CN118660960A

CN118660960A - Polynucleotides, compositions and methods for genome editing

Info

Publication number: CN118660960A
Application number: CN202280078591.7A
Authority: CN
Inventors: S·穆勒帕蒂; L·J·斯特雷茨
Original assignee: Intellia Therapeutics Inc
Current assignee: Intellia Therapeutics Inc
Priority date: 2021-11-03
Filing date: 2022-11-02
Publication date: 2024-09-17

Abstract

Compositions and methods for gene editing are provided. In some embodiments, a polynucleotide encoding an RNA-guided DNA binder such as Neisseria meningitidis Cas9 is provided that can provide one or more of improved editing efficiency, reduced immunogenicity, or other benefits.

Description

Polynucleotides, compositions and methods for genome editing

The present application claims the benefit of priority from U.S. provisional application No. 63/275,425, filed on month 11, 3 of 2021, and U.S. provisional application No. 63/352,158, filed on month 6, 14 of 2022, the contents of each of which are incorporated by reference in their entirety.

The present application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created at 1 of 11 of 2022, named 01155-0049-00PCT_ST26 and is 11,070,539 bytes in size.

The present disclosure relates to polynucleotides, compositions, and methods for genome editing involving RNA-guided DNA binding agents such as CRISPR-Cas systems and subunits thereof.

RNA-guided DNA binding agents such as CRISPR-Cas systems can be used for targeted genome editing, including in eukaryotic cells as well as in vivo. Such editing has been shown to inactivate certain deleterious alleles or correct certain deleterious point mutations. For example, neisseria meningitidis (NEISSERIA MENINGITIDIS) Cas9 (NmeCas 9) has an advantageously low off-target cleavage rate. RNA-guided DNA binding agents can be generated in situ by contacting a cell with a polynucleotide, such as mRNA or an expression construct. However, existing methods, for example in certain cell types or organisms such as mammals, may provide less than desirable robust expression, or may have undesirable immunogenicity, for example may cause undesirable increases in cytokine levels.

Thus, there is a need for polynucleotides, compositions, and methods for expressing polypeptides such as NmeCas. The present disclosure is directed to compositions and methods for polypeptide expression that provide one or more benefits, such as at least one of the following: improving expression levels, increasing activity of the encoded polypeptide, or reducing immunogenicity (e.g., reducing cytokine elevation following administration), or at least providing the public with a useful choice. In some embodiments, a polynucleotide encoding an RNA-guided DNA binding agent (e.g., nmeCas) is provided, wherein one or more of its coding sequence, codon usage, non-coding sequence (e.g., UTR), or heterologous domain (e.g., NLS) differs from an existing polynucleotide in the manner disclosed herein. Such features have been found to provide benefits as described above. In some embodiments, the improved editing efficiency occurs in or is specific to an organ or cell type of a mammal, such as a liver or liver cell.

The present disclosure provides the following embodiments.

In some embodiments, a polynucleotide is provided, the polynucleotide comprising an Open Reading Frame (ORF), the ORF comprising: a nucleotide sequence encoding a C-terminal neisseria meningitidis (Nme) Cas9 polypeptide that is at least 90% identical to SEQ ID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、 or any one of 301-303 and 317-321, wherein Nme Cas9 is Nme2 Cas9, nmel Cas9, or Nme3 Cas9; and a nucleotide sequence encoding a first Nuclear Localization Signal (NLS).

In some embodiments, the ORF further comprises a nucleotide sequence encoding a second NLS. In some embodiments, the first NLS and the second NLS are independently selected from the group consisting of SEQ ID NOs: 388 and 410-422. In some embodiments, the polynucleotide further comprises a poly-a sequence or a polyadenylation signal sequence. In some embodiments, the ORF further comprises a nucleotide sequence encoding a linker sequence between the first NLS and the second NLS. In some embodiments, the ORF further comprises a nucleotide sequence encoding a linker spacer between the Nme Cas9 coding sequence and the NLS proximal to the Nme Cas9 coding sequence. In some embodiments, the ORF Nme Cas9 has double stranded endonuclease activity. In some embodiments, the ORF Nme Cas9 has nickase activity. In some embodiments, the ORF Nme Cas9 comprises a dCas9 DNA binding domain.

The following numbered embodiments provide additional support and description of embodiments herein.

Embodiment 1 is a polynucleotide comprising an Open Reading Frame (ORF), said ORF comprising: a nucleotide sequence encoding a C-terminal neisseria meningitidis (Nme) Cas9 polypeptide that is at least 90% identical to any one of SEQ ID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、301-303 or 316-321, wherein the Nme Cas9 is Nme2 Cas9, nme1 Cas9, or Nme3 Cas9; and a nucleotide sequence encoding a first Nuclear Localization Signal (NLS).

Embodiment 2 is the polynucleotide of embodiment 1, wherein the ORF further comprises a nucleotide sequence encoding a second NLS.

Embodiment 3 is the polynucleotide of embodiment 1, wherein the first NLS and the second NLS are independently selected from the group consisting of SEQ ID NOs: 388 and 410-422.

Embodiment 4 is the polynucleotide of any one of embodiments 1-3, wherein the polynucleotide further comprises a poly-a sequence or a polyadenylation signal sequence.

Embodiment 5 is the polynucleotide of embodiment 4, wherein the poly-a sequence comprises a non-adenine nucleotide.

Embodiment 6 is the polynucleotide of any one of embodiments 4-5, wherein the poly-a sequence comprises 100-400 nucleotides.

Embodiment 7 is the polynucleotide of any one of embodiments 4-6, wherein the poly-a sequence comprises the amino acid sequence of SEQ ID NO: 409.

Embodiment 8 is the polynucleotide of any one of embodiments 1-7, wherein the ORF further comprises a nucleotide sequence encoding a linker sequence between the first NLS and the second NLS.

Embodiment 9 is the polynucleotide of any one of embodiments 1-8, wherein the ORF further comprises a nucleotide sequence encoding a linker spacer sequence between the Nme Cas9 coding sequence and the NLS proximal to the Nme Cas9 coding sequence.

Embodiment 10 is the polynucleotide of any one of embodiments 8-9, wherein the linker comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids.

Embodiment 11 is the polynucleotide of any one of embodiments 8-10, wherein the linker sequence comprises GGG or GGGs, optionally wherein the GGG or GGGs sequence is located at the N-terminus of the spacer sequence.

Embodiment 12 is the polynucleotide of any one of embodiments 8-11, wherein the linker sequence comprises the sequence of SEQ ID NO:61-122 one sequence.

Embodiment 13 is the polynucleotide of any one of embodiments 1-12, wherein the ORF further comprises one or more additional heterologous functional domains.

Embodiment 14 is the polynucleotide of any one of embodiments 1-13, wherein the Nme Cas9 has double-stranded endonuclease activity.

Embodiment 15 is the polynucleotide of any one of embodiments 1-14, wherein the Nme Cas9 has nickase activity.

Embodiment 16 is the polynucleotide of any one of embodiments 1-14, wherein the Nme Cas9 comprises a dCas9 DNA binding domain.

Embodiment 17 is the polynucleotide of any one of embodiments 1-16, wherein the NmeCas comprises a sequence that hybridizes to SEQ ID NO:1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, 297 or any of 310-315 has an amino acid sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

Embodiment 18 is the polynucleotide of any one of embodiments 1-17, wherein the NmeCas comprises the sequence set forth in SEQ ID NO:1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, 297 or 310-315.

Embodiment 19 is the polynucleotide of any one of embodiments 1-18, wherein the sequence encoding the NmeCas comprises a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of any one of SEQ ID NO：15、18-27、29、32-41、221-226、228-233、235-240、242-247、249-254、256-261、263-268、270-275、277-282、284-289、291-296、298-303、304-309 or 316-321.

Embodiment 20 is the polynucleotide of any one of embodiments 1-19, wherein the sequence encoding the NmeCas comprises the nucleotide sequence of any one of SEQ ID NO：15、18-27、29、32-41、221-226、228-233、235-240、242-247、249-254、256-261、263-268、270-275、277-282、284-289、291-296、298-303、304-309 or 316-321.

Embodiment 21 is a polynucleotide comprising an Open Reading Frame (ORF) encoding a polypeptide comprising: cytidine deaminase, optionally apodec 3A deaminase; a nucleotide sequence encoding a C-terminal neisseria meningitidis (Nme) Cas9 nickase polypeptide that hybridizes with SEQ ID NO:1 and any one of 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, or 297 is at least 90% identical, wherein the Nme Cas9 nickase is Nme2 Cas9 nickase, nme1 Cas9 nickase, or Nme3 Cas9 nickase; and a nucleotide sequence encoding a first Nuclear Localization Signal (NLS); wherein the polypeptide does not comprise a Uracil Glycosidase Inhibitor (UGI).

Embodiment 22 is the polynucleotide of embodiment 21, wherein the ORF further comprises a nucleotide sequence encoding a second NLS.

Embodiment 23 is the polynucleotide of any one of embodiments 21-22, wherein the deaminase is located at the N-terminus of an NLS in the polypeptide.

Embodiment 24 is the polynucleotide of any one of embodiments 21-23, wherein the cytidine deaminase is located at the N-terminus of the first NLS and the second NLS in the polypeptide.

Embodiment 25 is the polynucleotide of any one of embodiments 21-22, wherein the cytidine deaminase is located at the C-terminus of an NLS in the polypeptide.

Embodiment 26 is the polynucleotide of any one of embodiments 23-25, wherein the cytidine deaminase is located at the C-terminus of the first NLS and the second NLS in the polypeptide.

Embodiment 27 is the polynucleotide of any one of embodiments 21-26, wherein the ORF does not comprise a coding sequence of an NLS located at the C-terminus of the ORF encoding Nme Cas 9.

Embodiment 28 is the polynucleotide of any one of embodiments 21-26, wherein the ORF does not comprise a coding sequence located at the C-terminus of the ORF encoding the Nme Cas 9.

Embodiment 29 is the polynucleotide of any one of embodiments 1-28, wherein the cytidine deaminase comprises a nucleotide sequence that hybridizes to SEQ ID NO:151 has an amino acid sequence of at least 87% identity.

Embodiment 30 is the polynucleotide of any one of embodiments 1-28, wherein the cytidine deaminase comprises a nucleotide sequence that hybridizes to SEQ ID NO:152-216 have an amino acid sequence with at least 80% identity.

Embodiment 31 is the polynucleotide of any one of embodiments 1-28, wherein the cytidine deaminase comprises a nucleotide sequence that hybridizes to SEQ ID NO:14 having an amino acid sequence having at least 80% identity.

Embodiment 32 is the polynucleotide of any one of embodiments 1-31, wherein the ORF comprises a nucleotide sequence identical to SEQ ID NO:42, a nucleotide sequence having at least 80% identity.

Embodiment 33 is the polynucleotide of any one of embodiments 1-32, wherein the polynucleotide comprises a sequence that hybridizes to SEQ ID NO:391-398 have a 5' UTR with at least 90% identity.

Embodiment 34 is the polynucleotide of any one of embodiments 1-33, wherein the polynucleotide comprises a nucleotide sequence comprising SEQ ID NO:391-398 one 5' UTR.

Embodiment 35 is the polynucleotide of any one of embodiments 1-34, wherein the polynucleotide comprises a sequence that hybridizes to SEQ ID NO:399-406 have a 3' UTR with at least 90% identity.

Embodiment 36 is the polynucleotide of any one of embodiments 1-35, wherein the polynucleotide comprises a nucleotide sequence comprising SEQ ID NO:399-306 one 3' UTR.

Embodiment 37 is the polynucleotide of any one of embodiments 1-36, wherein the polynucleotide comprises a 5'utr and a 3' utr from the same source.

Embodiment 38 is the polynucleotide of any one of embodiments 1-37, wherein the polynucleotide comprises a 5 'Cap, optionally wherein the 5' Cap is Cap0, cap1, or Cap2.

Embodiment 39 is the polynucleotide of any one of embodiments 1-38, wherein at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons of the ORF are minimal adenine codons or minimal uridine codons.

Embodiment 40 is the polynucleotide of any one of embodiments 1-39, wherein the ORF comprises or consists of codons that increase translation of mRNA in the mammal.

Embodiment 41 is the polynucleotide of any one of embodiments 1-40, wherein the ORF comprises or consists of codons that increase translation of mRNA in a human.

Embodiment 42 is the polynucleotide of any one of embodiments 1-41, wherein the polynucleotide is mRNA.

Embodiment 43 is the polynucleotide of embodiment 42, wherein the ORF comprises a sequence having at least 90%, 95%, 98% or 100% identity to any one of SEQ ID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、301-303 or 316-321.

Embodiment 44 is the polynucleotide of any one of embodiments 42-43, wherein at least 10% of uridine in said mRNA is replaced by modified uridine.

Embodiment 45 is the polynucleotide of any one of embodiments 42-43, wherein less than 10% of the uridine in the mRNA is replaced by modified uridine.

Embodiment 46 is the polynucleotide of embodiment 45, wherein the modified uridine is one or more of N1-methyl-pseudouridine, 5-methoxyuridine, or 5-iodouridine.

Embodiment 47 is the polynucleotide of embodiment 45, wherein the modified uridine is one or both of N1-methyl-pseudouridine or 5-methoxyuridine.

Embodiment 48 is the polynucleotide of any one of embodiments 45-47, wherein the modified uridine is N1-methyl-pseudouridine.

Embodiment 49 is the polynucleotide of any one of embodiments 45-47, wherein the modified uridine is 5-methoxyuridine.

Embodiment 50 is the polynucleotide of any one of embodiments 44 and 46-49, wherein 15% to 45% of the uridine is replaced by modified uridine.

Embodiment 51 is the polynucleotide of embodiment 50, wherein at least 20% or at least 30% of the uridine is replaced by modified uridine.

Embodiment 52 is the polynucleotide of embodiment 51, wherein at least 80% or at least 90% of the uridine is replaced by modified uridine.

Embodiment 53 is the polynucleotide of embodiment 52, wherein 100% of the uridine is replaced by modified uridine.

Embodiment 54 is the polynucleotide of embodiment 42, wherein less than 10% of the nucleotides in the mRNA are substituted with modified nucleotides.

Embodiment 55 is a composition comprising the polynucleotide of any one of embodiments 1-54 and at least one guide RNA (gRNA).

Embodiment 56 is a composition comprising a first polynucleotide comprising a first Open Reading Frame (ORF) encoding a polypeptide comprising a cytidine deaminase, optionally an apodec 3A deaminase, and NmeCas nickase, and a second polynucleotide comprising a second open reading frame encoding a Uracil Glycosidase Inhibitor (UGI), wherein the second polynucleotide is different from the first polynucleotide, and optionally further comprising a guide RNA (gRNA).

Embodiment 57 is the composition of embodiment 55 or 56, wherein the gRNA is a single guide RNA.

Embodiment 58 is the composition of embodiment 55 or 56, wherein the gRNA is a dual guide RNA.

Embodiment 59 is a composition comprising the polynucleotide of any one of embodiments 1-57, further comprising a single guide RNA, wherein the single guide RNA comprises a guide region and a conserved region, wherein the conserved region comprises one or more of:

(a) A shortened repeat/anti-repeat region, wherein the shortened repeat/anti-repeat region lacks 2-24 nucleotides, wherein

(I) Relative to SEQ ID NO:500, one or more of nucleotides 37-48 and 53-64 are deleted, and optionally one or more of nucleotides 37-64 are substituted; and

(Ii) Nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b)

(B) A shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2-10, optionally 2-8 nucleotides, wherein

(I) Relative to SEQ ID NO:500, one or more of nucleotides 82-86 and 91-95 are deleted, and optionally one or more of positions 82-96 are substituted; and

(Ii) Nucleotide 81 is linked to nucleotide 96 by at least 4 nucleotides; or (b)

(C) A shortened hairpin 2 region, wherein the shortened hairpin 2 lacks 2-18, optionally 2-16 nucleotides, wherein

(I) Relative to SEQ ID NO:500, one or more of nucleotides 113-121 and 126-134 is deleted, and optionally one or more of nucleotides 113-134 is substituted; and

(Ii) Nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides;

Wherein one or both of nucleotides 144-145 are relative to SEQ ID NO:500 is optionally deleted;

wherein at least 10 nucleotides are modified nucleotides.

Embodiment 60 is a composition comprising the polynucleotide of any one of embodiments 1-57, further comprising a single guide RNA, wherein the single guide RNA comprises a guide region and a conserved region, wherein the conserved region comprises one or more of:

(I) Relative to SEQ ID NO:500, one or more of nucleotides 37-64 are deleted and optionally substituted; and

(Ii) Nucleotide 36 is linked to nucleotide 65 by: (i) A first internal linker that replaces 4 nucleotides, alone or in combination with nucleotides, or (ii) at least 4 nucleotides; or (b)

(I) Relative to SEQ ID NO:500, one or more of nucleotides 82-95 is deleted and optionally substituted; and

(Ii) Nucleotide 81 is linked to nucleotide 96 by: (i) A second internal linker that replaces 4 nucleotides, alone or in combination with nucleotides, or (ii) at least 4 nucleotides; or (b)

(I) Relative to SEQ ID NO:500, one or more of nucleotides 113-134 is deleted and optionally substituted; and

(Ii) Nucleotide 112 is linked to nucleotide 135 by: (i) A third internal linker that replaces 4 nucleotides, alone or in combination with nucleotides, or (ii) at least 4 nucleotides;

wherein one or both of nucleotides 144-145 are compared to SEQ ID NO:500 is optionally deleted;

wherein the gRNA includes at least one of the first internal linker, the second internal linker, and the third internal linker.

Embodiment 61 is a polypeptide encoded by the polynucleotide of any one of embodiments 1-60.

Embodiment 62 is a vector comprising the polynucleotide of any one of embodiments 1-60.

Embodiment 63 is an expression construct comprising a promoter operably linked to a sequence encoding the polynucleotide of any one of embodiments 1-60.

Embodiment 64 is the expression construct of embodiment 63, wherein the promoter is an RNA polymerase promoter, optionally a bacterial RNA polymerase promoter.

Embodiment 65 is the expression construct of embodiment 63 or 64, further comprising a poly-a tail sequence or a polyadenylation signal sequence.

Embodiment 66 is the expression construct of embodiment 65, wherein the poly-a tail sequence is an encoded poly-a tail sequence.

Embodiment 67 is a plasmid comprising the expression construct of any one of embodiments 63-66.

Embodiment 68 is a host cell comprising the vector of embodiment 62, the expression construct of any one of embodiments 63-66, or the plasmid of embodiment 67.

Embodiment 69 is a pharmaceutical composition comprising the polynucleotide, composition, or polypeptide of any one of embodiments 1-61 and a pharmaceutically acceptable carrier.

Embodiment 70 is a kit comprising the polynucleotide, composition, or polypeptide of any one of embodiments 1-61.

Embodiment 71 is a use of the polynucleotide, composition or polypeptide of any one of embodiments 1-61 for modifying a gene of interest in a cell.

Embodiment 72 is the use of the polynucleotide, composition or polypeptide of any one of embodiments 1-61 for the manufacture of a medicament for modifying a gene of interest in a cell.

Embodiment 73 is the polynucleotide or composition of any one of embodiments 1-60, wherein the polynucleotide or composition is formulated as a lipid nucleic acid assembly composition, optionally a lipid nanoparticle.

Embodiment 74 is a method of modifying a gene of interest, comprising delivering to a cell a polynucleotide, polypeptide, or composition of any one of embodiments 1-61.

Embodiment 75 is a method of modifying a gene of interest, comprising delivering to a cell one or more lipid nucleic acid assembly compositions, optionally lipid nanoparticles, comprising the polynucleotide of any one of embodiments 1-60 and one or more guide RNAs.

Embodiment 76 is the method of any one of embodiments 74-75, wherein at least one of the lipid nucleic acid assembly compositions comprises a Lipid Nanoparticle (LNP), optionally wherein all of the lipid nucleic acid assembly compositions comprise LNP.

Embodiment 77 is the method of any one of embodiments 74-75, wherein at least one of the lipid nucleic acid assembly compositions is a liposome complex (lipoplex) composition.

Embodiment 78 is the composition or method of any one of embodiments 75-77, wherein the lipid nucleic acid assembly composition comprises an ionizable lipid.

Embodiment 79 is a method of producing the polynucleotide of any one of embodiments 1-54, comprising contacting the expression construct of embodiments 63-66 with an RNA polymerase and an NTP comprising at least one modified nucleotide.

Embodiment 80 is the method of embodiment 79, wherein the NTP comprises one modified nucleotide.

Embodiment 81 is the method of embodiment 79 or 80, wherein the modified nucleotide comprises a modified uridine.

Embodiment 82 is the method of embodiment 81, wherein at least 80% or at least 90% or 100% of the uridine positions are modified uridine.

Embodiment 83 is the method of embodiment 81 or 82, wherein the modified uridine comprises or is a substituted uridine, pseudouridine, or a substituted pseudouridine, optionally N1-methyl-pseudouridine.

Embodiment 84 is the method of any one of embodiments 79-83, wherein the expression construct comprises an encoded poly-a tail sequence.

Drawings

Figure 1 shows the average percent editing at TTR locus in PMH at increasing doses of Nme2Cas9 mRNA and chemically modified sgrnas.

Fig. 2A shows the average percent editing at TTR locus in PMH using different ratios of sgRNA and Nme2Cas9 mRNA.

Fig. 2B shows the average percent editing at TTR locus in PMH using different ratios of pgRNA and Nme2Cas9 mRNA.

Figure 3 shows the average percent editing at TTR locus in PMH for pgRNA and Nme2Cas9 mRNA.

Fig. 4 shows the average percent editing at the PCSK9 locus in PMH.

FIG. 5A shows the average editing results at the VEGFA locus in HEK cells treated with mRNA C

FIG. 5B shows the average editing results at the VEGFA locus in HEK cells treated with mRNA I

FIG. 5C shows the average editing results at the VEGFA locus in HEK cells treated with mRNA J

FIG. 5D shows the average editing results at the VEGFA locus in PHH cells treated with mRNA C

FIG. 5E shows the average editing results at the VEGFA locus in PHH cells treated with mRNA I

FIG. 5F shows the average editing results at the VEGFA locus in PHH cells treated with mRNA J

Figure 6 shows the average percent editing at the mouse TTR locus in PMH cells treated with NmeCas constructs designed to have 1 or 2 nuclear localization sequences.

Figure 7 shows the average percent editing at the mouse TTR locus in PMH cells treated with pgRNA and various Nme2Cas9 mRNA.

Figure 8 shows fold change in Nme2Cas9 protein expression compared to SpyCas9 protein expression in PMH, PRH, PCH and PHH cells.

Figures 9A-9F show fold-change in Nme2Cas9 protein expression compared to SpyCas9 protein expression in T cells from 2 donors measured 24 hours, 48 hours, and 72 hours post-treatment.

Figure 10 shows the average percent editing at TTR locus in mouse livers treated with sgRNA and Nme2Cas 9.

Fig. 11A shows the average percent editing at TTR locus in mouse liver after treatment with pgRNA and Nme2Cas 9.

Fig. 11B shows the mean serum TTR protein after treatment with pgRNA and Nme2Cas 9.

Fig. 11C shows the average TTR gene knockdown percentage after treatment with pgRNA and Nme2Cas 9.

Fig. 11D shows the average percent editing at TTR loci in mouse liver after treatment with pgRNA and various Nme2Cas 9.

Fig. 11E shows serum TTR protein knockdown after treatment with pgRNA and various Nme2Cas 9.

Figure 12 shows the average percent editing in mouse liver after treatment with various Nme2Cas9 constructs.

Figure 13 shows the average percent editing in mouse liver after treatment with pgRNA and various Nme2Cas 9.

Figure 14 shows the average percent editing in mouse liver after treatment with various base editors.

Fig. 15 shows an exemplary schematic of Nme2 sgrnas in a possible secondary structure comprising repeat/anti-repeat regions and hairpin regions (which include hairpin 1 and hairpin 2 regions), and further indicating guide (or targeting) regions (indicated with grey fill with dashed outline), bases unsuitable for single or paired deletions (indicated with grey fill with solid outline), bases suitable for single or paired deletions (open circles).

Figure 16 shows the average percentage of CD3 negative T cells after TRAC editing with Nme1Cas 9.

Figure 17 shows the average percentage of CD3 negative T cells after TRAC editing with Nme3Cas 9.

FIG. 18 shows the expression of Nme-HiBiT construct in T cells at 24 hours.

FIG. 19 shows a CD3 negative cell population as a function of NmeCas mRNA amounts.

Figure 20 shows the dose response curve for selected grnas in PCH.

Figure 21 shows the dose response curve of the LNP dilution series in PCH.

Fig. 22 shows serum TTR levels in mice.

Fig. 23 shows the percent editing at TTR loci in mouse liver samples.

FIG. 24 shows dose response curves for selected gRNAs in PMH.

FIG. 25 shows a dose response curve for selected gRNAs in PMH.

Figure 26 shows the average percent editing at PCSK9 locus in PMH under modified sgRNA.

Figure 27 shows the average percent editing in PMH of several Nme2Cas9 mRNA versus modified sgrnas.

Fig. 28 shows the percent editing at TTR loci in primary mouse hepatocytes.

Fig. 29 shows serum TTR levels in mice.

Figure 30 shows the percent editing at TTR locus in mouse liver samples.

Fig. 31 shows serum TTR measurements after treatment in mice.

Fig. 32 shows the percent editing at TTR loci in mouse liver samples.

FIG. 33 shows an exemplary sgRNA in a possible secondary structure (G021536; SEQ ID NO: 139). Methylation is shown in bold; phosphorothioate linkages are indicated by '×'. Watson-Crick base pairing is indicated by a '-' between nucleotides in the duplex portion. non-Watson-Crick base pairing is indicated by a'. Cndot.between nucleotides in the duplex portion.

FIG. 34 shows an exemplary sgRNA in a possible secondary structure (G032572; SEQ ID NO: 528). Unmodified nucleotides are shown in bold and methylation is shown in light; phosphorothioate linkages are indicated by '×'. Watson-Crick base pairing is indicated by a '-' between nucleotides in the duplex portion. non-Watson-Crick base pairing is indicated by a'. Cndot.between nucleotides in the duplex portion.

FIG. 35 shows an exemplary sgRNA in a possible secondary structure (G031771; SEQ ID NO: 529). Unmodified nucleotides are shown in bold and methylation is shown in light; phosphorothioate linkages are indicated by '×'. Watson-Crick base pairing is indicated by a '-' between nucleotides in the duplex portion. non-Watson-Crick base pairing is indicated by a'. Cndot.between nucleotides in the duplex portion.

Brief description of the disclosed sequences

The transcript sequence may typically include GGG as the first three nucleotides for use with ARCA, or AGG as the first three nucleotides for use with CleanCap ^TM. Thus, the first three nucleotides can be modified for use with other capping methods such as vaccinia (Vaccinia) capping enzymes. The promoter and poly-A sequences are not included in the transcript sequence. A promoter such as the U6 promoter (SEQ ID NO: 389) or the CMV promoter (SEQ ID NO: 390) and a promoter such as the SEQ ID NO:409 can be attached at the 5 'and 3' ends, respectively, to the disclosed transcript sequences. Most nucleotide sequences are provided in DNA form, but can be easily converted to RNA by converting T to U.

Detailed Description

Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the invention as defined by the appended claims.

Before the present teachings are described in detail, it is to be understood that this disclosure is not limited to particular compositions or method steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates and reference to "a cell" includes a plurality of cells, and so forth.

Numerical ranges include numbers defining the ranges. In view of the significant figures and errors associated with measurements, measured and measurable values should be understood as approximations. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

The term "about" is used herein to mean within typical tolerances in the art. For example, "about" may be understood as about 2 standard deviations from the mean. In certain embodiments, about +10% is meant. In certain embodiments, about means +5%, +2% or +1%. When an amount of a particular number or range is present, it is understood that the amount of the particular number or range is not necessarily the same. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

The use of "include/comprise/include", "contain/contain" and "include/include" is not intended to be limiting. It is to be understood that both the foregoing general description and the detailed description are exemplary and explanatory only and are not restrictive of the teachings.

The term "at least" preceding a number or a series of numbers is to be understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included as is apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 17 nucleotides of a 20 nucleotide nucleic acid molecule" means that 17, 18, 19 or 20 nucleotides have the indicated properties. When at least before a series of numbers or ranges, it is to be understood that "at least" can modify each of the numbers in the series or ranges.

As used herein, "no more than" or "less than" is to be understood as the value adjacent to the phrase, and the logical lower value or integer to zero as indicated by the context logic. For example, a duplex region of "no more than 2 nucleotide base pairs" has 2, 1, or 0 nucleotide base pairs. When "no more than" or "less than" is present before a series of numbers or ranges, it is understood that each of the numbers in the series or ranges is modified.

As used herein, a range includes both upper and lower limits.

As used herein, it is understood that when the maximum amount of a value is represented by 100% (e.g., 100% inhibition), the value is interpreted according to the detection method. For example, 100% inhibition and the like are understood to be inhibition at levels below the level at which detection is determined.

Unless specifically indicated above in the specification, embodiments in this specification that "comprise" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components; embodiments described in this specification as "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components; and embodiments in this specification that "consist essentially of the various components are also contemplated as" consisting of "or" comprising "the recited components (this interchangeability is not applicable to the use of these terms in the claims).

The section headings used herein are for organizational purposes only and are not to be construed as limiting the required subject matter in any way. In the event that any document incorporated by reference contradicts the expression (including but not limited to definition) of the specification, the expression of the specification is in control. While the teachings of the present invention are described in conjunction with various embodiments, it is not intended that the teachings of the present invention be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

I. Definition of the definition

Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings:

As used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C or a combination thereof" is intended to include at least one of: A. b, C, AB, AC, BC or ABC, and BA, CA, CB, ACB, CBA, BCA, BAC or CAB if the order is important in a particular case. Continuing with this example, explicitly including repeated combinations comprising one or more items or terms, such as BB, AAA, AAB, BBC, AAABC, CBBA, BABB, etc. The skilled artisan will appreciate that there is generally no limit to the number of items or items in any combination unless otherwise apparent from the context.

As used herein, the term "kit" refers to a packaged set of related components such as one or more polynucleotides or compositions and one or more related materials such as a delivery device (e.g., syringe), solvent, solution, buffer, instructions, or desiccant.

Unless otherwise specified herein, "or" is used in an inclusive sense, i.e., equivalent to "and/or".

"Polynucleotide (polynucleotide)" and "nucleic acid" are used herein to refer to polymeric compounds comprising nucleosides or nucleoside analogs that have nitrogen-containing heterocyclic bases or base analogs linked together along the backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. The nucleic acid "backbone" may be comprised of a plurality of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid may be ribose, deoxyribose, or similar compounds having a substitution (e.g., a 2 'methoxy or 2' halo substitution). The nitrogenous base can be a conventional base (A, G, C, T, U), an analog thereof (e.g., a modified uridine such as 5-methoxyuridine, pseudouridine or N1-methylpseuduridines or other modified uridine), inosine, a purine or derivative of pyrimidine (e.g., N4-methyldeoxyguanosine, deazapurine or azapurine, deazapyrimidine or azapyrimidine, a pyrimidine base having a substituent at the 5-or 6-position (e.g., 5-methylcytosine), a purine base having a substituent at the 2-, 6-or 8-position, 2-amino-6-methylaminopurine, O6-methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O4-alkyl-pyrimidine; U.S. Pat. Nos. 5,378,825 and PCT WO 93/13121). For a general discussion, see The Biochemistry of the Nucleic Acids-36, adams et al, 11 th edition, 1992). The nucleic acid may include one or more "abasic" residues, wherein the backbone does not include a nitrogenous base for the polymer position (U.S. Pat. No. 5,585,481). The nucleic acid may comprise only conventional RNA or DNA sugars, bases and linkages, or may comprise conventional components with substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA), analogs containing one or more LNA nucleotide monomers with bicyclic furanose units locked in RNA in a simulated sugar conformation that enhance the hybrid affinity for complementary RNA and DNA sequences (Vester and Wengel,2004,Biochemistry 43 (42): 13233-41). RNA and DNA have different sugar moieties and may differ in the presence of uracil or an analog thereof in RNA and thymine or an analog thereof in DNA.

