein containing intein before the intein-mediated protein splicing) comes from two genes. Such intein is referred to herein as a fracture intein (e.g., fracture intein-N and fracture intein-C). Therefore, the first polypeptide sequence and the second polypeptide sequence can be linked together using an intein-based method. For example, in cyanobacteria, the catalytic subunit a (i.e., DnaE) of DNA polymerase III is encoded by two separate genes dnaE-n and dnaE-c. When positioned as a part of the first polypeptide sequence, the intein-N domain (e.g., encoded by the dnaE-n gene) can connect the first polypeptide sequence to the second polypeptide sequence, wherein the second polypeptide sequence comprises an intein-C domain (e.g., encoded by the dnaE-c gene). Thus, in some embodiments, a protein can be prepared by providing nucleic acids encoding first and second polypeptide sequences (e.g., wherein the first nucleic acid molecule encodes the first polypeptide sequence and the second nucleic acid molecule encodes the second polypeptide sequence), and introducing the nucleic acids into a cell under conditions that allow for the production of the first and second polypeptide sequences and for the first polypeptide sequence to be linked to the second polypeptide sequence by an intein-based mechanism.

The use of inteins to link heterologous protein fragments is described in, for example, Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014) (which is incorporated herein by reference in its entirety). For example, when fused to separate protein fragments, inteins IntN and IntC can recognize each other, self-cleave, and/or simultaneously link to the flanking N-terminal and C-terminal exteins of the protein fragments to which they are fused, thereby reconstructing a full-length protein from two protein fragments.

In some embodiments, synthetic inteins based on the dnaE intein are used, namely, the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pairs. Examples of such inteins are described, for example, in Stevens et al., J Am Chem Soc. 2016 Feb 24;138(7):2162-5 (which is incorporated herein by reference in its entirety). Non-limiting examples of intein pairs that can be used according to the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, which is incorporated herein by reference).

In some embodiments involving the split Cas9, the intein-N domain and the intein-C domain can be fused to the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9, respectively, so as to connect the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9. For example, in some embodiments, the intein-N is fused to the C-terminal end of the N-terminal portion of the split Cas9, i.e., forming a structure of N-[N-terminal portion of the split Cas9]-[intein-N]~C. In some embodiments, the intein-C is fused to the N-terminal end of the C-terminal portion of the split Cas9, i.e., forming a structure of N-[intein-C]~[C-terminal portion of the split Cas9]-C. The intein-mediated protein splicing mechanism for connecting a protein fused with an intein (e.g., a split Cas9) is described in the following: Shah et al., Chem Sci. [Chemical Science] 2014; 5(1): 446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art, and are described, for example, by WO 2020051561, WO2014004336, WO 2017132580, US 20150344549, and US 20180127780, each of which is incorporated herein by reference in its entirety.

In some embodiments, the break refers to being divided into two or more fragments. In some embodiments, the break Cas9 protein or the break Cas9 comprises a Cas9 protein, which is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. The polypeptide corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein can be spliced to form a reconstructed Cas9 protein. In an embodiment, the Cas9 protein is divided into two fragments within the disordered region of the protein, for example, as described in: Nishimasu et al., Cell [Cell], Vol. 156, No. 5, pp. 935-949, 2014, or Jiang et al. (2016) Science [Science] 351: 867-871 and PDB file: 5F9R (each of which is incorporated herein by reference in its entirety). The disordered region can be determined by one or more protein structure determination techniques known in the art, including but not limited to X-ray crystallography, NMR spectroscopy, electron microscopy (e.g., cryoEM) and/or computer protein modeling. In some embodiments, the protein is split into two fragments at any C, T, A, or S in the region of SpCas9, for example, between amino acids A292-G364, F445-K483, or E565-T637, or at corresponding positions in any other Cas9, Cas9 variants (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting a protein into two fragments is referred to as cleavage of the protein.

In some embodiments, the length of the protein fragment ranges from about 2-1000 amino acids (e.g., 2-10, 10-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids). In some embodiments, the length of the protein fragment ranges from about 5-500 amino acids (e.g., 5-10, 10-50, 50-100, 100-200, 200-300, 300-400, or 400-500 amino acids). In some embodiments, the length of the protein fragment ranges from about 20-200 amino acids (e.g., 20-30, 30-40, 40-50, 50-100, or 100-200 amino acids).

In some embodiments, a portion or fragment of a genetically modified polypeptide is fused to an intein. A nuclease may be fused to the N-terminus or C-terminus of an intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. Inteins, nucleases, and capsid proteins may be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of an intein is fused to the C-terminus of a fusion protein, and the C-terminus of an intein is fused to the N-terminus of an AAV capsid protein.

In some embodiments, an endonuclease domain (e.g., a nickase Cas9 domain) is fused to intein-N, and a polypeptide comprising a RT domain is fused to intein-C.

Exemplary nucleotide and amino acid sequences of intein-N domains and compatible intein-C domains are provided below:

DnaE Intein-N DNA:

DnaE Intein-N Protein:

DnaE Intein-C DNA:

DnaE Intein-C Protein:

Cfa-N DNA:

Cfa-N Protein:

Cfa-C DNA:

Cfa-C Protein:

Additional domains

Genetic modification polypeptides can bind to target DNA sequences and template nucleic acids (e.g., template RNA), cut target sites and write (e.g., reverse transcribe) templates into DNA, thereby producing modifications of target sites. In some embodiments, additional domains can be added to the polypeptide to improve the efficiency of the process. In some embodiments, the gene modification polypeptide may include additional DNA connection domains to connect reverse transcribed DNA to the DNA of the target site. In some embodiments, the polypeptide may include a heterologous RNA binding domain. In some embodiments, the polypeptide may include a domain with 5' to 3' exonuclease activity (e.g., wherein the 5' to 3' exonuclease activity increases the repair of changes in the target site, such as facilitating changes in the original genomic sequence). In some embodiments, the polypeptide may include a domain with 3' to 5' exonuclease activity, such as a proofreading activity. In some embodiments, a writing domain, such as an RT domain, has a 3' to 5' exonuclease activity, such as a proofreading activity.

Template nucleic acid

Gene modification systems as described herein can modify host target DNA sites using template nucleic acid sequences. In certain embodiments, gene modification systems as described herein transcribe RNA sequence templates into host target DNA sites by reverse transcription (TPRT) triggered by target. Modify one or more DNA sequences by directly reverse transcription of RNA sequence templates into host genome, gene modification systems can insert object sequences into target genomes, without the need to introduce exogenous DNA sequences into host cells (different from, for example, CRISPR systems) and eliminate exogenous DNA insertion steps. Gene modification systems can also delete sequences from target genomes or introduce substitutions using object sequences. Therefore, gene modification systems provide platforms using customized RNA sequence templates, which templates include object sequences, for example, sequences including heterologous gene encoding and/or functional information.

In some embodiments, the template nucleic acid comprises one or more sequences (eg, 2 sequences) that bind to a gene modifying polypeptide.

In some embodiments, the systems or methods described herein include a single template nucleic acid (e.g., template RNA). In some embodiments, the systems or methods described herein include multiple template nucleic acids (e.g., template RNA). For example, the system described herein includes a first RNA and a second RNA (e.g., template RNA), the first RNA includes (e.g., from 5' to 3') a sequence (e.g., DNA binding domain and/or endonuclease domain, e.g., gRNA) binding to a gene-modified polypeptide and a sequence binding to a target site (e.g., the second strand of a site in a target genome), the second RNA includes (e.g., from 5' to 3') optionally binding to a gene-modified polypeptide (e.g., specifically binding to an RT domain), a heterologous object sequence, and a PBS sequence. In some embodiments, when the system includes multiple nucleic acids, each nucleic acid includes a conjugated domain. In some embodiments, the conjugated domain enables nucleic acid molecules to be associated, e.g., by hybridization of complementary sequences. For example, in some embodiments, the first RNA includes a first conjugated domain and the second RNA includes a second conjugated domain, and the first and second conjugated domains can hybridize to each other, e.g., under stringent conditions. In some embodiments, stringent conditions for hybridization include hybridization in 4x sodium chloride/sodium citrate (SSC) at about 65°C, followed by a wash in 1x SSC at about 65°C.

In some embodiments, the template nucleic acid comprises RNA. In some embodiments, the template nucleic acid comprises DNA (e.g., single-stranded or double-stranded DNA).

In some embodiments, the template nucleic acid comprises one or more (e.g., 2) homology domains having homology to the target sequence. In some embodiments, the length of the homology domain is about 10-20, 20-50, or 50-100 nucleotides.

In some embodiments, the template RNA may include a gRNA sequence, for example, to guide the gene-modifying polypeptide to the target site of interest. In some embodiments, the template RNA includes (e.g., from 5' to 3'): (i) a gRNA spacer that optionally binds to a target site (e.g., the second strand of a site in a target genome), (ii) a gRNA scaffold that optionally binds to a polypeptide described herein (e.g., a gene-modifying polypeptide or a Cas polypeptide), (iii) a heterologous subject sequence that includes a mutation region (optionally, the heterologous subject sequence includes a first homology region, a mutation region, and a second homology region from 5' to 3'), and (iv) a primer binding site (PBS) sequence that includes a 3' target homology domain.

The template nucleic acid (e.g., template RNA) component of the genome editing system described herein is generally capable of binding to the gene-modified polypeptide of the system. In some embodiments, the template nucleic acid (e.g., template RNA) has a 3′ region that is capable of binding to the gene-modified polypeptide. The binding region (e.g., 3′ region) can be a structured RNA region, for example, having at least 1, 2, or 3 hairpin loops that are capable of binding to the gene-modified polypeptide of the system. The binding region can associate the template nucleic acid (e.g., template RNA) with any polypeptide module. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) can be associated with the RNA binding domain in the polypeptide. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) can be associated with the reverse transcription domain of the gene-modified polypeptide (e.g., specifically binding to the RT domain). In some embodiments, the template nucleic acid (e.g., template RNA) can be associated with the DNA binding domain of the polypeptide, for example, gRNA is associated with the DNA binding domain of the Cas9 source. In some embodiments, the binding region can also provide DNA target recognition, such as gRNA hybridizing with the target DNA sequence and binding to the polypeptide, such as the Cas9 domain. In some embodiments, a template nucleic acid (eg, a template RNA) can be associated with multiple components of a polypeptide (eg, a DNA binding domain and a reverse transcription domain).

In some embodiments, the template RNA has a poly A tail at the 3' end. In some embodiments, the template RNA does not have a poly A tail at the 3' end.

In some embodiments, the template nucleic acid is a template RNA. In some embodiments, the template RNA comprises one or more modified nucleotides. For example, in some embodiments, the template RNA comprises one or more deoxyribonucleotides. In some embodiments, a region of the template RNA is replaced by a DNA nucleotide, for example, to enhance the stability of the molecule. For example, the 3′ end of the template may comprise a DNA nucleotide, and the rest of the template comprises an RNA nucleotide that can be reverse transcribed. For example, in some embodiments, the heterologous object sequence is mainly or completely composed of RNA nucleotides (e.g., at least 90%, 95%, 98% or 99% RNA nucleotides). In some embodiments, the PBS sequence is mainly or completely composed of DNA nucleotides (e.g., at least 90%, 95%, 98% or 99% DNA nucleotides). In other embodiments, the heterologous object sequence for writing into the genome may comprise DNA nucleotides. In some embodiments, the DNA nucleotides in the template are copied into the genome by a domain capable of having DNA-dependent DNA polymerase activity. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in a polypeptide. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain, which is also capable of DNA-dependent DNA polymerization, such as second-strand synthesis. In some embodiments, the template molecule consists solely of DNA nucleotides.

In some embodiments, the system described herein comprises two nucleic acids, which together constitute the sequence of the template RNA described herein. In some embodiments, the two nucleic acids are associated with each other in a non-covalent manner, for example, directly associated with each other (e.g., by base pairing), or indirectly associated as part of a complex comprising one or more additional molecules.

The template RNA described herein may comprise, from 5' to 3': (1) a gRNA spacer; (2) a gRNA scaffold; (3) a heterologous target sequence; (4) a primer binding site (PBS) sequence. Each of these components will now be described in more detail.

gRNA spacers and gRNA scaffolds

The template RNA described herein may include a gRNA spacer that guides the gene modification system to the target nucleic acid, and a gRNA scaffold that promotes the association of the template RNA with the Cas domain of the gene modification polypeptide. The system described herein may also include a gRNA that is not a part of the template nucleic acid. For example, a gRNA comprising a gRNA spacer and a gRNA scaffold but not comprising a heterologous object sequence or a PBS sequence may be used, for example, to induce a second strand nick, for example, as described in the section entitled "Second Strand Nicking" herein.

In some embodiments, gRNA is a short synthetic RNA consisting of a scaffold sequence involved in CRISPR-associated protein binding and a user-defined targeting sequence of about 20 nucleotides for a genomic target. Nishimasu et al. Cell [Cell] 156, pp. 935-949 (2014) describes the structure of a complete gRNA. gRNA (also known as sgRNA of a single guide RNA) consists of sequences derived from crRNA and tracrRNA, which are connected by artificial tetraloops. The crRNA sequence can be divided into a guide region (20nt) and a repeat sequence region (12nt), while the tracrRNA sequence can be divided into an anti-repeat sequence region (14nt) and three tracrRNA stem loops (Nishimasu et al. Cell [Cell] 156, pp. 935-949 (2014)). In practice, the guide RNA sequence is typically designed to have a length of 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and is complementary to the target nucleic acid sequence. Custom gRNA generators and algorithms are commercially available for designing effective guide RNAs. In some embodiments, gRNA comprises two RNA components from a natural CRISPR system, such as crRNA and tracrRNA. As is known in the art, gRNA may also include a chimeric single guide RNA (sgRNA) containing sequences from tracrRNA (to bind nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing/binding). Chemically modified sgRNAs have also been shown to be effective for use with CRISPR-associated proteins; see, for example, Hendel et al. (2015) Nature Biotechnol. [Nature Biotechnology], 985-991. In some embodiments, the gRNA spacer comprises a nucleic acid sequence complementary to a DNA sequence associated with a target gene.

In some embodiments, a region of a template nucleic acid (e.g., template RNA) comprising a gRNA adopts an underwinding ribbon structure of a gRNA bound to a target DNA (e.g., as described in: Mulepati et al. Science 2014 Sep 19: Vol. 345, No. 6203, pp. 1479-1484). Without wishing to be bound by theory, this atypical structure is believed to be promoted by rotating out of the RNA-DNA hybrid every six nucleotides. Thus, in some embodiments, a region of a template nucleic acid (e.g., template RNA) comprising a gRNA can tolerate increased mismatches with a target site at certain intervals (e.g., every six bases). In some embodiments, a region of a template nucleic acid (e.g., template RNA) comprising a gRNA homologous to a target site can have wobble positions at regular intervals (e.g., every six bases) that do not require base pairing with the target site.

In some embodiments, the template nucleic acid (e.g., template RNA) has at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 bases with at least 80%, 85%, 90%, 95%, 99%, or 100% homology to the target site, e.g., at the 5' end, e.g., comprising a gRNA spacer sequence of a length suitable for the Cas9 domain (Table 8) of the gene modifying polypeptide.

In some embodiments, Cas9 derivatives with enhanced activity can be used in gene-modified polypeptides. In some embodiments, the Cas9 derivatives can include mutations that improve the activity of the HNH endonuclease domain, such as SpyCas9R221K, N394K, or mutations that improve R-loop formation, such as SpyCas9 L1245V, or combinations of such mutations, such as SpyCas9 R221K/N394K, SpyCas9 N394K/L1245V, SpyCas9 R221K/L1245V, or SpyCas9 R221K/N394K/L1245V (see, e.g., Spencer and Zhang Sci Rep [Science Report] 7: 16836 (2017), Cas9 derivatives and their contents containing mutations are incorporated herein by reference). In some embodiments, the Cas9 derivative may include one or more types of mutations described herein, e.g., PAM modification mutations, protein stabilization mutations, activity enhancement mutations, and/or mutations that partially or completely inactivate one or two endonuclease domains relative to the parent enzyme (e.g., one or more mutations to eliminate endonuclease activity against one or both strands of the target DNA, e.g., nickase or catalytic inactivation enzyme). In some embodiments, the Cas9 enzyme used in the system described herein may include a mutation that imparts enzyme nickase activity (e.g., SpyCas9N863A or H840A) in addition to mutations that improve catalytic efficiency (e.g., SpyCas9R221K, N394K, and/or L1245V). In some embodiments, the Cas9 enzyme used in the system described herein is a SpyCas9 enzyme or derivative that further includes an N863A mutation that imparts nickase activity in addition to R221K and N394K mutations that improve catalytic efficiency.

Table 12 provides parameters defining the components for designing gRNA and/or template RNA to apply the Cas variants listed in Table 8 to gene modification. The cleavage site indicates the verified or predicted original spacer adjacent motif (PAM) requirement, verified or predicted cleavage site position (relative to the most upstream base of the PAM site). The gRNA of a given enzyme can be assembled by connecting crRNA, tetraloop and tracrRNA sequences, and further adding a 5' spacer with a length matching the original spacer of the target site in the spacer (min) and spacer (max). In addition, the predicted position of the ssDNA nick on the target is important for designing the PBS sequence of the template RNA (which can be immediately annealed with the sequence of the nick 5' to start the reverse transcription initiated by the target). In some embodiments, the gRNA scaffold described herein comprises a nucleic acid sequence comprising the following in the 5' to 3' direction, or a sequence having at least 70%, 80%, 85%, 90%, 95% or 99% identity thereto: a crRNA of Table 12, a tetraloop from the same row of Table 12, and a tracrRNA from the same row of Table 12. In some embodiments, the gRNA or template RNA comprising the scaffold further comprises a gRNA spacer having a length within the spacer (min) and spacer (max) indicated in the same row of Table 12. In some embodiments, the system further comprising a gene modifying polypeptide comprises a gRNA or template RNA having a sequence according to Table 12, wherein the gene modifying polypeptide comprises a Cas domain described in the same row of Table 12.

Heterogeneous object sequences

The template RNA described herein may include a heterologous subject sequence, which the gene-modifying polypeptide may use as a template for reverse transcription to write the desired sequence into the target nucleic acid. In some embodiments, the heterologous subject sequence includes a post-editing homology region, a mutation region, and a pre-editing homology region from 5' to 3'. Without wishing to be bound by theory, the RT that reverse transcribes the template RNA first reverse transcribes the pre-editing homology region, then the mutation region, and then the post-editing homology region, thereby generating a DNA strand containing the desired mutation with homology regions on both sides.

In some embodiments, the length of the heterologous subject sequence is at least 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 , 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, or 1,000 nucleotides (nt) or a length of at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases. In some embodiments, the length of the heterogeneous object sequence is no more than 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 7, 8, 9, 10, 15, 10, 15, 20, 25, 30, 35, 40, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, 1,000, or 2,000 nucleotides (nt) or a length of no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 kilobases. In some embodiments, the length of the heterogeneous object sequence is 30-1000, 40-1000, 50-1000, 60-1000, 70-1000, 74-1000, 75-1000, 76-1000, 77-1000, 78-1000, 79-1000, 80-1000, 85-1000, 90-1000, 100-1000, 120-1000, 140-1000, 160-1000, 180-1000, 200-1000, 500-1000, 30-500, 40-500, 50-500, 60-500, 70-500, 74-500, 75-500, 76-500, 77-500, 78-500, 79-500, 80-500, 85-500, 90-500, 100-500, 120-500, 140-500, 160-500, 180-500, 200-500, 30- 200, 40-200, 50-200, 60-200, 70-200, 74-200, 75-200, 76-200, 77-200, 78-200, 79-200, 80-200, 85-200, 90-200, 100-20 0, 120-200, 140-200, 160-200, 180-200, 30-100, 40-100, 50-100, 60-100, 70-100, 74-100, 75-100, 76-100, 77-100, 78-100, 79-100, 80-100, 85-100 or 90-100 nucleotides (nt) in length, or 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-15, 6-10, 6-9, 6-8, 6-7, 7-20, 7-15, 7-10, 7-9, 7-8, 8-20, 8-15, 8-10, 8-9, 9-20, 9-15, 9-10, 10-15, 10-20, or 15-20 kilobases. In some embodiments, the length of the heterologous subject sequence is 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10-20 nt, such as 10-80, 10-50, or 10-20 nt in length, such as about 10-20 nt in length. In some embodiments, the length of the heterologous subject sequence is 8-30, 9-25, 10-20, 11-16, or 12-15 nucleotides in length, such as 11-16 nt in length. Without wishing to be bound by theory, in some embodiments, a larger insert size, a larger edit region (e.g., the distance between the first edit/substitution and the second edit/substitution in the target region), and/or a greater number of desired edits (e.g., mismatches of the heterologous subject sequence with the target genome) can result in a longer optimal heterologous subject sequence.

In certain embodiments, the template nucleic acid comprises a customized RNA sequence template, which can identify, design, engineer and construct a customized RNA sequence template to include a sequence that changes or specifies the function of the host genome, such as by introducing a heterologous coding region into the genome; affecting or causing exon structure/alternative splicing, such as causing exon skipping of one or more exons; causing endogenous gene disruption, such as causing gene knockout; causing transcriptional activation of endogenous genes; causing epigenetic regulation of endogenous DNA; causing upregulation of one or more operably connected genes, such as causing gene activation or overexpression; causing upregulation of one or more operably connected genes, such as causing gene knockout; etc. In certain embodiments, the customized RNA sequence template can be engineered to include sequences encoding exons and/or transgenes, providing binding sites for transcription factor activators, repressors, enhancers, etc. and combinations thereof. In some embodiments, a customized template can be engineered to encode a nucleic acid or peptide tag to be expressed in an endogenous RNA transcript or endogenous protein operably connected to a target site. In other embodiments, the coding sequence can be further customized with a splice donor site, a splice acceptor site, or a poly A tail.

The template nucleic acid (e.g., template RNA) of the system generally includes an object sequence (e.g., a heterologous object sequence) for writing the desired sequence into the target DNA. The object sequence may be coded or non-coded. The template nucleic acid (e.g., template RNA) may be designed to produce an insertion, mutation, or deletion at the target DNA locus. In some embodiments, the template nucleic acid (e.g., template RNA) may be designed to cause insertion into the target DNA. For example, the template nucleic acid (e.g., template RNA) may contain a heterologous sequence, wherein reverse transcription will cause the heterologous sequence to be inserted into the target DNA. In other embodiments, the RNA template may be designed to introduce deletions into the target DNA. For example, the template nucleic acid (e.g., template RNA) may match the target DNA upstream and downstream of the desired deletion, wherein reverse transcription will cause replication of upstream and downstream sequences from the template nucleic acid (e.g., template RNA), without intervening sequences, such as causing the deletion of intervening sequences. In other embodiments, the template nucleic acid (e.g., template RNA) may be designed to introduce editing into the target DNA. For example, the template RNA may match the target DNA sequence except for one or more nucleotides, wherein reverse transcription will cause these editing to be copied into the target DNA, such as causing mutations, such as transposition or transversion mutations.

In some embodiments, writing the subject sequence into the target site results in substitution of nucleotides, e.g., where the full length of the subject sequence corresponds to the matched length of the target site with one or more mismatched bases. In some embodiments, the heterologous subject sequence can be designed so that a combination of sequence changes can occur, e.g., simultaneous additions and deletions, additions and substitutions, or deletions and substitutions.

In some embodiments, the heterologous object sequence may comprise an open reading frame or a fragment of an open reading frame. In some embodiments, the heterologous object sequence has a Kozak sequence. In some embodiments, the heterologous object sequence has an internal ribosome entry site. In some embodiments, the heterologous object sequence has a self-cleaving peptide, such as a T2A or P2A site. In some embodiments, the heterologous object sequence has a start codon. In some embodiments, the template RNA has a splice acceptor site. In some embodiments, the template RNA has a splice donor site. Exemplary splice acceptors and splice donor sites are described in WO 2016044416, which is incorporated herein by reference in its entirety. Exemplary splice acceptor site sequences are known to those skilled in the art. In some embodiments, the template RNA has a microRNA binding site downstream of the stop codon. In some embodiments, the template RNA has a poly A tail downstream of the stop codon of the open reading frame. In some embodiments, the template RNA comprises one or more exons. In some embodiments, the template RNA comprises one or more introns. In some embodiments, the template RNA comprises a eukaryotic transcription terminator. In some embodiments, the template RNA comprises an enhanced translation element or a translation enhancing element. In some embodiments, the RNA comprises a human T-cell leukemia virus (HTLV-1) R region. In some embodiments, the RNA comprises a post-transcriptional regulatory element that enhances nuclear export, such as a post-transcriptional regulatory element of hepatitis B virus (HPRE) or woodchuck hepatitis virus (WPRE).

In some embodiments, the heterologous subject sequence can contain non-coding sequences. For example, the template nucleic acid (e.g., template RNA) can include regulatory elements, e.g., promoter or enhancer sequences or miRNA binding sites. In some embodiments, the integration of the subject sequence at the target site will result in the upregulation of endogenous genes. In some embodiments, the integration of the subject sequence at the target site will result in the downregulation of endogenous genes. In some embodiments, the template nucleic acid (e.g., template RNA) includes a tissue-specific promoter or enhancer, each of which can be unidirectional or bidirectional. In some embodiments, the promoter is an RNA polymerase I promoter, an RNA polymerase II promoter, or an RNA polymerase III promoter. In some embodiments, the promoter includes a TATA element. In some embodiments, the promoter includes a B recognition element. In some embodiments, the promoter has one or more binding sites for a transcription factor.

In some embodiments, the template nucleic acid (e.g., template RNA) comprises a site for coordinating epigenetic modifications. In some embodiments, the template nucleic acid (e.g., template RNA) comprises a chromatin insulator. For example, the template nucleic acid (e.g., template RNA) comprises a CTCF site or a site targeted for DNA methylation.

In some embodiments, the template nucleic acid (e.g., template RNA) comprises a gene expression unit consisting of at least one regulatory region operably linked to an effector sequence. The effector sequence can be a sequence transcribed into RNA (e.g., a coding sequence or a non-coding sequence, such as a sequence encoding a microRNA).

In some embodiments, a heterologous subject sequence of a template nucleic acid (e.g., a template RNA) is inserted into an endogenous intron of a target genome. In some embodiments, a heterologous subject sequence of a template nucleic acid (e.g., a template RNA) is inserted into a target genome, thereby acting as a new exon. In some embodiments, insertion of a heterologous subject sequence into a target genome results in replacement of a native exon or skipping of a native exon.

Template nucleic acid (e.g., template RNA) can be designed to produce insertion, mutation or deletion at the target DNA locus. In some embodiments, template nucleic acid (e.g., template RNA) can be designed to cause insertion of target DNA. For example, template nucleic acid (e.g., template RNA) can contain heterologous object sequence, wherein reverse transcription will cause heterologous object sequence to be inserted into target DNA. In other embodiments, RNA template can be designed to write deletion into target DNA. For example, template nucleic acid (e.g., template RNA) can match target DNA upstream and downstream of desired deletion, wherein reverse transcription will cause replication of upstream and downstream sequences from template nucleic acid (e.g., template RNA), without intervening sequence, such as causing the deletion of intervening sequence. In other embodiments, template nucleic acid (e.g., template RNA) can be designed to write editing into target DNA. For example, template RNA can match target DNA sequence except one or more nucleotides, wherein reverse transcription will cause these editing to be copied into target DNA, such as causing mutation, such as transposition or transversion mutation.

In some embodiments, the pre-editing homology domain comprises a nucleic acid sequence having at least 100% sequence identity to a nucleic acid sequence contained in a target nucleic acid molecule.

In some embodiments, the post-editing homology domain comprises a nucleic acid sequence having at least 100% sequence identity to a nucleic acid sequence contained in a target nucleic acid molecule.

PBS sequence

In some embodiments, the template nucleic acid (e.g., template RNA) comprises a PBS sequence. In some embodiments, the PBS sequence is located 3' of the heterologous subject sequence and is complementary to a sequence adjacent to a site to be modified by the system described herein, or a sequence complementary to a sequence adjacent to a site to be modified by a system/gene modification polypeptide comprises no more than 1, 2, 3, 4, or 5 mismatches. In some embodiments, the PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nicking site in the target nucleic acid molecule. In some embodiments, the binding of the PBS sequence to the target nucleic acid molecule allows for the initiation of target-triggered reverse transcription (TPRT), for example, the 3' homology domain acts as a primer for TPRT. In some embodiments, the length of the PBS sequence is 3-5, 5-10, 10-30, 10-25, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-30, 11-25, 11-20, 11-19, 11-18, 11- 17,11-16,11-15,11-14,11-13,11-12,12-30,12-25,12-20,12-19,12-18,12-17,12-16,12-15,12-14,12-13,13-30,13-25,13-20,13-19,13-18 ,13-17,13 -16, 13-15, 13-14, 14-30, 14-25, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-30, 15-25, 15-20, 15-19, 15-18, 15-17, 15-16, 16-30, 16-25, 16-2 0, 16-19, 1 In some embodiments, the PBS sequence is 5-20, 8-16, 8-14, 8-13, 9-13, 9-12 or 10-12 nucleotides in length, for example, 9-12 nucleotides in length.

The template nucleic acid (e.g., template RNA) may have some homology with the target DNA. In some embodiments, the PBS sequence domain of the template nucleic acid (e.g., template RNA) may be used as an annealing zone for the target DNA so that the target DNA is positioned to initiate reverse transcription of the template nucleic acid (e.g., template RNA). In some embodiments, the template nucleic acid (e.g., template RNA) has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases completely homologous to the target DNA at the 3' end of the RNA. In some embodiments, the template nucleic acid (e.g., template RNA) has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200, or more bases that are at least 50%, 60%, 70%, 80%, 90, 100, 110, 120, 130, 140, 150, 175, 200, or more bases at the 5′ end of the template nucleic acid (e.g., template RNA) that are at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% homologous to the target DNA.

Exemplary Template Sequences

In some embodiments of the systems and methods herein, the template RNA comprises a gRNA spacer comprising a core nucleotide of a gRNA spacer sequence of Table 1. In some embodiments, the gRNA spacer further comprises one or more (e.g., 2, 3 or all) consecutive nucleotides starting from the 3' end of the flanking nucleotides of the gRNA spacer. In some embodiments, a template RNA comprising a sequence in Table 1 is included in a system further comprising a gene modifying polypeptide having an RT domain listed in the same row of Table 1. The RT domain amino acid sequence can be found, for example, in Table 6 herein.

Table 1: Exemplary gRNA spacer Cas pairs

Table 1 provides a gRNA database for correcting pathogenic EV6 mutations in HBB. A list of spacers, PAMs, and Cas variants for creating cuts at appropriate locations to install the desired genome editing using a gene modification system. The spacers in this table are designed for use with gene modification polypeptides containing nickase variants of the Cas species shown in the table. Tables 2, 3, and 4 detail the other components of the system and are organized so that the ID number shown in column 1 ("ID") here corresponds to the same ID number in the subsequent tables.

In the exemplary template sequences provided herein, capital letters represent "core nucleotides", while lowercase letters represent "flanking nucleotides". Herein, when it is considered that an RNA sequence (e.g., a template RNA sequence) comprises a specific sequence containing thymine (T) (e.g., a sequence of Table 1 or a portion thereof), it is of course understood that the RNA sequence can (and does often) comprise uracil (U) in place of T. For example, the RNA sequence can comprise U at each position shown as T in the sequence in Table 1. More particularly, the present disclosure provides an RNA sequence according to each gRNA spacer sequence shown in Table 1, wherein the RNA sequence has U in place of each T in the sequence in Table 1.

In some embodiments of the systems and methods herein, the heterologous object sequence comprises the core nucleotides of the RT template sequence of Table 3. In some embodiments, the heterologous object sequence further comprises one or more (e.g., 2, 3, 4, 5, 10, 20, 30, 40 or all) consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence. In some embodiments, the heterologous object sequence comprises the core nucleotides of the RT template sequence of Table 3 corresponding to the gRNA spacer sequence. In the context of the sequence table, when two components have the same ID number in the reference table, the first component "corresponds to" the second component. For example, for a gRNA spacer of ID#1, the corresponding RT template will be an RT template also having ID#1. In some embodiments, the heterologous object sequence further comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence.

In some embodiments, the primer binding site (PBS) sequence has a sequence comprising core nucleotides from a PBS sequence in the same row as the RT template sequence in Table 3. In some embodiments, the PBS sequence further comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or all) consecutive nucleotides starting from the 5' end of the flanking nucleotides of the primer region.

Table 3: Exemplary RT sequence (heterologous subject sequence) and PBS sequence pairs

Table 3 provides exemplary PBS sequences and heterologous object sequences (reverse transcription template regions) for correcting template RNAs for pathogenic EV6 mutations in HBB. The gRNA spacers in Table 1 are filtered, for example, by appearing within the 15nt range of the desired editing position and filtering using a grade 1 Cas enzyme. PBS sequences and heterologous object sequences (reverse transcription template regions) are designed relative to the incision sites guided by the homologous gRNA in Table 1, as described in the present application. For illustration, these regions are designed to extend beyond the editing position (RT) 8-17nt (initiate) and 1-50nt. It is not desirable to be limited by the examples, and considering the change in length, a sequence using a maximum length parameter and containing all templates of shorter length within a given parameter is provided. The sequence is shown as follows: the core sequence is represented by uppercase letters, and the flanking sequences that may be truncated within the length parameter are represented by lowercase letters.

Capital letters represent "core nucleotides", while lowercase letters represent "flanking nucleotides". Herein, when an RNA sequence (e.g., a template RNA sequence) is considered to contain a specific sequence containing thymine (T) (e.g., a sequence of Table 3 or a portion thereof), it is of course understood that the RNA sequence can (and does often) contain uracil (U) in place of T. For example, the RNA sequence can contain a U at each position shown as a T in the sequence in Table 3. More specifically, the present disclosure provides an RNA sequence according to each heterologous subject sequence and a PBS sequence shown in Table 3, wherein the RNA sequence has a U in place of each T in the sequence of Table 3.

In some embodiments of the systems and methods herein, the template RNA comprises a gRNA scaffold (e.g., which binds a gene modifying polypeptide, such as a Cas polypeptide) comprising a sequence of a gRNA scaffold in Table 12. In some embodiments, the gRNA scaffold comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a gRNA scaffold in Table 12. In some embodiments, the gRNA scaffold comprises a sequence of a scaffold region in Table 12 corresponding to an RT template sequence, a spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

In some embodiments of the systems and methods herein, the system further comprises a second strand targeting gRNA that guides the nick to the second strand of the human HBB gene. In some embodiments, the second strand targeting gRNA comprises a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2. In some embodiments, the gRNA spacer further comprises one or more (e.g., 2, 3 or all) consecutive nucleotides that start from the 3' end of the flanking nucleotides of the left gRNA spacer sequence or the right gRNA spacer sequence. In some embodiments, the second strand targeting gRNA comprises a sequence of core nucleotides containing the second nick gRNA sequence in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the second nick gRNA sequence further comprises one or more consecutive nucleotides that start from the 3' end of the flanking nucleotides of the second nick gRNA sequence. In some embodiments, the second nick gRNA comprises a gRNA scaffold sequence orthogonal to the Cas domain of the gene modifying polypeptide. In some embodiments, the second nicked gRNA comprises the gRNA scaffold sequence of Table 12.

