WO2025045247A1

WO2025045247A1 - Nucleic acids encoding crispr-associated proteins and uses thereof

Info

Publication number: WO2025045247A1
Application number: PCT/CN2024/116339
Authority: WO
Inventors: Bang Wang
Original assignee: Geneditbio Limited
Priority date: 2023-08-31
Filing date: 2024-09-02
Publication date: 2025-03-06

Abstract

Provides are artificial nucleic acids, in particular RNA. A composition and kit of parts comprising the same are also provided.

Description

Nucleic acids encoding CRISPR-associated proteins and uses thereof

The present invention claims the priorities of the PCT/CN2023/116171, filed on August 31, 2023, and the PCT/CN2024/074906 filed on January 31, 2024, the contents of which are incorporated herein by their entirety.

Technical Field

The present invention relates to artificial nucleic acids, in particular RNAs, encoding polypeptides or proteins of interest, especially CRISPR-associated polypeptides or CRISPR-associated proteins, and (pharmaceutical) compositions and kit-of-parts comprising the same. Said artificial nucleic acids, in particular RNAs, (pharmaceutical) composition and kits are for use in medicine, for instance in gene therapy, and in particular in the treatment and/or prophylaxis of disease amenable to treatment with CRISPR-associated proteins, e.g. by gene editing, knock-in, knock-out or modulating the expression of target genes of interest.

Background

With the development of mRNA-based drug technologies, therapeutic mRNA has been applied in many fields, such as cancer immunotherapies, infectious disease vaccines, allergy tolerization, protein-replacement and supplementation therapies, genome engineering, gene therapy and genetic reprogramming etc., particularly in CRISPR-associated gene therapy. In the field of gene therapy, a prolonged presence of editing enzymes translated from DNA-based vectors resulted in off-target effect. However, the transient expression of the nucleases from mRNA would minimize thus nonspecific effect. Engineered mRNA encoding ZFNs, TALENs, Cas9 have been applied successfully to edit genomes by disrupting or integrating sequences ex vivo and in vivo. (Sahin, U., Karikó, K. &Türeci, mRNA-based therapeutics -developing a new class of drugs. Nat Rev Drug Discov 13, 759–780 (2014) . doi. org/10.1038/nrd4278) .

However, unresolved issues such as the control and regulation of mRNA translation need to be addressed. Complying with these needs is the object of the present invention.

Summary

The present invention solves the above-mentioned technical problems through the following technical solutions:

In one aspect, the present invention provides an mRNA comprising an open reading frame encoding an RNA-guided DNA-binding agent, wherein the open reading frame comprises a sequence having at least 90%identity to any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232, and 247.

In some embodiments, the open reading frame comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to the sequence of any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232, and 247.

In some embodiments, the mRNA further comprises a 5’ UTR with at least 90%identity to any one of SEQ ID NOs: 110-171 or 1-62.

In some embodiments, the mRNA further comprises a 5’ UTR with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to any one of SEQ ID NOs: 110-171 or 1-62.

In some embodiments, the mRNA further comprises a 3’ UTR with at least 90%identity to any one of SEQ ID NOs: 172-210 or 63-101.

In some embodiments, the mRNA further comprises a 3’ UTR with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to any one of SEQ ID NOs: 172-210 or 63-101.

In some embodiments, the mRNA further comprises a poly-adenylated (poly-A) tail with at least 90%identity to SEQ ID NO: 243, 244, 245 or 246.

In some embodiments, the mRNA further comprises a poly-adenylated (poly-A) tail with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 243, 244, 245 or 246.

In another aspect, the present invention provides an mRNA comprising a sequence with at least 98%identity to any one of SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252, and 254.

In some embodiments, the mRNA comprises a sequence selected from any one of SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252, and 254.

In some embodiments, at least 10%of the uridine is substituted with a modified uridine, wherein the modified uridine is one or more of N1-methyl-pseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine.

In yet another aspect, the present invention provides an expression construct comprising a promoter operably linked to a sequence encoding an mRNA mentioned above, wherein the expression construct is optionally a plasmid expression construct.

In still yet another aspect, the present invention provides an isolated host cell comprising the expression construct mentioned above.

In one aspect, the present invention provides a composition comprising the mRNA mentioned above and at least one guide RNA.

In another aspect, the present invention provides a lipid nanoparticle comprising the mRNA mentioned above.

In yet another aspect, the present invention provides a pharmaceutical composition comprising the mRNA mentioned above and a pharmaceutically acceptable carrier.

In still yet another aspect, the present invention provides a method of genome editing or modifying a target gene comprising contacting a cell with the mRNA mentioned above.

In one aspect, the present invention provides an artificial nucleic acid molecule comprising a. at least one coding region encoding at least one CRISPR-associated protein; b. at least one 5’ untranslated region (5’UTR) element; and c. at least one 3' untranslated region (3’ UTR) element, wherein said coding region comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleic acid sequence selected from any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232 and 247.

In some embodiments, said coding region further comprises at least one nuclear localization signal.

In some embodiments, said artificial nucleic acid molecule is an RNA.

In some embodiments, the RNA comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252, and 254.

In some embodiments, said artificial nucleic acid molecule is a DNA.

In some embodiments, the DNA comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 217-224, and 249-251.

In another aspect, the present invention provides a recombinant expression vector comprising the artificial nucleic acid molecule mentioned above.

In some embodiments, the artificial nucleic acid molecule is operably linked to a promoter.

In some embodiments, the promoter is an inducible promoter.

In some embodiments, the artificial nucleic acid molecule further comprising a multiple cloning site.

In yet another aspect, the present invention provides an in vitro genetically modified host cell comprising the artificial nucleic acid according to any one of claims 18 to 23 or the recombinant expression vector mentioned above.

In still yet another aspect, the present invention provides a composition comprising the artificial nucleic acid molecule mentioned above or the recombinant expression vector mentioned above and a pharmaceutically acceptable carrier and/or excipient.

In some embodiments, the composition further comprises a guide RNA or a nucleic acid encoding the same, said gRNA being capable of targeting the CRISPR-associated protein to a target DNA sequence of interest.

In some embodiments, the guide RNA is a single guide (sgRNA) .

In some embodiments, the guide RNA is a dual guide (dgRNA) .

In one aspect, the present invention provides a kit comprising the artificial nucleic acid molecule mentioned above or the recombinant expression vector mentioned above, and optionally a liquid vehicle and/or optionally technical instructions with information on the administration and dosage of the artificial nucleic acid molecule or the composition.

In some embodiments, the kit further comprises a guide RNA (gRNA) or a nucleic acid encoding the same, said gRNA being capable of targeting the CRISPR-associated protein to a target DNA sequence of interest, or a regulatory element operably linked thereto.

In some embodiments, the guide RNA is a single guide (sgRNA) .

