TTR-targeted gene editing composition

Abstract

The application relates to a composition and method for TTR gene editing and treatment of ATTR amyloidosis. By mutating Cas9 nuclease, the fidelity of a TTR gene editing system is improved, the off-target rate is reduced, and the specificity and safety of the gene editing system are greatly improved.

Description

Gene editing composition targeting TTR Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a gene editing tool with high efficiency and low off-target effects, and a composition comprising the gene editing tool. Background Art

[0002] CRISPR-Cas9 is the third-generation gene editing technology, following the introduction of ZFNs, TALENs, and other gene editing technologies. Since Zhang Feng first reported the high editing efficiency of Cas9 in mammalian cells in 2013, CRISPR-Cas9 has rapidly developed, becoming one of the most efficient, simplest, lowest-cost, and most versatile technologies for gene editing and modification, and the most mainstream gene editing system today.

[0003] CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) is a natural immune system in prokaryotes, found in 40% of sequenced bacteria and 90% of sequenced archaea. After being invaded by a virus, some bacteria can "store" a small fragment of the viral gene within their own DNA. Upon encountering another viral attack, the bacteria can recognize the virus based on this "stored" fragment and cut the viral DNA, rendering it ineffective.

[0004] Currently, the classic CRISPR-Cas9 system consists of two parts: sgRNA and Cas9 protein. sgRNA is composed of crRNA and tracrRNA, and is currently mostly combined into a single RNA. The Cas9 protein binds to the sgRNA to form an RNA-protein complex (RNP), which locates and recognizes the PAM and target sequence. The DNA double helix will be unwound to form an R-Loop. The sgRNA hybridizes with the complementary chain, while the other chain remains free. Subsequently, the Cas9 protein's precise blunt-end cleavage site is located 3 nucleotides upstream of the PAM, forming a blunt-end product. The HNH domain of the Cas9 protein is responsible for cutting the DNA chain that is complementary to the crRNA, while the RuvC domain is responsible for cutting the other non-complementary DNA chain. Ultimately, under the action of Cas9, a DNA double-strand break (DSB) is generated, and gene knockout is achieved through frameshift mutations during the genome repair process.

[0005] Although the CRISPR-Cas9 system can efficiently edit target genes, the wild-type Cas9 protein has a high off-target rate, with an average of approximately 100 off-target sites. This high off-target frequency can lead to serious risks of genomic mutations and chromosomal translocations, hindering the development of gene editing.

[0006] Studies have shown that the specificity of the CRISPR / Cas9 system mainly depends on the recognition sequence of the sgRNA. Since the designed sgRNA may form mismatches with non-target DNA sequences, it may lead to unexpected gene mutations, which are called off-target effects. Since off-target mutations may cause genomic instability and disrupt the functions of other normal genes, researchers are committed to studying the factors affecting the off-target effect of the CRISPR / Cas9 system. Specifically, the factors affecting off-target effect include: (1) the effect of sgRNA and DNA pairing sequence on off-target effect: there may be base mismatches between the sgRNA sequence and the DNA binding region; (2) the effect of PAM on off-target effect: generally, different PAM sequences have different off-target effects; generally speaking, the more nucleotides required, the lower the off-target effect; (3) the influence of Cas9 and other factors: by modifying the wild-type Cas9 to improve the specificity of the CRISPR / Cas9 system, the use of mutant nucleases can induce efficient gene editing in human cells and greatly reduce the level of off-target mutations.

[0007] Regarding the off-target issue, the current CRISPR / Cas9 gene editing technology has the following problems: 1) The off-target rate of Cas9 protein is high, resulting in a higher risk of genome damage and chromosomal aberrations; 2) The current mainstream gene editing delivery vector is AVV. AVV delivery allows the editing tool to be expressed for a long time, increasing the off-target risk; 3) Viral vector delivery is immunogenic and is not suitable for repeated administration.

[0008] Transthyretin (TTR), also known as vitamin A-binding protein, is an important component of plasma proteins and is widely distributed in various cells, plasma, and tissue fluids. As a carrier protein, TTR is mainly synthesized in the liver and the choroid plexus in the brain, secreted into the blood and cerebrospinal fluid, and carries thyroxine and retinol (i.e., vitamin A) to various tissues and cells throughout the body. As a carrier protein, the function of TTR can often be replaced by thyroxine-binding globulin and albumin in plasma. Under physiological conditions, TTR is a stable protein. After TTR breaks down into monomers, it can cause amyloidosis. In recent years, more and more TTR-targeted drugs are gradually entering the clinic for use in neurological diseases, endocrine and metabolic diseases, and so on.

[0009] Among TTR-related diseases, the most studied are TTR-related amyloidosis, such as transthyretin cardia amyloidosis myocardiopathy (ATTR-CM), transthyretin amyloid polyneuropathy (ATTR-PN), and Alzheimer's disease (AD). TTR has also been linked to tumors, with significant differences in its expression levels in different tumor cells, suggesting its potential role in promoting or inhibiting malignant tumors.

[0010] As a rare disease highly associated with the TTR protein, ATTR-CM is characterized by a low incidence, progressive severity, diverse clinical manifestations, and challenging clinical and differential diagnosis. It rapidly leads to heart failure and death, with a survival period of only 2-7 years after diagnosis, and a five-year survival rate of less than 50%. ATTR-CM was initially reported in only a few endemic countries or regions, but subsequent studies have reported cases in 29 countries to date. Transthyretin amyloidosis (ATTR) is categorized by etiology as either mutant (ATTRv) or wild-type (ATTRwt). Worldwide, there are approximately 50,000 cases of ATTRv and 200,000-500,000 cases of ATTRwt. Currently, there is no accurate epidemiological data available for ATTR in China, and the clinical diagnosis rate is less than 1%. Due to a lack of understanding of the disease and the heterogeneity of its symptoms, the disease remains underdiagnosed, and its incidence is significantly underestimated.

[0011] Therefore, it is necessary to develop a novel CRISPR-Cas9 system to treat TTR-related diseases.

[0012] Summary of the Invention

[0013] Based on the defects of the existing technology, this application targets the transthyretin gene (TTR) and achieves efficient TTR gene knockout by optimizing sgRNA, Cas9 and LNP delivery system to treat cardiomyopathy (ATTR-CM) and neuropathy (ATTR-PN) caused by transthyretin amyloidosis (ATTR).

[0014] Specifically, this application involves the following technical solutions:

[0015] 1. A gene editing composition comprising:

[0016] 1) Cas9 nuclease or an enzymatically active fragment thereof, or a nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof; and

[0017] 2) a guide RNA (gRNA) targeting the transthyretin (TTR) gene or a nucleic acid molecule encoding the gRNA, wherein the Cas9 nuclease comprises a mutation at one or more amino acid residue positions selected from the group consisting of K526, N692, Q695, H698, N497, Y450, Q926, K377, E387, D397, R400, D406, A421, L423, R424, Q426, Y430, K442, P449, V452, A456, R457, W464, M465, K468, E470, T474, P475, W476, F478, K4 84. S487, A488, T496, F498, L502, N504, K506, P509, F518, N522, E523, L540, S541, I548, D550, F553, V561, K562, E573, A589, L598, D605, L607, N609, N612, E617, D618, D628, R629, R635, K6 37. L651, K652, R654, T657, G658, L666, K673, S675, I679, L680, L683, N690, R691, F693, S701, F704, Q712, G715, Q716, H723, I724, L727, I733, L738, Q739, N803, Q805, Q807, K810, Y812, D8 29. N831, R832, ​​S834, D835, Q844, S845, K848, R859, K862, R864, K866, K890, T893, Q894, D898, N899, K902, K913, K918, Q920, T924, R925, T928, K929, H930, S960, K961, S964, K968, R976, H982, H983, Y1013, K1031, T1033, S1106, K1107, S1109, Y1237, Y1242, K1244 and K1246, wherein the amino acid residue positions correspond to the amino acid numbering in the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence SEQ ID NO: 38 or are defined with reference to the amino acid numbering in the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence SEQ ID NO: 38.

[0018] 2. The gene editing composition of claim 1 , wherein the Cas9 nuclease comprises or is selected from the group consisting of one or more mutations relative to SEQ ID NO. 38: K526X, Q695X, H698X, and R691X, wherein each X is independently selected from the group consisting of glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), serine (S), threonine (T), lysine (K), arginine (R), phenylalanine (F), or tyrosine (Y).

[0019] 3. The gene editing composition according to item 2, wherein each X is independently selected from any one of the following: glycine (G), alanine (A), aspartic acid (D) and glutamic acid (E).

[0020] 4. The gene editing composition according to claim 3, wherein the Cas9 nuclease comprises or is a combination of mutations selected from any one of the following groups relative to the mutation of SEQ ID NO. 38:

[0021] K526A+R691A+Q695A+H698A, K526A+R691A+N692A+Q695A+H698A, K526G+R691G+Q695G+H698G, K526D+R691A+Q695A+H698A, K526A+R69 1D+Q695A+H698A, K526A+R691A+Q695A+H698D, K526E+R691A+Q695A+H698A, K526A+R691E+Q695A+H698A and K526A+R691A+Q695A+H698E.

[0022] 5. A gene editing composition according to any one of items 1 to 4, wherein the Cas9 nuclease or its enzymatically active fragment further comprises a nuclear localization signal located at the N-terminus and / or C-terminus.

[0023] 6. The gene editing composition according to claim 5, wherein:

[0024] The amino acid sequence of the nuclear localization signal is as shown in SEQ ID NO.39 or SEQ ID NO.40, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO.39 or SEQ ID NO.40;

[0025] Optionally, the amino acid sequence of the nuclear localization signal at the N-terminus is as shown in SEQ ID NO.40, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO.40; and / or the amino acid sequence of the nuclear localization signal peptide at the C-terminus is as shown in SEQ ID NO.39, and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO.39.

[0026] 7. A gene editing composition according to any one of items 1 to 6, wherein the amino acid sequence of the Cas9 nuclease is as shown in SEQ ID NO.36, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.36.

[0027] 8. A gene editing composition according to any one of items 1 to 7, wherein the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof comprises a 5' non-coding region sequence (5'UTR) and a 3' non-coding region sequence (3'UTR); optionally, the 5'UTR comprises or is the 5'UTR of the tobacco phagocytic virus (TEV) gene, and / or the 3'UTR comprises or is the 3'UTR of the human hemoglobin alpha 1 (hHBA1) gene.

[0028] 9. The gene editing composition according to claim 8, wherein the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO. 6 or 7, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence as shown in SEQ ID NO. 6 or 7; and / or

[0029] The nucleotide sequence of the 3'UTR is as shown in SEQ ID NO.9, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO.9.

[0030] 10. A gene editing composition according to item 8 or 9, wherein the nucleic acid molecule encoding the Cas9 nuclease further comprises a polyadenosine tail (Poly (A) tail) sequence or a tailing signal sequence, and preferably the Poly (A) tail sequence comprises or is a nucleic acid sequence as shown in SEQ ID NO.8.