Table 2: Exemplary left gRNA spacer and right gRNA spacer pairs

Table 2 provides optional exemplary second nicking gRNA species for correcting pathogenic E6V mutations in HBB. The gRNA spacers in Table 1 were filtered, for example, by appearing within 15nt of the desired editing position and using a level 1 Cas enzyme. The second nicking gRNA was generated by searching the opposite strand of DNA in the region -40 to -140 ("left") and +40 to +140 ("right") relative to the first nicking site defined by the first gRNA to find the PAM used by the corresponding Cas variant. An exemplary spacer is shown on each side of the target nicking site.

Capital letters represent "core nucleotides", while lowercase letters represent "flanking nucleotides". Herein, when it is considered that an RNA sequence (e.g., a gRNA that produces a second nick) contains a specific sequence containing thymine (T) (e.g., a sequence in Table 2 or a portion thereof), it is of course understood that the RNA sequence can (and does often) contain uracil (U) in place of T. For example, the RNA sequence can contain U at each position shown as T in the sequence in Table 2. More particularly, the present disclosure provides an RNA sequence according to each gRNA spacer sequence shown in Table 2, wherein the RNA sequence has U replacing each T in the sequence in Table 2.

In some embodiments, the systems and methods provided herein can include the template sequences listed in Table 4. Table 4 provides exemplary template RNA sequences (column 4) and optionally second nicking gRNA sequences (column 5) designed to be paired with a gene modifying polypeptide to correct a mutation in the HBB gene. The templates in Table 4 are intended to illustrate the following overall sequences: (1) a gRNA spacer (e.g., for targeting a first strand nick), (2) a gRNA scaffold, (3) a heterologous subject sequence, and (4) a PBS sequence (e.g., for initiating TPRT at a first strand nick).

The template RNA sequences shown in Tables 1-4, 5A-5D and 6A can be customized according to the cells being targeted. For example, in some embodiments, it is desirable to inactivate the PAM sequence during editing (e.g., using a "PAM-killing" modification) to reduce the possibility of further gene editing after initial editing (e.g., retargeting by Cas). Therefore, some template RNAs described herein are designed to write mutations (e.g., substitutions) into the PAM of the target site so that after editing, the PAM site will mutate to a sequence that the gene-modified polypeptide no longer recognizes. Therefore, the mutation region within the heterologous object sequence of the template RNA may include a PAM-killing sequence. Without wishing to be bound by theory, in some embodiments, the PAM-killing sequence may prevent the re-engagement of the gene-modified polypeptide after completing the gene modification, or reduce re-engagement relative to a template RNA lacking a PAM-killing sequence. In some embodiments, the PAM-killing sequence does not change the amino acid sequence encoded by the gene, for example, the PAM-killing sequence produces a silent mutation. In other embodiments, it is desirable to keep the PAM sequence intact (without PAM-killing).

Similarly, in some embodiments, in order to reduce the possibility of further gene editing after initial editing (e.g., by Cas re-targeting), it may be desirable to change the first three nucleotides of the RT template sequence by a "seed killing" motif. Therefore, certain template RNAs described herein are designed to write mutations (e.g., substitutions) into the target site portion corresponding to the first three nucleotides of the RT template sequence, so that after editing, the target site will mutate to a sequence with lower homology to the RT template sequence. Therefore, the mutation region within the heterologous object sequence of the template RNA may include a seed killing sequence. Without wishing to be bound by theory, in some embodiments, the seed killing sequence may prevent the rejoining of the gene modified polypeptide after the gene modification is completed, or reduce the rejoining relative to a template RNA that is otherwise similar but lacks a seed killing sequence. In some embodiments, the seed killing sequence does not change the amino acid sequence encoded by the gene, for example, the seed killing sequence produces a silent mutation. In other embodiments, it is desirable to keep the seed region intact and not use a seed killing sequence.

In other embodiments, in order to optimize or improve gene editing efficiency, it may be necessary to escape the mismatch repair or nucleotide repair pathway of the target cell, or to bias the repair pathway of the target cell to preserve the edited chain. In some embodiments, multiple silent mutations (e.g., silent substitutions) can be introduced into the RT template sequence to escape the mismatch repair or nucleotide repair pathway of the target cell, or to bias the repair pathway of the target cell to preserve the edited chain.

Table 7A provides exemplary silent mutations at various positions within the HBB gene.

Table 7A. Exemplary silent mutation codons of HBB gene

In some embodiments, the template RNA comprises one or more silent mutations.

In some embodiments, the silent mutation comprises a mutation in the codon encoding the sixth amino acid (proline) of the HBB gene that includes an initial methionine, such as a mutation to CCC or CCG.

In some embodiments, the template RNA comprises one or more silent substitutions as shown in Tables X1-X4 herein.

It should be understood that the silent mutations shown in Table 7A can be used alone or in any combination in the template RNA sequences described herein.

gRNA with inducible activity

In some embodiments, gRNA as described herein (e.g., gRNA as a part of template RNA or gRNA for second strand nick) has induction activity. Induction activity can be achieved by template nucleic acid such as template RNA, and the template nucleic acid (except gRNA) also includes a blocking domain, wherein the sequence of part or all of the blocking domain is at least partially complementary to a part or all of the gRNA. Therefore, the blocking domain can hybridize or substantially hybridize with a part or all of the gRNA. In some embodiments, the blocking domain and the induction activity gRNA are arranged on a template nucleic acid such as template RNA, so that the gRNA can adopt a first conformation (wherein the blocking domain hybridizes or substantially hybridizes with the gRNA) and a second conformation (wherein the blocking domain does not hybridize or substantially does not hybridize with the gRNA). In some embodiments, in the first conformation, the gRNA cannot bind to a gene modification polypeptide (e.g., a template nucleic acid binding domain, a DNA binding domain, or a nucleic acid endonuclease domain (e.g., CRISPR/Cas protein)) or bind in a manner that significantly reduces affinity compared to a template RNA similar to other aspects lacking a blocking domain. In some embodiments, in the second conformation, the gRNA can bind to a gene-modifying polypeptide (e.g., a template nucleic acid binding domain, a DNA binding domain, or a nuclease domain (e.g., a CRISPR/Cas protein)). In some embodiments, whether the gRNA is in the first conformation or the second conformation can affect whether the DNA binding or nuclease activity of the gene-modifying polypeptide (e.g., a CRISPR/Cas protein contained in the gene-modifying polypeptide) is active.

In some embodiments, the gRNA coordinating the second incision has induction activity. In some embodiments, the gRNA coordinating the second incision is induced after the template is reverse transcribed. In some embodiments, the hybridization of gRNA and the blocking domain can be destroyed using open molecules. In some embodiments, the open molecule comprises a medicament that is combined with part or all of the gRNA or the blocking domain and inhibits the hybridization of gRNA and the blocking domain. In some embodiments, the open molecule comprises a nucleic acid, for example, comprising a sequence partially or completely complementary to the gRNA, the blocking domain or both. By selecting or designing a suitable open molecule, the open molecule provided can promote the change of gRNA conformation, so that it can be associated with CRISPR/Cas protein and provide the related functions (for example, DNA binding and/or endonuclease activity) of CRISPR/Cas protein. It is not desirable to be bound by theory, providing an open molecule at a selected time and/or position can allow the activity of gRNA, CRISPR/Cas protein or a gene modification system comprising them to be controlled in space and time. In some embodiments, the open molecule comprises an exogenous cell comprising a gene modification polypeptide and/or a template nucleic acid. In some embodiments, the open molecule comprises an endogenous agent (e.g., endogenous for cells comprising a gene modification polypeptide and/or a template nucleic acid (which comprises a gRNA and a blocking domain). For example, an inducible gRNA, a blocking domain, and an open molecule can be selected so that the open molecule is an endogenous agent expressed in a target cell or tissue, for example, so as to ensure the activity of the gene modification system in the target cell or tissue. As another example, an inducible gRNA, a blocking domain, and an open molecule can be selected so that the open molecule does not exist or is not substantially expressed in one or more non-target cells or tissues, for example, so as to ensure that the activity of the gene modification system does not occur or does not substantially occur in one or more non-target cells or tissues, or occurs at a reduced level compared to the target cell or tissue. Exemplary blocking domains, open molecules, and uses thereof are described in PCT Application Publication WO 2020044039 A1, which is incorporated herein by reference in its entirety. In some embodiments, a template nucleic acid (e.g., a template RNA) may include one or more sequences or structures for being combined by one or more components (e.g., a reverse transcriptase or an RNA binding domain and a gRNA) of a gene modification polypeptide. In some embodiments, gRNA promotes interaction with a template nucleic acid binding domain (e.g., RNA binding domain) of a gene-modified polypeptide. In some embodiments, gRNA guides the gene-modified polypeptide to a matching target sequence, e.g., in a target cell genome.

Circular RNA and ribozymes in gene modification systems

It is expected that the use of circular and/or linear RNA states during the preparation, delivery or gene modification reaction in the target cell may be useful. Therefore, in some embodiments of any aspect described herein, the gene modification system comprises one or more circular RNAs (circRNA). In some embodiments of any aspect described herein, the gene modification system comprises one or more linear RNAs. In some embodiments, nucleic acids described herein (e.g., template nucleic acids, nucleic acid molecules encoding gene modification polypeptides, or both) are circRNAs. In some embodiments, circular RNA molecules encode gene modification polypeptides. In some embodiments, circRNA molecules encoding gene modification polypeptides are delivered to host cells. In some embodiments, circular RNA molecules encode recombinases, such as described herein. In some embodiments, circRNA molecules encoding recombinases are delivered to host cells. In some embodiments, circRNA molecules encoding gene modification polypeptides are linearized (e.g., in host cells, e.g., in the nucleus of host cells) before translation.

It has been found that circular RNA (circRNA) is naturally present in cells, and it has been found that it has different functions, including non-coding and protein coding in human cells. It has been shown that circRNA can be engineered by incorporating self-splicing introns into RNA molecules (or DNA encoding RNA molecules), resulting in RNA cyclization, and the engineered circRNA can have enhanced protein production and stability (Wesselhoeft et al. Nature Communications [Nature Communications] 2018). In some embodiments, the gene-modified polypeptide is encoded by circRNA. In certain embodiments, the template nucleic acid is DNA, such as dsDNA or ssDNA. In certain embodiments, circDNA comprises template RNA.

In some embodiments, circRNA comprises one or more ribozyme sequences. In some embodiments, the ribozyme sequence is activated for, for example, self-cutting in a host cell, for example, thereby causing the linearization of circRNA. In some embodiments, when the magnesium concentration reaches a level sufficient for cutting, the ribozyme is activated, for example, in a host cell. In some embodiments, before being delivered to the host cell, the circRNA remains in a low magnesium environment. In some embodiments, the ribozyme is a protein reactive ribozyme. In some embodiments, the ribozyme is a nucleic acid reactive ribozyme. In some embodiments, the circRNA comprises a cleavage site. In some embodiments, the circRNA comprises a second cleavage site.

In certain embodiments, circRNA is linearized in the nucleus of target cells. In certain embodiments, the linearization of circRNA in the nucleus of cells relates to the components present in the nucleus of cells, for example, to activate the cleavage event. In certain embodiments, a ribozyme (for example, a ribozyme from B2 or ALU elements) that reacts to a nuclear element (for example, a nucleoprotein, for example, a genome interacting protein, for example, an epigenetic modifier, for example, EZH2) is incorporated into the circRNA of a gene modification system. In certain embodiments, the nuclear localization of circRNA causes the autocatalytic activity of the ribozyme to increase and the linearization of the circRNA.

In some embodiments, the ribozyme is heterologous to one or more other components of the gene modification system. In some embodiments, the inducible ribozyme (e.g., in a circRNA described herein) is synthetically produced, for example, by designing an aptamer that is reactive to a protein ligand. A system utilizing satellite RNA of a tobacco ringspot virus hammerhead ribozyme and an MS2 coat protein aptamer has been described (Kennedy et al. Nucleic Acids Res 42(19): 12306-12321 (2014), which is incorporated herein by reference in its entirety), which results in activation of ribozyme activity in the presence of MS2 coat protein. In embodiments, such a system responds to a protein ligand that is localized to the cytoplasm or nucleus. In some embodiments, the protein ligand is not MS2. Methods for generating RNA aptamers for target ligands have been described, for example, based on systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk and Gold, Science 249(4968):505-510 (1990); Ellington and Szostak, Nature 346(6287):818-822 (1990); the methods of each of which are incorporated herein by reference) and in some cases aided by computer design (Bell et al. PNAS 117(15):8486-8493, the methods of which are incorporated herein by reference). Thus, in some embodiments, aptamers for target ligands are generated and incorporated into synthetic ribozyme systems, for example, to induce ribozyme-mediated cleavage and linearization of circRNAs, for example in the presence of a protein ligand. In some embodiments, circRNA linearization is initiated in the cytoplasm, for example, using an aptamer associated with a ligand in the cytoplasm. In some embodiments, circRNA linearization is initiated in the nucleus, for example, using an aptamer associated with a ligand in the nucleus. In embodiments, the ligand in the nucleus comprises an epigenetic modifier or a transcription factor. In some embodiments, the ligand that initiates linearization is present in the on-target cells at a higher level than in the off-target cells.

It is also expected that nucleic acid-reactive ribozyme systems can be used for circRNA linearization. For example, in, for example, Penchovsky (Biotechnology Advances [Biological Technology Progress] 32 (5): 1015-1027 (2014), incorporated herein by reference), a biosensor that senses a determined target nucleic acid molecule to trigger ribozyme activation is described. By these methods, ribozymes are naturally folded into an inactive state and are activated only in the presence of a determined target nucleic acid molecule (e.g., an RNA molecule). In some embodiments, the circRNA of the gene modification system comprises a nucleic acid-reactive ribozyme that is activated in the presence of a determined target nucleic acid (e.g., RNA, such as mRNA, miRNA, guide RNA, gRNA, sgRNA, ncRNA, lncRNA, tRNA, snRNA, or mtRNA). In some embodiments, the nucleic acid that triggers linearization is present in the target cell at a level higher than that of the off-target cell.

In some embodiments of any aspect of this invention, the gene modification system incorporates one or more ribozymes with inducible specificity to the target tissue or target cell, for example, ribozymes activated by ligands or nucleic acids present at higher levels in the target tissue or target cell. In some embodiments, the gene modification system incorporates ribozymes with inducible specificity to subcellular compartments (such as nuclei, nucleoli, cytoplasm or mitochondria). In some embodiments, ribozymes are activated by ligands or nucleic acids present at higher levels in the target subcellular compartment. In some embodiments, the RNA component of the gene modification system is provided in the form of circRNA, for example, it is activated by linearization. In some embodiments, the linearization of the circRNA encoding the gene modification polypeptide activates the molecule for translation. In some embodiments, the signal of the circRNA component activating the gene modification system exists at a higher level in the target cell or tissue, for example, so that the system is specifically activated in these cells.

In some embodiments, the RNA component of the gene modification system is provided in the form of circRNA, for example, it is inactivated by linearization. In some embodiments, the circRNA encoding the gene modification polypeptide is inactivated by cutting and degradation. In some embodiments, the circRNA encoding the gene modification polypeptide is inactivated by cutting that separates the translation signal from the coding sequence of the polypeptide. In some embodiments, the signal of the circRNA component of the inactivation gene modification system is present at a higher level in off-target cells or tissues, so that the system is specifically inactivated in these cells.

Target nucleic acid site

In some embodiments, after genetic modification, the target site surrounding the editing sequence contains a limited number of insertions or deletions, for example, in less than about 50% or 10% of the editing events, for example, as determined by long read amplicon sequencing of the target site, for example, as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated herein by reference in its entirety). In some embodiments, the target site does not show multiple consecutive editing events, such as head-to-tail or head-to-head duplications, for example, as determined by long read amplicon sequencing of the target site, for example, as described in Karst et al. bioRxivdoi.org/10.1101/645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site comprises an integration sequence corresponding to the template RNA. In some embodiments, the target site does not contain an insertion generated by an endogenous RNA in more than about 1% or 10% of the events, for example, as determined by long read amplicon sequencing of the target site, for example, as described in Karst et al. bioRxiv doi.org/10.1101/645903 (2020) (which is incorporated herein by reference in its entirety). In some embodiments, the target site comprises an integration sequence corresponding to a template RNA.

In certain aspects of the invention, the host DNA binding site integrated by the gene modification system can be in a gene, in an intron, in an exon, in an ORF, outside the coding region of any gene, in the regulatory region of a gene, or outside the regulatory region of a gene. In other aspects, the polypeptide can be combined with one or more than one host DNA sequence.

In some embodiments, the gene modification system is used to edit the target locus in multiple alleles. In some embodiments, the gene modification system is designed to edit a specific allele. For example, the gene modification polypeptide can be directed to a specific sequence present only on one allele, such as a template RNA containing homology with the target allele (e.g., gRNA or annealing domain), but not for a second homologous allele. In some embodiments, the gene modification system can change haplotype-specific alleles. In some embodiments, the gene modification system targeting a specific allele preferentially targets the allele, for example, the target allele has at least 2, 4, 6, 8 or 10 times of preference.

Second strand cut

In some embodiments, the gene modification system described herein includes a nickase activity (e.g., in a gene modification polypeptide) that nicks the first strand, and a nickase activity (e.g., in a polypeptide separated from the gene modification polypeptide) that nicks the second strand of the target DNA. As discussed herein, without wishing to be bound by theory, nicking the first strand of the target site DNA is thought to provide a 3′OH of a sequence (e.g., a heterologous object sequence) that can be used by the RT domain for reverse transcription of the template RNA. Without wishing to be bound by theory, it is believed that introducing additional nicks to the second strand may bias the cellular DNA repair mechanism toward using sequences based on heterologous object sequences more frequently than the original genomic sequence. In some embodiments, the additional nicks of the second strand are produced by the same endonuclease domain (e.g., a nickase domain) as the nicks of the first strand. In some embodiments, the same gene modification polypeptide nicks both the first strand and the second strand. In some embodiments, the gene modification polypeptide comprises a CRISPR/Cas domain and the additional nicks of the second strand are guided by additional nucleic acids (e.g., a second gRNA that comprises a guide CRISPR/Cas domain to nick the second strand). In other embodiments, the other second strand nick is produced by an endonuclease domain (e.g., a nickase domain) different from the nick of the first strand. In certain embodiments, the different endonuclease domains are located in other polypeptides (e.g., the system of the present invention further comprises other polypeptides), separated from the genetically modified polypeptide. In certain embodiments, other polypeptides comprise endonuclease domains (e.g., a nickase domain) as described herein. In certain embodiments, other polypeptides comprise DNA binding domains such as described herein.

It is contemplated herein that the position at which the second strand nick occurs relative to the first strand nick may affect the extent to which one or more of the following are achieved: the desired genetic modification DNA modification is achieved, an undesirable double strand break (DSB) occurs, an undesirable insertion occurs, or an undesirable deletion occurs. Without wishing to be bound by theory, the second strand nick may occur in two general orientations: inward nicks and outward nicks.

In some embodiments, in the inward cut orientation, the RT domain polymerizes (e.g., using a template RNA (e.g., a heterologous subject sequence)) away from the second strand cut. In some embodiments, in the inward cut orientation, the position of the cut of the first strand and the position of the cut of the second strand are located between the first PAM site and the second PAM site (e.g., in the case where both cuts are produced by a polypeptide (e.g., a gene modifying polypeptide) comprising a CRISPR/Cas domain). When there are two PAMs on the outside and two cuts on the inside, this inward cut orientation can also be referred to as "PAM-outside". In some embodiments, in the inward cut orientation, the position of the cut of the first strand and the position of the cut of the second strand are between the site where the polypeptide and the additional polypeptide bind to the target DNA. In some embodiments, in the inward cut orientation, the position of the cut of the second strand is located between the binding site of the polypeptide and the additional polypeptide, and the cut of the first strand is also located between the binding site of the polypeptide and the additional polypeptide. In some embodiments, in the inward cut orientation, the position of the cut of the first strand and the position of the cut of the second strand are located between the PAM site and the binding site of the second polypeptide at a distance from the target site.

Examples of gene modification systems that provide an inward incision orientation include: a gene modification polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that guides incision of a target site DNA on a first strand, and an additional nucleic acid (which comprises an additional gRNA that guides incision at a site at a distance from the first incision site), wherein the location of the first incision and the location of the second incision are between the PAM sites of the sites to which the two gRNAs guide the gene modification polypeptides. As another example, another gene modification system that provides an inward incision orientation comprises a gene modification polypeptide comprising a zinc finger molecule and a first nickase domain, wherein the zinc finger molecule binds to the target DNA in a manner that guides the first nickase domain to incision the first strand of the target site; an additional polypeptide comprising a CRISPR/Cas domain, and an additional nucleic acid comprising a gRNA that guides the additional polypeptide to incision at a site on the second strand that is a distance from the target site DNA, wherein the location of the first incision and the location of the second incision are located between the PAM site and the site to which the zinc finger molecule binds. As another example, another gene modification system providing an inward incision orientation comprises a gene modification polypeptide comprising a zinc finger molecule and a first nickase domain, wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to incise the first chain of the target site; and another polypeptide comprising a TAL effector molecule and a second nickase domain, wherein the TAL effector molecule binds to a site a certain distance from the target site in a manner that directs the other polypeptide to incise the second chain, wherein the position of the first incision and the position of the second incision are between the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds.

In some embodiments, in the outward nick orientation, the RT domain polymerizes (e.g., using a template RNA (e.g., a heterologous subject sequence)) toward the second strand nick. In some embodiments, in the outward nick orientation, when both the first and second nicks are produced by a polypeptide (e.g., a gene modifying polypeptide) comprising a CRISPR/Cas domain, the first PAM site and the second PAM site are located between the position of the nick of the first strand and the position of the nick of the second strand. When there are two PAMs on the inside and two nicks on the outside, this outward nick orientation may also be referred to as "PAM-inside". In some embodiments, in the outward nick orientation, the polypeptide (e.g., a gene modifying polypeptide) and the additional polypeptide bind to a site on the target DNA between the position of the nick of the first strand and the position of the nick of the second strand. In some embodiments, in the outward nick orientation, the position of the nick of the second strand is located on the opposite side of the binding site of the polypeptide and the additional polypeptide relative to the position of the nick of the first strand. In some embodiments, in the outward orientation, the PAM site and the binding site of the second polypeptide at a distance from the target site are located between the position of the nick of the first strand and the position of the nick of the second strand.

Examples of gene modification systems that provide an outward nicking orientation include: a gene modification polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that guides nicking of a target site DNA on a first strand, and an additional nucleic acid (which comprises an additional gRNA that guides nicking at a site a certain distance from the first nicking site), wherein the position of the first nick and the position of the second nick are outside the PAM site of the site to which the two gRNAs guide the gene modification polypeptide (i.e., the PAM site is located between the position of the first nick and the position of the second nick). As another example, another gene modification system that provides an outward nicking orientation comprises a gene modification polypeptide comprising a zinc finger molecule and a first nickase domain, wherein the zinc finger molecule binds to the target DNA in a manner that guides the first nickase domain to nick the first strand of the target site; another polypeptide comprising a CRISPR/Cas domain, and another nucleic acid comprising a gRNA that guides the other polypeptide to nick a site on the second strand that is a distance away from the target site DNA, wherein the position of the first nick and the second nick are outside the PAM site and the site to which the zinc finger molecule binds (i.e., the site to which the PAM site and the zinc finger molecule binds are between the position of the first nick and the position of the second nick). As another example, another gene modification system with an outward nicking orientation is provided, which comprises a gene modification polypeptide containing a zinc finger molecule and a first nickase domain, wherein the zinc finger molecule binds to the target DNA in a manner that guides the first nickase domain to nick the first chain of the target site; and another polypeptide containing a TAL effector molecule and a second nickase domain, wherein the TAL effector molecule binds to a site at a certain distance from the target site in a manner that guides the other polypeptide to nick the second chain, wherein the position of the first nick and the position of the second nick are outside the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds (i.e., the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds are between the position of the first nick and the position of the second nick).

Without wishing to be bound by theory, it is believed that for a genetic modification system that provides a second strand nick, an outward nick orientation is preferred in some embodiments. As described herein, an inward nick can produce a greater number of double-strand breaks (DSBs) compared to an outward nick orientation. DSBs can be recognized by the DSB repair pathway in the nucleus, which may result in undesirable insertions and deletions. An outward nick orientation can provide a reduced risk of DSB formation, and a correspondingly smaller amount of undesirable insertions and deletions. In some embodiments, undesirable insertions and deletions are insertions and deletions that are not encoded by a heterologous object sequence, such as insertions or deletions produced by a double-strand break repair pathway that is unrelated to the modification encoded by the heterologous object sequence. In some embodiments, the desired genetic modification comprises a change (e.g., substitution, insertion, or deletion) to a target DNA encoded by a heterologous object sequence (e.g., and achieved by genetic modification by writing a heterologous object sequence into a target site). In some embodiments, the first strand nick and the second strand nick are in an outward orientation.

In addition, the distance between the first chain nick and the second chain nick may affect the extent of one or more of the following: obtaining the desired genetic modification system DNA modification, the occurrence of undesirable double-strand breaks (DSBs), the occurrence of undesirable insertions, or the occurrence of undesirable deletions. Without wishing to be bound by theory, it is believed that the benefit of the second chain nick, that is, DNA repair is biased towards incorporating heterologous object sequences into the target DNA, increases as the distance between the first chain nick and the second chain nick decreases. However, it is believed that the risk of DSB formation also increases as the distance between the first chain nick and the second chain nick decreases. Accordingly, it is believed that the number of undesirable insertions and/or deletions may increase as the distance between the first chain nick and the second chain nick decreases. In some embodiments, the distance between the first chain nick and the second chain nick is selected to balance the benefit of DNA repair biased towards incorporating heterologous object sequences into the target DNA and the risk of DSB formation and undesirable deletions and/or insertions. In some embodiments, a system where the first strand nick and the second strand nick are separated by at least a threshold distance has an increased level of desired gene modification system modification results, a reduced level of undesired deletions, and/or a reduced level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are separated by less than a threshold distance. In some embodiments, one or more threshold distances are given below.

In some embodiments, the first nick and the second nick are separated by at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides. In some embodiments, the first nick and the second nick are separated by no more than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 250 nucleotides. In some embodiments, the first incision and the second incision are 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 1 80-190, 20-180, 30-180, 40-180, 50-180, 60-180, 70-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 20-170, 30-170, 40-170, 50-170, 6 0-170, 70-170, 80-170, 90-170, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 20-160, 30-160, 40-160, 50-160, 60-160, 70-160, 80-160, 90-160, 100-160, 110-160, 12 0-160, 130-160, 140-160, 150-160, 20-1 50,30-150,40-150,50-150,60-150,70-150,80-150,90-150,100-150,110-150,120-150,130-150,140-150,20-140,30-140,40-140,50-140,6 0-140, 70-140, 80-140, 90-140, 100-140, 110-140, 120-140, 130-140, 20-130, 30-130, 40-130, 50-130, 60-130, 70-130, 80-130, 90-130, 100-130, 110-130, 120-130, 20-120, 30-120, 40- 120, 50-120, 60-120, 70-120, 80-120, 90- 120, 100-120, 110-120, 20-110, 30-110, 40-110, 50-110, 60-110, 70-110, 80-110, 90-110, 100-110, 20-100, 30-100, 40-100, 50-100, 60-100, 70 -100, 80-100, 90-100, 20-90, 30-90, 40-9 In some embodiments, the first nick and the second nick are separated by 40-100 nucleotides.

Do not wish to be bound by theory, think that for providing the second chain nick and selecting the gene modification system of inward nick orientation, increasing the distance between the first chain nick and the second chain nick can be preferred.As described herein, inward nick orientation can produce more number of DSBs than outward nick orientation, and can cause more undesirable insertion and deletion than outward nick orientation, but increase the distance between the nick and can alleviate this increase of DSB, undesirable deletion and/or undesirable insertion.In certain embodiments, relative to the first nick and the second nick spaced apart less than the threshold distance in other aspects similar inward nick orientation system, wherein the first nick and the second nick spaced apart at least the threshold distance inward nick orientation has the desired gene modification system modification result of increase level, the undesirable deletion of reduction level and/or the undesirable insertion of reduction level.In certain embodiments, the threshold distance is provided below.

In some embodiments, the first strand nick and the second strand nick are in an inward orientation. In some embodiments, the first strand nick and the second strand nick are in an inward orientation, and the first strand nick and the second strand nick are at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, or 500 nucleotides apart, such as at least 100 nucleotides, (and optionally no more than 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, or 120 nucleotides apart). In some embodiments, the first and second chain cutouts are in an inward orientation and the first and second chain cutouts are 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 1 150-160, 100-150, 110-150, 120-150, 130-150, 140-150, 100-140, 110-140, 120-140, 130-140, 100-130, 110-130, 120-130, 100-120, 110-120, or 100-110 nucleotides.

Chemically modified nucleic acids and nucleic acid termini characteristics

The nucleic acids described herein (e.g., template nucleic acids, such as template RNA; or nucleic acids encoding gene-modified polypeptides (e.g., mRNA); or gRNA) may comprise unmodified or modified nucleobases. Naturally occurring RNA is synthesized from four basic ribonucleotides: ATP, CTP, UTP, and GTP, but may contain nucleotides that are modified post-transcriptionally. In addition, approximately one hundred different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. [RNA Modification Database: 1999 Update] Nucl Acids Res [Nucleic Acids Research] 27: 196-197). RNA may also comprise completely synthetic nucleotides that do not occur in nature.

In some embodiments, the chemical modification is a chemical modification provided in WO/2016/183482, U.S. Patent Publication No. 20090286852, International Application Nos. WO/2012/019168, WO/2012/045075, WO/2012/135805, WO/2012/158736, WO/2013/039857, WO/2013/039861, WO/2013/052523, WO/2013/090648, WO/2013/096709, WO/2013/101690, WO/2013/106496, WO/2013/130161, WO/2013/158736. 69. WO/2013/151736, WO/2013/151672, WO/2013/151664, WO/2013/151665, WO/2013/151668, WO/2013/151671, WO/2013/151667, WO/2013/151670, WO/2013 /151666、WO/2013/151663、WO/2014/028429、WO/2014/081507、WO/2014/093924、WO/2014/093574、WO/2014/113089、WO/2014/144711、WO/2014/144767 , WO/2014/144039, WO/2014/152540, WO/2014/152030, WO/2014/152031, WO/2014/152027, WO/2014/152211, WO/2014/158795, WO/2014/159813, WO/2014/16 W 6/014846, WO/2016/022914, WO/2016/036902, WO/2016/077125, or WO/2016/077123, each of which is incorporated herein by reference in its entirety. It should be understood that the incorporation of chemically modified nucleotides into a polynucleotide can result in the incorporation of modifications into the nucleobase, the backbone, or both, depending on the position of the modification in the nucleotide. In some embodiments, the backbone modification is a modification provided in EP 2813570, which is incorporated herein by reference in its entirety. In some embodiments, the modified cap is a cap provided in U.S. Patent Publication No. 20050287539 (which is incorporated herein by reference in its entirety).

In some embodiments, the chemically modified nucleic acid (e.g., RNA, e.g., mRNA) comprises one or more ARCA: anti-reverse cap analog (m27.3′-OGP3G), GP3G (unmethylated cap analog), m7GP3G (monomethylated cap analog), m32.2.7GP3G (trimethylated cap analog), m5CTP (5′-methyl-cytidine triphosphate), m6ATP (N6-methyl-adenosine-5′-triphosphate), s2UTP (2-thio-uridine triphosphate), and Ψ (pseudouridine triphosphate).

In some embodiments, the chemically modified nucleic acid comprises a 5′ cap, such as: a 7-methylguanosine cap (e.g., an O-Me-m7G cap); a hypermethylated cap analog; an NAD+-derived cap analog (e.g., as described in Kiledjian, Trends in Cell Biology 28, 454-464 (2018)); or a modified, such as a biotinylated, cap analog (e.g., as described in Bednarek et al., Phil Trans R Soc B 373, 20180167 (2018)).

In some embodiments, the chemically modified nucleic acid comprises a 3′ feature selected from one or more of the following: a poly A tail; a 16-nucleotide long stem-loop structure flanked by 5 unpaired nucleotides (e.g., as described in Mannironi et al., Nucleic Acid Research 17, 9113-9126 (1989)); a triple helix structure (e.g., as described in Brown et al., PNAS 109, 19202-19207 (2012)); a tRNA, Y RNA, or vault RNA structure (e.g., as described in Labno et al., Biochemica et Biophysica Acta [Biochimica et Biophysica Sinica] 1863, 3125-3147 (2016)); incorporation of one or more deoxyribonucleotide triphosphates (dNTPs), 2′O-methylated NTPs, or phosphorothioate-NTPs; chemical modification of single nucleotides (e.g., oxidation of the 3′ terminal ribose to a reactive aldehyde followed by conjugation of an aldehyde-reactive modified nucleotide); or chemical linkage to another nucleic acid molecule.

In some embodiments, the nucleic acid (e.g., template nucleic acid) comprises one or more modified nucleotides, for example, selected from dihydrouridine, inosine, 7-methylguanosine, 5-methylcytidine (5mC), 5′-phosphoribosylthymidine, 2′-O-methylribothymidine, 2′-O-ethylribothymidine, 2′-fluororibothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl-deoxyuridine (pdU), C-5 propynylcytidine (pC), C-5 propynyluridine (pU), 5-methylcytidine, 5-methyluridine, 5-methyldeoxycytidine, 5-methyldeoxyur ...cytidine, 5-methyldeoxyuridine, 5-methyldeoxyuridine, 5-methyldeoxyuridine, 5-methyldeoxyuridine, 5-methyldeoxyuridine, 5-methyldeoxycytidine, 5-methyldeoxyuridine, 5-methyldeoxyuridine, 5-methyldeoxyuridine, 5-methyldeoxyuridine, 5-methyldeoxycytidine, 5-methyldeoxyuridine, 5-methyldeoxyuridine, 5-methyldeoxyuridine, 5-methyldeoxycytidine Oxy, 2,6-diaminopurine, 5′-dimethoxytrityl-N4-ethyl-2′-deoxycytidine, C-5 propynyl-f-cytidine (pfC), C-5 propynyl-f-uridine (pfU), 5-methyl f-cytidine, 5-methyl f-uridine, C-5 propynyl-m-cytidine (pmC), C-5 propynyl-f-uridine (pmU), 5-methyl m-cytidine, 5-methyl m-uridine, LNA (locked nucleic acid), MGB (minor groove binder) pseudouridine (Ψ), 1-N-methyl pseudouridine (1-Me-Ψ), or 5-methoxyuridine (5-MO-U).

In some embodiments, the nucleic acid comprises a backbone modification, such as a modification to a sugar or phosphate group in the backbone. In some embodiments, the nucleic acid comprises a nucleobase modification.

In some embodiments, the nucleic acid comprises one or more chemically modified nucleotides of Table 13, one or more chemical backbone modifications of Table 14, and one or more chemically modified caps of Table 15. For example, in some embodiments, the nucleic acid comprises two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of chemical modifications. For example, the nucleic acid can comprise two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of modified nucleobases, e.g., as described herein, e.g., in Table 13. Alternatively or in combination, the nucleic acid can comprise two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of backbone modifications, e.g., as described herein, e.g., in Table 14. Alternatively or in combination, the nucleic acid can comprise one or more modified caps, e.g., as described herein, e.g., as described in Table 15. For example, in some embodiments, the nucleic acid comprises one or more types of modified nucleobases and one or more types of backbone modifications; one or more types of modified nucleobases and one or more modified caps; one or more types of modified caps and one or more types of backbone modifications; or one or more types of modified nucleobases, one or more types of backbone modifications, and one or more types of modified caps.