As used herein, "polypeptide" refers to a multimeric compound comprising amino acid residues that can adopt a three-dimensional conformation. Polypeptides include, but are not limited to, enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid binding proteins, antibodies, and the like. The polypeptide may not necessarily comprise post-translational modifications, unnatural amino acids, prosthetic groups, etc.

As used herein, "cytidine deaminase" means a polypeptide or polypeptide complex capable of having cytidine deaminase activity that catalyzes the hydrolytic deamination of cytidine or deoxycytidine, typically producing uridine or deoxyuridine. Cytidine deaminase encompasses enzymes in the cytidine deaminase superfamily, and in particular enzymes of the apobic family (enzyme subgroup apobic 1, apobic 2, apobic 4 and apobic 3), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminase (see e.g. Conticello et al mol. Biol. Evol.22:367-77, 2005;Conticello,Genome Biol.9:229, 2008; muramatsu et al, j. Biol. Chem.274:18470-6, 1999; carrington et al, cells 9:1690 (2020)). In some embodiments, variants of any known cytidine deaminase or apodec protein are contemplated. Variants include proteins having a sequence that differs from the wild-type protein by one or several mutations (i.e., substitutions, deletions, insertions), such as one or several single point substitutions. For example, the shortened sequences may be used, for example, by deleting the N-terminal, C-terminal or internal amino acids, preferably one to four amino acids at the C-terminal end of the sequence. As used herein, the term "variant" refers to allelic variants, splice variants, and natural or artificial mutations that are homologous to the reference sequence. The variant is "functional" in that it shows catalytic activity for DNA editing.

As used herein, the term "apodec 3A" refers to a cytidine deaminase, e.g., a protein expressed by the human a3A gene. Apodec 3A may have catalytic DNA editing activity. The amino acid sequence of APOBEC3A has been described (UniPROT accession ID: p 31941) and is shown as SEQ ID NO:151 are included herein. In some embodiments, the apodec 3A protein is a human apodec 3A protein or a wild-type protein. Variants include proteins having a sequence that differs from the wild-type apodec 3A protein by one or several mutations (i.e., substitutions, deletions, insertions), such as one or several single point substitutions. For example, a shortened apodec 3A sequence may be used, e.g. by deleting N-terminal, C-terminal or internal amino acids, preferably one to four amino acids at the C-terminal end of the sequence. As used herein, the term "variant" refers to allelic variants, splice variants, and natural or artificial mutants that are homologous to the apodec 3A reference sequence. The variant is "functional" in that it shows catalytic activity for DNA editing. In some embodiments, apodec 3A (e.g., human apodec 3A) has wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, apodec 3A (e.g., human apodec 3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence).

Several Cas9 orthologs have been obtained from neisseria meningitidis (Esvelt et al, nat. Methods, volume 10, 2013, 1116-1121; hou et al, PNAS, volume 110, 2013, pages 15644-15649) (Nme 1Cas9, nme2Cas9, and Nme3Cas 9). The Nme2Cas9 ortholog functions efficiently in mammalian cells, recognizes N4CCPAM, and is useful for in vivo editing (Ran et al, NATURE, volume 520, 2015, pages 186-191; kim et al, nat. Commun., volume 8, 2017, page 14500). Nme2Cas9 has been shown to be naturally resistant to off-target editing (Lee et al, MOL. THER., 24 th Vol., 2016, pages 645-654; kim et al, 2017). See also, e.g., WO/2020081568 (e.g., pages 28 and 42), which describes Nme2Cas9D16A nickases, the contents of which are hereby incorporated by reference in their entirety. In addition, nmeCas variants are known in the art, see, e.g., huang et al, nature biotech.2022, doi.org/10.1038/s41587-022-01410-2, which describe Cas9 variants targeting mononucleotide-pyrimidine PAM. Throughout, "NmeCas" or "Nme Cas9" is generic and encompasses any type of NmeCas, including Nme1Cas9, nme2Cas9, and Nme3Cas9.

As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising polypeptides from at least two different proteins or sources. One polypeptide may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein, thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein", respectively. Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising a peptide linker. Methods of recombinant protein expression and purification are well known and include those described by Green and Sambrook, molecular Cloning: a Laboratory Manual (4 th edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (2012)) which is incorporated herein by reference in its entirety.

As used herein, the term "uracil glycosidase inhibitor", "uracil DNA glycosidase inhibitor" or "UGI" refers to a protein capable of inhibiting uracil-DNA glycosidase (UDG) base-excision repair enzymes (e.g., uniProt ID: P14739; SEQ ID NO: 3).

The term "linker" as used herein refers to a chemical group or molecule that connects two adjacent molecules or moieties. Typically, the linker is located between or flanked by two groups, molecules or other moieties, and is linked to each other via a covalent bond. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., peptide or protein). Exemplary peptide linkers are disclosed elsewhere herein.

"Modified uridine" is used herein to refer to nucleosides other than thymidine that have the same hydrogen bond acceptor as uridine and that differ from uridine in one or more structural differences. In some embodiments, the modified uridine is a substituted uridine, i.e., a uridine in which one or more aprotic substituents (e.g., alkoxy groups such as methoxy groups) replace protons. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more aprotic substituents (e.g., alkyl groups such as methyl) replace protons. In some embodiments, the modified uridine is any of a substituted uridine, a pseudouridine, or a substituted pseudouridine, such as N1-methyl-pseudouridine.

As used herein, "uridine position" refers to a position in a polynucleotide occupied by uridine or a modified uridine. Thus, for example, a polynucleotide in which "100% of the uridine positions are modified uridine" contains modified uridine at each position of uridine in a conventional RNA that would be the same sequence (where all bases are standard A, U, C or G bases). Unless otherwise indicated, U in the polynucleotide sequence of the sequence table/sequence listing in or accompanying the present disclosure may be uridine or a modified uridine.

As used herein, a first sequence is considered to "comprise a sequence having at least X% identity to a second sequence" if an alignment of the first sequence to the second sequence reveals that X% or more of the entire second sequence matches the first sequence. For example, sequence AAGA comprises a sequence that has 100% identity to sequence AAG, since there are matches at all three positions of the second sequence, and thus an alignment will result in 100% identity. The difference between RNA and DNA (typically, uridine is replaced with thymidine or vice versa) and the presence of nucleoside analogues such as modified uridine does not result in a difference in identity or complementarity between polynucleotides, provided that the relevant nucleotides (e.g., thymidine, uridine or modified uridine) have the same complementary sequence (e.g., adenosine for thymidine, uridine or modified uridine; another example is cytosine and 5-methylcytosine, both with guanosine as the complementary sequence). Thus, for example, the sequence 5'-AXG (where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine) is considered to be 100% identical to AUG, since both are fully complementary to the same sequence (5' -CAU). Exemplary alignment algorithms are the Smith-Waterman (Smith-Waterman) and the Needman-Wunsch algorithm, which are well known in the art. Those skilled in the art will understand what algorithm selection and parameter settings are appropriate for a given sequence pair to be aligned; the nidman-tumbler algorithm with default settings provided by the EBI at www.ebi.ac.uk website server is generally appropriate for sequences with generally similar lengths and > 50% expected identity for amino acids or > 75% expected identity for nucleotides.

"MRNA" is used herein to refer to a polynucleotide that is RNA or modified RNA and that comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for ribosome and aminoacylating tRNA translation). The mRNA may comprise a phosphate-sugar backbone comprising ribose residues or analogs thereof, such as 2' -methoxy ribose residues. In some embodiments, the sugar of the mRNA phosphate-sugar backbone consists essentially of ribose residues, 2' -methoxy ribose residues, or combinations thereof. Typically, the mRNA does not contain a significant amount of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). The mRNA may contain modified uridine at some or all of its uridine positions.

As used herein, "RNA-guided DNA binding agent" means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or DNA binding subunits of such complexes, wherein the DNA binding activity is sequence specific and depends on the RNA sequence. Exemplary RNA-guided DNA binding agents include Cas lyase/nickase and its inactive forms ("dCas DNA binding agents"). As used herein, "Cas nuclease" is also referred to as "Cas protein" which encompasses Cas lyase, cas nickase, and dCas DNA binding agents. The dCas DNA binding agent may be a dead nuclease comprising a non-functional nuclease domain (RuvC or HNH domain). In some embodiments, the Cas lyase or Cas nickase encompasses dCas DNA binding agents modified to allow DNA cleavage, e.g., via fusion with a fokl domain. Exemplary nucleotide and polypeptide sequences for Cas9 molecules are provided below. Methods for identifying alternative nucleotide sequences encoding Cas9 polypeptide sequences (including alternative naturally occurring variants) are known in the art. Sequences having at least 75%, 80%, 85%, preferably 90%, 95%, 96%, 97%, 98% or 99% identity to any of the Cas9 nucleic acid sequences, amino acid sequences, or nucleic acid sequences encoding the amino acid sequences provided herein are also contemplated. An exemplary open reading frame for Cas9 is provided in table 39A.

As used herein, a "minimum uridine codon" for a given amino acid is the codon with the least uridine (typically 0 or 1, except for the phenylalanine codon, where the minimum uridine codon has 2 uridine). For the purpose of assessing uridine content, modified uridine residues are considered equivalent to uridine.

As used herein, the "uridine dinucleotide (UU) content" of an ORF may be expressed in absolute terms as a count of UU dinucleotides in the ORF or as a percentage of positions occupied by uridine based on the ratio expressed as uridine dinucleotides (e.g., the uridine dinucleotide content of AUUAU will be 40% because uridine of uridine dinucleotides occupies 2 out of 5 positions). For the purpose of assessing uridine dinucleotide content, modified uridine residues are considered equivalent to uridine.

As used herein, a "minimal adenine codon" for a given amino acid is a codon with minimal adenine (typically 0 or 1, except for the codons for lysine and asparagine, where the minimal adenine codon has 2 adenine). Modified adenine residues are considered equivalent to adenine for the purpose of evaluating adenine content.

As used herein, the "adenine dinucleotide content" of an ORF may be expressed in absolute terms as the AA dinucleotide count in the ORF or as a percentage of the positions occupied by adenine expressed as adenine dinucleotide based on the ratio (e.g., the adenine dinucleotide content of UAAUA will be 40% because adenine of adenine dinucleotide occupies 2 out of 5 positions). Modified adenine residues are considered equivalent to adenine for the purpose of evaluating adenine dinucleotide content.

As used herein, the "minimum repeat content" of a given Open Reading Frame (ORF) is the smallest sum possible of the occurrences of AA, CC, GG and TT (or TU, UT or UU) dinucleotides in an ORF encoding the same amino acid sequence as the given ORF. The repeat content may be expressed in absolute terms as the count of AA, CC, GG and TT (or TU, UT or UU) dinucleotides in the ORF or as the count of AA, CC, GG and TT (or TU, UT or UU) dinucleotides in the ORF divided by the nucleotide length of the ORF based on the ratio (e.g., UAAUA will have a repeat content of 20% because one repeat occurs in a sequence of 5 nucleotides). To evaluate the minimal repeat content, modified adenine, guanine, cytosine, thymine, and uracil residues are considered equivalent to adenine, guanine, cytosine, thymine, and uracil residues.

"Guide RNA," "gRNA," and "guide" are used interchangeably herein to refer to crRNA (also known as CRISPR RNA), or a combination of crRNA and trRNA (also known as tracrRNA). crrnas and trRNA can associate as a single RNA molecule (single guide RNA, sgRNA) or as two separate RNA molecules (double guide RNA, dgRNA). "guide RNA" or "gRNA" refers to each type. trRNA can be a naturally occurring sequence, or trRNA sequence having modifications or variations as compared to a naturally occurring sequence. The guide RNA can include a modified RNA as described herein. The guide RNAs described herein are suitable for use with Nme Cas9, such as Nmel, nme2, or Nme3 Cas9, unless the context clearly indicates otherwise. For example, fig. 15 shows an exemplary schematic of Nme2 sgrnas in a possible secondary structure.

As used herein, "guide sequence" or "guide region" or "spacer sequence" or the like refers to a sequence within a guide RNA that is complementary to a target sequence and that is used to direct the guide RNA to the target sequence for binding or modification (e.g., cleavage) by NmeCas. The guide sequence may be 20-25 nucleotides in length, for example in the case of Nme Cas9 and related Cas9 homologs/orthologs. Shorter or longer sequences may also be used as guides, for example 20, 21, 22, 23, 24 or 25 nucleotides in length. In the case of Nme Cas9, the guide sequence may be at least 22, 23, 24, or 25 nucleotides in length. In the case of Nme Cas9, the guide sequence may form a duplex of 22, 23, 24, or 25 consecutive base pairs with its target sequence, e.g., a duplex of 24 consecutive base pairs.

The target sequence for the Cas protein includes both the positive and negative strands of genomic DNA (i.e., the given sequence and the reverse complement of the sequence) because the nucleic acid substrate of the Cas protein is a double-stranded nucleic acid. Thus, where the guide sequence is said to be "complementary to" the target sequence, it is understood that the guide sequence may direct binding of the guide RNA to the reverse complement of the target sequence. Thus, in some embodiments, where the guide sequence binds to the reverse complement of the target sequence, the guide sequence has identity to certain nucleotides of the target sequence (e.g., the target sequence that does not include PAM) except that in the guide sequence U replaces T.

As used herein, "insertion/deletion" refers to an insertion/deletion mutation consisting of a plurality of nucleotides that are inserted or deleted at Double Strand Break (DSB) sites in a nucleic acid.

As used herein, "knock-down" refers to a decrease in expression of a particular gene product (e.g., protein, mRNA, or both). Protein knockdown can be measured by detecting proteins secreted by a tissue or cell population (e.g., in serum or cell culture medium) or by detecting the total cellular amount of protein from the tissue or cell population of interest. Methods for measuring knockdown of mRNA are known and include sequencing mRNA isolated from a tissue or cell population of interest. In some embodiments, "knockdown" may refer to some loss of expression of a particular gene product, such as a decrease in the amount of transcribed mRNA or a decrease in the amount of protein expressed or secreted by a cell population (including in vivo populations such as those found in tissues).

As used herein, "gene knockout" refers to the lack of expression of a particular protein in a cell. Gene knockout can be measured by detecting the amount of protein secreted by a tissue or cell population (e.g., in serum or cell culture medium) or by detecting the total cellular amount of protein of a tissue or cell population. In some embodiments, the methods of the present disclosure "knock out" a protein of interest in one or more cells (e.g., in a population of cells, including in vivo populations, such as those found in tissues). In some embodiments, the gene knockout does not result in the formation of a mutant of the protein of interest, for example, by insertion/deletion, but rather the complete loss of expression of the protein of interest in the cell.

As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to guide RNAs as well as RNA-guided DNA binding agents, such as Cas lyase, nickase or dCasDNA binding agents (e.g., cas 9). In some embodiments, the guide RNA directs an RNA-guided DNA binding agent, such as Cas9, to the target sequence, and the guide RNA hybridizes to the target sequence and the binding agent binds to the target sequence; in the case where the binding agent is a lyase or a nicking enzyme, the binding is followed by cleavage or nicking.

As used herein, "target sequence" refers to a nucleic acid sequence in a target gene that is complementary to the guide sequence of the gRNA. The interaction of the target sequence with the guide sequence directs RNA-guided DNA binding agent binding and potentially nicking or cleavage within the target sequence (depending on the activity of the agent).

In some embodiments, the sequence of interest may be adjacent to PAM. In some embodiments, PAM may be adjacent to or within 1,2, 3, or 4 nucleotides of the 3' end of the target sequence. The length and sequence of PAM may depend on the Cas protein used. For example, PAM may be selected from a specific Nme Cas9 protein or a consensus of Nme Cas9 orthologs or a specific PAM sequence (Edraki et al, 2019). In some embodiments, PAM may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NCC, N4GAYW, N4GYTT, N4GTCT, NNNNCC (a), NNNNCAAA (where N is defined as any nucleotide, W is defined as A or T, and R is defined as A or G; and (a) is preferred, but not required, A follows the second C). In some embodiments, the PAM sequence may be NCC.

As used herein, "treating" refers to the administration or application of a therapeutic agent for a disease or disorder in a subject, and includes slowing or arresting the development or progression of the disease, alleviating one or more signs or symptoms of the disease, curing the disease, or preventing the recurrence of one or more symptoms of the disease.

As used herein, the term "lipid nanoparticle" (LNP) refers to a particle comprising a plurality (i.e., more than one) of lipid molecules that are physically associated with each other by intermolecular forces. LNP can be, for example, microspheres (including unilamellar and multilamellar vesicles, e.g., "liposomes," in some embodiments, substantially spherical lamellar phase lipid bilayers, and in more particular embodiments can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules), a dispersed phase in an emulsion, micelles, or an internal phase in suspension. Emulsions, micelles, and suspensions may be compositions suitable for topical or surface delivery. See also e.g. WO2017173054A1, the content of which is hereby incorporated by reference in its entirety. Any LNP known to those of skill in the art capable of delivering nucleotides to a subject can be used with the guide RNAs described herein and nucleic acids encoding RNA-guided DNA binding agents.

As used herein, the term "nuclear localization signal" (NLS) or "nuclear localization sequence" refers to an amino acid sequence that induces the transport of molecules comprising or linked to such sequences to the nucleus of eukaryotic cells. The nuclear localization signal may form part of the molecule to be transported. In some embodiments, the NLS may be attached to the remainder of the molecule by covalent bonds, hydrogen bonds, or ionic interactions.

As used herein, "delivery" and "administration" are used interchangeably and include in vitro and in vivo applications.

As used herein, co-administration means that the multiple substances are administered together in close enough temporal proximity to act together with the agent. Co-administration encompasses both administration of substances together in a single formulation and administration of substances close enough in time in separate formulations that the agents act together.

As used herein, the phrase "pharmaceutically acceptable" means useful in preparing a pharmaceutical composition that is generally non-toxic and not biologically undesirable and not otherwise unacceptable for pharmaceutical use. Pharmaceutically acceptable generally refers to non-pyrolyzing substances. Pharmaceutically acceptable may refer to sterile substances, particularly medical substances for injection or infusion.

Exemplary polynucleotides and compositions

In some embodiments, a polynucleotide is provided, the polynucleotide comprising an Open Reading Frame (ORF), the ORF comprising:

A nucleotide sequence encoding a C-terminal neisseria meningitidis (Nme) Cas9 polypeptide that is at least 90% identical to any one of SEQ ID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、301-303 or 316-321; and

A nucleotide sequence encoding a first Nuclear Localization Signal (NLS); and

In some embodiments, nme Cas9 is Nme2 Cas9. In some embodiments, nme Cas9 is Nme1 Cas9. In some embodiments, nme Cas9 is Nme3Cas9. In some embodiments, the ORF further comprises a nucleotide sequence encoding a second NLS. In some embodiments, the polynucleotide is mRNA.

In some embodiments, the ORF comprises a sequence having at least 90%, 95%, 98% or 100% identity to the sequence of any one of SEQ ID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、301-303 or 316-321. In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:29 or 32-41 has a sequence that is at least 90%, 95%, 98% or 100% identical. In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:32 has a sequence of at least 90%, 95%, 98% or 100% identity. In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:33 has a sequence having at least 90%, 95%, 98% or 100% identity. In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:34, has a sequence that is at least 90%, 95%, 98% or 100% identical. In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:35 has a sequence having at least 90%, 95%, 98% or 100% identity. In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:36, has a sequence of at least 90%, 95%, 98% or 100% identity. In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:38 has a sequence having at least 90%, 95%, 98% or 100% identity. In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:39 has a sequence having at least 90%, 95%, 98% or 100% identity. In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:41 has a sequence having at least 90%, 95%, 98% or 100% identity.

In some embodiments, the ORF comprises a nucleotide sequence that hybridizes to SEQ ID NO:38 or 41 has a sequence having at least 90%, 95%, 98% or 100% identity.

In some embodiments, a polynucleotide is provided comprising an ORF as disclosed herein. In some embodiments, a polynucleotide encoding an Nme Cas9 polypeptide that is at least 90% identical to any one of SEQ ID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、301-303 or 316-321 is provided, wherein Nme Cas9 is Nme2 Cas9, nme3 Cas9, or Nme1 Cas9; a first Nuclear Localization Signal (NLS); and a second NLS, wherein the encoded first NLS and second NLS are N-terminal to NmeCas polypeptides.

In some embodiments, a polypeptide comprising a sequence that hybridizes to SEQ ID NO:1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, or 297, or any of 310-315 has an Nme Cas9 polypeptide that is at least 90% identical to any of the amino acid sequences of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, wherein Nme Cas9 is Nme2 Cas9, nme3 Cas9, or Nme1 Cas9; a first Nuclear Localization Signal (NLS); and a second NLS, wherein the encoded first NLS and second NLS are N-terminal to NmeCas polypeptides.

In some embodiments, a method of modifying a gene of interest is provided, the method comprising administering a composition described herein. In some embodiments, the method comprises delivering to the cell a polynucleotide comprising an Open Reading Frame (ORF), the ORF comprising: a nucleotide sequence encoding a C-terminal neisseria meningitidis (Nme) Cas9 polypeptide that is at least 90% identical to any one of SEQ ID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、301-303 or 316-321, wherein Nme Cas9 is Nme2 Cas9 or Nme1 Cas9 or Nme3 Cas9; a nucleotide sequence encoding a first Nuclear Localization Signal (NLS); and optionally a nucleotide sequence encoding a second NLS. In some embodiments, the polynucleotide is delivered to the cell in vitro. In some embodiments, the polynucleotide is delivered to a cell in vivo.

In some embodiments, the compositions described herein further comprise at least one gRNA. In some embodiments, a composition is provided that comprises an mRNA as described herein and at least one gRNA. In some embodiments, the gRNA is a single guide gRNA (sgRNA). In some embodiments, the gRNA is a dual guide gRNA (dgRNA).

In some embodiments, the composition is capable of effecting genome editing upon administration to a subject. In some embodiments, the subject is a human.

A. RNA-guided DNA binding agents; nmeCas 9A 9

RNA-guided DNA binding agents described herein encompass neisseria meningitidis Cas9 (NmeCas 9) and modifications and variants thereof. In some embodiments, nmeCas is Nme2Cas9. In some embodiments, nmeCas is Nme1 Cas9. In some embodiments, nmeCas is Nme3 Cas9.

A modified version with one inactive catalytic domain (RuvC or HNH) is called "nicking enzyme". Nicking enzymes cleave only one strand of the target DNA, thereby creating a single strand break. Single strand breaks may also be referred to as "nicks". In some embodiments, the compositions and methods comprise a nicking enzyme. In some embodiments, the compositions and methods comprise a nicking enzyme RNA-guided DNA binding agent, such as nicking enzyme Cas, e.g., nicking enzyme Cas9, that induces nicking rather than double strand breaks in the DNA of interest.

In some embodiments, nmeCas nuclease may be modified to contain only one functional nuclease domain. For example, an RNA-guided DNA binding agent may be modified such that one of the nuclease domains is mutated or deleted, either entirely or partially, to reduce its nucleic acid cleavage activity.

In some embodiments, nmeCas nicking enzymes with reduced activity RuvC domains are used. In some embodiments, nmeCas nicking enzymes with inactive RuvC domains are used. In some embodiments, nmeCas nicking enzymes with reduced activity HNH domains are used. In some embodiments, nmeCas nicking enzymes with inactive HNH domains are used.

In some embodiments, conservative amino acids within the NmeCas nuclease domain are substituted to reduce or alter nuclease activity. Wild-type Cas9 has two nuclease domains: ruvC and HNH. RuvC domains cleave non-target DNA strands, and HNH domains cleave target DNA strands. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain or more than one HNH domain. In some embodiments, the Cas9 nuclease is a wild-type Cas9. In some embodiments, cas9 is capable of inducing a double strand break in the DNA of interest. In certain embodiments, the Cas nuclease can cleave dsDNA, which can cleave one strand of dsDNA, or which can have no DNA cleaving enzyme or nickase activity. In some embodiments NmeCas may comprise amino acid substitutions in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in RuvC or RuvC-like nuclease domains include H588A (based on neisseria meningitidis Cas9 protein). In some embodiments, the Cas protein may comprise amino acid substitutions in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include D16A (based on NmeCas proteins).

In some embodiments, chimeric Cas proteins are used in which one domain or region of a protein is replaced with a portion of a different protein. In some embodiments, nmeCas nuclease domains can be replaced with domains from a different nuclease (e.g., fok 1). In some embodiments, nmeCas protein may be a modified NmeCas nuclease.

In some embodiments, the nuclease may be modified to induce a point mutation or base change, such as deamination.

In some embodiments, the Cas protein comprises a fusion protein comprising a Cas nuclease (e.g., nmeCas) linked to a heterologous functional domain, which Cas nuclease is a nickase or is catalytically inactive. In some embodiments, the Cas protein comprises a fusion protein comprising a catalytically inactive Cas nuclease (e.g., nmeCas) linked to a heterologous functional domain (see, e.g., WO 2014152432). In some embodiments, the catalytically inactive Cas9 is from neisseria meningitidis Cas9. In some embodiments, the catalytically inactive Cas comprises a mutation that inactivates the Cas.

In some embodiments, the heterologous functional domain is a domain that modulates gene expression, histone or DNA. In some embodiments, the heterologous functional domain is a transcriptional activation domain or a transcriptional repression domain. In some embodiments, the nuclease is a catalytically inactive Cas nuclease, such as dCas9.

In some embodiments, the heterologous domain is a deaminase, such as a cytidine deaminase or an adenine deaminase. In certain embodiments, the heterologous domain is a C-to-T base converter (cytidine deaminase), such as an apolipoprotein B mRNA editor (apodec) deaminase. For example, the heterologous functional domain of the deaminase may be part of a fusion protein containing a Cas nuclease having nickase activity or a catalytically inactive Cas nuclease, discussed further below.

In some embodiments, nme Cas9 has double stranded endonuclease activity.

In some embodiments, nme Cas9 has nickase activity.

In some embodiments, nme Cas9 comprises dCas9 DNA binding domain.

In some embodiments, nme Cas9 comprises the amino acid sequence set forth in SEQ ID NO:1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290 or 297 or any of 310-315, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (as shown in table 39A). In some embodiments, nme Cas9 comprises SEQ ID NO:1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290 or 297 or any of 310-315 (as shown in table 39A).

In some embodiments, the sequence encoding NmeCas9 comprises a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO：15、18-27、29、32-41、221-226、228-233、235-240、242-247、249-254、256-261、263-268、270-275、277-282、284-289、291-296、298-303、304-309 or 316-321 (as shown in table 39A). In some embodiments, the sequence encoding NmeCas9 comprises the nucleotide sequence of any one of SEQ ID NO：15、18-27、29、32-41、221-226、228-233、235-240、242-247、249-254、256-261、263-268、270-275、277-282、284-289、291-296、298-303、304-309 or 316-321 (as shown in table 39A).

In some embodiments, any of the foregoing levels of identity is at least 95%, at least 98%, at least 99%, or 100%.

B. Exemplary coding sequences

In any of the embodiments set forth herein, the polynucleotide is an mRNA comprising an ORF encoding the RNA-guided DNA binding agent disclosed above. In any of the embodiments set forth herein, the polynucleotide is an mRNA comprising an ORF encoding NmeCas. In any of the embodiments set forth herein, the polynucleotide may be an expression construct comprising a promoter operably linked to an ORF encoding an RNA-guided DNA binding agent (e.g., nmeCas).

For each polypeptide molecule produced by an mRNA molecule, some ORFs translate more efficiently in vivo than others. Codon pair usage of such efficiently translated ORFs may contribute to translation efficiency. Further description of the ORF coding sequence, codon pair usage, improvements in codon repetition content are disclosed in WO 2019/0067910 and WO 2020/198641, the respective contents of which are hereby incorporated by reference in their entirety.

For example, in some embodiments, at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons of the ORF are minimal adenine codons or minimal uridine codons. In some embodiments, the ORF comprises or consists of codons that increase translation of mRNA in the mammal. In some embodiments, the ORF comprises or consists of codons that increase translation of the mRNA in a human. The increase in translation in a mammal, a cell type, an organ of a mammal, a human, an organ of a human, etc., can be determined relative to the extent of translation of the wild-type sequence of the ORF or relative to an ORF having a codon distribution that matches the codon distribution of the organism from which the ORF originates or the organism containing the most similar ORF at the amino acid level.

In some embodiments, the GC content of the ORF is greater than or equal to 56%. In some embodiments, the GC content of the ORF is greater than or equal to 56.5%. In some embodiments, the GC content of the ORF is greater than or equal to 57%. In some embodiments, the GC content of the ORF is greater than or equal to 57.5%. In some embodiments, the GC content of the ORF is greater than or equal to 58%. In some embodiments, the GC content of the ORF is greater than or equal to 58.5%. In some embodiments, the GC content of the ORF is greater than or equal to 59%. In some embodiments, the GC content of the ORF is less than or equal to 63%. In some embodiments, the GC content of the ORF is less than or equal to 62.6%. In some embodiments, the GC content of the ORF is less than or equal to 62.1%. In some embodiments, the GC content of the ORF is less than or equal to 61.6%. In some embodiments, the GC content of the ORF is less than or equal to 61.1%. In some embodiments, the GC content of the ORF is less than or equal to 60.6%. In some embodiments, the GC content of the ORF is less than or equal to 60.1%.

In some embodiments, the ORF consists of a set of codons, wherein at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are those listed in table 1.

Table 1.

Amino acids	Low A/U
		Gly	GGC
Glu	GAG
		Asp	GAC
Val	GTG
		Ala	GCC
Arg	CGG
		Ser	AGC
Lys	AAG
		Asn	AAC
Met	ATG
		Ile	ATC
Thr	ACC
		Trp	TGG
Cys	TGC
		Tyr	TAC
Leu	CTG
		Phe	TTC
Gln	CAG
		His	CAC

1. ORF with low uridine content

In some embodiments, the uridine content of the ORF encoding the polypeptide is in the range of about 150% of its minimum uridine content to its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum uridine content. In some embodiments, the uridine content of the ORF is equal to its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 150% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 145% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 140% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 135% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 130% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 125% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 120% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 115% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 110% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 105% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 104% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 103% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 102% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 101% of its minimum uridine content.

In some embodiments, the uridine dinucleotide content of the ORF is in the range of from its minimum uridine dinucleotide content to 200% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 195％、190％、185％、180％、175％、170％、165％、160％、155％、150％、145％、140％、135％、130％、125％、120％、115％、110％、105％、104％、103％、102％ or 101% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is equal to its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 200% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 195% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 190% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 185% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 180% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 175% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 170% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 165% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 160% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 155% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is equal to its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 150% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 145% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 140% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 135% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 130% of its minimum uridine dinucleotide content. in some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 125% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 120% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 115% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 110% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 105% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 104% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 103% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 102% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 101% of its minimum uridine dinucleotide content.

In some embodiments, the uridine dinucleotide content of the ORF is in the range of its minimum uridine dinucleotide content to 90% or less of the maximum uridine dinucleotide content of a reference sequence encoding the same protein as the mRNA in question. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the maximum uridine dinucleotide content of a reference sequence encoding the same protein as the mRNA in question.

In some embodiments, the uridine trinucleotide content of the ORF is in the range of 0 uridine trinucleotide to 1, 2,3,4,5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 uridine trinucleotide (wherein the longer strings of uridine count is the number of unique trinucleoside segments therein, e.g. uridine tetranucleotide contains two uridine trinucleotide, uridine pentanucleotide contains three uridine trinucleotide, etc.). In some embodiments, the uridine trinucleotide content of the ORF ranges from 0% uridine trinucleotide to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5% or 2% uridine trinucleotide, wherein the uridine trinucleotide content is calculated as a percentage of positions in the sequence of uridine forming part of a uridine trinucleotide (or longer strings of uridine), such that sequences UUUAAA and UUUUAAAA will each have a uridine trinucleotide content of 50%. For example, in some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 2%. For example, in some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 1.5%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 1%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.9%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.8%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.7%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.6%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.5%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.4%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.3%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.2%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.1%. In some embodiments, the ORF does not have uridine trinucleotide.

In some embodiments, the uridine trinucleotide content of the ORF is in the range of its minimum uridine trinucleotide content to 90% or less of the maximum uridine trinucleotide content of the reference sequence encoding the same protein as the polynucleotide in question. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the maximum uridine trinucleotide content of a reference sequence encoding the same protein as the polynucleotide in question.