[0031] 11. A gene editing composition according to any one of items 8 to 10, wherein the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof is an mRNA molecule.

[0032] 12. The gene editing composition according to item 11, wherein the mRNA molecule comprises a 5' cap structure, and optionally the 5' cap structure is m7G(5')ppp(5')(2'OMeA)pG.

[0033] 13. A gene editing composition according to any one of items 1 to 12, wherein the nucleotide sequence of the nucleic acid molecule encoding the Cas9 nuclease is as shown in SEQ ID NO: 2, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2.

[0034] 14. A gene editing composition according to any one of items 1 to 13, wherein the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof is an mRNA molecule, which contains base-modified uridine (U); optionally, the base-modified U is 1-methylpseudouridine; optionally, each U in the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof is 1-methylpseudouridine.

[0035] 15. A gene editing composition according to any one of items 1-14, wherein the gRNA comprises a spacer sequence, and the spacer sequence is as shown in any one of SEQ ID NO.20-24, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NO.20-24.

[0036] 16. The gene editing composition of claim 15, wherein the gRNA consists of a crRNA and a tracrRNA comprising the spacer sequence; optionally, the nucleotide sequence of the crRNA comprises or is the nucleotide sequence shown in SEQ ID NO.18, and the tracrRNA comprises or is the nucleotide sequence shown in SEQ ID NO.19.

[0037] 17. The gene editing composition of claim 15, wherein the gRNA is a single guide RNA (sgRNA); optionally, the nucleic acid sequence of the sgRNA is as shown in any one of SEQ ID NOs: 10-16, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in any one of SEQ ID NOs: 10-16.

[0038] 18. The gene editing composition of claim 17, wherein the nucleotide sequence of the sgRNA is as shown in SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 16, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 16.

[0039] 19. The gene editing composition according to any one of items 15 to 18, wherein the sgRNA, crRNA and tracrRNA are chemically modified, preferably the chemical modification comprises or is a 2'-O-methylation modification of the 1st to 3rd nucleotides and the last 1 to 3 nucleotides from the 5' end of the sgRNA, crRNA or tracrRNA, and a phosphorothioate modification of the 1st to 3rd internucleotide bonds and the last 1-3 internucleotide bonds from the 5' end.

[0040] 20. A gene editing composition according to any one of items 1 to 19, wherein the mass ratio of the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof to the gRNA or the nucleic acid molecule encoding the gRNA is about 1:4 to about 4:1, for example about 1:1.

[0041] 21. The gene editing composition of any one of items 1 to 20, comprising a lipid nanoparticle (LNP), the lipid component of the LNP comprising or consisting of 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% helper lipids, and 0.5-15 mol% surfactant; and:

[0042] The structural lipid is selected from cholesterol and cholesterol derivatives;

[0043] The cationic lipid is selected from the group consisting of: SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA, preferably SM-102;

[0044] The helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC; and / or

[0045] The surfactant is selected from PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, and is preferably PEG2000-DMG.

[0046] 22. A Cas9 nuclease or an enzymatically active fragment thereof, comprising or comprising one or more mutations selected from the group consisting of K526X, Q695X, H698X, and R691X relative to SEQ ID NO. 38, wherein each X is independently selected from the group consisting of glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), serine (S), threonine (T), lysine (K), arginine (R), phenylalanine (F), or tyrosine (Y), wherein the amino acid residue positions correspond to the amino acid numbering of the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence in SEQ ID NO: 38 or are defined with reference to the amino acid numbering of the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence in SEQ ID NO: 38.

[0047] 23. The Cas9 nuclease or an enzymatically active fragment thereof according to item 22, wherein the Cas9 nuclease comprises or is a mutation selected from any one of the following groups relative to the mutation of SEQ ID NO. 38:

[0048] K526A+R691A+Q695A+H698A, K526A+R691A+N692A+Q695A+H698A, K526G+R691G+Q695G+H698G, K526D+R691A+Q695A+H698A, K526A+R69 1D+Q695A+H698A, K526A+R691A+Q695A+H698D, K526E+R691A+Q695A+H698A, K526A+R691E+Q695A+H698A and K526A+R691A+Q695A+H698E.

[0049] 24. The Cas9 nuclease or enzymatically active fragment thereof according to item 22 or 23, wherein the Cas9 nuclease is SpCas9, and optionally, the amino acid sequence of the Cas9 nuclease is as shown in SEQ ID NO: 36, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence as shown in SEQ ID NO: 36.

[0050] 25. A gRNA targeting the TTR gene, which is an sgRNA, and whose nucleotide sequence is as shown in any one of SEQ ID NOs: 10-16 or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence as shown in any one of SEQ ID NOs: 10-16; or

[0051] It consists of crRNA and tracrRNA, wherein the crRNA is as shown in SEQ ID NO.18 or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence as shown in SEQ ID NO: 18, and the tracrRNA is as shown in SEQ ID NO.19 or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence as shown in SEQ ID NO.19.

[0052] 26. A composition, a ribonucleoprotein complex or a protein lipid complex comprising the Cas9 nuclease or an enzymatically active fragment thereof according to any one of items 22-23, or the gRNA according to item 24.

[0053] 27. An engineered vector encoding the Cas9 nuclease or enzymatically active fragment thereof according to any one of items 21-23 and / or the gRNA according to item 24.

[0054] 28. A method for editing a transthyretin gene in a host cell, comprising introducing into the host cell the gene editing composition of any one of items 1-21, the ribonucleoprotein complex or protein lipid complex of item 26, or the engineered vector of item 27.

[0055] 29. A method for preventing or treating a transthyretin gene-related disease or condition, comprising introducing into a subject the gene editing composition of any one of items 1-21, the ribonucleoprotein complex or protein lipid complex of item 26, or the engineered vector of item 27.

[0056] 30. The method of claim 28 or 29, wherein the TTR-related disease or condition comprises transthyretin amyloidosis.

[0057] 31. The method of claim 30, wherein the TTR-related disease or condition comprises cardiomyopathy and neuropathy resulting from transthyretin amyloidosis.

[0058] 32. Use of the gene editing composition described in any one of items 1 to 21, the ribonucleoprotein complex or protein lipid complex described in item 26, or the engineered vector described in item 27 in the preparation of a drug for editing the transthyretin gene in a host cell.

[0059] 33. Use of the gene editing composition described in any one of items 1 to 20, the ribonucleoprotein complex or protein lipid complex described in item 26, or the engineered vector described in item 27 in the preparation of a drug for preventing or treating transthyretin gene-related diseases or conditions.

[0060] 34. The use of claim 32 or 33, wherein the TTR-related disease or condition comprises transthyretin amyloidosis.

[0061] 35. The use of claim 34, wherein the TTR-related disease or condition comprises cardiomyopathy and neuropathy caused by transthyretin amyloidosis.

[0062] 36. A pharmaceutical composition comprising the gene editing composition of any one of items 1-21, the ribonucleoprotein complex or protein lipid complex of item 26, or the engineered vector of item 27, and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1: Bioanalyzer analysis of the integrity of the mRNA encoding the Cas9 protein.

[0064] Figure 2: Experimental results showing normal translation of Cas9 mRNA in HEK293T cells.

[0065] Figure 3: HPLC-RP analysis of sgRNA purity.

[0066] Figure 4: T7E1 enzyme digestion reaction and ELISA detection of TTR gene editing efficiency and protein knockdown effect of different sgRNAs targeting the TTR gene. Figure A: T7E1 enzyme digestion detection image; Figure B: ELISA detection of TTR protein content in HepG2 cell supernatant.

[0067] Figure 5: GUIDE-seq analysis of off-target rates for sgTTR-0 and sgTTR-1. A shows the number of off-targets detected for sgTTR-0 and sgTTR-1 in combination with SpCas9 mRNA. B shows the distribution of chromosomal cleavage sites for the sgTTR-0 and Cas9 mRNA combinations. C shows the distribution of chromosomal cleavage sites for the sgTTR-1 and Cas9 mRNA combinations.

[0068] Figure 6: T7E1 enzyme digestion reaction to detect the cutting efficiency of sgRNA with different modifications and mutations on the TTR gene.

[0069] Figure 7: Off-target rate (safety) experiment of GUIDE-seq Cas9-mut5. Figure A: Specific off-target site counts for different Cas9 mutants combined with sgTTR-0 as measured by GUIDE-seq. Figure B: Statistical off-target frequency (number of off-target reads / number of on-target reads) for different Cas9 mutants combined with sgTTR-0. Figure C: Knockdown effect of sgTTR-0+SpCas9-mut5 on TTR protein in humanized mouse serum at different dosages.

[0070] Figure 8: Gene editing effects of different SpCas9 mutants combined with sgTTR-0. Panel A: ELISA assay for TTR protein levels in mouse serum one week after dosing (1 mg / kg); Panel B: Amplicon sequencing assay for TTR gene editing efficiency in mouse liver one month after dosing (1 mg / kg); Panel C: GUIDE-seq assay for off-target sites and their frequencies for Cas9-WT+sgTTR-0 and Cas9-mut5+sgTTR-0.

[0071] Figure 9: Gene editing effects of sgTTR-0 and SpCas9 mRNA using different LNP delivery formulations. Figure A: TTR protein levels in mouse serum measured by ELISA one week after dosing (0.3 mg / kg); Figure B: TTR gene editing effects in the liver measured one month after dosing (0.3 mg / kg); Figure C: TTR protein levels in mouse serum measured by ELISA one week after dosing (1 mg / kg) using different delivery formulations.

[0072] Figure 10: Gene editing effects of different mass ratios of sgTTR-0 and SpCas9 mRNA. Panel A: ELISA assay for TTR protein levels in mouse serum one week after dosing; Panel B: Amplicon sequencing assay for TTR gene editing efficiency in mouse liver one month after dosing.

[0073] Figure 11: GUIDE-seq analysis of safety experiments at different sgRNA to mRNA mass ratios. Figure A: GUIDE-seq analysis of the number of off-target sites at different sgRNA to mRNA mass ratios; Figure B: Statistical analysis of off-target frequencies (number of off-target reads / number of on-target reads) at different sgRNA to mRNA mass ratios.

[0074] Figure 12: Editing efficiency of different Cas9 mutants at the TTR locus in HEK293T cells and HepG2 cells. Figure A: Editing efficiency of different Cas9 mutants at the TTR locus in HEK293T cells; Figure B: Editing efficiency of different Cas9 mutants at the TTR locus in HepG2 cells; Figure C: TTR protein knockdown efficiency of different Cas9 mutants in HepG2 cells.