In some embodiments, the guide RNA is a dual guide (dgRNA) .

In another aspect, the present invention provides a method of inducing a double stranded break (DSB) within a gene of interest, and/or modifying the gene of interest, comprising delivering the artificial nucleic acid molecule mentioned above, the recombinant expression vector mentioned above, the composition mentioned above, or the kit mentioned above to a cell.

In yet another aspect, the present invention provides a method of treating, preventing or diagnosing diseases associated with a gene of interest comprising administering the artificial nucleic acid molecule mentioned above, the recombinant expression vector mentioned above, the composition mentioned above, or the kit mentioned above to a subject in need thereof.

In still yet another aspect, the present invention provides use of the artificial nucleic acid molecule mentioned above, the recombinant expression vector mentioned above, the composition mentioned above, or the kit mentioned above in the preparation of medicament.

In one aspect, the present invention provides use of the artificial nucleic acid molecule mentioned above, the recombinant expression vector mentioned above, the composition mentioned above, or the kit mentioned above for gene therapy.

In another aspect, the present invention provides use of the artificial nucleic acid molecule mentioned above, the recombinant expression vector mentioned above, the composition mentioned above, or the kit mentioned above for inducing a double-stranded break (DSB) within a gene of interest, modifying the gene of interest, and/or modulating the expression of a gene of interest in a cell or subject.

In yet another aspect, the present invention provides the artificial nucleic acid molecule mentioned above, the recombinant expression vector mentioned above, the composition mentioned above, or the kit mentioned above for use in inducing a double-stranded break (DSB) within a gene of interest, modifying the gene of interest, and/or modulating the expression of a gene of interest in a cell or subject.

In still yet another aspect, the present invention provides the artificial nucleic acid molecule mentioned above, the recombinant expression vector mentioned above, the composition mentioned above, or the kit mentioned above for use in treating, preventing or diagnosing diseases associated with a gene of interest in a subject.

In one aspect, the present invention provides an artificial nucleic acid molecule comprising

i. at least one coding region encoding at least one peptide of interest;

ii. at least one 5’ untranslated region (5’UTR) element comprising a DNA sequence with at least 90%identity to a sequence selected from any one of SEQ ID NOs: 110-171 or an RNA sequence with at least 90%identity to a sequence selected from any one of SEQ ID NOs: 1-62;

iii. at least one 3’ untranslated region (3’ UTR) element comprising a DNA sequence with at least 90%identity to a sequence selected from any one of SEQ ID NOs: 172-210; or an RNA sequence with at least 90%identity to a sequence selected from any one of SEQ ID NOs: 63-101.

In some embodiments, the 5’ untranslated region (5’UTR) element comprising a DNA sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a sequence selected from any one of SEQ ID NOs: 110-171, or an RNA sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a sequence selected from any one of SEQ ID NOs: 1-62.

In some embodiments, the 3’ untranslated region (3’UTR) element comprising a DNA sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a sequence selected from any one of SEQ ID NOs: 172-210, or an RNA sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a sequence selected from any one of SEQ ID NOs: 63-101.

Sequence Listing-1

Sequence Listing-2

Sequence Listing-3

Table 1. Detailed description of sequence listing 1-3

Brief description of the drawings

Figure 1 shows percent editing following transfection of PHH cells with lipoplex comprising SpCas9 sgRNA (sg5815-3, SEQ ID NO: 242) and SpCas9 mRNA (m5815-1, SEQ ID NO: 233/CAS001D, SEQ ID NO: 236/CAS001F, SEQ ID NO: 238) .

Figure 2 shows percent editing following transfection of PHH cells with lipoplex comprising SpCas9 sgRNA (sg5815-3, SEQ ID NO: 242) and SpCas9 mRNA (m5815-1, SEQ ID NO: 233/CAS001C, SEQ ID NO: 235/CAS001F, SEQ ID NO: 238) .

Figure 3 shows percent editing following transfection of WT mice with LNP (ALC-0315) comprising SpCas9 sgRNA (sg5815-1, SEQ ID NO: 241) and SpCas9 mRNA (m5815-1, SEQ ID NO: 233/m5815-9, SEQ ID NO: 240/CAS001F, SEQ ID NO: 238) .

Figure 4 shows percent editing following transfection of WT mice with LNP (ALC-0315) comprising SpCas9 sgRNA (sg5815-1, SEQ ID NO: 241) and SpCas9 mRNA (m5815-1, SEQ ID NO: 233/m5815-9, SEQ ID NO: 240/CAS001F, SEQ ID NO: 238) .

Figure 5 shows the activity of SpCas9 (CAS001F) mRNA with different 5’UTR (A) and 5’UTR and 3’UTR combinations (B) in HepG2 cells, and the corresponding SEQ ID NOs of the names from left to right in the horizontal line are shown in the table 12. And the different SpCas9 mRNAs are combined with the sgRNA of SEQ ID NO: 109 respectively to form the different cargos.

Table 2

Figure 6A shows the activity of SpCas9 (CAS001F) mRNA with different 5’UTR (A&B) and 5’UTR and 3’UTR combinations (C) in HepG2 cells, and the corresponding SEQ ID NOs of the names from left to right in the horizontal line are shown in the table 14. And the different SpCas9 mRNAs are combined with the sgRNA of SEQ ID NO: 109 respectively to form the different cargos.

Table 3

Figure 6B shows the activity of SpCas9 (CAS001F) mRNA with different 5’UTR (A&B) and 5’UTR and 3’UTR combinations (C) in HepG2 cells, and the corresponding SEQ ID NOs of the names from left to right in the horizontal line are shown in the table 15. And the different SpCas9 mRNAs are combined with the sgRNA of SEQ ID NO: 109 respectively to form the different cargos.

Table 4

Figure 6C shows the activity of SpCas9 (CAS001F) mRNA with different 5’UTR (A&B) and 5’UTR and 3’UTR combinations (C) in HepG2 cells, and the corresponding SEQ ID NOs of the names from left to right in the horizontal line are shown in the table 16. And the different SpCas9 mRNAs are combined with the sgRNA of SEQ ID NO: 109 respectively to form the different cargos.

Table 5

Figure 7A shows the activity of SpCas9 (CAS001F) mRNA with different 5’UTR and 3’UTR combinations in HepG2 cells. And the different SpCas9 mRNAs are combined with the sgRNA of SEQ ID NO: 109 respectively to form the different cargos.

Table 6

Figure 7B shows the activity of SpCas9 (CAS001F) mRNA with different 5’UTR and 3’UTR combinations in HepG2 cells. And the different SpCas9 mRNAs are combined with the sgRNA of SEQ ID NO: 109 respectively to form the different cargos.