In some embodiments, the nucleic acid comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) modified nucleobases. In some embodiments, all nucleobases of the nucleic acid are modified. In some embodiments, the nucleic acid is modified at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) positions in the backbone. In some embodiments, all backbone positions of the nucleic acid are modified.

Table 13. Modified nucleotides

Table 14. Main chain modifications

Table 15. Modified caps

The nucleotides constituting the template of the gene modification system can be natural bases or modified bases, or a combination thereof. For example, the template can include pseudouridine, dihydrouridine, inosine, 7-methylguanosine or other modified bases. In some embodiments, the template can include locked nucleic acid nucleotides. In some embodiments, the modified bases used in the template do not inhibit reverse transcription of the template. In some embodiments, the modified bases used in the template can improve reverse transcription, such as specificity or fidelity.

In some embodiments, the RNA component of the system (e.g., template RNA or gRNA) comprises one or more nucleotide modifications. In some embodiments, the modification pattern of gRNA can significantly affect in vivo activity compared to unmodified or terminally modified guides (e.g., as shown below: Figure 1D from Finn et al. Cell Rep [Cell Report] 22 (9): 2227-2235 (2018); which is incorporated herein by reference in its entirety). Without wishing to be bound by theory, the process may be at least partially due to the RNA stability conferred by the modification. Non-limiting examples of such modifications may include 2′-O-methyl (2′-O-Me), 2′-0-(2-methoxyethyl) (2′-0-MOE), 2′-fluoro (2′-F), phosphorothioate (PS) bonds between nucleotides, G-C substitutions, and reverse abasic connections between nucleotides and their equivalents.

In some embodiments, the template RNA (e.g., at the portion where it binds the target site) or guide RNA comprises a 5' terminal region. In some embodiments, the template RNA or guide RNA does not comprise a 5' terminal region. In some embodiments, the 5' terminal region comprises a gRNA spacer region, for example, as described in Briner AE et al., Molecular Cell [Molecular Cell] 56: 333-339 (2014) for sgRNA (incorporated herein by reference in its entirety; applicable herein, for example, for all guide RNAs). In some embodiments, the 5' terminal region comprises a 5' end modification. In some embodiments, a 5' terminal region with or without a spacer region may be associated with crRNA, trRNA, sgRNA, and/or dgRNA. In some cases, the gRNA spacer region may comprise a guide region, a guide domain, or a targeting domain.

In some embodiments, the template RNA described herein (e.g., at the portion where it binds the target site) or guide RNA comprises any sequence shown in Table 4 of WO 2018107028 A1 (incorporated herein in its entirety by reference). In some embodiments, when the sequence shows a guide region and/or a spacer region, the composition may or may not include the region. In some embodiments, the guide RNA comprises one or more modifications of any sequence shown in Table 4 of WO 2018107028 A1 (e.g., as represented by SEQ IDNO therein). In an embodiment, the nucleotides may be the same or different, and/or the modification pattern shown may be the same or similar to the modification pattern of the guide sequence shown in Table 4 of WO 2018107028 A1. In some embodiments, the modification pattern includes the relative position and identity of the modification of the gRNA or gRNA region (e.g., 5' terminal region, lower stem region, bulge region, upper stem region, junction region, hairpin 1 region, hairpin 2 region, 3' terminal region). In some embodiments, the modification pattern comprises at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the modification of any of the sequences shown in the sequence column of Table 4 of WO 2018107028 A1 and/or the modification on one or more regions of the sequence. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the modification pattern of any of the sequences shown in the sequence column of Table 4 of WO 2018107028 A1. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more regions of the sequence shown in Table 4 of WO 2018107028A1 (e.g., at the 5' terminal region, lower stem region, bulge region, upper stem region, junction region, hairpin 1 region, hairpin 2 region, and/or 3' terminal region). In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the modification pattern of the sequence at the 5' terminal region. In certain embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical on the lower stem. In certain embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical on the projection. In certain embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical on the upper stem. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical on the joint. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical on hairpin 1. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical on hairpin 2. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical at the 3' end. In some embodiments, the modification pattern is different from the modification pattern of the sequence of Table 4 of WO 2018107028A1 or the region of such sequence (e.g., 5' end, lower stem, bulge, upper stem, connection, hairpin 1, hairpin 2, 3' end), for example, at 0, 1, 2, 3, 4, 5, 6 or more nucleotides. In some embodiments, the gRNA comprises modifications, which are different from the modifications of the sequences of Table 4 of WO 2018107028A1, for example, at 0, 1, 2, 3, 4, 5, 6 or more nucleotides. In some embodiments, the gRNA comprises modifications that differ from modifications in a region of the sequence of Table 4 of WO2018107028A1 (e.g., 5′ end, lower stem, bulge, upper stem, junction, hairpin 1, hairpin 2, 3′ end), for example, at 0, 1, 2, 3, 4, 5, 6 or more nucleotides.

In some embodiments, the template RNA (e.g., in the portion where it binds to the target site) or gRNA comprises 2′-O-methyl (2′-O-Me) modified nucleotides. In some embodiments, the gRNA comprises 2′-O-(2-methoxyethyl) (2′-O-moe) modified nucleotides. In some embodiments, the gRNA comprises 2′-fluoro (2′-F) modified nucleotides. In some embodiments, the gRNA comprises phosphorothioate (PS) bonds between nucleotides. In some embodiments, the gRNA comprises 5′ end modifications, 3′ end modifications, or 5′ and 3′ end modifications. In some embodiments, the 5′ end modification comprises phosphorothioate (PS) bonds between nucleotides. In some embodiments, the 5′ end modification comprises 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-MOE) and/or 2′-fluoro (2′-F) modified nucleotides. In some embodiments, the 5' end modification comprises at least one phosphorothioate (PS) bond and 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-MOE) and/or 2'-fluoro (2'-F) modified nucleotides. The end modification may comprise phosphorothioate (PS), 2'-O-methyl (2'-O-Me), 2'--O--(2-methoxyethyl) (2'-O-MOE) and/or 2'-fluoro (2'-F) modifications. Equivalent end modifications are also included in the embodiments described herein. In some embodiments, the template RNA or gRNA comprises an end modification combined with a modification of one or more regions of the template RNA or gRNA. Other exemplary modifications and methods for protecting RNA (e.g., gRNA) and formulas thereof are described in WO 2018126176 A1 (which is incorporated herein by reference in its entirety).

In some embodiments, the template RNA described herein comprises three phosphorothioate connections at the 5' end and three phosphorothioate connections at the 3' end. In some embodiments, the template RNA described herein comprises three 2'-O-methyl ribonucleotides at the 5' end and three 2'-O-methyl ribonucleotides at the 3' end. In some embodiments, the three nucleotides at the 5' end of the template RNA are 2'-O-methyl ribonucleotides, the connection between the three nucleotides at the 5' end of the template RNA is a phosphorothioate connection, the three nucleotides at the 3' end of the template RNA are 2'-O-methyl ribonucleotides, and the connection between the three nucleotides at the 3' end of the template RNA is a phosphorothioate connection. In some embodiments, the template RNA comprises alternating segments of ribonucleotides and 2'-O-methyl ribonucleotides, such as segments with a length between 12 and 28 nucleotides. In some embodiments, the central portion of the template RNA comprises alternating segments, and the 5' and 3' ends each comprise three 2'-O-methyl ribonucleotides and three phosphorothioate connections.

In some embodiments, a structure-guided and systematic approach is used to introduce modifications (e.g., 2′-OMe-RNA, 2′-F-RNA, and PS modifications) into a template RNA or guide RNA, e.g., as described in Mir et al. Nat Commun [Nature Communications] 9: 2641 (2018) (incorporated herein by reference in its entirety). In some embodiments, incorporation of 2′-F-RNA increases the thermal and nuclease stability of the RNA: RNA or RNA: DNA duplex, e.g., while minimizing interference with the C3′-internal sugar fold. In some embodiments, 2′-F may be better tolerated than 2′-OMe at positions where 2′-OH is important for RNA: DNA duplex stability. In some embodiments, the crRNA comprises one or more modifications that do not reduce Cas9 activity, e.g., C10, C20, or C21 (fully modified), e.g., as described in Supplementary Table 1 of Mir et al. Nat Commun [Nature Communications] 9: 2641 (2018) (incorporated herein by reference in its entirety). In some embodiments, the tracrRNA comprises one or more modifications that do not reduce Cas9 activity, such as T2, T6, T7, or T8 (fully modified) as described in Supplementary Table 1 of Mir et al. Nat Commun [Nature Communications] 9: 2641 (2018). In some embodiments, a crRNA comprising one or more modifications (e.g., as described herein) can be paired with a tracrRNA comprising one or more modifications (e.g., C20 and T2). In some embodiments, the gRNA comprises, for example, a chimera of a crRNA and a tracrRNA (e.g., Jinek et al. Science [Science] 337(6096): 816-821 (2012)). In embodiments, modifications from crRNA and tracrRNA are mapped onto a single guide chimera, for example, to generate a modified gRNA with enhanced stability.

In some embodiments, the gRNA molecule can be modified by adding or reducing naturally occurring structural components such as hairpins. In some embodiments, the gRNA may include a gRNA lacking one or more 3' hairpin elements, for example, as described in WO2018106727 (incorporated herein in its entirety by reference). In some embodiments, the gRNA may include an added hairpin structure, for example, a hairpin structure added in the spacer region, which is shown in the teachings of Kocak et al. Nat Biotechnol [Nature Biotechnology] 37 (6): 657-666 (2019) to increase the specificity of the CRISPR-Cas system. Additional modifications, including shortened gRNAs and examples of specific modifications that increase in vivo activity can be found in US 20190316121 (incorporated herein in its entirety by reference).

In some embodiments, a structure-guided and systematic approach (e.g., as described in Mir et al. Nat Commun 9:2641 (2018); incorporated herein by reference in its entirety) is used to find modifications of the template RNA. In embodiments, modifications are identified by including or excluding a guide region of the template RNA. In some embodiments, the structure of a polypeptide bound to the template RNA is used to determine the non-protein contacting nucleotides of the RNA, which can then be selected for modification, e.g., where the risk of disrupting RNA binding to the polypeptide is low. Secondary structure in the template RNA can also be predicted in silico by software tools, such as the RNAstructure tool available at rna.urmc.rochester.edu/RNAstructureWeb (Bellaousov et al. Nucleic Acids Res 41:W471-W474 (2013); incorporated herein by reference in its entirety), e.g., to determine secondary structures, such as hairpins, stems, and/or protrusions, for selection of modifications.

Composition and system generation

As will be appreciated by those skilled in the art, methods for designing and constructing nucleic acid constructs and proteins or polypeptides (e.g., the systems, constructs, and polypeptides described herein) are routine in the art. Typically, recombinant methods can be used. Generally, see Smales and James (eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar, and Meibohm (eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013). Methods for designing, preparing, evaluating, purifying, and manipulating nucleic acid compositions are described in Green and Sambrook (eds.), Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

The present disclosure provides, in part, nucleic acids (e.g., vectors) encoding gene-modified polypeptides as described herein, template nucleic acids as described herein, or both. In some embodiments, the vector comprises a selective marker, e.g., an antibiotic resistance marker. In some embodiments, the antibiotic resistance marker is a kanamycin resistance marker. In some embodiments, the antibiotic resistance marker does not confer resistance to β-lactam antibiotics. In some embodiments, the vector does not comprise an ampicillin resistance marker. In some embodiments, the vector comprises a kanamycin resistance marker and does not comprise an ampicillin resistance marker. In some embodiments, the vector encoding the gene-modified polypeptide is integrated into the target cell genome (e.g., after being applied to a target cell, tissue, organ, or subject). In some embodiments, the vector encoding the gene-modified polypeptide is not integrated into the target cell genome (e.g., after being applied to a target cell, tissue, organ, or subject). In some embodiments, the vector encoding the template nucleic acid (e.g., template RNA) is not integrated into the target cell genome (e.g., after being applied to a target cell, tissue, organ, or subject). In some embodiments, if the vector is integrated into the target site in the target cell genome, the selective marker is not integrated into the genome. In some embodiments, if the vector is integrated into the target site in the target cell genome, the genes or sequences involved in the maintenance of the vector (e.g., plasmid maintenance genes) are not integrated into the genome. In some embodiments, if the vector is integrated into the target site in the target cell genome, the transfer regulatory sequence (e.g., inverted terminal repeat sequence, e.g., from AAV) is not integrated into the genome. In some embodiments, administration of a vector (e.g., a vector encoding a gene modified polypeptide as described herein, a template nucleic acid as described herein, or both) to a target cell, tissue, organ, or subject may allow a portion of the vector to be integrated into one or more target sites in one or more genomes of the target cell, tissue, organ, or subject. In some embodiments, less than 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the target sites (e.g., no target sites) containing integrated material include a selective marker (e.g., an antibiotic resistance gene) from the vector, a transfer regulatory sequence (e.g., an inverted terminal repeat sequence, e.g., from AAV), or both.

Exemplary methods for producing therapeutic drug proteins or polypeptides described herein involve expression in mammalian cells, although insect cells, yeast, bacteria, or other cells may also be used to produce recombinant proteins under the control of appropriate promoters. Mammalian expression vectors may contain non-transcribed elements, such as an origin of replication, a suitable promoter, and other 5' or 3' flanking non-transcribed sequences; and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early promoter, splice and polyadenylation sites, can be used to provide other genetic elements required for expression of heterologous DNA sequences. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in the following literature: Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

Various mammalian cell culture systems can be used to express and manufacture recombinant proteins. Examples of mammalian expression systems include CHO, COS, HEK293, HeLA and BHK cell lines. The process of host cell culture for producing protein therapeutics is described in the following literature: Zhou and Kantardjieff (editor), Mammalian Cell Cultures for Biologics Manufacturing [mammalian cell culture for biologics manufacturing] (Advances in Biochemical Engineering/Biotechnology [progress in biochemical engineering/biotechnology]), Springer [Springer Press] (2014). Compositions described herein may include vectors, such as viral vectors encoding recombinant proteins, such as lentiviral vectors. In certain embodiments, vectors, such as viral vectors, may include nucleic acids encoding recombinant proteins.

Purification of protein therapeutics is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).

The present disclosure also provides compositions and methods for producing template nucleic acid molecules (e.g., template RNA) specific to gene-modified polypeptides and/or genomic target sites. In one aspect, the method includes producing RNA segments, including upstream homologous segments, heterologous object sequence segments, gene-modified polypeptide binding motifs, and gRNA segments.

Therapeutic applications

In some embodiments, the gene modification system as described herein can be used to modify cells (e.g., animal cells, plant cells, or fungal cells). In some embodiments, the gene modification system as described herein can be used to modify mammalian cells (e.g., human cells). In some embodiments, the gene modification system as described herein can be used to modify cells from livestock animals (e.g., cattle, horses, sheep, goats, pigs, llamas, alpacas, camels, yaks, chickens, ducks, geese, or ostriches). In some embodiments, the gene modification system as described herein can be used as a laboratory tool or research tool, or in a laboratory method or research method, for example, to modify animal cells such as mammalian cells (e.g., human cells), plant cells, or fungal cells.

By integrating the coding gene into the RNA sequence template, the gene modification system can meet the treatment needs, for example, by providing the expression of therapeutic transgenes in individuals with loss-of-function mutations, by replacing gain-of-function mutations with normal transgenes, by providing regulatory sequences to eliminate gain-of-function mutation expression, and/or by controlling the expression of operably connected genes, transgenes and their systems. In certain embodiments, the RNA sequence template encodes a promoter region that has specificity for the treatment of the host cell, such as a tissue-specific promoter or enhancer. In yet other embodiments, the promoter can be operably linked to the coding sequence.

Thus, provided herein are methods for treating sickle cell disease (SCD) (eg, sickle cell anemia) in a subject in need thereof. In some embodiments, treatment results in improvement of one or more symptoms associated with SCD.

In some embodiments, the systems herein are used to treat a subject having an E6 (eg, E6V) mutation.

In some embodiments, treatment with a system disclosed herein corrects E6V mutations in about 60%-70% (e.g., about 60%-65% or about 65%-70%) of cells. In some embodiments, treatment with a system disclosed herein corrects E6V mutations in about 60%-70% (e.g., about 60%-65% or about 65%-70%) of DNA isolated from treated cells.

In some embodiments, treatment using the gene modification system described herein can result in one or more of the following outcomes:

(a) The number of sickle cells decreases;

(b) decreased production of abnormal forms of β-globulins (e.g., hemoglobin S);

(c) reduction in pain and/or organ damage associated with sickle cell-related vascular occlusion; and/or

(d) Increase in normal blood flow,

These results were compared to SCD subjects who were not treated with the gene modification system described herein.

Administration and delivery

The compositions and systems described herein can be used in vitro or in vivo. In some embodiments, for example, the system or components of the system are delivered to cells (e.g., mammalian cells, such as human cells) in vitro or in vivo. In some embodiments, the cell is a eukaryotic cell, such as a cell of a multicellular organism, such as an animal, such as a mammal (e.g., a human, a pig, a cow), a bird (e.g., poultry, such as a chicken, a turkey, or a duck) or a fish. In some embodiments, the cell is a non-human animal cell (e.g., an experimental animal, a livestock animal, or a companion animal). In some embodiments, the cell is a stem cell (e.g., a hematopoietic stem cell), a fibroblast, or a T cell. In some embodiments, the cell is an immune cell, for example, a T cell (e.g., a Treg, a CD4, a CD8, a γδ or a memory T cell), a B cell (e.g., a memory B cell or a plasma cell) or a NK cell. In some embodiments, the cell is a non-dividing cell, such as a non-dividing fibroblast or a non-dividing T cell. In some embodiments, the cell is an HSC and p53 is not upregulated or is upregulated by less than 10%, 5%, 2% or 1%, for example, as determined according to the method described in Example 30 of PCT/US2019/048607. The skilled artisan will appreciate that the components of the gene modification system can be delivered in the form of polypeptides, nucleic acids (e.g., DNA, RNA), and combinations thereof.

In one embodiment, the system and/or components of the system are delivered in the form of nucleic acids. For example, a genetically modified polypeptide can be delivered in the form of a DNA or RNA encoding the polypeptide, and a template RNA can be delivered in the form of RNA or its complementary DNA to be transcribed into RNA. In some embodiments, the system or components of the system are delivered on 1, 2, 3, 4 or more different nucleic acid molecules. In some embodiments, the system or components of the system are delivered as a combination of DNA and RNA. In some embodiments, the system or components of the system are delivered as a combination of DNA and protein. In some embodiments, the system or components of the system are delivered as a combination of RNA and protein. In some embodiments, the genetically modified polypeptide is delivered as a protein.

In some embodiments, a system or a component of a system is delivered to a cell, such as a mammalian cell or a human cell, using a vector. The vector can be, for example, a plasmid or a virus. In some embodiments, delivery is in vivo, in vitro, ex vivo or in situ. In some embodiments, the virus is an adeno-associated virus (AAV), a lentivirus or an adenovirus. In some embodiments, a system or a component of a system is delivered to a cell together with a virus-like particle or a virion. In some embodiments, delivery uses more than one virus, virus-like particle or virion.

In one embodiment, compositions and systems described herein can be formulated in liposomes or other similar vesicles. Liposomes are spherical vesicle structures, which are composed of a monolayer or multilayer lipid bilayer around an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. Liposomes can be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their loads across biomembranes and blood-brain barriers (BBB) (for review, see, e.g., Spuch and Navarro, Journal of Drug Delivery [Drug Delivery Magazine], Vol. 2011, Article ID 469679, p. 12, 2011.doi: 10.1155/2011/469679).

Vesicles can be made of several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. Patent No. 6,693,086, which is incorporated herein by reference for its teachings on the preparation of multilamellar vesicle lipids). Although vesicle formation can be spontaneous when the lipid film is mixed with an aqueous solution, vesicle formation can also be accelerated by applying force in the form of oscillations using a homogenizer, sonicator, or extrusion device (for review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, Vol. 2011, Article ID 469679, p. 12, 2011. doi: 10.1155/2011/469679). Extruded lipids can be prepared by extrusion through a filter of decreasing size as described in Templeton et al., Nature Biotech, 15:647-652, 1997, which is incorporated herein by reference for its teachings on the preparation of extruded lipids.

A variety of nanoparticles can be used for delivery, such as liposomes, lipid nanoparticles, cationic lipid nanoparticles, ionizable lipid nanoparticles, polymer nanoparticles, gold nanoparticles, dendrimers, cyclodextrin nanoparticles, micelles, or combinations thereof.

Lipid nanoparticles are examples of carriers that provide biocompatibility and biodegradable delivery systems for pharmaceutical compositions described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs), which retain the characteristics of SLNs, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively guide drug delivery to specific targets and improve drug stability and controlled drug release. Lipopolymer nanoparticles (PLNs), i.e., a carrier that combines liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs are composed of core-shell structures; polymer cores provide stable structures, and phospholipid shells provide good biocompatibility. In this way, these two components increase drug encapsulation efficiency, promote surface modification, and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi: 10.3390/nano7060122.

Exosomes can also be used as drug delivery vehicles for the compositions and systems described herein. For a review, see Ha et al., July 2016. Acta Pharmaceutica Sinica B, Vol. 6, No. 4, pp. 287-296; https://doi.org/10.1016/j.apsb.2016.02.001.

Fusogens interact and fuse with target cells, and can therefore be used as delivery vehicles for a variety of molecules. They are generally composed of an amphipathic lipid bilayer that closes the lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer. Fusogen components have been shown to be engineered to give target cell specificity for fusion and load delivery, thereby allowing the creation of delivery vehicles with programmable cell specificity (see, for example, patent application WO 2020014209, which relates to the design, preparation and use of fusogens, which are incorporated herein by reference).

In some embodiments, one or more protein components of the gene modification system can be pre-associated with a template nucleic acid (e.g., a template RNA). For example, in some embodiments, a gene modification polypeptide can first be combined with a template nucleic acid (e.g., a template RNA) to form a ribonucleoprotein (RNP) complex. In some embodiments, RNPs can be delivered to cells by, for example, transfection, nuclear transfection, viruses, vesicles, LNPs, exosomes, fusions.

Genetic modification systems can be introduced into cells, tissues, and multicellular organisms. In some embodiments, the system or components of the system are delivered to the cell via mechanical or physical means.

The formulation of protein therapeutics is described in Meyer (ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

Tissue-specific activity/administration

In some embodiments, the systems described herein can utilize one or more features (eg, promoter or microRNA binding site) to limit activity in off-target cells or tissues.

In some embodiments, nucleic acid described herein (e.g., template RNA or DNA encoding template RNA) comprises a promoter sequence, such as a tissue-specific promoter sequence. In some embodiments, tissue-specific promoters are used to increase the target cell specificity of the gene modification system. For example, a promoter can be selected based on the fact that the promoter is active in the target cell type but inactive (or active at a lower level) in the non-target cell type. Therefore, even if the promoter is integrated into the genome of the non-target cell, it will not drive the expression of the integrated gene (or only drive low-level expression). As described herein, a system with a tissue-specific promoter sequence in the template RNA can also be used in combination with a microRNA binding site (e.g., in a template RNA or a nucleic acid encoding a gene modification protein, for example, as described herein). A system with a tissue-specific promoter sequence in the template RNA can also be used in combination with a DNA encoding a gene modification polypeptide driven by a tissue-specific promoter, for example, to obtain a higher level of gene modification protein in the target cell than in the non-target cell. In some embodiments, for example, for liver indications, a tissue-specific promoter is selected from Table 3 of WO 2020014209 (incorporated herein by reference).

In some embodiments, nucleic acid as described herein (e.g., template RNA or DNA encoding template RNA) comprises microRNA binding site. In some embodiments, microRNA binding site is used to increase the target cell specificity of gene modification system. For example, microRNA binding site can be selected based on the recognition of miRNA present in non-target cell type but not present in target cell type (or present at a reduced level relative to non-target cell). Therefore, when template RNA is present in non-target cell, it will be combined with miRNA, and when template RNA is present in target cell, it will not be combined with niRNA (or combined, but combined at a reduced level relative to non-target cell). Although it is not desired to be bound by theory, the combination of miRNA and template RNA can interfere with its activity, for example, heterologous object sequence can be interfered with to insert into genome. Therefore, the system will edit the genome of target cell more effectively than its editing of non-target cell genome, for example, heterologous object sequence will be more effectively inserted into the genome of target cell than inserting into the genome of non-target cell, or insertion or deletion is more effectively produced in target cell than in non-target cell. Systems with microRNA binding sites in the template RNA (or DNA encoding it) can also be used in combination with nucleic acids encoding gene-modified polypeptides, wherein the expression of the gene-modified polypeptide is regulated by a second microRNA binding site, for example as described herein. In some embodiments, for example, for liver indications, the miRNA is selected from Table 4 of WO 2020014209 (incorporated herein by reference).

In some embodiments, the template RNA comprises a microRNA sequence, a siRNA sequence, a guide RNA sequence, or a piwiRNA sequence.

Promoter

In some embodiments, one or more promoters or enhancer elements are operably connected to nucleic acids or template nucleic acids encoding gene modification proteins, for example, they control the expression of heterologous subject sequences. In certain embodiments, the one or more promoters or enhancer elements comprise cell type or tissue specific elements. In some embodiments, the promoter or enhancer is identical or derived from a promoter or enhancer that naturally controls the expression of heterologous subject sequences. For example, the ornithine transcarbamylase promoter and enhancer can be used to control the expression of the ornithine transcarbamylase gene in the system or method provided by the invention to correct the ornithine transcarbamylase defect. In some embodiments, the promoter is a promoter in Table 16 or 17 or a functional fragment or variant thereof.

For example, commercially available exemplary tissue-specific promoters can be found in the uniform resource locator (e.g., www.invivogen.com/tissue-specific-promoters). In some embodiments, the promoter is a natural promoter or a minimal promoter, for example, consisting of a single fragment from the 5' region of a given gene. In some embodiments, the natural promoter includes a core promoter and its natural 5'UTR. In some embodiments, the 5'UTR includes an intron. In other embodiments, these include composite promoters that combine promoter elements with different starting points, or are produced by assembling a distal enhancer with the same minimal promoter as the starting point.

Exemplary cell or tissue specific promoters are provided in the table below, and exemplary nucleic acid sequences encoding them are known in the art and can be readily obtained using a variety of resources, such as the NCBI database, including RefSeq, and the Eukaryotic Promoter Database (//epd.epfl.ch//index.php).

Table 16. Exemplary cell or tissue specific promoters

Table 17. Additional exemplary cell or tissue specific promoters

Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements may be used in the expression vector, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like (see, e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544; which is incorporated herein by reference in its entirety).

In some embodiments, the nucleic acid encoding the gene modification protein or template nucleic acid is operably linked to a control element (e.g., a transcriptional control element, such as a promoter). In some embodiments, the transcriptional control element may function in: a eukaryotic cell such as a mammalian cell; or a prokaryotic cell (e.g., a bacterial or archaeal cell). In some embodiments, the nucleotide sequence encoding the polypeptide is operably linked to a plurality of control elements that allow, for example, the nucleotide sequence encoding the polypeptide to be expressed in prokaryotic and eukaryotic cells.

For purposes of illustration, examples of spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, and the like. Neuron-specific spatially restricted promoters include, but are not limited to, the neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956); the aromatic amino acid decarboxylase (AADC) promoter, the neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); the synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); the thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat. Med. 16(10):1161-1166); the serotonin receptor promoter (see, e.g., GenBank S62283); the tyrosine hydroxylase promoter (TH) (see, e.g., Oh et al. (2009) Gene Ther [Gene Therapy] 16: 437; Sasaoka et al. (1992) Mol. Brain =The promoters of the DNMT promoters are shown in Table 1. The promoters of the DNMT promoters are shown in Table 2. The promoters of the DNMT promoters are shown in Table 3. The promoters of the DNMT promoters are shown in Table 4. The promoters of the DNMT promoters are shown in Table 5. J. [Journal of the European Molecular Biology Association] 17: 3793-3805); myelin basic protein (MBP) promoter; Ca2+-calmodulin-dependent protein kinase II-α (CamKHα) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 93: 13250; and Casanova et al. (2001) Genesis [Genetics] 31: 37); CMV enhancer/platelet-derived growth factor-β promoter (see, e.g., Liu et al. (2004) Gene Therapy [Gene Therapy] 11: 52-60); etc.

Adipocyte-specific spatially restricted promoters include, but are not limited to: the aP2 gene promoter/enhancer, e.g., the -5.4 kb to +21 bp region of the human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol [Endocrinology]. 138:1604; Ross et al. (1990) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 87:9590; and Pavjani et al. (2005) Nat. Med. [Nature Medicine] 11:797); glucose transporter- 4 (GLUT4) promoter (see, e.g., Knight et al. (2003) Proc. Natl. Acad. Sci. USA 100:14725); fatty acid translocase (FAT/CD36) promoter (see, e.g., Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2002) J. Biol. Chem. 277:15703); stearoyl-CoA desaturase-1 (S CD1) promoter (Tabor et al. (1999) J. Biol. Chem. 274:20603); leptin promoter (see, e.g., Mason et al. (1998) Endocrinol. 139:1013; and Chen et al. (1999) Biochem. Biophys. Res. Comm. 262:187); adiponectin promoter (see, e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 264:187). es. Comm. [Biochemical and Biophysical Research Communications] 331: 484; and Chakrabarti (2010) Endocrinol. [Endocrinology] 151: 2408); lipoprotein promoter (see, e.g., Platt et al. (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 86: 7490); anti-insulin protein promoter (see, e.g., Seo et al. (2003) Molec. Endocrinol. [Molecular Endocrinology] 17: 1522); etc.

Cardiomyocyte-specific, spatially restricted promoters include, but are not limited to, control sequences derived from genes such as myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C, cardiac actin, and the like. Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. N.Y. Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Mol. Cell. Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.

Smooth muscle-specific spatially restricted promoters include, but are not limited to, the SM22α promoter (see, e.g., Akyürek et al. (2000) Mol. Med. 6:983; and U.S. Pat. No. 7,169,874); the smooth muscle cell differentiation-specific antigen (smoothelin) promoter (see, e.g., WO 2001/018048); the α-smooth muscle actin promoter; etc. For example, a 0.4 kb region of the SM22α promoter, which contains two CArG elements, has been shown to mediate vascular smooth muscle cell-specific expression (see, e.g., Kim, et al. (1997) Mol. Cell. Biol. 17, 2266-2278; Li, et al. (1996) J. Cell Biol. 132, 849-859; and Moessler, et al. (1996) Development 122, 2415-2425).

Photoreceptor-specific, spatially restricted promoters include, but are not limited to, the rhodopsin promoter; the rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); the beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); the retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); the interphotoreceptor retinoid binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); the IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225); etc.

In some embodiments, a gene modification system, such as a DNA encoding a gene modification polypeptide, a DNA encoding a template RNA, or a DNA or RNA encoding a heterologous object sequence, is designed so that one or more elements are operably connected to a tissue-specific promoter, such as a promoter active in a T cell. In another embodiment, a T cell active promoter is inactive in other cell types such as B cells and NK cells. In some embodiments, a T cell active promoter is derived from a promoter of a gene encoding a T cell receptor component (such as TRAC, TRBC, TRGC, TRDC). In some embodiments, a T cell active promoter is derived from a promoter of a gene encoding a component of a T cell specific differentiation protein cluster (such as CD3, such as CD3D, CD3E, CD3G, CD3Z). In some embodiments, a T cell specific promoter in a gene modification system is found by comparing publicly available gene expression data across cell types and selecting a promoter from a gene with enhanced expression in a T cell. In some embodiments, a promoter can be selected according to a desired expression width, such as a promoter active only in a T cell, a promoter active only in a NK cell, or a promoter active in both a T cell and a NK cell.

Cell-specific promoters known in the art can be used to direct the expression of the gene-modified protein, for example, as described herein. Non-limiting exemplary mammalian cell-specific promoters have been characterized and used in mice expressing Cre recombinase in a cell-specific manner. Certain non-limiting exemplary mammalian cell-specific promoters are listed in Table 1 of US 9845481, which is incorporated herein by reference.

In some embodiments, the vector as described herein comprises an expression cassette. Typically, the expression cassette comprises a nucleic acid molecule operably connected to a promoter sequence of the present invention. For example, when a promoter can affect the expression of a coding sequence, the promoter is operably connected to the coding sequence (for example, the coding sequence is under the transcriptional control of the promoter). The coding sequence can be operably connected to a regulatory sequence in a sense or antisense orientation. In certain embodiments, the promoter is a heterologous promoter. In certain embodiments, the expression cassette may comprise additional elements, for example, introns, enhancers, polyadenylation sites, woodchuck response elements (WREs) and/or other elements known to affect the expression level of the coding sequence. The promoter typically controls the expression of a coding sequence or a functional RNA. In certain embodiments, the promoter sequence comprises proximal and more distal upstream elements, and may further comprise an enhancer element. An enhancer typically can stimulate the activity of a promoter, and may be an intrinsic element of a promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. In certain embodiments, the promoter as a whole is derived from a natural gene. In certain embodiments, the promoter is composed of different elements derived from different naturally occurring promoters. In certain embodiments, the promoter comprises a synthetic nucleotide sequence. It will be appreciated by those skilled in the art that different promoters will direct the expression of genes in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions or in response to the presence or absence of drugs or transcriptional cofactors. Ubiquitous, cell type-specific, tissue-specific, developmental stage-specific and conditional promoters, for example, drug-responsive promoters (e.g., tetracycline-responsive promoters) are well known to those skilled in the art. Exemplary promoters include, but are not limited to, phosphoglycerate kinase (PKG) promoter, CAG (complex of CMV enhancer, chicken beta actin promoter (CBA) and rabbit beta globin intron), NSE (neuron-specific enolase), synapsin or NeuN promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), SFFV promoter, Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, etc. Other promoters may be of human origin or from other species including from mice. Common promoters include, for example, human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, Rous sarcoma virus long terminal repeat, [β]-actin, rat insulin promoter, phosphoglycerate kinase promoter, human α-1 antitrypsin (hAAT) promoter, thyroxine transporter promoter, TBG promoter and other liver-specific promoters, desmin promoter and similar muscle-specific promoters, EF1-α promoter, hybrid promoters with multi-tissue specificity, promoters specific to neurons (such as synaptophysin) and glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are well known and readily available to those skilled in the art, and can be used to obtain high-level expression of the target coding sequence. In addition, sequences derived from non-viral genes (such as mouse metallothionein genes) will also find use herein. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA). Additional exemplary promoter sequences are described, for example, in WO 2018213786 A1 (which is incorporated herein by reference in its entirety).

In some embodiments, the apolipoprotein E enhancer (ApoE) or a functional fragment thereof is used, for example, to promote expression in the liver. In some embodiments, two copies of the ApoE enhancer or a functional fragment thereof are used. In some embodiments, the ApoE enhancer or a functional fragment thereof is used in combination with a promoter (e.g., human alpha-1 antitrypsin (hAAT) promoter).