[0075] Figure 13: Editing efficiency of Cas9 and its mutants at high-frequency off-target sites (i.e., off-target rate). Figure A: Editing efficiency of wild-type Cas9 and different Cas9 mutants at off-target site 1 (9216122); Figure B: Editing efficiency of wild-type Cas9 and different Cas9 mutants at off-target site 2 (192658579); Figure C: Editing efficiency of wild-type Cas9 and different Cas9 mutants at off-target site 3 (168377982); Figure D: Editing efficiency of wild-type Cas9 and different Cas9 mutants at off-target site 4 (5671351). DETAILED DESCRIPTION

[0076] definition:

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0078] As used herein, "coding sequence" may refer to a ribonucleotide sequence in a mature mRNA that can be translated into a protein, or may refer to the complementary sequence of a deoxyribonucleotide (DNA) sequence that serves as a template for transcribing the ribonucleotide (RNA) sequence. Furthermore, the "coding sequence" of the present application may further include polynucleotide sequences encoding functional nucleic acids, such as miRNA, shRNA, dsRNA, and the like.

[0079] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0080] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. It will also be understood that the skilled artisan can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecule to reflect the codon usage of any particular host organism in which the polypeptide is expressed. Therefore, unless otherwise indicated, "polynucleotides encoding the protein or immunogenic fragment of the present application" include all polynucleotide sequences that are degenerate to each other and encode the same amino acid sequence.

[0081] The term "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA and an RNA-guided DNA binder, such as a Cas nuclease, e.g., a Cas cleavage enzyme, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binder, such as Cas9, to a target sequence, and the guide RNA hybridizes to the target sequence and the agent binds to the target sequence; where the agent is a cleavage enzyme or nickase, binding may be followed by cleavage or nicking.

[0082] The term "TTR", or transthyretin, refers to transthyretin, the gene product of the TTR gene, also known as vitamin A-binding protein. It is an important component of plasma proteins and is widely distributed in various cells, plasma, and tissue fluids. As a carrier protein, TTR is mainly synthesized in the liver and the choroid plexus in the brain, secreted into the blood and cerebrospinal fluid, and carries thyroxine and retinol (vitamin A) to various tissues and cells throughout the body. As a carrier protein, the function of TTR can often be replaced by thyroxine-binding globulin and albumin in plasma. Under physiological conditions, TTR is a stable protein. When TTR breaks down into monomers, it can cause amyloidosis. In recent years, more and more TTR-targeted drugs are gradually entering the clinic for use in neurological diseases, endocrine and metabolic diseases, and so on.

[0083] The terms "nuclear localization signal," "NLS," "nuclear localization signal peptide," or "nuclear localization sequence" refer to an amino acid sequence or peptide that induces transport of a molecule comprising or linked to such a sequence into the nucleus of a eukaryotic cell. The nuclear localization signal can form part of the molecule to be transported. In some embodiments, the NLS can be attached to the molecule via a covalent bond, a hydrogen bond, or an ionic interaction.

[0084] As used herein, "mRNA" (messenger RNA) is any RNA of a naturally occurring, non-naturally occurring, or modified amino acid polymer that encodes at least one protein and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. It will be appreciated by those skilled in the art that, unless otherwise indicated, the polynucleotide sequences described herein may use "T" to refer to thymine when representing a DNA sequence, but when the polynucleotide sequence represents RNA (e.g., mRNA), the "T" will be replaced by "U" (uracil). Thus, any DNA disclosed and identified by a particular sequence number (SEQ ID NO) herein also discloses an RNA (e.g., mRNA) sequence that is complementary or corresponding to the DNA, wherein each "T" of the DNA sequence is replaced by a "U."

[0085] In some embodiments, the sugar of the nucleic acid phosphate-sugar backbone consists essentially of ribose residues, 2'-methoxyribose residues, or a combination thereof. Generally, 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%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). mRNA may contain modified uridines at some or all of its uridine positions.

[0086] Nucleic Acids

[0087] The term "nucleic acid" or "nucleic acid molecule" will be recognized and understood by those of ordinary skill in the art. As used herein, the term "nucleic acid" or "nucleic acid molecule" preferably refers to a DNA (molecule) or RNA (molecule). It is preferably used synonymously with the term polynucleotide. Preferably, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other via phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid molecule" also includes modified nucleic acid molecules, such as base-modified, sugar-modified, or backbone-modified DNA or RNA molecules as defined herein.

[0088] Unless otherwise specified, "nucleotide" herein refers not only to naturally occurring ribonucleotides or deoxyribonucleotide monomers, but also to their related structural variants, including derivatives and analogs, which are functionally equivalent in the specific context in which the nucleotide is used, unless the context clearly indicates otherwise. For example, "nucleotide" refers to a deoxyribonucleotide or a ribonucleotide. Nucleotides can be standard nucleotides (i.e., adenosine (A), guanosine (G), cytidine (C), thymidine (T) and uridine (U), nucleotide isomers or nucleotide analogs, for example, U or T in the nucleotide sequence used to represent mRNA can represent natural uridine and pseudouridine, such as 1-methylpseudouridine. Nucleotide analogs refer to nucleotides with modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications on the sugar or base portion of the nucleotide include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl and thiol groups, and substitution of the carbon and nitrogen atoms of the base with other atoms (e.g., 7-deazapurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino oligonucleotides.

[0089] In some embodiments, the nucleic acid comprises at least one heterologous untranslated region (UTR). The term "untranslated region" or "UTR" or "UTR element" will be recognized and understood by those of ordinary skill in the art to mean a portion of a nucleic acid molecule, typically located 5' or 3' to a coding sequence. The 5' end is referred to as a 5'UTR, and the 3' end is referred to as a 3'UTR. Generally speaking, UTRs are not translated into proteins; UTRs can be part of a nucleic acid, such as DNA or RNA. UTRs can comprise elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be ribosome binding sites, miRNA binding sites, etc.; RNA (e.g., mRNA) can further comprise a 5'UTR, a 3'UTR, a 3'-poly(A) and / or a 5' cap analog.

[0090] In some embodiments, the 5'UTR is a heterologous UTR, i.e., a UTR found in nature that is associated with a different ORF; in another embodiment, the 5'UTR is a synthetic UTR; the 5'UTR is a region of the mRNA that is located upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 5'UTR does not encode a protein. The natural 5'UTR has characteristics that play a role in translation initiation, such as the Kozak sequence, which has a consensus CCR(A / G)CCAUGG; exemplary 5'UTRs also include tobacco etch virus, African clawed frog or human α-globin or β-globin, human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, and alpha-1-globin 5'UTR, etc.

[0091] In some embodiments, the 3'UTR can be heterologous or synthetic; for example: HBA1 (human Hemoglobin Subunit Alpha 1), globin UTR, including African clawed frog β-globin UTR and human β-globin UTR; other 3'UTRs can also be CYBA (cytochrome b-245alpha chain), rabbit β-globin, hepatitis B virus (HBV), α-globin 3'UTR and VEEV (Venezuelan equine encephalitis virus) virus 3'UTR sequences. In some embodiments, rps9 (Ribosomal Protein S9) 3'UTR, FIG4 (FIG4 Phosphoinositide 5-Phosphatase), gp130, DH143 and human albumin hHBB (human hemoglobin subunit beta) 3'UTR can also be used.

[0092] In some embodiments, the 3'-poly (A) tail is also called a poly (A) tail; the poly (A) tail is located downstream of the 3' UTR, for example, the mRNA region immediately downstream (i.e., 3'), which contains multiple consecutive adenosine monophosphates. The poly (A) tail may contain 10 to 300 adenosine monophosphates, and may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some preferred embodiments, the poly (A) tail contains 50 to 250 adenosine monophosphates, more preferably 50-100 adenosine monophosphates; most preferably 100 adenosine monophosphates; in relevant biological environments (e.g., in cells, in vivo), the function of the 3'-poly (A) tail is to protect the mRNA from enzymatic degradation, for example in the cytoplasm, and to facilitate transcription termination and / or export of the mRNA from the nucleus and translation.

[0093] In some embodiments, the RNA (e.g., mRNA) further comprises a 5' guanosine cap; the 5' guanosine cap is a eukaryotic mRNA transcript, and the 5' cap is composed of an inverted 7-methylguanosine, connected to the rest of the eukaryotic mRNA via a 5'-5' triphosphate bridge, namely cap 0 (cap 0), which primarily serves as a quality control for correct mRNA processing and helps stabilize the eukaryotic mRNA; based on cap 0, the first nucleotide is methylated with 2'-OH, referred to as cap 1 (cap 1); in addition to cap 0 and cap 1, the second nucleotide can also be further methylated, referred to as cap 2; generally speaking, the 5'-cap can be synthesized by different synthetic routes of 5'-capped mRNA based on enzymatic, chemical, or chemoenzymatic methods;

[0094] In some embodiments, during in vitro transcription, a cap analog is directly added to the in vitro transcription (IVT) system, and the 5' cap analog includes but is not limited to: m 7 Gppp(2'OMeA)pG、m 7 GpppApA、m 7 GpppApC、m 7 GpppApG、m 7 GpppApU、m 7 GpppCpA、m 7 GpppCpC、m 7 GpppCpG、m 7 GpppCpU、m 7 GpppGpA、m 7 GpppGpC、m7 GpppGpG、m 7 GpppGpU、m 7 GpppUpA、m 7 GpppUpC、m 7 GpppUpG、m 7 GpppUpU、m 7 Gpppm 6 ApG, m 7 G 3’Ome pppApA、m 7 G 3’Ome pppApC、m 7 G 3’Ome pppApU、m 7 G 3’Ome pppApG、m 7 G 3’Ome pppCpA、m 7 G 3’Ome pppCpC、m 7 G 3’Ome pppCpG、m 7 G 3’Ome pppCpU、m 7 G 3’Ome pppUpA、m 7 G 3’Ome pppUpC、m 7 G 3’Ome pppUpG、m 7 G 3’Ome pppUpU、m 7 G 3’Ome pppA 2’Ome pG、m 7 G 3’Ome pppA 2’Ome pC、m 7 G 3’Ome pppA 2’Ome pU、m 7 G 3’Ome pppA 2’Ome pA、m 7 G 3’Ome pppC 2’Ome pA、m 7 G 3’Ome pppC 2’Ome pU、m 7 G 3’Ome pppC 2’Ome pG、m 7 G 3’Ome pppC 2’Ome pC、m 7 G 3’Ome ppppG2’Ome pA, m 7 G 3’Ome pppG 2’Ome pU、m 7 G 3’Ome pppG 2’Ome pG、m 7 G 3’Ome pppG 2’Ome pC、m 7 G 3’Ome pppU 2’Ome pA, m 7 G 3’Ome pppU 2’Ome pU、m 7 G 3’Ome pppU 2’Ome pG、m 7 G 3’Ome pppU 2’Ome pC etc.

[0095] In some embodiments, the capped analogs may also be other structures, such as tetramers, pentamers, hexamers, heptamers, octamers, nonamers, or decamers, etc. The specific sequence thereof may be determined according to the template.