Table 7

Figure 7C shows the activity of SpCas9 (CAS001F) mRNA with different 5’UTR and 3’UTR combinations in HepG2 cells. And the different SpCas9 mRNAs are combined with the sgRNA of SEQ ID NO: 109 respectively to form the different cargos.

Table 8

Figure 8A shows the activity of Fluc mRNA with different 5’UTRs (A, B, C&D) and 3’UTRs (E) in HEK293T cells.

Table 9

Figure 8B shows the activity of Fluc mRNA with different 5’UTRs (A, B, C&D) and 3’UTRs (E) in HEK293T cells.

Table 10

Figure 8C shows the activity of Fluc mRNA with different 5’UTRs (A, B, C&D) and 3’UTRs (E) in HEK293T cells.

Table 11

Figure 8D shows the activity of Fluc mRNA with different 5’UTRs (A, B, C&D) and 3’UTRs (E) in HEK293T cells.

Table 12

Figure 8E shows the activity of Fluc mRNA with different 5’UTRs (A, B, C&D) and 3’UTRs (E) in HEK293T cells.

Table 13

Figure 9A shows the editing efficiency of mRNA samples tailed with different poly (A) structures, segmented A60+A60 and A95 respectively, in primary human hepatocyte (PHH) cell. mRNA-790 and mRNA-784 were generated from the same IVT DNA template with poly (A) A60+A60, but different IVT reaction -A and -B. mRNA-768 and mRNA-791 were generated from the same IVT DNA template with poly (A) A95, but different IVT reaction -A and -B. All the mRNA samples tested contain CDS sequence of SpCas9-encoding CAS001F. sgRNA which is named as sg5815-3 (SEQ ID NO: 242) was used as the guide RNA in this experiment.

Table 14

Figure 9B shows the editing efficiency of mRNA tailed with different poly (A) structures, segmented A60+A60 and A95 respectively, in primary human hepatocyte (PHH) cell. mRNA-788 and mRNA-802 tailed with poly (A) A60+A60, while with different NLS included. mRNA-791 and mRNA-792 tailed with poly (A) A95 and they were generated from the same IVT DNA template. All the mRNA samples tested contain CDS sequence of SpCas9-encoding CAS001F. sgRNA which is named as sg5815-3 (SEQ ID NO: 242) was used as the guide RNA in this experiment.

Table 15

Figure 9C shows the editing efficiency of mRNA tailed with different poly (A) structures, segmented A60+A60 and A95 respectively, in CD-1 mice. mRNA used in this experiment are the same as used in Figure 9B. CD1-mice were administrated with two doses, 0.03 mpk (light gray bar) and 0.1 mpk (dark gray bar) , as displayed in the figure. sgRNA which is named as sg5815-1 (SEQ ID NO: 241) was used as the guide RNA in this experiment.

Table 16

Figure 10 shows percent editing following transfection of WT mice with LNP (ALC-0315) comprising SpCas9 sgRNA (sg5815-1, SEQ ID NO: 241) and SpCas9 mRNA (CAS001F, SEQ ID NO: 238, mRNA-788/SEQ ID NO: 252) .

Detailed description

Definitions

The term "UTR" refers to an "untranslated region" flanking the coding sequence of an artificial nucleic acid as defined herein. In this context, an "UTR element" comprises or consists of a nucleic acid sequence, which is derived from the (naturally occurring, wild-type) UTR of a particular gene, preferably as exemplified herein.

When referring to UTR elements “derived from” a particular UTR, reference is made to nucleic acid sequences corresponding to the sequence of said UTR ( "parent UTR" ) or a homolog, variant or fragment of said UTR. The term includes sequences corresponding to the entire (full-length) wild-type sequence of said UTR, or a homolog, variant or fragment thereof, including full-length homologs and variants, as well as fragments of said full-length wild-type sequences, homologs and variants, and variants of said fragments. The term "corresponds to" means that the nucleic acid sequence derived from the "parent UTR" may be an RNA sequence (e.g. equal to the RNA sequence used for defining said parent UTR sequence) , or a DNA sequence (both sense and antisense strand and both mature and immature) , which corresponds to such RNA sequence.

When referring to an UTR element derived from an UTR of a gene, “or a homolog, fragment or variant thereof” , the expression “or a homolog, fragment or variant thereof” may refer to the gene, or the UTR, or both.

The term "homolog" in the context of genes (or nucleic acid sequences derived therefrom or comprised by said gene, like a UTR) refers to a gene (or a nucleic acid sequence derived therefrom or comprised by said gene) related to a second gene (or such nucleic acid sequence) by descent from a common ancestral DNA sequence. The term, "homolog" includes genes separated by the event of speciation ( "ortholog" ) and genes separated by the event of genetic duplication ( "paralog" ) .

Artificial nucleic acid molecule: An artificial nucleic acid molecule may typically be understood to be a nucleic acid molecule, e.g. a DNA or an RNA, that does not occur naturally. In other words, an artificial nucleic acid molecule may be understood as a non-natural nucleic acid molecule. Such nucleic acid molecule may be non-natural due to its individual sequence (which does not occur naturally) and/or due to other modifications, e.g. structural modifications of nucleotides, which do not occur naturally. An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule or a hybrid-molecule comprising DNA and RNA portions. Typically, artificial nucleic acid molecules may be designed and/or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence) . In this context an artificial sequence is usually a sequence that may not occur naturally, i.e. it differs from the wild type sequence by at least one nucleotide. The term "wild type" may be understood as a sequence occurring in nature. Further, the term "artificial nucleic acid molecule" is not restricted to mean "one single molecule" but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.

Nucleic acid molecule: A nucleic acid molecule is a molecule comprising, preferably consisting of nucleic acid components. The term nucleic acid molecule preferably refers to DNA or RNA molecules. It is preferably used synonymous with the term "polynucleotide" . Preferably, a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone. The term "nucleic acid molecule" also encompasses modified nucleic acid molecules, such as base-modified, sugar-modified or backbone-modified etc. DNA or RNA molecules.

DNA: DNA is the usual abbreviation for deoxy-ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotides. These nucleotides are usually deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate and deoxy-cytidine-monophosphate monomers which are-by themselves-composed of a sugar moiety (deoxyribose) , a base moiety and a phosphate moiety, and polymerise by a characteristic backbone structure. The backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, i.e. deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the DNA sequence. DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g. by A/T-base-pairing and G/C-base-pairing.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic-acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotides. These nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific succession of the monomers is called the RNA-sequence. Usually RNA may be obtainable by transcription of a DNA-sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DNA usually results in the so-called premature RNA which has to be processed into so-called messenger-RNA, usually abbreviated as mRNA. Processing of the premature RNA, e.g. in eukaryotic organisms, comprises a variety of different posttranscriptional-modifications such as splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA. The mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino-acid sequence of a particular peptide or protein. Typically, a mature mRNA comprises a 5'-cap, a 5'-UTR, an open reading frame, a 3'-UTR and a poly (A) sequence. Aside from messenger RNA, several non-coding types of RNA exist which may be involved in regulation of transcription and/or translation.