In certain embodiments, regulatory sequences confer tissue-specific gene expression capability. In some cases, tissue-specific regulatory sequences are combined with tissue-specific transcription factors that induce transcription in a tissue-specific manner. Various tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: liver-specific thyroxine binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synaptophysin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, alpha-myosin heavy chain (a-MHC) promoter, or cardiac troponin T (cTnT) promoter. Other exemplary promoters include: β-actin promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther. [Gene Ther.], 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther. [Human Gene Ther.], 7:1503-14 (1996)), osteocalcin promoter (Stein et al., Mol. Biol. Rep. [Molecular Biology Reports], 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res. [Journal of Bone and Mineral Research] 11: 654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol. [Journal of Immunology], 161: 1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor alpha chain promoter, neurons such as neuron-specific enolase (NSE) promoter (Andersen et al. Cell. Mol. Neurobiol. [Cellular and Molecular Neurobiology], 13: 503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron [Neuron], 15:373-84 (1995)), among others. Additional exemplary promoter sequences are described, for example, in U.S. Patent No. 10300146 (which is incorporated herein by reference in its entirety). In some embodiments, the tissue-specific regulatory element (e.g., tissue-specific promoter) is selected from one known to be operably linked to a gene that is highly expressed in a given tissue, for example, as measured by RNA-seq or protein expression data, or a combination thereof. Methods for analyzing tissue specificity by expression are taught in Fagerberg et al. Mol Cell Proteomics [Molecular and Cellular Proteomics] 13 (2): 397-406 (2014), which is incorporated herein by reference in its entirety.

In certain embodiments, carrier as described herein is a polycistronic expression construct.Polycistronic expression constructs include, for example, a construct carrying a first expression cassette and a second expression cassette, the first expression cassette for example comprising a first promoter and a first encoding nucleic acid sequence, and the second expression cassette for example comprising a second promoter and a second encoding nucleic acid sequence.In some cases, such polycistronic expression constructs can be particularly useful for delivering non-translated gene products (e.g., hairpin RNA) and polypeptides (e.g., genetically modified polypeptides and genetically modified templates).In certain embodiments, polycistronic expression constructs can show the expression level of one or more included transgenic reductions, for example, because promoters interfere or there are very close incompatible nucleic acid elements.If polycistronic expression constructs are a part of a viral vector, then in some cases, the presence of self-complementary nucleic acid sequences may interfere with the formation of viral propagation or packaging required structure.

In some embodiments, the sequence encodes a hairpin-containing RNA. In some embodiments, the hairpin RNA is a guide RNA, a template RNA, a shRNA, or a microRNA. In some embodiments, the first promoter is an RNA polymerase I promoter. In some embodiments, the first promoter is an RNA polymerase II promoter. In some embodiments, the second promoter is an RNA polymerase III promoter. In some embodiments, the second promoter is a U6 or H1 promoter.

Without wishing to be bound by theory, multicistronic expression constructs may not achieve optimal expression levels compared to expression systems containing only one cistron. One of the proposed reasons for the reduced expression levels achieved with polycistronic expression constructs containing two or more promoter elements is the phenomenon of promoter interference (see, e.g., Curtin JA, Dane AP, Swanson A, Alexander IE, Ginn SL. Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct. Gene Ther. 2008 Mar;15(5):384-90; and Martin-Duque P, Jezzard S, Kaftansis L, Vassaux G. Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes. Hum Gene Ther. 2008 Mar;15(5):384-90; and Martin-Duque P, Jezzard S, Kaftansis L, Vassaux G. Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes. Hum Gene Ther. 2008 Mar;15(5):384-90; and Ther. [Human Gene Therapy] 2004 Oct; 15(10): 995-1002; both references are incorporated herein by reference for their disclosure of the promoter interference phenomenon). In some embodiments, the problem of promoter interference can be overcome by, for example, generating a polycistronic expression construct comprising only one promoter that promotes transcription of multiple encoding nucleic acid sequences separated by an internal ribosome entry site; or by separating cistrons comprising their own promoters with transcription insulator elements. In some embodiments, single promoter-driven expression of multiple cistrons may result in uneven expression levels of the cistrons. In some embodiments, the promoter cannot be effectively separated and the separation element may be incompatible with some gene transfer vectors (e.g., some retroviral vectors).

MicroRNA

MicroRNA (miRNA) and other small interfering nucleic acids usually regulate gene expression via target RNA transcript cutting/degradation or translation repression of target messenger RNA (mRNA). In some cases, miRNA can be naturally expressed, typically as the final 19-25 non-translated RNA products. MiRNA usually exhibits their activity through sequence-specific interactions with the 3' untranslated region (UTR) of the target mRNA. These endogenously expressed miRNAs can form hairpin precursors, which are subsequently processed into miRNA duplexes and further processed into mature single-stranded miRNA molecules. This mature miRNA usually guides the multiprotein complex miRISC, which recognizes the 3'UTR region of the target mRNA according to the complementarity of the target mRNA and the mature miRNA. Useful transgenic products can include, for example, miRNA or miRNA binding sites that regulate the expression of the polypeptide connected. A non-limiting list of miRNA genes; for example, in methods such as those listed in US 10300146, 22: 25-25: 48 (which is incorporated herein by reference), the products of these genes and their homologs can be used as transgenes or as targets for small interfering nucleic acids (e.g., miRNA sponges, antisense oligonucleotides). In some embodiments, one or more binding sites for one or more of the aforementioned miRNAs are incorporated into a transgene (e.g., a transgene delivered by a rAAV vector), for example to inhibit the expression of the transgene in one or more tissues of an animal carrying the transgene. In some embodiments, the binding site can be selected so as to control the expression of the transgene in a tissue-specific manner. For example, a binding site for liver-specific miR-122 can be incorporated into a transgene to inhibit the expression of the transgene in the liver. Additional exemplary miRNA sequences are described, for example, in U.S. Pat. No. 10,300,146 (which is incorporated herein by reference in its entirety).

MiR inhibitors or miRNA inhibitors are generally agents that block miRNA expression and/or processing. Examples of such agents include, but are not limited to, microRNA antagonists, microRNA-specific antisense, microRNA sponges, and microRNA oligonucleotides (double-stranded, hairpins, short oligonucleotides) that inhibit the interaction of miRNA with the Drosha complex. MicroRNA inhibitors, such as miRNA sponges, can be expressed in cells from transgenics (e.g., as described in Ebert, M.S. Nature Methods [Natural Methods], August 12, 2007, electronic publication; it is incorporated herein by reference in its entirety). In some embodiments, microRNA sponges or other miR inhibitors are used together with AAV. MicroRNA sponges typically specifically inhibit miRNAs through complementary heptamer seed sequences. In some embodiments, a single sponge sequence can be used to silence the entire miRNA family. Other methods for silencing miRNA function (de-repression of miRNA targets) in cells will be apparent to those of ordinary skill in the art.

In some embodiments, the gene modification systems, template RNAs, or polypeptides described herein are administered to a target tissue (e.g., a first tissue) or are active in a target tissue (e.g., a first tissue) (e.g., more active therein). In some embodiments, the gene modification systems, template RNAs, or polypeptides are not administered to non-target tissues or are less active in non-target tissues (e.g., not active therein). In some embodiments, the gene modification systems, template RNAs, or polypeptides described herein can be used to modify DNA in a target tissue (e.g., a first tissue) (e.g., and not modify DNA in non-target tissues).

In some embodiments, the gene modification system comprises (a) a polypeptide described herein or a nucleic acid encoding the same, (b) a template nucleic acid described herein (e.g., a template RNA), and (c) one or more first tissue-specific expression control sequences specific for a target tissue, wherein the one or more first tissue-specific expression control sequences specific for a target tissue are operably associated with (a), (b), or (a) and (b), wherein, when associated with (a), (a) comprises a nucleic acid encoding a polypeptide.

In some embodiments, the nucleic acid in (b) comprises RNA.

In some embodiments, the nucleic acid in (b) comprises DNA.

In some embodiments, the nucleic acid in (b): (i) is a single-stranded segment or comprises a single-stranded segment, for example, is a single-stranded DNA or comprises a single-stranded segment and one or more double-stranded segments; (ii) has an inverted terminal repeat sequence; or (iii) both (i) and (ii).

In some embodiments, the nucleic acid in (b) is or comprises a double-stranded segment.

In some embodiments, (a) comprises a nucleic acid encoding a polypeptide.

In some embodiments, the nucleic acid in (a) comprises RNA.

In some embodiments, the nucleic acid in (a) comprises DNA.

In some embodiments, the nucleic acid in (a): (i) is a single-stranded segment or comprises a single-stranded segment, for example, is a single-stranded DNA or comprises a single-stranded segment and one or more double-stranded segments; (ii) has an inverted terminal repeat sequence; or (iii) both (i) and (ii).

In some embodiments, the nucleic acid in (a) is or comprises a double-stranded segment.

In some embodiments, the nucleic acid in (a), (b), or (a) and (b) is linear.

In some embodiments, the nucleic acid in (a), (b), or (a) and (b) is circular, such as a plasmid or a minicircle.

In some embodiments, the heterologous subject sequence is operably associated with a first promoter.

In some embodiments, the one or more first tissue-specific expression control sequences comprise a tissue-specific promoter.

In some embodiments, the tissue-specific promoter comprises a first promoter operably associated with: (i) a heterologous subject sequence, (ii) a nucleic acid encoding a retroviral RT, or (iii) both (i) and (ii).

In some embodiments, the one or more first tissue-specific expression control sequences comprise a tissue-specific microRNA recognition sequence operably associated with: (i) a heterologous subject sequence, (ii) a nucleic acid encoding a retroviral RT domain, or (iii) (i) and (ii).

In some embodiments, the system comprises a tissue-specific promoter, and the system further comprises one or more tissue-specific microRNA recognition sequences, wherein: (i) the tissue-specific promoter is operably associated with: (I) a heterologous subject sequence, (II) a nucleic acid encoding a retroviral RT domain, or (III) (I) and (II); and/or (ii) the one or more tissue-specific microRNA recognition sequences are operably associated with: (I) a heterologous subject sequence, (II) a nucleic acid encoding a retroviral RT, or (III) (I) and (II).

In some embodiments, where (a) comprises a nucleic acid encoding a polypeptide, the nucleic acid comprises a promoter operably associated with the nucleic acid encoding the polypeptide.

In some embodiments, the nucleic acid encoding the polypeptide comprises one or more second tissue-specific expression control sequences specific for the target tissue operably associated with the polypeptide encoding sequence.

In some embodiments, the one or more second tissue-specific expression control sequences comprise a tissue-specific promoter.

In some embodiments, a tissue-specific promoter is a promoter operably associated with a nucleic acid encoding a polypeptide.

In some embodiments, the one or more second tissue-specific expression control sequences comprise a tissue-specific microRNA recognition sequence.

In some embodiments, the promoter operably associated with the nucleic acid encoding the polypeptide is a tissue-specific promoter, and the system further comprises one or more tissue-specific microRNA recognition sequences.

In some embodiments, the nucleic acid component sequences of the systems provided herein (e.g., encoding polypeptides or comprising heterologous subject sequences) are flanked by untranslated regions (UTRs) that modify protein expression levels. Various 5' and 3' UTRs can affect protein expression. For example, in some embodiments, the coding sequence may be preceded by a 5' UTR that modifies RNA stability or protein translation. In some embodiments, the sequence may be followed by a 3' UTR that modifies RNA stability or translation. In some embodiments, the sequence may be preceded by a 5' UTR followed by a 3' UTR that modifies RNA stability or translation. In some embodiments, the 5′ and/or 3′ UTR may be selected from the 5′ and 3′ UTRs of complement factor 3 (C3) (CACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACC; SEQ ID NO: 11,004) or orosomucoid 1 (ORM1) (CAGGACACAGCCTTGGATCAGGACAGAGACTTGGGGGCCATCCTGCCCCTCCAACCCGACATGTGTACCTCAGCTTTTTCCCTCACTTGCATCAATAAAGCTTCTGTGTTTGGAACAGCTAA; SEQ ID NO: 11,005) (Asrani et al. RNA Biology 2018). In certain embodiments, the 5′ UTR is the 5′ UTR from C3 and the 3′ UTR is the 3′ UTR from ORM1. In certain embodiments, the 5′UTR and 3′UTR for protein expression (e.g., mRNA (or DNA encoding RNA) of a genetically modified polypeptide or heterologous subject sequence) comprise optimized expression sequences. In some embodiments, the 5′UTR comprises GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 11,006) and/or the 3′UTR comprises UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA (SEQ ID NO: 11,007), e.g., as described in Richner et al. Cell 168(6): P1114-1125 (2017), the sequences of which are incorporated herein by reference. In some embodiments, the 5′ and/or 3′UTR can be selected to enhance protein expression. In some embodiments, 5' and/or 3' UTRs can be selected to modify protein expression to minimize overproduction inhibition. In some embodiments, the UTR is around the coding sequence, for example, outside the coding sequence and in other embodiments, near the coding sequence. In some embodiments, additional regulatory elements (e.g., miRNA binding sites, cis-regulatory sites) are included in the UTR.

In some embodiments, the open reading frame of the gene modification system, e.g., an ORF of an mRNA (or DNA encoding an mRNA) encoding a gene modification polypeptide or one or more ORFs of an mRNA (or DNA encoding an mRNA) of a heterologous subject sequence, is flanked by 5′ and/or 3′ untranslated regions (UTRs) that enhance expression thereof. In some embodiments, the 5′UTR of the mRNA component of the system (or a transcript produced from the DNA component) comprises the sequence 5′-GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC-3′; SEQ ID NO: 11,008). In some embodiments, the 3′UTR of the mRNA component of the system (or a transcript produced from the DNA component) comprises the sequence 5′-UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA-3′ (SEQ ID NO: 11,009). This combination of 5′UTR and 3′UTR has been demonstrated to result in the desired expression of an operably linked ORF by Richner et al. Cell 168(6): P1114-1125 (2017), the teachings and sequences of which are incorporated herein by reference. In some embodiments, the system described herein comprises DNA encoding a transcript, wherein the DNA comprises corresponding 5′UTR and 3′UTR sequences, wherein T replaces U in the sequences listed above. In some embodiments, the DNA vector used to produce the RNA component of the system further comprises a promoter upstream of the 5′UTR for initiating in vitro transcription, such as a T7, T3, or SP6 promoter. The above 5′UTR begins with GGG, which is a suitable start for optimizing transcription using T7 RNA polymerase. For adjusting transcription levels and changing the transcription start site nucleotide to accommodate alternative 5′UTRs, the teachings of Davidson et al. Pac Symp Biocomput 433-443 (2010) describe T7 promoter variants that meet these two characteristics and methods for discovering them.

Viral vectors and their components

In addition to sources of relevant enzymes or domains described herein, for example, as a source of polymerases and polymerase functions (e.g., DNA-dependent DNA polymerases, RNA-dependent RNA polymerases, RNA-dependent DNA polymerases, DNA-dependent RNA polymerases, reverse transcriptases) used herein, viruses are also useful sources of delivery vehicles for the systems described herein. Some enzymes, such as reverse transcriptases, may have multiple activities, such as being capable of RNA-dependent DNA polymerization and DNA-dependent DNA polymerization, such as first and second strand synthesis. In some embodiments, viruses used as a source of gene modification delivery systems or components thereof can be selected from the group described in Baltimore Bacteriol Rev [Bacterial Review] 35(3): 235-241 (1971).

In some embodiments, the virus is selected from group I viruses, for example, the virus is a DNA virus and packages dsDNA into virions. In some embodiments, the group I virus is selected from, for example, adenovirus, herpes virus, poxvirus.

In some embodiments, the virus is selected from a group II virus, e.g., the virus is a DNA virus and packages ssDNA into virions. In some embodiments, the group II virus is selected from, e.g., a parvovirus. In some embodiments, the parvovirus is a dependent parvovirus, e.g., an adeno-associated virus (AAV).

In some embodiments, the virus is selected from a group III virus, e.g., the virus is an RNA virus and packages the dsRNA into a virion. In some embodiments, the group III virus is selected from, e.g., a reovirus. In some embodiments, one or both strands of the dsRNA contained in such a virion are coding molecules that can be used directly as mRNA after transduction into a host cell, e.g., can be directly translated into a protein after transduction into a host cell without any intervening nucleic acid replication or polymerization steps.

In some embodiments, the virus is selected from a group IV virus, e.g., the virus is an RNA virus and packages the ssRNA(+) into a virion. In some embodiments, the group IV virus is selected from, e.g., a coronavirus, a picornavirus, a togavirus. In some embodiments, the ssRNA(+) contained in such a virion is a coding molecule that can be directly used as an mRNA after transduction into a host cell, e.g., can be directly translated into a protein after transduction into a host cell without any intervening nucleic acid replication or polymerization steps.

In some embodiments, the virus is selected from group V viruses, for example, the virus is an RNA virus and packages ssRNA (-) into virions. In some embodiments, group V viruses are selected from, for example, orthomyxoviruses, rhabdoviruses. In some embodiments, RNA viruses with ssRNA (-) genomes also carry enzymes in the virion that are transduced into host cells with viral genomes, such as RNA-dependent RNA polymerases that can copy ssRNA (-) into ssRNA (+) that can be directly translated by the host.

In some embodiments, the virus is selected from group VI virus, for example, the virus is a retrovirus and ssRNA (+) is packaged into a virion. In some embodiments, group VI virus is selected from, for example, a retrovirus. In some embodiments, the retrovirus is a slow virus, for example, HIV-1, HIV-2, SIV, BIV. In some embodiments, the retrovirus is a spumavirus, for example, a foamy virus, for example, HFV, SFV, BFV. In some embodiments, the ssRNA (+) contained in such virions is a coding molecule that can be directly used as mRNA after being transduced to a host cell, for example, after being transduced to a host cell, it can be directly translated into protein without any intervening nucleic acid replication or polymerization step. In some embodiments, ssRNA (+) is first reverse transcribed and copied to produce a dsDNA genome intermediate, and mRNA can be transcribed in a host cell by the genome intermediate. In some embodiments, the RNA virus with a ssRNA (+) genome also carries an enzyme in the virus body that is transduced into a host cell with the viral genome, such as an RNA-dependent DNA polymerase that is capable of copying the ssRNA (+) to dsDNA that can be transcribed into mRNA and translated by the host. In some embodiments, a reverse transcriptase from a Group VI retrovirus is incorporated as a reverse transcriptase domain of a gene modifying polypeptide.

In some embodiments, the virus is selected from group VII virus, for example, the virus is a retrovirus and dsRNA is packaged into a virion. In some embodiments, group VII virus is selected from, for example, hepadnavirus. In some embodiments, one or two chains of the dsRNA contained in such virions are coding molecules, which can be directly used as mRNA after being transduced to a host cell, for example, can be directly translated into protein without any intervening nucleic acid replication or polymerization step after being transduced to a host cell. In some embodiments, one or two chains of the dsRNA contained in such virions are first reverse transcribed and copied to produce a dsDNA genome intermediate, and mRNA can be transcribed in a host cell by the genome intermediate. In some embodiments, RNA viruses with dsRNA genomes also carry enzymes in virions, which are transduced to host cells with viral genomes, such as RNA-dependent DNA polymerases, which can copy dsRNA to dsDNA that can be transcribed into mRNA and translated by the host. In some embodiments, the reverse transcriptase from group VII retroviruses is incorporated as the reverse transcriptase domain of a gene-modified polypeptide.

In some embodiments, the virion used to deliver nucleic acid in the present invention can also carry enzymes involved in the gene modification process. For example, a retroviral virion can include a reverse transcriptase domain delivered to a host cell together with the nucleic acid. In some embodiments, the RNA template can be associated with the gene modification polypeptide in the virion, so that the two are co-delivered to the target cell after the nucleic acid is transduced from the viral particle. In some embodiments, the nucleic acid in the virion can include DNA, such as linear ssDNA, linear dsDNA, circular ssDNA, circular dsDNA, small ring DNA, dbDNA, ceDNA. In some embodiments, the nucleic acid in the virion can include RNA, such as linear ssRNA, linear dsRNA, circular ssRNA, circular dsRNA. In some embodiments, the viral genome can be cyclized after being transduced to the host cell, for example, a linear ssRNA molecule can undergo covalent bonding to form a circular ssRNA, and a linear dsRNA molecule can undergo covalent bonding to form a circular dsRNA or one or more circular ssRNAs. In some embodiments, the viral genome can be replicated by rolling circle replication in a host cell. In some embodiments, the viral genome can include a single nucleic acid molecule, for example, including a non-segmented genome. In some embodiments, the viral genome may comprise two or more nucleic acid molecules, e.g., comprising a segmented genome. In some embodiments, the nucleic acid in the virion may be associated with one or more proteins. In some embodiments, one or more proteins in the virion may be delivered to the host cell after transduction. In some embodiments, the native virus may be adapted for nucleic acid delivery by adding a virion packaging signal to the target nucleic acid, wherein the host cell is used to package the target nucleic acid containing the packaging signal.

In some embodiments, the virosome used as a delivery vehicle may comprise a commensal human virus. In some embodiments, the virosome used as a delivery vehicle may comprise an anellovirus, the use of which is described in WO 2018232017 A1, which is incorporated herein by reference in its entirety.

AAV administration

In some embodiments, adeno-associated virus (AAV) is used in combination with the systems, template nucleic acids, and/or polypeptides described herein. In some embodiments, AAV is used to deliver, administer, or package the systems, template nucleic acids, and/or polypeptides described herein. In some embodiments, AAV is a recombinant AAV (rAAV).

In some embodiments, the system comprises (a) a polypeptide described herein or a nucleic acid encoding the same, (b) a template nucleic acid described herein (e.g., a template RNA), and (c) one or more first tissue-specific expression control sequences specific for a target tissue, wherein the one or more first tissue-specific expression control sequences specific for a target tissue are operably associated with (a), (b), or (a) and (b), wherein, when associated with (a), (a) comprises a nucleic acid encoding a polypeptide.

In some embodiments, the system described herein further comprises a first recombinant adeno-associated virus (rAAV) capsid protein; wherein at least one of (a) or (b) is associated with a first rAAV capsid protein, wherein at least one of (a) or (b) is flanked by AAV inverted terminal repeat sequences (ITRs).

In some embodiments, (a) and (b) are associated with a first rAAV capsid protein.

In some embodiments, (a) and (b) are on a single nucleic acid.

In some embodiments, the system further comprises a second rAAV capsid protein, wherein at least one of (a) or (b) is associated with the second rAAV capsid protein, and wherein at least one of (a) or (b) associated with the second rAAV capsid protein is different from at least one of (a) or (b) associated with the first rAAV capsid protein.

In some embodiments, at least one of (a) or (b) is associated with a first or second rAAV capsid protein and is dispersed within the first or second rAAV capsid protein, which is in the form of an AAV capsid particle.

In some embodiments, the system further comprises a nanoparticle, wherein the nanoparticle is associated with at least one of (a) or (b).

In some embodiments, (a) and (b) are respectively associated with: a) a first rAAV capsid protein and a second rAAV capsid protein; b) a nanoparticle and a first rAAV capsid protein; c) a first rAAV capsid protein; d) a first adenovirus capsid protein; e) a first nanoparticle and a second nanoparticle; or f) a first nanoparticle.

Viral vectors can be used to deliver all or part of the system provided by the present invention, for example, in the method provided by the present invention. Systems derived from different viruses have been used to deliver polypeptides or nucleic acids; for example: integrase-deficient lentivirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus and baculovirus (in Hodge et al. Hum Gene Ther [Human Gene Therapy] 2017; Narayanavari et al. Crit Rev Biochem Mol Biol [Review of Biochemistry and Molecular Biology] 2017; Boehme et al. Curr Gene Ther [Current Gene Therapy] 2015) were reviewed.

Adenoviruses are common viruses that have been used as gene delivery vehicles due to their well-defined biological properties, genetic stability, high transduction efficiency, and ease of large-scale production (e.g., see Lee et al., Genes & Diseases, 2017 for a review). They have a linear dsDNA genome and are available in multiple serotypes that differ in tissue and cell tropism. To prevent infectious virus replication in recipient cells, adenoviral genomes used for packaging are deleted for some or all endogenous viral proteins, which are provided in trans in viral production cells. This renders the genomes dependent on helper functions, meaning that they can only be replicated and packaged into viral particles in the presence of the missing components provided by the so-called helper functions. Helper-dependent adenoviral systems that have all viral ORFs removed are compatible with packaging of up to about 37 kb of foreign DNA (Parks et al., J Virol, 1997). In some embodiments, adenoviral vectors are used to deliver DNA corresponding to polypeptides or template components of a gene modification system, or both are contained on separate or the same adenoviral vector. In some embodiments, the adenovirus is a helper-dependent adenovirus (HD-AdV) that is not self-packaging. In some embodiments, the adenovirus is a high-capacity adenovirus (HC-AdV) that has deleted all or most of the endogenous viral ORFs while retaining the sequence components required for packaging into adenoviral particles. For this type of vector, the only adenoviral sequences required for genome packaging are non-coding sequences: inverted terminal repeats (ITRs) at both ends and a packaging signal at the 5′ end (Jager et al. Nat Protoc [Natural Experiment Manual] 2009). In some embodiments, the adenovirus genome also contains filler DNA to meet the minimum genome size for optimal production and stability (see, e.g., Hausl et al. Mol Ther [Molecular Therapy] 2010). In some embodiments, adenovirus is used to deliver gene modification systems to the liver.

In some embodiments, adenovirus is used to deliver the gene modification system to HSC, such as HDAd5/35++. HDAd5/35++ is an adenovirus with modified serotype 35 fibers that de-target the vector from the liver (Wang et al. Blood Adv 2019). In some embodiments, adenoviruses that deliver the gene modification system to HSC utilize receptors specifically expressed on primitive HSCs, such as CD46.

Adeno-associated virus (AAV) belongs to the Parvoviridae family, more specifically to the genus Dependaviridovirus. The AAV genome consists of a linear single-stranded DNA molecule that contains approximately 4.7 kilobases (kb) and consists of two major open reading frames (ORFs) encoding nonstructural Rep (replication) and structural Cap (capsid) proteins. The second ORF within the cap gene was identified as encoding assembly activation protein (AAP). The DNA flanking the AAV coding region is two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with intermittent palindromic sequences that can fold into energy-stable hairpin structures that serve as primers for DNA replication. In addition to their role in DNA replication, ITR sequences have been shown to be involved in the integration of viral DNA into the cellular genome, rescue from the host genome or plasmid, and packaging of viral nucleic acids into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. [Current Topics in Microbiology and Immunology] 158: 97-129). In some embodiments, the flanks of one or more genetically modified nucleic acid components are AAV-derived ITRs for viral packaging. See, for example, WO 2019113310.

In some embodiments, one or more components of the gene modification system are carried by at least one AAV vector. In some embodiments, at least one AAV vector is selected for the tropism of specific cells, tissues, and organisms. In some embodiments, the AAV vector is a pseudotype, such as AAV2/8, wherein AAV2 describes the design of the construct, but the capsid protein is replaced by the protein from AAV8. It should be understood that any of the vectors can be a pseudotype derivative, wherein the capsid protein used to package the AAV genome is derived from the capsid protein of different AAV serotypes. It is not desirable to be limited to vector selection, and a list of exemplary AAV serotypes can be found in Table 18. In some embodiments, the AAV for gene modification can be evolved for novel cell or tissue tropism, as has been demonstrated in the literature (e.g., Davidsson et al. Proc Natl Acad Sci U S A [Proceedings of the National Academy of Sciences of the United States] 2019).

In some embodiments, the AAV delivery vector is a vector having two AAV inverted terminal repeats (ITRs) and a nucleotide sequence of interest (e.g., a sequence encoding a gene-modifying polypeptide or a DNA template, or both), each of the ITRs having an interrupted (or non-continuous) palindromic sequence, i.e., a sequence consisting of three segments: the first segment and the last segment are identical when read 5′→3′, but hybridize when placed relative to each other, and a different segment separates the identical segments. See, e.g., WO 2012123430.

Typically, AAV virions with capsids are produced by introducing one or more plasmids encoding rAAV or scAAV genomes, Rep proteins, and Cap proteins (Grimm et al., 1998). After the introduction of these helper plasmids in trans, the AAV genome is "rescued" (i.e., released and subsequently recovered) from the host genome and further packaged to produce infectious AAV. In some embodiments, one or more genetically modified nucleic acids are packaged into AAV particles by introducing nucleic acids flanked by ITRs together with helper functions into packaging cells.

In some embodiments, the AAV genome is a so-called self-complementary genome (referred to as scAAV), such that the sequence between the ITRs contains the desired nucleic acid sequence (e.g., DNA encoding a gene-modified polypeptide or template, or both) and the reverse complement of the desired nucleic acid sequence, so that the two components can fold and self-hybridize. In some embodiments, the self-complementary modules are separated by intervening sequences that allow the DNA to fold itself, for example, to form a stem loop. The advantage of scAAV is that it is ready for transcription after entering the nucleus, rather than first relying on ITR initiation and second-strand synthesis to form dsDNA. In some embodiments, one or more gene modification components are designed as scAAV, wherein the sequence between the AAV ITRs contains two reverse complementary modules that can self-hybridize to produce dsDNA.

In some embodiments, the nucleic acid delivered to the cell (e.g., encoding a polypeptide or a template, or both) is a linear duplex DNA with closed ends (CELiD DNA or ceDNA). In some embodiments, ceDNA is derived from a replicative form of the AAV genome (Li et al. PLoS One [Public Library of Science Comprehensive] 2013). In some embodiments, the nucleic acid (e.g., encoding a polypeptide or a template DNA, or both) is flanked by ITRs, such as AAV ITRs, wherein at least one ITR comprises a terminal dissociation site and a replication protein binding site (sometimes referred to as a replication protein binding site). In some embodiments, the ITR is derived from an adeno-associated virus, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. In some embodiments, the ITR is symmetrical. In some embodiments, the ITR is asymmetrical. In some embodiments, at least one Rep protein is provided to enable the construct to replicate. In some embodiments, at least one Rep protein is derived from an adeno-associated virus, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. In some embodiments, ceDNA is produced by providing (i) DNA flanked by ITRs (e.g., AAV ITRs), and (ii) components required for ITR-dependent replication, such as AAV proteins Rep78 and Rep52 (or nucleic acids encoding the proteins) to production cells. In some embodiments, ceDNA does not contain any capsid protein, for example, it is not packaged into infectious AAV particles. In some embodiments, ceDNA is formulated into LNPs (see, for example, WO 2019051289 A1).

In some embodiments, the ceDNA vector consists of two self-complementary sequences, such as asymmetric or symmetric or substantially symmetric ITRs as defined herein, flanking the expression cassette, wherein the ceDNA vector is not associated with a capsid protein. In some embodiments, the ceDNA vector comprises two self-complementary sequences found in the AAV genome, wherein at least one ITR comprises an operable Rep binding element (RBE) of AAV (sometimes also referred to herein as "RBS") and a terminal dissociation site (trs) or a functional variant of the RBE. See, e.g., WO 2019113310.

In some embodiments, the AAV genome comprises two genes encoding four replication proteins and three capsid proteins, respectively. In some embodiments, there are 145-bp inverted terminal repeats (ITRs) on either side of the flanks of the gene. In some embodiments, the virion comprises up to three capsid proteins (Vp1, Vp2, and/or Vp3), for example, produced at a ratio of 1:1:10. In some embodiments, the capsid proteins are produced from the same open reading frame and/or differential splicing (Vp1) and alternative translation start sites (Vp2 and Vp3, respectively). Typically, Vp3 is the most abundant subunit in the virion and is involved in receptor recognition on the cell surface, thereby defining the tropism of the virus. In some embodiments, Vp1 comprises a phospholipase domain, for example, that plays a role in viral infectivity, at the N-terminus of Vp1.

In some embodiments, the packaging capacity of the viral vector limits the size of the genetic modification system that can be packaged into the vector. For example, the packaging capacity of AAV can be about 4.5 kb (e.g., about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0 kb), for example, including one or two inverted terminal repeats (ITRs), for example, 145 base ITRs.

In some embodiments, recombinant AAV (rAAV) comprises a cis-acting 145-bp ITR flanking the vector transgene cassette, for example, providing up to 4.5 kb for packaging of exogenous DNA. After infection, in some cases, rAAV can express the fusion protein of the present invention and persist in the form of an episome in a circular head-to-tail concatemer without integrating into the host genome. rAAV can be used, for example, in vitro and in vivo. In some embodiments, AAV-mediated gene delivery requires that the length of the coding sequence of the gene is equal to or greater than the wild-type AAV genome in size.

AAV delivery of genes exceeding this size and/or the use of large physiological regulatory elements can be accomplished, for example, by dividing the protein or proteins to be delivered into two or more fragments. In some embodiments, the N-terminal fragment is fused to an intein-N sequence. In some embodiments, the C-terminal fragment is fused to an intein-C sequence. In an embodiment, the fragments are packaged into two or more AAV vectors.

In some embodiments, a dual AAV vector is generated by dividing a large transgene expression cassette into two separate halves (5' and 3' ends, or head and tail), for example, wherein each half of the cassette is packaged in a single AAV vector (which is <5 kb). In some embodiments, reassembly of the full-length transgene expression cassette can then be achieved upon co-infection of the same cell by two dual AAV vectors. In some embodiments, co-infection is followed by one or more of the following: (1) homologous recombination (HR) between the 5' and 3' genomes (dual AAV overlapping vectors); (2) ITR-mediated tail-to-head concatemerization of the 5' and 3' genomes (dual AAV trans-splicing vectors); and/or (3) a combination of these two mechanisms (dual AAV hybrid vectors). In some embodiments, in vivo use of the dual AAV vector results in expression of the full-length protein. In some embodiments, the use of dual AAV vector platforms represents an effective and feasible gene transfer strategy for transgenes greater than about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 kb in size. In some embodiments, AAV vectors can also be used to transduce cells with target nucleic acids, for example, in the in vitro production of nucleic acids and peptides. In some embodiments, AAV vectors can be used for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160: 38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5: 793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994); each of which is incorporated herein by reference in its entirety). The construction of recombinant AAV vectors is described in many publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat and Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989) (each of which is incorporated herein by reference in its entirety).

In some embodiments, the genetically modified polypeptides described herein (e.g., with or without one or more guide nucleic acids) can be delivered using AAV, lentivirus, adenovirus, or other plasmid or viral vector types, particularly using formulations and dosages from the following literature: for example, U.S. Pat. No. 8,454,972 (formulations, dosages for adenovirus), U.S. Pat. No. 8,404,658 (formulations, dosages for AAV), and U.S. Pat. No. 5,846,946 (formulations, dosages for DNA plasmids) and from clinical trials and disclosures on clinical trials involving lentivirus, AAV, and adenovirus. For example, for AAV, the route of administration, formulation, and dosage may be as described in U.S. Pat. No. 8,454,972 and clinical trials involving AAV. For adenovirus, the route of administration, formulation, and dosage may be as described in U.S. Pat. No. 8,404,658 and clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation, and dosage may be as described in U.S. Pat. No. 5,846,946 and clinical studies involving plasmids. Dosages can be based on or extrapolated to an average 70 kg individual (e.g., male adult) and can be adjusted for patients, subjects, mammals of different weights and species. The frequency of administration is within the scope of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on conventional factors, including the age, sex, general health, other conditions, and specific conditions or symptoms addressed by the patient or subject. In some embodiments, the viral vector can be injected into the target tissue. For specific genetic modification of cell types, in some embodiments, the expression of the genetically modified polypeptide and the optional guide nucleic acid can be driven by a specific promoter of the cell type.