In some embodiments, the ORF has a minimum nucleotide homopolymer, e.g., a repeating string of identical nucleotides. For example, in some embodiments, when the smallest uridine codon is selected from the codons listed in table 2, the polynucleotide is constructed by selecting the smallest uridine codon that reduces the number and length of nucleotide homopolymers (e.g., GCA for alanine, GCC for glycine, GGA for GGG, or AAG for lysine, AAA).

The uridine content or uridine dinucleotide content or uridine trinucleotide content of a given ORF can be reduced, for example, by using the smallest uridine codon in a sufficient portion of the ORF. For example, the amino acid sequence of a polypeptide encoded by an ORF described herein can be translated back into the ORF sequence by converting the amino acid to codons, wherein some or all of the ORFs use the exemplary minimal uridine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons in the ORF are the codons listed in table 2.

TABLE 2 exemplary minimum uridine codons

In some embodiments, the ORF consists of a set of codons, wherein at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are those listed in table 2.

2. ORF with low adenine content

In some embodiments, the adenine content of the ORF is in the range of from its minimum adenine content to about 150% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum adenine content. In some embodiments, the adenine content of the ORF is equal to its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 150% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 145% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 140% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 135% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 130% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 125% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 120% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 115% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 110% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 105% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 104% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 103% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 102% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 101% of its minimum adenine content.

In some embodiments, the adenine dinucleotide content of the ORF is in the range of from its minimum adenine dinucleotide content to 200% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 195％、190％、185％、180％、175％、170％、165％、160％、155％、150％、145％、140％、135％、130％、125％、120％、115％、110％、105％、104％、103％、102％ or 101% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is equal to its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 200% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 195% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 190% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 185% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 180% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 175% of its minimum adenine dinucleotide content. in some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 170% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 165% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 160% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 155% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is equal to its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 150% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 145% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 140% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 135% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 130% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 125% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 120% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 115% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 110% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 105% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 104% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 103% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 102% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 101% of its minimum adenine dinucleotide content.

In some embodiments, the adenine dinucleotide of the ORF is in a range from its minimum adenine dinucleotide content to 90% or less of the maximum adenine dinucleotide content of a reference sequence encoding the same protein as the polynucleotide in question. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the maximum adenine dinucleotide content of a reference sequence encoding the same protein as the polynucleotide in question.

In some embodiments, the adenine trinucleotide content of the ORF ranges from 0 adenine trinucleotide to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 adenine trinucleotides (wherein the longer strand adenine is counted by the number of unique trinucleotide segments therein, e.g., adenine tetranucleotide contains two adenine trinucleotides, adenine pentanucleotide contains three adenine trinucleotides, etc.). In some embodiments, the adenine trinucleotide content of the ORF ranges from 0% adenine trinucleotide to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5% or 2% adenine trinucleotide, wherein the adenine trinucleotide percentage content is calculated as a percentage of positions in the sequence of adenine forming part of the adenine trinucleotide (or longer strings of adenine) such that sequences UUUAAA and UUUUAAAA each have an adenine trinucleotide content of 50%. For example, in some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 2%. For example, in some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 1.5%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 1%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.9%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.8%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.7%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.6%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.5%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.4%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.3%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.2%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.1%. In some embodiments, the ORF does not have adenine trinucleotide.

In some embodiments, the adenine trinucleotide of the ORF is in the range of its minimum adenine trinucleotide content to 90% or less of the maximum adenine trinucleotide content of a reference sequence encoding the same protein as the polynucleotide in question. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the maximum adenine trinucleotide content of a reference sequence encoding the same protein as the polynucleotide in question. In some embodiments, the ORF has a minimum nucleotide homopolymer, e.g., a repeating string of identical nucleotides. For example, in some embodiments, when the smallest adenine codon is selected from the codons listed in table 3, the polynucleotide is constructed by selecting the smallest adenine codon that reduces the number and length of nucleotide homopolymers (e.g., GCA for alanine, GCC for glycine, GGA for GGG, or AAG for lysine, AAA). The adenine content or adenine dinucleotide content or adenine trinucleotide content in a given ORF can be reduced, for example, by using a minimum number of adenine codons in a sufficient portion of the ORF. For example, the amino acid sequence of a polypeptide encoded by an ORF described herein can be translated back into the ORF sequence by converting the amino acid into codons, wherein some or all of the ORFs use the exemplary minimal adenine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons in the ORF are the codons listed in table 3.

TABLE 3 exemplary minimal adenine codons

In some embodiments, the ORF consists of a set of codons, wherein at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are those listed in table 3.

3. ORF with low adenine and low uridine content

In terms of feasibility, any of the features described above with respect to low adenine content may be combined with any of the features described above with respect to low uridine content. For example, the uridine content of the ORF is in the range of from its minimum uridine content to about 150% of its minimum uridine content (e.g., the uridine content of the ORF is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum uridine content) and the adenine content is in the range of from its minimum adenine content to about 150% of its minimum adenine content (e.g., less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum adenine content). The same is true for uridine and adenine dinucleotide. Similarly, the content of uridine nucleotides and adenine dinucleotides in an ORF can be as set forth above. Similarly, the content of uridine dinucleotides and adenine nucleotides in an ORF can be as set forth above.

The uridine and adenine nucleotide or dinucleotide content of a given ORF can be reduced, for example, by using minimal uridine and adenine codons in a sufficient portion of the ORF. For example, the amino acid sequence of a polypeptide encoded by an ORF described herein can be translated back into the ORF sequence by converting the amino acid to codons, wherein some or all of the ORFs use the exemplary minimal uridine and adenine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons in the ORF are the codons listed in table 4.

TABLE 4 exemplary minimum uridine and adenine codons

	Amino acids	Minimum uridine codons
			A	Alanine (Ala)	GCC or GCG
G	Glycine (Gly)	GGC or GGG
			V	Valine (valine)	GUC or GUG
D	Aspartic acid	GAC
			E	Glutamic acid	GAG
I	Isoleucine (Ile)	AUC
			T	Threonine (Thr)	ACC or ACG
N	Asparagine derivatives	AAC
			K	Lysine	AAG
S	Serine (serine)	AGC or UCC or UCG
			R	Arginine (Arg)	CGC or CGG
L	Leucine (leucine)	CUG or CUC
			P	Proline (proline)	CCG or CCC
H	Histidine	CAC
			Q	Glutamine	CAG
F	Phenylalanine (Phe)	UUC
			Y	Tyrosine	UAC
C	Cysteine (S)	UGC
			W	Tryptophan	UGG
M	Methionine	AUG

In some embodiments, the ORF consists of a set of codons, wherein at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are those listed in table 4. As can be seen in table 4, each of the three listed serine codons contains one a or one U. In some embodiments, uridine minimization is performed for serine by prioritization using AGC codons. In some embodiments, adenine minimization is performed for serine by using UCC or UCG codon prioritization.

4. Codons that enhance translation or correspond to highly expressed trnas; exemplary codon sets

In some embodiments, the ORF has codons that increase translation in a mammal (e.g., a human). In other embodiments, the ORF has codons that increase translation in an organ (e.g., liver) of a mammal (e.g., a human). In other embodiments, the ORF has codons that increase translation in a mammalian (e.g., human) cell type (e.g., hepatocyte). The increase in translation in a mammal, a cell type, an organ of a mammal, a human, an organ of a human, etc., can be determined relative to the extent of translation of the wild-type sequence of the ORF or relative to an ORF having a codon distribution that matches the codon distribution of the organism from which the ORF originates or the organism containing the most similar ORF at the amino acid level.

In some embodiments, the polypeptide encoded by the ORF is a Cas9 nuclease derived from a prokaryote described below, and the increase in translation in a mammal, cell type, mammalian organ, human organ, etc., can be determined relative to the extent of translation of the wild-type sequence of the ORF or relative to the ORF of interest (e.g., an ORF encoding a human protein or transgene for expression in a human cell). For example, the ORF may be an ORF having the following codon distribution: the codon distribution is the same as that of the organism from which the ORF was derived or an organism containing the most similar ORF at the amino acid level, e.g., neisseria meningitidis, or with respect to translation of the Cas9 ORF contained in SEQ ID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、301-303 or 316-321, all other conditions being equal, including any applicable point mutations, heterologous domains, etc. Codons suitable for use in increasing expression in humans (including human livers and human hepatocytes) can be codons corresponding to highly expressed trnas in human livers/hepatocytes, which are discussed in DITTMAR KA, PLOS Genetics 2 (12): e221 (2006). In some embodiments, at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the ORF are codons corresponding to highly expressed trnas (e.g., highest expressed trnas for each amino acid) in a mammal (e.g., human). In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the ORF are codons corresponding to highly expressed trnas (e.g., highest expressing trnas for each amino acid) in a mammalian organ (e.g., human organ). In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the ORF are codons corresponding to highly expressed trnas (e.g., highest expressing trnas for each amino acid) in the mammalian liver (e.g., human liver). In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the ORF are codons corresponding to highly expressed trnas (e.g., highest expressing trnas for each amino acid) in mammalian hepatocytes (e.g., human hepatocytes).

Alternatively, codons corresponding to highly expressed tRNA in an organism (e.g., a human) can be used in general.

Any of the foregoing codon usage methods may be combined with selecting codons that help reduce repeat content; or using the codon set of table 1 as shown above; the minimum uridine or adenine codons shown above, e.g., in tables 2, 3, or 4, are used, and then when more than one option is available, codons corresponding to higher expressed tRNA in a general organism (e.g., human), or in an organ or cell type of interest, e.g., liver or hepatocyte (e.g., human liver or human hepatocyte).

C. nuclear Locating Signal (NLS)

The Nuclear Localization Signals (NLS) disclosed herein may facilitate RNA-guided transport of DNA binding agents into the nucleus. The first NLS and (when present) second NLS disclosed herein can be linked at the N-terminus to an RNA-guided DNA binding agent sequence, i.e., the RNA-guided DNA binding agent is the C-terminal domain in the encoded polypeptide. The first NLS and (when present) the second NLS disclosed herein can be linked at the N-terminus to a NmeCas coding sequence. Additional NLS may be ligated at the N-terminus of NmeCas coding sequences. In some embodiments, the encoded polypeptide comprises three NLS located at the N-terminus of the NmeCas coding sequence. In some embodiments, at least one NLS is provided at the C-terminus of the RNA-guided DNA binding agent sequence (e.g., with or without an intervening spacer between the NLS and the previous domain). In some embodiments, the first NLS and the second NLS are provided at the C-terminus of the RNA-guided DNA binding agent sequence (e.g., with or without an intervening spacer between the NLS and the preceding domain).

Thus, in some embodiments, the ORF encoding the polypeptides disclosed herein comprises a coding sequence of a first NLS and a coding sequence of a second NLS such that the encoded first NLS and second NLS are located at the N-terminus of NmeCas polypeptides. In some embodiments, the ORF further comprises a coding sequence of a third NLS located at the C-terminus of the ORF encoding Nme Cas 9.

In some embodiments, the NLS may be a single-part (monopartite) sequence, such as SV40NLS, PKKKRKV (SEQ ID NO: 388) or PKKKRRV (SEQ ID NO: 421). In some embodiments, the NLS may be a bipartite sequence, such as NLS, KRPAATKKAGQAKKKK (SEQ ID NO: 422) of a nucleoplasmin. In some embodiments, the NLS sequence may comprise LAAKRSRTT(SEQ ID NO：410)、QAAKRSRTT(SEQ ID NO：411)、PAPAKRERTT(SEQ ID NO：412)、QAAKRPRTT(SEQ ID NO：413)、RAAKRPRTT(SEQ ID NO：414)、AAAKRSWSMAA(SEQ ID NO：415)、AAAKRVWSMAF(SEQ ID NO：416)、AAAKRSWSMAF(SEQ ID NO：417)、AAAKRKYFAA(SEQ ID NO：418)、RAAKRKAFAA(SEQ ID NO：419)、 or RAAKRKYFAV (SEQ ID NO: 420). The NLS may be an intranuclear microribonucleoprotein input protein (snurportin) -1 internalization facilitator (importin) - β (IBB domain, e.g., SPN1-impβ sequence see Huber et al, 2002, J.cell Bio.,156, 467-479. In one particular embodiment, a single PKKKRKV (SEQ ID NO: 388.) in some embodiments, the first NLS and the second NLS are independently selected from the group consisting of SV40NLS, nucleoplasmin NLS, bipartite NLS, c-myc-like NLS, and NLS comprising sequence KTRAD. In certain embodiments, the first NLS and the second NLS may be identical (e.g., two SV40 NLS). In certain embodiments, the first NLS and the second NLS may be different.

In some embodiments, the first NLS is an SV40NLS and the second NLS is a nucleoplasmin NLS.

In some embodiments, the SV40 NLS comprises the sequence PKKKRKVE (SEQ ID NO: 383) or KKKRKVE (SEQ ID NO: 384). In some embodiments, the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 422). In some embodiments, the bipartite NLS comprises the sequence of KRTADGSEFESPKKKRKVE (SEQ ID NO: 385). In some embodiments, the c-myc-like NLS comprises the sequence PAAKKKKLD (SEQ ID NO: 386).

In some embodiments, one or more NLSs according to any of the preceding embodiments are present in an RNA-guided DNA binding agent in combination with one or more additional heterologous functional domains, such as any of the heterologous functional domains described below.

D. Other heterologous functional domains

In some embodiments, the polypeptide encoded by the ORF described herein (e.g., an RNA-guided DNA binding agent) comprises one or more additional heterologous functional domains (e.g., is or comprises a fusion polypeptide). In some embodiments, the ORF further comprises a nucleotide sequence encoding one or more additional heterologous functional domains.

In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain can serve as a signal peptide for protein degradation. In some embodiments, protein degradation may be mediated by proteolytic enzymes, such as proteasome, lysosomal proteases, or calpain (calpain proteases). In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA binding agent may be modified by the addition of ubiquitin or polyubiquitin chains. In some embodiments, the ubiquitin can be ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon stimulatory gene-15 (ISG 15)), ubiquitin-related modifier-1 (URM 1), down-regulated protein-8 of neuronal-precursor-cell expression (NEDD 8, also known as Rub1 in saccharomyces cerevisiae (s. Cerevisae)), human leukocyte antigen F-related (FAT 10), autophagy-8 (ATG 8) and autophagy-12 (ATG 12), fau ubiquitin-like protein (FUB 1), membrane anchored UBL (MUB), ubiquitin fold modifier-1 (UFM 1) and ubiquitin-like protein-5 (UBL 5).

In some embodiments, the heterologous functional domain may be a tag domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent protein (e.g., GFP-2, tagGFP, turboGFP, sfGFP, EGFP, emerald, azami Green, monomers Azami Green, copGFP, aceGFP, zsGreenl), yellow fluorescent protein (e.g., YFP, EYFP, citrine, venus, YPet, phiYFP, zsYellowl), blue fluorescent protein (e.g., EBFP2, azurite, mKalamal, GFPuv, sapphire, T-sapphire), cyan fluorescent protein (e.g., ECFP, cerulean, cyPet, amCyanl, midoriishi-Cyan), red fluorescent protein (mKate, mKate2, mPlum, dsRed monomer, mCherry, mRFP1, dsRed-Express, dsRed2, dsRed-monomer, hcRed-Tandmem, hcRed1, asRed2, eqFP, mRasberry, mStrawberry, jred), and Orange fluorescent protein (mOrange, mKO, kusabira-Orange, monomers Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the tag domain may be a purification tag or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin Binding Protein (CBP), maltose Binding Protein (MBP), thioredoxin (TRX), poly (NANP), tandem Affinity Purification (TAP) tag 、myc、AcV5、AU1、AU5、E、ECS、E2、FLAG、HA、nus、Softag 1、Softag 3、Strep、SBP、Glu-Glu、HSV、KT3、S、S1、T7、V5、VSV-G、6xHis、8xHis、 Biotin Carboxyl Carrier Protein (BCCP), polyHis, calmodulin, and HiBiT. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol Acetyl Transferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

In other embodiments, the heterologous functional domain may target an RNA-guided DNA binding agent to a particular organelle, cell type, tissue, or organ.

In other embodiments, the heterologous functional domain may be an effector domain. The effector domain may modify or affect the target sequence when the RNA-guided DNA binding agent is directed to its target sequence, e.g., when the Cas nuclease is directed to the target sequence by the gRNA. In some embodiments, the effector domain may be selected from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain. In some embodiments, the heterologous domain is a nuclease, such as a fokl nuclease. See, for example, U.S. patent No. 9,023,649. In some embodiments, the heterologous domain is a transcriptional activator or repressor. See, e.g., qi et al ,"Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression",Cell 152：1173-83(2013);Perez-Pinera et al ,"RNA-guided gene activation by CRISPR-Cas9-based transcription factors",Nat.Methods10：973-6(2013);Mali et al ,"CAS9transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering",Nat.Biotechnol.31：833-8(2013);Gilbert et al ,"CRI SPR-mediated modular RNA-guided regulation of transcription in eukaryotes",Cell 154：442-51(2013)., and therefore, RNA-guided DNA binding agents essentially become transcription factors that can be guided using guide RNA to bind to a desired target sequence. In certain embodiments, the DNA modification domain is a methylation domain, such as a demethylation or methyltransferase domain. In certain embodiments, the effector domain is a DNA modification domain, such as a base editing domain. In particular embodiments, the DNA modification domain is a nucleic acid editing domain, such as a deaminase domain, that introduces specific modifications into DNA, which is discussed further below.

In some embodiments, the ORF further comprises a nucleotide sequence encoding a linker sequence between the first NLS and the second NLS.

In some embodiments, the ORF further comprises a nucleotide sequence encoding a linker sequence between the Nme Cas9 coding sequence and the NLS proximal to the Nme Cas9 coding sequence.

In some embodiments, the spacer comprises at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more amino acids. In some embodiments, the spacer comprises at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

In some embodiments, the peptide linker is a 16 residue "XTEN" linker or variant thereof (see, e.g., examples; and Schellenberger et al ,A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner.Nat.Biotechnol.27,1186-1190(2009)). in some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 58), SGSETPGTSESA (SEQ ID NO: 59), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 60).

In some embodiments, the peptide linker comprises a (GGGGS)_n(SEQ ID NO：62)、(G)_n、(EAAAK)_n(SEQ ID NO：63)、(GGS)_n(SEQ ID NO：61) or SGSETPGTSESATPES (SEQ ID NO: 58) motif (see, e.g., Guilinger J P,Thompson D B,Liu D R.Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.Nat.Biotechnol.2014;32(6)：577-82;, incorporated herein by reference in its entirety), or a (XP) n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. See WO2015089406, e.g., paragraph [0012], the entire contents of which are incorporated herein by reference.

In some embodiments, the peptide linker comprises a sequence selected from the group consisting of SEQ ID NOs: 61-122, one of the or multiple sequences.

E.UTR; kozak sequence

In some embodiments, the polynucleotide comprises at least one UTR from hydroxysteroid 17-beta dehydrogenase 4 (HSD 17B4 or HSD), e.g., a 5' UTR from HSD. In some embodiments, the polynucleotide comprises at least one UTR from a globin mRNA, such as human alpha globin (HBA) mRNA, human beta globin (HBB) mRNA, or Xenopus Beta Globin (XBG) mRNA. In some embodiments, the polynucleotide comprises a 5'utr, 3' utr, or 5 'and 3' utr from a globin mRNA, e.g., HBA, HBB, or XBG. In some embodiments, the polynucleotide comprises a 5' utr from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, albumin gene, hba, HBB, or XBG. In some embodiments, the polynucleotide comprises a 3' utr from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, albumin gene, hba, HBB, or XBG. In some embodiments, the polynucleotide comprises 5 'and 3' utrs from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin genes, HBA, HBB, XBG, heat shock protein 90 (Hsp 90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β -actin, α -tubulin, tumor protein (p 53), or Epidermal Growth Factor Receptor (EGFR).

In some embodiments, the polynucleotide comprises 5 'and 3' utrs from the same source, e.g., constitutively expressed mRNA, e.g., actin, albumin, or globin, such as HBA, HBB, or XBG.

In some embodiments, a polynucleotide disclosed herein comprises a nucleotide sequence that hybridizes to SEQ ID NO:391-398 have a 5' UTR with at least 90% identity. In some embodiments, a polynucleotide disclosed herein comprises a nucleotide sequence that hybridizes to SEQ ID NO:399-406 have a 3' UTR with at least 90% identity. In some embodiments, any of the foregoing levels of identity is at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the mRNA disclosed herein comprises a polypeptide having SEQ ID NO:391-398, any of which are shown in phantom the 5' UTR of the sequence of the same. In some embodiments, a polynucleotide disclosed herein comprises a nucleotide sequence having SEQ ID NO:399-406 3' UTR of the sequence of the same.

In some embodiments, the polynucleotide does not comprise a 5'utr, e.g., no additional nucleotides are present between the 5' cap and the initiation codon. In some embodiments, the mRNA comprises a Kozak sequence (described below) between the 5 'cap and the start codon, but does not have any additional 5' utr. In some embodiments, the mRNA does not comprise a 3' utr, e.g., no additional nucleotides are present between the stop codon and the poly-a tail.

In some embodiments, the mRNA comprises a Kozak sequence. The Kozak sequence can affect translation initiation and overall yield of polypeptides translated from mRNA. The Kozak sequence includes a methionine codon that may serve as an initiation codon. The minimum Kozak sequence is NNNRUGN, where at least one of the following holds: the first N is a or G and the second N is G. In the case of nucleotide sequences, R means purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG or RNNAUGG. In some embodiments, the Kozak sequence is rccRUGg with zero mismatches or up to one or two mismatches at positions in the lower case form. In some embodiments, the Kozak sequence is rccAUGg with zero mismatches or up to one or two mismatches at positions in the lower case form. In some embodiments, the Kozak sequence is gccRccAUGG (nucleotides 4-13 of SEQ ID NO: 408; SEQ ID NO: 407) with zero mismatches or up to one, two, or three mismatches at positions in lower case. In some embodiments, the Kozak sequence is gccAccAUG with zero mismatches or up to one, two, three, or four mismatches at positions in lower case letters. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG (SEQ ID NO: 408) with zero mismatches or up to one, two, three or four mismatches at positions in lower case form.

5' Cap

In some embodiments, a polynucleotide (e.g., mRNA) disclosed herein comprises a 5' Cap, such as Cap0, cap1, or Cap2.

The 5' cap is typically a 7-methylguanine ribonucleotide (which may be further modified, as discussed below, for example, with respect to ARCA) that is linked via a 5' -triphosphate to the 5' position of the first nucleotide of the 5' to 3' strand of the nucleic acid, i.e., the first cap proximal nucleotide. In Cap0, the ribose sugar of both the first and second Cap proximal nucleotides of the mRNA contain a 2' -hydroxyl group. In Cap1, the ribose of the first and second transcribed nucleotides of mRNA contain 2 '-methoxy and 2' -hydroxy, respectively. In Cap2, ribose of the first and second Cap proximal nucleotides of mRNA contain 2' -methoxy. See, e.g., katibah et al, (2014) Proc NATL ACAD SCI USA 111 (33): 12025-30; abbas et al (2017) Proc NATL ACAD SCI USA 114 (11): E2106-E2115. Most endogenous higher eukaryotic nucleic acids, including mammalian nucleic acids (e.g., human nucleic acids), comprise Cap1 or Cap2. Cap0, as well as other Cap structures different from Cap1 and Cap2, can be immunogenic in mammals (e.g., humans) due to the recognition by components of the innate immune system such as IFIT-1 and IFIT-5 as "non-self," which can result in elevated levels of cytokines (including type I interferons). Components of the innate immune system such as IFIT-1 and IFIT-5 can also compete with eIF4E for binding to nucleic acids having caps other than Cap1 or Cap2, potentially inhibiting nucleic acid translation.

The cap may be included in a co-transcribed manner. For example, ARCA (anti-reverse cap analogue; thermo FISHER SCIENTIFIC catalog AM 8045) is a cap analogue comprising 7-methylguanine 3' -methoxy-5 ' -triphosphate linked to the 5' position of guanine ribonucleotides, which can be incorporated into transcripts in vitro at the beginning. ARCA produces Cap0 caps or Cap 0-like caps, wherein the 2' position of the nucleotide proximal to the first Cap is a hydroxyl group. See, e.g., STEPINSKI et al ,(2001)"Synthesis and properties of mRNAs containing the novel'anti-reverse'cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′deoxy)GpppG",RNA 7：1486-1495. below for an ARCA structure.

CleanCap ^TM AG (m 7G (5 ') ppp (5') (2 'OMeA) pG; triLink Biotechnologies catalog number N-7113) or CleanCap ^TM GG (m 7G (5') ppp (5 ') (2' OMeG) pG; triLink Biotechnologies catalog number N-7133) may be used to provide Cap 1 structure in a co-transcriptional manner. 3' -O-methylated versions of CleanCap ^TM AG and CleanCap ^TM GG are also available under the catalogue numbers N-7413 and N-7433, respectively, from TriLink Biotechnologies. CleanCap ^TM AG structure is shown below. The CleanCap ^TM structure is sometimes referred to herein using the last three digits of the catalog numbers listed above (e.g., for the TriLink Biotechnologies catalog number N-7113, the use of "CleanCap ^TM" designation).

Alternatively, the cap may be added to the RNA in a post-transcriptional fashion. For example, vaccinia capping enzymes are commercially available (NEW ENGLAND Biolabs catalog number M2080S) and have RNA triphosphatase and guanylate transferase activities provided by their D1 subunits, and guanine methyltransferases provided by their D12 subunits. Thus, 7-methylguanine can be added to RNA in the presence of S-adenosylmethionine and GTP to produce Cap0. See, e.g., guo, p. And Moss, b. (1990) proc.Natl. Acad.Sci.USA 87, 4023-4027; mao, x. And Shuman, s. (1994) j.biol. Chem.269, 24472-24479. For additional discussion of caps and capping methods, see, e.g., WO2017/053297 and Ishikawa et al, nucleic acids sylp.ser. (2009) stage 53, 129-130.

Poly-A tail

In some embodiments, the polynucleotide is an mRNA encoding a polypeptide disclosed herein comprising an ORF, and the mRNA further comprises a polyadenylation (poly-a) tail.

In some embodiments, the polynucleotides disclosed herein further comprise a poly-A tail sequence or a polyadenylation signal sequence. In some embodiments, the poly-A tail sequence comprises 100-400 nucleotides.

In some embodiments, the poly-A sequence comprises non-adenine nucleotides. In some cases, the poly-A tail is "interrupted" at one or more positions within the poly-A tail by one or more non-adenine nucleotide "anchors". The poly-A tail may comprise at least 8 consecutive adenine nucleotides, but also one or more non-adenine nucleotides. As used herein, "non-adenine nucleotide" refers to any natural or non-natural nucleotide that does not contain adenine. Guanine, thymine and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tail on an mRNA described herein can comprise consecutive adenine nucleotides located 3' of the nucleotides encoding the polypeptides disclosed herein. In some cases, the poly-a tail on the mRNA comprises a non-contiguous adenine nucleotide located 3' of a nucleotide encoding an RNA-guided DNA binding agent or sequence of interest, wherein the non-adenine nucleotides interrupt the adenine nucleotides at regular or irregular intervals.

In some embodiments, the poly-A tail is encoded in a plasmid for in vitro transcription of mRNA and becomes part of the transcript. The number of consecutive adenine nucleotides in the poly-A sequence encoded in the plasmid, i.e., the poly-A sequence, may not be precise, e.g., a 100poly-A sequence in the plasmid may not produce exactly 100poly-A sequences in the transcribed mRNA. In some embodiments, the poly-A tail is not encoded in a plasmid and is added by PCR tailing or enzymatic tailing, e.g., using E.coli poly (A) polymerase.

In some embodiments, one or more non-adenine nucleotides are positioned to disrupt consecutive adenine nucleotides, such that a poly (a) binding protein can bind to a stretch of consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides follow at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides follow at least 8-50 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides follow at least 8-100 consecutive adenine nucleotides. In some embodiments, the non-adenine nucleotides follow one, two, three, four, five, six, or seven adenine nucleotides and are followed by at least 8 consecutive adenine nucleotides.

The poly-A tail of the present disclosure may comprise one of the following sequences: consecutive adenine nucleotides are followed by one or more non-adenine nucleotides, optionally followed by additional adenine nucleotides.

In some embodiments, the poly-A tail comprises or contains one non-adenine nucleotide or a contiguous stretch of 2-10 non-adenine nucleotides. In some embodiments, the non-adenine nucleotide follows at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some cases, one or more non-adenine nucleotides follow at least 8-50 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides follow at least 8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49 or 50 consecutive adenine nucleotides.

In some embodiments, the non-adenine nucleotide is guanine, cytosine, or thymine. In some cases, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is thymine nucleotide. In some cases where more than one non-adenine nucleotide is present, the non-adenine nucleotide may be selected from: a) Guanine and thymine nucleotides; b) Guanine and cytosine nucleotides; c) Thymine and cytosine nucleotides; or d) guanine, thymine and cytosine nucleotides. An exemplary poly-A tail comprising a non-adenine nucleotide is provided as SEQ ID NO:409.

In some embodiments, the poly-a tail sequence comprises SEQ ID NO: 409.

G. modified nucleotides

In some embodiments, a nucleic acid comprising an ORF encoding a polypeptide disclosed herein comprises a modified uridine at some or all uridine positions.

In some embodiments, the modified uridine is a uridine modified at position 5, e.g., with halogen or C1-C3 alkoxy. In some embodiments, the modified uridine is a pseudouridine modified at position 1, e.g., with a C1-C3 alkyl group. The modified uridine can be, for example, pseudouridine, N1-methyl pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methylpseuduridines. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions in the polynucleotides according to the present disclosure are modified uridine. In some embodiments, at least 10% of uridine at uridine positions in the polynucleotides according to the present disclosure are replaced by modified uridine. In some embodiments, at least 20% of uridine at uridine positions in the polynucleotides according to the present disclosure are replaced by modified uridine. In some embodiments, at least 30% of uridine at uridine positions in the polynucleotides according to the present disclosure are replaced by modified uridine. In some embodiments, at least 80% of uridine at uridine positions in the polynucleotides according to the present disclosure are replaced with modified uridine. In some embodiments, at least 90% of the uridine at the uridine positions in the polynucleotides according to the present disclosure are replaced by modified uridine.

In some embodiments, 10% -25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a polynucleotide according to the present disclosure are modified uridine. In some embodiments, 15% to 45% of uridine in uridine positions in polynucleotides according to the present disclosure are replaced with modified uridine.

In some embodiments, 100% of the uridine at the uridine positions in the polynucleotides according to the present disclosure are replaced with modified uridine.

In some embodiments, the modified uridine is one or more of N1-methyl-pseudouridine, 5-methoxyuridine, or 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is one or both of N1-methyl-pseudouridine or 5-methoxyuridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is 5-methoxyuridine.

In some embodiments, 10% -25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a polynucleotide according to the present disclosure are 5-methoxyuridine. In some embodiments, 10% -25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a polynucleotide according to the present disclosure are pseudouridine. In some embodiments, 10% -25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a polynucleotide according to the present disclosure are N1-methylpseuduridines. In some embodiments, 10% -25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a polynucleotide according to the present disclosure are 5-iodouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in the polynucleotides according to the present disclosure are 5-methoxyuridine, and the remainder are N1-methyl pseudouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in the polynucleotides according to the present disclosure are 5-iodouridine, and the remainder are N1-methyl pseudouridine. In some embodiments, 15% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in a polynucleotide according to the present disclosure are substituted with a modified uridine, optionally wherein the modified uridine is N1-methyl-pseudouridine. In some embodiments, 15% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in the polynucleotides according to the present disclosure are substituted with N1-methyl-pseudouridine. In some embodiments, 85%, 90%, 95%, or 100% of uridine positions in polynucleotides according to the present disclosure are substituted with N1-methyl-pseudouridine. In some embodiments, 100% of the uridine is replaced with N1-methyl-pseudouridine. In some embodiments, 15% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in a polynucleotide according to the present disclosure are substituted with a modified uridine, optionally wherein the modified uridine is a pseudouridine. In some embodiments, 15% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in a polynucleotide according to the present disclosure are substituted with pseudouridine. In some embodiments, 85%, 90%, 95%, or 100% of the uridine positions in the polynucleotides according to the present disclosure are substituted with pseudouridine. In some embodiments, 100% of the uridine is replaced with pseudouridine.