[0096] Messenger RNA (mRNA) is any RNA that encodes (at least one) protein (a naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein. The skilled artisan will understand that, unless otherwise indicated, the nucleic acid sequences listed in this application may reference "T" in the representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" will be replaced with a "U." Therefore, any DNA disclosed and identified herein by a particular sequence identification number also discloses the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each "T" of the DNA sequence is replaced by a "U."

[0097] open reading frame

[0098] An open reading frame (ORF) is a continuous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). Generally speaking, an ORF typically encodes a protein. It will be understood that the sequences disclosed herein may also contain additional elements, such as 5' and 3' UTRs, but unlike ORFs, these elements are not necessarily present in the RNA polynucleotides of the present application.

[0099] In some embodiments, the composition comprises RNA (e.g., mRNA) comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the nucleotide sequence of SEQ ID NO: 2.

[0100] In some embodiments, the open reading frame is preferably at least partially codon optimized. Codon optimization is based on such discovery: translation efficiency can be determined by the different frequencies of transfer RNA (tRNA) occurring in the cell. Therefore, if there is so-called "rare codon" of increasing degree in the coding region of the nucleic acid of the application defined herein, the translation of the corresponding modified nucleotide sequence is less efficient than when there is a codon encoding relatively "common" tRNA. Those skilled in the art can carry out codon optimization for sequence to be translated with the characteristics of its in vitro expression system.

[0101] Chemically modified or unmodified nucleotides

[0102] In some embodiments, RNA (e.g., mRNA) is not chemically modified, but rather comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides disclosed herein comprise standard nucleoside residues, such as those present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, the nucleotides and nucleosides disclosed herein include standard deoxyribonucleosides, such as those present in DNA (e.g., dA, dG, dC, or dT);

[0103] In some embodiments, the composition of the present application comprises an RNA having an open reading frame encoding a modified Cas9 protein, such as a modified SpCas9 protein, wherein the nucleic acid comprises standard (unmodified) or modified nucleotides and / or nucleosides known in the art. In some embodiments, the nucleotides and nucleosides of the present application include modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include modifications of the sugar, backbone or core base moiety of nucleotides and / or nucleosides known in the art.

[0104] In some embodiments, the modified nucleic acid base in the nucleic acid (e.g., RNA nucleic acid, e.g., mRNA nucleic acid) includes 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methoxy-uridine, 5-methyl-cytidine and / or pseudouridine, pseudouridine.

[0105] In vitro transcription system (IVT)

[0106] In vitro transcription is the process of using DNA as a template in an in vitro cell-free system containing components such as RNA polymerase and NTP to mimic the in vivo transcription process to generate mRNA. Generally speaking, the capped RNA synthesized in the in vitro transcription reaction can be used for subsequent experiments such as microinjection, in vitro translation, and transfection. The in vitro transcription system usually includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. NTPs can be synthesized by oneself or selected from a supplier. NTPs can be natural or non-natural NTPs. Optional polymerases include, but are not limited to, phage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or polymerase mutants thereof, such as, but not limited to, polymerases capable of incorporating modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides. Some embodiments exclude the use of DNA enzymes. In some embodiments, the RNA contains a 5' guanosine cap.

[0107] In addition to synthesis by in vitro transcription systems, chemical synthesis methods can also be used, including solid-phase chemical synthesis and liquid-phase chemical synthesis; with respect to solid-phase chemical synthesis, the nucleic acids disclosed in this application can be prepared in whole or in part using solid-phase technology; solid-phase chemical synthesis of nucleic acids is an automated method in which molecules are fixed on a solid support and synthesized stepwise in a reactant solution. Solid-phase synthesis can be used for site-specific introduction of chemical modifications in nucleic acid sequences; with respect to liquid-phase chemical synthesis, the nucleic acids of this application can be synthesized in liquid phase by sequentially adding monomer constructs. In addition, the above-mentioned synthesis methods can also be used in combination, because the synthesis methods discussed above each have their own advantages and limitations, and attempts can be made to combine these methods to overcome the above-mentioned limitations. Combinations of these methods are within the scope of this application.

[0108] The term "identity" refers to the relationship between the sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids) determined by comparing sequences. Identity also refers to the degree of sequence relatedness between or among sequences determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, where gap comparisons (if any) are solved by a specific mathematical model or computer program (e.g., an "algorithm"). The identities of the related antigens or nucleic acids can be easily calculated by known methods. "Percentage (%) identity" for polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid sequence or the nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to obtain maximum percentage identity. Methods and computer programs for comparison are well known in the art. It is understood that identity depends on the calculation of percentage identity, but its value may vary due to gaps and penalties introduced in the calculation. Typically, variants of a particular polynucleotide or polypeptide have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a particular reference polynucleotide or polypeptide as determined by the sequence alignment programs and parameters described herein and known to those of skill in the art.

[0109] Lipid nanoparticles (LNPs)

[0110] The RNA of the present application (for example, mRNA) is formulated in lipid nanoparticle (LNP).Lipid nanoparticle generally comprises ionizable cationic lipid, helper lipid, cholesterol and PEG lipid component and nucleic acid of interest.Lipid nanoparticle of the present application can use component generally known in the art, and composition and method generate.

[0111] pharmaceutical preparations

[0112] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for preventing or treating, for example, gene modification or editing in humans and other mammals. The term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, such that the composition is particularly suitable for in vivo or in vitro diagnostic or therapeutic use. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects after administration to a subject or after administration to a subject. The carrier in the pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient and can stabilize it. One or more solubilizing agents can be used as a pharmaceutical carrier for delivering the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible carriers, adjuvants, additives, and diluents to obtain a composition that can be used as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Other suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for them, are described in Remington's Pharmaceutical Sciences.

[0113] sgRNA

[0114] In each of the embodiments of the compositions, uses, and methods described herein, the guide RNA may comprise a single RNA molecule as a "single guide RNA" or "sgRNA". The sgRNA may comprise a crRNA or portion thereof containing a guide sequence covalently linked to the tracrRNA. In some embodiments, the crRNA and tracrRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure through base pairing between the crRNA and the respective portions of the tracrRNA. In some embodiments, the crRNA and tracrRNA are covalently linked via one or more bonds that are not phosphodiester bonds. In some embodiments, approximately 20 bases at the 5' end of the sgRNA are complementary to the genome, and bases from 21-100 nt are interacting sequences with Cas9.

[0115] In a first aspect, the present application relates to a gene editing composition comprising

[0116] 1) Cas9 nuclease or an enzymatically active fragment thereof, or a nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof; and

[0117] 2) a guide RNA (gRNA) targeting the transthyretin (TTR) gene or a nucleic acid molecule encoding the gRNA, wherein the Cas9 nuclease comprises a mutation at one or more amino acid residue positions selected from the group consisting of K526, N692, Q695, H698, N497, Y450, Q926, K377, E387, D397, R400, D406, A421, L423, R424, Q426, Y430, K442, P449, V452, A456, R457, W464, M465, K466 8. E470, T474, P475, W476, F478, K484, S487, A488, T496, F498, L502, N504, K506, P509, F518, N522, E523, L540, S541, I54 8. D550, F553, V561, K562, E573, A589, L598, D605, L607, N609, N612, E617, D618, D628, R629, R635, K637, L651, K652, R654 , T657, G658, L666, K673, S675, I679, L680, L683, N690, R691, F693, S701, F704, Q712, G715, Q716, H723, I724, L727, I733 , L738, Q739, N803, Q805, Q807, K810, Y812, D829, N831, R832, ​​S834, D835, Q844, S845, K848, R859, K862, R864, K866, K890, T893, Q894, D898, N899, K902, K913, K918, Q920, T924, R925, T928, K929, H930, S960, K961, S964, K968, R976, H982, H983, Y1013, K1031, T1033, S1106, K1107, S1109, Y1237, Y1242, K1244 and K1246, wherein the amino acid residue positions correspond to the amino acid numbering in the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence SEQ ID NO: 38 or are defined with reference to the amino acid numbering in the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence SEQ ID NO: 38.

[0118] In some embodiments, the Cas9 nuclease comprises or is selected from the group consisting of one or more mutations relative to SEQ ID NO. 38: K526X, Q695X, H698X, and R691X, wherein each X is independently selected from the group consisting of glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), serine (S), threonine (T), lysine (K), arginine (R), phenylalanine (F), or tyrosine (Y).

[0119] In some embodiments, each X is independently selected from any one of the following: glycine (G), alanine (A), aspartic acid (D), and glutamic acid (E).

[0120] In some embodiments, the Cas9 nuclease includes or is a combination of mutations relative to SEQ ID NO. 38 selected from any one of the following groups:

[0121] K526A+R691A+Q695A+H698A, K526A+R691A+N692A+Q695A+H698A, K526G+R691G+Q695G+H698G, K526D+R691A+Q695A+H698A, K526A+R69 1D+Q695A+H698A, K526A+R691A+Q695A+H698D, K526E+R691A+Q695A+H698A, K526A+R691E+Q695A+H698A and K526A+R691A+Q695A+H698E.

[0122] In some embodiments, the Cas9 nuclease includes or is a combination of mutations relative to wild-type SpCas9 selected from any one of the following groups:

[0123] K526A+R691A+Q695A+H698A, K526A+R691A+N692A+Q695A+H698A, K526G+R691G+Q695G+H698G, K526D+R691A+Q695A+H698A, K526A+R69 1D+Q695A+H698A, K526A+R691A+Q695A+H698D, K526E+R691A+Q695A+H698A, K526A+R691E+Q695A+H698A and K526A+R691A+Q695A+H698E.

[0124] In some embodiments, the Cas9 nuclease or enzymatically active fragment thereof further comprises a nuclear localization signal at its N-terminal side and / or C-terminal side.

[0125] In some embodiments, the amino acid sequence of the nuclear localization signal is as shown in SEQ ID NO.39 or SEQ ID NO.40, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.39 or SEQ ID NO.40.

[0126] In some embodiments, the amino acid sequence of the nuclear localization signal of the Cas9 nuclease or its enzymatically active fragment at its N-terminus is as shown in SEQ ID NO.40, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO.40; and / or the amino acid sequence of the nuclear localization signal of the nuclear localization signal peptide at its C-terminus is as shown in SEQ ID NO.39, and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO.39.

[0127] In some embodiments, the amino acid sequence of the Cas9 nuclease is as shown in SEQ ID NO.36, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO.36.

[0128] In some embodiments, the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof comprises a 5' non-coding region sequence (5'UTR) and a 3' non-coding region sequence (3'UTR);

[0129] In some embodiments, the 3'UTR can also be selected from the 3'UTR of PSMB3, ALB7, α-globin, CASP1, COX6B1, GNAS, NDUFA1, DH143, gp130, hHBB, hHBA1, CYBA (cytochrome b-245alpha chain), rabbit β-globin, hepatitis B virus (HBV), VEEV (Venezuelan equine encephalitis virus) virus, rps9 (ribosomal protein S9), FIG4 (FIG4 phosphoinositide 5-phosphatase), human albumin hHBB (human hemoglobin β subunit), HBA1 (human hemoglobin α1 subunit), or a homolog, fragment or variant from any of these genes.