In some embodiments, AAV allows for low toxicity, for example, because the purification method does not require ultracentrifugation of cell pellets that can activate an immune response. In some embodiments, AAV allows for a low likelihood of causing insertional mutagenesis, for example, because it does not substantially integrate into the host genome.

In some embodiments, AAV has a packaging limit of about 4.4, 4.5, 4.6, 4.7, or 4.75 kb. In some embodiments, the gene modified polypeptide coding sequence, promoter, and transcription terminator can be assembled into a single viral vector. In some cases, SpCas9 (4.1 kb) may be difficult to package into AAV. Therefore, in some embodiments, a gene modified polypeptide coding sequence having a length shorter than other gene modified polypeptide coding sequences or base editors is used. In some embodiments, the gene modified polypeptide coding sequence is less than about 4.5 kb, 4.4 kb, 4.3 kb, 4.2 kb, 4.1 kb, 4 kb, 3.9 kb, 3.8 kb, 3.7 kb, 3.6 kb, 3.5 kb, 3.4 kb, 3.3 kb, 3.2 kb, 3.1 kb, 3 kb, 2.9 kb, 2.8 kb, 2.7 kb, 2.6 kb, 2.5 kb, 2 kb, or 1.5 kb.

AAV can be AAV1, AAV2, AAV5, or any combination thereof. In some embodiments, the type of AAV is selected based on the cells to be targeted; for example, AAV serotype 1, 2, 5 or hybrid capsid AAV1, AAV2, AAV5, or any combination thereof can be selected for targeting brain or neuronal cells; or AAV4 can be selected for targeting cardiac tissue. In some embodiments, AAV8 is selected for delivery to the liver. Exemplary AAV serotypes for these cells are described in, for example, Grimm, D. et al., J. Virol. [Journal of Virology] 82: 5887-5911 (2008) (which is incorporated herein by reference in its entirety). In some embodiments, AAV refers to all serotypes, subtypes, and naturally occurring AAVs as well as recombinant AAVs. AAV can be used to refer to the virus itself or its derivatives. In some embodiments, AAV includes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrhlO, AAVLK03, AV10, AAV11, AAV 12, rhlO, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various AAV serotypes, as well as the sequences of natural terminal repeats (TR), Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. Additional exemplary AAV serotypes are listed in Table 18.

Table 18. Exemplary AAV serotypes.

In some embodiments, a pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV as described herein) has less than 10% empty capsids, less than 8% empty capsids, less than 7% empty capsids, less than 5% empty capsids, less than 3% empty capsids, or less than 1% empty capsids. In some embodiments, the pharmaceutical composition has less than about 5% empty capsids. In some embodiments, the number of empty capsids is below the limit of detection. In some embodiments, it is advantageous for a pharmaceutical composition to have a small amount of empty capsids because, for example, empty capsids may produce adverse reactions (e.g., immune, inflammatory, liver, and/or cardiac reactions) that have little or no substantial therapeutic benefit.

In some embodiments, the residual host cell protein (rHCP) in the pharmaceutical composition is less than or equal to 100 ng/ml rHCP/1 x 10 ¹³ vg/ml, for example, less than or equal to 40 ng/ml rHCP/1 x 10 ¹³ vg/ml or 1-50 ng/ml rHCP/1 x 10 ¹³ vg/ml. In some embodiments, the pharmaceutical composition comprises less than 10 ng rHCP/1.0 x 10 ¹³ vg, or less than 5 ng rHCP/1.0 x 10 ¹³ vg, less than 4 ng rHCP/1.0 x 10 ¹³ vg, or less than 3 ng rHCP/1.0 x 10 ¹³ vg, or any concentration therebetween. In some embodiments, the residual host cell DNA (hcDNA) in the pharmaceutical composition is less than or equal to 5 x 10 ⁶ pg/ml hcDNA/1 x 10 ¹³ vg/ml, less than or equal to 1.2 x 10 ⁶ pg/ml hcDNA/1 x 10 ¹³ vg/ml, or 1 x 10 ⁵ pg/ml hcDNA/1 x 10 ¹³ vg/ml. In some embodiments, the residual host cell DNA in the pharmaceutical composition is less than 5.0 x 10 ⁵ pg/1 x 10 ¹³ vg, less than 2.0 x 10 ⁵ pg/1.0 x 10 ¹³ vg, less than 1.1 x 10 ⁵ pg/1.0 x 10 ¹³ vg, less than 1.0 x 10 ⁵ pg hcDNA/1.0 x 10 ¹³ vg, less than 0.9 x 10 ⁵ pg hcDNA/1.0 x 10 ¹³ vg, less than 0.8 x 10 ⁵ pg hcDNA/1.0 x 10 ¹³ vg, or any concentration therebetween.

In some embodiments, the residual plasmid DNA in the pharmaceutical composition is less than or equal to 1.7 x 10 ⁵ pg/ml/1.0 x 10 ¹³ vg/ml, or 1 x 10 ⁵ pg/ml/1 x 1.0 x 10 ¹³ vg/ml, or 1.7 x 10 ⁶ pg/ml/1.0 x 10 ¹³ vg/ml. In some embodiments, the residual DNA plasmid in the pharmaceutical composition is less than 10.0 x 10 ⁵ pg/1.0 x 10 ¹³ vg, less than 8.0 x 10 ⁵ pg/1.0 x 10 ¹³ vg, or less than 6.8 x 10 ⁵ pg/1.0 x 10 ¹³ vg. In embodiments, the pharmaceutical composition comprises less than 0.5 ng/1.0 x 10 ¹³ vg, less than 0.3 ng/1.0 x 10 ¹³ vg, less than 0.22 ng/1.0 x 10 ¹³ vg, or less than 0.2 ng/1.0 x 10 ¹³ vg, or any intermediate concentration of bovine serum albumin (BSA). In embodiments, the benzonase in the pharmaceutical composition is less than 0.2 ng/1.0 x 10 ¹³ vg, less than 0.1 ng/1.0 x 10 ¹³ vg, less than 0.09 ng/1.0 x 10 ¹³ vg, less than 0.08 ng/1.0 x 10 ¹³ vg, or any intermediate concentration. In embodiments, the poloxamer 188 in the pharmaceutical composition is about 10 to 150 ppm, about 15 to 100 ppm, or about 20 to 80 ppm. In embodiments, the cesium in the pharmaceutical composition is less than 50 pg/g (ppm), less than 30 pg/g (ppm), or less than 20 pg/g (ppm), or any intermediate concentration.

In embodiments, the pharmaceutical composition comprises less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or any percentage in between of total impurities, e.g., as determined by SDS-PAGE. In embodiments, for example, the total purity as determined by SDS-PAGE is greater than 90%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or any percentage in between. In embodiments, for example, as measured by SDS-PAGE, there is no single unnamed related impurity greater than 5%, greater than 4%, greater than 3% or greater than 2%, or any percentage in between. In embodiments, the pharmaceutical composition comprises a percentage of filled capsid relative to total capsid (e.g., peak 1 + peak 2 as measured by analytical ultracentrifugation) of greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 91.9%, greater than 92%, greater than 93%, or any percentage in between. In embodiments of the pharmaceutical composition, the percentage of filled capsid measured in peak 1 by analytical ultracentrifugation is 20-80%, 25-75%, 30-75%, 35-75%, or 37.4-70.3%. In embodiments of the pharmaceutical composition, the percentage of filled capsid measured in peak 2 by analytical ultracentrifugation is 20%-80%, 20%-70%, 22%-65%, 24%-62%, or 24.9%-60.1%.

In one embodiment, the pharmaceutical composition comprises 1.0 to 5.0 x 10 ¹³ vg/mL, 1.2 to 3.0 x 10 ¹³ vg/mL or 1.7 to 2.3 x 10 ¹³ vg/ml genomic titer. In one embodiment, the pharmaceutical composition shows less than 5CFU/mL, less than 4CFU/mL, less than 3CFU/mL, less than 2CFU/mL or less than 1CFU/mL or any intermediate concentration of bioburden. In an embodiment, according to USP, such as USP<85> (incorporated by reference in its entirety) the amount of endotoxin is less than 1.0EU/mL, less than 0.8EU/mL or less than 0.75EU/mL. In an embodiment, according to USP, such as USP<785> (incorporated by reference in its entirety) the osmotic pressure molarity of the pharmaceutical composition is 350 to 450mOsm/kg, 370 to 440mOsm/kg or 390 to 430mOsm/kg. In embodiments, the pharmaceutical composition contains less than 1200 particles/container greater than 25 μm, less than 1000 particles/container greater than 25 μm, less than 500 particles/container greater than 25 μm, or any intermediate value. In embodiments, the pharmaceutical composition contains less than 10,000 particles/container greater than 10 μm, less than 8000 particles/container greater than 10 μm, or less than 600 particles/container greater than 10 μm.

In one embodiment, the pharmaceutical composition has a genomic titer of 0.5 to 5.0 x 10 ¹³ vg/mL, 1.0 to 4.0 x 10 ¹³ vg/mL, 1.5 to 3.0 x 10 ¹³ vg/ml, or 1.7 to 2.3 x 10 ¹³ vg/ml. In one embodiment, the pharmaceutical composition described herein comprises one or more of the following: less than about 0.09 ng of full-potency nuclease/1.0 x 10 ¹³ vg, less than about 30 pg/g (ppm) of cesium, about 20 to 80 ppm of poloxamer 188, less than about 0.22 ng of BSA/1.0 x 10 ¹³ vg, less than about 6.8 x 10 ⁵ pg of residual DNA plasmid/1.0 x 10 ¹³ vg, less than about 1.1 x 10 ⁵ pg of residual hcDNA/1.0 x 10 ¹³ vg, less than about 4 ng of rHCP/1.0 x 10 ¹³ vg, pH 7.7 to 8.3, about 390 to 430 mOsm/kg, less than about 600 particles/container with a size of >25 μm, less than about 6000 particles/container with a size of >10 μm, about 1.7 x 10 ^13-2.3 x10 ¹³ vg/mL genome titer, infectious titer of about 3.9 x 10 ⁸ to 8.4 x 10 ¹⁰ IU/1.0 x 10 ¹³ vg, total protein of about 100-300 pg/1.0 x 10 ¹³ vg, mean survival of A7SMA mice >24 days at a viral vector dose of about 7.5 x 10 ¹³ vg/kg, about 70% to 130% relative potency based on an in vitro cell-based assay and/or less than about 5% empty capsids. In various embodiments, the pharmaceutical compositions described herein contain any of the viral particles discussed herein, the pharmaceutical compositions retaining potency within ±20%, ±15%, ±10%, or ±5% of a reference standard. In some embodiments, potency is measured using a suitable in vitro cell assay or in vivo animal model.

Additional methods for preparing, characterizing, and administering AAV particles are taught in WO 2019094253, which is incorporated herein by reference in its entirety.

Other rAAV constructs that may be used in accordance with the present invention include those described in Wang et al. 2019, available at: //doi.org/10.1038/s41573-019-0012-9, including Table 1 thereof, which is incorporated by reference in its entirety.

Lipid Nanoparticles

The method and system provided herein can adopt any suitable carrier or delivery form, including lipid nanoparticles (LNP) in certain embodiments. In certain embodiments, lipid nanoparticles include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic or zwitterionic lipids); one or more conjugated lipids (PEG conjugated lipids or conjugated to the lipid of polymer as described in Table 5 of WO2019217941; it is incorporated herein in its entirety by citing); one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or a combination of the foregoing.

Lipids that can be used to form nanoparticles (e.g., lipid nanoparticles) include, for example, those described in Table 4 of WO 2019217941 (incorporated by reference) - for example, the lipid-containing nanoparticles may comprise one or more lipids in Table 4 of WO 2019217941. The lipid nanoparticles may include additional elements, such as polymers, such as those described in Table 5 of WO 2019217941, incorporated by reference.

In some embodiments, the conjugated lipid, when present, may include one or more of: PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerol (PEGS-DAG) (such as 4-0-(2′,3′-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO 2019051289 (incorporated by reference), and combinations of the foregoing.

In some embodiments, the sterol that can be incorporated into the lipid nanoparticles includes one or more of cholesterol or cholesterol derivatives, such as those in WO2009/127060 or US 2010/0130588, which are incorporated by reference. Additional exemplary sterols include plant sterols, including those described in Eygeris et al. (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, which are incorporated by reference herein.

In some embodiments, lipid particles include ionizable lipids, non-cationic lipids, conjugated lipids and sterols that inhibit particle aggregation. The amounts of these components can be changed independently to obtain desired properties. For example, in some embodiments, lipid nanoparticles include: ionizable lipids in an amount of about 20 mol% to about 90 mol% of total lipids (in other embodiments, it can be 20%-70% (mol), 30%-60% (mol) or 40%-50% (mol) of total lipids present in lipid nanoparticles; about 50 mol% to about 90 mol%); non-cationic lipids in an amount of about 5 mol% to about 30 mol% of total lipids; conjugated lipids in an amount of about 0.5 mol% to about 20 mol% of total lipids, and sterols in an amount of about 20 mol% to about 50 mol% of total lipids. The ratio of total lipids to nucleic acids (e.g., encoding gene modification polypeptides or template nucleic acids) can be varied as desired. For example, the ratio of total lipids to nucleic acids (mass or weight) can be about 10: 1 to about 30: 1.

In some embodiments, the ionizable lipid can be a cationic lipid, an ionizable cationic lipid, such as a cationic lipid that can exist in a positively charged form or a neutral form according to pH, or an amine-containing lipid that can be easily protonated. In some embodiments, the cationic lipid is, for example, a lipid that can be positively charged under physiological conditions. Exemplary cationic lipids include one or more positively charged amine groups. In some embodiments, the lipid particles include cationic lipids and neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (such as sterols), PEG, cholesterol and polymer conjugated lipids are formulated together. In some embodiments, the cationic lipid can be an ionizable cationic lipid. Exemplary cationic lipids as disclosed herein can have an effective pKa exceeding 6.0. In an embodiment, the lipid nanoparticle can include a second cationic lipid with an effective pKa different from the first cationic lipid (e.g., greater than the first effective pKa). Lipid nanoparticles can include 40mol% to 60mol% of cationic lipids, neutral lipids, steroids, polymer-conjugated lipids and therapeutic agents, such as nucleic acids (e.g., RNA) as described herein (e.g., template nucleic acids or nucleic acids encoding gene-modified polypeptides), encapsulated in lipid nanoparticles or associated with lipid nanoparticles. In certain embodiments, nucleic acids and cationic lipids are co-formulated. Nucleic acids can be adsorbed onto the surface of LNPs (e.g., LNPs comprising cationic lipids). In certain embodiments, nucleic acids can be encapsulated in LNPs (e.g., LNPs comprising cationic lipids). In certain embodiments, lipid nanoparticles can include targeting moieties, such as targeting moieties coated with targeting agents. In certain embodiments, LNP formulations are biodegradable. In some embodiments, lipid nanoparticles comprising one or more lipids described herein (e.g., formula (i), (ii), (vii), and/or (ix)) encapsulate at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or 100% of RNA molecules, e.g., template RNA and/or mRNA encoding a gene modifying polypeptide.

In some embodiments, the ratio of lipid to nucleic acid (mass/mass ratio; w/w ratio) can be in the following range: about 1: 1 to about 25: 1, about 10: 1 to about 14: 1, about 3: 1 to about 15: 1, about 4: 1 to about 10: 1, about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1. The amount of lipid and nucleic acid can be adjusted to provide a desired N/P ratio, such as 3, 4, 5, 6, 7, 8, 9, 10 or higher N/P ratios. Typically, the total lipid content of the lipid nanoparticle formulation can be in the range of about 5 mg/ml to about 30 mg/mL.

Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of WO 2019051289, which is incorporated herein by reference. Additional exemplary lipids include, but are not limited to, one or more of the following formulae: X of US 2016/0311759; I of US 20150376115 or US 2016/0376224; I, II, or III of US 20160151284; I, IA, II, or IIA of US 20170210967; I-c of US 20150140070; A of US 2013/0178541; I of US 2013/0303587 or US 2013/0123338; I of US 2015/0141678; II, III, IV, or V of US 2015/0239926; I of US 2017/0119904; I or II of US 2017/117528; A of US 2012/0149894; A of US 2015/0057373; A of WO 2013/116126; A of US 2013/0090372; A of US 2013/0274523; A of US 2013/0274504; A of US 2013/0053572; A of WO2013/016058; A of WO2012/162210; I of US 2008/042973; I, II, III or IV of US 2012/01287670; I or II of US 2014/0200257; I, II or III of US 2015/0203446; I or III of US 2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID or III-XXIV of US 2014/0308304; US 2013/0338210; I, II, III or IV of WO2009/132131; A of US 2012/01011478; I or XXXV of US 2012/0027796; XIV or XVII of US 2012/0058144; of US 2013/0323269; I of US 2011/0117125; I, II or III of US 2011/0256175; US I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV or XVI of US 2011/0076335; I or II of US 2006/008378; I of US 2013/0123338; I or X-A-Y-Z of US 2015/0064242; XVI, XVII or XVIII of US 2013/0022649; I, II or III of US 2013/0116307; I, II or III of US 2013/0116307; I or II of US 2010/0062967; I-X of 2013/0189351; I of US2014/0039032; V of US 2018/0028664; I of US 2016/0317458; I of US 2013/0195920; 5, 6 or 10 of US10,221,127; III-3 of WO 2018/081480; I-5 or I-8 of WO 2020/081938; 18 or 25 of US 9,867,888; A of US 2019/0136231; II of WO 2020/219876; 1 of US2012/0027803; OF-02 of US 2019/0240349; US 23 of 10,086,013; cKK-E12/A6 of Miao et al. (2020); C12-200 of WO 2010/053572; 7C1 of Dahlman et al. (2017); 304-O13 or 503-O13 of Whitehead et al.; TS-P4C2 of US 9,708,628; I of WO 2020/106946; I of WO 2020/106946.

In some embodiments, the ionizable lipid is MC3 (6Z, 9Z, 28Z, 31Z) - heptatriacontane-6, 9, 28, 31-tetraen-19-yl-4- (dimethylamino) butyrate (DLin-MC3-DMA or MC3), for example, as described in Example 9 of WO 2019051289 A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is lipid ATX-002, for example, as described in Example 10 of WO 2019051289 A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is (13Z, 16Z) -A, A-dimethyl-3-nonyldocosa-13, 16-dien-1-amine (compound 32), for example, as described in Example 11 of WO 2019051289 A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is compound 6 or compound 22, for example, as described in Example 12 of WO 2019051289 A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); for example, as described in Example 1 of US 9,867,888 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadec-9,12-dienoate (LP01), for example, as synthesized in Example 13 of WO 2015/095340 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is di((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), for example, as synthesized in Examples 7, 8, or 9 of US 2012/0027803, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), for example, as synthesized in Examples 14 and 16 of WO 2010/053572, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is; imidazolyl cholesteryl ester (ICE) lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(1H-imidazol-4-yl)propanoate, for example, structure (I) from WO 2020/106946 (which is incorporated herein by reference in its entirety).

Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for delivering a composition described herein, e.g., a nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene-modifying polypeptide) include:

In some embodiments, LNPs comprising formula (i) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

In some embodiments, LNPs comprising formula (ii) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

In some embodiments, LNPs comprising formula (iii) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

In some embodiments, LNPs comprising formula (v) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

In some embodiments, LNPs comprising formula (vi) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

In some embodiments, LNPs comprising formula (viii) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

In some embodiments, LNPs comprising formula (ix) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

in

^X1 is O, ^NR1 or a direct bond, ^X2 is C2-5 alkylene, ^X3 is C(＝0) or a direct bond, ^R1 is H or Me, ^R3 is C1-3 alkyl, ^R2 is C1-3 alkyl, or ^R2 together with the nitrogen atom to which it is attached and 1-3 carbon atoms of ^X2 form a 4-, 5- or 6-membered ring, or ^X1 is ^NR1 , ^R1 and ^R2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or ^R2 and ^R3 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered ring, ^Y1 is C2-12 alkylene, and ^Y2 is selected from

n is 0 to 3, ^R4 is C1-15 alkyl, ^Z1 is C1-6 alkylene or a direct bond,

^Z2 is

(in either orientation) or absent, provided that if Z ¹ is a direct bond, then Z ² is absent;

^R5 is C5-9 alkyl or C6-10 alkoxy, ^R6 is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and ^R7 is H or Me, or a salt thereof, provided that if ^R3 and ^R2 are C2 alkyl, ^X1 is O, ^X2 is a straight chain C3 alkylene, ^X3 is C(=0), ^Y1 is a straight chain Ce alkylene, ( ^Y2 ) ^nR4 is

, ^R4 is a linear C5 alkyl, ^Z1 is a C2 alkylene, ^Z2 is absent, W is a methylene, and ^R7 is H, then ^R5 and ^R6 are not Cx alkoxy.

In some embodiments, LNPs comprising formula (xii) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

In some embodiments, LNPs comprising formula (xi) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

in

In some embodiments, the LNP comprises a compound of formula (xiii) and a compound of formula (xiv).

In some embodiments, LNPs comprising formula (xv) are used to deliver the gene modification compositions described herein to the liver and/or hepatocytes.

In some embodiments, LNPs comprising the formulation of formula (xvi) are used to deliver the gene modification compositions described herein to lung endothelial cells.

in

In some embodiments, lipid compounds used to form lipid nanoparticles for delivering a composition described herein, such as a nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene-modifying polypeptide) are prepared by one of the following reactions:

Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g. 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (D The phospholipids of the present invention are preferably phospholipids of the present invention, such as phospholipids, ... In certain embodiments, additional exemplary lipids include, but are not limited to, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, which is incorporated herein by reference. In some embodiments, such lipids include plant lipids (e.g., DGTS) found to improve liver transfection with mRNA. In some embodiments, the non-cationic lipid may have the following structure,

Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids, such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, ricinoleic acid glyceride, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, polyethoxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, etc. Other non-cationic lipids are described in WO2017/099823 or U.S. Patent Publication US 2018/0028664, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the non-cationic lipid is oleic acid or a compound of formula I, II or IV of US 2018/0028664, which is incorporated herein by reference in its entirety. The non-cationic lipid may account for, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5%-20% (mol) or 10%-15% (mol) of the total lipid present in the lipid nanoparticle. In an embodiment, the molar ratio of ionizable lipid to neutral lipid is about 2: 1 to about 8: 1 (e.g., about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1 or 8: 1).

In some embodiments, the lipid nanoparticles do not comprise any phospholipids.

In some aspects, the lipid nanoparticles may further include components such as sterols to provide membrane integrity. An exemplary sterol that can be used in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether and 6-ketocholestanol; non-polar analogs such as 5a-cholestanes, cholesterenone, 5a-cholestanone, 5p-cholestanone and cholesterol decanoate; and mixtures thereof. In some embodiments, cholesterol derivatives are polar analogs, for example, cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication WO 2009/127060 and U.S. Patent Publication US 2010/0130588, each of which is incorporated herein by reference in its entirety.

In some embodiments, components that provide membrane integrity, such as sterols, may account for 0-50% (mol) of the total lipid present in the lipid nanoparticle (e.g., 0-10%, 10%-20%, 20%-30%, 30%-40% or 40%-50%). In some embodiments, such components are 20%-50% (mol), 30%-40% (mol) of the total lipid content of the lipid nanoparticle.

In certain embodiments, lipid nanoparticles may include polyethylene glycol (PEG) or conjugated lipid molecules. Typically, these are used to suppress the aggregation of lipid nanoparticles and/or provide spatial stabilization. Exemplary conjugated lipids include but are not limited to PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates and mixtures thereof. In certain embodiments, conjugated lipid molecules are PEG-lipid conjugates, such as (methoxypolyethylene glycol) conjugated lipids.

Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG-2K), P PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US Pat. In some embodiments, the PEG-lipid is a compound of formula III, III-a-1, III-a-2, III-b-1, III-b-2 or V of US 2018/0028664, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-lipid has US 2018/0028664, the contents of which are incorporated herein by reference in their entirety. 20150376115 or Formula II of US 2016/0376224, the contents of both of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of the following: PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-distearylglycerol, PEG-dilaurylglyceramide, PEG- Dimyristylglyceramide, PEG-dipalmitoylglyceramide, PEG-distearylglyceramide, PEG-cholesterol (1-[8′-(cholest-5-ene-3[β]-oxy)formamido-3′,6′-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from the group consisting of:

In some embodiments, lipids conjugated to molecules other than PEG can also be used in place of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates) and cationic polymer lipid (GPL) conjugates can be used in place of PEG-lipids or together with PEG-lipids.

Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO 2019051289 A9 and WO 2020106946 A1, the contents of all of which are incorporated herein by reference in their entirety.

In some embodiments, the LNP comprises a compound of formula (xix), a compound of formula (xxi), and a compound of formula (xxv). In some embodiments, LNPs comprising formulations of formula (xix), formula (xxi), and formula (xxv) are used to deliver the gene modification compositions described herein to the lung or lung cells.

In some embodiments, the lipid nanoparticle may comprise one or more cationic lipids selected from formula (i), formula (ii), formula (iii), formula (vii) and formula (ix). In some embodiments, the LNP may further comprise one or more neutral lipids, such as DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, steroids, such as cholesterol, and/or one or more polymer-conjugated lipids, such as PEGylated lipids, such as PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or PEG dialkoxypropylcarbamate.

In some embodiments, PEG or conjugated lipids can account for 0-20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the content of PEG or conjugated lipids is 0.5%-10% or 2%-5% (mol) of the total lipids present in the lipid nanoparticles. The molar ratio of ionizable lipids, non-cationic lipids, sterols and PEG/conjugated lipids can be changed as needed. For example, lipid particles can include 30%-70% ionizable lipids by mole or total weight of the composition, 0-60% cholesterol by mole or total weight of the composition, 0-30% non-cationic lipids by mole or total weight of the composition and 1%-10% conjugated lipids by mole or total weight of the composition. Preferably, the composition includes 30%-40% ionizable lipids by mole or total weight of the composition, 40%-50% cholesterol by mole or total weight of the composition, and 10%-20% non-cationic lipids by mole or total weight of the composition. In some other embodiments, the composition is 50%-75% ionizable lipids by mole or total weight of the composition, 20%-40% cholesterol by mole or total weight of the composition, and 5% to 10% non-cationic lipids by mole or total weight of the composition, and 1%-10% conjugated lipids by mole or total weight of the composition. The composition may contain 60%-70% ionizable lipids by mole or total weight of the composition, 25%-35% cholesterol by mole or total weight of the composition, and 5%-10% non-cationic lipids by mole or total weight of the composition. The composition may also contain up to 90% ionizable lipids by mole or total weight of the composition and 2% to 15% non-cationic lipids by mole or total weight of the composition. The formulation can also be a lipid nanoparticle formulation, for example comprising 8%-30% ionizable lipid by mole or total weight of the composition, 5%-30% non-cationic lipid by mole or total weight of the composition, and 0-20% cholesterol by mole or total weight of the composition; 4%-25% ionizable lipid by mole or total weight of the composition, 4%-25% non-cationic lipid by mole or total weight of the composition, 2% to 25% cholesterol by mole or total weight of the composition, 10% to 35% conjugated lipid by mole or total weight of the composition, and 0% to 20% cholesterol by mole or total weight of the composition; 5% cholesterol; or 2%-30% ionizable lipids by mole or total weight of the composition, 2%-30% non-cationic lipids by mole or total weight of the composition, 1% to 15% cholesterol by mole or total weight of the composition, 2% to 35% conjugated lipids by mole or total weight of the composition, and 1%-20% cholesterol by mole or total weight of the composition; or even up to 90% ionizable lipids by mole or total weight of the composition and 2%-10% non-cationic lipids by mole or total weight of the composition, or even 100% cationic lipids by mole or total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipids, phospholipids, cholesterol and PEGylated lipids in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipids, cholesterol and PEGylated lipids in a molar ratio of 60:38.5:1.5.

In some embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, wherein the lipid molar ratio of the ionizable lipid is in the range of 20 to 70 mol%, with a target of 40-60 mol%, the molar percentage of the non-cationic lipid is in the range of 0 to 30 mol%, with a target of 0 to 15 mol%, the molar percentage of the sterol is in the range of 20 to 70 mol%, with a target of 30 to 50 mol%, and the molar percentage of the PEGylated lipid is in the range of 1 to 6 mol%, with a target of 2 to 5 mol%.

In some embodiments, the lipid particle comprises a molar ratio of ionizable lipid/non-cationic lipid/sterol/conjugated lipid of 50:10:38.5:1.5.

In one aspect, the present disclosure provides lipid nanoparticle formulations comprising phospholipids, phosphatidylcholine, phosphatidylcholine, and phosphatidylethanolamine.

In certain embodiments, one or more additional compounds may also be included. Those compounds may be administered alone, or additional compounds may be included in the lipid nanoparticles of the present invention. In other words, except nucleic acid or at least the second nucleic acid, the lipid nanoparticles may contain other compounds that are different from the first nucleic acid. In a non-limiting manner, other additional compounds may be selected from the group consisting of: small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptide mimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biomaterials, or any combination thereof.

In some embodiments, lipid nanoparticles (or formulations comprising lipid nanoparticles) lack reactive impurities (e.g., aldehydes or ketones), or include reactive impurities (e.g., aldehydes or ketones) below a preselected level. Although not wishing to be bound by theory, in some embodiments, lipid reagents are used to prepare lipid nanoparticle formulations, and lipid reagents may include contaminating reactive impurities (e.g., aldehydes or ketones). Lipid reagents for manufacture can be selected based on reactive impurities (e.g., aldehydes or ketones) below a preselected level. Not wishing to be bound by theory, in some embodiments, aldehydes may cause modification and damage to RNA, e.g., crosslinking between bases and/or covalent conjugation of lipids to RNA (e.g., forming lipid-RNA adducts). In some cases, this may result in reverse transcriptase reaction failure and/or, for example, incorporation of inappropriate bases at one or more sites of one or more lesions, such as mutations in newly synthesized target DNA.

In some embodiments, the lipid nanoparticle formulations are produced using lipid reagents comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of the total reactive impurity (e.g., aldehyde) content. In some embodiments, the lipid nanoparticle formulations are produced using lipid reagents comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, lipid nanoparticle formulations are produced using lipid reagents that include: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of total reactive impurities (e.g., aldehydes) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of any single reactive impurity (e.g., aldehyde) material. In some embodiments, lipid nanoparticle formulations are produced using a variety of lipid reagents, and each of the multiple lipid reagents independently meets one or more criteria described in this paragraph. In some embodiments, each of the multiple lipid reagents meets the same criteria, such as the criteria of this paragraph.

In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of the total reactive impurity (e.g., aldehyde) content. In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, the lipid nanoparticle formulation comprises: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.

In some embodiments, one or more or optionally all lipid reagents used for lipid nanoparticles or their formulations as described herein comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of total reactive impurity (e.g., aldehyde) content. In some embodiments, one or more or optionally all lipid reagents used for lipid nanoparticles or their formulations as described herein comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of any single reactive impurity (e.g., aldehyde) substance. In some embodiments, one or more or optionally all lipid agents used in the lipid nanoparticles or formulations thereof described herein comprise: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.

In some embodiments, the total aldehyde content and/or the amount of any single reactive impurity (e.g., aldehyde) species is determined by liquid chromatography (LC), e.g., coupled with tandem mass spectrometry (MS/MS), e.g., according to the method described in Example 40 of PCT/US21/20948. In some embodiments, the reactive impurity (e.g., aldehyde) content and/or the amount of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of nucleic acid molecules (e.g., RNA molecules, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in lipid reagents. In some embodiments, the reactive impurity (e.g., aldehyde) content and/or the amount of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of nucleotides or nucleosides (e.g., ribonucleotides or ribonucleosides, e.g., contained in or isolated from a template nucleic acid as described herein), e.g., in lipid reagents associated with the presence of reactive impurities (e.g., aldehydes), e.g., according to the method described in Example 41 of PCT/US21/20948. In an embodiment, chemical modification of a nucleic acid molecule, nucleotide or nucleoside is detected by assaying for the presence of one or more modified nucleotides or nucleosides, e.g., using LC-MS/MS analysis, e.g., according to the method described in Example 41 of PCT/US21/20948.

In some embodiments, the nucleic acids (e.g., RNA) described herein (e.g., template nucleic acids or nucleic acids encoding gene-modified polypeptides) do not comprise aldehyde modifications, or comprise less than a preselected amount of aldehyde modifications. In some embodiments, the nucleic acids have less than 50, 20, 10, 5, 2, or 1 aldehyde modifications per 1000 nucleotides on average, for example, where a single crosslink of two nucleotides is a single aldehyde modification. In some embodiments, the aldehyde modification is an RNA adduct (e.g., a lipid-RNA adduct). In some embodiments, the aldehyde-modified nucleotides are crosslinks between bases. In some embodiments, the nucleic acids (e.g., RNA) described herein comprise less than 50, 20, 10, 5, 2, or 1 crosslinks between nucleotides.

In some embodiments, LNPs are directed to specific tissues by adding targeting domains. For example, biological ligands can be displayed on the surface of LNPs to enhance interaction with cells displaying cognate receptors, thereby promoting association with tissues in which cells express receptors and cargo delivery therein. In some embodiments, the biological ligand can be a ligand that drives delivery to the liver, for example, LNPs displaying GalNAc promote delivery of nucleic acid cargo to hepatocytes displaying asialoglycoprotein receptors (ASGPR). The work of Akinc et al. Mol Ther [Molecular Therapy] 18(7): 1357-1364 (2010) teaches conjugating trivalent GalNAc ligands to PEG-lipids (GalNAc-PEG-DSG) to produce LNPs that rely on ASGPR to obtain observable LNP cargo effects (see, e.g., FIG. 6 therein). Other LNP formulations displaying ligands, such as those incorporating folic acid, transferrin, or antibodies, are discussed in WO 2017223135, which is incorporated herein by reference in its entirety, along with the references used therein: namely, Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; and Peer and Lieberman, Gene Ther. 2011 18:1127-1133.

In some embodiments, LNPs are selected for tissue-specific activity by adding Selective ORgan Targeting (SORT) molecules to formulations containing traditional components such as ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and poly(ethylene glycol) (PEG). The teachings of Cheng et al. Nat Nanotechnol 15(4): 313-320 (2020) demonstrate that the addition of supplemental "SORT" components can precisely alter the in vivo RNA delivery profile and mediate tissue-specific (e.g., lung, liver, spleen) gene delivery and editing based on the percentage and biophysical properties of the SORT molecules.

In certain embodiments, LNP comprises biodegradable ionizable lipids. In certain embodiments, LNP comprises (9Z, 12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecane-9,12-dienoate, also referred to as 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadecane-9,12-dienoate) or another ionizable lipid. See, e.g., WO 2019/067992, WO/2017/173054, WO 2015/095340 and WO 2014/136086, and the lipids of the references provided therein. In some embodiments, the terms cationic and ionizable are interchangeable in the context of LNP lipids, for example, where the ionizable lipid is cationic depending on pH.

In some embodiments, the LNPs described herein comprise the lipids described in Table 19.