Exemplary polynucleotides and compositions comprising deaminase and RNA-guided nicking enzyme

The RNA-guided DNA binding agents disclosed herein can also comprise a base editing domain that introduces specific modifications to the target nucleic acid, e.g., deaminase domain.

In some embodiments, a nucleic acid is provided comprising an open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., A3A) and a C-terminal NmeCas nickase, and a first Nuclear Localization Signal (NLS), wherein the polypeptide does not comprise a Uracil Glycosidase Inhibitor (UGI).

In some embodiments, the second NLS is located N-terminal to the Nme Cas9 nickase. In some embodiments, the deaminase is located at the N-terminus of the NLS (i.e., the first NLS or the second NLS). In some embodiments, the deaminase is located at the N-terminus of all NLS in the polypeptide. In some embodiments, and wherein the polypeptide does not comprise a Uracil Glycosidase Inhibitor (UGI).

In some embodiments, the polynucleotide is DNA or RNA. In some embodiments, the polynucleotide is mRNA. In some embodiments, a polypeptide encoded by an mRNA is provided.

In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, an optional NLS, a cytidine deaminase (e.g., apodec 3A), an optional linker, a D16A NmeCas nickase. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, an optional NLS, a cytidine deaminase (e.g., apodec 3A), an optional linker, a D16A Nme2Cas9 nickase. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a first NLS and a second NLS, a cytidine deaminase (e.g., apodec 3A), an optional linker, a D16A NmeCas nickase. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a first NLS and a second NLS, a cytidine deaminase (e.g., apodec 3A), an optional linker, D16ANme2Cas9 nickase. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a first NLS, a cytidine deaminase (e.g., apodec 3A), a second NLS, an optional linker, a D16A NmeCas nickase. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a first NLS, a cytidine deaminase (e.g., apodec 3A), a second NLS, an optional linker, a D16A Nme2Cas9 nickase.

In some embodiments, the polypeptide comprises A3A and the RNA-guided nicking enzyme does not comprise a Uracil Glycosidase Inhibitor (UGI).

In some embodiments, a composition is provided comprising a first polypeptide or mRNA encoding a first polypeptide comprising a cytidine deaminase, optionally apodec 3A deaminase (a 3A); c-terminal NmeCas nickase; a first Nuclear Localization Signal (NLS); and optionally a second NLS; wherein the first NLS and (when present) the second NLS are located N-terminal to the sequence encoding NmeCas nickase, wherein the first polypeptide does not comprise a Uracil Glycosidase Inhibitor (UGI); and a second polypeptide, or mRNA encoding a second polypeptide, comprising a Uracil Glycosidase Inhibitor (UGI), wherein the second polypeptide is different from the first polypeptide.

In some embodiments, a method of modifying a gene of interest is provided, the method comprising administering a composition described herein. In some embodiments, the method comprises delivering to the cell a first nucleic acid comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase, optionally apodec 3A deaminase (a 3A); c-terminal NmeCas nickase; a first Nuclear Localization Signal (NLS); and optionally a second NLS; wherein the first NLS and (when present) the second NLS are N-terminal to a sequence encoding NmeCas nicking enzyme, wherein the first polypeptide does not comprise a Uracil Glycosidase Inhibitor (UGI), and a second nucleic acid comprising a second open reading frame encoding a Uracil Glycosidase Inhibitor (UGI), wherein the second nucleic acid is different from the first nucleic acid.

In some embodiments, the method comprises delivering to the cell a polypeptide comprising a deaminase, optionally apodec 3A deaminase (a 3A); c-terminal NmeCas nickase; a first Nuclear Localization Signal (NLS); and a second NLS; wherein the first NLS and the second NLS are N-terminal to a sequence encoding NmeCas nicking enzyme, wherein the first polypeptide does not comprise a Uracil Glycosidase Inhibitor (UGI), or a nucleic acid encoding a polypeptide, and delivering the Uracil Glycosidase Inhibitor (UGI), or the nucleic acid encoding UGI, to a cell.

In some embodiments, the molar ratio of the mRNA encoding UGI to the mRNA encoding APOBEC3A deaminase (e.g., A3A) and RNA directed nicking enzyme is about 1:35 to about 30:1. In some embodiments, the molar ratio is from about 1:25 to about 25:1. In some embodiments, the molar ratio is from about 1:20 to about 25:1. In some embodiments, the molar ratio is from about 1:10 to about 22:1. In some embodiments, the molar ratio is from about 1:5 to about 25:1. In some embodiments, the molar ratio is from about 1:1 to about 30:1. In some embodiments, the molar ratio is from about 2:1 to about 10:1. In some embodiments, the molar ratio is from about 5:1 to about 20:1. In some embodiments, the molar ratio is from about 1:1 to about 25:1. In some embodiments, the molar ratio may be about 1∶35、1∶34、1∶33、1∶32、1∶31、1∶30、1∶32、1∶31、1∶30、1∶29、1∶28、1∶27、1∶26、1∶25、1∶24、1∶23、1∶22、1∶21、1∶20、1∶19、1∶18、1∶17、1∶16、1∶15、1∶14、1∶13、1∶12、1∶11、1∶10、1∶9、1∶8、1∶7、1∶6、1∶5、1∶4、1∶3、1∶2、1∶1、2∶1、3∶1、4∶1、5∶1、6∶1、7∶1、8∶1、9∶1、10∶1、11∶1、12∶1、13∶1、14∶1、15∶1、16∶1、17∶1、18∶1、19∶1、20∶1、21∶1、22∶1、23∶1、24∶1、25∶1、26∶1、27∶1、28∶1、29∶1 or 30:1. In some embodiments, the molar ratio is equal to or greater than about 1:1. In some embodiments, the molar ratio is about 1:1. In some embodiments, the molar ratio is about 2:1. In some embodiments, the molar ratio is about 3:1. In some embodiments, the molar ratio is about 4:1. In some embodiments, the molar ratio is about 5:1. In some embodiments, the molar ratio is about 6:1. In some embodiments, the molar ratio is about 7:1. In some embodiments, the molar ratio is about 8:1. In some embodiments, the molar ratio is about 9:1. In some embodiments, the molar ratio is about 10:1. In some embodiments, the molar ratio is about 11:1. In some embodiments, the molar ratio is about 12:1. In some embodiments, the molar ratio is about 13:1. In some embodiments, the molar ratio is about 14:1. In some embodiments, the molar ratio is about 15:1. In some embodiments, the molar ratio is about 16:1. In some embodiments, the molar ratio is about 17:1. In some embodiments, the molar ratio is about 18:1. In some embodiments, the molar ratio is about 19:1. In some embodiments, the molar ratio is about 20:1. In some embodiments, the molar ratio is about 21:1. In some embodiments, the molar ratio is about 22:1. In some embodiments, the molar ratio is about 23:1. In some embodiments, the molar ratio is about 24:1. In some embodiments, the molar ratio is about 25:1.

Similarly, in some embodiments, if the protein is delivered, the molar ratio discussed above with respect to the mRNA encoding the UGI protein is similar to the mRNA encoding apodec 3A deaminase (a 3A) and RNA guided nicking enzyme.

In some embodiments, the composition is capable of effecting genome editing after administration to a subject.

A. Cytidine deaminase; APOBEC3A deaminase

Cytidine deaminase encompasses enzymes in the cytidine deaminase superfamily, and in particular enzymes of the apobic family (enzyme subgroup apobic 1, apobic 2, apobic 4 and apobic 3), activity-induced cytidine deaminase (AID or AICDA) and CMP deaminase (see e.g. Conticello et al mol. Biol. Evol.22:367-77, 2005;Conticello,Genome Biol.9:229, 2008; muramatsu et al, j. Biol. Chem.274:18470-6, 1999; and Carrington et al, cells 9:1690 (2020)).

In some embodiments, the cytidine deaminase disclosed herein is an enzyme of the apodec family. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of the apodec 1, apodec 2, apodec 4, and apodec 3 subgroup. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of the apodec 3 subgroup. In some embodiments, the cytidine deaminase disclosed herein is apodec 3A deaminase (a 3A). In some embodiments, the deaminase comprises apodec 3A deaminase.

In some embodiments, the apodec 3A deaminase (a 3A) disclosed herein is human a3A. In some embodiments, the apodec 3A deaminase (a 3A) disclosed herein is human a3A. In some embodiments, A3A is wild-type A3A.

In some embodiments, A3A is an A3A variant. The A3A variant shares homology with wild type A3A or a fragment thereof. In some embodiments, the A3A variant has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with wild-type A3A. In some embodiments, the A3A variant may have 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、21、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50 or more amino acid changes compared to wild-type A3A. In some embodiments, the A3A variant comprises a fragment of A3A such that the fragment is at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a corresponding fragment of wild-type A3A.

In some embodiments, the A3A variant is a protein having a sequence that differs from the wild-type A3A protein by one or several mutations, such as substitutions, deletions, insertions, one or several single point substitutions. In some embodiments, the shortened A3A sequence may be used, for example, by deleting N-terminal, C-terminal, or internal amino acids. In some embodiments, a shortened A3A sequence is used, wherein one to four amino acids at the C-terminus of the sequence are deleted. In some embodiments, apodec 3A (e.g., human apodec 3A) has wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, apodec 3A (e.g., human apodec 3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence).

In some embodiments, wild-type A3A is human A3A (UniProt accession ID: p319411, SEQ ID NO: 151).

In some embodiments, A3A disclosed herein comprises a nucleotide sequence that hybridizes to SEQ ID NO:151 has an amino acid sequence having at least 80% identity. In some embodiments, the level of identity is at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, A3A comprises a nucleotide sequence that hybridizes to SEQ ID NO:151 has an amino acid sequence of at least 87% identity. In some embodiments, A3A comprises a nucleotide sequence that hybridizes to SEQ ID NO:151 has an amino acid sequence having at least 90% identity. In some embodiments, A3A comprises a nucleotide sequence that hybridizes to SEQ ID NO:151 has an amino acid sequence of at least 95% identity. In some embodiments, A3A comprises a nucleotide sequence that hybridizes to SEQ ID NO:151 has an amino acid sequence of at least 98% identity. In some embodiments, A3A comprises a nucleotide sequence that is identical to A3A ID NO:151 has an amino acid sequence of at least 99% identity. In some embodiments, A3A comprises SEQ ID NO: 151.

In some embodiments, a cytidine deaminase disclosed herein comprises a nucleotide sequence that hybridizes to SEQ ID NO:151-216 has an amino acid sequence that is at least 80% identical. In some embodiments, the level of identity is at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the cytidine deaminase comprises SEQ ID NO:151-216 amino acid sequence of the same.

B.UGI

Without being bound by any theory, providing UGI and polypeptides comprising deaminase may facilitate the methods described herein by inhibiting cellular DNA repair mechanisms (e.g., UDG and downstream repair effectors) that would recognize uracil in DNA as a form of DNA damage or would otherwise cleave or modify uracil or surrounding nucleotides. It is understood that the use of UGI can increase the efficiency of editing enzymes capable of deaminating C residues.

Suitable UGI proteins and nucleotide sequences are provided herein and additional suitable UGI sequences are known to those skilled in the art and include, for example, those disclosed in: wang et al ,Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase.J.Biol.Chem.264：1163-1171(1989);Lundquist et al ,Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein.Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase.J.Biol.Chem.272：21408-21419(1997);Ravishankar et al ,X-ray analysis ofa complex of Escherichia coli uracil DNA glycosylase(EcUDG)with a proteinaceous inhibitor.The structure elucidation of a prokaryotic UDG.Nucleic Acids Res.26：4880-4887(1998); and Putnam et al ,Proteinmimicry of DNA from crystal structures of the uracil-DNA glycosylaseinhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase.J.Mol.Biol.287：331-346(1999), are each incorporated herein by reference in their entirety. It is understood that any protein capable of inhibiting uracil-DNA glycosidase base excision repair enzymes is within the scope of the present disclosure. In addition, any protein that blocks or inhibits base excision repair is also within the scope of the present disclosure. In some embodiments, the uracil glycosidase inhibitor is a protein that binds uracil. In some embodiments, the uracil glycosidase inhibitor is a protein that binds uracil in DNA. In some embodiments, the uracil glycosidase inhibitor is a single chain binding protein. In some embodiments, the uracil glycosidase inhibitor is a catalytically inactive uracil DNA-glycosidase protein. In some embodiments, the uracil glycosidase inhibitor is a catalytically inactive uracil DNA-glycosidase protein that does not cleave uracil from DNA. In some embodiments, the uracil glycosidase inhibitor is a catalytically inactive UDG.

In some embodiments, the Uracil Glycosidase Inhibitors (UGIs) disclosed herein comprise an amino acid sequence that hybridizes to SEQ ID NO:3 has an amino acid sequence of at least 80%. In some embodiments, any of the foregoing levels of identity is at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the UGI comprises a sequence that is identical to SEQ ID NO:3 having an amino acid sequence with at least 90% identity. In some embodiments, the UGI comprises a sequence that is identical to SEQ ID NO:3 having an amino acid sequence of at least 95% identity. In some embodiments, the UGI comprises a sequence that is identical to SEQ ID NO:3 having an amino acid sequence of at least 98% identity. In some embodiments, the UGI comprises a sequence that is identical to SEQ ID NO:3 having an amino acid sequence of at least 99% identity. In some embodiments, the UGI comprises SEQ ID NO:3, and a sequence of amino acids.

C. Joint

In some embodiments, the polypeptide comprising a deaminase and an RNA-guided nicking enzyme described herein further comprises a linker that links the deaminase and the RNA-guided nicking enzyme. In some embodiments, the linker is a peptide linker. In some embodiments, the nucleic acid encoding a polypeptide comprising a deaminase and an RNA-guided nicking enzyme further comprises a sequence encoding a peptide linker. In some embodiments, mRNA encoding a deaminase-linker-fusion protein is provided.

In some embodiments, the peptide linker is any amino acid stretch having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more amino acids.

In some embodiments, the peptide linker comprises a (GGGGS)_n(SEQ ID NO：62)、(G)_n、(EAAAK)_n(SEQ ID NO：63)、(GGS)_n(SEQ ID NO：61) or SGSETPGTSESATPES (SEQ ID NO: 58) motif (see, e.g., Guilinger J P,Thompson D B,Liu D R.Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.Nat.Biotechnol.2014;32(6)：577-82;, incorporated herein by reference in its entirety), or a (XP) _n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. See WO2015089406, e.g., paragraph [0012], the entire contents of which are incorporated herein by reference.

In some embodiments, the peptide linker comprises a sequence selected from the group consisting of SEQ ID NOs: 58-122, one of the or multiple sequences. In some embodiments, the peptide linker comprises a sequence selected from the group consisting of SEQ ID NOs: 58. SEQ ID NO: 59. SEQ ID NO: 60. SEQ ID NO: 118. SEQ ID NO: 119. SEQ ID NO: 120. SEQ ID NO:121 and SEQ ID NO:122, and one or more sequences of 122.

D. Compositions comprising apobic 3A deaminase and RNA-guided nicking enzyme

In some embodiments, an mRNA encoding a polypeptide comprising cytidine deaminase (e.g., A3A) and RNA-guided nicking enzyme is provided. In some embodiments, the polypeptide comprises a human deaminase (e.g., A3A) and a C-terminal RNA-guided nicking enzyme; and a nucleotide sequence encoding the first NLS and optionally the second NLS. In certain embodiments, the deaminase is located at the N-terminus of the NLS. In certain embodiments, the deaminase is located at the N-terminus of all NLS.

In some embodiments, the polypeptide comprises a wild-type deaminase (e.g., A3A) and a C-terminal RNA-guided nicking enzyme. In some embodiments, the polypeptide comprises an A3A variant and an RNA-guided nicking enzyme. In some embodiments, the polypeptide comprises a deaminase (e.g., A3A) and a Cas9 nickase. In some embodiments, the polypeptide comprises a deaminase (e.g., A3A) and a D16ANmeCas nickase. In some embodiments, the polypeptide comprises a human deaminase (e.g., A3A) and D16A NmeCas nickase. In some embodiments, the polypeptide comprises an A3A variant and a D16A NmeCas nickase. In some embodiments, the polypeptide lacks UGI. In some embodiments, the deaminase (e.g., A3A) and RNA-guided nicking enzyme are linked via a linker. In some embodiments, the polypeptide further comprises one or more additional heterologous functional domains. In some embodiments, the polypeptide further comprises a Nuclear Localization Sequence (NLS) (described herein).

In some embodiments, the polypeptide comprises a human deaminase (e.g., A3A) and a C-terminal D16A NmeCas nickase, wherein the human deaminase (e.g., A3A) and D16ANmeCas are fused via a linker. In some embodiments, the polypeptide comprises human A3A and C-terminal D16A NmeCas nickase and an NLS located at the N-terminus of the fusion polypeptide. In some embodiments, the polypeptide comprises human A3A and C-terminal D16A NmeCas nickase, wherein human A3A and D16A NmeCas9 are fused via a linker, and the NLS is optionally fused to the N-terminus of human A3A via a linker.

Polypeptides may be organized in a variety of ways to form single chains. The first NLS and (when present) the second NLS are located at the N-terminus of the sequence encoding Cas9 nickase. The additional NLS may be located at the N-terminus of the Cas9 nickase. A3A may be N-terminal or C-terminal compared to NLS. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a first NLS, optionally a second NLS, deaminase, optionally a linker, RNA-guided nicking enzyme, and optionally an NLS. In some embodiments, the linker is independently present between the first and second NLSs and the NLS and deaminase. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a deaminase, a first NLS, optionally a second NLS, a C-terminal RNA-guided nicking enzyme. In some embodiments, the linker is independently present between the deaminase and the first NLS, between the first NLS and the second NLS, and between the NLS and the C-terminal nicking enzyme.

In any of the preceding embodiments, the polypeptide may comprise a sequence that hybridizes to SEQ ID NO:14 having an amino acid sequence having at least 80% identity. In some embodiments, any of the foregoing levels of identity are at least 85%, 90%, 95%, 98%, or 99%, or 100% identical. In some embodiments, a polypeptide disclosed herein may comprise a sequence that hybridizes to SEQ ID NO:14 having an amino acid sequence having at least 90% identity. In some embodiments, a polypeptide disclosed herein may comprise a sequence that hybridizes to SEQ ID NO:3 or 6 has an amino acid sequence having at least 95% identity. In some embodiments, a polypeptide disclosed herein may comprise a sequence that hybridizes to SEQ ID NO:14 has an amino acid sequence having at least 98% identity. In some embodiments, a polypeptide disclosed herein may comprise a sequence that hybridizes to SEQ ID NO:14 having an amino acid sequence of at least 99% identity. In some embodiments, a polypeptide disclosed herein may comprise SEQ ID NO:14, and a sequence of amino acids.

In any of the preceding embodiments, the nucleic acid sequence comprising an open reading frame encoding a polypeptide disclosed herein may comprise a sequence that hybridizes to SEQ ID NO:42, and a nucleic acid sequence having at least 80% identity. In some embodiments, any of the foregoing levels of identity are at least 85%, 90%, 95%, 98%, or 99%, or 100% identical.

In any of the foregoing embodiments, the mRNA sequence encoding a polypeptide disclosed herein can comprise a sequence identical to SEQ ID NO:28, a nucleic acid sequence having at least 80% identity. In some embodiments, any of the foregoing levels of identity are at least 85%, 90%, 95%, 98%, or 99%, or 100% identical.

In any of the preceding embodiments, A3A may comprise a sequence identical to SEQ ID NO:151 has an amino acid sequence having at least 80% identity. In some embodiments, the level of identity is at least 85%, 87%, 90%, 95%, 98%, or 99%, or 100% identical. In some embodiments, A3A comprises SEQ ID NO: 151.

In any of the preceding embodiments, nmeCas a nickase may comprise a sequence that hybridizes to SEQ ID NO: 220. 248 or 276, or a fragment thereof, has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical. In some embodiments, the level of identity is at least 85%, 87%, 90%, 95%, 98%, or 99%, or 100% identical. In some embodiments, the RNA guided nicking enzyme comprises SEQ ID NO: 220. 248 or 276. In some embodiments, the RNA guided nicking enzyme comprises SEQ ID NO: 220. 248 or 276. In some embodiments, the RNA guided nicking enzyme comprises SEQ ID NO: 220. 248 or 276.

Guide RNA

In some embodiments, at least one guide RNA is provided in combination with a polynucleotide disclosed herein, e.g., a polynucleotide encoding an RNA-guided DNA binding agent. In some embodiments, the guide RNA is provided as a molecule that is separate from the polynucleotide. In some embodiments, the guide RNA is provided as part of a polynucleotide disclosed herein, e.g., part of a UTR.

In some embodiments, the compositions comprising the polynucleotides disclosed herein further comprise at least one guide RNA (or "gRNA").

In some embodiments, the gRNA is a single guide gRNA (or "sgRNA").

In some embodiments, the gRNA is a double guide RNA.

In some embodiments, the guide RNA comprises a modified sgRNA. The sgrnas may be modified to improve their in vivo stability.

In some embodiments, the gRNA described herein is a neisseria meningitidis Cas9 (NmeCas 9) gRNA comprising a conserved portion that contains a repeat/anti-repeat region, a hairpin 1 region, and a hairpin 2 region, wherein one or more of the repeat/anti-repeat region, the hairpin 1 region, and the hairpin 2 region are shortened. An exemplary wild-type NmeCas guide RNA comprises the sequence of (N)_20-25GUUGUAGCUCCCUUUCUCAUUUCGGAAACGAAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCGCAACGCUCUGCCCCUUAAAGCUUCUGCUUUAAGGGGCAUCGUUUA(SEQ ID NO：500). As used herein, (N) _20-25 denotes 20-25, i.e. 20, 21, 22, 23, 24 or 25 abuts N. A. C, G and U represent nucleotides having adenine, cytosine, guanine and uracil bases, respectively. In some embodiments, (N) _20-25 is 24 nucleotides in length. N is any natural or unnatural nucleotide, and wherein the population of N comprises a guide sequence.

In some embodiments, a single guide RNA comprises a guide region and a conserved region, wherein the conserved region comprises one or more of:

(Ii) Nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b)

(Ii) Nucleotide 81 is linked to nucleotide 96 by at least 4 nucleotides; or (b)

(Ii) Nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides;

Wherein relative to SEQ ID NO:500, one or two nucleotides 144-145 are optionally deleted; and wherein at least 10 nucleotides are modified nucleotides.

In some embodiments, the shortened repeat/anti-repeat region of the gRNA lacks 18 nucleotides. In some embodiments, the shortened repeat/anti-repeat region of the gRNA lacks 22 nucleotides.

In some embodiments, nucleotide 36 is linked to nucleotide 65 by 6 nucleotides in the shortened repeat/anti-repeat region of the gRNA. In some embodiments, nucleotide 36 is linked to nucleotide 65 by 7 nucleotides in the shortened repeat/anti-repeat region of the gRNA. In some embodiments, nucleotide 36 is linked to nucleotide 65 by 8 nucleotides in the shortened repeat/anti-repeat region of the gRNA. In some embodiments, nucleotide 36 is linked to nucleotide 65 by 9 nucleotides in the shortened repeat/anti-repeat region of the gRNA. In some embodiments, nucleotide 36 is linked to nucleotide 65 by 10 nucleotides in the shortened repeat/anti-repeat region of the gRNA.

In some embodiments, in the shortened repeat/anti-repeat region of the gRNA, nucleotides 38-48 and 53-63 are compared to SEQ ID NO:500 is missing. In some embodiments, in the shortened repeat/anti-repeat region of the gRNA, nucleotides 38, 41-48, 53-60, and 63 are compared to SEQ ID NO:500 is missing.

In some embodiments, nucleotide 36 is linked to nucleotide 65 by 6 nucleotides in the shortened repeat/anti-repeat region of the gRNA. In some embodiments, in the shortened repeat/anti-repeat region of the gRNA, nucleotides 38-48 and 53-63 are compared to SEQ ID NO:500, and nucleotide 36 is linked to nucleotide 65 by nucleotides 37, 49-52 and 64.

In some embodiments, nucleotide 36 is linked to nucleotide 65 by 10 nucleotides in the shortened repeat/anti-repeat region of the gRNA. In some embodiments, in the shortened repeat/anti-repeat region of the gRNA, nucleotides 38, 41-48, 53-60, and 63 are compared to SEQ ID NO:500, and nucleotide 36 is linked to nucleotide 65 by nucleotides 37, 39, 40, 49-52, 61, 62 and 64.

In some embodiments, all nucleotides 38-48 and nucleotides 53-63 of the upper stem of the shortened repeat/anti-repeat region are identical to the amino acid sequence of SEQ ID NO:500 is missing.

In some embodiments, all nucleotides 39-48 and nucleotides 53-62 of the upper stem of the shortened repeat/anti-repeat region are identical to the amino acid sequence of SEQ ID NO:500, and nucleotides 38 and 63 are substituted.

In some embodiments, the shortened repeat/anti-repeat region has 14 modified nucleotides. In some embodiments, the shortened repeat/anti-repeat region has 15 modified nucleotides. In some embodiments, the shortened repeat/anti-repeat region has 16 modified nucleotides. In some embodiments, the shortened repeat/anti-repeat region has 17 modified nucleotides. In some embodiments, the shortened repeat/anti-repeat region has 18 modified nucleotides. In some embodiments, the shortened repeat/anti-repeat region has 19 modified nucleotides. In some embodiments, the shortened repeat/anti-repeat region has 20 modified nucleotides.

In some embodiments, the shortened hairpin 1 region lacks 2 nucleotides. In some embodiments, the shortened hairpin 1 region lacks 21 nucleotides. In some embodiments, the shortened hairpin 1 region lacks 2 nucleotides, and nucleotides 86 and 91 are relative to SEQ ID NO:500 is missing. In some embodiments, the shortened hairpin 1 region lacks 2 nucleotides, and nucleotides 85 and 92 are relative to SEQ ID NO:500 is missing. In some embodiments, in the shortened hairpin 1 region, nucleotide 81 is connected to nucleotide 96 by 12 nucleotides. In some embodiments, in the shortened hairpin 1 region, nucleotide 81 is connected to nucleotide 96 by 12 nucleotides. In some embodiments, in the shortened hairpin 1 region, nucleotides 86 and 91 are relative to SEQ ID NO:500, and nucleotide 81 is linked to nucleotide 96 by nucleotides 82-85, 87-90 and 92-95. In some embodiments, in the shortened hairpin 1 region, nucleotides 85 and 92 relative to SEQ ID NO:500, and nucleotide 81 is linked to nucleotide 96 by nucleotides 82-84, 86-91 and 93-95.

In some embodiments, the shortened hairpin 1 region has a duplex portion of 7 base pairing nucleotides in length. In some embodiments, the shortened hairpin 1 region has a duplex portion of 8 base pairing nucleotides in length.

In the stem of the shortened hairpin 1 region, the length is seven base pairing nucleotides. In some embodiments, nucleotides 85-86 and nucleotides 91-92 of the shortened hairpin 1 region are deleted.

In some embodiments, the shortened hairpin 1 region has 13 modified nucleotides.

In some embodiments, shortened hairpin 2 lacks 18 nucleotides. In some embodiments, shortened hairpin 2 has 24 nucleotides. In some embodiments, in shortened hairpin 2, nucleotides 113-121 and 126-134 correspond to SEQ ID NO:500 is missing. In some embodiments, shortened hairpin 2 lacks 18 nucleotides and nucleotides 113-121 and 126-134 are compared to SEQ ID NO:500 is missing. In some embodiments, in the shortened hairpin 2 region, nucleotide 112 is linked to nucleotide 135 by 4 nucleotides. In some embodiments, in the shortened hairpin 2 region, nucleotides 113-121 and 126-134 correspond to SEQ ID NO:500 is deleted and nucleotide 112 is linked to nucleotide 135 by nucleotides 122-125.

In some embodiments, the shortened repeat/anti-repeat region has a length of 28 nucleotides. In some embodiments, the shortened repeat/anti-repeat region has a length of 32 nucleotides.

In some embodiments, the upper stem of the shortened repeat/anti-repeat region comprises no more than one base pair. In some embodiments, the upper stem of the shortened repeat/anti-repeat region comprises no more than three base pairs.

In some embodiments, the shortened hairpin 2 region has 8 modified nucleotides.

In some embodiments, a guide RNA (gRNA) comprises a guide region and a conserved region comprising:

(a) A shortened repeat/anti-repeat region, wherein the shortened repeat/anti-repeat region is relative to SEQ ID NO:500 lacks 18-22 nucleotides, wherein

(I) Nucleotides 38-48 and 53-63 deleted; and

(Ii) Nucleotide 36 is linked to nucleotide 65 by 6-10 nucleotides;

(b) A shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2 nucleotides, wherein relative to SEQ ID NO:500, nucleotide 86 and 91 deletions or nucleotide 85 and 92 deletions; and

(C) A shortened hairpin 2 region, wherein the shortened hairpin 2 lacks 18 nucleotides, wherein nucleotides 113-121 and 126-134 are relative to SEQ ID NO:500 deletions; and wherein nucleotides 144-145 correspond to SEQ ID NO:500 deletions; wherein at least 10 nucleotides are modified nucleotides.

(I) Nucleotides 38, 41-48, 53-60 and 63 are deleted; and

(Ii) Nucleotide 36 is linked to nucleotide 65 by 6-10 nucleotides;

(b) A shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2 nucleotides, wherein relative to SEQ ID NO:500, nucleotide 86 and 91 deletions or nucleotide 85 and 92 deletions;

(c) A shortened hairpin 2 region, wherein the shortened hairpin 2 lacks 18 nucleotides, wherein relative to SEQ ID NO:500, nucleotides 113-121 and 126-134 are deleted; and

Wherein relative to SEQ ID NO:500, nucleotide 144-145 deletion;

wherein at least 10 nucleotides are modified nucleotides.

In some embodiments, a guide RNA (gRNA) is provided that includes a guide region and a conserved region that includes one or more of:

(I) Nucleotides 37-48 and 53-64 deleted; and

(Ii) Nucleotide 36 is linked to nucleotide 65 by 6-10 nucleotides; or (b)

(B) A shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2 nucleotides, wherein relative to SEQ ID NO:500, nucleotide 86 and 91 deletions or nucleotide 85 and 92 deletions; or (b)

Wherein relative to SEQ ID NO:500, nucleotide 144-145 deletion;

wherein at least 10 nucleotides are modified nucleotides.

In other embodiments, the shortened repeat/anti-repeat region, wherein the shortened repeat/anti-repeat region is compared to SEQ ID NO:500 lacks 22 nucleotides. In other embodiments, nucleotide 36 is linked to nucleotide 65 by a sequence comprising nucleotide sequence UGAAAC. In other embodiments, nucleotide 36 is linked to nucleotide 65 by 10 nucleotides. In other embodiments, nucleotide 36 is linked to nucleotide 65 by a sequence comprising nucleotide sequence UUCGAAAGAC.

In some embodiments, the guide RNA (gRNA) of the preceding embodiments comprises a guide region and a conserved region comprising:

(a) A shortened repeat/anti-repeat region, wherein the shortened repeat/anti-repeat region lacks 18-22 nucleotides, wherein

(I) Nucleotides 37 to 48 and 53 to 64 relative to SEQ ID NO:500 deletions; and

(Ii) Nucleotide 36 is linked to nucleotide 65 by 6-10 nucleotides;

(b) A shortened hairpin 1 region, wherein the shortened hairpin 1 is relative to SEQ ID NO:500 lacks 2 nucleotides, wherein nucleotides 86 and 91 are deleted or nucleotides 85 and 92 are deleted;

(D) Wherein relative to SEQ ID NO:500, nucleotide 144-145 deletion;

wherein at least 10 nucleotides are modified nucleotides.

FIGS. 33-35 show exemplary sgRNAs in possible secondary structures.