Heterologous sequence: Two sequences are typically understood to be 'heterologous' if they are not derivable from the same gene. i.e., although heterologous sequences may be derivable from the same organism, they naturally (in nature) do not occur in the same nucleic acid molecule, such as in the same mRNA.

Open reading frame: An open reading frame (ORF) in the context of the invention may typically be a sequence of several nucleotide triplets, which may be translated into a peptide or protein. An open reading frame preferably contains a start codon, i.e. a combination of three subsequent nucleotides coding usually for the amino acid methionine (ATG) , at its 5'-end and a subsequent region, which usually exhibits a length which is a multiple of 3 nucleotides. An ORF is preferably terminated by a stop-codon (e.g., TAA, TAG, TGA) . Typically, this is the only stop-codon of the open reading frame. Thus, an open reading frame in the context of the present invention is preferably a nucleotide sequence, consisting of a number of nucleotides that may be divided by three, which starts with a start codon (e.g. ATG) and which preferably terminates with a stop codon (e.g., TAA, TGA, or TAG) . The open reading frame may be isolated or it may be incorporated in a longer nucleic acid sequence, for example in a vector or an mRNA. An open reading frame may also be termed " (protein) coding sequence" or, preferably, "coding sequence" .

Codon: As used herein, the term "codon" refers to a sequence of three nucleotides that together form a unit of genetic code in a DNA or RNA molecule. A codon is operationally defined by the initial nucleotide from which translation starts and sets the frame for a run of successive nucleotide triplets, which is known as an "open reading frame" (ORF) . For example, the string GGGAAACCC, if read from the first position, contains the codons GGG, AAA, and CCC; if read from the second position, it contains the codons GGA and AAC; and if read from the third position, GAA and ACC. Thus, every nucleic sequence read in its 5'→ 3' direction comprises three reading frames, each producing a possibly distinct amino acid sequence (in the given example, Gly-Lys-Pro, Gly-Asn, or Glu-Thr, respectively) . DNA is double-stranded defining six possible reading frames, three in the forward orientation on one strand and three reverse on the opposite strand. Open reading frames encoding polypeptides are typically defined by a start codon, usually the first AUG codon in the sequence.

Initiation Codon: As used herein, the term "initiation codon" , used interchangeably with the term "start codon" , refers to the first codon of an open reading frame that is translated by the ribosome and is comprised of a triplet of linked adenine-uracil-guanine nucleobases. The initiation codon is depicted by the first letter codes of adenine (A) , uracil (U) , and guanine (G) and is often written simply as "AUG" . Although natural mRNAs may use codons other than AUG as the initiation codon, which are referred to herein as "alternative initiation codons" , the initiation codons of polynucleotides described herein use the AUG codon. During the process of translation initiation, the sequence comprising the initiation codon is recognized via complementary base-pairing to the anticodon of an initiator tRNA (Met-tRNAi^Met) bound by the ribosome. Open reading frames may contain more than one AUG initiation codon, which are referred to herein as "alternate initiation codons" .

The initiation codon plays a critical role in translation initiation. The initiation codon is the first codon of an open reading frame that is translated by the ribosome. Typically, the initiation codon comprises the nucleotide triplet AUG, however, in some instances translation initiation can occur at other codons comprised of distinct nucleotides. The initiation of translation in eukaryotes is a multistep biochemical process that involves numerous protein-protein, protein-RNA, and RNA-RNA interactions between messenger RNA molecules (mRNAs) , the 40S ribosomal subunit, other components of the translation machinery (e.g., eukaryotic initiation factors; eIFs) . The current model of mRNA translation initiation postulates that the pre-initiation complex (alternatively "43S pre-initiation complex" ; abbreviated as "PIC" ) translocates from the site of recruitment on the mRNA (typically the 5' cap) to the initiation codon by scanning nucleotides in a 5' to 3' direction until the first AUG codon that resides within a specific translation-promotive nucleotide context (the Kozak sequence) is encountered (Kozak (1989) J Cell Biol 108: 229-241) . Scanning by the PIC ends upon complementary base-pairing between nucleotides comprising the anticodon of the initiator Met-tRNAi^Met transfer RNA and nucleotides comprising the initiation codon of the mRNA. Productive base-pairing between the AUG codon and the Met-tRNAi^Met anticodon elicits a series of structural and biochemical events that culminate in the joining of the large 60S ribosomal subunit to the PIC to form an active ribosome that is competent for translation elongation.

Sequence identity: Two or more sequences are identical if they exhibit the same length and order of nucleotides or amino acids. The percentage of identity typically describes the extent to which two sequences are identical, i.e. it typically describes the percentage of nucleotides that correspond in their sequence position with identical nucleotides of a reference-sequence. For determination of the degree of identity ( "%identity) , the sequences to be compared are typically considered to exhibit the same length, i.e. the length of the longest sequence of the sequences to be compared. This means that a first sequence consisting of 8 nucleotides is 80%identical to a second sequence consisting of 10 nucleotides comprising the first sequence. In other words, in the context of the present invention, identity of sequences preferably relates to the percentage of nucleotides or amino acids of a sequence which have the same position in two or more sequences having the same length. Specifically, the "%identity" of two amino acid sequences or two nucleic acid sequences may be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in either sequence for best alignment with the other sequence) and comparing the amino acids or nucleotides at corresponding positions. Gaps are usually regarded as non-identical positions, irrespective of their actual position in an alignment. The " best alignment" is typically an alignment of two sequences that results in the highest percent identity. The percent identity is determined by the number of identical nucleotides in the sequences being compared (i.e., %identity = #of identical positions/total #of positions x 100) . The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art.

The term "variant" in the context of nucleic acid sequences of genes refers to nucleic acid sequence variants, i.e. nucleic acid sequences or genes comprising a nucleic acid sequence that differs in at least one nucleic acid from a reference (or "parent" ) nucleic acid sequence of a reference (or "parent" ) nucleic acid or gene. Variant nucleic acids or genes may thus preferably comprise, in their nucleic acid sequence, at least one mutation, substitution, insertion or deletion as compared to their respective reference sequence. Preferably, the term "variant" as used herein includes naturally occurring variants, and engineered variants of nucleic acid sequences or genes. Therefore, a "variant" as defined herein can be derived from, isolated from, related to, based on or homologous to the reference nucleic acid sequence/Variants" may preferably have a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90%and most preferably of at least 95%or even 97%, to a nucleic acid sequence of the respective naturally occurring (wild-type) nucleic acid sequence or gene, or a homolog, fragment or derivative thereof.