[0130] In some embodiments, the 5'UTR is selected from the 5'UTR of tobacco etch virus, African clawed frog or human α-globin or β-globin, human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, alpha-1-globin, HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or a homolog, fragment or variant from any of these genes.

[0131] Optionally, the 5'UTR comprises or is the 5'UTR of a tobacco epithelium virus (TEV) gene, and / or the 3'UTR comprises or is the 3'UTR of a human hemoglobin alpha 1 (hHBA1) gene.

[0132] In some embodiments, the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO. 6 or 7, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO. 6 or 7.

[0133] In some embodiments, the nucleotide sequence of the 3'UTR is as shown in SEQ ID NO.9, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO.9.

[0134] In some embodiments, the nucleic acid molecule encoding the Cas9 nuclease further comprises a polyadenosine tail (Poly (A) tail) sequence or a tailing signal sequence, and preferably the Poly (A) tail sequence comprises or is a nucleic acid sequence as shown in SEQ ID NO.8.

[0135] In some embodiments, the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof is an mRNA molecule.

[0136] In some embodiments, the mRNA further comprises a 5' guanosine cap selected from the group consisting of: m7Gppp (2'OMeA) pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7Gppp GpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GpppUpC, m7GpppUpG, m7GpppUpU, m7Gpppm6ApG, m7G 3’Ome pppApA、m7G 3’Ome pppApC、m7G 3’Ome pppApU、m7G 3’Ome pppApG、m7G 3’Ome pppCpA、m7G 3’Ome pppCpC、m7G 3’Ome pppCpG、m7G 3’Ome pppCpU、m7G 3’Ome pppUpA、m7G 3’Ome pppUpC、m7G 3’Ome pppUpG、m7G 3’Ome pppUpU、m7G 3’Ome pppA 2’Ome pG、m7G 3’Ome pppA 2’Ome pC、m7G 3’Ome pppA 2’Ome pU、m7G 3’Ome pppA 2’Ome pA、m7G 3’Ome ppC 2’Ome pA、m7G 3’Ome ppC 2’Ome pU、m7G 3’Ome ppC 2’Ome pG、m7G 3’Ome ppC 2’Ome pC、m7G 3’Ome pppG2’Ome pA、m7G 3’Ome pppG 2’Ome pU、m7G 3’Ome pppG 2’Ome pG、m7G 3’Ome pppG 2’Ome pC、m7G 3’Ome pppU 2’Ome pA、m7G 3’Ome pppU 2’Ome pU、m7G 3’Ome pppU 2’Ome pG、m7G 3’Ome pppU 2’Ome pC.

[0137] In some embodiments, the mRNA comprises chemically modified bases or analogs, including 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP), and pseudouridine; preferably, 1-methyl pseudouridine partially replaces uridine; and most preferably, 1-methyl pseudouridine completely replaces uracil, so that each uracil in the sequence is replaced by 1-methyl pseudouridine.

[0138] In some embodiments, the mRNA molecule comprises a 5' cap structure, optionally wherein the 5' cap structure is m7G(5')ppp(5')(2'OMeA)pG.

[0139] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the Cas9 nuclease is as shown in SEQ ID NO:2, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO:2.

[0140] In some embodiments, the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof is an mRNA molecule comprising base-modified uridine (U); optionally, the base-modified U is 1-methylpseudouridine; optionally, each U in the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof is 1-methylpseudouridine.

[0141] In some embodiments, the gRNA comprises a spacer sequence, which is as shown in any one of SEQ ID NO.20-24, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NO.20-24.

[0142] In some embodiments, the gRNA consists of a crRNA and a tracrRNA comprising the spacer sequence; optionally, the nucleotide sequence of the crRNA comprises or is the nucleotide sequence shown in SEQ ID NO.18, and the tracrRNA comprises or is the nucleotide sequence shown in SEQ ID NO.19.

[0143] In some embodiments, the gRNA is a single guide RNA (sgRNA); optionally, the spacer nucleic acid sequence of the sgRNA is as shown in any one of SEQ ID NOs: 20-24, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in any one of SEQ ID NOs: 20-24.

[0144] In some embodiments, the sgRNA has a nucleotide sequence as shown in SEQ ID NO: 10, SEQ ID NO: 15, or SEQ ID NO: 16, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence as shown in SEQ ID NO: 10, SEQ ID NO: 15, or SEQ ID NO: 16.

[0145] In some embodiments, the sgRNA, crRNA, and tracrRNA are chemically modified, preferably the chemical modification comprises or is 2'-O-methylation of the first to third nucleotides and the last 1 to 3 nucleotides from the 5' end of the sgRNA, crRNA, or tracrRNA, and phosphorothioate modification of the first to third internucleotide linkages and the last 1-3 internucleotide linkages from the 5' end.

[0146] In some embodiments, the mass ratio of the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof to the gRNA or the nucleic acid molecule encoding the gRNA is about 1:4 to about 4:1, for example about 1:1.

[0147] In some embodiments, the gene editing composition of the present application comprises lipid nanoparticles (LNPs), the lipid component of the LNP comprising 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% auxiliary lipids and 0.5-15 mol% surfactants, or consisting of 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% auxiliary lipids and 0.5-15 mol% surfactants.

[0148] In some embodiments, the structured lipid is selected from cholesterol and cholesterol derivatives.

[0149] In some embodiments, the cationic lipid is selected from the group consisting of: SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, DlinDMA, preferably SM-102.

[0150] The structure of SM-102 is as follows:

[0151] In some embodiments, the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC.

[0152] In some embodiments, the surfactant is selected from PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, preferably PEG2000-DMG.

[0153] In some embodiments, the lipid nanoparticles of the present application comprise an N:P ratio of about 2:1 to about 30:1.

[0154] In some embodiments, the lipid nanoparticles of the present application comprise an N:P ratio of about 6:1.

[0155] In some embodiments, the lipid nanoparticles of the present application comprise an N:P ratio of about 3:1.

[0156] In some embodiments, the lipid nanoparticles of the present application comprise a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 10:1 to about 100:1.

[0157] In some embodiments, the lipid nanoparticles of the present application comprise a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 20:1.

[0158] In some embodiments, the lipid nanoparticles of the present application comprise a wt / wt ratio of an ionizable cationic lipid component to RNA of about 10: 1. In some embodiments, the lipid nanoparticles of the present application have an average diameter of from about 50 nm to about 150 nm.

[0159] In some embodiments, the lipid nanoparticles of the present application have an average diameter of about 70 nm to about 120 nm, preferably 100-120 nm, and most preferably 100 nm.

[0160] In some embodiments, the present application relates to an LNP composition comprising Cas nuclease mRNA and at least one sgRNA.

[0161] In some embodiments, the LNP composition comprises a ratio of sgRNA to Cas nuclease mRNA of about 25:1 to about 1:25.

[0162] In some embodiments, the LNP composition comprises a ratio of sgRNA to Cas nuclease mRNA of about 10:1 to about 1:10.

[0163] In some embodiments, the LNP composition comprises a ratio of sgRNA to Cas nuclease mRNA of about 8: 1 to about 1 : 8. The ratio is by weight.

[0164] In some embodiments, the LNP composition comprises a ratio of sgRNA to Cas nuclease mRNA of about 5:1 to about 1:5.

[0165] In some embodiments, the ratio ranges from about 3:1 to 1:3, about 2:1 to 1:2, about 5:1 to 1:2, about 5:1 to 1:1, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1:1.

[0166] In some embodiments, the sgRNA to mRNA ratio is about 3: 1 or about 2: 1; in some embodiments, the sgRNA to Cas nuclease mRNA ratio is about 1: 1. The ratio can be about 25: 1, 10: 1, 5: 1, 3: 1, 1: 1, 1:3, 1:5, 1: 10, or 1:25.

[0167] In some embodiments, the Cas nuclease is preferably a class II Cas nuclease, including Cas9, Cpf1, C2c1, C2c2, C2c3, HFCas9, HypaCas9, eSPCas9 proteins and modified forms thereof, more preferably Cas9 nuclease.

[0168] In some embodiments, the Cas9 nuclease comprises Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogenes, Rhodospirillum rubrum), Nocardiopsis dassonvillei.

[0169] In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is a Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is a Cas9 nuclease from Staphylococcus aureus.

[0170] In some embodiments, the LNP composition comprises: sgRNA, mRNA encoding Cas9, and lipid nanoparticles (LNP);

[0171] In some embodiments, the ratio of the sgRNA to the Cas9 mRNA is 1:1 (mass ratio), and the lipid nanoparticles (LNP) contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.

[0172] In some embodiments, the present application relates to a composition comprising gRNA and Cas9mRNA, wherein the spacer sequence of the sgRNA comprises SEQ ID NO: 20 or is shown as SEQ ID NO. 20, and the amino acid sequence of the Cas9 nuclease is shown as SEQ ID NO: 36.

[0173] In some embodiments, the present application relates to a composition comprising gRNA and Cas9 nuclease, wherein the spacer sequence of the sgRNA comprises SEQ ID NO: 20 or is shown as SEQ ID NO. 20, and the amino acid sequence of the Cas9 nuclease is shown as SEQ ID NO: 36.

[0174] In some embodiments, the present application relates to a composition comprising gRNA and Cas9mRNA, wherein the spacer sequence of the sgRNA comprises SEQ ID NO: 20 or is shown as SEQ ID NO. 20, and the Cas9mRNA sequence is shown as SEQ ID NO: 2.

[0175] In some embodiments, the present application relates to a composition comprising gRNA, Cas9 mRNA and lipid nanoparticles (LNP), wherein the spacer sequence of the sgRNA comprises SEQ ID NO: 20, the amino acid sequence of the Cas9 nuclease is shown in SEQ ID NO: 36, and the lipid nanoparticles (LNP) contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG, and the ratio of the sgRNA to the encoding Cas9 mRNA is 1: 1 (mass ratio).

[0176] In some embodiments, the present application relates to a composition comprising gRNA, Cas9mRNA and lipid nanoparticles (LNP), wherein the spacer sequence of the sgRNA comprises SEQ ID NO: 20 or as shown in SEQ ID NO. 20, the Cas9mRNA sequence is as shown in SEQ ID NO: 2, and the lipid nanoparticles (LNP) contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG, and the ratio of the sgRNA to the encoding Cas9mRNA is 1: 1 (mass ratio).

[0177] In some embodiments, the present application relates to a composition comprising gRNA and Cas9 nuclease, wherein:

[0178] The gRNA is an sgRNA, whose nucleotide sequence is shown in SEQ ID NO: 10, 15 or 16, or the gRNA is composed of crRNA and tracrRNA, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO.18, and the nucleotide sequence of the tracrRNA is shown in SEQ ID NO.19; the amino acid sequence of the Cas9 nuclease is shown in SEQ ID NO: 36.