In some embodiments, nmeCas9 short sgrnas comprise one of the following sequences in a 5 'to 3' orientation ：(N)_20-25GUUGUAGC UCCCUGAAACCGUU GCUACAAUAAGGCCGUCGAAAGAUGUGCCGCAACGCUCUGCCUUCUGGCAUCGUU(SEQ ID NO：501);

Wherein N is a nucleotide encoding a leader sequence. In some embodiments, N is equal to 24. In some embodiments, N is equal to 25.N represents a nucleotide having any base (e.g., A, C, G or U). (N) _20-25 represents 20-25, i.e., 20, 21, 22, 23, 24, or 25 consecutive N.

In some embodiments, at least 10 nucleotides of the conserved portion of NmeCas short sgrnas are modified nucleotides.

In some embodiments, nmeCas short sgrnas comprise a conserved region that contains one of the following sequences in a 5 'to 3' orientation:

mGUUGmUmAmGmCUCCCmUmUmCmGmAmAmAmGmAmCmCGUUmGmCUAmCAAU*AAGmGmCCmGmUmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU(SEQ ID NO：515).NmeCas9 Additional examples of short gRNAs (e.g., SEQ ID NOS: 512-530) are provided in Table 39B.

In some embodiments, nmeCas 'to 3' directed short gRNA comprises one of the following sequences:

Wherein N represents a nucleotide having any base (e.g., A, C, G or U). (mN x) ₃ denotes three consecutive nucleotides each having any base, 2'-OMe and 3' ps linkage to the next nucleotide, respectively. Nucleotide modifications are denoted m as 2' -OMe modifications and are PS linkages. In the case of modified nucleotide sequences, N, A, C, G and U are, in certain embodiments, unmodified RNA nucleotides, i.e., 2'-OH and phosphodiesterase linkages to 3' nucleotides.

The shortened NmeCas g rna may comprise an internal linker as disclosed herein.

An "internal linker" as used herein describes a non-nucleotide segment that joins two nucleotides within a guide RNA. If the gRNA contains a spacer, the internal linker is located outside the spacer (e.g., in the backbone or conserved region of the gRNA). For a V-type guide, it is understood that the last hairpin is the only hairpin in the structure, i.e., the repeat-anti-repeat region. In some embodiments, the internal linker comprises a PEG-linker as disclosed herein. In some embodiments, the internal linker comprises a PEG-linker as disclosed herein.

Exemplary positions of the joints are shown below:

(N)_20-25 GUUGUAGCUCCCUUC(L1)GACCGUUGCUACAAUAAGGCCGUC(L1)GAUGUGCCGCAACGCUCUGCC(L1)GGCAUCGUU(SEQ ID NO：506). As used herein, (L1) refers to an internal linker having a bridging length of about 15-21 atoms.

In some embodiments, the shortened NmeCas guide RNA comprising an internal linker may be chemically modified. Exemplary modifications include modification patterns of the following sequences:

In some embodiments, the sgRNA comprises SEQ ID NO:141 and 143-150 (Nme PEG guide), wherein N is any natural or unnatural nucleotide, and wherein all N comprises a guide sequence.

IV. delivery

In some embodiments, the polynucleotides or compositions disclosed herein are formulated in or administered via lipid nanoparticles; see, for example, WO2017173054, the contents of which are hereby incorporated by reference in their entirety.

Lipid

Disclosed herein are various embodiments of assembling a composition using a lipid nucleic acid comprising a nucleic acid or composition described herein. In some embodiments, the lipid nucleic acid assembly composition comprises a nucleic acid (e.g., mRNA) comprising an open reading frame encoding a polynucleotide comprising an Open Reading Frame (ORF) comprising a nucleotide sequence encoding a C-terminal neisseria meningitidis (Nme) Cas9 polypeptide disclosed herein and a nucleotide sequence encoding a first Nuclear Localization Signal (NLS). In some embodiments, nmeCas is Nme2Cas9, nme1Cas9, or Nme3Cas9.

As used herein, "lipid nucleic acid assembly composition" refers to lipid-based delivery compositions, including Lipid Nanoparticles (LNPs) and liposome complexes. LNP refers to lipid nanoparticles < 100 nM. LNP is formed by precisely mixing a lipid component (e.g., in ethanol) with an aqueous nucleic acid component, and the LNP is uniform in size. Liposome complexes are particles formed by mixing lipid and nucleic acid components in large amounts and are between about 100nm and 1 micron in size. In certain embodiments, the lipid nucleic acid assembly is an LNP. As used herein, a "lipid nucleic acid assembly" comprises a plurality (i.e., more than one) of lipid molecules that are physically associated with each other by intermolecular forces. The lipid nucleic acid assembly may comprise a bioavailable lipid having a pKa value of < 7.5 or < 7. The lipid nucleic acid assembly is formed by mixing an aqueous solution containing nucleic acid with an organic solvent-based lipid solution (e.g., 100% ethanol). Suitable solutions or solvents include or may contain: water, PBS, tris buffer, naCl, citrate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. The pharmaceutically acceptable buffer may optionally be included in a pharmaceutical formulation comprising the lipid nucleic acid assembly, e.g., for ex vivo therapy. In some embodiments, the aqueous solution comprises RNA, e.g., mRNA or gRNA. In some embodiments, the aqueous solution comprises mRNA encoding an RNA-guided DNA binding agent (e.g., cas 9).

As used herein, a Lipid Nanoparticle (LNP) refers to a particle that comprises a plurality (i.e., more than one) of lipid molecules that are physically associated with each other by intermolecular forces. LNP can be, for example, microspheres (including unilamellar and multilamellar vesicles, e.g., "liposomes," in some embodiments, substantially spherical lamellar phase lipid bilayers, and in more particular embodiments can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules), a dispersed phase in an emulsion, micelles, or an internal phase in suspension. Emulsions, micelles, and suspensions may be compositions suitable for topical and/or surface delivery. See also e.g. WO2017173054A1, the content of which is hereby incorporated by reference in its entirety. Any LNP known to those of skill in the art capable of delivering nucleotides to a subject can be used with the guide RNA and nucleic acids encoding NmeCas and NLS described herein.

In some embodiments, the aqueous solution comprises a nucleic acid encoding a polypeptide comprising A3A and an RNA-guided nicking enzyme. The pharmaceutical formulation comprising the lipid nucleic acid assembly composition may optionally comprise a pharmaceutically acceptable buffer.

In some embodiments, the lipid nucleic acid assembly composition includes an "amine lipid" (sometimes described herein or elsewhere as an "ionizable lipid" or "biodegradable lipid"), and optionally a "helper lipid", "neutral lipid", and a stealth lipid, such as a PEG lipid. In some embodiments, the amine lipid or ionizable lipid is cationic depending on pH.

1. Amine lipids

In some embodiments, the lipid nucleic acid assembly composition comprises an "amine lipid," which is, for example, an ionizable lipid, such as lipid a or an equivalent thereof, including acetal analogs of lipid a.

In some embodiments, the amine lipid is lipid a, which is octadecyl-9, 12-dienoic acid (9 z,12 z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- ((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester, also known as (9 z,12 z) -octadecyl-9, 12-dienoic acid 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- ((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester. Lipid a can be depicted as:

lipid A can be synthesized according to WO2015/095340 (e.g., pages 84-86). In some embodiments, the amine lipid is an equivalent of lipid a.

In some embodiments, the amine lipid is an analog of lipid a. In some embodiments, the lipid a analog is an acetal analog of lipid a. In certain lipid nucleic acid assembly compositions, the acetal analogue is a C4-C12 acetal analogue. In some embodiments, the acetal analogue is a C5-C12 acetal analogue. In additional embodiments, the acetal analogue is a C5-C10 acetal analogue. In other embodiments, the acetal analogue is selected from the group consisting of C4, C5, C6, C7, C9, C10, C11 and C12 acetal analogues.

Amine lipids and other "biodegradable lipids" suitable for use in the lipid nucleic acid assemblies described herein are biodegradable in vivo or ex vivo. Amine lipids have low toxicity (e.g., are tolerated in animal models in amounts greater than or equal to 10mg/kg without adverse effects). In some embodiments, the lipid nucleic acid assemblies comprising amine lipids include lipid nucleic acid assemblies in which at least 75% of the amine lipids are cleared from plasma or engineered cells within 8, 10, 12, 24, or 48 hours or 3, 4, 5,6,7, or 10 days. In some embodiments, lipid nucleic acid assemblies comprising amine lipids include lipid nucleic acid assemblies in which at least 50% of the nucleic acid, e.g., mRNA or gRNA, is cleared from plasma within 8, 10, 12, 24, or 48 hours or 3, 4, 5,6,7, or 10 days. In some embodiments, lipid nucleic acid assemblies comprising amine lipids include those in which at least 50% of the lipid nucleic acid assemblies are cleared from plasma within 8, 10, 12, 24, or 48 hours or 3, 4, 5,6,7, or 10 days, for example, by measuring lipids (e.g., amine lipids), nucleic acids (e.g., RNA/mRNA), or other components. In some embodiments, lipid encapsulation of the lipid nucleic acid assemblies is measured relative to free lipid, RNA, or nucleic acid components.

Biodegradable lipids include, for example, biodegradable lipids of WO/2020/219876, WO/2020/118041, WO/2020/072605, WO/2019/067992, WO/2017/173054, WO2015/095340, and WO2014/136086, and LNP includes LNP compositions described therein, the lipids and compositions of which are hereby incorporated by reference.

Lipid clearance can be measured as described in the literature. See Maier, m.a. et al ,Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics.Mol.Ther.2013,21(8),1570-78("Maier")., for example, in Maier, LNP-siRNA systems containing sirnas targeting luciferases were administered at 0.3mg/kg via lateral tail vein to six to eight week old male C57B1/6 mice by intravenous bolus injection. Blood, liver and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2,4, 8, 24, 48, 96 and 168 hours post-dose. The mice were perfused with physiological saline and blood samples were treated to obtain plasma prior to tissue collection. All samples were processed and analyzed by LC-MS. Furthermore Maier describes a procedure for assessing toxicity after administration of LNP-siRNA formulations. For example, male Shi Boge-dolichous rats (Sprague-DAWLEY RAT) were administered with sirnas targeting luciferases at 0, 1,3, 5 and 10mg/kg (5 animals/group) via single intravenous bolus injection at a dose volume of 5 mL/kg. After 24 hours, about 1mL of blood was obtained from the jugular vein of the conscious animal and serum was isolated. At 72 hours post-dose, all animals were euthanized for necropsy. Clinical symptoms, body weight, serum chemistry, organ weight and histopathology were assessed. Although Maier describes methods for evaluating siRNA-LNP formulations, these methods can be adapted to evaluate clearance, pharmacokinetics, and toxicity of administration of the lipid nucleic acid assembly compositions of the present disclosure.

Ionizable and bioavailable lipids for LNP delivery of nucleic acids known in the art are suitable. Lipids can be ionized according to the pH of the medium in which they are located. For example, in a weakly acidic medium, lipids, such as amine lipids, may be protonated and thus positively charged. Conversely, in a weakly alkaline medium, such as blood at a pH of about 7.35, lipids, such as amine lipids, may not be protonated and thus uncharged.

The ability of a lipid to carry a charge is related to its inherent pKa. In some embodiments, the amine lipids of the present disclosure may each independently have a pKa in the range of about 5.1 to about 7.4. In some embodiments, the bioavailable lipids of the present disclosure may each independently have a pKa in the range of about 5.1 to about 7.4, e.g., about 5.5 to about 6.6, about 5.6 to about 6.4, about 5.8 to about 6.2, or about 5.8 to about 6.5. For example, amine lipids of the present disclosure may each independently have a pKa in the range of about 5.8 to about 6.5. Lipids having pKa in the range of about 5.1 to about 7.4 are useful for in vivo delivery of cargo (cargo), for example, to the liver. Furthermore, lipids having pKa in the range of about 5.3 to about 6.4 have been found to be effective for in vivo delivery to, for example, tumors. See, for example, WO2014/136086.

2. Additional lipids

"Neutral lipids" suitable for use in the lipid nucleic acid assembly compositions of the present disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to: 5-heptadecylphenyl-1, 3-diol (resorcinol), dicarboxyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine such as 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), phosphocholine (DOPC), dimyristoyl-phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), lecithin phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1-myristoyl-2-soft-phosphatidylcholine (MPPC), 1-soft-phosphatidyl-2-myristoyl phosphatidylcholine (PMPC), 1-soft-phosphatidyl-2-stearoyl phosphatidylcholine (PSPC), 1, 2-distearoyl-sn-glycero-3-Phosphocholine (PC), 1-stearoyl-2-soft phosphatidylcholine (DAPC), 1-stearoyl-2-Phosphatidylethanolamine (PE), phosphatidylcholine (POPC), 1-myristoyl-2-phosphatidylcholine (POPC), phosphatidylcholine (POE-phosphatidylcholine (POPC), lysophosphatidylcholine (POE-phosphatidylcholine (POPC), di-oleoyl phosphatidylcholine di-stearoyl phosphatidylethanolamine (DSPE), di-myristoyl phosphatidylethanolamine (DMPE), di-soft acyl phosphatidylethanolamine (DPPE), soft acyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of: distearoyl phosphatidylcholine (DSPC) and dimyristoyl phosphatidylethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoyl phosphatidylcholine (DSPC).

"Helper lipids" include steroids, sterols and alkyl resorcinol. Auxiliary lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecyl resorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be cholesterol hemisuccinate.

A "stealth lipid" is a lipid that alters the length of time a nanoparticle can be present in the body (e.g., in blood). Stealth lipids may aid the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids as used herein may modulate the pharmacokinetic properties of the lipid nucleic acid assemblies or aid in nanoparticle ex vivo stability. Stealth lipids suitable for use in the lipid nucleic acid assembly compositions of the present disclosure include, but are not limited to, stealth lipids having a hydrophilic head group attached to a lipid moiety. Stealth lipids suitable for use in the lipid nucleic acid assembly compositions of the present disclosure and information regarding the biochemistry of such lipids can be found in Romberg et al, pharmaceutical Research, vol.25, stage 1, 2008, pages 55-71 and Hoekstra et al, biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG lipids are disclosed, for example, in WO 2006/007712.

In one embodiment, the hydrophilic head group of the stealth lipid comprises a polymer moiety selected from PEG-based polymers. The stealth lipid may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG lipid.

In one embodiment, the stealth lipid comprises a polymer moiety selected from the group consisting of polymers based on: PEG (sometimes referred to as poly (ethylene oxide)), poly (oxazoline), poly (vinyl alcohol), poly (glycerol), poly (N-vinylpyrrolidone), polyamino acids, and poly [ N- (2-hydroxypropyl) methacrylamide ].

In one embodiment, the PEG lipid comprises a PEG-based (sometimes referred to as poly (ethylene oxide)) polymer moiety.

PEG lipids also comprise a lipid moiety. In some embodiments, the lipid moiety may be derived from diacylglycerols or diacylglycerol amides, including those comprising a dialkylglycerol or dialkylglyceroamide group having an alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups, such as an amide or ester. In some embodiments, the alkyl chain length comprises about C10 to C20. The dialkylglycerol or dialkylglyceramide groups may also contain one or more substituted alkyl groups. The chain length may be symmetrical or asymmetrical.

As used herein, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer, unless otherwise indicated. In one embodiment, the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In one embodiment, PEG is unsubstituted. In one embodiment, PEG is substituted with, for example, one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the term includes PEG copolymers such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, poly (ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); in another embodiment, the term excludes PEG copolymers. In one embodiment, the molecular weight of the PEG is from about 130 to about 50,000, in one sub-embodiment from about 150 to about 30,000, in one sub-embodiment from about 150 to about 20,000, in one sub-embodiment from about 150 to about 15,000, in one sub-embodiment from about 150 to about 10,000, in one sub-embodiment from about 150 to about 6,000, in one sub-embodiment from about 150 to about 5,000, in one sub-embodiment from about 150 to about 4,000, in one sub-embodiment from about 150 to about 3,000, in one sub-embodiment from about 300 to about 3,000, in one sub-embodiment from about 1,000 to about 3,000, and in one sub-embodiment from about 1,500 to about 2,500.

In some embodiments, the PEG (e.g., conjugated to a lipid moiety or lipid such as a stealth lipid) is "PEG-2K", also referred to as "PEG 2000", which has an average molecular weight of about 2,000 daltons. PEG-2K is herein represented by the following formula (I), wherein n is 45, meaning that the number average degree of polymerization comprises about 45 subunitsHowever, other PEG embodiments known in the art may be used, including, for example, those wherein the number average degree of polymerization comprises about 23 subunits (n=23) and/or 68 subunits (n=68). In some embodiments, n may be in the range of about 30 to about 60. In some embodiments, n may be in the range of about 35 to about 55. In some embodiments, n may be in the range of about 40 to about 50. In some embodiments, n may be in the range of about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.

In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (e.g., 1, 2-dimyristoyl-rac-glycerol-3-methylpolyoxyethyleneglycol 2000 (PEG 2 k-DMG) or PEG-DMG (catalog No. GM-020, from NOF, tokyo, japan), PEG-dipalmitoylglycerol, PEG-distearylglycerol (PEG-DSPE) (catalog No. DSPE-020cn, NOF, tokyo, japan), PEG-dilauryl glyceramide, PEG-dimyristoylglyceride, PEG-dipalmitoyl glyceramide and PEG-distearoyl glyceramide, PEG-cholesterol (1- [8' - (cholest-5-en-3 [ beta ] -oxy) carboxamide-3 ',6' -dioxaoctyl ] carbamoyl- [ omega ] -methyl-poly (ethylene glycol), PEG-DMB (3, 4-ditetradecyloxybenzyl- [ omega ] -methyl-poly (ethylene glycol) ether), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (PEG 2 k-DMG) (catalog number 880150P, From Avanti Polar Lipids, alabaster, alabasra, USA), 1, 2-distearoyl-sn-glycero-3-phosphate ethanolamine-N- [ methoxy (polyethylene glycol) -2000] (PEG 2 k-DSPE) (catalog No. 880120C, from Avanti Polar Lipids, alabaster, alabasra, USA), 1, 2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG 2k-DSG; GS-020,NOF Tokyo,Japan), poly (ethylene glycol) -2000-dimethacrylate (PEG 2 k-DMA) and 1, 2-distearyloxypropyl-3-amine-N- [ methoxy (polyethylene glycol) -2000] (PEG 2 k-DSA). In one embodiment, the PEG lipid may be 1, 2-dimyristoyl-rac-glycerol-3-methylpolyoxyethyleneglycol 2000 (PEG 2 k-DMG). In one embodiment, the PEG lipid may be PEG2k-DMG. In one embodiment, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid can be PEG2k-DSG. In one embodiment, the PEG lipid may be PEG2k-DSPE. In one embodiment, the PEG lipid may be PEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DMA. In one embodiment, the PEG lipid may be compound S027, which is disclosed in WO2016/010840 (paragraphs [00240] to [00244 ]). In one embodiment, the PEG lipid may be PEG2k-DSA. In one embodiment, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid can be PEG2k-C14. In some embodiments, the PEG lipid can be PEG2k-C16. In some embodiments, the PEG lipid can be PEG2k-C18.

In a preferred embodiment, the PEG lipid comprises a glyceryl group. In a preferred embodiment, the PEG lipid comprises a dimyristoyl glycerol (DMG) group. In a preferred embodiment, the PEG lipid comprises PEG-2k. In a preferred embodiment, the PEG lipid is PEG-DMG. In a preferred embodiment, the PEG lipid is PEG-2k-DMG. In a preferred embodiment, the PEG lipid is 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000. In a preferred embodiment, PEG-2k-DMG is 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000.

LNP

Lipid Nanoparticles (LNPs) are well known means for delivering nucleotide and protein cargo, and can be used to deliver polynucleotides, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, the LNP delivers the nucleic acid, the protein, or the nucleic acid along with the protein.

As used herein, a Lipid Nanoparticle (LNP) refers to a particle that comprises a plurality (i.e., more than one) of lipid molecules that are physically associated with each other by intermolecular forces. LNP may be, for example, microspheres (including unilamellar and multilamellar vesicles, e.g., "liposomes", which in some embodiments are substantially spherical lamellar phase lipid bilayers, and in more particular embodiments may comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules), a dispersed phase in an emulsion, a micelle, or an internal phase in suspension (see, e.g., WO2017173054, the contents of which are hereby incorporated by reference in their entirety). Any LNP known to those of skill in the art that is capable of delivering a nucleotide to a subject can be utilized.

In some embodiments, the LNP comprises a cationic lipid. In some embodiments, the LNP comprises (9 z,12 z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl octadec-9, 12-dienoate, also known as 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- ((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl (9 z,12 z) -octadec-9, 12-dienoate), referred to herein as lipid a. In some embodiments, the LNP comprises a molar ratio (N: P) of cationic lipid amine to RNA phosphate of about 4.5. In some embodiments, the LNP comprises 8- ((7, 7-bis (octyloxy) heptyl) (2-hydroxyethyl) amino) octanoate. In some embodiments, the LNP comprises a molar ratio of cationic lipid amine to RNA phosphate (N: P) of about 4.5 to 6.5. In some embodiments, the LNP comprises a molar ratio (N: P) of cationic lipid amine to RNA phosphate of about 4.5. In some embodiments, the LNP comprises a molar ratio (N: P) of cationic lipid amine to RNA phosphate of about 6.0.

In some embodiments, the disclosure comprises a method for delivering a polynucleotide or composition disclosed herein to a subject, wherein the polynucleotide is associated with an LNP. In some embodiments, the disclosure comprises a method for delivering a first polynucleotide and a second polynucleotide to a subject, or a composition for delivering a first polynucleotide and a second polynucleotide to a subject, wherein the first polynucleotide and the second polynucleotide are associated with, e.g., co-formulated with, the same LNP. In some embodiments, the disclosure comprises a method for delivering a first polynucleotide and a second polynucleotide to a subject, or a composition for delivering a first polynucleotide and a second polynucleotide to a subject, wherein the first polynucleotide and the second polynucleotide are each associated with an independent LNP, e.g., each polynucleotide is associated with an independent LNP for administration to or use with a subject, e.g., for co-administration. In some embodiments, the first polynucleotide and the second polynucleotide encode NmeCas nicking enzymes and UGIs. In some embodiments, the composition further comprises one or more guide RNAs. In some embodiments, the method further comprises delivering one or more guide RNAs.

In some embodiments, provided herein is a method of delivering any of the polynucleotides or compositions described herein to a cell or population of cells or a subject, including in vivo to a cell or population of cells of a subject, wherein any one or more of the components is associated with LNP. In some embodiments, the composition further comprises one or more guide RNAs. In some embodiments, the method further comprises delivering one or more guide RNAs.

In some embodiments, provided herein is a composition comprising any of the polynucleotides or compositions described herein or donor constructs disclosed herein, alone or in combination with an LNP. In some embodiments, the composition further comprises one or more guide RNAs. In some embodiments, the method further comprises delivering one or more guide RNAs.

In some embodiments, LNPs associated with the polynucleotides or compositions disclosed herein are used to prepare medicaments for use in treating a disease or disorder.

In some embodiments, a method of modifying a gene of interest is provided, the method comprising delivering to a cell one or more lipid nucleic acid assembly compositions comprising a polynucleotide as disclosed herein and one or more guide RNAs, optionally a lipid nanoparticle.

In some embodiments, at least one of the lipid nucleic acid assembly compositions comprises a Lipid Nanoparticle (LNP), optionally wherein all of the lipid nucleic acid assembly compositions comprise LNP. In some embodiments, at least one of the lipid nucleic acid assembly compositions is a lipid complex composition. In some embodiments, the lipid nucleic acid composition comprises an ionizable lipid.

Electroporation is a well known method for delivering cargo, and any electroporation method may be used to deliver the polynucleotides or compositions disclosed herein. In some embodiments, electroporation may be used to deliver any of the polynucleotides or compositions disclosed herein.

In some embodiments, the disclosure comprises a method of delivering a polynucleotide, polypeptide, or composition disclosed herein to an ex vivo cell, wherein the polynucleotide or composition is associated with or not associated with an LNP. In some embodiments, the LNP is also associated with one or more guide RNAs. See, for example, PCT/US2021/029446, incorporated herein by reference.

In some embodiments, a kit is provided comprising a polynucleotide, polypeptide, or composition disclosed herein.

In some embodiments, a pharmaceutical formulation is provided comprising a polynucleotide, polypeptide, or composition disclosed herein. The pharmaceutical formulation may also comprise a pharmaceutically acceptable carrier, such as water or a buffer. The pharmaceutical formulation may also comprise one or more pharmaceutically acceptable excipients, such as stabilizers, preservatives, extenders, and the like. The pharmaceutical formulation may also comprise one or more pharmaceutically acceptable salts, such as sodium chloride. In some embodiments, the pharmaceutical formulation is formulated for intravenous administration. In some embodiments, the pharmaceutical formulation is non-pyrogenic. In some embodiments, the pharmaceutical formulation is sterile, particularly for injection or infusion.

Exemplary uses, methods, and treatments

In some embodiments, the polynucleotides, expression constructs, compositions, lipid Nanoparticles (LNPs), or pharmaceutical compositions disclosed herein are used in gene therapy, e.g., of a gene of interest.

In some embodiments, there is provided the use of a polynucleotide, composition or polypeptide disclosed herein for modifying a gene of interest in a cell.

In some embodiments, there is provided the use of a polynucleotide, composition or polypeptide disclosed herein for the manufacture of a medicament for modifying a gene of interest in a cell.

In some embodiments, the polynucleotide or composition is formulated as a lipid nucleic acid assembly composition, optionally a lipid nanoparticle.

In some embodiments, a method of modifying a gene of interest is provided, the method comprising delivering to a cell a polynucleotide, polypeptide, or composition disclosed herein.

In some embodiments, the polynucleotide, expression construct, composition, lipid Nanoparticle (LNP), or pharmaceutical composition is used for genome editing, e.g., editing a gene of interest in which the polynucleotide encodes an RNA-guided DNA binding agent (e.g., nmeCas 9).

In some embodiments, a polynucleotide, expression construct, composition, lipid Nanoparticle (LNP), or pharmaceutical composition disclosed herein encoding a polypeptide disclosed herein is used to express the polypeptide in a heterologous cell, e.g., a human cell or a mouse cell.

In some embodiments, the polynucleotide, expression construct, composition, lipid Nanoparticle (LNP), or pharmaceutical composition is used to modify a gene of interest, e.g., alter its sequence or epigenetic state, wherein the polynucleotide encodes an RNA-guided DNA binding agent (e.g., nmeCas 9).

In some embodiments, the polynucleotide, expression construct, composition, lipid Nanoparticle (LNP), or pharmaceutical composition is used to induce a Double Strand Break (DSB) within a gene of interest. In some embodiments, the polynucleotide, expression construct, composition, lipid Nanoparticle (LNP), or pharmaceutical composition is used to induce an insertion/deletion in a gene of interest. In some embodiments, there is provided the use of a polynucleotide, expression construct, composition, lipid Nanoparticle (LNP) or pharmaceutical composition disclosed herein for the preparation of a medicament for genome editing, e.g., editing, of a target gene in which the polynucleotide encodes an RNA-guided DNA binding agent (e.g., nmeCas 9). In some embodiments, there is provided the use of a polynucleotide, expression construct, composition, lipid Nanoparticle (LNP) or pharmaceutical composition disclosed herein encoding a polypeptide disclosed herein for the preparation of a medicament for expressing the polypeptide or increasing expression of the polypeptide in a heterologous cell, e.g., a human cell or a mouse cell. In some embodiments, there is provided the use of a polynucleotide, expression construct, composition, lipid Nanoparticle (LNP) or pharmaceutical composition disclosed herein for the preparation of a medicament for modifying a gene of interest, e.g., for altering its sequence or epigenetic status. In some embodiments, there is provided the use of a polynucleotide, expression construct, composition, lipid Nanoparticle (LNP) or pharmaceutical composition disclosed herein for the preparation of a medicament for inducing a Double Strand Break (DSB) within a gene of interest. In some embodiments, there is provided the use of a polynucleotide, expression construct, composition, lipid Nanoparticle (LNP) or pharmaceutical composition disclosed herein for the preparation of a medicament for inducing an insertion/deletion in a gene of interest.

In some embodiments, the gene of interest is a transgene. In some embodiments, the gene of interest is an endogenous gene. The gene of interest may be in a subject, e.g., a mammal, e.g., a human. In some embodiments, the gene of interest is in an organ, such as the liver, e.g., a mammalian liver, e.g., a human liver. In some embodiments, the gene of interest is in a liver cell, e.g., a mammalian liver cell, e.g., a human liver cell. In some embodiments, the gene of interest is in a hepatocyte, e.g., a mammalian hepatocyte, e.g., a human hepatocyte. In some embodiments, the liver cells or hepatocytes are in situ. In some embodiments, the liver cells or hepatocytes are isolated, for example, in culture, for example, in primary culture. In some embodiments, the cells of interest are Peripheral Blood Mononuclear Cells (PBMCs), such as mammalian PBMCs, e.g., human PBMCs. In some embodiments, the PBMCs are immune cells, e.g., T cells, B cells, NK cells. In some embodiments, the cell is a pluripotent cell, e.g., a mammalian pluripotent cell, e.g., a human pluripotent cell. In some embodiments, the target cell is a stem cell, e.g., a mammalian stem cell, e.g., a human stem cell. In some embodiments, the stem cells are present in bone marrow. In some embodiments, the stem cells are induced pluripotent stem cells (iPCS). In some embodiments, the cells are isolated, e.g., in ex vivo culture.

Also provided are methods corresponding to the uses disclosed herein, comprising administering to a subject a polynucleotide, expression construct, composition, lipid Nanoparticle (LNP) or pharmaceutical composition disclosed herein or contacting a cell (e.g., a cell as described above) with a polynucleotide, LNP or pharmaceutical composition disclosed herein, e.g., to express a polypeptide disclosed herein or to increase expression of a polypeptide disclosed herein in, e.g., a heterologous cell, e.g., a human cell or a mouse cell.

In any of the foregoing embodiments involving a subject, the subject may be a mammal. In any of the foregoing embodiments involving a subject, the subject may be a human.

In some embodiments, a polynucleotide, expression construct, composition, lipid Nanoparticle (LNP), or pharmaceutical composition disclosed herein is administered intravenously or for intravenous administration.

In some embodiments, a single administration of a polynucleotide, LNP, or pharmaceutical composition disclosed herein is sufficient to knock down expression of a gene product of interest. In some embodiments, a single administration of a polynucleotide, LNP, or pharmaceutical composition disclosed herein is sufficient to knock out expression of a gene product of interest. In other embodiments, more than one administration of a polynucleotide, LNP, or pharmaceutical composition disclosed herein may be beneficial for maximizing editing, modification, insertion/deletion formation, DSB formation, etc. via cumulative effects.

Exemplary DNA molecules, vectors, expression constructs, host cells, and methods of production

In certain embodiments, the present disclosure provides DNA molecules comprising ORF sequences encoding polypeptides disclosed herein. In some embodiments, the DNA molecule comprises a nucleic acid that does not encode a polypeptide disclosed herein in addition to the ORF sequence. Nucleic acids that do not encode polypeptides include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids that encode guide RNAs.

In some embodiments, the DNA molecule further comprises a nucleotide sequence encoding crRNA, trRNA, or crRNA and trRNA. In some embodiments, the nucleotide sequence encoding crRNA, trRNA, or crRNA and trRNA comprises or consists of: a guide sequence flanked by all or a portion of a repeat sequence from a naturally occurring CRISPR/Cas system. The nucleic acid comprising or consisting of crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence, wherein the vector sequence comprises or consists of: nucleic acids that do not naturally occur with crrnas, trrnas, or crrnas and trRNA. In some embodiments, crrnas and trRNA are encoded by non-contiguous nucleic acids within one vector. In other embodiments, crrnas and trRNA may be encoded by contiguous nucleic acids. In some embodiments, crrnas and trRNA are encoded by opposite strands of a single nucleic acid. In other embodiments, crRNA and trRNA are encoded by the same strand of a single nucleic acid.