The term "fragment" in the context of nucleic acid sequences or genes refers to a continuous subsequence of the full-length reference (or "parent" ) nucleic acid sequence or gene. In other words, a "fragment" may typically be a shorter portion of a full-length nucleic acid sequence or gene. Accordingly, a fragment, typically, consists of a sequence that is identical to the corresponding stretch within the full-length nucleic acid sequence or gene. The term includes naturally occurring fragments as well as engineered fragments. A preferred fragment of a sequence in the context of the present invention, consists of a continuous stretch of nucleic acids corresponding to a continuous stretch of entities in the nucleic acid or gene the fragment is derived from, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80%of the total (i.e. full-length) nucleic acid sequence or gene from which the fragment is derived. A sequence identity indicated with respect to such a fragment preferably refers to the entire nucleic acid sequence or gene. Preferably, a "fragment" may comprise a nucleic acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90%and most preferably of at least 95%or even 97%, to a reference nucleic acid sequence or gene that it is derived from.

The following examples further illustrate the present disclosure, but the present disclosure is not limited thereto.

Below presents preferred embodiments of the present disclosure based on the drawings in order to illustrate the technical schemes of the present disclosure in detail.

Example 1. LNP formulation and analytics

The Cas9 mRNA and sgRNA were encapsulated within lipid nanoparticles (LNP) using a Precision MPF-L2^TM AITESEN. In general, the lipid nanoparticle components were dissolved in 100%ethanol with the lipid component (LP01, DSPC, cholesterol and DMG-PEG2000 in molar ratio of 50: 9: 38: 3 or ALC-0315, DSPC, cholesterol and ALC-0159 in molar ratio of 46.3: 42.7: 9.4: 1.6) . The RNA cargos were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. The LNPs were formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6, with the ratio of mRNA to gRNA at 2: 1 by weight. RNA in aqueous buffer conditions is mixed with ethanolic lipid mix, respectively in the volume ratio of 3 parts of RNA and 1 part of lipid mix. After mixing, the LNPs were collected, and then remaining buffer was exchanged into PH7.5 TSS (contain 5%w/v (g/L) sucrose, 45 mM NaCl, 50 mM Tris , 100-fold excess of sample volume) , overnight at 4℃ under gentle stirring using a 10 kDa dialysis bag. The resulting mixture was then filtered using a 0.2 um sterile filter. The resulting filtrate was stored at 2-8 ℃.

Lp01 is (9Z, 12Z) -3- ( (4, 4-Bis (octyloxy) butanoyl) oxy) -2- ( ( ( (3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl octadeca-9, 12-dienoate and Lp01 has the structure shown in the below:

ALC-0315 is 6- ( (2-hexyldecanoyl) oxy) -N- (6- ( (2-hexyldecanoyl) oxy) hexyl) -N- (4-hydroxybutyl) hexan-1-aminium and ALC-0315 has the structure shown in the below:

Table 17 Prescription of LNP-01

Table 18 Prescription of LNP-0315

Dynamic Light Scattering ( "DLS" ) is used to determine the average particle size and polydispersity index ( "PDI" ) and of LNP samples. Average particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. Dilute LNP samples in 1x PBS at volumetric ratios of 1: 99 prior to testing. Measure the mean hydrodynamic diameter of each sample by reporting average particle size along with PDI. Zeta potential of the LNP is also measured by Malvern Zetasizer. Samples are diluted at volumetric ratios of 1: 19 in 10mM NaCl prior to measurement.

A fluorescence-based assay (ThermoFisher Scientific) is used to determine the encapsulation efficiency, which is calculated as (Total RNA -Free RNA) /Total RNA. LNP samples are diluted with1x TE buffer containing 1%Triton-X 100 to a proper concentration to determine total RNA or 1x TE buffer to determine free RNA. Standard curves are prepared by utilizing the starting RNA solution used to make the formulations according to the manufacturer's instructions) . stain is then added to each of the standards and samples and allowed stain to incubate for approximately 5 minutes at room temperature, in the absence of light. A Tecan INFINITE 200 PRO is used to read the samples with excitation and emission wavelength at 480nm, 520nm respectively. Total RNA and free RNA are calculated from the appropriate standard curves after subtracting the fluorescence value of the reagent blank from that of each of the samples.

Typically, when preparing LNPs, encapsulation was >80%, particle size was <120 nm, and pdi was < 0.2

Example 2. SpCas9 mRNA sequence optimization

Primary human liver hepatocytes (PHH) cells were thawed and resuspended in hepatocyte thawing medium with supplements (Lonza, Cat. MCHT50) followed by centrifugation at 100 g for 10 minutes. The supernatant was discarded and the pelleted cells resuspended in hepatocyte plating medium (Lonza, Cat. MP100) plus 10%fetal bovine serum. Cells were counted and plated on Ultra Low Adsorption Cell Culture 96-well plates (Liver Biotech, Cat. LV-ULA002-96W) at a density of 40,000 cells/well. Plated cells were allowed to settle and adhere for 24 hours in a tissue culture incubator at 37℃ and 5%CO₂ atmosphere. After incubation cells were checked for monolayer formation and media was replaced with hepatocyte culture medium (Lonza, Cat. CC-3198) plus 10%fetal bovine serum. Cells were sequentially transfected with a lipoplex containing SpCas9 mRNA/sgRNA (6.25/1.56 nM sgRNA sg5815-3, mRNA/sgRNA=2: 1 w/w) , Lipofectamine RNAiMax (0.3 μL/well, Invitrogen^TM) and OptiMem per the manufacturer's protocol. The plate was placed at 37℃ and 5%CO₂ and cells were harvested on day three post-transfection. Total genomic DNA were extracted separately using the QuickExtract^TM DNA Extraction Soln 1.0 according to the manufacturer’s instructions. Next-generation sequencing (NGS) was used to measure the editing efficiency.

Figure 1 and Figure 2 shows the editing efficiency in PHH cells for SpCas9 mRNA indicated m5815-1, SEQ ID NO: 233/CAS001D (327) , SEQ ID NO: 236/CAS001F (331) , SEQ ID NO: 238. The result indicated the highest editing efficiency for SpCas9 mRNA CAS001F.

Example 3. LNP delivery to mice having WT (murine) TTR.

Selected guide RNA, mRNA and LNP formulation were further tested in mice. mice from, ranging 6-12 weeks of age, were used in each study involving mice. LNPs were dosed via the lateral tail vein injection. The animals were observed post dose for adverse effects. Body weight was measured at twenty-four hours before-administration and animals euthanized post dose via exsanguination under isoflurane anesthesia or CO₂ asphyxiation. Blood was collected from inner canthal veins into serum separator tubes. The LNPs were made as described in Example 1.

For studies involving in vivo editing, liver tissue was collected from the left lateral lobe from each animal. Genomic DNA was isolated using Nucleic acid extraction Kit (Denogen, DNS033-48) . All DNA samples were prepared for Sanger-seq and NGS analysis as described in Example 2.