Table 19: Exemplary lipids

In some embodiments, multiple components of the gene modification system can be prepared as a single LNP formulation, for example, the LNP formulation includes mRNA and RNA templates encoding gene modification polypeptides. The ratio of nucleic acid components can be changed to maximize the characteristics of the therapeutic agent. In some embodiments, the ratio of RNA template to mRNA encoding gene modification polypeptide is about 1: 1 to 100: 1 in molar ratio, for example, about 1: 1 to 20: 1, about 20: 1 to 40: 1, about 40: 1 to 60: 1, about 60: 1 to 80: 1 or about 80: 1 to 100: 1. In other embodiments, a system of multiple nucleic acids can be prepared by a separate formulation, for example, a LNP formulation comprising template RNA and a second LNP formulation comprising mRNA encoding gene modification polypeptides. In some embodiments, the system can include more than two nucleic acid components formulated into LNP. In some embodiments, the system can include a protein (e.g., gene modification polypeptide) and a template RNA formulated into at least one LNP formulation.

In some embodiments, the average LNP diameter of the LNP formulation can be between tens of nm and hundreds of nm, for example, as measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of LNP formulations may be from about 50nm to about 100nm, from about 50nm to about 90nm, from about 50nm to about 80nm, from about 50nm to about 70nm, from about 50nm to about 60nm, from about 60nm to about 100nm, from about 60nm to about 90nm, from about 60nm to about 80nm, from about 60nm to about 70nm, from about 70nm to about 100nm, from about 70nm to about 90nm, from about 70nm to about 80nm, from about 80nm to about 100nm, from about 80nm to about 90nm, or from about 90nm to about 100nm. In some embodiments, the average LNP diameter of LNP formulations may be from about 70nm to about 100nm. In specific embodiments, the average LNP diameter of LNP formulations may be about 80nm. In some embodiments, the average LNP diameter of LNP formulations may be about 100nm. In some embodiments, the LNP formulation has an average LNP diameter ranging from about 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

In some cases, LNP can be relatively homogeneous.Polydispersity index can be used for indicating the homogeneity of LNP, for example the size distribution of lipid nanoparticles.Small (for example, less than 0.3) polydispersity index usually indicates narrow size distribution.The polydispersity index of LNP can be about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25.In certain embodiments, the polydispersity index of LNP can be about 0.10 to about 0.20.

The zeta potential of LNP can be used to indicate the electrokinetic potential of the composition. In certain embodiments, the zeta potential can describe the surface charge of LNP. Lipid nanoparticles with relatively low charge (positive or negative charge) are generally desirable because higher charged materials may not interact with cells, tissues and other elements in the body undesirably. In some embodiments, the zeta potential of the LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

The encapsulation efficiency of protein and/or nucleic acid (e.g., genetically modified polypeptide or mRNA encoding the polypeptide) describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with LNP after preparation relative to the initial amount provided. Encapsulation efficiency is ideally higher (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after the lipid nanoparticles are broken with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence can be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For lipid nanoparticles described herein, the encapsulation efficiency of proteins and/or nucleic acids can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In some embodiments, the encapsulation efficiency can be at least 90%. In some embodiments, the encapsulation efficiency can be at least 95%.

The LNP may optionally include one or more layers of coating. In some embodiments, the LNP may be formulated in a capsule, film or tablet having a coating. The capsule, film or tablet comprising the composition described herein may have any available size, tensile strength, hardness or density.

Additional exemplary lipids, formulations, methods, and LNP characterizations are taught by WO 2020061457, which is incorporated herein by reference in its entirety.

In some embodiments, in vitro or ex vivo cell lipid transfection is performed using Lipofectamine MessengerMax (ThermoFisher) or TransIT-mRNA transfection reagent (Mirus Bio). In certain embodiments, LNPs are formulated using GenVoy_ILM ionizable lipid mixtures (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl [German Applied Chemistry] 51(34): 8529-8533 (2012), which is incorporated herein by reference in its entirety.

LNP formulations optimized for delivery of CRISPR-Cas systems (e.g., Cas9-gRNA RNPs, gRNA, Cas9 mRNA) are described in WO 2019067992 and WO 2019067910, both of which are incorporated by reference.

Additional specific LNP formulations useful for delivery of nucleic acids are described in US 8,158,601 and US 8,168,775, both of which are incorporated by reference, including the formulation sold under the name ONPATTRO used in patisiran.

Exemplary dosing of genetically modified LNPs may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg/kg (RNA). Exemplary dosing of AAVs comprising nucleic acids encoding one or more components of the system may include MOIs of about 10 ¹¹ , 10 ¹² , 10 ¹³ , and 10 ¹⁴ vg/kg.

Kits, preparations and pharmaceutical compositions

On the one hand, the present disclosure provides a kit comprising a genetically modified polypeptide or a genetically modified system, for example, as described herein. In some embodiments, the kit comprises a genetically modified polypeptide (or a nucleic acid encoding a polypeptide) and a template RNA (or a DNA encoding a template RNA). In some embodiments, the kit further comprises reagents for introducing the system into cells, such as transfection reagents, LNPs, etc. In some embodiments, the kit is suitable for any method described herein. In some embodiments, the kit comprises one or more elements, compositions (e.g., pharmaceutical compositions), genetically modified polypeptides and/or genetically modified systems, or functional fragments or components thereof, which are, for example, arranged in articles. In some embodiments, the kit comprises instructions for use thereof.

In one aspect, the disclosure provides an article of manufacture, eg, having disposed therein a kit described herein or components thereof.

In one aspect, the present disclosure provides a pharmaceutical composition comprising a genetically modified polypeptide or a genetically modified system, for example, as described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a template RNA and/or an RNA encoding a polypeptide. In an embodiment, the pharmaceutical composition has one or more (e.g., 1, 2, 3, or 4) of the following features:

(a) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) of DNA template relative to template RNA and/or RNA encoding a polypeptide, e.g., on a molar basis;

(b) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) uncapped RNA relative to template RNA and/or RNA encoding a polypeptide, e.g., on a molar basis;

(c) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) of partial-length RNA relative to the template RNA and/or RNA encoding the polypeptide, e.g., on a molar basis;

(d) Substantially lacking unreacted cap dinucleotide.

Chemistry, Manufacturing and Controls (CMC)

Purification of protein therapeutics is described, for example, in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).

In some embodiments, the genetic modification system, polypeptide and/or template nucleic acid (e.g., template RNA) meets certain quality standards. In some embodiments, the genetic modification system, polypeptide and/or template nucleic acid (e.g., template RNA) produced by the methods described herein meets certain quality standards. Therefore, in some aspects, the disclosure relates to methods for manufacturing genetic modification systems, polypeptides and/or template nucleic acids (e.g., template RNA) that meet certain quality standards, for example, wherein the quality standards have been determined. In some aspects, the disclosure also relates to methods for determining the quality standards in genetic modification systems, polypeptides and/or template nucleic acids (e.g., template RNA). In some embodiments, quality standards include, but are not limited to, one or more of the following (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 items):

(i) the length of the template RNA, e.g., whether the length of the template RNA is greater than or within a reference length, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the template RNAs present are greater than 100, 125, 150, 175 or 200 nucleotides in length;

(ii) the presence, absence and/or length of a poly A tail on the template RNA, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the template RNA present contains a poly A tail (e.g., a poly A tail of at least 5, 10, 20, 30, 50, 70, 100 nucleotides in length);

(iii) the presence, absence and/or type of a 5' cap on the template RNA, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the template RNAs present contain a 5' cap, e.g., whether the cap is a 7-methylguanosine cap, e.g., an O-Me-m7G cap;

(iv) the presence, absence and/or type of one or more modified nucleotides (e.g., selected from pseudouridine, dihydrouridine, inosine, 7-methylguanosine, 1-N-methylpseudouridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U), 5-methylcytidine (5mC) or locked nucleotide) in the template RNA, for example, whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the template RNA present contains one or more modified nucleotides;

(v) stability of the template RNA (e.g., over time and/or under pre-selected conditions), such as whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the template RNA remains intact (e.g., greater than 100, 125, 150, 175, or 200 nucleotides in length) after stability testing;

(vi) the efficacy of the template RNA in a system for modifying DNA, e.g., whether at least 1% of the target sites are modified after determining the efficacy of a system comprising the template RNA;

(vii) the length of the polypeptide, the first polypeptide, or the second polypeptide, for example, whether the length of the polypeptide, the first polypeptide, or the second polypeptide exceeds the reference length or is within the reference length range, for example, whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the polypeptides, the first polypeptide, or the second polypeptide are greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more in length. , 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids (and optionally, no more than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids in length);

(viii) the presence, absence and/or type of post-translational modification on the polypeptide, the first polypeptide or the second polypeptide, such as whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the polypeptide, the first polypeptide or the second polypeptide contains phosphorylation, methylation, acetylation, myristoylation, palmitoylation, prenylation, glipyatyon or fatty acylation, or any combination thereof;

(ix) the presence, absence and/or type of one or more artificial, synthetic or atypical amino acids (e.g., selected from ornithine, β-alanine, GABA, δ-aminolevulinic acid, PABA, D-amino acids (e.g., D-alanine or D-glutamic acid), aminoisobutyric acid, dehydroalanine, cystathionine, lanthionine, methionine, diaminopimelate, homoalanine, norvaline, norleucine, homonorleucine (Homonorleucine), homoserine, O-methyl-homoserine and O-ethyl-homoserine, ethionine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, tellurocysteine or telluromethionine) in the polypeptides, the first polypeptide or the second polypeptide, such as whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the polypeptides, the first polypeptide or the second polypeptide contain the one or more artificial, synthetic or atypical amino acids;

(x) the stability of the polypeptide, the first polypeptide, or the second polypeptide (e.g., over time and/or under preselected conditions), such as whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the polypeptide, the first polypeptide, or the second polypeptide remains intact after the stability test (e.g., a polypeptide greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 120 ... 0, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids in length (and optionally, no more than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids in length);

(xi) the efficacy of the polypeptide, the first polypeptide, or the second polypeptide in a system for modifying DNA, such as whether at least 1% of the target sites are modified after determining the efficacy of a system comprising the polypeptide, the first polypeptide, or the second polypeptide; or

(xii) the presence, absence, and/or level of one or more of pyrogens, viruses, fungi, bacterial pathogens, or host cell proteins, e.g., whether the system is free or substantially free of pyrogens, viruses, fungi, bacterial pathogens, or host cell protein contamination.

In some embodiments, the systems or pharmaceutical compositions described herein are free of endotoxin.

In some embodiments, the presence, absence, and/or level of one or more of a pyrogen, a virus, a fungus, a bacterial pathogen, and/or a host cell protein is determined. In embodiments, it is determined whether the system is free of or substantially free of pyrogen, virus, fungus, bacterial pathogen, and/or host cell protein contamination.

In some embodiments, a pharmaceutical composition or system as described herein has one or more (e.g., 1, 2, 3, or 4) of the following features:

(a) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) of DNA template relative to template RNA and/or RNA encoding a polypeptide, e.g., on a molar basis;

(b) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) uncapped RNA relative to template RNA and/or RNA encoding a polypeptide, e.g., on a molar basis;

(c) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) of partial-length RNA relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;

(d) Substantially lacking unreacted cap dinucleotide.

Examples

Example 1: Screening for template RNA conformations that correct sickle cell disease associated with mutations in the human cell genome landing pad

This example describes the use of a gene modification system to quantify the activity of a template RNA to correct the HBB:E6V mutation (also known as E7V or HbS variant; NC_000011.10:g.5227002T>A), the gene modification system comprising a gene modification polypeptide and a template RNA comprising a heterologous subject sequence and a PBS sequence of varying lengths. In this example, the template RNA comprises:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

One or more template RNAs described in Tables 1-4 can be tested as described in this example. The heterologous subject sequence and PBS sequence are designed to correct the SCD mutation in the landing pad by replacing the "A" nucleotide with a "T" nucleotide at the mutation site through gene editing to reverse the E6V mutation in the corresponding protein.

Cell lines are established with a "landing pad" or stable integrant that mimics the region of the HBB gene containing the E6V mutation site and flanking sequences. In some embodiments, the cell line used for screening may contain one or more additional SNPs in the HBB locus relative to a patient or reference sequence (e.g., hg38 human genome reference sequence), and the landing pad containing the target mutation is optionally designed to carry one or more non-pathogenic SNPs to match the endogenous cell line HBB locus, for example, designed to carry mutations that recapitulate SNPs present in the endogenous HBB locus in HEK293T cells. Without wishing to be limited by example, it should be understood that a template RNA sequence that is found to successfully edit a target mutation at a site containing an additional SNP relative to a reference sequence will be different from the therapeutic template RNA in any region overlapping with the additional SNP. For example, a successful template RNA for a HEK293T-based screening assay in which a genomic landing pad contains a target mutation (corresponding to an endogenous E6V mutation caused by the DNA substitution NC_000011.10:g.5227002T>A) and an additional substitution in the protospacer relative to hg38 (corresponding to the NC_000011.10:g.5227013T>C mutation at the endogenous HBB locus in HEK293T cells) can provide a candidate composition, wherein the corresponding therapeutic template RNA will therefore have a substitution (C>T) in the spacer region relative to the corresponding spacer region of the screening template RNA, so as to enable therapeutic correction of an E6V mutation at a target site that lacks the additional substitution, such as at a target site that contains a pathogenic E6V mutation but otherwise matches the hg38 reference sequence. In this example, a screening cell line containing a target site landing pad that contains a pathogenic mutation with an additional T>C substitution in the protospacer region may be used to contain a spacer sequence The screening template RNA is corrected, and the corresponding therapeutic template RNA may contain a spacer sequence Where underlined nucleotides indicate positions that were altered to match the screening cell target sequence or the hg38 target sequence. In some embodiments, the spacer, PBS, and/or RT template regions may need to be adjusted in this manner to account for any differences between the screening and reference target sequences. It is also contemplated that a given patient or patient population may have one or more SNPs at the target locus relative to hg38 in addition to the pathogenic E6V mutation, and thus similar adaptability of the candidate template RNA molecules may be used to generate template RNA sequences specific to the patient or patient population.

The DNA of the landing pad was chemically synthesized and cloned into the pLenti-N-tGFP vector. The landing pad sequence cloned into the lentiviral expression vector was confirmed and sequence-verified by Sanger sequencing of the landing pad. Sequence-verified plasmids (9 μg) and lentiviral packaging mixtures (9 μg, Bosetta) were transfected into the packaging cell line LentiX-293T (Takara Bio) using Lipofectamine2000 ^TM according to the manufacturer's instructions. The transfected cells were incubated at 37°C, 5% _CO2 for 48 hours (including changing the culture medium once every 24 hours), and then the culture medium containing viral particles was collected from the cell culture dish. The collected culture medium was filtered through a 0.2 μm filter to remove cell debris and prepare for transduction of HEK293T cells. The culture medium containing the virus was diluted in DMEM and mixed with polybrene to prepare a dilution series for transduction of HEK293T cells, wherein the final concentration of polybrene was 8 μg/ml. HEK293T cells were grown in virus-containing medium for 48 hours and then split with fresh medium. Split cells were grown to confluence and transduction efficiency of different virus dilutions was measured by detecting GFP expression of genomic integrated lentivirus (containing GFP and HBB:E6V landing pad) via flow cytometry and ddPCR.

A gene modification system comprising (i) a compatible gene modification polypeptide described herein, e.g., having: an NLS of Table 11, a compatible Cas9 domain having a sequence of Table 8, a linker of Table 10, an RT sequence of Table 6 (e.g., MLVMS_P03355_PLV919), and a second NLS of Table 11, and (ii) a template RNA of any one of Tables 1-4 is transfected into a HEK293T landing pad cell line. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 1 μg of gene modification polypeptide mRNA is combined with 10 μM template RNA. The mRNA and template RNA are added to 25 μL SF buffer containing 250,000 HEK293T landing pad cells, and the cells are nuclear transfected using program DS-150. After nuclear transfection, the cells are cultured at 37°C, 5% _CO2 for 3 days, followed by cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the HBB:E6V site were used to amplify across the locus. Amplicons were analyzed by short read sequencing using Illumina MiSeq. In some embodiments, the assay converts at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the copies of the HBB gene in the indicated sample to the desired wild-type sequence.

Example 2: Selection of gene-modified peptides by pooled screening in HEK293T and U20S cells

This example describes the use of an RNA gene modification system to target editing of coding sequences in the human genome. More specifically, this example describes infection of HEK293T and U2OS cells with a library of gene modification candidates, followed by transfection of template guide RNA (tgRNA) for in vitro gene modification in cells, e.g., as a means of evaluating the editing activity of new gene modification polypeptides in human cells by a pooled screening approach.

The gene modification polypeptide library candidates determined herein each contain: 1) a Streptococcus pyogenes (Spy) Cas9 nickase containing an N863A mutation, which inactivates an endonuclease active site; 2) one of the 122 peptide linkers described in Table 10; and 3) a reverse transcriptase (RT) domain from a retroviral source in Table 6. If the specific retroviral RT domain utilized is expected to function as a monomer, these domains are selected. For each selected RT domain, the wild-type sequence was tested, as well as a version in which a point mutation was installed in the main wild-type sequence. In particular, 143 RT domains were tested, either wild-type or containing various mutations. A total of 17,446 Cas-linker-RT gene modification polypeptides were tested.

The system described here is a two-component system comprising: 1) an expression plasmid encoding a human codon-optimized gene-modified polypeptide library candidate within a lentiviral cassette, 2) a tgRNA expression plasmid expressing a non-coding tgRNA sequence that is recognized by Cas and localized to the genomic locus of interest and also serves as a template for reverse transcription of the desired edit into the genome via an RT domain driven by a U6 promoter. The lentiviral cassette comprises: (i) a CMV promoter for expression in mammalian cells; (ii) a gene-modified polypeptide library candidate as indicated; (iii) a self-cleaving T2A polypeptide; (iv) a puromycin resistance gene that enables selection in mammalian cells; and (v) a poly A tail termination signal.

To prepare a cell pool expressing a candidate for a gene-modified polypeptide library, HEK293T or U2OS cells were transduced with a pooled lentiviral preparation of a candidate plasmid library for gene modification. HEK293 Lenti-X cells were seeded in 15 cm plates (12 x10 ⁶ cells) before lentiviral plasmid transfection. Lentiviral plasmid transfection was performed using a lentiviral packaging mixture (Bosetta, 27 ug), and plasmid DNA (27 ug) of the candidate library for gene modification was transfected using Lipofectamine 2000 and Opti-MEM medium on the second day according to the manufacturer's protocol. Extracellular DNA was removed by replacing the complete medium on the second day, and the virus-containing medium was harvested after 48 hours. The lentiviral culture medium was concentrated using Lenti-x concentrate (Takara Biosciences), and 5 mL lentiviral aliquots were prepared and stored at -80 °C. Lentiviral titer determination was performed by counting colony forming units after puromycin selection. HEK293T or U2OS cells carrying a genomic landing pad expressing BFP were seeded in culture plates at 6x107 cells and transduced at a multiplicity of infection (MOI) of 0.3 to minimize multiple infections per cell. Puromycin (2.5ug/mL) was added 48 hours after infection to select infected cells. Cells were maintained under puromycin selection for at least 7 days and then expanded for tgRNA electroporation.

To determine the genome editing capabilities of the gene modification library candidates in the assay, infected HEK293T or U2OS cells expressing BFP were then transfected by electroporation at 250,000 cells/well with 200 ng of tgRNA (g4 or g10) plasmid designed to convert BFP to GFP, with cell counts sufficient to achieve >1000x coverage for each library candidate.

The g4tgRNA (5′ to 3′) is as follows: a 20-nucleotide spacer region (GCCGAAGCACTGCACGCCGT; SEQ ID NO: 11,011), a scaffold region (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC; SEQ ID NO: 11,012), a template region encoding a single base pair substitution that changes BFP to GFP (bold) and a synonymous point mutation (NGG to NCG) introduced in the SpyCas9 PAM to prevent reengagement of the gene modification polypeptide after the functional gene modification reaction is completed (underlined). and a 13-nucleotide PBS (GCGTGCAGTGCTT; SEQ ID NO: 11,014).

Similarly, the g10 tgRNA (5′ to 3′) is as follows: a 20-nucleotide spacer region (AGAAGTCGTGCTGCTTCATG; SEQ ID NO: 11,015), a scaffold region (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC; SEQ ID NO: 11,016), a template region encoding a single base pair substitution that changes BFP to GFP (bold) and a synonymous point mutation (NGG to NGA) in the SpyCas9 PAM to prevent re-engagement of the functional gene modification polypeptide after completion of the gene modification reaction ( underlined ). and a 13-nucleotide PBS (GAAGCAGCACGAC; SEQ ID NO: 11,018).

To evaluate the genome editing capabilities of various constructs in the assay, cells were sorted by fluorescence activated cell sorting (FACS) 6-7 days after electroporation to detect GFP expression. Cells were sorted and harvested into different populations of unedited (BFP+) cells, edited (GFP+) cells, and imperfectly edited (BFP-, GFP-) cells. Unsorted cell samples were also harvested as input populations to determine enrichment during analysis.

To determine which gene modification library candidates have genome editing capabilities in the assay, genomic DNA (gDNA) was harvested from sorted and unsorted cell populations and analyzed by sequencing the gene modification library candidates in each population. Briefly, the gene modification sequence was amplified from the genome using specific primers for the lentiviral cassette, amplified in a second round of PCR to dilute the genomic DNA, and then sequenced using Oxford Nanopore Sequencing Technology according to the manufacturer's protocol.

After quality control of the sequencing reads, reads of at least 1500 and no more than 3200 nucleotides are mapped to the gene modification polypeptide library sequence, and those reads that match at least 80% of the library sequence are considered to have been successfully aligned with a given candidate. In order to identify gene modification candidates capable of gene editing in the assay, the read counts of each library candidate in the editing population are compared with the read counts in the initial unsorted population. For the purpose of this pooled screening, gene modification candidates with genome editing capabilities are selected as those candidates that are enriched in the transformed (GFP+) population relative to unsorted (input) cells, and where the enrichment is determined to be equal to or higher than the enrichment level of the reference (element ID number: 17380).

A large number of gene-modified polypeptide candidates were determined to be enriched in the GFP+ cell population. For example, of the 17,446 candidates tested, more than 3,300 showed enrichment in the GFP+ sorted population (relative to the unsorted population), which was at least comparable to the reference values under similar experimental conditions (HEK293T using g4 tgRNA; HEK293T cells using g10tgRNA; or U2OS cells using g4 tgRNA), as shown in Table D. Although 17,446 candidates were also tested in U2OS cells using g10tgRNA, under this experimental condition, the pooled screening did not generate candidates that were enriched in the transformed (GFP+) population relative to the unsorted (input) cells; further studies are needed to explain these results.

Table D. Combinations of linkers and RT sequences screened. The amino acid sequence of each RT in this table is provided in Table 6.

Example 3: Screening for template RNA configurations that install SCD mutations in the endogenous HBB gene in human cells

This example describes the use of a gene modification system containing a gene modification polypeptide and a template RNA to identify favorable configurations for editing the endogenous HBB gene in human cells, the template RNA comprising heterologous target sequences of varying lengths and primer binding site sequences. In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

The template RNA was designed to contain an 8-17nt PBS sequence and a 9-20nt heterologous subject sequence. Two different gRNA spacer sequences were used to target sites close to the SCD mutation in the endogenous HBB genomic site. The heterologous subject sequence and PBS sequence were designed to install the SCD mutation (E6V mutation) into the endogenous gene by replacing the "A" nucleotide with a "T" nucleotide at the mutation site using the gene modification system described herein. The template RNA sequences used were those shown in Table A (HBB5 sequence) and Table B (HBB8 sequence), except for the following. First, the mutation region of the RT template sequence was designed to install the mutation (A→T) rather than correcting back to the wild-type sequence. In particular, the RT template region for installing the SCD using template HBB5 comprises at least a portion of the following sequence: Installing RT Template (PAM-killing): AACGGCAGACTTCTCTACAG (SEQ ID NO: 21672), where the no PAM-killing equivalent would be: Installing RT Template (no PAM-killing): AACGGCAGACTTCTCCACAG (SEQ ID NO: 21673). Additionally, the installed version of the HBB8 spacer has the following sequence, which differs from the HBB8 mutation-correcting spacer due to the SCD mutation falling within the target protospacer, resulting in a single nt difference relative to the WT sequence without the SCD mutation: GTAACGGCAGACTTCTCCTC (SEQ ID NO: 21674). In particular, the RT template region for installing the SCD mutation using template HBB8 comprises at least a portion of the following sequence: Installation RT template (293T SNP): TGGTGCACCTGACTCCTGTG (SEQ ID NO: 21676), wherein the equivalent template lacking the 293T SNP and targeting the hg38 reference sequence would be: Installation RT template (no SNP): TGGTGCATCTGACTCCTGTG (SEQ ID NO: 21675).

A gene modification system comprising a gene modification polypeptide (see Table C) and a template RNA was transfected into HEK293T cells. The gene modification polypeptide and template RNA were delivered in RNA form by nuclear transfection. In particular, 1 μg of gene modification polypeptide mRNA was combined with 10 μM template RNA. mRNA and template RNA were added to 25 μL SF buffer containing 250,000 HEK293T cells, and the cells were nuclear transfected using program DS-150. After nuclear transfection, the cells were cultured at 37 ° C, 5% CO ₂ for 3 days, followed by cell lysis and genomic DNA extraction. In order to analyze gene editing activity, primers flanking the HBB genomic target site were used to amplify across the locus. The amplicons were analyzed by short read sequencing using Illumina MiSeq. Gene editing activity with high editing efficiency was detected in a configuration with a 9-12nt PBS sequence and a 13-16nt heterologous object sequence. These results indicate that the template RNA comprising the gRNA spacer and gRNA scaffold described herein successfully guides the gene-modifying polypeptide to the endogenous HBB gene in human cells for specific gene editing. The results are shown in Table E.

Although this experiment demonstrated installation of mutations rather than correction of mutations, it indicates that editing can be performed at the native HBB locus.

Table E. HBB5 and HBB8 sequences used to install mutations. The columns indicate from left to right: 1) the name of the HBB5 template RNA; 2) the full HBB5 template RNA sequence depicted as RNA, further showing chemical modifications as used in Example 3; 3) the observed activity of the template RNA as defined in Example 3 in column 2; 4) the name of the HBB8 template RNA; 5) the full HBB8 template RNA sequence depicted as RNA, further showing chemical modifications as used in Example 3; 6) the observed activity of the template RNA as defined in Example 3 in column 5.

Example 4: Screening for template RNA configurations that correct SCD mutations in human cell genome landing pads

This example describes the use of a gene modification system to identify favorable configurations for correcting SCD mutations, the gene modification system comprising a gene modification polypeptide and a template RNA comprising heterologous subject sequences and PBS sequences of varying lengths. In this example, the template RNA comprises:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

The template RNA was designed to contain an 8-17nt PBS sequence and a 9-20nt heterologous subject sequence (Tables A and B). Two different gRNA spacer sequences, designated HBB5 (see Table A) and HBB8 (see Table B), were used to target sites close to SCD mutations in conventional genomic landing pads in human cells. The heterologous subject sequence and PBS sequence were designed to correct the SCD mutation in the landing pad by replacing "T" nucleotides with "A" nucleotides at the mutation site using the gene modification system described herein.

Cell lines were established with a "landing pad" or stable integrant that mimics a region of the HBB gene containing sequences flanking the SCD mutation site. The DNA for the landing pad was chemically synthesized and cloned into the pLenti-N-tGFP vector. The landing pad cloned into the lentiviral expression vector was confirmed and the sequence was verified by Sanger sequencing of the landing pad. Sequence-verified plasmids (9 μg) and lentiviral packaging mix (9 μg, obtained from Bosetta) were transfected into the packaging cell line LentiX-293T (Takara Biotech Co., Ltd.) using Lipofectamine2000 ^TM according to the manufacturer's instructions. The transfected cells were incubated at 37°C, 5% _CO2 for 48 hours (including a medium change every 24 hours), and the medium containing viral particles was then collected from the cell culture dishes. The collected medium was filtered through a 0.2 μm filter to remove cell debris and prepared for transduction of HEK293T cells. The virus-containing medium was diluted in DMEM and mixed with polybrene to prepare a dilution series for transduction of HEK293T cells, wherein the final concentration of polybrene was 8 μg/ml. HEK293T cells were grown in medium containing virus for 48 hours and then split with fresh medium. The split cells were grown to confluence, and the transduction efficiency of different virus dilutions was measured by detecting GFP expression of genomic integrated lentivirus (containing GFP and HBB landing pad) via flow cytometry and ddPCR.

The gene modification system comprising a gene modification polypeptide (see Table C) and a template RNA was transfected into the HEK293T landing pad cell line. The gene modification polypeptide and the template RNA were delivered in RNA form by nuclear transfection. In particular, 1 μg of the gene modification polypeptide mRNA was combined with 10 μM template RNA. The mRNA and template RNA were added to 25 μL SF buffer containing 250,000 HEK293T landing pad cells, and the cells were nuclear transfected using program DS-150. After nuclear transfection, the cells were cultured at 37 ° C, 5% CO ₂ for 3 days, followed by cell lysis and genomic DNA extraction. In order to analyze gene editing activity, primers flanking the HBB genomic target site were used to amplify across the locus. The amplicons were analyzed by short read sequencing using Illumina MiSeq. Gene editing activity with high editing efficiency was detected in configurations with 9-12nt PBS sequences and 12-14nt heterologous object sequences, and is shown in Tables A and B. In particular, in Table A and Table B, "+" indicates an editing frequency of <3%, "++" indicates an editing frequency of 3%-7%, and "+++" indicates an editing frequency of >=7%.

It should be understood that the template RNA sequences shown in Table A can be customized according to the cells being targeted. For example, HEK293T cells have a SNP in the HBB gene (NC_000011.10: g.5227013A>G (T>C in the HBB coding strand), relative to the human hg38 reference genome), and therefore the template RNA sequences shown in Tables A and B are suitable for cells with this SNP. Template RNAs suitable for cells with different sequences at the SNP position ("no SNP") can utilize the following sequences, where capital letters indicate core sequences and lowercase letters indicate flanking sequences, and underscores indicate mutation regions. Similarly, in some embodiments, it is desired to inactivate the PAM sequence ("PAM-killing") upon editing, and in other embodiments, it is preferred to keep the PAM sequence intact (no "PAM-killing"). The RT template can be designed, for example, as shown below in a "PAM-killing" or "no PAM-killing" form.

HBB5 spacer (no SNP): CATGGTGCATCTGACTCCTG (SEQ ID NO: 21668)

HBB5 PBS (no SNP):GAGTCAGAtgcaccatg (SEQ ID NO: 21669)

HBB5 RT template (no PAM-killing): aacggcagactTCTCG T CAG (SEQ ID NO: 21670)

HBB8 RT template (no SNP): tggtgcatctgACTCCTG A G (SEQ ID NO: 21671)

Example 5: Quantifying the activity of gene-edited peptides and templates rewriting the endogenous B-globulin locus in 293T cells and CD34+ primary human hematopoietic stem cells (HSCs)

This example demonstrates the use of a gene modification system containing a gene modification polypeptide and a template RNA to convert the glutamic acid codon (GAG) at amino acid position 7 in the endogenous B-globin locus in primary human HSCs to alanine (GCA), thereby rewriting a non-pathogenic sequence to position 7. The conversion involves a change of two base pairs (i.e., replacing the DNA bases adenine and guanine at nucleotide positions 20 and 21 with the bases cytosine and adenine, respectively).

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the template RNA comprises the following sequence from 5' to 3', wherein the first 3 and last 3 bases have 2'-O-methyl phosphorothioate chemical modifications.

FYF tgRNA11

FYF tgRNA12

FYF tgRNA13

FYF tgRNA14

FYF tgRNA15

FYF tgRNA16

FYF tgRNA17

FYF tgRNA18

FYF tgRNA19

FYF tgRNA20

The genetically modified polypeptide tested comprised the sequence labeled RNAV209 described in Example 8.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into 293T cells and human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 1000 or 2000ng gene modification polypeptide RNA and 1000 or 2000ng template RNA are all combined in RNA form at a 1:1 ratio. The RNA mixture is added to 200,000 293T cells or 200,000 primary human HSC in a total of 20 μL of Lonza SF buffer (293T) or Lonza P3 buffer (HSC), and cells are nuclear transfected in 16-well nuclear transfection boxes using program DS-150 (293T) or DZ-100 (HSC). After nuclear transfection, the cells were incubated at room temperature for 10 minutes and transferred to 24-well plates containing 500 μL DMEM + 10% fetal bovine serum (293T) or 500 μL StemSpan-XF + SCF, 100 ng / mL Flt3-L and 100 ng / mL TPO (HSC) in each well, and cultured at 37 ° C, 5% CO ₂ for 3 days, followed by cell lysis and genomic DNA extraction. In order to analyze gene editing activity, primers flanking the locus of the target insertion site were used to amplify across the locus. The amplicons were analyzed by short read sequencing using Illumina MiSeq. The DNA bases adenine and guanine at nucleotide positions 20 and 21 were replaced with bases cytosine and adenine, respectively, downstream of the transcription start site within the endogenous B-globulin locus to indicate successful editing.

The tested gene modification system achieves up to 18.5% average perfect rewriting in 293T cells and achieves up to 7.9% perfect rewriting in primary human HSC. As shown in Figure 2, when screened with template gRNA, the average perfect rewriting level detected in 293T cells (single incision under 2000ng/RNA) is 4%-18% and 0%-2.5% in primary human HSC (single incision under 2000ng/RNA). As shown in Figure 3, using the gRNA shown, the average perfect rewriting level detected in 293T cells (single incision under 2000ng/RNA) is 6%-18.5% and 0%-7.9% in primary human HSC (single incision under 2000ng/RNA). These results show that the non-pathogenic sequence is rewritten to the clinically relevant codons in the endogenous B-globulin locus in primary human HSC using the gene modification system.

Example 6: Quantifying the activity of gene-editing polypeptides and templates rewriting the endogenous B-globin locus in primary human fibroblasts

This example demonstrates the use of a gene modification system containing a gene modification polypeptide and a template RNA to convert the glutamic acid codon (GAG) at amino acid position 7 in the endogenous B-globin locus in human primary fibroblasts to alanine (GCA). The conversion involves a change of two base pairs (i.e., replacing the DNA bases adenine and guanine at nucleotide positions 20 and 21 with the bases cytosine and adenine, respectively).

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the template RNA comprises the sequence of tgRNA14 as described in the previous examples.

The genetically modified polypeptide tested comprised the sequence labeled RNAV209 described in Example 8.

The system further comprises a gRNA sequence designed to generate a second nick, wherein the first 3 bases and the last 3 bases have a 2′-O-methyl phosphorothioate modification and comprise the following sequence: 5′-CCUUGAUACCAACCUGCCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3′ (SEQ ID NO: 20613).

The gene modification system comprising the gene modification polypeptide described above and template RNA is electroporated into human primary fibroblasts.Gene modification polypeptide and template RNA are delivered in RNA form by electroporation and include the sequence described in detail above.In particular, two doses (1000ng and 2000ng) are delivered, wherein each gene editing component is delivered as RNA with a specified dose.For example, for a 1000ng dosage, 1000ng gene modification polypeptide RNA is combined with 1000ng template RNA in RNA form and 1000ng second nick gRNA in RNA form in 10 μL electroporation mixtures comprising 200,000 primary human fibroblasts resuspended in buffer R (Invitrogen).The electroporation mixture is then aspirated into 10 μL neon electroporation tips (Invitrogen), transferred to neon electroporation system (Invitrogen), and electroporated with a pulse 20mS at 1700mV. The cells were then transferred to a well of a 12-well plate (Corning), cultured in 1 mL of Glutamax containing DMEM (supplemented with 15% fetal bovine serum, 1% non-essential amino acids, 1% sodium pyruvate, and 1% HEPES) (all from Gibco), and cultured at 37 ° C, 5% CO ₂ for 3 days, followed by cell lysis and genomic DNA extraction. In order to analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Illumina MiSeq was used to analyze amplicons by short read sequencing. The DNA bases adenine and guanine at nucleotide positions 20 and 21 were replaced with bases cytosine and adenine, respectively, downstream of the transcription start site in the endogenous B-globulin locus to indicate successful editing.