In some embodiments, the DNA molecule further comprises a promoter operably linked to a sequence encoding any one of the ORFs encoding the polypeptides disclosed herein. In some embodiments, the DNA molecule is an expression construct suitable for expression in a mammalian cell, such as a human cell or a mouse cell, such as a human hepatocyte or a rodent (e.g., mouse) hepatocyte. In some embodiments, the DNA molecule is an expression construct suitable for expression in cells of a mammalian organ, such as a human liver or rodent (e.g., mouse) liver. In some embodiments, the DNA molecule is a plasmid or episome. In some embodiments, the DNA molecule is contained in a host cell, such as a bacterium or a cultured eukaryotic cell. Exemplary bacteria include Proteus, e.g., E.coli. Exemplary cultured eukaryotic cells include primary hepatocytes, including hepatocytes of rodent (e.g., mouse) or human origin; hepatocyte cell lines, including hepatocytes of rodent (e.g., mouse) or human origin; a human cell line; rodent (e.g., mouse) cell lines; CHO cells; microbial fungi, such as schizosaccharomyces, or budding yeasts, such as saccharomyces cerevisiae; and insect cells.

In some embodiments, a method of making an mRNA disclosed herein is provided. In some embodiments, such methods comprise contacting a DNA molecule described herein with an RNA polymerase under conditions that allow transcription. In some embodiments, the contacting is performed in vitro, e.g., in a cell-free system. In some embodiments, the RNA polymerase is a phage-derived RNA polymerase, such as a T7 RNA polymerase. In some embodiments, there is provided an NTP comprising at least one modified nucleotide as discussed above. In some embodiments, the NTP comprises at least one modified nucleotide as discussed above and does not comprise UTP.

In some embodiments, a method of producing a polynucleotide disclosed herein is provided. In some embodiments, such methods comprise contacting an expression construct disclosed herein with an RNA polymerase and an NTP comprising at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a modified uridine. In other embodiments, at least 80% of the uridine positions are modified uridine. In other embodiments, at least 90% of the uridine positions are modified uridine. In other embodiments, 100% of the uridine positions are modified uridine. In other embodiments, the modified uridine comprises or is a substituted uridine, a pseudouridine, or a substituted pseudouridine. In other embodiments, the modified uridine comprises or is N1-methyl-pseudouridine. In some embodiments, the expression construct comprises a encoded poly-A tail sequence.

In some embodiments, polynucleotides disclosed herein may be contained within or delivered by a vector system of one or more vectors. In some embodiments, one or more or all of the vectors may be DNA vectors. In some embodiments, one or more or all of the vectors may be RNA vectors. In some embodiments, one or more or all of the vectors may be circular. In other embodiments, one or more or all of the vectors may be linear. In some embodiments, one or more or all of the carriers may be encapsulated in a lipid nanoparticle, a liposome, a non-lipid nanoparticle, or a viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vectors, lentiviral vectors, adenovirus vectors, helper-dependent adenovirus vectors (HDAd), herpes simplex virus (HSV-1) vectors, phage T4, baculovirus vectors, and retroviral vectors. In some embodiments, the viral vector may be an AAV vector. In other embodiments, the viral vector may be a lentiviral vector. In some embodiments, the lentivirus may be non-integrated. In some embodiments, the viral vector may be an adenovirus vector. In some embodiments, the adenovirus may be a high cloning capacity or "enteroless" adenovirus in which all viral coding regions except the 5 'and 3' Inverted Terminal Repeats (ITRs) and the packaging signal ('I') are deleted from the virus to increase its packaging capacity. In other embodiments, the viral vector may be an HSV-1 vector. In some embodiments, the HSV-1-based vector is helper-dependent, and in other embodiments, it is non-helper-dependent. For example, an amplicon vector that retains only packaging sequences requires helper virus with structural components for packaging, whereas a 30kb HSV-1 vector that lacks non-essential viral functions does not require helper virus. In other embodiments, the viral vector may be phage T4. In some embodiments, phage T4 may be able to package any linear or circular DNA or RNA molecule when the viral head is emptied. In other embodiments, the viral vector may be a baculovirus vector. In yet other embodiments, the viral vector may be a retroviral vector. In embodiments using AAV or lentiviral vectors with smaller cloning capacity, it may be desirable to use more than one vector to deliver all components of the vector system as disclosed herein. For example, one AAV vector may contain a sequence encoding a Cas protein, while a second AAV vector may contain one or more guide sequences.

In some embodiments, the vector may be capable of driving expression of one or more coding sequences in a cell, e.g., the coding sequences of the mRNA disclosed herein. In some embodiments, the cell may be a prokaryotic cell, such as a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, such as a yeast, plant, insect, or mammalian cell. In some embodiments, the eukaryotic cell may be a mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters for driving expression in different types of cells are known in the art. In some embodiments, the promoter may be wild-type. In other embodiments, the promoter may be modified for more efficient or effective expression. In yet other embodiments, the promoter may be truncated but retain its function. For example, the promoter may have a normal size or a reduced size suitable for proper packaging of the vector in a virus.

In some embodiments, the vector system may comprise a copy of a nucleotide sequence comprising an ORF encoding a polypeptide disclosed herein. In other embodiments, the vector system may comprise more than one copy of a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, a nucleotide sequence encoding a polypeptide disclosed herein is operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one promoter.

In some embodiments, the promoter may be constitutive, inducible, or tissue specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting exemplary constitutive promoters include the cytomegalovirus immediate early promoter (CMV), the simian virus (SV 40) promoter, the adenovirus Major Late Promoter (MLP), the Rous Sarcoma Virus (RSV) promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor- α (EF 1 a) promoter, the ubiquitin promoter, the actin promoter, the tubulin promoter, the immunoglobulin promoter, functional fragments thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include inducible promoters that can be induced by thermal shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohols. In some embodiments, the inducible promoter may be an inducible promoter having a low basal (non-inducible) level of expression, e.g.Promoter (Clontech).

In some embodiments, the promoter may be a tissue specific promoter, such as a promoter specific for expression in the liver.

The vector may also comprise a nucleotide sequence encoding at least one guide RNA. In some embodiments, the vector comprises one copy of the guide RNA. In other embodiments, the vector comprises more than one copy of the guide RNA. In embodiments with more than one guide RNA, the guide RNAs may be different such that they target different target sequences, or may be the same such that they target the same target sequence. In some embodiments in which the vector comprises more than one guide RNA, each guide RNA may have other different properties, such as activity or stability in ribonucleoprotein complexes with RNA-guided DNA binders (e.g., nmeCas 9). In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or translational control sequence, e.g., a promoter, 3'utr, or 5' utr. In one embodiment, the promoter can be a tRNA promoter, e.g., tRNA ^Lys3 or a tRNA chimera. See Mefferd et al, rna.201218: 1683-9; scherer et al, nucleic Acids res.200735:2620-2628. In some embodiments, the promoter is recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include the U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human H1 promoter. In embodiments with more than one guide RNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotide encoding the crRNA of the guide RNA and the nucleotide encoding trRNA of the guide RNA may be provided on the same vector. In some embodiments, the nucleotide encoding the crRNA and the nucleotide encoding trRNA may be driven by the same promoter. In some embodiments, crRNA and trRNA can be transcribed as a single transcript. For example, crrnas and trRNA can be treated with a single transcript to form a bimolecular guide RNA. Alternatively crrnas and trRNA may be transcribed as single molecule guide RNAs. In other embodiments, crrnas and trRNA may be driven by their corresponding promoters on the same vector. In still other embodiments, crrnas and trRNA may be encoded by different vectors.

In some embodiments, the composition comprises a carrier system, wherein the system comprises more than one carrier. In some embodiments, the carrier system may comprise one single carrier. In other embodiments, the carrier system may comprise two carriers. In other embodiments, the carrier system may comprise three carriers. When different polynucleotides are used for multiplexing (multiplexing) or when multiple copies of the polynucleotide are used, the vector system may comprise more than three vectors.

In some embodiments, a host cell is provided that comprises a vector, expression construct, or plasmid disclosed herein.

In some embodiments, the vector system may comprise an inducible promoter to initiate expression only after delivery thereof to the target cell. Non-limiting exemplary inducible promoters include inducible promoters that can be induced by thermal shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohols. In some embodiments, the inducible promoter may be an inducible promoter having a low basal (non-inducible) level of expression, e.g.Promoter (Clontech).

In additional embodiments, the vector system may comprise a tissue specific promoter to initiate expression only after its delivery into a particular tissue.

Determination of ORF efficacy

When the polypeptide is expressed with a function of interest or other component of a system, the efficacy of a polynucleotide comprising an ORF encoding a polypeptide disclosed herein can be determined, e.g., using any of those recognized in the art to detect the presence, expression level, or activity of a particular polypeptide, e.g., by an enzyme-linked immunosorbent assay (ELISA), other immunological methods, western blot, liquid chromatography-mass spectrometry (LC-MS), FACS analysis, hiBiT peptide assay (Promega), or other assay described herein; or a method for determining the level of enzymatic activity in a biological sample (e.g., a cell, cell lysate or extract, conditioned medium, whole blood, serum, plasma, urine, or tissue), such as an in vitro activity assay. Exemplary assays described herein for activity of various encoded polypeptides, e.g., RNA-guided DNA binding agents, include assays for insertion/deletion formation, deamination, or mRNA or protein expression. In some embodiments, the efficacy of a polynucleotide comprising an ORF encoding a polypeptide disclosed herein is determined based on an in vitro model.

1. Determining efficacy of ORFs encoding RNA-directed DNA binding agents

In some embodiments, the efficacy of mRNA is determined when expressed with other components of RNP, such as at least one gRNA, e.g., TTR-targeted gRNA.

RNA-guided DNA binding agents (e.g., nmeCas 9) with lyase activity can result in double strand breaks in DNA. Non-homologous end joining (NHEJ) is a method by which Double Strand Breaks (DSBs) in DNA are repaired by reconnecting the broken ends, which may generate errors in the form of insertion/deletion (indel) mutations. Before religating the ends, the DNA ends of the DSBs are often subjected to an enzymatic treatment that causes the addition or removal of nucleotides at one or both strands. These additions or deletions prior to re-ligation cause the presence of insertion or deletion (indel) mutations at the NHEJ repair site in the DNA sequence. A variety of mutations due to insertions/deletions can alter the reading frame or introduce stop codons prematurely, and thus produce nonfunctional proteins.

In some embodiments, the efficacy of the mRNA encoding the nuclease is determined based on an in vitro model. In some embodiments, the in vitro model is HEK293 cells. In some embodiments, the in vitro model is a HUH7 human hepatoma cell. In some embodiments, the in vitro model is a primary hepatocyte, e.g., a primary human or mouse hepatocyte.

In some embodiments, gene editing events are detected, such as the formation of insertion/deletion ("indel") mutations using linear amplification of labeled primers and isolation of labeled amplification products (hereinafter referred to as "LAM-PCR" or "Linear Amplification (LA)" Methods, as described in WO2018/067447 or Schmidt et al, nature Methods 4:1051-1057 (2007), or next generation sequencing ("NGS"; e.g., using Illumina NGS platform), as described below), or other Methods known in the art for detecting insertion/deletion mutations.

For example, in order to quantitatively determine editing efficiency at a target location in a genome, in NGS methods, genomic DNA is isolated and deep sequencing is utilized to identify the presence of insertions and deletions introduced by gene editing. PCR primers are designed around the target site (e.g., TTR) and the genomic region of interest is amplified. Additional PCR was performed according to the manufacturer's protocol (Illumina) to add the chemistry required for sequencing. Amplicons were sequenced on an Illumina MiSeq instrument. After elimination of reads with low quality scores, the reads are aligned with a reference genome (e.g., mm 10). The resulting file containing reads is mapped to a reference genome (BAM file), where reads that overlap with the target region of interest are selected and the number of wild-type reads is calculated relative to the number of reads containing insertions, substitutions or deletions. The percent editing (e.g., "editing efficiency" or "percent editing") is defined as the total number of sequence reads with insertions or deletions relative to the total number of sequence reads including wild-type.

Examples

The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.

Example 1 materials and methods

In vitro transcription of nuclease mRNA ("IVT")

Capped and polyadenylation mRNA containing N1-methyl pseudoU is produced by in vitro transcription using conventional methods. For example, plasmid DNA containing the T7 promoter, sequences for transcription and polyadenylation regions was linearized with XbaI according to the manufacturer's protocol. XbaI was deactivated by heating. Linearized plasmids were purified from enzymes and buffer salts. The IVT reaction for producing modified mRNA was performed by incubating at 37 ℃ each of the following: 50 ng/. Mu.L of linearized plasmid; GTP, ATP, CTP and N1-methyl pseudo-UTP (Trilink) at 2-5mM each; 10-25mM ARCA (Trilink); 5U/. Mu. L T7 RNA polymerase; 1U/. Mu.L murine RNase inhibitor (NEB); 0.004U/. Mu.L of inorganic E.coli pyrophosphatase (NEB); and 1 x reaction buffer. TURBO DNase (Thermo Fisher) was added to a final concentration of 0.01U/. Mu.L and the reaction incubated at 37℃to remove the DNA template.

MRNA was purified using MEGACLEAR TRANSCRIPTION CLEAN-up kit (Thermo Fisher) or RNeasy Maxi kit (Qiagen) according to the manufacturer's protocol. Alternatively, mRNA is purified via a precipitation scheme, in some cases followed by HPLC-based purification. Briefly, after dnase digestion, mRNA was purified using LiCl precipitation, ammonium acetate precipitation, and sodium acetate precipitation. For HPLC purified mRNA, after LiCl precipitation and reconstitution, the mRNA is purified by RP-IP HPLC (see, e.g., kariko et al, nucleicAcids Research,2011, vol.39, stage 21 el 42). Fractions selected for pooling were pooled and desalted by sodium acetate/ethanol precipitation as described above. In another alternative, the mRNA is purified by LiCl precipitation, followed by further purification by tangential flow filtration. RNA concentration was determined by measuring absorbance at 260nm (Nanodrop) and transcripts were analyzed by capillary electrophoresis with Bioanalyzer (Agilent).

When the sequences cited in this paragraph are referred to below for RNA, it is understood that T should be replaced with U (which may be a modified nucleoside as described above). Messenger RNAs used in the examples include 5 'caps and 3' polyadenylation sequences, e.g., up to 100nt. Guide RNAs are synthesized chemically with modified nucleotides by commercial suppliers or using standard in vitro synthesis techniques.

According to SEQ ID No:43-47 and 49 (see sequences in table 39A), cas9 mRNA was generated from plasmid DNA encoding the open reading frame (Streptococcus pyogenes) ("Spy") Cas 9.

Hepatocyte preparation

Primary Mouse Hepatocytes (PMH), primary Rat Hepatocytes (PRH), primary Human Hepatocytes (PHH), and primary cynomolgus monkey hepatocytes (PCH) were prepared as follows. PMH (Gibco, MCM837, unless specified otherwise), PRH (Gibco, rs977, unless specified otherwise), PCH (In Vitro ADMET Laboratories,10136011, unless specified otherwise), PHH (Gibco, hu8284, unless specified otherwise) were thawed and resuspended in 50mL of Cryopreserved Hepatocyte Recovery Medium (CHRM) (Invitrogen, CM 7000) followed by centrifugation. Cells were resuspended in hepatocyte medium by the following seeding supplements: williams E medium with FBS content was inoculated with a supplement (Gibco, catalog number A13450). Cells were pelleted by centrifugation, resuspended in medium, and plated at densities of 20,000 cells/well (for PMH) and 30,000 cells/well (for PHH) on a Bio-coat collagen I coated 96-well plate (Corning # 354407). The inoculated cells were allowed to stand and adhere in a tissue culture incubator at 37℃and 5% CO ₂ atmosphere for 4-6 hours. After incubation, cell monolayer formation was checked and washed once, and inoculated with 100 μl of hepatocyte maintenance medium: williams' E medium (Gibco, catalog number A12176-01) plus a supplement package (Gibco, catalog number CM 3000).

HEK cell preparation

HEK-293 cells (ATCC, CRL-1573, unless otherwise specified) were thawed and resuspended in serum-free Dulbecco's modified eagle's medium (Dulbecco's Modified Eagle Medium) (Corning # 10-013-CV) with 10% FBS content (Gibco # A31605-02) and 1% penicillin-streptomycin (Gibco # 15070063). Cells were counted and inoculated into Dulbecco's modified eagle's medium (Corning # 10-013-CV) with 10% FBS content (Gibco #A 31605-02) on 96 well tissue culture plates (Falcon, # 353072). The inoculated cells were allowed to stand and adhere in a tissue culture incubator at 37℃under 5% CO ₂ atmosphere for 18 hours.

Preparation of LNP formulations containing sgrnas and Cas9 mRNA

In general, the lipid nanoparticle component is dissolved in 100% ethanol at various molar ratios. RNA cargo (e.g., cas9 mRNA and gRNA) was dissolved in 25mM citrate, 100mM NaCl (pH 5.0), resulting in an RNA cargo concentration of approximately 0.45 mg/mL. The LNP used contained the ionizable lipid (octadeca-9, 12-dienoic acid (9 z,12 z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester, also known as (9 z,12 z) -octadeca-9, 12-dienoic acid 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester) (also referred to herein as lipid a), cholesterol, distearylphospholipid choline (DSPC) and 1, 2-dimyristoyl-rac-glycerol-3-methylpolyoxyethyleneglycol 2000 (PEG 2 k-DMG) in a molar ratio of 50% lipid a, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. The LNP used comprises a single RNA species, such as Cas9 mRNA or sgRNA. LNP was similarly prepared with a mixture of Cas9 mRNA and guide RNA.

LNP was prepared using a cross-flow technique using lipid-containing ethanol mixed with two volumes of RNA solution and an impinging jet of one volume of water. First, lipids in ethanol were mixed with two volumes of RNA solution via a mixing cross. The fourth water stream is then mixed with the output stream of the cross via an in-line tee (see WO2016010840, fig. 2). LNP was kept at room temperature for 1 hour and further diluted with water (approximately 1:1 v/v). The diluted LNP buffer was exchanged into 50mM Tris, 45mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) and concentrated as necessary by methods known in the art. The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was characterized to determine encapsulation efficiency, polydispersity index, and average particle size. The final LNP was stored at 4℃or-80℃until further use.

SgRNA and C as9 mRNA Liposome transfection

Liposome transfection of Cas9 mRNA and gRNA was performed using a pre-mixed lipid formulation. The liposome transfection reagent contained ionizable lipid A, cholesterol, DSPC and PEG2k-DMG in a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2k-DMG. This mixture was reconstituted in 100% ethanol and then mixed with RNA (e.g., cas9 mRNA and gRNA) at a molar ratio of lipid amine to RNA phosphate (N: P) of about 6.0.

Next generation sequencing ("NGS") and editing efficiency analysis

Genomic DNA was extracted using commercial kits according to manufacturer's protocol, for example QuickExtract ^TM DNA extraction solution (Lucigen, catalog No. QE 09050). In order to quantitatively determine editing efficiency at a target location in a genome, deep sequencing was used to identify the presence of insertions and deletions introduced by gene editing. PCR primers are designed around a target site within a gene of interest (e.g., TRAC), and genomic regions of interest are amplified. Primer sequence design was performed according to the standards in the art.

Additional PCR was performed according to the manufacturer's protocol (Illumina) to add chemicals for sequencing. Amplicons were sequenced on an Illumina MiSeq instrument. After elimination of reads with low quality scores, the reads are aligned with a human reference genome (e.g., hg 38). Reads overlapping the target region of interest are realigned with the local genomic sequence to improve the alignment. The number of wild-type reads was then counted relative to the number of reads containing C to T mutations, C to a/G mutations or insertions/deletions. Insertions and deletions were scored in a 20bp region centered on the predicted Cas9 cleavage site. Percent insertions/deletions are defined as the total number of sequencing reads that insert or delete one or more bases within the 20bp scoring region divided by the total number of sequencing reads (including wild-type). The C to T mutation or C to A/G mutation was scored in a 40bp region comprising 10bp upstream and 10bp downstream of the 20bp sgRNA target sequence. The percent C to T editing is defined as the total number of sequencing reads having one or more C to T mutations within the 40bp region divided by the total number of sequencing reads (including wild type). The percentage of C to A/G mutations was similarly calculated.

Example 2 in vitro editing using selected leads in Primary Mouse Hepatocytes (PMH)

Modified sgrnas screening was performed to evaluate the editing efficiency of 95 different sgrnas targeting different sites within the mouse TTR gene. Based on the study, two sgrnas (G021320 and G021256) were selected for evaluation in a dose response assay. The two test primers were compared to the mouse TTR SPYCAS primer (G000502) with a 20 nucleotide primer sequence. The tested NmeCas sgrnas targeting the mouse TTR gene included a 24 nucleotide guide sequence (as represented by N) and guide backbone ：mN*mNNNNNNNNmNNNmNNNNNNNNNNNNmGUUGmUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAU*AAGmGmCCmGmUmCmGmAmAmAmGmAmUGUGCmCGCmAmAmCmGCUCUmGmCCmUmUmCmUGmGCmAmUC*mG*mU*mU(SEQ ID NO：508), where A, C, G, U and N are adenine, cytosine, guanine, uracil and any ribonucleotide, respectively, unless otherwise specified. m indicates a 2' o-methyl modification, and indicates phosphorothioate linkage between nucleotides. Unmodified and modified versions of the guide are provided in table 39B.

The guide and Cas9 mRNA liposomes were transfected into Primary Mouse Hepatocytes (PMHs) as described below. PMH (In Vitro ADMETLaboratories MCM) was prepared as described in example 1. Liposome transfection was performed as described in example 1 with dose response of sgRNA and mRNA. Briefly, cells were incubated at 37℃for 24 hours with 5% CO ₂, followed by treatment with liposome complexes. The liposome complexes were incubated at 37℃for 10 min in maintenance medium containing 10% Fetal Bovine Serum (FBS). After incubation, the liposome complexes were added to mouse hepatocytes in an 8-point, 3-fold dose response assay starting with a maximum dose of 300ng Cas9 mRNA and 50nM sgRNA. Under each condition, the messenger RNA dose was proportional to the gRNA dose, although only gRNA doses are listed in table 5. Cells were lysed 72 hours after treatment and NGS analysis was performed as described in example 1.

Dose response of edit efficiency to guide concentration was performed in triplicate samples. Table 5 shows the mean percent editing and Standard Deviation (SD) at each guide concentration and calculated EC ₅₀ values. The mean and Standard Deviation (SD) are depicted in fig. 1.

TABLE 5 average percent editing in primary mouse hepatocytes

Example 3 sgrnas versus sgrnas or pgrnas using LNP: mRNA ratio

A study was performed to evaluate the editing efficiency of the sgRNA design containing PEG linkers (pgRNA). The study compared two TTR-targeted grnas with identical guide sequences, one of which included three PEG linkers in the constant region of the guide (pgRNA, G021846), and one of which did not (G021845), as shown in table 39B. The guide and mRNA were formulated in LNP alone and mixed to the required ratio for delivery to Primary Mouse Hepatocytes (PMH) via Lipid Nanoparticles (LNP).

PMH cells were prepared, treated and analyzed as described in example 1, unless otherwise indicated. PMH cells (lot MCM 114) from In Vitro ADMET Laboratories were seeded at a density of 15,000 cells/well. Cells were treated with LNP as described below. LNP was prepared generally as described in example 1. LNP was prepared with a lipid composition of 50/9/38/3, expressed as the molar ratio of ionizable lipid A/cholesterol/DSPC/PEG, respectively. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. LNP encapsulates a single RNA material, gRNA G021845, gRNA G021846, or mRNA (mRNA M), as described in example 1.

PMH cells were treated with varying amounts of LNP at a ratio of gRNA to mRNA of 1:4, 1:2, 1:1, 2:1, 4:1 or 8:1 based on the weight of RNA cargo. Duplicate samples were included in each assay. The guide was assayed at 8-point 3-fold dose response curves starting at 1 ng/. Mu.L total RNA concentration, as shown in Table 6. The average percent edit results are shown in table 6. Fig. 2A shows the average percent editing of sgRNA G021845, and fig. 2B shows the average percent editing of sgRNAG 021846. "ND" in the table indicates a value that could not be detected due to experimental failure.

Table 6 average percent editing in pmh

Example 3.1 in vitro editing of modified PEGylated guide (pgRNA) in PMH using LNP

Modified pgrnas with the same targeting sites in the mouse TTR gene were assayed to evaluate editing efficiency in PMH cells.

PMH cells were prepared, treated and analyzed as described in example 1, unless otherwise indicated. PMH cells from In Vitro ADMET Laboratories (lot number MC 148) were used and seeded at a density of 15,000 cells/well. LNP formulations were prepared as described in example 1. LNP was prepared with a lipid composition of 50/9/38/3, expressed as the molar ratio of ionizable lipid A/cholesterol/DSPC/PEG, respectively. LNP was formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and with gRNA or with mRNA indicated in table 7.

PMH in 100 μl medium was treated with 30ng LNP of total mRNA (mRNA P) by weight and LNP of gRNA in the amounts indicated in table 7. Samples were run in duplicate. The average editing results of PMH are shown in table 7 and fig. 3.

Table 7 average percent edit in pmh

EXAMPLE 4 Nme2-mRNA Studies

Example 4.1 in vitro editing in primary mouse hepatocytes

The editing efficiency of messenger mRNA encoding Nme2Cas9 ORF with different NLS layouts in Primary Mouse Hepatocytes (PMHs) was determined.

PMH was prepared as described in example 1. Liposome transfection was performed using Lipofectamine MessengerMAX transfection reagent (Invitrogen LMRNA 001) according to the manufacturer's protocol to transform cells with 100nM sgRNA G020361 targeted to mouse PCSK9 and mRNA at the concentrations listed in Table 8. Triplicate samples were included in the assay. After 72 hours of incubation in maintenance medium at 37 ℃, cells were collected and NGS analysis was performed as described in example 1. The average editing results with Standard Deviation (SD) are shown in table 8 and fig. 4.

Table 8-average percent editing at PCSK9 Gene locus in PMH

Example 4 dose response of 2-Nme2 ORF variants and primers with chemically modified variations

The efficiency of editing messenger mRNA encoding Nme2Cas9 ORF with different NLS configurations in Primary Human Hepatocytes (PHH) and HEK-293 cells was determined. Assays were performed using grnas with the same guide sequence targeting VEGFA locus TS47, and grnas with different lengths and chemical modification patterns. PHH cells were prepared as described in example 1. HEK293 cells were thawed and seeded in DMEM (Corning, 10-013-CV) with 10% fbs in 96-well plates at a density of 30,000 cells/well and incubated for 24 hours. Liposome transfection was performed using Lipofectamine MessengerMAX transfection reagent (Invitrogen LMRNA 001) according to the manufacturer's protocol. Dose response 1 starting with the highest dose of 100nM gRNA and 1 ng/. Mu.L mRNA: the 3 dilution series was used to transform cells with the concentrations of gRNA listed in tables 9-10. Repeated samples were included in the assay. After 72 hours incubation at 37 ℃, cells were collected and NGS analysis was performed as described in example 1. Average editing results with Standard Deviation (SD) are shown in table 9 and fig. 5A-5C (for HEK cells) and table 10 and fig. 5D-5F (for PHH).

Table 9 mean percent editing in hek cells

TABLE 10 average percent editing in PHH cells

Example 4.3 dose response using Nme2 NLS variant of LNP in PMH

The editing efficiency of messenger mRNA encoding Nme2Cas9 ORF with different NLS layouts in Primary Mouse Hepatocytes (PMHs) was determined. The assay tests for a primer targeting the mouse TTR locus and includes both sgRNA and pgRNA designs.

PMH was prepared as in example 1. LNP was typically prepared as described in example 1 with a single RNA material as cargo, as indicated in table 11. LNP was prepared with a lipid composition of 50/9/38/3, expressed as the molar ratio of ionizable lipid A/cholesterol/DSPC/PEG, respectively. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6.

Cells were treated with 60ng/100 μl LNP containing gRNA and LNP containing mRNA as indicated in table 11 by RNA weight. Cells were incubated in Williams E medium (Gibco, A1217601) containing maintenance supplements and 10% fetal bovine serum at 37℃for 72 hours. After 72 hours incubation at 37 ℃, cells were collected and edited by NGS evaluation as described in example 1. The average percent edit data is shown in table 11 and fig. 6.

Table 11 mean percent editing at the mouse TTR locus in primary mouse hepatocytes.

Example 4.4 dose response using Nme2 NLS variant of LNP in PMH

PMH (Gibco, MC 148) was prepared as described in example 1. LNP is typically prepared as described in example 1 using a single RNA species as cargo. LNP was prepared with a lipid composition of 50/9/38/3, expressed as the molar ratio of ionizable lipid A/cholesterol/DSPC/PEG, respectively. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6.

Cells were treated with 30ng/100 μl LNP containing gRNA G021844 and LNP containing mRNA as indicated in table LS4 by RNA weight. Cells were incubated for 24 hours in Williams E medium (Gibco, A1217601) containing maintenance supplements and 10% fetal bovine serum. After 72 hours of incubation, cells were collected and edited by NGS evaluation as described in example 1. The average percent edit data is shown in table 12 and fig. 7.

Table 12-average percent editing in PMH treated with LNP.

EXAMPLES 5-NmeCas protein expression

EXAMPLE 5.1 protein expression in Primary human hepatocytes

To quantify the expression of each mRNA construct, mRNA and protein expression levels were measured after LNP delivered mRNA encoding SpyCas9 or NmeCas to primary human hepatocytes.

PHH cells were prepared as described in example 1. LNP is typically prepared as described in example 1 using a single RNA species as cargo. LNP was prepared at a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6.

Cells are given one LNP containing mRNA (mRNA only), or two LNPs containing mRNA or gRNA. Each LNP was applied to cells at 16.7ng total RNA cargo per 100. Mu.l. After treatment with LNP, cells were incubated for 24 hours in williams E medium (Gibco, a 1217601) containing maintenance supplements and 10% fetal bovine serum. After 24 hours incubation at 37 ℃, cells were collected and quantitatively expressed via the Nano-Glo HiBiT lysis detection system (Promega, N3030) following the manufacturer's instructions. The original luminescence was normalized to the standard curve using HiBiT control protein (Promega, N3010). Protein expression of the different Cas9 variants shown in table 13 and fig. 8 was normalized to the SpyCas9 expression lines measured in corresponding hepatocytes delivered with SpyCas9mRNA alone. Consistent with the data shown in table 13, protein expression from these same constructs was higher for NmeCas9 constructs than for SpyCas9 constructs when measured by western blot detection with anti-HiBiT antibodies from PHH cell extracts or by detection in PMH, PCH, PHH and HiBiT in PRH cells.

Table 13-mean fold expression of Cas9 variants expressed as compared to SpyCas9 in corresponding hepatocytes delivered with SpyCas9 mRNA alone, as measured by HiBiT assay

Example 5.2: protein expression in T cells

To quantify the expression of each mRNA construct, protein expression levels were measured after LNP delivered mRNA encoding SpyCas9 or Nme2Cas9 to T cells.

Healthy human donor blood apheresis is commercially available (Hemacare). T cells from both donors (W106 and W864) were isolated by negative selection on MultiMACS Cell Separator Plus apparatus using EasySep human T cell isolation kit (Stem Cell Technology, cat. 17951) according to manufacturer's instructions. Isolated T cells were cryopreserved in CS10 freezing medium (Cryostor, cat# 07930) for future use.

After thawing, T cells were cultured in complete T cell growth medium consisting of CTSOpTmizer basal medium (CTS OpTmizer medium (Gibco, a 1048501) containing 1X GlutaMAX, 10mM HEPES buffer, 1% penicillin/streptomycin) supplemented with cytokines (200 IU/mL IL2, 5ng/mL IL7 and 5ng/mL IL 15) and 2.5% human serum (Gemini, 100-512). After overnight standing at 37℃T cells at a density of 1e6/mL were activated with T cell TransAct reagent (1:100 dilution, miltenyi) and incubated for 48 hours in a tissue culture incubator.

Activated T cells were treated with LNP delivering mRNA encoding Nme2-mRNA or Spy mRNA with HiBiT tags. LNP was prepared generally as in example 1. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. LNP encapsulating Nme2Cas9mRNA used lipid a, cholesterol, DSPC, and PEG2k-DMG at a molar ratio of 50% lipid a, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG. LNP encapsulating SpyCas 9mRNA used lipid a, cholesterol, DSPC, and PEG2k-DMG at a molar ratio of 50% lipid a, 38.5% cholesterol, 10% DSPC, and 1.5% PEG2k-DMG.