The data shown in Figure 3 and Figure 4 are from WT mice (n=5 per group) administered LNP0315, containing SpCas9 mRNA m5815-1, SEQ ID NO: 233/m5815-9, SEQ ID NO: 240/CAS001F, SEQ ID NO: 238 and sgRNA sg5815-1 (SEQ ID NO: 241) at 0.3 MPK (total RNA content, mRNA/sgRNA=2: 1 w/w) and euthanized at 6 days post dose. Samples were processed as described above.

Example 4

Methods

In vitro transcription (IVT)

1. Preparation of template DNA

1.1 Plasmid construction

mRNA was IVT synthesized from a linear plasmid DNA template. The plasmid template was constructed with pVAX1 as backbone. In the plasmid, T7 promoter was fused to a 5’ UTR, kozak sequence, SpCas9 CDS or SaCas9 CDS or Firefly luciferase reporter gene, a 3’ UTR and poly (A) of less than A100 or separated poly (A) of A60+6dNTP+A60. Plasmid construction was performed by either Gibson assembly or Golden gate assembly.

1.2 Plasmid isolation

Plate the E. coli strains carrying the plasmid DNA template from a glycerol stock onto a fresh LB plate containing corresponding antibiotic (ampicillin 100 μg/mL, or kanamycin 100 μg/mL) and incubate at 37℃ overnight in an incubator.

Inoculate 20/200 mL LB liquid medium with a fresh single colony from the plate and cultivate at 37℃, 220 rpm overnight.

Plasmid was isolated using commercial plasmid prep kit ( (TIANGEN endo-free plasmid midi prep kit (DP108) or (Macherey-Nagel) NucleoBond Xtra Midi EF, Midi kit for endotoxin-free plasmid DNA (740420.10) ) .

1.3 Plasmid linearization

Plasmid template was linearized with BsaI or BspQI or Esp3I or NdeI (NEB) overnight at 37℃according to manufacturer’s instruction.

1.4 Proteinase K treatment

Linearized plasmid DNA template was treated with proteinase K (100–200 μg/mL) and 0.5%SDS for 30 min at 50℃ to remove potential inhibitors of transcription introduced during plasmid prep, such as RNases. The reaction was followed with incubation at 65℃ for 10 min to heat inactivate the enzyme .

1.5 Purification of linearized plasmid

Following up, the RE digestion reaction is purified with phenol/chloroform extraction (using an equal volume) and ethanol precipitation.

1) Add 1/10 volume of 3 M sodium acetate, mix well.

2) Add 1 volume of DNA extraction reagent (phenol: chloroform: isoamyl alcohol= 25: 24: 1) , mix well, sit at room temperature for 3 min.

3) Centrifuge at 12000 rpm for 15 min.

4) Carefully transfer the supernatant to a fresh EP tube.

5) Add an equal volume of isopropanol, precipitate at -20℃ for at least 30 min.

6) Centrifuge at 12000 rpm for 15 min to pellet the linear plasmid DNA. Carefully discard the supernatant.

7) Wash the DNA pellet with 1 mL 70%ethanol, centrifuge at 12000 rpm for 1 min.

8) Repeat the wash step.

9) Carefully remove any residual fluid with a fine-tipped pipette.

10) Air dry the pellet for around 10 min, or longer if needed.

11) Resuspend the pellet using 30-50 μL NF water (if 20 μg plasmid was digested) .

12) Check the concentration of the linear plasmid using Qubit or NanoDrop.

1.6 In vitro transcription (IVT)

Co-transcriptional capping was performed using IVT reagents and GAG cap from Hongene. The 20μL IVT reaction contains ATP (10 mM) , UTP (10 mM) , GTP (10 mM) , VTP (10 mM) , GAG or GAG 3’ oMe cap (10 mM) , DNA template (1 μg) , 5X Reaction buffer (1X) , and T7 enzyme mix (1.5 μL) . The reaction was assembled at room temperature, mixed well by gently flicking the tube and incubated at 37℃ for 4 h.

In the case of co-transcriptional capping with ARCA cap, ARCA cap instead of GAG cap was added to the IVT reaction at final concentration of 8 mM, and GTP was used at final concentration 2 mM. All other components kept the same as in co-transcriptional capping with GAG cap.

1.7 DNase digestion

After 4 h incubation at 37℃, the IVT reaction was treated with DNase I (2 U for 20 μL reaction) and incubated at 37℃ for 15 min.

1.8 mRNA purification

mRNA product was purified through lithium chloride precipitation. Briefly,

Stop the reaction (20+1 μL) and precipitate the RNA by adding 30 μL Nuclease-free Water and 30 μL LiCl Precipitation Solution (7.5 M lithium chloride, 50 mM EDTA, final concentration 2.778 M (2.5-2.8 M) ) .

Mix thoroughly. Chill for ≥30 min at –20℃.

Centrifuge at 4℃ for 15 min at maximum speed to pellet the RNA.

Carefully remove the supernatant. Wash the pellet once with ～1 mL pre-cooled 70%ethanol, and re-centrifuge to maximize the removal of unincorporated nucleotides.

Carefully remove the 70%ethanol, and resuspend the RNA in a solution orappropriate for your application. Determine the RNA concentration and store frozen at –20℃ or –70℃.

1.9 mRNA quantitation

The final mRNA product was quantified by either fluorescent dye (Qubit) or UV light absorbance (Nanodrop) . The purity of mRNA product was checked on both agarose gel and by capillary electrophoresis using 5300 fragment analyzer (Agilent) .

1.10 mRNA activity assay in HepG2

HepG2 cells were cultured in Advanced Dulbecco’s Modified Eagle’s Medium (Advanced DMEM, Gibco) containing 2 mM L-glutamine (GlutaMAXTM-l, Gibco) , supplemented with 10%fetal bovine serum (FETAL BOVINE SERUM, GEMINI) at 37 ℃ in 5%CO₂-buffered incubators. For transfection of mRNAs, 2.0*10⁵ HepG2 cells were seeded in each well of a 24-well plate. 12.5 nM or 3.125 nM or 1.56 nM gRNA and corresponding mRNA with cargo ratio 1: 1 (w/w) were transfected using Lipofectamine RNAiMAX Reagent as per the manufacturer’s instructions (Life Technologies) . After 72 hours of incubation, the cells were harvest and gDNA was isolated from the cells. The editing efficiency at the target was analyzed via NGS or Sanger seq with the PCR product at the target site.