As shown in Figure 4, when the gene editing polypeptide is combined with the template guide RNA and the second nick gRNA is not added, the perfect rewriting level is 3.7% and 10.6% at 1000ng and 2000ng doses, respectively. The addition of the second nick increases the perfect rewriting from 3.7% to 44.5% at a dose of 1000ng and from 10.6% to 56.5% at a dose of 2000ng. In this experiment, insertion and deletion levels in the range of 1.5%-1.65% (single nick; 1000ng, 2000ng) and 7.9%-5.8% (second nick; 1000ng, 2000ng) were observed. These results show that the non-pathogenic sequence is rewritten into the clinically relevant codons in the endogenous B-globulin locus in human primary fibroblasts using the gene modification system. In addition, the introduction of the second nick gRNA increases the perfect rewriting by five to ten times, depending on the dose administered.

Example 7: Comparison of the activity of gene editing polypeptides and multiple templates to rewrite different sequences to the same position within the endogenous B-globin locus in wild-type human primary fibroblasts and fibroblasts containing a sickle cell mutation.

This example demonstrates similar efficacy when different mutations are installed into the same genomic locus by changing the sequence within the reverse transcriptase (RT) domain of the template guide RNA and keeping the design of the gene-modified polypeptide, template RNA, primer binding site (PBS), and template guide RNA scaffold unchanged. In this example, two adjacent DNA bases, one of which is located at the sickle cell disease mutation site within the B-globin locus, are replaced in wild-type fibroblasts, thereby converting the glutamate codon (GAG) at amino acid position 7 in the endogenous B-globin locus in human primary fibroblasts to alanine (GCA). At the same time, the valine codon (GTG) present in fibroblasts containing the sickle mutation is converted to a synonymous glutamate codon (GAA) at the same amino acid position.

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the template RNA used in wild-type fibroblasts comprised the sequence of tgRNA14 as described in the previous examples.

The template RNA used in sickle fibroblasts contained the following sequence and contained 2'-O-methyl phosphorothioate modifications at the first 3 and last 3 bases: 5'-CAUGGUGCACCUGACUCCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCAGACUUCUCUUCAGGAGUCAGGUG-3' (SEQ ID NO: 20614)

The genetically modified polypeptide tested comprised the sequence labeled RNAV209 described in Example 8.

The system further comprises a gRNA sequence designed to generate a second nick, wherein the gRNA has the following sequence: 5'-CCUUGAUACCAACCUGCCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO: 20615).

In this example, the same gRNA comprising the above sequence was used for both wild-type and sickle cell second incision conditions.

The gene modification system comprising the gene modification polypeptide and template RNA described above is electroporated into wild-type and human primary fibroblasts containing sickle mutations. The gene modification polypeptide and template RNA are delivered in RNA form by electroporation and include the sequence described in detail above. A dose (1000ng) is delivered, wherein each gene editing component is delivered as RNA at a specified dose. In particular, 1000ng of gene modification polypeptide RNA is combined with 1000ng of template RNA in RNA form and 1000ng of second nick gRNA in RNA form in a 10 μL electroporation mixture comprising 200,000 primary human fibroblasts resuspended in buffer R (Invitrogen). The electroporation mixture is then aspirated into 10 μL neon electroporation tips (Invitrogen), transferred to a neon electroporation system (Invitrogen), and electroporated with a pulse at 1700mV for 20mS. The cells were then transferred to a well of a 12-well plate (Corning), cultured in 1 mL of Glutamax containing DMEM (supplemented with 15% fetal bovine serum, 1% non-essential amino acids, 1% sodium pyruvate, and 1% HEPES) (all from Gibco), and cultured at 37 ° C, 5% CO ₂ for 3 days, followed by cell lysis and genomic DNA extraction. In order to analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. The amplicons were analyzed by Sanger sequencing and then analyzed using the TIDER algorithm. The DNA bases adenine and guanine at nucleotide positions 20 and 21 were replaced with bases cytosine and adenine, respectively, downstream of the transcription start site in the endogenous B-globulin locus, indicating successful editing in wild-type cells. In contrast, the DNA bases thymine and guanine at nucleotide positions 20 and 21 were replaced with bases adenine, respectively, downstream of the transcription start site in the endogenous B-globulin locus, indicating successful editing in fibroblasts containing sickle mutations.

As shown in Figure 5, when the gene editing polypeptide was combined with the template guide RNA, perfect rewriting levels of 10.8% and 6.1% were detected in wild-type and sickle fibroblasts, respectively. Adding a second nick increased perfect rewriting to 75.6% in wild-type cells and 74.6% in sickle fibroblasts. These results indicate the use of a gene modification system to correct pathogenic mutations in human primary fibroblasts with sickle mutations and install non-pathogenic mutations into wild-type human primary fibroblasts. In addition, the introduction of a second nick gRNA increased perfect rewriting by more than 7-fold in wild-type primary fibroblasts and more than ten-fold in sickle primary fibroblasts.

Example 8: Quantifying the activity of gene-edited peptides and templates rewriting the endogenous FAH locus in primary mouse hepatocytes

This example demonstrates the use of a gene modification system comprising a gene modification polypeptide and a template RNA to convert A nucleotides in the endogenous Fah locus in mouse primary hepatocytes derived from Fah5981SB mice to G nucleotides. The Fah5981SB mouse model contains a G to A point mutation in the last nucleotide of exon 8 of the Fah gene, resulting in aberrant mRNA splicing and subsequent mRNA degradation without the production of Fah protein, and thus can be used as a mouse model for hereditary tyrosinemia type I.

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the template RNA (including chemical modification patterns) comprises the following sequence:

FAH1_R14_P12 heavy RNACS048

FAH1_R15_P10_Heavy RNACS049

FAH2_R19_P11_MUT_Heavy RNACS052

FAH2_R19_P13_MUT_Heavy RNACS053

Other exemplary template RNAs that can be used in this experiment include the following:

FAH1 RNACS050

FAH1 RNACS051

In the above sequence, m = 2'-O-methyl ribonucleotide, r = ribose, * = phosphorothioate bond.

The gene-modifying polypeptides tested include the following sequences: RNAV209 (nCas9-RT) and RNAV214 (wtCas9-RT). In particular, nCas9-RT and wtCas9-RT have the following amino acid sequences:

nCas9-RT(RNAV209):

wtCas9-RT(RNAV214):

Underlining indicates residues that differ between the nickase and wild-type sequences.

The gene modification system comprising the gene modification polypeptide listed above and the above-mentioned template RNA is transfected into mouse primary hepatocytes. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 4 μg of gene modification polypeptide mRNA is combined with 10 μg of chemically synthesized template RNA in 5 μL of water. The transfection mixture is added to 100,000 mouse primary hepatocytes in buffer P3 [Lonza], and then the cells are nuclear transfected using program DG-138. After nuclear transfection, the cells are cultured at 37 ° C, 5% CO ₂ for 3 days, followed by cell lysis and genomic DNA extraction. In order to analyze gene editing activity, primers flanking the target insertion site locus are used to amplify across the locus. Illumina MiSeq is used to analyze the amplicon by short read sequencing. The conversion of the A to G sequence at the end of exon 8 of the fah gene indicates successful editing.

As shown in Figure 2, for the FAH2 template, a perfect rewriting level of 4%-8% (conversion of A to G, no unwanted mutations detected) was detected using RNAV209, but not using RNAV214-040. Indel levels of 4.4% to 6.6% were observed using RNAV209. In addition, the amount of WT FahmRNA was measured by quantitative RT-PCR using primers that bind to exons 7 and 8. As shown in Figure 3, when the FAH2 template was tested using RNAV209-013mRNA, the FAH2 template increased the abundance of Fah mRNA by up to 12% relative to WT. These results indicate that the use of a gene modification system to reverse mutations in the Fah gene resulted in partial restoration of wild-type Fah mRNA expression.

Example 9: Quantification of the activity of in vivo gene-editing polypeptides and templates rewriting the endogenous FAH locus in mouse liver

This example demonstrates the use of a gene modification system comprising a gene modification polypeptide and a template RNA to convert A nucleotides to G nucleotides in the endogenous Fah locus in mouse liver in the Fah5981SB mouse model. The Fah5981SB mouse model contains a G to A point mutation in the last nucleotide of exon 8 of the Fah gene, resulting in aberrant mRNA splicing and subsequent mRNA degradation without production of Fah protein, and serves as a mouse model for hereditary tyrosinemia type I.

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the template RNA comprises the following sequence:

FAH1_R14_P12 heavy RNACS048

FAH1_R15_P10_Heavy RNACS049

FAH2_R19_P11_MUT_Heavy RNACS052

FAH2_R19_P13_MUT_Heavy RNACS053

The genetically modified polypeptides tested included the following sequences: RNAV209 and RNAV214, each of which sequences is provided in Example 3.

The gene modification system comprising the above-mentioned gene modification polypeptide and template RNA is formulated in LNP and delivered to mice. In particular, 2 mg/kg total RNA equivalents (combined with template RNA and mRNA at 1:1 (w/w)) formulated in LNP were intravenously administered to 7-9 week-old, male and female mixed Fah5981SB mice. Six hours or 6 days after administration, the animals were killed and their livers were collected for analysis. In order to determine the expression distribution of gene modification polypeptides in the liver, 6-hour liver samples were subjected to immunohistochemical analysis using anti-Cas9 antibodies. After staining, the quantification of Cas9-positive hepatocytes was determined by QuPath Markup. As shown in Figure 4, the expression of gene modification polypeptides was observed in 82%-91% of hepatocytes.

To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify the loci of genomic DNA from liver samples collected 6 days after administration. The amplicon was analyzed by short read sequencing using Illumina MiSeq. The conversion of A nucleotides to G nucleotides indicated successful editing. As shown in Figure 5, a perfect rewrite level of 0.1%-1.9% (conversion of A to G, no unwanted mutations were detected) was detected in different groups. The level of indels was in the range of 0.2%-0.4%.

To determine the phenotypic correction caused by gene editing activity, the restoration of wild-type FAH mRNA was determined by real-time qRT-PCR, and the restoration of Fah protein expression was determined by immunohistochemical analysis using anti-Fah antibodies. As shown in Figure 6, 0.1%-6% wild-type mRNA recovery was detected in different groups relative to heterozygous littermates. As shown in Figure 7, Fah protein was detected in 0.1%-7% of the liver cross-sectional area in different groups. These results indicate that reversal of the Fah gene mutation using the gene modification system in the in vivo mouse model of hereditary tyrosinemia type I resulted in partial restoration of wild-type Fah mRNA and Fah protein expression.

Example 10. Gene Editing at the TTR Locus in an In Vivo Mouse Model

This example demonstrates the successful delivery of mRNA and guides using Cas9-mediated gene editing in C57Blk/6 mice using the protospacer sequence ACACAAAUACCAGUCCAGCG targeting the TTR locus using gene modifying polypeptides and RNA.

RNA was prepared as follows. An mRNA encoding a gene-modified polypeptide having a sequence shown in Table 10A below was produced by in vitro transcription, and the purified mRNA was dissolved in 1 mM sodium citrate (pH 6) to a final concentration of 1-2 mg/mL of RNA. Similarly, a guide RNA having a sequence shown in Table 10A below was produced by chemical synthesis and dissolved in water or an aqueous buffer to a final concentration of 1-2 mg/mL of RNA.

Table 10A. Sequences of Example 10

Lipid nanoparticle (LNP) components (ionizable lipids, helper lipids, sterols, PEG) were dissolved in 100% ethanol along with lipid components at a molar ratio of 47:8:43.5:1.5, respectively. RNA (guide and mRNA) were combined at a weight ratio of 1:1 and diluted to a concentration of 0.05-0.2 mg/mL in sodium acetate buffer at pH 5. RNA was formulated into different LNPs with a molar ratio of lipid amine to total RNA phosphate (N:P) of 4.0. LNPs were formed by microfluidic or turbulent mixing of lipid and RNA solutions. During the mixing process using different flow rates, the ratio of aqueous solvent to organic solvent was maintained at 3:1. After mixing, the LNPs were diluted, collected, and buffer exchanged to 50 mM Tris, 9% sucrose buffer using tangential flow filtration. The formulation was concentrated to 1.0 mg/mL or higher and then filtered through a 0.2 μm sterile filter. The final LNPs were stored at -80°C until further use.

The LNP formulation was delivered intravenously to C57Blk/6 mice of about 8 weeks of age at a concentration of 1-0.1 mg/kg by tail vein bolus injection. The animals were euthanized 6 hours after injection and the liver was collected at autopsy to measure the expression of Cas9-RT. The animals were euthanized 5 days after injection, and the liver was collected at autopsy to evaluate the gene editing activity of the TTR locus. The expression of Cas9-RT gene editing polypeptide in the liver was measured by Western blotting, wherein Cas9 was detected by mouse monoclonal antibody (7A9-3A3, Cell Signaling Technology), and GAPDH (Cell Signaling Technology) was used as a load control. (Figure 12). The editing of the TTR locus was quantified by Sanger sequencing, and then the amplicon of the TTR locus near the original spacer binding site was subjected to TIDE analysis. The editing of the TTR locus was observed, as shown in Figure 13. The TTR protein level in serum was quantified by ELISA using a standard curve (Aviva Biosciences). As shown in Figure 14, the TTR protein level in the serum of the treated animals was reduced. These experiments show that the Cas9-RT polypeptide can be expressed in vivo and can edit the TTR locus, resulting in a decrease in the TTR protein level in the serum.

Example 11. Gene Editing at the TTR Locus in an In Vivo Cynomolgus Monkey Model

This example demonstrates successful delivery of mRNA and guides using Cas9-mediated gene editing in a cynomolgus macaque model using the protospacer sequence ACACAAAUACCAGUCCAGCG (SEQ ID NO: 20630) targeting the TTR locus, using gene modifying polypeptides and RNA.

RNA was prepared as follows. mRNA encoding a gene-modified polypeptide having a sequence shown in Table 11A below was produced by in vitro transcription, and the purified mRNA was dissolved in 1 mM sodium citrate (pH 6) to a final concentration of 1-2 mg/mL of RNA. Similarly, a guide RNA having a sequence shown in Table 11A below was produced by chemical synthesis and dissolved in water or an aqueous buffer to a final concentration of 1-2 mg/mL of RNA.

Table 11A. Sequences of Example 11

Lipid nanoparticle (LNP) components (ionizable lipids, helper lipids, sterols, PEG) were dissolved in 100% ethanol along with lipid components at a molar ratio of 47:8:43.5:1.5, respectively. RNA (guide and mRNA) were combined at a weight ratio of 1:1 and diluted to a concentration of 0.05-0.2 mg/mL in sodium acetate buffer at pH 5. RNA was formulated into different LNPs with a molar ratio of lipid amine to total RNA phosphate (N:P) of 4.0. LNPs were formed by microfluidic or turbulent mixing of lipid and RNA solutions. During the mixing process using different flow rates, the ratio of aqueous solvent to organic solvent was maintained at 3:1. After mixing, the LNPs were diluted, collected, and buffer exchanged to 50 mM Tris, 9% sucrose buffer using tangential flow filtration. The formulation was concentrated to 1.0 mg/mL or higher and then filtered through a 0.2 μm sterile filter. The final LNPs were stored at -80°C until further use. LNP formulations were delivered intravenously at 2 mg/kg within 1 hour by infusion, with an infusion volume of 5 ml/kg. Macaques from mainland Asia were given dexamethasone 2 mg/kg bolus by intramuscular injection 1.5-2 h before intravenous infusion using a syringe pump. The animals were monitored after infusion, and the expression of Cas9-RT was measured by laparoscopic biopsies collected from the liver 8-12 h, 24 h, and 48 h after infusion. The animals were euthanized 14 days after infusion, and the liver was harvested by dividing the organ into 8 different fragments to evaluate the gene editing activity of the TTR locus. The expression of Cas9-RT gene editing polypeptides in the liver was quantified by capillary electrophoresis western blotting using the ProteinSimple Jess system (bio-techne company), wherein Cas9 was detected by mouse monoclonal antibodies (7A9-3A3, Cell Signaling Technology Company).

The relative expression of Cas9-RT gene editing polypeptides was measured by area under the curve analysis, as shown in Figure 15. Editing of the TTR locus was quantified by sequencing the amplicon of the TTR locus near the protospacer binding site. Editing of the TTR locus was observed, as shown in Figure 16. These experiments demonstrate that Cas9-RT polypeptides can be expressed in vivo and can edit the TTR locus in a non-human primate model.

Example 12: Quantifying the activity of gene-modified polypeptides and template RNA editing of the endogenous B-globin locus in CD34+ primary human hematopoietic stem cells (HSCs)

This example demonstrates the use of a gene modification system containing an exemplary gene modification polypeptide and a template RNA to convert a glutamic acid codon (GAG) at amino acid position 7 in the endogenous B-globin locus in primary human HSCs to alanine (GCG), thereby demonstrating targeting a sequence position associated with sickle cell disease (SCD) and editing the sequence to encode a non-pathogenic amino acid at position 7. The "C" residue installed by this process is referred to as the "Makassar" variant and is a non-pathogenic sequence variant that occurs in the human population. The conversion comprises a one base pair change (i.e., replacing the DNA base adenine at nucleotide position 20 with the base cytosine in SEQ ID NO: 20633).

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

An exemplary template RNA comprises the following sequence from 5' to 3', wherein the first 3 and last 3 bases have 2'-O-methyl phosphorothioate chemical modifications as indicated. In the sequence below, m = 2'-O-methyl ribonucleotide, r = ribose, * = phosphorothioate bond.

Unmodified versions of these sequences are shown below in Table BB. In some embodiments, the sequences used in this table may be used without chemical modification.

Table BB. tg34, tg35 and tg36 without nucleotide modifications.

The genetically modified polypeptide tested comprised the sequence labeled RNAV209 described in Example 8.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 2000ng of mRNA encoding the gene modification polypeptide is combined with 2000ng of template RNA. The RNA mixture is added to 200,000 primary human HSCs in a total of 20 μL of Lonza P3 buffer, and HSCs are nuclear transfected in a 16-well nuclear transfection box using program DZ-100. After nuclear transfection, the cells are incubated at room temperature for 10 minutes and transferred to a 24-well plate containing 500 μL StemSpan-XF+SCF of 100ng/mL, Flt3-L of 100ng/mL, and TPO of 100ng/mL in each well, and cultured for 3 days at 37°C, 5% _CO2 , followed by cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Amplicons were analyzed by short read sequencing using Illumina MiSeq. Replacement of the DNA base adenine at nucleotide position 20 with the base cytosine downstream of the transcription start site within the endogenous B-globin locus indicated successful editing.

As shown in Figure 17, when primary human HSCs were treated with exemplary template gRNAs and mRNAs encoding exemplary gene-modified polypeptides, an average perfect rewriting level of 1.3% to 1.8% was detected in primary human HSCs, corresponding to replacement of C nucleotides with A at SCD codons. These results indicate that non-pathogenic sequences were edited into clinically relevant codons in the endogenous B-globulin locus in primary human HSCs using the gene modification system. The results further indicate that several exemplary template RNAs can be used to achieve the desired editing.

Example 13: Comparison of the activity of different second-strand targeting gRNAs in combination with gene-modifying polypeptides and template RNAs in editing the endogenous B-globulin locus in CD34+ primary human hematopoietic stem cells (HSCs).

This example demonstrates the use of an exemplary gene modification system containing a gene modification polypeptide, a template RNA, and one of several different exemplary second strand targeting gRNAs to convert a glutamic acid codon (GAG) at amino acid position 7 in an endogenous B-globin locus in primary human HSCs to an alanine (GCA or GCG), thereby demonstrating targeting of a sequence position associated with sickle cell disease (SCD) and editing the sequence to encode a non-pathogenic amino acid at position 7. The conversion includes two base pair changes of an exemplary template RNA comprising an exemplary HBB5 spacer (i.e., DNA bases adenine and guanine at nucleotide positions 20 and 21 are replaced with bases cytosine and adenine, respectively); and one base pair change of an exemplary template RNA comprising an exemplary HBB8 spacer (i.e., DNA base adenine at nucleotide position 20 is replaced with base cytosine).

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

The template RNA comprises the nucleic acid sequence described in Example 5, labeled as FYF tgRNA14 for the exemplary HBB5 template RNA or as tg34 for the exemplary HBB8 template RNA, respectively.

The genetically modified polypeptide comprises the amino acid sequence labeled RNAV209 described in Example 8.

The second strand targeting gRNA sequences designed to produce the second nick include the sequences listed in Table X1.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 3000ng of mRNA encoding the gene modification polypeptide is combined with 2000ng of template RNA and 2000ng (for a system comprising HBB5 template RNA) or 3000ng (for a system comprising HBB8 template RNA) of the second strand targeting gRNA. The RNA mixture is added to 200,000 primary human HSCs in a total of 20 μL of Lonza P3 buffer, and HSCs are nuclear transfected in a 16-well nuclear transfection box using program DZ-100. After nuclear transfection, the cells were incubated at room temperature for 10 minutes and transferred to a 24-well plate containing 500 μL StemSpan-XF+SCF, 100 ng/mL Flt3-L, and 100 ng/mL TPO in each well, and cultured at 37 ° C, 5% CO ₂ for 3 days, followed by cell lysis and genomic DNA extraction. In order to analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. The amplicons were analyzed by short read sequencing using Illumina MiSeq. The DNA bases adenine and guanine at nucleotide positions 20 and 21 were replaced with bases cytosine and adenine (HBB5 template RNA) or the DNA base adenine at nucleotide position 20 was replaced with base cytosine (HBB8 template RNA) downstream of the transcription start site within the endogenous B-globulin locus, indicating successful editing.

As shown in Figure 18A, when HSCs were treated with an exemplary gene modification system comprising an exemplary HBB5 template RNA tg14 and various second-strand targeting gRNAs, an average perfect rewriting level of 4.5%-21.3% was detected in primary human HSCs, corresponding to the replacement of adenine and guanine (respectively) at nucleotide positions 20 and 21 with the bases cytosine and adenine at SCD codons. As shown in Figure 18B, when HSCs were treated with an exemplary gene modification system comprising an exemplary HBB8 template gRNA tg34 and various second-strand targeting gRNAs, an average perfect rewriting level of 2.9%-24.6% was detected in primary human HSCs, corresponding to the replacement of adenine at nucleotide position 20 with the base cytosine at SCD codons.

These results indicate that the editing activity of an exemplary gene modification system targeting clinically relevant codons in the endogenous B-globulin locus in primary human HSCs is increased using a second strand targeting gRNA. The results further indicate that the positioning of the second strand targeting gRNA (e.g., relative to the sequence targeted by the spacer of the exemplary template RNA) increases the enhancement of editing activity, e.g., more than 9 times higher than perfect rewriting in the absence of a second strand targeting gRNA.

Example 14: Characterization of the configuration of template RNA comprising silent substitutions for editing the endogenous B-globin locus in CD34+ primary human hematopoietic stem cells (HSCs).

This example demonstrates the use of a gene modification system containing an exemplary gene modification polypeptide and various template RNAs containing different silent substitutions to convert a glutamic acid codon at amino acid position 7 in the endogenous B-globulin locus in primary human HSCs to alanine, thereby demonstrating targeting of a sequence position associated with sickle cell disease (SCD) and editing of the sequence to encode a non-pathogenic sequence into position 7. The conversion comprises a two base pair change of the exemplary HBB5 template RNA (i.e., replacement of the DNA bases adenine and guanine at nucleotide positions 20 and 21 with the bases cytosine and adenine) plus the inclusion of additional relevant silent substitutions that alter the nucleic acid sequence of the DNA but not the protein sequence by using different synonymous codons.

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the exemplary template RNA comprises the following sequence from 5' to 3', wherein the first 3 and last 3 bases have 2'-O-methyl phosphorothioate chemical modifications. In the sequence below, m = 2'-O-methyl ribonucleotide, r = ribose, * = phosphorothioate bond. Different combinations of substitutions and RT/PBS lengths are included (Table X2).

Selected corresponding template RNA sequences that do not include silent substitutions are provided in Example 5 (eg, FYF tgRNA14 is the corresponding template RNA sequence of tg14h, FYF tgRNA19 is the corresponding template RNA sequence of tg19h, etc.).

The genetically modified polypeptide used comprised the sequence labeled RNAV209 described in Example 8.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 3000ng of mRNA encoding the gene modification polypeptide is combined with 2000ng of template RNA. The RNA mixture is added to 200,000 primary human HSCs in a total of 20 μL of Lonza P3 buffer, and HSCs are nuclear transfected in a 16-well nuclear transfection box using program DZ-100. After nuclear transfection, the cells are incubated at room temperature for 10 minutes and transferred to 24-well plates containing 500 μL StemSpan-XF+SCF of 100ng/mL, Flt3-L of 100ng/mL, and TPO of 100ng/mL in each well, and cultured for 3 days at 37°C, 5% _CO2 , followed by cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Amplicons were analyzed by short read sequencing using Illumina MiSeq. The DNA bases adenine and guanine at nucleotide positions 20 and 21 were replaced with the bases cytosine and adenine (HBB5 template RNA) downstream of the transcription start site within the endogenous B-globin locus, plus the inclusion of the expected silent substitution indicated successful editing.

As shown in Figure 19A, when HSCs were treated with an exemplary gene modification system containing an exemplary HBB5 template RNA containing various silent substitutions, an average perfect rewriting level of 0.2%-7.3% was detected in primary human HSCs, corresponding to the replacement of adenine and guanine (respectively) at nucleotide positions 20 and 21 with the bases cytosine and adenine at the SCD codon. The results show that in some cases, one or more silent substitutions can increase editing activity across several different template RNAs, such as the exemplary silent substitution hs1. In particular, replacing the codon encoding the 6th amino acid (proline) of the HBB gene counting the initial methionine with CCC or CCG increases editing.

These results show that when targeting clinically relevant codons in the endogenous B-globin locus in primary human HSCs, the introduction of silent substitutions within the exemplary template RNA increased the editing activity of the gene modification system comprising the template RNA by up to 5-fold. The results further show that modulating one or more consistency of silent substitutions can increase the enhancement of editing activity.

Example 15: Characterization of the configuration of template RNA comprising silent substitutions for editing the endogenous B-globin locus in CD34+ primary human hematopoietic stem cells (HSCs).

This example demonstrates the use of a gene modification system containing an exemplary gene modification polypeptide and various template RNAs containing different silent substitutions to convert a glutamic acid codon at amino acid position 7 in the endogenous B-globulin locus in primary human HSCs to alanine, thereby demonstrating targeting of a sequence position associated with sickle cell disease (SCD) and editing of the sequence to encode a non-pathogenic sequence into position 7. The conversion comprises a one base pair change of the exemplary HBB8 template RNA (i.e., replacement of the DNA base adenine at nucleotide position 20 with the base cytosine) plus the inclusion of an additional relevant silent substitution that alters the nucleic acid sequence of the DNA but does not alter the protein sequence by using a different synonymous codon.

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the exemplary template RNA comprises the following sequence from 5' to 3', wherein the first 3 and last 3 bases have 2'-O-methyl phosphorothioate chemical modifications. In the sequence below, m = 2'-O-methyl ribonucleotide, r = ribose, * = phosphorothioate bond. Different combinations of substitutions and RT/PBS lengths are included (Table X3).

The genetically modified polypeptide used comprised the sequence labeled RNAV209 described in Example 8.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 3000ng of mRNA encoding the gene modification polypeptide is combined with 3000ng of template RNA. The RNA mixture is added to 200,000 primary human HSCs in a total of 20 μL of Lonza P3 buffer, and HSCs are nuclear transfected in a 16-well nuclear transfection box using program DZ-100. After nuclear transfection, the cells are incubated at room temperature for 10 minutes and transferred to 24-well plates containing 500 μL StemSpan-XF+SCF of 100ng/mL, Flt3-L of 100ng/mL, and TPO of 100ng/mL in each well, and cultured for 3 days at 37°C, 5% _CO2 , followed by cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Amplicons were analyzed by short read sequencing using Illumina MiSeq. The DNA base adenine at nucleotide position 20 was replaced with the base cytosine (HBB8 template RNA) downstream of the transcription start site within the endogenous B-globin locus plus the inclusion of the expected silent substitution indicated successful editing.

As shown in Figure 19B, when HSC was treated with an exemplary gene modification system comprising an exemplary HBB8 template RNA containing various silent substitutions, an average perfect rewrite level of 0.1%-13.1% was detected in primary human HSC, corresponding to the replacement of adenine and guanine (respectively) at nucleotide positions 20 and 21 with the bases cytosine and adenine at the SCD codon. These results further indicate that when targeting clinically relevant codons in the endogenous B-globulin locus in primary human HSC, the introduction of silent substitutions in the exemplary template gRNA increased the editing activity of the gene modification system comprising the template RNA by more than 9 times. The results further indicate that regulating one or more consistencies of silent substitutions can increase the enhancement of editing activity.

Example 16: Evaluation of the effects of second strand targeting gRNA and silencing substitutions on the activity of gene-modified polypeptides and template RNA editing of the endogenous B-globin locus achieved in CD34+ primary human hematopoietic stem cells (HSCs).

This example demonstrates that the glutamic acid codon at amino acid position 7 in the endogenous B-globin locus in primary human HSCs is converted to alanine using a gene modification system containing or not containing various second-strand targeting gRNAs, exemplary gene modification polypeptides, and template RNAs, thereby rewriting the non-pathogenic sequence to position 7. The conversion comprises a change of 2 base pairs of the exemplary HBB5 template RNA (i.e., DNA bases thymidine, adenine, and guanine at nucleotide positions 18, 20, and 21 are replaced with bases guanine, cytosine, and adenine). For the exemplary HBB8 template RNA, the conversion comprises DNA bases thymidine and adenine at nucleotide positions 18 and 20 are changed to bases cytosine and cytosine, respectively (e.g., using template RNA tg34_HBB8_hs13) or DNA bases thymidine, adenine, and guanine at nucleotide positions 18, 20, and 21 are replaced with bases cytosine, cytosine, and cytosine, respectively (e.g., using tg34_HBB8_hs10). This example demonstrates editing using systems containing various second-strand targeting gRNAs with: an exemplary HBB5 template RNA containing a silencing substitution ( FIG. 20A ), or one of two exemplary HBB8 template RNAs, each containing a different silencing substitution ( FIG. 20B ).

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the template RNA comprises the sequence labeled tg14_hs1 described in Example 14 or the sequences labeled tg34_HBB8hs10 and tg34_HBB8hs13 described in Example 15.

The system further comprises a second strand targeting gRNA comprising the sequence in Table X1.

The genetically modified polypeptide tested comprised the sequence labeled RNAV209 described in Example 8.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 3000ng of mRNA encoding gene modification polypeptide is combined with 3000ng template RNA with or without 2000ng second-chain targeting gRNA. The RNA mixture is added to 200,000 primary human HSCs in a total of 20 μL of Lonza P3 buffer, and HSCs are nuclear transfected in a 16-well nuclear transfection box using program DZ-100. After nuclear transfection, the cells are incubated at room temperature for 10 minutes and transferred to 24-well plates containing 500 μL StemSpan-XF+SCF of 100ng/mL, Flt3-L of 100ng/mL, and TPO of 100ng/mL in each well, and cultured for 3 days at 37°C, 5% _CO2 , followed by cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. The amplicons were analyzed by short read sequencing using Illumina MiSeq. DNA bases thymidine, adenine, and guanine at nucleotide positions 18, 20, and 21 were replaced with bases guanine, cytosine, and adenine, indicating successful editing of the HBB5 spacer. Thymidine and adenine at nucleotide positions 18 and 20 were replaced with bases cytosine and cytosine, respectively (tg34_HBB8_hs13) or DNA bases thymidine, adenine, and guanine at nucleotide positions 18, 20, and 21 were replaced with bases cytosine, cytosine, and cytosine, respectively (tg34_HBB8_hs10), indicating successful editing of the HBB8 spacer.

Figure 20A shows a graph of % editing in HSCs treated with gene modification systems comprising exemplary HBB5 template RNA tg14_hs1 (comprising exemplary silent substitutions) with or without various second strand targeting gRNAs. The results indicate the additive effect of second strand targeting gRNAs and template gRNAs for HBB5 template RNAs containing silent substitutions used to rewrite non-pathogenic sequences into clinically relevant codons in the endogenous B-globulin locus.

Figure 20B shows a graph of % editing in HSCs treated with gene modification systems comprising one of two exemplary HBB8 template RNAs, tg34_hs13 or tg34_hs10, each comprising a different exemplary silent substitution, and with or without various second-strand targeting gRNAs. The results further demonstrate the additive effect of second-strand targeting gRNAs and template gRNAs for HBB8 template RNAs containing silent substitutions for rewriting non-pathogenic sequences into clinically relevant codons in the endogenous B-globulin locus.

Example 17: Evaluation of the effects of second strand targeting gRNA and silencing substitutions on the activity of gene-modified polypeptides and template RNA editing of the endogenous B-globin locus achieved in CD34+ primary human hematopoietic stem cells (HSCs).

This example demonstrates the use of a gene modification system with or without a second strand targeting gRNA, exemplary gene modifying polypeptides, and various template RNAs (some containing silent substitutions) to convert a glutamic acid codon at amino acid position 7 in the endogenous B-globin locus in primary human HSCs to alanine, thereby demonstrating targeting of a sequence position associated with sickle cell disease (SCD) and editing of the sequence to encode a non-pathogenic sequence into position 7. The conversion comprised a 2 base pair change of the exemplary HBB5 template RNA (i.e., replacement of the DNA bases adenine and guanine at nucleotide positions 20 and 21 with the bases cytosine and adenine) plus or minus an additional replacement of thymidine at nucleotide position 18 with guanine (silent substitution).

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the template RNA comprises a sequence labeled tg14h, tg14_hs1, tg19h or tg19_hs1 as described in Example 14.

The system further comprises a gRNA sequence designed to produce a second nick, wherein the gRNA has a sequence labeled HBB5_g37 in Table X1.

The genetically modified polypeptide tested comprised the sequence labeled RNAV209 described in Example 8.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 3000ng of mRNA encoding gene modification polypeptide is combined with 2000ng template RNA with or without 2000ng second-chain targeting gRNA. The RNA mixture is added to 200,000 primary human HSCs in a total of 20 μL of Lonza P3 buffer, and the cells are nuclear transfected in a 16-well nuclear transfection box using program DZ-100. After nuclear transfection, the cells are incubated at room temperature for 10 minutes and transferred to a 24-well plate containing 500 μL StemSpan-XF+SCF of 100ng/mL, Flt3-L of 100ng/mL, and TPO of 100ng/mL, and cultured for 3 days at 37°C, 5% _CO2 , followed by cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Illumina MiSeq was used to analyze amplicons by short read sequencing. The DNA bases adenine and guanine at nucleotide positions 20 and 21 were replaced with bases cytosine and adenine (tg14h or tg19h) or the DNA bases thymidine, adenine and guanine at nucleotide positions 18, 20, and 21 were replaced with bases guanine, cytosine, and adenine (tg14_hs1 or tg19_hs1) downstream of the transcription start site in the endogenous B-globulin locus, indicating successful editing.