Immediately prior to LNP treatment of T cells, LNP was preincubated with 10ug/mL of ApoE3 (Peprotech, cat# 350-02) at 37℃for 5 min at an LNP concentration of 13.33ug/mL total RNA in complete T cell culture medium supplemented with cytokines (200 IU/mL IL2 (Peprotech, cat# 200-02), 5ng/mL IL7 (Peprotech, cat# 200-07) and 5ng/mL IL15 (Peprotech, cat# 200-15) and 2.5% human serum (Gemini, 100-512.) after incubation, then LNP was mixed with T cells in complete T cell culture medium at a volume of 1:1, cytokines were used for ApoE incubation, T cells were collected for protein expression analysis 24, 48 and 72 hours after LNP treatment byHiBiT LYTIC ASSAY (Promega) lyse T cells and viaThe Nano-Glo HiBiT extracellular detection system (Promega, catalog No. N2420) followed the manufacturer's instructions to quantify Cas9 protein levels. Luminescence was measured using a Biotek Neo2 plate reader. Linear regression was drawn on GraphPad using the number of proteins and luminescence readings from the standard control, forcing the line through x=0, y=0. The number of proteins per lysate was calculated using the y=ax+0 equation.

Samples were normalized to the average of SpyCas9 at an LNP dose of 0.83 μg/ml. Tables 14A-14B and fig. 9A-9F show relative Cas9 protein expression of mRNA in activated cells at 24, 48, and 72 hours after LNP treatment for donor 1 or donor 2. Cas9 is expressed in activated T cells in a dose dependent manner. Protein expression was higher for Nme2Cas9 samples compared to SpyCas9 samples in activated T cells.

Table 14A: protein expression of donor 1 normalized to average SpyCas 9.83 μg/ml sample

Table 14B: protein expression of donor 2 normalized to average spycas90.83 μg/ml sample

Example 6 in vivo editing in mouse liver Using Lipid Nanoparticles (LNPs)

LNP formulated for all in vivo studies was performed as described in example 1. Deviations from the regimen are noted in the respective embodiments. Transport and Storage Solutions (TSS) for LNP preparation were dosed as vehicle-only negative controls in the experiments.

In vivo editing in a mouse model

The in vivo editing efficiency of the selected guide design was tested. CD-1 female mice in the 6-10 week old range were used in each study involving mice. Animals were weighed prior to dosing. LNP was formulated generally as described in example 1. LNP contains 50% ionizable lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG in a molar ratio. The lipid nucleic acid assemblies were formulated with a molar ratio of lipid amine to RNA phosphate (N: P) of about 6.

LNP was administered via the lateral tail vein in a volume of 0.2mL per animal (about 10mL per kilogram body weight). Body weight was measured twenty-four hours after administration. Animals were euthanized by exsanguination about 6-7 days after LNP delivery, under isoflurane anesthesia following dosing. Blood was collected via cardiac puncture into a serum separation tube. For studies involving in vivo editing, liver tissue was collected from the left inside leaf from each animal for DNA extraction and analysis.

For in vivo studies, genomic DNA was extracted from tissues using a bead-based extraction kit, such as the Zymo Quick-DNA 96 kit (Zymo Research, catalog number D3010) according to the manufacturer's protocol. NGS analysis was performed as described in example 1.

Transthyretin (TTR) ELISA assay for animal studies

Blood was collected and serum was isolated as described above. Total TTR serum levels were determined using a mouse prealbumin (transthyroxine) ELISA kit (AVIVA SYSTEMS Biology, catalog No. OKIA 00111). Kit reagents and standards were prepared according to the manufacturer's protocol. The mouse serum was diluted to a final dilution of 10,000 fold by 1x assay diluent. Standard curve dilutions (100 μl each) and diluted serum samples were added to each well of ELISA plates pre-coated with capture antibodies. The plates were incubated for 30 minutes at room temperature and then washed. Enzyme-antibody conjugate (100 μl per well) was added and incubated for 20 minutes. The unbound antibody conjugate is removed and the plate is washed again, then the chromogenic substrate solution is added. The plates were incubated for 10 minutes, then 100 μl of stop solution, such as sulfuric acid (about 0.3M), was added. The plates were read on Clariostar plate reader at an absorbance of 450 nm. Serum TTR levels were calculated by SoftMax Pro software version 6.4.2 or Mars software version 3.31 using a four parameter logistic curve fit off of the standard curve. The final serum values were adjusted according to the assay dilutions. Percent protein knockdown (KD%) values were determined relative to controls, which are typically vehicle (TSS) sham-treated animals unless indicated otherwise. The TSS percentage was calculated by dividing each sample TTR value by the average of the TSS group and then adjusting to the percentage value.

Example 6.1 in vivo editing using coformulated LNP

The editing efficiency of the modified sgrnas tested in example 4.2 was further evaluated in a mouse model. Guide RNA designs with the same guide sequence targeting mouse PCSK9 but with different lengths of conserved regions were tested and LNPs were prepared as described in example 1. LNP was prepared using ionizable lipid a, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 50% lipid a, 38% cholesterol, 9% DSPC, and 3% PEG2 k-DMG. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. The gRNA and mRNA C targeting the PCSK9 gene as indicated in table 15 were co-formulated in LNP at 1:2 by weight of gRNA and mRNA. LNP was administered to female CD-1 mice (n=5) at a dose of 1mg/kg total RNA as described above. Mice were euthanized 7 days after dosing. The editing efficiency of LNP containing the indicated sgrnas is shown in table 15 and plotted in fig. 10.

Table 15 average percent editing in mouse liver.

Guide body	Dosage (mg/kg)	Average edit%	SD
				Vehicle body	-	0.0	0.0
G017564	1	2.5	0.9
				G017565	1	2.2	1.0
G017566	1	2.2	1.2

Example 6.2 in vivo editing Using pgRNA and mRNA LNP

The editing efficiency of the modified pgrnas was evaluated in vivo. In addition to the guide modifications specified in the previous study of example 6.1, the four nucleotides in each loop of repeat/anti-repeat region, hairpin 1 and hairpin 2 were also replaced with spacer-18 PEG linkers.

LNP is typically prepared as described in example 1 using a single RNA species as cargo. LNP contains lipid A, cholesterol, DSPC and PEG2k-DMG in a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2k-DMG. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6.

LNP containing TTR gene-targeted gRNA indicated in table 16 was administered to female CD-1 mice (n=5) as described above at a dose of 0.1mg/kg or 0.3mg/kg total RNA. LNP containing mRNA (mRNA M; SEQ ID NO: 23) and LNP containing pgRNA (G021846 or G021844), respectively, are expressed as 1 by weight of RNA: 2 is delivered simultaneously. Mice were euthanized 7 days after dosing.

The editing efficiency, serum TTR knockdown, and TSS percentages of LNP containing the indicated pgrnas are shown in table 16 and are depicted in fig. 11A-11C, respectively.

TABLE 16 liver editing, serum TTR protein and TTR protein knockdown

PgRNA (G021844) from the study described above was evaluated in mice at different dose levels of alternative mRNA. LNP is typically prepared as described in example 1 using a single RNA species as cargo. LNP containing pgRNA (G21844) or mRNA (mRNA P or mRNA M) were formulated as described in example 1. LNP used was prepared with ionizable lipid a, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 50% lipid a, 38% cholesterol, 9% DSPC, and 3% PEG2 k-DMG. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. Both G000502 and G021844 target exon 3 of the mouse TTR gene. LNP containing pgRNA and LNP containing mRNA are based on combined RNA weight, respectively at 2 by RNA weight: 1: mRNA ratios were administered simultaneously. The additional LNP is co-formulated with G000502 and SpyCas9 mRNA, respectively, in a 1:2 ratio by weight (preferably SpyCas9 guide: mRNA ratio).

LNP indicated in table 17 was administered to female CD-1 mice (n=4) at a dose of 0.1mg/kg or 0.03mg/kg total RNA. The editing efficiency of LNP containing the specified gRNA is shown in table 17 and plotted in fig. 11D and 11E.

TABLE 17 liver editing and serum TTR protein knockdown

Example 6.3 in vivo editing Using sgRNA and mRNA LNP

LNP is typically prepared as described in example 1 using a single RNA species as cargo. LNP used was prepared with lipid A, cholesterol, DSPC and PEG2k-DMG in a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. The sgrnas were designed to target either the pcsk9 gene (G020361) or the Rosa26 gene (G020848).

LNP containing sgRNA or mRNA was administered to female CD-1 mice (n=5) at a dose of 1mg/kg total RNA. The mrnas tested (mRNA C, mRNA J, mRNAQ, mRNA N) were designed with different numbers and arrangements of NLS. LNP is administered simultaneously at a gRNA to mRNA ratio of 1:1 by weight of RNA based on the combined weight of RNA cargo. The average percent editing is shown in table 18 and plotted in fig. 12.

Table 18 average percent editing in mouse liver.

Example 7 in vivo editing Using NmeCas and sgRNA or pgRNA

The editing efficiency of the modified pgrnas tested with Nme2Cas9 was tested in a mouse model. All Nme sgrnas tested contained the same 24nt guide sequence targeting mTTR.

LNP is typically prepared as described in example 1 using a single RNA species as cargo. LNP used was prepared with lipid A, cholesterol, DSPC and PEG2k-DMG in a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. LNP was mixed in a 2:1 ratio by weight of gRNA to mRNA cargo. The dose is calculated based on the combined RNA mass of the gRNA and mRNA. Transport and Storage Solutions (TSS) for LNP preparation were dosed as vehicle-only negative controls in the experiments.

CD-1 female mice in the 6-10 week old range were used in each study involving mice (n=5/group except for n=4 for TSS control). Formulations were administered intravenously via tail vein injection according to the dosages listed in table 19. Adverse reactions in animals were observed periodically at least 24 hours after dosing. Six days after treatment, animals were euthanized by cardiac puncture under isoflurane anesthesia; liver tissue was collected for downstream analysis. Liver perforations weighing between 5 and 15mg were collected to isolate genomic DNA and total RNA. Genomic DNA samples were analyzed by NGS sequencing as described in example 1. The editing efficiency of LNP containing the specified mRNA and gRNA is shown in table 19 and plotted in fig. 13.

TABLE 19 average percent edit in mouse liver

Example 8 in vivo base editing using Nme2Cas9 gRNA

Editing efficiency of modified grnas with different mrnas was tested with Nme base editor constructs in a mouse model. This experiment was performed in parallel with example 7 and using the same control sample. LNP is typically prepared as described in example 1 using a single RNA species as cargo. The LNP used was prepared with lipid A, cholesterol, DSPC and PEG2k-DMG in a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. The LNP used was formulated as described in example 1, except that each component, guide RNA or mRNA was formulated individually in the LNP, and the LNP was mixed prior to administration, as described in table 20. For Nme2Cas9 and Nme2Cas9 base editor samples, LNP was mixed at a ratio of 2:1 by weight of gRNA to editor mRNA cargo. For the SpyCas9 base editor sample, LNP is 1 by weight of gRNA and editor mRNA cargo: 2. The doses as indicated in table 20 and fig. 14 were calculated based on the combined RNA weight of gRNA and editor mRNA. The base editor sample was treated with an additional 0.03mpk UGI mRNA. Transport and Storage Solutions (TSS) for LNP preparation were dosed as vehicle-only negative controls in the experiments.

CD-1 female mice in the 6-10 week old range were used in each study involving mice (n=5/group except for n=4 for TSS control). Formulations were administered intravenously via tail vein injection according to the dosages listed in table 20. Adverse reactions in animals were observed periodically at least 24 hours after dosing. Six days after treatment, animals were euthanized by cardiac puncture under isoflurane anesthesia; liver tissue was collected for downstream analysis. Liver perforations weighing between 5 and 15mg were collected to isolate genomic DNA and total RNA. Genomic DNA was extracted using DNA isolation kit (ZymoResearch, D3010) and samples were analyzed by NGS sequencing as described in example 1. The editing efficiency of LNP containing the specified gRNA is shown in table 20 and plotted in fig. 14.

Table 20-average percent editing in mouse liver.

Example 9 guide screening using Nme1Cas9 and Nme3Cas9 mRNA in T cells

Editing efficiency of a modified gRNA scaffold was tested in T cells with Nme1Cas9 or Nme3Cas9 mRNA using a guide with 9 different target sequences in the TRAC locus.

Healthy human donor blood apheresis is commercially available (Hemacare, donor 3786), and cells are washed and resuspended inTreatment was performed in PBS/EDTA buffer (Miltenyi Biotec catalog 130-070-525) and in MultiMACS ^TM Cell 24Separator Plus apparatus (Miltenyi Biotec). Using Stright fromCD4/CD8 MicroBead kit, human (Miltenyi Biotec catalog No. 130-122-352) isolated T cells via positive selection. T cells were aliquoted and cryopreserved to be used inCS10 (StemCell Technologies catalog number 07930). After thawing, T cells were seeded at a density of 1.0×10++ ⁶ cells/ml in T Cell Growth Medium (TCGM) consisting of: CTS OpTmizer T cell expansion SFM and T cell expansion supplements (Thermo Fisher catalog A1048501), 5% human AB serum (GeminiBio catalog 100-512), 1 Xpenicillin-streptomycin, 1 XGlutamax, 10mM HEPES, 200U/mL recombinant human interleukin-2 (Peprotech, catalog 200-02), 5ng/mL recombinant human interleukin-7 (Peprotech, catalog 200-07), and 5ng/mL recombinant human interleukin-15 (Peprotech, catalog 200-15). T cells were allowed to stand in this medium for 24 hours, at which time they were activated by T CELL TRANSACT ^TM human reagents (Miltenyi, catalogue No. 130-111-160) added in a 1:100 volume ratio.

For Nme1Cas9 guide screening, solutions containing mRNA encoding Nme1Cas9 (mRNA AB) were prepared in P3 buffer. Guide RNAs targeting various sites in the TRAC locus were denatured at 95 ℃ for 2 min and incubated for 5 min at room temperature. Forty-eight hours after activation, T cells were collected, centrifuged, and resuspended in P3 electroporation buffer (Lonza) at a concentration of 12.5x10- ⁶ cells/ml. For each well to be electroporated, 1x10 ⁵ T cells were mixed with 600ng Nme1Cas9 mRNA and 5. Mu.M gRNA in a final volume of 20. Mu.L of P3 electroporation buffer. This mixture was transferred in duplicate into a 96-well nucleic acid ^TM plate and electroporated using the manufacturer's pulse code. The electroporated T cells were then allowed to stand in CTS OpTmizer T cell growth medium without cytokines for 15 minutes and then transferred to a new flat bottom 96 well plate containing CTSOpTmizer T cell growth medium additionally supplemented with cytokines. The resulting plates were incubated for 3 days at 37 ℃. On day 3 after electroporation, cells were split 1:2 in 2U-shaped plates.

On day 7 after electroporation, the seeded T cells were assayed by flow cytometry to determine surface expression of T cell receptors. Briefly, T cells were incubated with antibodies to CD3 (BioLegend, catalog No. 317336), CD4 (BioLegend, catalog No. 317434), CD8 (BioLegend, catalog No. 301046) and Viakrome (Beckman Coulter, catalog No. C36628). The cells were then washed, resuspended in cell staining buffer and processed on a Cytoflex flow cytometer (Beckman Coulter). Flow cytometry data were analyzed using the FlowJo software package. T cells are gated based on size, shape, viability and expression of CD8 and CD 3. Samples were run in duplicate.

CD3 is a cell surface component of the T cell receptor complex and its presence at the cell surface is used as a surrogate marker for TRAC protein expression. The CD3 negative cell populations and corresponding Standard Deviations (SDs) for each of the designated grnas are shown in table 21 and plotted in fig. 16.

Table 21 average CD3 negative T cell percentages after TRAC editing with Nme1Cas9

For screening the guide with Nme3Cas9 mRNA, T cells were prepared as described in the examples. Solutions containing mRNA encoding Nme3Cas9 (mRNA Z), as well as NmelCas (mRNA AB) and Nme2Cas9 (mRNA O) controls were prepared in P3 buffer. Electroporation of NmeCas9 (e.g., nmelCas, nme2Cas9, or Nme3Cas 9) gRNA and mRNA was performed as described above. Samples were electroporated in triplicate. On day 3 post electroporation, cell determination via flow cytometry was performed as described above.

The CD3 negative cell populations and corresponding Standard Deviations (SDs) for each of the designated grnas are shown in table 22 and plotted in fig. 17.

Table 22 average percentage of CD3 negative T cells after TRAC editing with Nme3Cas 9.

Guide ID	Average value of	SD
			G028844	2.99	0.49
G028845	2.97	0.30
			G028846	22.83	1.65
G028847	8.71	1.16
			G028848	95.6	0.74
G028849	6.24	0.02
			G028850	69.63	3.57
G028851	1.49	0.53
			G028852	79.13	3.34
G028853 (Nme 1 control)	97.43	0.20
			G021469 (Nme 2 control)	92.46	2.00

Example 10 codon optimization NmeCas expression of mRNA

To quantify the expression of each mRNA construct, protein expression levels were measured after electroporation of mRNA encoding Nme1Cas9, nme2Cas9, or Nme3Cas9 to T cells. All NmeCas mRNA constructs have the same general structure with a contiguous SV40 and nucleoplasmin nuclear localization signal coding sequence at the N-terminus of the NmeCas open reading frame. The construct includes the coding sequence of the HiBiT tag at the C-terminus of the NmeCas open reading frame. The components are linked by a linker and specific sequences are provided herein.

Healthy human donor blood apheresis is commercially available (Hemacare, donor 3786), and cells are washed and resuspended inTreatment was performed in PBS/EDTA buffer (Miltenyi Biotec catalog 130-070-525) and in MultiMACS ^TM Cell 24Separator Plus apparatus (Miltenyi Biotec). Using Stright fromCD4/CD8 MicroBead kit, human (Miltenyi Biotec catalog No. 130-122-352) isolated T cells via positive selection. Aliquoting T cells and inCS10 (StemCell Technologies catalog number 07930) is cold stored for future use. After thawing, T cells were seeded at a density of 1.0×10++ ⁶ cells/ml in T Cell Growth Medium (TCGM) consisting of: CTS OpTmizer T cell expansion SFM and T cell expansion supplements (ThermoFisher catalog A1048501), 5% human AB serum (GeminiBio catalog 100-512), 1 Xpenicillin-streptomycin, 1XGlutamax, 10mM HEPES, 200U/mL recombinant human interleukin-2 (Peprotech, catalog 200-02), 5ng/mL recombinant human interleukin-7 (Peprotech, catalog 200-07), and 5ng/mL recombinant human interleukin-15 (Peprotech, catalog 200-15). T cells were allowed to stand in this medium for 24 hours, at which time they were activated by T CELL TRANSACT ^TM human reagents (Miltenyi, catalogue No. 130-111-160) added in a 1:100 volume ratio.

Solutions containing mRNA encoding NmeCas were prepared in P3 buffer. Guide RNAs targeting the TRAC locus were removed from storage and denatured at 95 ℃ for 2 min and incubated for 5min at room temperature. Forty-eight hours after activation, T cells were collected, centrifuged, and resuspended in P3 electroporation buffer (Lonza) at a concentration of 12.5x10- ⁶ cells/ml. Each well to be electroporated contains 1x10 ⁵ cells, nmeCas mRNA specified in Table 23, and 1 μM gRNA specified in Table 23 (G028853 for NmelCas; G021469 for Nme2Cas 9; G028848 for Nme3Cas 9), with a final volume of 20 μLP3 electroporation buffer. NmeCas9mRNA was tested using three-fold, five-point serial dilutions starting from 600ng mRNA. The appropriate gRNA and mRNA mixtures were transferred in triplicate into 96-well nucleic acid ^TM plates and electroporated using the manufacturer's pulse code. The electroporated T cells were then allowed to stand in CTS OpTmizerT cell growth medium without cytokines for 15 minutes and then transferred to a new flat bottom 96 well plate containing CTS OpTmizer T cell growth medium additionally supplemented with cytokines. The resulting plates were incubated at 37℃for 24 hours, then subjected to HiBiT luminometry, or 96 hours, then subjected to flow cytometry.

T cells were collected 24 hours after electroporation for protein expression analysis. T cell passageHiBiT LYTIC ASSAY (Promega) was dissolved. Luminescence was measured using a Biotek Neo2 plate reader. Table 23 and fig. 18 show Cas9 protein expression and corresponding Standard Deviation (SD) as Relative Luminescence Units (RLU) in activated cells.

Table 23. Mean luminescence (RLU) as a relative measure of Cas9 protein expression in 24 hour T cells.

On day 4 post-editing, T cells were assayed by flow cytometry to determine surface protein expression. Briefly, T cells were incubated with a mixture of antibodies diluted in cell staining buffer (BioLegend, cat# 420201) for 30 minutes at 4 ℃.1, the method comprises the following steps: antibodies to CD3 (BioLegend, catalog No. 317336), CD4 (BioLegend, catalog No. 317434), CD8 (BioLegend, catalog No. 301046) and Viakrome (beckmacoulter, catalog No. C36628) were diluted 100. The cells were then washed, resuspended in 100uL of cell staining buffer and processed on a Cytoflex flow cytometer (Beckman Coulter). Flow cytometry data were analyzed using the FlowJo software package. T cells are gated based on size, shape, viability, CD8 and CD 3. Samples were run in triplicate. The CD3 negative cell populations and corresponding Standard Deviations (SDs) for each of the designated grnas are shown in table 24 and plotted in fig. 19.

Table 24. CD3 negative cell percentages of T cells after trac editing.

Example 11 in vitro editing Using selected guide in Primary cynomolgus macaque liver cells (PCH)

Three NmeCas sgrnas (G024739, G024741 and G024743) were selected for evaluation in a dose response assay. The NmeCas sgRNA tested for targeting the cynomolgus monkey TTR gene included a 24 nucleotide guide sequence.

Unmodified and modified versions of the guide are provided in table 25.

TABLE 25 unmodified and modified versions of selected gRNAs

As described below, gRNA and Cas9 mRNA liposomes were transfected into primary cynomolgus monkey hepatocytes (PCH). Prepared as described in example 1 (In Vitro ADMETLaboratories 10136011). PCH was seeded at a density of 40,000 cells/well. LNP formulations were prepared as described in example 1. LNP was prepared with a lipid composition of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2k-DMG in molar ratios. LNP was formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and with the gRNA indicated in table 25. The PCH in 100. Mu.L of medium was treated with LNP containing sgRNA at 8-point, 4-fold dilution series (starting from 70 ng) and 30ng doses of encapsulated mRNA O by weight of mRNA. The sgRNA concentration in each well is indicated in table 26. Cells were lysed 72 hours after treatment and NGS analysis was performed as described in example 1. Dose response of edit efficiency to guide concentration was measured in triplicate samples. Table 26 and fig. 20 show the mean percent edit and Standard Deviation (SD) at each guide concentration.

Table 26 average percent insertions/deletions at TTR after editing in PCH.

Example 12 in vitro editing of LNP using mRNA dilution series in PCH

Modified sgrnas with the same targeting sites in cynomolgus monkey TTR gene were determined to evaluate the editing efficiency of different mRNA (mRNA O, mRNA AA) and formulation ratios in PCH. Unless otherwise indicated, the preparation, processing and analysis of PCH (In Vitro ADMETLaboratories, 10136011) in this example were the same as described in example 1. PCH was used and seeded at a density of 50,000 cells/well. LNP formulations were prepared as described in example 1. LNP was prepared with a lipid composition having a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated at a lipid amine to RNA phosphate (N: P) molar ratio of about 6 and with the gRNA indicated in table 27. PCH in 100. Mu.L medium was treated with 8-point, 3-fold serial dilutions of LNP with various gRNA: mRNA ratios mixed (formulated alone) or co-formulated. The highest dose was 3 ng/. Mu.L total RNA by weight, and the gRNA to mRNA ratios of the dilution series are indicated in Table 27. Samples were run in triplicate. Average percent editing, standard Deviation (SD) and calculated EC50 are shown in table 27 and figure 21.

Table 27 average percent insertions/deletions at TTR locus after editing in PCH.

Example 13 in vivo editing Using NmeCas g RNA

Editing efficiency of modified grnas was tested in mice with Nme2Cas9 constructs. All Nme sgrnas tested contained the same 24nt guide sequence targeting the mouse TTR gene (mTTR).

LNP is generally described as in example 1 as 1 by weight of gRNA and mRNA O: 2. The LNP used was prepared in a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. Dosages were calculated based on the combined RNA weight of gRNA and mRNA. Transport and Storage Solutions (TSS) for LNP preparation were dosed as vehicle-only negative controls in the experiments.

CD-1 female mice of about 6-8 weeks of age were used in each study involving mice. The animals were fed conventional feed and standard maintenance was performed. Animals were weighed prior to dosing. TSS and LNP formulations were administered intravenously via tail vein injection at a dose of 0.03 mpk. Adverse reactions in animals were observed periodically at least 24 hours after dosing. Fourteen days after treatment, animals were euthanized by cardiac exsanguination under isoflurane anesthesia; blood and liver tissue for serum preparation were collected for downstream analysis.

Serum TTR levels shown in Table 28 and FIG. 22 were generated and compared to negative controls (TSS) for all experimental groups using serum TTR ELISA-prealbumin ELISA (AVIVA SYSTEMS; catalog No. OKIA 00111) according to the manufacturer's protocol.

Table 28 serum TTR levels (. Mu.g/ml).

Guide ID	Serum TTR (mug/ml)	SD	TSS％	N
					TSS	673.7	44.13	100	5
G021536	378.2	83.0	56.1	7
					G029377	419.3	83.5	62.2	9
G029384	270.1	63.90	40.1	4
					G029392	375.4	58.23	55.7	4
G029391	509.1	115.3	75.6	4
					G029390	623.2	144.3	92.5	4

Liver punch biopsies weighing between 5 and 15mg were collected to isolate genomic DNA. Genomic DNA was extracted using DNA isolation kit (ZymoResearch, D3012) and samples were analyzed by NGS sequencing as described in example 1. The editing efficiency of LNP containing the specified gRNA is shown in table 29 and plotted in fig. 23.

TABLE 29 average percent insertions/deletions at TTR loci in mouse liver samples

Guide body	Average edit%	SD	N
				TSS	0.1	0	5
G021536	27.2	4.58	7
				G029377	25.7	6.77	9
G029384	34.9	4.05	4
				G029392	20.9	6.14	4
G029391	5.4	2.60	4
				G029390	5.5	3.66	4

Example 14 dose response curve of nmecass 9 gRNA in PMH with Nme2Cas9

Editing efficiency of modified grnas was tested with Nme2Cas9 constructs in Primary Mouse Hepatocytes (PMHs). All Nme sgrnas tested contained the same 24nt guide sequence targeting the mouse TTR gene (mTTR).

PMH (Gibco, lot MC 931) was thawed and resuspended in hepatocyte thawing medium, followed by centrifugation. Supernatant was discarded and pelleted cells were resuspended in hepatocyte plate medium (Williams E medium (Gibco, cat. No. A12176-01)) with seed supplements dexamethasone+mix supplement (Gibco, cat. No. A15563, cat. No. 2459010) and FBS content (Gibco, cat. No. A13450, cat. No. 2486425). Cells were counted and seeded at a concentration of 15,000 cells/well on a Bio-coat collagen I coated 96-well plate (Corning, reference number 356407, lot number 08722018). The inoculated cells were allowed to stand and adhere in a tissue culture incubator at 37℃and 5% CO2 atmosphere for 4-6 hours. After incubation, cell monolayer formation was examined and washed once with hepatocyte maintenance medium (williams E medium) with plate medium supplements (Gibco, cat No. a15564, lot No. 2459014).

LNPs are generally prepared as described in example 1 with 1:2 cargo by weight of gRNA to mRNA O. The LNP used was prepared in a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated with a molar ratio of lipid amine to RNA phosphate (N: P) of 6. As shown in table 30, each LNP was applied to cells at the highest dose (300 ngmRNA O and 46.5nM gRNA (about 150ng gRNA) using an 8-point 3-fold serial dilution starting from 450ng total cargo per 100 μl well. After treatment with LNP, cells were incubated for 24 hours at 37 ℃ in williams E medium containing plate medium supplements (Gibco, cat No. a15564, lot No. 2459014) and 3% fetal bovine serum. After 72 hours, cells were collected and analyzed by NGS as described in example 1.

The editing efficiency of LNPs containing the specified grnas and the respective corresponding EC50 are shown in table 30 and plotted in fig. 24.

Table 30 average percent insertions/deletions at the TTR locus in primary mouse hepatocytes.

Example 15 dose response curve of nmecass 9 gRNA in PMH with Nme2Cas9

LNPs are generally prepared as described in example 1 with 1:2 cargo by weight of gRNA to mRNA O. The LNP used was prepared in a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated with a molar ratio of lipid amine to RNA phosphate (N: P) of 6. As shown in table 31, each LNP was applied to cells using 8-point 3-fold serial dilutions starting from 450ng total cargo per 100 μl well at the highest dose (300 ngmRNA O and 46.5nM gRNA (i.e., 150ng gRNA). After treatment with LNP, cells were incubated for 24 hours at 37 ℃ in williams E medium containing plate medium supplements (Gibco, cat No. a15564, lot No. 2459014) and 3% fetal bovine serum. Samples were run in triplicate. After 72 hours, cells were collected and analyzed by NGS as described in example 1.

The editing efficiency of LNP containing the specified gRNA and the respective corresponding EC50 are shown in table 31 and plotted in fig. 25.

Example 16 in vitro editing in Primary Mouse Hepatocytes (PMH) Using dilution Curve

A. EXAMPLE 16.1 evaluation of modified sgRNA Using dilution series

Modified sgrnas with various backbone structures were designed as shown in tables 1-2, all of which target previously published sites in the mouse pcsk9 gene (see WO 2019094791), and were tested for editing efficiency using Primary Mouse Hepatocytes (PMH). Cells were prepared as described in example 1 using PMH cells (In Vitro ADMET Laboratories) and seeded at a density of 20,000 cells/well. Cells were transfected with MessengerMax (Invitrogen) at 1 ng/. Mu.l Nme2 Cas9 mRNA (mRNA U) and the concentrations of sgRNA indicated in Table 32 according to the manufacturer's protocol. Repeated samples were included in the assay. Cells were collected 72 hours post-transfection and analyzed by NGS as described in example 1. The average percent edits with standard deviation are shown in table 32 and fig. 26.

Table 32 average percent edit in pmh

B. EXAMPLE 16.2 evaluation of mRNA poly-A tail modification and cargo ratio

The sgrnas targeting the mouse psck gene were selected from table 32 to evaluate modifications by the poly-a tail and the sgrnas: specific combinations of mRNA ratios resulted in lead editing efficiencies. The PMH cells used were prepared, treated and analyzed as described in example 1, unless otherwise indicated. PMH (Gibco) was seeded at a density of 15,000 cells/well.

LNP was prepared generally as described in example 1. LNP was prepared with a lipid composition of 50/9/38/3, expressed as the molar ratio of ionizable lipid A/cholesterol/DSPC/PEG, respectively. LNP was formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. LNP encapsulates gRNAG017566 or one of three mrnas encoding the same Nme2Cas9 Open Reading Frame (ORF) but with different encoded poly-a tails, as indicated in table 33. Preliminary experiments to keep sgRNA application constant and vary mRNA application amount showed 1 by weight: 1, sgRNA: mRNA ratios produced the highest percent editing. In the current example, increasing doses of mRNA LNP and gRNA LNP were applied to cells in 100 μl of medium as described in table 33, maintaining 1 by weight: 1, sgRNA: mRNA ratio. Table 33 and fig. 27 show the average percent edit and Standard Deviation (SD).

Table 33 average percent edit in pmh

Example 17 dose response curve of nmecass 9 gRNA in PMH with Nme2Cas9

PMH (Gibco, lot number MC 931) was thawed and resuspended in hepatocyte thawing medium containing an seeding supplement (williams E medium (Gibco, cat No. a 12176-01)) with dexamethasone + mixed fluid supplement (Gibco, cat No. a15563, lot No. 2019842) and seeding supplement with FBS content (Gibco, cat No. a13450, lot No. 1970698), followed by centrifugation. The supernatant was discarded and the pelleted cells were resuspended in hepatocyte plate medium plus supplement package (Invitrogen, catalog nos. a1217601 and Gibco, catalog No. CM 3000). Cells were counted and seeded at a concentration of 15,000 cells/well on a Bio-coat collagen I coated 96-well plate (Thermo Fisher, cat. 877272). The inoculated cells were allowed to stand and adhere in a tissue culture incubator at 37℃and 5% CO2 atmosphere for 4-6 hours. After incubation, cell monolayer formation was examined and washed once with hepatocyte maintenance medium (Invitrogen, catalog nos. a1217601 and Gibco, catalog No. CM 4000).