1.11 Fluc mRNA activity assay in HEK293T

HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, CORNING) containing 2 mM L-glutamine (GlutaMAXTM-l, Gibco) , supplemented with 10%fetal bovine serum (FETAL BOVINE SERUM, GEMINI) at 37℃ in 5%CO₂-buffered incubators. For transfection of Fluc mRNAs, 3.0*10⁴ HEK293T cells were seeded in each well of a 96-well plate. 50 ng Fluc mRNA was transfected using Lipofectamine RNAiMAX Reagent as per the manufacturer’s instructions (Life Biotechnologies) . At different time points post-transfection, e.g., 4 h, 6 h, 9 h, 21 h and/or 24 h, the cells in each well were treated with Bright-Lumi^TM Firefly luciferase Reporter Gene Assay kit (Beyotime Technology, China) according to manufacturer’s introduction. Briefly, 1/1 volume of the Luciferase assay reagent was added into 100 μL cell culture in the well and mix well. After 2 min reaction at room temperature (around 25℃) , the luminescence signal was detected using a multi-mode microplate reader (INFINITE 200 PRO, TECAN) .

Example 5

The cap utilized in the mRNA sequences named GEB_mRNA_p318, GEB_mRNA_p917, and GEB_mRNA_p676 was sourced from SyngeneBio. The product name is CAP 5 m7G (5') vppp (5') (2'OMeA) pG, with the item number CAP5011. And the cap has the following structure:

Primary human liver hepatocytes (PHH) cells were thawed and resuspended in hepatocyte thawing medium with supplements (Lonza, Cat. MCHT50) followed by centrifugation at 100 g for 10 minutes. The supernatant was discarded and the pelleted cells resuspended in hepatocyte plating medium (Lonza, Cat. MP100) plus 10%fetal bovine serum. Cells were counted and plated on Ultra Low Adsorption Cell Culture 96-well plates (Liver Biotech, Cat. LV-ULA002-96W) at a density of 40,000 cells/well. Plated cells were allowed to settle and adhere for 24 hours in a tissue culture incubator at 37℃ and 5%CO₂ atmosphere. After incubation cells were checked for monolayer formation and media was replaced with hepatocyte culture medium (Lonza, Cat. CC-3198) plus 10%fetal bovine serum. Cells were sequentially transfected with a lipoplex containing SpCas9 mRNA/sgRNA (0.12 nM sgRNA sg5815-3, mRNA/sgRNA=1: 1 w/w) , Lipofectamine RNAiMax (0.3 μL/well, Invitrogen^TM) and OptiMem per the manufacturer's protocol. The plate was placed at 37℃ and 5%CO₂ and cells were harvested on day three post-transfection. Total genomic DNA were extracted separately using the QuickExtract^TM DNA Extraction Soln 1.0 according to the manufacturer’s instructions. Next-generation sequencing (NGS) was used to measure the editing efficiency. Figure 9A and Figure 9B shows the editing efficiency of mRNA tailed with different poly (A) structures, segmented A60+A60 and A95 respectively, in PHH cells.

Selected guide RNA, mRNA and LNP formulation were further tested in mice. mice from, ranging 6-12 weeks of age, were used in each study involving mice. LNPs were dosed via the lateral tail vein injection. The animals were observed post dose for adverse effects. Body weight was measured at twenty-four hours before-administration and animals euthanized post dose via exsanguination under isoflurane anesthesia or CO₂ asphyxiation. Blood was collected from inner canthal veins into serum separator tubes. The LNPs were made as described in Example 1. For studies involving in vivo editing, liver tissue was collected from the left lateral lobe from each animal. Genomic DNA was isolated using Nucleic acid extraction Kit (Denogen, DNS033-48) . All DNA samples were prepared for Sanger-seq and NGS analysis as described in Example 2. Figure 9C shows the editing efficiency of mRNA tailed with different poly (A) structures, segmented A60+A60 and A95 respectively, in CD-1 mice. The data shown in Figure 9C are from WT mice (n=5 per group) administered LNP0315, containing SpCas9 mRNA and sgRNA sg5815-1 (SEQ ID NO: 241) at 0.03 MPK (total RNA content, mRNA/sgRNA=2: 1 w/w) or 0.1 MPK (total RNA content, mRNA/sgRNA=2: 1 w/w) , and euthanized at 6 days post dose. Samples were processed as described above.

Example 6

Selected guide RNA, mRNA and LNP formulation were further tested in mice. mice from, ranging 6-12 weeks of age, were used in each study involving mice. LNPs were dosed via the lateral tail vein injection. The animals were observed post dose for adverse effects. Body weight was measured at twenty-four hours before-administration and animals euthanized post dose via exsanguination under isoflurane anesthesia or CO₂ asphyxiation. Blood was collected from inner canthal veins into serum separator tubes. The LNPs were made as described in Example 1. For studies involving in vivo editing, liver tissue was collected from the left lateral lobe from each animal. Genomic DNA was isolated using Nucleic acid extraction Kit (Denogen, DNS033-48) . All DNA samples were prepared for Sanger-seq and NGS analysis as described in Example 2. Figure 10 shows the editing efficiency of mRNA with different UTRs, in CD-1 mice. The data shown in Figure 10 are from WT mice (n=5 per group) administered LNP0315, containing SpCas9 mRNA and sgRNA sg5815-1 (SEQ ID NO: 241) at 0.1 MPK (total RNA content, mRNA/sgRNA=2: 1 w/w) , and euthanized at 6 days post dose. Samples were processed as described above.

Claims

An mRNA comprising an open reading frame encoding an RNA-guided DNA-binding agent, wherein the open reading frame comprises a sequence having at least 90%identity to any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232, and 247.
The mRNA of claim 1, wherein the open reading frame comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to the sequence of any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232, and 247.
The mRNA of claim 1 or 2, wherein the mRNA further comprises a 5’ UTR with at least 90%identity to any one of SEQ ID NOs: 110-171 or 1-62.
The mRNA of any one of the preceding claims, wherein the mRNA further comprises a 5’ UTR with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to any one of SEQ ID NOs: 110-171 or 1-62.
The mRNA of any one of the preceding claims, wherein the mRNA further comprises a 3’ UTR with at least 90%identity to any one of SEQ ID NOs: 172-210 or 63-101.
The mRNA of any one of the preceding claims, wherein the mRNA further comprises a 3’ UTR with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to any one of SEQ ID NOs: 172-210 or 63-101.
The mRNA of any one of the preceding claims, wherein the mRNA further comprises a poly-adenylated (poly-A) tail with at least 90%identity to SEQ ID NO: 243, 244, 245 or 246.
The mRNA of any one of the preceding claims, wherein the mRNA further comprises a poly-adenylated (poly-A) tail with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 243, 244, 245 or 246.
An mRNA comprising a sequence with at least 98%identity to any one of SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252, and 254.
The mRNA of claim 9, wherein the mRNA comprises a sequence selected from any one of SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252, and 254.
The mRNA of any one of claims 1 to 10, wherein at least 10%of the uridine is substituted with a modified uridine, wherein the modified uridine is one or more of N1-methyl-pseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine.
An expression construct comprising a promoter operably linked to a sequence encoding an mRNA according to any one of claims 1-11, wherein the expression construct is optionally a plasmid expression construct.
An isolated host cell comprising the expression construct of claim 12.
A composition comprising the mRNA according to any one of claims 1-11 and at least one guide RNA.
A lipid nanoparticle comprising the mRNA according to any one of claims 1-11.
A pharmaceutical composition comprising the mRNA according to any one of claims 1-13 and a pharmaceutically acceptable carrier.
A method of genome editing or modifying a target gene comprising contacting a cell with the mRNA according to any one of claims 1-13;