[0179] In some embodiments, the present application relates to a composition comprising gRNA and Cas9 mRNA, wherein:

[0180] The gRNA is an sgRNA, whose nucleotide sequence is shown in SEQ ID NO: 10, 15 or 16, or the gRNA is composed of crRNA and tracrRNA, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO.18, and the nucleotide sequence of the tracrRNA is shown in SEQ ID NO.19; the amino acid sequence of the Cas9 nuclease is shown in SEQ ID NO: 36.

[0181] In some embodiments, the present application relates to a composition comprising gRNA and Cas9 mRNA, wherein:

[0182] The gRNA is an sgRNA, whose nucleotide sequence is shown in SEQ ID NO: 10, 15 or 16, or the gRNA is composed of crRNA and tracrRNA, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO.18, and the nucleotide sequence of the tracrRNA is shown in SEQ ID NO.19; the Cas9 mRNA sequence is shown in SEQ ID NO: 2.

[0183] In some embodiments, the present application relates to a composition comprising gRNA, Cas9 mRNA, and lipid nanoparticles (LNPs), wherein:

[0184] The gRNA is an sgRNA, the nucleotide sequence of which is shown in SEQ ID NO: 10, 15 or 16, or the gRNA consists of a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO. 18, and the nucleotide sequence of the tracrRNA is shown in SEQ ID NO. 19;

[0185] The amino acid sequence of the Cas9 nuclease is shown in SEQ ID NO: 36, the lipid nanoparticles (LNP) contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG, and the ratio of the sgRNA to the encoding Cas9 mRNA is 1:1 (mass ratio).

[0186] In some embodiments, the present application relates to a composition comprising gRNA, Cas9 mRNA, and lipid nanoparticles (LNPs), wherein:

[0187] The gRNA is an sgRNA, the nucleotide sequence of which is shown in SEQ ID NO: 10, 15 or 16, or the gRNA consists of a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO. 18, and the nucleotide sequence of the tracrRNA is shown in SEQ ID NO. 19;

[0188] The Cas9 mRNA sequence is shown in SEQ ID NO: 2, the lipid nanoparticles (LNP) contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG, and the ratio of the sgRNA to the encoding Cas9 mRNA is 1:1 (mass ratio).

[0189] In some embodiments, the present application relates to a composition comprising gRNA, Cas9 mRNA, and lipid nanoparticles (LNPs), wherein:

[0190] The gRNA is an sgRNA, the nucleotide sequence of which is shown in SEQ ID NO: 10, 15 or 16, or the gRNA consists of a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO. 18, the nucleotide sequence of the tracrRNA is shown in SEQ ID NO. 19, and the sgRNA, crRNA and tracrRNA have the following modifications: the first to third nucleotides and the last 1 to 3 nucleotides from the 5' end are modified by 2'-O-methylation, and the first to third internucleotide bonds and the last 1-3 internucleotide bonds from the 5' end are phosphorothioate modified;

[0191] The amino acid sequence of the Cas9 nuclease is shown in SEQ ID NO: 36, the lipid nanoparticles (LNP) contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG, and the ratio of the sgRNA to the encoding Cas9 mRNA is 1:1 (mass ratio).

[0192] In some embodiments, the present application relates to a composition comprising gRNA, Cas9 mRNA, and lipid nanoparticles (LNPs), wherein:

[0193] The gRNA is an sgRNA, the nucleotide sequence of which is shown in SEQ ID NO: 10, 15 or 16, or the gRNA consists of a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO. 18, the nucleotide sequence of the tracrRNA is shown in SEQ ID NO. 19, and the sgRNA, crRNA and tracrRNA have the following modifications: the first to third nucleotides and the last 1 to 3 nucleotides from the 5' end are modified by 2'-O-methylation, and the first to third internucleotide bonds and the last 1-3 internucleotide bonds from the 5' end are phosphorothioate modified;

[0194] The Cas9 mRNA sequence is shown in SEQ ID NO: 2, the lipid nanoparticles (LNP) contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG, and the ratio of the sgRNA to the encoding Cas9 mRNA is 1:1 (mass ratio).

[0195] In addition, those skilled in the art know that the anti-off-target property of Cas9 mutants is based on the fact that the mutation reduces the electrostatic and hydrophobic interactions between Cas9 and the genomic DNA phosphate backbone, and has little to do with the specific base sequence. Therefore, the anti-off-target ability of Cas9 mutants is often independent of the DNA base sequence. For example, HF1-Cas9 (Nature. 2016 Jan 28; 529 (7587): 490-5.) and HiFi-Cas9 (Nat Med. 2018 Aug; 24 (8): 1216-1224.) show specificity at many target sites, and there is no sequence-specific anti-off-target property.

[0196] In some embodiments, the Cas9 molecule can form a gene editing composition with sgRNAs targeting multiple targets, wherein the targets are selected from: TTR, PCSK9, kallikrein B1, and SERPINA1.

[0197] In a second aspect, the present application relates to a Cas9 nuclease or an enzymatically active fragment thereof, comprising or relative to a mutation of SEQ ID NO. 38, one or more mutations selected from the group consisting of K526X, Q695X, H698X, and R691X, wherein each X is independently selected from the group consisting of glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), serine (S), threonine (T), lysine (K), arginine (R), phenylalanine (F), or tyrosine (Y), wherein the amino acid residue positions correspond to the amino acid numbering of the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence SEQ ID NO: 38 or are defined with reference to the amino acid numbering of the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence SEQ ID NO: 38.

[0198] In some embodiments, the Cas9 nuclease includes or is a mutation relative to SEQ ID NO. 38 selected from any one of the following groups of mutation combinations:

[0199] K526A+R691A+Q695A+H698A, K526A+R691A+N692A+Q695A+H698A, K526G+R691G+Q695G+H698G, K526D+R691A+Q695A+H698A, K526A+R691D+Q695A+H698A, K526A+R691A+Q695A+H698D, K526E+R691A+ Q695A+H698A, K526A+R691E+Q695A+H698A and K526A+R691A+Q695A+H698E.

[0200] In some embodiments, the amino acid sequence of the Cas9 nuclease is as shown in SEQ ID NO:36, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:36.

[0201] In a third aspect, the present application relates to a gRNA targeting the TTR gene, which is an sgRNA and whose nucleotide sequence is as shown in any one of SEQ ID NOs: 10-16 or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence as shown in any one of SEQ ID NOs: 10-16.

[0202] In some embodiments, the gRNA is chemically modified. The modified nucleosides and nucleotides can include one or more of the following: (i) changes in one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens in the phosphodiester backbone linkage, such as substitutions (exemplary backbone modifications); (ii) changes in the ribose moiety (e.g., the 2' hydroxyl group on the ribose), such as substitutions (exemplary sugar modifications); (iii) large-scale replacement of phosphate moieties with "dephospho" linkers (exemplary backbone modifications); (iv) modification or replacement of naturally occurring nucleobases (including with non-canonical nucleobases) (exemplary base modifications); (v) replacement or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3' or 5' end of the oligonucleotide, such as removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar and / or backbone modifications); and (vii) modification or replacement of sugars (exemplary sugar modifications).

[0203] Chemical modifications (such as those listed above) can be combined to provide modified gRNAs that include nucleosides and nucleotides (collectively referred to as "residues") that can have two, three, four or more modifications. For example, the modified residues can have modified sugars and modified core bases. In some embodiments, each base of the modified gRNA, for example, all bases, has a modified phosphate group, such as a thiophosphate group. In certain embodiments, all or substantially all of the phosphate groups in the phosphate groups of the gRNA molecule are replaced with thiophosphate groups. In some embodiments, the modified gRNA includes at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA includes at least one modified residue at or near the 3' end of the RNA.

[0204] In some embodiments, the sgRNA nucleotide sequence is preferably modified as follows: thiolation (*), 2'-O-methoxylation (m), or fluorolysis. These modifications can further improve sequence stability and prevent degradation.

[0205] In some embodiments, the sgRNA is preferably 50-150 nucleotides, more preferably 65-120 nucleotides.

[0206] In some embodiments, the gRNA includes one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%) of the positions in the modified gRNA are modified nucleosides or nucleotides.

[0207] In some embodiments, the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) linkage between nucleotides.

[0208] In some embodiments, the gRNA targeting the TTR gene consists of crRNA and tracrRNA, wherein the crRNA is as shown in SEQ ID NO.18 or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence shown in SEQ ID NO: 18, and the tracrRNA is as shown in SEQ ID NO.19 or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence shown in SEQ ID NO.19.

[0209] In a fourth aspect, the present application relates to a composition, a ribonucleoprotein complex or a protein lipid complex comprising the Cas9 nuclease or an enzymatically active fragment thereof according to the second aspect of the present application, or the gRNA according to the third aspect of the present application.

[0210] In a fifth aspect, the present application relates to an engineered vector encoding the Cas9 nuclease or its enzymatically active fragment of the second aspect of the present application and / or the gRNA of the third aspect of the present application.

[0211] In the sixth aspect, the present application relates to a method for editing the transthyretin gene in a host cell, comprising introducing the gene editing composition of the first aspect of the present application, the ribonucleoprotein complex or protein lipid complex of the fourth aspect of the present application, or the engineered vector of the fifth aspect of the present application into the host cell.

[0212] In the seventh aspect, the present application relates to a method for preventing or treating transthyretin gene-related diseases or conditions, comprising introducing the gene editing composition of the first aspect of the present application, the ribonucleoprotein complex or protein lipid complex of the third aspect of the present application, or the engineered vector of the fifth aspect of the present application into a subject.

[0213] In some embodiments, the TTR-related disease or disorder comprises transthyretin amyloidosis.

[0214] In some embodiments, the TTR-related disease or disorder comprises cardiomyopathy and neuropathy caused by transthyretin amyloidosis.

[0215] In the eighth aspect, the present application relates to the use of the gene editing composition of the first aspect of the present application, the ribonucleoprotein complex or protein lipid complex of the third aspect of the present application, or the engineered vector of the fifth aspect of the present application in the preparation of a drug for editing the transthyretin gene in a host cell.

[0216] In the ninth aspect, the present application relates to the use of the gene editing composition of the first aspect of the present application, the ribonucleoprotein complex or protein lipid complex of the third aspect of the present application, or the engineered vector of the fifth aspect of the present application in the preparation of a drug for preventing or treating transthyretin gene-related diseases or disorders.

[0217] In some embodiments, the TTR-related disease or disorder comprises transthyretin amyloidosis.

[0218] In some embodiments, the TTR-related disease or disorder comprises cardiomyopathy and neuropathy caused by transthyretin amyloidosis.