As shown in Figure 20C, when HSCs were treated with an exemplary gene modification system comprising an exemplary HBB5 template RNA tg14h or tg19h and no second strand targeting gRNA was added, average perfect rewriting levels of 1.8% and 3.4% were detected in human HSCs, corresponding to the replacement of adenine and guanine (respectively) at nucleotide positions 20 and 21 with the bases cytosine and adenine at SCD codons. Including hs1 silent substitutions (tg14_hs1 or tg19_hs1) in the template gRNA increased perfect rewriting to 9.1% and 6.3%.

Adding a second strand targeting gRNA increased the average perfect rewriting to 17.1% for tg14h and 30.2% for tg14_hs1. Similarly, adding a second strand targeting gRNA increased the average perfect rewriting to 20.2% for tg19h and 32.2% for tg19_hs1.

These results indicate that silent substitutions and second-strand targeting gRNAs can individually increase the editing activity of the gene modification system, and further show the additive effects of second-strand targeting gRNAs and silent substitutions within an exemplary HBB5 template RNA. The results show that when silent substitutions and second-strand targeting gRNAs were used in primary human HSCs to write non-pathogenic sequences into clinically relevant codons in the endogenous B-globulin locus, the cumulative increase in editing activity was more than 20-fold.

Example 18: Evaluation of the effects of a gene modification system that edits the endogenous B-globin locus on stemness markers in CD34+ primary human hematopoietic stem cells (HSCs).

This example demonstrates that editing using a gene modification system containing an exemplary gene modification polypeptide and a template RNA with or without a second strand targeting gRNA to convert a glutamic acid codon at amino acid position 7 in the endogenous B-globin locus in primary human HSCs to alanine does not significantly affect the levels of stem cell markers and the proportions of cell marker-characterized subpopulations.

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

More specifically, the template RNA comprises the nucleic acid sequence labeled FYF tgRNA14 described in Example 5.

The system further comprises a second strand targeting gRNA sequence designed to produce a second nick, wherein the gRNA has a sequence labeled HBB5_g37 in Table X1.

The genetically modified polypeptide tested comprised the amino acid sequence labeled RNAV209 described in Example 8.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 3000ng of mRNA encoding gene modification polypeptide is combined with 2000ng template RNA with or without 2000ng second-chain targeting gRNA. The RNA mixture is added to 200,000 primary human HSCs in a total of 20 μL of Lonza P3 buffer, and the cells are nuclear transfected in a 16-well nuclear transfection box using program DZ-100. After nuclear transfection, the cells are incubated at room temperature for 10 minutes and transferred to a 24-well plate containing 500 μL StemSpan-XF+SCF of 100ng/mL, Flt3-L of 100ng/mL, and TPO of 100ng/mL, and cultured for 3 days at 37°C, 5% _CO2 , followed by cell lysis and genomic DNA extraction. In order to analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Illumina MiSeq was used to analyze the amplicon by short read sequencing. The DNA bases adenine and guanine at nucleotide positions 20 and 21 were replaced with bases cytosine and adenine, respectively, downstream of the transcription start site in the endogenous B-globulin locus to indicate successful editing. To analyze cell surface markers representing different HSC subsets, cells were stained with fluorescently labeled anti-human CD90, CD133, CD34 antibodies and analyzed by flow cytometry 3 days after nuclear transfection.

As shown in Figure 21A, when human HSCs were treated with an exemplary gene editing polypeptide in combination with template guide RNA tg14 without or with a second strand targeting gRNA, respectively, editing activity levels of 6.3% and 34.4% were detected in the HSCs, corresponding to the replacement of adenine and guanine (respectively) at nucleotide positions 20 and 21 with the bases cytosine and adenine at SCD codons. Analysis of the distribution of hematopoietic subpopulations (CD34+CD133+CD90+, a combination of markers enriched in HSCs with long-term remodeling potential; CD34+CD133+CD90-, a combination of markers enriched in early progenitor cells; CD34+CD133-, a combination of markers enriched in committed progenitor cells; CD34-, no markers enriched in differentiated cells) revealed that there was no deviation from the subpopulation ratio when comparing samples treated with the exemplary gene modification system (with or without the addition of a second strand targeting gRNA) with mock-treated controls (Figure 21B).

These results indicate that editing that introduces non-pathogenic sequences into clinically relevant codons in the endogenous B-globulin locus does not affect the phenotype of primary human HSCs, and in particular does not affect markers indicative of differentiation potential in HSCs.

Example 19: Evaluation of Editing of a HSC Subpopulation with Long-term Remodeling Capacity Using Genetically Modified Polypeptides and Template RNA for Editing of the Endogenous B-Globin Locus Achieved.

This example demonstrates that editing using a gene modification system containing an exemplary gene modification polypeptide and template RNA and a second strand targeting gRNA to convert a glutamic acid codon (GAG) at amino acid position 7 in the endogenous B-globin locus in primary human HSCs to alanine (GCA or GCG) effectively targets a subpopulation of HSCs associated with long-term remodeling, as well as other subpopulations, thereby rewriting a non-pathogenic sequence to position 7 of stem cells with longevity and differentiation potential. The conversion comprises a two-base pair change of the exemplary HBB5 template RNA (i.e., replacing the DNA bases adenine and guanine at nucleotide positions 20 and 21 with the bases cytosine and adenine, respectively) and a one-base pair change of the exemplary HBB8 template RNA (i.e., replacing the DNA base adenine at nucleotide position 20 with the base cytosine).

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

The template RNA comprises the sequence described in Example 5, which is labeled as FYFtgRNA14 for the HBB5 template RNA or as tgRNA34 for the HBB8 template RNA.

The genetically modified polypeptide tested comprised the sequence labeled RNAV209 described in Example 8.

The system further comprises a second strand targeting gRNA comprising the sequences listed as HBB5_g37 and HBB8_256fw in Table X1.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 3000ng of mRNA encoding gene modification polypeptide is combined with 2000ng template RNA with or without 2000ng second-chain targeting gRNA. The RNA mixture is added to 200,000 primary human HSCs in a total of 20 μL of Lonza P3 buffer, and the cells are nuclear transfected in a 16-well nuclear transfection box using program DZ-100. After nuclear transfection, the cells are incubated at room temperature for 10 minutes and transferred to a 24-well plate containing 500 μL StemSpan-XF+SCF of 100ng/mL, Flt3-L of 100ng/mL, and TPO of 100ng/mL, and cultured at 37°C, 5% _CO2 . Three days after nuclear transfection, cells were stained with fluorescently labeled anti-human CD90, CD133, CD34 antibodies, and CD34+CD133+CD90+ and CD34+CD133+CD90 parts were sorted by FACS, and cell lysis and genomic DNA extraction were performed. In order to analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Illumina MiSeq was used to analyze amplicons by short read sequencing. The DNA bases adenine and guanine at nucleotide positions 20 and 21 were replaced with bases cytosine and adenine (HBB5 template RNA) or the DNA base adenine at nucleotide position 20 was replaced with base cytosine (HBB8 template RNA) downstream of the transcription start site in the endogenous B-globulin locus to indicate successful editing.

As shown in Figure 22A, after treatment with a gene modification system comprising HBB5 template RNA or HBB8 template RNA, 19.3% and 29.8% editing activity levels were detected in the CD34+CD133+CD90+HSC subpopulation. CD34+CD133+CD90+ cells are enriched in HSCs with long-term reconstruction potential. 23.73% and 31.5% editing activity levels were detected in all remaining HSC populations (not CD34+CD133+CD90+) treated with the same exemplary gene modification system comprising HBB5 template RNA and HBB8 template RNA, respectively. The experiment was repeated using exemplary HBB5 template RNA tg14_hs1 (Table X1) and second-chain targeting gRNA (Figure 22B), and the results showed that the editing activity in the CD34+CD133+90+HSC enriched portion was 56% and the editing activity in the CD34+90-progenitor enriched portion was 52.9%. The results show that adding silencing substitutions to the template RNA (compare tg14_hs1 in FIG. 22B with FYF tgRNA14 in FIG. 22A ) significantly increases the editing activity of the gene modification system when used in long-term primary human HSCs.

These results indicate that the editing activity of the exemplary gene modification system can write non-pathogenic sequences into clinically relevant codons in the endogenous B-globin locus in phenotypically long-term primary human HSCs. The results further indicate that the level of editing activity in phenotypically long-term primary human HSCs is comparable to that achieved in the rest of the HSC population. The results further indicate high editing levels (greater than 50%) in long-term and progenitor HSCs.

Example 20: Evaluation of the effect of rewriting the endogenous B-globin locus of CD34+ primary human hematopoietic stem cells (HSCs) on differentiation capacity using gene-editing polypeptides and template RNA.

This example demonstrates that editing using a gene modification system containing an exemplary gene modification polypeptide and a template RNA with or without a second strand targeting gRNA to convert a glutamic acid codon (GAG) at amino acid position 7 in the endogenous B-globin locus in primary human HSCs to alanine (GCA or GCG) (thereby rewriting a non-pathogenic sequence into position 7) does not significantly alter the differentiation capacity of human HSCs. The conversion comprises a change of two base pairs of the exemplary HBB5 template RNA (i.e., replacing the DNA bases adenine and guanine at nucleotide positions 20 and 21 with the bases cytosine and adenine, respectively).

In this example, the template RNA contains:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

The template RNA comprises the sequence labeled FYFtgRNA14 for the HBB5 template RNA described in Example 5.

The genetically modified polypeptide tested comprised the sequence labeled RNAV209 described in Example 8.

The system further comprises a second strand targeting gRNA comprising the sequence listed as HBB5_g27 in Table X1.

The gene modification system comprising the gene modification polypeptide and template RNA described above is transfected into human HSC. The gene modification polypeptide and template RNA are delivered in RNA form by nuclear transfection. In particular, 3000ng of mRNA encoding gene modification polypeptide RNA is combined with 2000ng template RNA with or without 2000ng second-chain targeting gRNA. The RNA mixture is added to 200,000 primary human HSCs in a total of 20 μL of Lonza P3 buffer, and the cells are nuclear transfected in a 16-well nuclear transfection box using program DZ-100. After nuclear transfection, the cells are incubated at room temperature for 10 minutes and transferred to a 24-well plate containing 500 μL StemSpan-XF+SCF of 100ng/mL, Flt3-L of 100ng/mL, and TPO of 100ng/mL, and cultured at 37°C, 5% _CO2 . Two days after nucleofection, cells were cultured in semisolid Methcult medium for colony formation assays.

To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. The amplicons were analyzed by short read sequencing using Illumina MiSeq. Successful editing was indicated by replacing the DNA bases adenine and guanine at nucleotide positions 20 and 21 with the bases cytosine and adenine (HBB5 template RNA) downstream of the transcription start site within the endogenous B-globulin locus.

As shown in Figure 23A, the total colony CFU number after HSC obtained from 3 different donors is treated with an exemplary gene modification system with or without a second-strand targeting gRNA is comparable to the total colony CFU number when HSC is subjected to simulation treatment. These results show that the viability of treated HSC is not significantly reduced by treatment with an exemplary gene modification system. As shown in Figure 23B, the number of CFU-E, BFU-E, CFU-M, CFU-GM and CFU-G produced by CD34+ cells transfected with an exemplary gene modification system after 14 days of clonal growth in methylcellulose is comparable to the corresponding CFU number when CD34+ cells are subjected to simulation treatment. Figure 23C shows a graph of the percentage of enucleated CD235+ cells after HSC treated with an exemplary gene modification system begins in vitro differentiation. The results show that HSC treated with an exemplary gene modification system produces similar percentages of erythroid cells at a rate similar to that of simulated HSC.

These results show that editing of non-pathogenic sequences into clinically relevant codons in the endogenous B-globulin locus using the exemplary gene modification system described herein has no significant effect on the differentiation capacity of human HSCs.

Example 21: Screening for the configuration of template RNA that corrects SCD mutations in human CD34+ cells harboring SCD mutations

This example describes the use of an exemplary gene modification system to identify favorable configurations for correcting SCD mutations, the gene modification system comprising a gene modification polypeptide and a template RNA comprising heterologous subject sequences and PBS sequences of varying lengths. In this example, the template RNA comprises:

(1) gRNA spacer;

(2) gRNA scaffold;

(3) heterogeneous object sequences; and

(4) Primer binding site (PBS) sequence.

Template RNAs were designed to contain 8-17 nucleotide PBS sequences and 9-20 nucleotide heterologous subject sequences (Table X4). Template RNAs with two different gRNA exemplary spacer sequences, HBB5 and HBB8, were used to target SCD mutations in CD34+SCD human cells. The heterologous subject and PBS sequences were designed to correct SCD mutations by replacing "T" nucleotides with "A" nucleotides (wild type) or with "C" (Makassar installation) at the mutation site using the gene modification system described herein. Template RNAs were also designed to generate either or both of the following: 1) PAM-killing mutations or 2) one or more silent substitutions.

An exemplary gene modification system comprising an mRNA encoding a gene-modified polypeptide and a template RNA from Table X4 with or without a second strand targeting gRNA (e.g., from Table X1) is used to transfect human HSCs containing SCD mutations. The gene modification system is used to correct SCD mutations by replacing "T" nucleotides with "A" (wild type) or "C" (Makassar) nucleotides at the mutation site in the endogenous B-globin locus in primary human HSCs. Amplicon sequencing will be used to show editing at the mutation site in the endogenous B-globin locus in primary human HSCs.

The results will demonstrate that the exemplary gene modification system has editing activity when correcting SCD mutations in the endogenous B-globin locus in primary human HSCs.

Herein, when an RNA sequence (e.g., a template RNA sequence) is considered to include a specific sequence containing thymine (T) (e.g., a sequence of Table A or Table B or a portion thereof), it is of course understood that the RNA sequence can (and does often) include uracil (U) in place of T. For example, the RNA sequence can include U at each position shown as T in the sequence in Table A or Table B. More specifically, the present disclosure provides an RNA sequence according to each template sequence shown in Table A and Table B, wherein the RNA sequence has U in place of each T in the sequence of Table A and Table B.

It should be understood that for all numerical limits describing a parameter in this application, such as "about," "at least," "less than," and "greater than," the description also necessarily encompasses any range bounded by the recited values. Thus, for example, the description "at least 1, 2, 3, 4, or 5" also specifically describes ranges of 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, etc.

For all patents, applications or other references cited herein, such as non-patent literature and reference sequence information, it should be understood that for all purposes and for stated claims, they are incorporated herein by reference in their entirety. If there is any conflict between the document incorporated by reference and the present application, the present application shall prevail. All information related to the reference gene sequence disclosed in the present application, such as GeneID or accession number (usually with reference to NCBI accession number), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants and reference mRNA (including, for example, exon boundaries or response elements) and protein sequences (such as conserved domain structures), and chemical references (such as PubChem compounds, PubChem substances or PubChem bioassay entries, including annotations therein, such as structures and determinations, etc.), are incorporated herein by reference in their entirety.

The headings used in this application are for convenience only and do not affect the interpretation of this application.

Claims (85)

1. A template RNA comprising, from 5 'to 3':

(i) A gRNA spacer complementary to a first portion of a human HBB gene, wherein the gRNA spacer has a sequence comprising a core nucleotide of a gRNA spacer sequence of table 1, and optionally comprises one or more consecutive nucleotides starting from the 3' end of a flanking nucleotide of the gRNA spacer, or wherein the gRNA spacer has a sequence of a spacer selected from table a, table AA, table B1, tables 5A-5D, table X4, or table X4A;

(ii) A gRNA scaffold that binds to a genetically modified polypeptide (e.g., binds to a Cas domain of the genetically modified polypeptide),

(Iii) A heterologous subject sequence comprising a mutation region for introducing a mutation into a second portion of the human HBB gene (e.g., correcting a mutation therein) (wherein, optionally, the heterologous subject sequence comprises from 5 'to 3' a post-editing homologous region, a mutation region, and a pre-editing homologous region), and

(Iv) A Primer Binding Site (PBS) sequence comprising at least 5, 6, 7 or 8 bases having 100% identity to a third portion of the human HBB gene.

2. The template RNA of claim 1, wherein the heterologous subject sequence comprises a core nucleotide of the RT template sequence in table 3, and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence, or wherein the heterologous subject sequence comprises a sequence of the RT template sequence in table a, table AA, table B1, tables 5A-5D, table X4, or table X4A.

3. The template RNA of claim 1, wherein the heterologous subject sequence comprises a core nucleotide of the RT template sequence in table 3 corresponding to the gRNA spacer sequence, and optionally comprises one or more consecutive nucleotides starting from the 3' end of flanking nucleotides of the RT template sequence, or wherein the heterologous subject sequence comprises a sequence of the RT template sequence in table a, table AA, table B1, tables 5A-5D, table X4, or table X4A corresponding to the gRNA spacer sequence.

4. A template RNA comprising, from 5 'to 3':

(i) A gRNA spacer complementary to a first part of the human HBB gene,

(Ii) A gRNA scaffold that binds to a genetically modified polypeptide (e.g., binds to a Cas domain of the genetically modified polypeptide),

(Iii) A heterologous subject sequence comprising a mutation region for introducing a mutation into a second portion of the human HBB gene, wherein the heterologous subject sequence comprises a core nucleotide of the RT template sequence in table 3 and optionally comprises one or more contiguous nucleotides starting from the 3' end of the flanking nucleotides of the RT template sequence, or wherein the heterologous subject sequence comprises an RT template sequence in table a, table AA, table B1, tables 5A-5D, table X4 or table X4A; and

(Iv) A PBS sequence comprising at least 5, 6, 7 or 8 bases having 100% identity to a third portion of the human HBB gene.

5. The template RNA of claim 4, wherein the gRNA spacer comprises a core nucleotide of a gRNA spacer sequence in table 1, and optionally comprises one or more consecutive nucleotides starting from the 3' end of a flanking nucleotide of the gRNA spacer sequence, or wherein the gRNA spacer comprises a gRNA spacer sequence in table a, table AA, table B1, tables 5A-5D, table X4, or table X4A.

6. The template RNA of any one of claims 1-5, wherein the gRNA spacer comprises CATGGTGCATCTGACTCCTG (SEQ ID NO: 21668) or CATGGTGCACCTGACTCCTG (SEQ ID NO: 19249).

7. The template RNA of any one of claims 1-5, wherein the gRNA spacer comprises GTAACGGCAGACTTCTCCAC (SEQ ID NO: 19971).

8. The template RNA of claim 4, wherein the heterologous subject sequence comprises a core nucleotide corresponding to a gRNA spacer sequence in table 1 of the RT template sequence, and optionally comprises one or more consecutive nucleotides starting from the 3' end of flanking nucleotides of the gRNA spacer sequence, or wherein the heterologous subject sequence comprises nucleotides corresponding to a gRNA spacer sequence in table a, table AA, table B1, tables 5A-5D, table X4, or table X4A of the RT template sequence.

9. The template RNA of any one of claims 1-8, wherein the PBS sequence has a sequence comprising core nucleotides from the same row of PBS sequence as the RT template sequence in table 3, and optionally comprising one or more consecutive nucleotides starting from the 5' end of the flanking nucleotides of the PBS sequence.

10. The template RNA of any one of claims 1-8, wherein the PBS sequence has a sequence comprising a core nucleotide corresponding to the PBS sequence in table 3 of the RT template sequence, the gRNA spacer sequence, or both, and optionally comprises one or more consecutive nucleotides starting at the 5' end of flanking nucleotides of the PBS sequence, or wherein the PBS sequence has a sequence comprising a core nucleotide corresponding to the PBS sequence in table a, table AA, table B1, table 5A-5D, table X4, or table X4A of the RT template sequence, the gRNA spacer sequence, or both.

11. The template RNA of any one of claims 1-10, wherein the gRNA scaffold comprises the sequence of the gRNA scaffold in table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

12. The template RNA of any one of claims 1-10, wherein the gRNA scaffold comprises a sequence corresponding to the RT template sequence, the gRNA spacer sequence, or a gRNA scaffold in table 12 of both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

13. The template RNA of any one of claims 1-12, wherein the mutation is a V6E mutation (e.g., correction of a pathogenic E6V mutation) of the HBB gene.

14. The template RNA of any one of claims 1-13, wherein the pre-edit sequence length comprises about 1 nucleotide to about 35 nucleotides (e.g., comprises about 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, or 30-35 nucleotides).

15. The template RNA of any one of claims 1-14, wherein the mutation region comprises a single nucleotide.

16. The template RNA of any one of claims 1-14, wherein the mutation region is at least two nucleotides in length.

17. The template RNA of any one of claims 1-14 or 16, wherein the mutation region is up to 32 (e.g., up to 5, 10, 15, 20, 25, 30, or 32) nucleotides in length and comprises one, two, or three sequence differences relative to the second portion of the human HBB gene.

18. The template RNA of any one of claims 1-14, 16 or 17, wherein the mutant region comprises two sequence differences relative to the second portion of the human HBB gene.

19. The template RNA of any one of claims 1-14 or 16-18, wherein the mutant region comprises a first region (e.g., a first nucleotide) designed to correct a pathogenic mutation in the HBB gene and a second region (e.g., a second nucleotide) designed to inactivate a PAM sequence (e.g., a "PAM-kill" mutation exemplified in table a, AA, B, or B1).

20. The template RNA of any one of claims 1-19, wherein the heterologous subject sequence has a sequence from the same row of RT template sequences as the gRNA spacer sequence in table a or B, or has 1,2, or 3 substitutions relative thereto, wherein optionally the bolded T shown in the RT template sequence in table a is replaced with G (e.g., a sequence without PAM-killing mutations), or wherein further optionally the bolded C shown in the RT template in table B is replaced with T or U (e.g., a sequence without SNP that is present in HEK293T cells but not present in hg38 human reference genome).

21. The template RNA of any one of claims 1-20, wherein the mutation region comprises less than 80%, 70%, 60%, 50%, 40% or 30% identity to the corresponding portion of the human HBB gene.

22. The template RNA of any one of claims 1-21, wherein the template RNA comprises one or more silent mutations (e.g., silent substitutions), e.g., as exemplified in table 7A, X4 or X4A.

23. The template RNA of example 22, wherein the one or more silent mutations comprises a silent substitution at the codon of the 6 th amino acid (proline) of the incorporated initial methionine encoding the HBB gene, e.g., a substitution of CCC or CCG.

24. The template RNA of any one of the preceding claims, wherein the mutant region comprises a first region designed to correct a pathogenic mutation in the HBB gene and a second region designed to introduce a silencing substitution.

25. The template RNA of any one of claims 1-24, comprising one or more chemically modified nucleotides.

26. A genetic modification system, comprising:

The template RNA of any one of claims 1-25, and

A genetically modified polypeptide or a nucleic acid (e.g., RNA) encoding the genetically modified polypeptide.

27. The genetic modification system of claim 26, wherein the genetic modification polypeptide comprises:

A Reverse Transcriptase (RT) domain (e.g., an RT domain from a retrovirus, or a polypeptide domain having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto); and

A Cas domain (e.g., cas9 domain) that binds to the target DNA molecule and is heterologous to the RT domain; and

Optionally, a linker disposed between the RT domain and the Cas domain.

28. The genetic modification system of claim 27, wherein the RT domain comprises:

(a) RT domain of table 6; or (b)

(B) RT domains from: murine Leukemia Virus (MMLV), porcine Endogenous Retrovirus (PERV), avian reticuloendotheliosis virus (AVIRE), feline Leukemia Virus (FLV), simian Foamy Virus (SFV) (e.g., SFV 3L), bovine Leukemia Virus (BLV), mersen-fei-henhouse monkey virus (MPMV), human Foamy Virus (HFV), or bovine foamy/syncytial virus (BFV/BSV).

29. The genetic modification system of claim 27 or 28, wherein the Cas domain comprises a Cas domain of table 7 or table 8.

30. The genetic modification system of claim 27 or 28, wherein the Cas domain:

(a) Is a Cas9 domain;

(b) Is a SpCas9 domain, blatCas domain, nme2Cas9 domain, pnpCas9 domain, sauCas domain, sauCas9-KKH domain, sauriCas9 domain, sauriCas9-KKH domain, scaCas9-Sc++ domain, spyCas9-NG domain, spyCas9-SpRY domain, or St1Cas9 domain; and/or

(C) Is a Cas9 domain comprising an N670A mutation, an N611A mutation, an N605A mutation, an N580A mutation, an N588A mutation, an N872A mutation, an N863 mutation, an N622A mutation, or an H840A mutation.

31. The genetic modification system of claim 30, wherein the Cas9 domain binds to a PAM sequence listed in table 7 or table 12.

32. The genetic modification system of claim 31, wherein the second portion of the human HBB gene overlaps with PAM recognized by the Cas domain, e.g., wherein the second portion of the human HBB gene is within the PAM or wherein the PAM is within the second portion of the human HBB gene.

33. The genetic modification system of any one of claims 26-32, wherein the gRNA spacer is a gRNA spacer according to table 1, and the Cas domain comprises or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a Cas domain listed in the same row of table 1.

34. The genetic modification system of any one of claims 26-32, wherein the template RNA comprises the sequence of the template RNA sequence in table 3, table a, table AA, table B1, table 5A-5D, table X4, or table X4A, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

35. The genetic modification system of any one of claims 26-32, wherein:

(a) The template RNA comprises the sequences of the template RNA sequences of Table 3, table A, table AA, table B1, tables 5A-5D, table X4 or Table X4A;

(b) The Cas domain comprises a Cas domain of table 7 or table 8;

(c) The linker comprises the linker sequence of Table 10 (e.g., the linker sequence of any one of SEQ ID NOS: 5217, 5106, 5190 and 5218); and

(D) The genetically modified polypeptide comprises one or two NLS sequences from Table 11 (e.g., NLS sequences of any one of SEQ ID NOs: 5245, 5290, 5323, 5330, 5349, 5350, 5351 and 4001).

36. The genetic modification system of any one of claims 26-35, which creates a first nick in the first strand of the human HBB gene.

37. The genetic modification system of claim 36, further comprising a second strand targeting gRNA that directs a second nick to a second strand of the human HBB gene.

38. The genetic modification system of claim 37, wherein the second-strand targeting gRNA comprises:

(i) A sequence comprising a core nucleotide of a left or right gRNA spacer sequence from table 2, and optionally comprising one or more consecutive nucleotides starting from the 3' end of a flanking nucleotide of the left or right gRNA spacer sequence; or (b)

(Ii) A second strand targeting gRNA comprising the spacer sequence of table 6A or having 1,2, or 3 substitutions of spacer sequences relative thereto.

39. The genetic modification system of claim 37, wherein the second strand targeting gRNA comprises a sequence comprising a core nucleotide of the left or right gRNA spacer sequence from table 2 that corresponds to the gRNA spacer sequence of (i), and optionally comprises one or more consecutive nucleotides starting from the 3' end of the flanking nucleotides of the left or right gRNA spacer sequence.

40. The genetic modification system of claim 37, wherein the second-strand targeting gRNA comprises:

(i) A sequence comprising a core nucleotide of a second nicked gRNA sequence from table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and optionally comprising one or more contiguous nucleotides starting from the 3' end of the flanking nucleotide of the second nicked gRNA sequence; or (b)

(Ii) A second strand targeting gRNA comprising a spacer sequence from table 6A or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

41. The genetic modification system of claim 37, wherein the second strand targeting gRNA comprises a sequence that comprises a core nucleotide of a second nicked gRNA sequence from table 4 that corresponds to the gRNA spacer sequence of (i), or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and optionally comprises one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the second nicked gRNA sequence.

42. The genetic modification system of any one of claims 37-41, wherein the second strand targeting gRNA has a "PAM-in orientation" with a template RNA of the genetic modification system, e.g., as exemplified in table 4, 6A, X4, or X4A.

43. The genetic modification system of any one of claims 37-42, wherein the second strand targeting gRNA targets a sequence that overlaps with a target mutation of the template RNA.

44. The genetic modification system of claim 43, wherein the second strand targeting gRNA comprises:

(i) Sequences complementary to sickle cell mutations (e.g., spacer sequences);

(ii) A sequence complementary to a wild-type sequence at a sickle cell locus (e.g., a spacer sequence);

(iii) A sequence complementary to Makassar sequences at a sickle cell locus (e.g., a spacer sequence);

(iv) A sequence (e.g., a spacer sequence) complementary to a SNP near the sickle cell locus, e.g., a SNP contained in genomic DNA of a subject (e.g., patient);

(v) Complementary to or comprising one or more silent substitutions near the sickle cell locus (e.g., a spacer sequence).

45. The template RNA or gene modification system of any one of the preceding claims, wherein the gRNA spacer comprises about 1,2, 3 or more flanking nucleotides of the gRNA spacer.

46. The template RNA or gene modification system of any one of the preceding claims, wherein the heterologous subject sequence comprises about 2, 3, 4, 5, 10, 20, 30, 40 or more flanking nucleotides of the RT template sequence.

47. The template RNA or gene modification system of any one of the preceding claims, wherein the heterologous subject sequence comprises about 8-30, 9-25, 10-20, 11-16, or 12-15 (e.g., about 11-16) nucleotides.

48. The template RNA or gene modification system of any one of the preceding claims, wherein the mutation region comprises a sequence difference of 1,2 or 3 nucleotide positions relative to the corresponding portion of the human HBB gene.

49. The template RNA or gene modification system of any one of the preceding claims, wherein the mutation region comprises a sequence difference of at least 2 nucleotide positions relative to the corresponding portion of the human HBB gene.

50. The template RNA or gene modification system of any one of the preceding claims, wherein the post-editing and/or pre-editing homology region comprises 100% identity to the HBB gene.

51. The template RNA or gene modification system of any one of the preceding claims, wherein the PBS sequence further comprises about 1,2,3,4, 5, 6, 7 or more flanking nucleotides.

52. The template RNA or gene modification system of any one of the preceding claims, wherein the PBS sequence comprises about 5-20, 8-16, 8-14, 8-13, 9-12, or 10-12 (e.g., about 9-12) nucleotides.

53. The template RNA or gene modification system of any one of the preceding claims, wherein the PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the HBB gene nick site.

54. The genetic modification system of any one of the preceding claims, wherein the domains of the genetically modified polypeptide are linked by a peptide linker.

55. The genetic modification system of claim 54, wherein the linker comprises the linker sequence of Table 10 (e.g., the linker sequence of any one of SEQ ID NOS: 5217, 5106, 5190 and 5218).

56. The genetic modification system of any one of the preceding claims, wherein the genetically modified polypeptide further comprises one or more Nuclear Localization Sequences (NLS).

57. The genetic modification system of claim 56, wherein the genetically modified polypeptide comprises a first NLS and a second NLS.

58. The genetic modification system of claim 56 or 57, wherein the NLS comprises the NLS sequence of table 11 (e.g., the sequence of any one of SEQ ID NOs: 5245, 5290, 5323, 5330, 5349, 5350, 5351 and 4001).

59. A template RNA comprising the sequence of the template RNA in table 4, table a, table AA, table B1, tables 5A-5D, table X4, or table X4A, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

60. A template RNA comprising the sequence of the template RNA in table 4, table a, table AA, table B1, tables 5A-5D, table X4 or table X4A.

61. A genetic modification system, comprising:

(iii) A template RNA comprising the sequence of the template RNA in table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto; and

(Iv) The second nicked gRNA sequence from table 4 in the same row as (i) has a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

62. A genetic modification system, comprising:

(iii) A template RNA comprising the sequence of the template RNA of table 4; and

(Iv) The second nicked gRNA sequence from table 4 in the same row as (i).

63. A DNA encoding the template RNA of any one of claims 1-25, 46-52, 59, or 60, or the genetic modification system of any one of claims 26-58, 60, or 61.

64. A pharmaceutical composition comprising the system of any one of claims 26-58, 60 or 61, or one or more nucleic acids encoding the system, and a pharmaceutically acceptable excipient or carrier.

65. The pharmaceutical composition of claim 64, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of: plasmid vectors, viral vectors, vesicles and lipid nanoparticles.

66. The pharmaceutical composition of claim 65, wherein the viral vector is an adeno-associated virus.

67. A host cell (e.g. a mammalian cell, e.g. a human cell) comprising the template RNA or the gene modification system of any one of the preceding claims.

68. A method of preparing the template RNA of any one of claims 1-25, 46-52, 59, or 60, the method comprising synthesizing the template RNA by: the DNA encoding the template RNA is introduced into the host cell by in vitro transcription (e.g., solid state synthesis) or by introducing the DNA into the host cell under conditions that allow for the production of the template RNA.

69. A method for modifying a target site in a human HBB gene in a cell, the method comprising contacting the cell with the genetic modification system of any one of claims 26-58, 60 or 61 or DNA encoding the genetic modification system, thereby modifying the target site in the human HBB gene in the cell.

70. A method for treating a subject having a disease or condition associated with a mutation in the human HBB gene, the method comprising administering the gene modification system or DNA encoding the gene modification system of any one of claims 26-58, 60 or 61 to the subject, thereby treating a subject having a disease or condition associated with a mutation in the human HBB gene.

71. The method of claim 69 or 70, wherein the disease or condition is Sickle Cell Disease (SCD).

72. The method of any one of claims 69-71 wherein the subject has an E6V mutation.

73. A method for treating a subject having SCD, the method comprising administering to the subject the genetic modification system or DNA encoding the genetic modification system of any one of claims 26-58, 60 or 61, thereby treating the subject having SCD.

74. The genetic modification system or method of any one of the preceding claims, wherein introduction of the system into a target cell corrects a pathogenic mutation of the HBB gene.

75. The genetic modification system or method of any one of the preceding claims, wherein the pathogenic mutation is an E6V mutation, and wherein the correction comprises an amino acid substitution of V6E.

76. The genetic modification system or method of any one of the preceding claims, wherein introduction of the system into a target cell results in a mutation that promotes restoration of function of the HBB gene.

77. The genetic modification system or method of any one of the preceding claims, wherein correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70% or more) of the target nucleic acid.

78. The genetic modification system or method of any one of the preceding claims, wherein correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70% or more) of the target cells.

79. The genetic modification system or method of any one of the preceding claims, wherein the genetic modification system comprises a second strand-targeted gRNA, and wherein correction of mutations in the target cell population is increased relative to a target cell population treated with a genetic modification system comprising a template RNA without the second strand-targeted gRNA.

80. The genetic modification system or method of any one of the preceding claims, wherein the template RNA comprises one or more silencing substitutions (e.g., as exemplified in tables 7A, X4 and X4A), and wherein correction of mutations in the target cell population is increased relative to a target cell population treated with a genetic modification system comprising a template RNA that does not comprise one or more silencing substitutions.

81. The method of any one of the preceding claims, wherein the cell is a mammalian cell, such as a human cell.

82. The method of any one of the preceding claims, wherein the subject is a human.

83. The method of any one of the preceding claims, wherein the contacting occurs ex vivo, e.g., wherein the DNA of the cell or subject is modified ex vivo.

84. The method of any one of the preceding claims, wherein the contacting occurs in vivo, e.g., wherein the DNA of the cell or subject is modified in vivo.

85. The method of any one of the preceding claims, wherein contacting the cell or the subject with the system comprises contacting the cell or the cell in the subject with a nucleic acid (e.g., DNA or RNA) encoding the genetically modified polypeptide under conditions that allow for production of the genetically modified polypeptide.