preferably, the method is in vitro or ex vivo, and/or, the method is not diagnostic, therapeutic, and surgical methods for the treatment of humans or animals.
An artificial nucleic acid molecule comprising a. at least one coding region encoding at least one CRISPR-associated protein; b. at least one 5’ untranslated region (5’ UTR) element; and c. at least one 3' untranslated region (3’ UTR) element, wherein said coding region comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleic acid sequence selected from any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232 and 247.
The artificial nucleic acid molecule according to claim 18, wherein said coding region further comprises at least one nuclear localization signal.
The artificial nucleic acid molecule according to claim 18 or 19, wherein said artificial nucleic acid molecule is an RNA.
The artificial nucleic acid molecule according to claim 20, wherein the RNA comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252, and 254.
The artificial nucleic acid molecule according to claim 18 or 19, wherein said artificial nucleic acid molecule is a DNA.
The artificial nucleic acid molecule according to claim 22, wherein the DNA comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 217-224, and 249-251.
A recombinant expression vector comprising the artificial nucleic acid molecule according to any one of claims to 18 to 23.
The recombinant expression vector according to claim 24, wherein the artificial nucleic acid molecule is operably linked to a promoter.
The recombinant expression vector according to claim 25, wherein the promoter is an inducible promoter.
The recombinant expression vector according to any one of the preceding claims, wherein the artificial nucleic acid molecule further comprising a multiple cloning site.
An in vitro genetically modified host cell comprising the artificial nucleic acid according to any one of claims 18 to 23 or the recombinant expression vector according to any one of claims 24 to 27.
A composition comprising the artificial nucleic acid molecule according to any one of claims 18 to 23 or the recombinant expression vector according to any one of claims 24 to 27 and a pharmaceutically acceptable carrier and/or excipient.
The composition according to claim 29, wherein the composition further comprises a guide RNA or a nucleic acid encoding the same, said gRNA being capable of targeting the CRISPR-associated protein to a target DNA sequence of interest.
The composition according to claim 30, wherein the guide RNA is a single guide (sgRNA) .
The composition according to claim 30, wherein the guide RNA is a dual guide (dgRNA) .
A kit comprising the artificial nucleic acid molecule according to any one of claims 18 to 23 or the recombinant expression vector according to any one of claims 24 to 27, and optionally a liquid vehicle and/or optionally technical instructions with information on the administration and dosage of the artificial nucleic acid molecule or the composition.
The kit according to claim 33, further comprising a guide RNA (gRNA) or a nucleic acid encoding the same, said gRNA being capable of targeting the CRISPR-associated protein to a target DNA sequence of interest, or a regulatory element operably linked thereto.
The kit according to claim 34, wherein the guide RNA is a single guide (sgRNA) .
The kit according to claim 34, wherein the guide RNA is a dual guide (dgRNA) .
A method of inducing a double stranded break (DSB) within a gene of interest, and/or modifying the gene of interest, comprising delivering the artificial nucleic acid molecule according to any one of claims 18-23, the recombinant expression vector according to any one of claims 24 to 27, the composition according to any one of claims 29 to 32, or the kit according to any one of claim 33 to 36 to a cell.
A method of treating, preventing or diagnosing diseases associated with a gene of interest comprising administering the artificial nucleic acid molecule according to any one of claims 18-23, the recombinant expression vector according to any one of claims 24 to 27, the composition according to any one of claims 29 to 32, or the kit according to any one of claim 33 to 36 to a subject in need thereof.
Use of the artificial nucleic acid molecule according to any one of claims 18-23, the recombinant expression vector according to any one of claims 24 to 27, the composition according to any one of claims 29 to 32, or the kit according to any one of claim 33 to 36 in the preparation of medicament.
Use of the artificial nucleic acid molecule according to any one of claims 18-23, the recombinant expression vector according to any one of claims 24 to 27, the composition according to any one of claims 29 to 32, or the kit according to any one of claim 33 to 36 for gene therapy.
Use of the artificial nucleic acid molecule according to any one of claims 18-23, the recombinant expression vector according to any one of claims 24 to 27, the composition according to any one of claims 29 to 32, or the kit according to any one of claim 33 to 36 for inducing a double-stranded break (DSB) within a gene of interest, modifying the gene of interest, and/or modulating the expression of a gene of interest in a cell or subject.
The artificial nucleic acid molecule according to any one of claims 18-23, the recombinant expression vector according to any one of claims 24 to 27, the composition according to any one of claims 29 to 32, or the kit according to any one of claim 33 to 36 for use in inducing a double-stranded break (DSB) within a gene of interest, modifying the gene of interest, and/or modulating the expression of a gene of interest in a cell or subject.
The artificial nucleic acid molecule according to any one of claims 18-23, the recombinant expression vector according to any one of claims 24 to 27, the composition according to any one of claims 29 to 32, or the kit according to any one of claim 33 to 36 for use in treating, preventing or diagnosing diseases associated with a gene of interest in a subject.
An artificial nucleic acid molecule comprising

i. at least one coding region encoding at least one peptide of interest;

ii. at least one 5’ untranslated region (5’ UTR) element comprising a DNA sequence with at least 90%identity to a sequence selected from any one of SEQ ID NOs: 110-171 or an RNA sequence with at least 90%identity to a sequence selected from any one of SEQ ID NOs: 1-62;

iii. at least one 3’ untranslated region (3’ UTR) element comprising a DNA sequence with at least 90%identity to a sequence selected from any one of SEQ ID NOs: 172-210; or an RNA sequence with at least 90%identity to a sequence selected from any one of SEQ ID NOs: 63-101.
The artificial nucleic acid molecule of claim 44, wherein the 5’ untranslated region (5’ UTR) element comprising a DNA sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a sequence selected from any one of SEQ ID NOs: 110-171, or an RNA sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a sequence selected from any one of SEQ ID NOs: 1-62.
The artificial nucleic acid molecule of claim 34, wherein the 3’ untranslated region (3’ UTR) element comprising a DNA sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a sequence selected from any one of SEQ ID NOs: 172-210, or an RNA sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a sequence selected from any one of SEQ ID NOs:63-101.