LNPs are generally prepared as described in example 1 with 1:2 cargo by weight of gRNA to mRNA O. The LNP used was prepared in a molar ratio of 50% lipid A, 38% cholesterol, 9% DSPC and 3% PEG2 k-DMG. LNP was formulated with a molar ratio of lipid amine to RNA phosphate of 6 (N: P). Each LNP was applied to cells using 8-point 4-fold serial dilutions starting from 300ng total RNA per 100 μl well (about 32.25nM gRNA concentration per well), as shown in table 32. After treatment with LNP, cells were incubated in williams E medium (Gibco, a 1217601) containing maintenance supplements and 3% fetal bovine serum at 37 ℃ for 24 hours. Samples were run in triplicate. After 72 hours, cells were collected and analyzed by NGS as described in example 1.

The editing efficiency of LNPs containing the specified grnas and the respective corresponding EC50 are shown in table 34 and plotted in fig. 28.

Table 34 average percent insertions/deletions at TTR loci in primary mouse hepatocytes.

Example 18 in vivo editing Using NmeCas g RNA

CD-1 female mice of about 6-8 weeks of age (all groups n=5) were used in each study involving mice. The animals were fed conventional feed and standard maintenance was performed. Animals were weighed prior to dosing. TSS and LNP formulations were administered intravenously via tail vein injection at a dose of 0.03 mpk. Adverse reactions in animals were observed periodically at least 24 hours after dosing. Seven days after treatment, animals were euthanized by cardiac exsanguination under isoflurane anesthesia; blood and liver tissue for serum preparation were collected for downstream analysis.

Serum TTR ELISA-prealbumin ELISA (AVIVA SYSTEMS; catalog number OKIA 00111) was used to generate the serum TTR levels shown in Table 35 and FIG. 29 according to the manufacturer's protocol. Serum TTR levels were significantly lower in all experimental groups compared to negative control (TSS).

Table 35 serum TTR levels (. Mu.g/ml).

Guide ID	Serum TTR (mug/ml)	SD	TSS％
				TSS	704.9	98.3	100％
G021844	150.0	84.9	21％
				G021536	371.1	95.6	53％
G027492	239.4	30.5	34％
				G027493	423.4	170.0	60％
G027494	496.3	89.8	70％
				G027495	263.6	68.9	37％
G027496	362.4	52.7	51％

Liver punch biopsies weighing between 5 and 15mg were collected to isolate genomic DNA. Genomic DNA was extracted using DNA isolation kit (ZymoResearch, D3012) and samples were analyzed by NGS sequencing (all groups n=5), as described in example 1. The editing efficiency of LNP containing the specified gRNA is shown in table 36 and plotted in fig. 30.

TABLE 36 average percent insertions/deletions at TTR loci in mouse liver samples

Guide body	Average value of	SD
			TSS	0.12	0.22
G021844	57.1	5.7
			G021536	31.5	4.9
G027492	51.3	10.4
			G027493	27.0	14.0
G027494	17.6	8.6
			G027495	43.2	7.2
G027496	23.5	8.6

Example 19 in vivo editing Using NmeCas g RNA

Editing efficiency of modified grnas was tested with Nme2Cas9 mRNA in mice. All Nme sgrnas tested contained the same 24nt guide sequence targeting mTTR.

CD-1 female mice of about 6 weeks of age (all groups n=5) were used in each study involving mice. Animals were weighed for dose calculation prior to dose administration and for monitoring 24 hours after administration. TSS and LNP formulations were administered intravenously via tail vein injection at doses of 0.01mpk or 0.03 mpk. Adverse reactions in animals were observed periodically at least 24 hours after dosing. Seven days after treatment, animals were euthanized by cardiac exsanguination under isoflurane anesthesia. Blood was collected by cardiac puncture for serum TTR ELISA and liver tissue was collected for downstream analysis.

Serum TTR results prepared according to the manufacturer's protocol using serum TTR ELISA-prealbumin ELISA (AVIVA SYSTEMS; catalog number OKIA 00111) are shown in FIG. 31 and Table 37.

Table 37 serum TTR measurements after treatment.

Liver punch biopsies weighing about 5mg-15mg were collected to isolate genomic DNA and total RNA. Genomic DNA was extracted using DNA isolation kit (ZymoResearch, D3012) and samples were analyzed by NGS sequencing (all groups n=5), as described in example 1. The editing efficiency of LNP containing the specified gRNA is shown in table 38 and plotted in fig. 32.

Table 38 average percent insertions/deletions at TTR loci in mouse liver samples.

Example 20 additional embodiment

The following numbered items provide additional support and description of embodiments herein.

Item 1 is a polynucleotide comprising an Open Reading Frame (ORF), said ORF comprising: a nucleotide sequence encoding a C-terminal neisseria meningitidis (Nme) Cas9 polypeptide that is at least 90% identical to any one of SEQID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、301-303 or 316-321, wherein the Nme Cas9 is Nme2 Cas9, nme1 Cas9, or Nme3 Cas9; and a nucleotide sequence encoding a first Nuclear Localization Signal (NLS).

Item 2 is the polynucleotide of item 1, wherein the ORF further comprises a nucleotide sequence encoding a second NLS.

Item 3 is the polynucleotide of item 1, wherein the first NLS and the second NLS are independently selected from the group consisting of SEQ ID NOs: 388 and 410-422.

Item 4 is the polynucleotide of any one of the preceding items, wherein the polynucleotide further comprises a polyA sequence or polyadenylation signal sequence.

Item 5 is the polynucleotide of item 4, wherein the polyA sequence comprises non-adenine nucleotides.

Item 6 is the polynucleotide of any one of items 4-5, wherein the polyA sequence comprises 100-400 nucleotides.

Item 7 is the polynucleotide of any one of items 4-6, wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 409.

Item 8 is the polynucleotide of any one of the preceding items, wherein the ORF further comprises a nucleotide sequence encoding a linker sequence between the first NLS and the second NLS.

Item 9 is the polynucleotide of any one of the preceding items, wherein the ORF further comprises a nucleotide sequence encoding a linker spacer sequence between the Nme Cas9 coding sequence and the NLS proximal to the Nme Cas9 coding sequence.

Item 10 is the polynucleotide of any one of items 8-9, wherein the linker comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids.

Item 11 is the polynucleotide of any one of items 8-10, wherein the linker sequence comprises GGG or GGGs, optionally wherein the GGG or GGGs sequence is located at the N-terminus of the spacer sequence.

Item 12 is the polynucleotide of any one of items 8-11, wherein the linker sequence comprises the sequence of SEQ ID NO:61-122 one sequence.

Item 13 is the polynucleotide of any one of the preceding items, wherein the ORF further comprises one or more additional heterologous functional domains.

Item 14 is the polynucleotide of any one of the preceding items, wherein the NmeCas has double stranded endonuclease activity.

Item 15 is the polynucleotide of any one of items 1-14, wherein the NmeCas has a nicking enzyme activity.

Item 16 is the polynucleotide of any one of items 1-14, wherein the NmeCas comprises a dCas9 DNA binding domain.

Item 17 is the polynucleotide of any one of the preceding items, wherein the NmeCas comprises a nucleotide sequence that hybridizes to SEQ ID NO:1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, 297 or any of 310-315 has an amino acid sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

Item 18 is the polynucleotide of any one of the preceding items, wherein the NmeCas comprises the sequence of SEQ ID NO:1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, 297 or 310-315.

Item 19 is the polynucleotide of any one of the preceding items, wherein the sequence encoding the NmeCas9 comprises a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of any one of SEQ ID NO：15、18-27、29、32-41、221-226、228-233、235-240、242-247、249-254、256-261、263-268、270-275、277-282、284-289、291-296、298-303、304-309 or 316-321.

Item 20 is the polynucleotide of any one of the preceding items, wherein the sequence encoding the NmeCas comprises the nucleotide sequence of any one of SEQ ID NO：15、18-27、29、32-41、221-226、228-233、235-240、242-247、249-254、256-261、263-268、270-275、277-282、284-289、291-296、298-303、304-309 or 316-321.

Item 21 is a polynucleotide comprising an Open Reading Frame (ORF) encoding a polypeptide comprising: cytidine deaminase, optionally apodec 3A deaminase; a nucleotide sequence encoding a C-terminal neisseria meningitidis (Nme) Cas9 nickase polypeptide that hybridizes with SEQ ID NO:1 and any one of 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, or 297 is at least 90% identical, wherein the Nme Cas9 nickase is Nme2 Cas9 nickase, nme1 Cas9 nickase, or Nme3 Cas9 nickase; and a nucleotide sequence encoding a first Nuclear Localization Signal (NLS); wherein the polypeptide does not comprise a Uracil Glycosidase Inhibitor (UGI).

Item 22 is the polynucleotide of item 21, wherein the ORF further comprises a nucleotide sequence encoding a second NLS.

Item 23 is the polynucleotide of any one of items 21-22, wherein the deaminase is located at the N-terminus of an NLS in the polypeptide.

Item 24 is the polynucleotide of any one of items 21-23, wherein the cytidine deaminase is located at the N-terminus of the first NLS and the second NLS in the polypeptide.

Item 25 is the polynucleotide of any one of items 21-22, wherein the cytidine deaminase is located at the C-terminus of the NLS in the polypeptide.

Item 26 is the polynucleotide of any one of items 23-25, wherein the cytidine deaminase is located at the C-terminus of the first NLS and the second NLS in the polypeptide.

Item 27 is the polynucleotide of any one of items 21-26, wherein the ORF does not comprise a coding sequence of an NLS located at the C-terminus of the ORF encoding the Nme Cas 9.

Item 28 is the polynucleotide of any one of items 21-26, wherein the ORF does not comprise a coding sequence located at the C-terminus of the ORF encoding the Nme Cas 9.

Item 29 is the polynucleotide of any one of the preceding items, wherein the cytidine deaminase comprises a nucleotide sequence that hybridizes with SEQ ID NO:151 has an amino acid sequence of at least 87% identity.

Item 30 is the polynucleotide of any one of the preceding items, wherein the cytidine deaminase comprises a nucleotide sequence that hybridizes with SEQ ID NO:152-216 have an amino acid sequence with at least 80% identity.

Item 31 is the polynucleotide of any one of the preceding items, wherein the cytidine deaminase comprises a nucleotide sequence that hybridizes with SEQ ID NO:14 having an amino acid sequence having at least 80% identity.

Item 32 is the polynucleotide of any one of the preceding items, the ORF comprising a nucleotide sequence that matches SEQ ID NO:42, a nucleotide sequence having at least 80% identity.

Item 33 is the polynucleotide of any one of the preceding items, wherein the polynucleotide comprises a nucleotide sequence that hybridizes to SEQ ID NO:391-398 have a 5' UTR with at least 90% identity.

Item 34 is the polynucleotide of any one of the preceding items, wherein the polynucleotide comprises a nucleotide sequence comprising SEQ ID NO:391-398 one 5' UTR.

Item 35 is the polynucleotide of any one of the preceding items, wherein the polynucleotide comprises a nucleotide sequence that hybridizes to SEQ ID NO:399-406 have a 3' UTR with at least 90% identity.

Item 36 is the polynucleotide of any one of the preceding items, wherein the polynucleotide comprises a nucleotide sequence comprising SEQ ID NO:399-306 one 3' UTR.

Item 37 is the polynucleotide of any one of the preceding items, wherein the polynucleotide comprises a 5'utr and a 3' utr from the same source.

Item 38 is the polynucleotide of any one of the preceding items, wherein the polynucleotide comprises a5 'Cap, optionally wherein the 5' Cap is Cap0, cap1, or Cap2.

Item 39 is the polynucleotide of any one of the preceding items, wherein at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons of the ORF are minimal adenine codons or minimal uridine codons.

Item 40 is the polynucleotide of any one of the preceding items, wherein the ORF comprises or consists of codons that increase translation of mRNA in the mammal.

Item 41 is the polynucleotide of any one of the preceding items, wherein the ORF comprises or consists of codons that increase translation of mRNA in a human.

Item 42 is the polynucleotide of any one of the preceding items, wherein the polynucleotide is mRNA.

Item 43 is the polynucleotide of item 42, wherein the ORF comprises a sequence having at least 90%, 95%, 98% or 100% identity to any one of SEQ ID NO：29、32-41、224-226、231-233、238-240、245-247、252-254、259-261、266-268、273-275、280-282、287-289、294-296、301-303 or 316-321.

Item 44 is the polynucleotide of any one of items 42-43, wherein at least 10% of uridine in the mRNA is replaced by modified uridine.

Item 45 is the polynucleotide of any one of items 42-43, wherein less than 10% of the uridine in the mRNA is replaced by modified uridine.

Item 46 is the polynucleotide of item 45, wherein the modified uridine is one or more of N1-methyl-pseudouridine, 5-methoxyuridine, or 5-iodouridine.

Item 47 is the polynucleotide of item 45, wherein the modified uridine is one or both of N1-methyl-pseudouridine or 5-methoxyuridine.

Item 48 is the polynucleotide of any one of items 45-47, wherein the modified uridine is N1-methyl-pseudouridine.

Item 49 is the polynucleotide of any one of items 45-47, wherein the modified uridine is 5-methoxyuridine.

Item 50 is the polynucleotide of any one of items 44 and 36-49, wherein 15% to 45% of the uridine is replaced by modified uridine.

Item 51 is the polynucleotide of item 50, wherein at least 20% or at least 30% of the uridine is replaced by modified uridine.

Item 52 is the polynucleotide of item 51, wherein at least 80% or at least 90% of the uridine is replaced by modified uridine.

Item 53 is the polynucleotide of item 52, wherein 100% of the uridine is replaced by modified uridine.

Item 54 is the polynucleotide of item 42, wherein less than 10% of the nucleotides in the mRNA are replaced by modified nucleotides.

Item 55 is a composition comprising the polynucleotide of any one of the preceding items and at least one guide RNA (gRNA).

Item 56 is a composition comprising a first polynucleotide comprising a first Open Reading Frame (ORF) encoding a polypeptide comprising a cytidine deaminase, optionally an apodec 3A deaminase, and NmeCas nicking enzyme, and a second polynucleotide comprising a second open reading frame encoding a Uracil Glycosidase Inhibitor (UGI), wherein the second polynucleotide is different from the first polynucleotide, and optionally further comprising a guide RNA (gRNA).

Item 57 is the composition of item 55 or 56, wherein the gRNA is a single guide RNA.

Item 58 is the composition of item 55 or 56, wherein the gRNA is a dual guide RNA.

Item 59 is a composition comprising the polynucleotide of any one of items 1-57, further comprising a single guide RNA, wherein the single guide RNA comprises a guide region and a conserved region, wherein the conserved region comprises one or more of:

(Ii) Nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b)

(Ii) Nucleotide 81 is linked to nucleotide 96 by at least 4 nucleotides; or (b)

(Ii) Nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides;

wherein relative to SEQ ID NO:500, one or both of nucleotides 144-145 are optionally deleted;

wherein at least 10 nucleotides are modified nucleotides.

Item 60 is a composition comprising the polynucleotide of any one of items 1-57, further comprising a single guide RNA, wherein the single guide RNA comprises a guide region and a conserved region, wherein the conserved region comprises one or more of:

Wherein compared to SEQ ID NO:500, one or both of nucleotides 144-145 are optionally deleted;

Item 61 is a polypeptide encoded by the polynucleotide of any one of items 1-60.

Item 62 is a vector comprising the polynucleotide of any one of items 1-60.

Item 63 is an expression construct comprising a promoter operably linked to a sequence encoding the polynucleotide of any one of items 1-60.

Item 64 is a plasmid comprising the expression construct of item 63.

Item 65 is a host cell comprising the vector of item 62, the expression construct of item 63, or the plasmid of item 64.

Item 66 is a pharmaceutical composition comprising the polynucleotide, composition, or polypeptide of any one of the preceding items and a pharmaceutically acceptable carrier.

Item 67 is a kit comprising the polynucleotide, composition, or polypeptide of any one of the preceding items.

Item 68 is the use of the polynucleotide, composition, or polypeptide of any one of the preceding items for modifying a gene of interest in a cell.

Item 69 is the use of a polynucleotide, composition, or polypeptide of any one of the preceding items for the manufacture of a medicament for modifying a gene of interest in a cell.

Item 70 is the polynucleotide or composition of any one of the preceding items, wherein the polynucleotide or composition is formulated as a lipid nucleic acid assembly composition, optionally a lipid nanoparticle.

Item 71 is a method of modifying a gene of interest, the method comprising delivering to a cell the polynucleotide, polypeptide, or composition of any one of the preceding items.

Item 72 is a method of modifying a gene of interest, the method comprising delivering to a cell one or more lipid nucleic acid assembly compositions comprising the polynucleotide of any one of items 1-60 and one or more guide RNAs, optionally lipid nanoparticles.

Item 73 is the method of any one of items 71-72, wherein at least one lipid nucleic acid assembly composition comprises Lipid Nanoparticles (LNPs), optionally wherein all lipid nucleic acid assembly compositions comprise LNPs.

Item 74 is the method of any one of items 71-72, wherein at least one lipid nucleic acid assembly composition is a liposome complex composition.

Item 75 is the composition or method of any one of items 72-74, wherein the lipid nucleic acid assembly composition comprises an ionizable lipid.

Claims

1. A polynucleotide comprising an open reading frame (ORF), the ORF comprising:

a nucleotide sequence encoding a C-terminal Neisseria meningitidis (Nme) Cas9 polypeptide that is at least 90% identical to any one of SEQ ID NOs: 29, 32-41, 224-226, 231-233, 238-240, 245-247, 252-254, 259-261, 266-268, 273-275, 280-282, 287-289, 294-296, 301-303, or 316-321, wherein the Nme Cas9 is Nme2Cas9, Nme1 Cas9, or Nme3 Cas9; and

A nucleotide sequence encoding the first nuclear localization signal (NLS).

2. The polynucleotide of claim 1, wherein the ORF further comprises a nucleotide sequence encoding a second NLS.

3. The polynucleotide of claim 1, wherein the first NLS and the second NLS are independently selected from SEQ ID NOs: 388 and 410-422.

4. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide further comprises a poly-A sequence or a polyadenylation signal sequence.

5. The polynucleotide of claim 4, wherein the poly-A sequence comprises non-adenine nucleotides.

6. The polynucleotide of any one of claims 4-5, wherein the poly-A sequence comprises 100-400 nucleotides.

7. The polynucleotide of any one of claims 4-6, wherein the poly-A sequence comprises the sequence of SEQ ID NO:409.

8. The polynucleotide of any one of claims 1-7, wherein the ORF further comprises a nucleotide sequence encoding a linker sequence between the first NLS and the second NLS.

9. The polynucleotide of any one of claims 1-8, wherein the ORF further comprises a nucleotide sequence encoding a linker spacer sequence between the Nme Cas9 coding sequence and the NLS proximal to the Nme Cas9 coding sequence.

10. The polynucleotide of any one of claims 8-9, wherein the linker comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids.

11. The polynucleotide of any one of claims 8-10, wherein the linker sequence comprises GGG or GGGS, optionally wherein the GGG or GGGS sequence is located at the N-terminus of the spacer sequence.

12. The polynucleotide of any one of claims 8-11, wherein the linker sequence comprises the sequence of any one of SEQ ID NOs: 61-122.

13. The polynucleotide of any one of claims 1-12, wherein the ORF further comprises one or more additional heterologous functional domains.

14. The polynucleotide of any one of claims 1-13, wherein the Nme Cas9 has double-stranded endonuclease activity.

15. The polynucleotide of any one of claims 1-14, wherein the Nme Cas9 has nickase activity.

16. The polynucleotide of any one of claims 1-14, wherein the Nme Cas9 comprises a dCas9 DNA binding domain.

17. The polynucleotide of any one of claims 1-16, wherein the NmeCas9 comprises an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, 297 or 310-315.

18. The polynucleotide of any one of claims 1-17, wherein the NmeCas9 comprises the amino acid sequence of SEQ ID NO: 1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, 297, or 310-315.

19. The polynucleotide of any one of claims 1-18, wherein the sequence encoding the NmeCas9 comprises a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 15, 18-27, 29, 32-41, 221-226, 228-233, 235-240, 242-247, 249-254, 256-261, 263-268, 270-275, 277-282, 284-289, 291-296, 298-303, 304-309 or 316-321.

20. The polynucleotide of any one of claims 1-19, wherein the sequence encoding the NmeCas9 comprises the nucleotide sequence of any one of SEQ ID NO: 15, 18-27, 29, 32-41, 221-226, 228-233, 235-240, 242-247, 249-254, 256-261, 263-268, 270-275, 277-282, 284-289, 291-296, 298-303, 304-309, or 316-321.

21. A polynucleotide comprising an open reading frame (ORF) encoding a polypeptide comprising:

a cytidine deaminase, which is optionally an APOBEC3A deaminase;

a nucleotide sequence encoding a C-terminal Neisseria meningitidis (Nme) Cas9 nickase polypeptide that is at least 90% identical to any one of SEQ ID NOs: 1 and 4-13, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, or 297, wherein the Nme Cas9 nickase is an Nme2 Cas9 nickase, an Nme1 Cas9 nickase, or an Nme3 Cas9 nickase; and

a nucleotide sequence encoding the first nuclear localization signal (NLS);

wherein the polypeptide does not comprise a uracil glycosidase inhibitor (UGI).

22. The polynucleotide of claim 21, wherein the ORF further comprises a nucleotide sequence encoding a second NLS.

23. The polynucleotide of any one of claims 21-22, wherein the deaminase is located N-terminally to the NLS in the polypeptide.

24. The polynucleotide of any one of claims 21-23, wherein the cytidine deaminase is located N-terminally to the first NLS and the second NLS in the polypeptide.

25. The polynucleotide of any one of claims 21-22, wherein the cytidine deaminase is located at the C-terminus of the NLS in the polypeptide.

26. The polynucleotide of any one of claims 23-25, wherein the cytidine deaminase is located C-terminally to the first NLS and the second NLS in the polypeptide.

27. The polynucleotide of any one of claims 21-26, wherein the ORF does not comprise a coding sequence for an NLS located at the C-terminus of the ORF encoding the NmeCas9.

28. The polynucleotide of any one of claims 21-26, wherein the ORF does not comprise a coding sequence located at the C-terminus of the ORF encoding the NmeCas9.

29. The polynucleotide of any one of claims 1-28, wherein the cytidine deaminase comprises an amino acid sequence at least 87% identical to SEQ ID NO: 151.

30. The polynucleotide of any one of claims 1-28, wherein the cytidine deaminase comprises an amino acid sequence at least 80% identical to SEQ ID NOs: 152-216.

31. The polynucleotide of any one of claims 1-28, wherein the cytidine deaminase comprises an amino acid sequence at least 80% identical to SEQ ID NO: 14.

32. The polynucleotide of any one of claims 1-31, wherein the ORF comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:42.

33. The polynucleotide of any one of claims 1-32, wherein the polynucleotide comprises a 5'UTR that is at least 90% identical to any one of SEQ ID NOs: 391-398.

34. The polynucleotide of any one of claims 1-33, wherein the polynucleotide comprises a 5'UTR comprising any one of SEQ ID NOs: 391-398.

35. The polynucleotide of any one of claims 1-34, wherein the polynucleotide comprises a 3'UTR that is at least 90% identical to any one of SEQ ID NOs: 399-406.

36. The polynucleotide of any one of claims 1-35, wherein the polynucleotide comprises a 3'UTR comprising any one of SEQ ID NOs: 399-306.

37. The polynucleotide of any one of claims 1-36, wherein the polynucleotide comprises a 5'UTR and a 3'UTR from the same source.

38. The polynucleotide of any one of claims 1-37, wherein the polynucleotide comprises a 5' cap, optionally wherein the 5' cap is Cap0, Cap1 or Cap2.

39. The polynucleotide of any one of claims 1-38, wherein at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons of the ORF are minimal adenine codons or minimal uridine codons.

40. The polynucleotide of any one of claims 1-39, wherein the ORF comprises or consists of a codon that increases translation of an mRNA in a mammal.

41. The polynucleotide of any one of claims 1-40, wherein the ORF comprises or consists of a codon that increases translation of an mRNA in humans.

42. The polynucleotide of any one of claims 1-41, wherein the polynucleotide is mRNA.

43. The polynucleotide of claim 42, wherein the ORF comprises a sequence that is at least 90%, 95%, 98% or 100% identical to any one of SEQ ID NOs: 29, 32-41, 224-226, 231-233, 238-240, 245-247, 252-254, 259-261, 266-268, 273-275, 280-282, 287-289, 294-296, 301-303 or 316-321.

44. The polynucleotide of any one of claims 42-43, wherein at least 10% of the uridines in the mRNA are substituted with modified uridines.

45. The polynucleotide of any one of claims 42-43, wherein less than 10% of the uridines in the mRNA are substituted with modified uridines.

46. The polynucleotide of claim 45, wherein the modified uridine is one or more of N1-methyl-pseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine.

47. The polynucleotide of claim 45, wherein the modified uridine is one or both of N1-methyl-pseudouridine or 5-methoxyuridine.

48. The polynucleotide of any one of claims 45-47, wherein the modified uridine is N1-methyl-pseudouridine.

49. The polynucleotide of any one of claims 45-47, wherein the modified uridine is 5-methoxyuridine.

50. The polynucleotide of any one of claims 44 and 46-49, wherein 15% to 45% of the uridines are substituted with the modified uridines.

51. The polynucleotide of claim 50, wherein at least 20% or at least 30% of the uridines are substituted with the modified uridines.

52. The polynucleotide of claim 51, wherein at least 80% or at least 90% of the uridines are substituted with the modified uridines.

53. The polynucleotide of claim 52, wherein 100% of the uridines are substituted with the modified uridines.

54. The polynucleotide of claim 42, wherein less than 10% of the nucleotides in the mRNA are substituted with modified nucleotides.

55. A composition comprising a polynucleotide according to any one of claims 1-54 and at least one guide RNA (gRNA).

56. A composition comprising a first polynucleotide comprising a first open reading frame (ORF) encoding a polypeptide comprising a cytidine deaminase, optionally an APOBEC3A deaminase and an NmeCas9 nickase, and a second polynucleotide comprising a second open reading frame encoding a uracil glycosidase inhibitor (UGI), wherein the second polynucleotide is different from the first polynucleotide and optionally further comprises a guide RNA (gRNA).

57. The composition of claim 55 or 56, wherein the gRNA is a single guide RNA.

58. The composition of claim 55 or 56, wherein the gRNA is a dual-guide RNA.

59. A composition comprising a polynucleotide according to any one of claims 1-57, further comprising a single guide RNA, wherein the single guide RNA comprises a guide region and a conserved region, wherein the conserved region comprises one or more of the following:

(i) relative to SEQ ID NO: 500, one or more of nucleotides 37-48 and 53-64 are deleted, and optionally one or more of nucleotides 37-64 are substituted; and

(ii) nucleotide 36 is linked to nucleotide 65 via at least 2 nucleotides; or

(i) relative to SEQ ID NO: 500, one or more of nucleotides 82-86 and 91-95 are deleted, and optionally one or more of positions 82-96 are substituted; and

(ii) nucleotide 81 is linked to nucleotide 96 via at least 4 nucleotides; or

(i) relative to SEQ ID NO: 500, one or more of nucleotides 113-121 and 126-134 are deleted, and optionally one or more of nucleotides 113-134 are substituted; and

(ii) nucleotide 112 is linked to nucleotide 135 via at least 4 nucleotides;

wherein relative to SEQ ID NO: 500, one or both of nucleotides 144-145 are optionally deleted;

At least 10 of the nucleotides are modified nucleotides.

60. A composition comprising a polynucleotide according to any one of claims 1-57, further comprising a single guide RNA, wherein the single guide RNA comprises a guide region and a conserved region, wherein the conserved region comprises one or more of the following:

(i) relative to SEQ ID NO: 500, one or more of nucleotides 37-64 are deleted and optionally substituted; and

(ii) nucleotide 36 is linked to nucleotide 65 by: (i) a first internal linker that replaces 4 nucleotides, either alone or in combination with a nucleotide, or (ii) at least 4 nucleotides; or

(i) relative to SEQ ID NO: 500, one or more of nucleotides 82-95 are deleted and optionally substituted; and

(ii) nucleotide 81 is linked to nucleotide 96 by: (i) a second internal linker that replaces 4 nucleotides, either alone or in combination with a nucleotide, or (ii) at least 4 nucleotides; or

(i) relative to SEQ ID NO: 500, one or more of nucleotides 113-134 are deleted and optionally substituted; and

(ii) nucleotide 112 is linked to nucleotide 135 by: (i) a third internal linker that replaces 4 nucleotides, either alone or in combination with nucleotides, or (ii) at least 4 nucleotides;

wherein one or both of nucleotides 144-145 are optionally deleted compared to SEQ ID NO: 500;

wherein the gRNA comprises at least one of the first internal linker, the second internal linker, and the third internal linker.

61. A polypeptide encoded by the polynucleotide of any one of claims 1-60.

62. A vector comprising the polynucleotide of any one of claims 1-60.

63. An expression construct comprising a promoter operably linked to a sequence encoding the polynucleotide of any one of claims 1-60.

64. An expression construct as claimed in claim 63, wherein the promoter is an RNA polymerase promoter, optionally a bacterial RNA polymerase promoter.

65. The expression construct of claim 63 or 64, further comprising a poly-A tail sequence or a polyadenylation signal sequence.

66. The expression construct of claim 65, wherein the poly-A tail sequence is an encoded poly-A tail sequence.

67. A plasmid comprising the expression construct of any one of claims 63-66.

68. A host cell comprising the vector of claim 62, the expression construct of any one of claims 63-66, or the plasmid of claim 67.

69. A pharmaceutical composition comprising the polynucleotide, composition or polypeptide of any one of claims 1-61 and a pharmaceutically acceptable carrier.

70. A kit comprising the polynucleotide, composition or polypeptide of any one of claims 1-61.

71. Use of the polynucleotide, composition or polypeptide of any one of claims 1-61 for modifying a target gene in a cell.

72. Use of the polynucleotide, composition or polypeptide of any one of claims 1-61 for the manufacture of a medicament for modifying a target gene in a cell.

73. The polynucleotide or composition of any one of claims 1-60, wherein the polynucleotide or composition is formulated as a lipid nucleic acid assembly composition, optionally a lipid nanoparticle.

74. A method for modifying a target gene, the method comprising delivering to a cell the polynucleotide, polypeptide or composition of any one of claims 1-61.

75. A method for modifying a target gene, the method comprising delivering to a cell one or more lipid nucleic acid assembly compositions comprising a polynucleotide according to any one of claims 1-60 and one or more guide RNAs, optionally lipid nanoparticles.

76. The method of any one of claims 74-75, wherein at least one lipid nucleic acid assembly composition comprises lipid nanoparticles (LNPs), optionally wherein all lipid nucleic acid assembly compositions comprise LNPs.

77. The method of any one of claims 74-75, wherein at least one lipid nucleic acid assembly composition is a liposome complex composition.

78. A composition or method as described in any one of claims 75-77, wherein the lipid nucleic acid assembly composition comprises an ionizable lipid.

79. A method of producing a polynucleotide as described in any one of claims 1-54, the method comprising contacting the expression construct of claims 63-66 with an RNA polymerase and an NTP comprising at least one modified nucleotide.

80. The method of claim 79, wherein the NTP comprises a modified nucleotide.

81. The method of claim 79 or 80, wherein the modified nucleotide comprises a modified uridine.

82. The method of claim 81, wherein at least 80% or at least 90% or 100% of the uridine positions are modified uridines.

83. The method of claim 81 or 82, wherein the modified uridine comprises or is a substituted uridine, a pseudouridine, or a substituted pseudouridine, optionally N1-methyl-pseudouridine.

84. The method of any one of claims 79-83, wherein the expression construct comprises an encoded poly-A tail sequence.