[0219] In the tenth aspect, the present application relates to a pharmaceutical composition comprising the gene editing composition of the first aspect of the present application, the ribonucleoprotein complex or protein lipid complex of the third aspect of the present application, or the engineered vector of the fifth aspect of the present application, and a pharmaceutically acceptable carrier.

[0220] Example

[0221] The embodiments of the present application will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if no specific conditions are specified, the conditions according to conventional conditions or manufacturer recommendations are used. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0222] The SpCas9 mutants (excluding the nuclear localization signal and signal peptide portion) used in the following Examples 1-7 relative to the amino acid mutation sites of the wild-type SpCas9 protein (SEQ ID NO.38), and the mRNA element structure encoding the mutants are shown in Table 1 below. All U (uridine) in the mRNA used in the examples of the present application are 1-methyl-pseudouridine. The mRNA sequences of SpCas9-WT (SEQ ID NO.1), SpCas9-Mut5 (SEQ ID NO.2), SpCas9-HF1 (SEQ ID NO.3) and HiFi-Cas9 (SEQ ID NO.4) used in the following examples are shown in the sequence table. In addition to the corresponding codons corresponding to the amino acid mutation sites in the mRNA sequences of other SpCas9 cleavage enzyme variants (SpCas9-Mut1 to Mut7, and SpCas9-HF4), the other partial sequences are the same as the mRNA sequence of SpCas9-HF1 (SEQ ID NO.3).

[0223] Table 1: Cas9 cleavage enzyme mutant mRNA structures

[0224] It should be understood that the protein and nucleic acid sequences used in the following examples are as shown in the sequence listing, or can be obtained by introducing the mutations described in Table 1 on the basis of the sequences shown in the sequence listing.

[0225] Example 1: Cas9 mRNA quality detection

[0226] 1.1 In vitro transcription (IVT) of Cas9 mRNA

[0227] 1. According to the instructions of the IVT kit (E131, Novoprotein), the IVT reaction system was prepared by mixing 10x Transcription Buffer, ATP, GTP, CTP, 1-N-Me-Pseudo UTP (Cat. No. WA0992, Zhaowei Technology), 5' cap analog m7G(5')ppp(5')(2'OMeA)pG (Cat. No. GAGNH23C2L1B, Zhaowei Technology), water for injection, linearized plasmid template with T7 promoter and DNA sequence encoding Cas9 mRNA (GenScript Biotech Co., Ltd.), and Enzyme Mix.

[0228] 2. The mixed reaction system was placed at 37°C for 40 minutes;

[0229] 3. Add the corresponding proportion of DNase I to terminate the reaction.

[0230] Cas9 protein mRNA was synthesized in vitro and then purified by hydrophobic chromatography and ultrafiltration. The purity of the mRNA encoding the Cas9 protein was analyzed by Bioanalyzer, confirming that high-purity mRNA (>95%) was obtained. The purity results of spCas9-WT mRNA are shown in Figure 1.

[0231] 1.2 Western blot analysis of Cas9 protein expression

[0232] HEK293T and HepG2 cells were transfected with mRNA encoding Cas9 as follows:

[0233] (1) Plating: HEK293T or HepG2 cells were digested and resuspended, and counted at 1.5×10 per well. 5 The cells were plated into 24-well plates.

[0234] (2) Transfection: 24 hours after plating, prepare two 1.5 ml EP tubes, add 250 μl Opti-MEM to each tube, add 500 ng Cas9 mRNA to EP tube No. 1 and mix well, add 2.5 μl Lipo2000 to EP tube No. 2 and mix well, then add the liquid in EP tube No. 2 to EP tube No. 1, mix well, centrifuge and let stand for 15 minutes before adding to the wells.

[0235] (3) Medium change: After 6 hours, remove the cell supernatant and add complete culture medium.

[0236] (4) Cell collection: 24 hours after mRNA transfection into HEK29T cells, the cells were collected and the expression of Cas9 protein was detected by western blot.

[0237] The results are shown in Figure 2, which shows that the Cas9 protein mRNA synthesized by the IVT step of Example 1 can be effectively expressed in HEK29T cells. It should be understood that the mRNA used in subsequent examples must undergo steps 1.1 and 1.2 before subsequent experiments can be carried out.

[0238] 1.3 gRNA chemical synthesis and purity testing

[0239] Step 1: Solid-Phase Synthesis: gRNA synthesis was performed using an automated synthesizer. Using the UnyLinker vector (Biocomma, 1000A CPG), synthesis was performed from the 3′ end to the 5′ end by sequentially coupling phosphoramidite nucleoside monomers. After the last monomer was coupled, the dimethoxytriphenyl (DMT) protecting group at the 5′ end was removed, and the protecting group (cyanoethyl) of the phosphate backbone was deprotected to obtain a solid-phase support intermediate.

[0240] Step 2: Cleavage / Aminolysis Deprotection: The solid support intermediate is cleaved from the solid support by aminolysis reaction to remove the protecting group, and the crude product is obtained after concentration and desilylation reaction.

[0241] Step 3: Chromatographic purification and ultrafiltration desalting: The crude product is purified by hydrophobic chromatography-acid hydrolysis-reverse phase chromatography to remove short chain sequences and other impurities. The collected qualified components are ultrafiltration desalted and pyrogen-free to obtain a gRNA aqueous solution without further freeze-drying.

[0242] Step 4: Aseptic filling: After the gRNA solution is sterile filtered twice, it is filled in the isolator to obtain the gRNA stock solution product.

[0243] The gRNA-related sequence structure involved in this application is as follows:

[0244] Table 2: gRNAs used in the implementation of this application

[0245] The purity of the relevant gRNA meets the requirements of subsequent experiments. The exemplary results are shown in Figure 3, which are the purity test results of sgTTR-0.

[0246] Example 2 Optimization of gRNA

[0247] 2.1 Editing efficiency test of different sRNAs on TTR gene and protein

[0248] HEK293T cells were transfected with different gRNAs (sgTTR-0, sgTTR-1, sgTTR-2, sgTTR-3, and sgTTR-7) in combination with SpCas9-WT mRNA (the mass ratio of gRNA to mRNA was 1:1). After 24 hours, the cells were lysed, and a DNA fragment of approximately 1 kb near the corresponding target site was amplified. The targeted cutting efficiency was detected by T7E1 enzyme digestion (the kit used was GeneArt™ Genomic Shearing Detection Kit, A24372, ThermoFisher).

[0249] The specific steps for detecting the targeted cutting efficiency by T7E1 enzyme cutting are as follows:

[0250] (1) Lyse the edited cells and PCR amplify the DNA bands approximately 1000 bp upstream and downstream of the editing site (i.e., design upstream primers for approximately 1000 bp upstream of the editing site, and design downstream primers for approximately 1000 bp downstream of the editing site);

[0251] (2) PCR products were purified by column and the concentration was determined by Nanodrop;

[0252] (3) Take 100 ng of purified DNA product and prepare the enzyme digestion reaction system according to the instructions. After annealing, add 1 μl of T7E1 enzyme and react at 37°C for 1 h.

[0253] (4) Agarose gel electrophoresis was used to identify the enzyme digestion results and detect the editing efficiency.

[0254] In addition, different gRNAs were combined with Cas9 mRNA to transfect HepG2 cells (the mass ratio of gRNA to mRNA was 1:1). After 72 hours, the cell supernatant was collected and the TTR protein content in the cell supernatant was detected by ELISA. The ELISA detection steps are as follows:

[0255] (1) Sample preparation and standard preparation: The sample to be tested (cell supernatant or animal serum) is diluted in an appropriate ratio, and standard solutions with gradient concentrations are prepared according to the instructions.

[0256] (2) Sample addition: 50 μL / well was added to a 96-well ELISA plate, sealed with a sealing film, and incubated at room temperature for 1 h.

[0257] (3) Add detection antibody: After washing the plate three times with PBST, add biotinylated detection antibody at 50 μl / well, seal the plate with sealing film, and incubate at room temperature for 1 h.

[0258] (4) Add secondary antibody: After washing the plate 3 times with PBST, add secondary antibody, 50ul / well, seal the plate with sealing film, and incubate at room temperature for 30min

[0259] (5) Color development: After washing the plate three times with PBST, add TMB color development solution (50 μl / well) and develop the color at room temperature in the dark for 10-15 minutes.

[0260] (6) Termination: Add 50 μl of stop solution to each well to stop color development.

[0261] (7) Plate reading: Read the OD value using a microplate reader at 450 nm.

[0262] The above test results (Figure 4) show that sgTTR-0, sgTTR-1, sgTTR-2, sgTTR-3, and sgTTR-7 all have certain cleavage activity, among which sgTTR-0 and sgTTR-1 have better effects. Further tests will be conducted on the above two sgTTRs in the future.

[0263] 2.2 GUIDE-seq analysis of off-target rates of sgTTR-0 and sgTTR-1

[0264] The examples of this application use the GUIDE-seq method to detect off-target rates. For details, see, for example (Tsai, Zheng et al. 2015, Malinin, Lee et al. 2021).

[0265] GUIDE-seq is a method for detecting off-target effects of gene editing. Its basic principle is to use a short double-stranded oligodeoxynucleotide tag (dsODN) to mark the breaks induced by CRISPR-Cas9 (that is, after the Cas9 enzyme in the CRISPR system cuts the genome to produce DNA double-strand breaks, there is a certain probability that the dsODN will be integrated into the genome during genome repair). The sequence of the gene region where the dsODN tag is located is then amplified (that is, upstream primers upstream of the genomic break site and downstream primers targeting the genomic break site are set up for sequence amplification) to construct a library and perform high-throughput sequencing. Finally, bioinformatics analysis is used to determine the location and mutation frequency of off-target mutations.

[0266] The steps for off-target rate detection are as follows:

[0267] (1) Cell transfection. gRNA (sg-TTR-0 and sgTTR-1), wild-type SpCas9 mRNA, and double-stranded oligodeoxynucleotides (dsODN) tags were simultaneously transfected into HepG2 cells by electroporation.

[0268] (2) Genomic DNA extraction: 72 hours after transfection, genomic DNA of cells was extracted.

[0269] (3) ODN integration rate detection: T7E1 enzyme digestion was used to detect DNA double-strand cutting efficiency, and Ndel enzyme digestion was used to detect dsODN tag insertion efficiency. Subsequent detection was performed after the Ndel / T7E1 integration efficiency reached 30%.

[0270] (4) Take appropriate quality genomic DNA to construct a library and use the MGI-2000 system for sequencing; (5) Bioinformatics analysis. The analysis results are shown in Figure 5, where:

[0271] (A) Shows the off-target amounts measured when sgTTR-0 and sgTTR-1 were combined with SpCas9-WT mRNA;

[0272] (B) shows the distribution of cleavage positions on chromosomes by the combination of sgTTR-0 and Cas9 mRNA;

[0273] (C) Shows the distribution of cleavage sites on chromosomes by the combination of sgTTR-1 and Cas9 mRNA.

[0274] It can be seen that different gRNAs show different off-target efficiencies, among which the off-target probability of sgTTR-0 is significantly lower than that of sgTTR-1.

[0275] 2.3 Further optimization exploration of sgTTR-0

[0276] 2.3.1 Western blot detection of TTR target protein

[0277] HEK293T cells were co-transfected with different sgTTR-0 variants (sgTTR-0-v02, sgTTR-0-v04, TTR-0-del (82 nt); and cr+tr, a combination of the crRNA and tracrRNA parts of sgTTR-0) and Cas9-HF1 mRNA. After 24 hours, the cells were lysed, and a DNA fragment of approximately 1 kb near the sgTTR-0 target site was amplified, and the cutting efficiency was detected by T7E1 enzyme digestion.

[0278] Among them, Figure 6 shows the results of the T7E1 enzyme digestion experiment, which shows that sgTTR-0-v02, sgTTR-0-v04, or splitting the sgRNA into crRNA and tracrRNA can maintain the editing effect on the TTR gene, while truncating the sgRNA to 82nt loses the editing effect on the TTR gene.

[0279] Example 3 Screening of Cas9 mutants with low off-target rates

[0280] 3.1 GUIDE-seq analysis of off-target rates (safety) of wild-type Cas9 (SpCas9-WT) and its mutants

[0281] After co-transfection of HepG2 cells with mRNA encoding SpCas9-WT and its mutants (SpCas9-WT, SpCas9-HF1, SpCas9-Mut1, SpCas9-Mut2, SpCas9-Mut3, SpCas9-Mut4, SpCas9-Mut5, and SpCas9-Mut6) and sgTTR-0, off-target efficiency was detected. The specific steps are as follows:

[0282] (1) Cell transfection. sgTTR-0, wild-type or mutant SpCas9 mRNA and double-stranded oligodeoxynucleotides (dsODN) tags were simultaneously transfected into HepG2 cells by electroporation.

[0283] (2) Genomic DNA extraction: 72 hours after transfection, genomic DNA of cells was extracted.

[0284] (3) ODN integration rate detection: T7E1 enzyme digestion was used to detect DNA double-strand cutting efficiency, and Ndel enzyme digestion was used to detect dsODN tag insertion efficiency. Subsequent detection was performed when the Ndel / T7E1 integration efficiency reached 30%.

[0285] (4) Take appropriate quality genomic DNA to construct a library and sequence it using the MGI-2000 system;

[0286] (5) Bioinformatics analysis.

[0287] The results are shown in Figure 7, where:

[0288] Part A of Figure 7 shows the specific number of off-target sites for different Cas9 mutants and sgTTR-0 combinations measured by GUIDE-seq. Among them, the combination of wild-type Cas9 and sgTTR-0 measured a total of 41 off-target sites, the reported low off-target mutant Cas9-HF1 and sgTTR-0 measured 11 off-target sites, and the combination of Cas9-mut5 and sgTTR-0 reduced the number of off-target sites to 6; Part B counted the off-target frequency (number of off-target reads / number of on-target reads) of different Cas9 mutants and sgTTR-0 combinations. The results of parts A and B of Figure 7 both show that the safety of Cas9-mut5 is not only much higher than that of wild-type Cas9, but also better than that of reported low off-target mutants and other mutants (such as Cas9-Mut1, Cas9-Mut2, Cas9-Mut3, Cas9-Mut4, and Cas9-Mut6).

[0289] Part C of Figure 7 shows the potential off-target sites of Cas9-mut5 and Cas9-WT across the entire genome.

[0290] 3.2 Detection of gene editing efficiency of different SpCas9 mutants combined with sgTTR-0

[0291] To test whether the editing efficiency of the aforementioned mutants with reduced off-target efficiency (e.g., SpCas9-Mut5) was affected by the mutation, the gene editing efficiency was tested at the TTR target site.

[0292] The mice used in this test are humanized TTR mouse models, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., with strain number T055186. This model mouse model utilizes gene editing technology to replace the mouse TTR gene coding region and regulatory sequences with corresponding human TTR gene fragments. It is commonly used in transthyretin amyloidosis disease research and drug screening.

[0293] The specific test steps are as follows:

[0294] (1) Preparation of test drugs. sgTTR-0 was encapsulated in LNPs with the mRNAs of SpCas9-WT and various mutants (SpCas9-WT, SpCas9-HF1, SpCas9-HF4, SpCas9-Mut1, SpCas9-Mut2, Cas9-Mut3, Cas9-Mut4, SpCas9-Mut5, and SpCas9-Mut6) at a mass ratio of 1:1. The encapsulation method is described in Example 4 below.

[0295] (2) Animal Grouping. 6-8 week old TTR humanized mice (either sex) were used. One week before dosing, submandibular blood was collected from the mice. TTR protein expression in the serum was determined using an ELISA kit (ab231920, abcam). Mice were grouped according to the initial level of TTR expression before dosing, with 4-6 mice per group, to ensure that the initial level of TTR expression in each group was comparable.

[0296] (3) Administration. The drug was administered once via tail vein injection. The day of administration was designated as day 0. The dosage was 1 mpk (1 mg / kg), 0.3 mg / kg, or 3 mg / kg, where mg represents the total mass of the nucleic acid in the drug.

[0297] (4) Regular blood sampling. Blood was collected from the submandibular area of ​​mice on days 4, 7, 14, 21, and 28 after administration, and serum was collected. TTR protein expression in the serum was detected using an ELISA kit (ab231920, abcam) to determine the knockdown level of TTR protein.

[0298] (5) Collection of samples from mice. 28 days after administration, all mice were killed, and the livers were quickly frozen in liquid nitrogen and stored at -80°C.

[0299] (6) Determination of liver editing efficiency. Liver genomic DNA was extracted, and PCR amplification was performed by designing primers near the target gene region. The PCR products were then subjected to high-throughput sequencing to obtain information on the mutation frequency of the target region. That is, the editing efficiency was determined by amplicon sequencing.

[0300] The experimental results are shown in Figure 8. Wild-type SpCas9 and various SpCas9 mutants combined with sgTTR-0 all showed some editing efficacy on the TTR gene in humanized TTR mice. Using the degree of TTR protein knockdown in the serum of humanized TTR mice as an indicator, the reported low-off-target mutant HF1 improved safety at the expense of efficacy, while Cas9-mut5 achieved even greater safety while maintaining comparable editing efficacy to wild-type Cas9.

[0301] Example 4 Optimization of LNP delivery formulation

[0302] 4.1 Gene Editing Trials of sgTTR-0 and SpCas9 mRNA in Different LNP Delivery Formulations

[0303] To obtain a more optimal LNP delivery formulation for in vivo genome editing, this example tested various LNP formulations as shown in Table 3.

[0304] The main steps are as follows:

[0305] (1) Preparation of test drugs. sgTTR-0 and SpCas9 mRNA (SpCas9-HF1 mRNA or SpCas9-Mut5 mRNA) were encapsulated in LNPs with different formulations.

[0306] (2) Animal Grouping. One week before dosing, serum TTR protein expression was measured using an ELISA kit (ab231920, Abcam). Mice were divided into groups of 4-6 mice per group based on the initial level of TTR expression before dosing, ensuring that the initial TTR expression levels of mice in each group were comparable. Mice were 6-8 weeks old, TTR humanized mice of either sex.

[0307] Table 3.

[0308] (3) Administration. Injection was performed via tail vein at a dose of 1 mpk or 0.3 mpk. The day of administration was recorded as day 0.

[0309] (4) One week after administration, the expression of TTR protein in serum was detected by ELISA to determine the knockdown level of TTR protein.

[0310] (5) Collection of samples from mice. 28 days after administration, the mice were killed and their livers were collected, quickly frozen in liquid nitrogen, and stored at -80°C.

[0311] (6) Determination of liver editing efficiency. Liver genomic DNA was extracted, the target gene region was amplified by PCR, and the PCR products were subjected to high-throughput sequencing to determine the editing efficiency.

[0312] The results showed (see Figure 9 ) that LNP-01 is a more suitable LNP formulation for delivery, and the combination of its encapsulated gRNA and Cas9mRNA (whether SpCas9-HF1 in AC or SpCas9-MUT5 in D in Figure 9 ) has the best gene editing effect.

[0313] Therefore, the LNP formulation used in all other embodiments of this application is LNP-01. Therefore, taking LNP-01 as an example, the packaging method of LNP in the embodiments of this application is as follows:

[0314] (1) Accurately weigh a certain amount of SM-102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG2000 lipids with a molar mass ratio of 50%, add appropriate amount of anhydrous ethanol to dissolve, and prepare a lipid working solution for use (final concentration of lipid working solution 20 mg / mL).

[0315] (2) Prepare citric acid buffer solution (10 mM, pH 4.0) containing 130 mM sodium chloride, Tris-NaOAc buffer solution (20 mM, 10.7 mM, pH 7.5), and Tris-NaOAc buffer solution (20 mM, 10.7 mM, pH 7.5) containing 60% sucrose respectively.

[0316] (3) Take an appropriate amount of mRNA stock solution and dilute it with the sodium chloride-citrate buffer solution prepared above to adjust the final concentration of the mRNA working solution to 0.18 mg / mL.

[0317] (4) Using a microfluidic instrument and a matching chip, the lipid working solution and the mRNA working solution were mixed in a volume ratio of 1:3 to prepare an mRNA-loaded LNP solution.

[0318] (5) Add 9 times the volume of Tris-NaOAc buffer solution to dilute the prepared LNP solution, and use TFF concentration and purification to remove the ethanol solution in the system.

[0319] (6) The mRNA content in the LNP solution was detected by ultraviolet spectrometry and an appropriate amount of Tris-NaOAc buffer solution (20 mM, 10.7 mM, pH 7.5) containing 60% sucrose was added to adjust the final mRNA concentration in the finished LNP solution to 100 μg / mL and the sucrose content in the external aqueous phase system to 8.7%.

[0320] The test results show that LNP encapsulation efficiency, particle size and other parameters meet the requirements of subsequent tests and can be used for lipid prescription screening.

[0321] 4.2 Detection of gene editing efficiency of sgTTR-0 and SpCas9 mRNA combinations at different mass ratios

[0322] To further optimize the editing efficiency of the gene editing system of this application, the ratio of gRNA and Cas9 mRNA in LNP liposomes was optimized based on the editing efficiency. The specific experimental steps are as follows:

[0323] (1) Preparation of test drugs. sgTTR-0 and SpCas9-HF1 mRNA were encapsulated in LNPs at different mass ratios, such as 1:1, 1:4, 1:8, 1:16, and 1:32. The encapsulation method is described in Section 4.1.

[0324] (2) Animal grouping. 6-8 week old TTR humanized mice (either sex) were used. One week before administration, serum TTR protein expression was measured using an ELISA kit (ab231920, abcam). Mice were then divided into groups based on the initial level of TTR expression before administration, with 4-6 mice per group to ensure that the initial TTR expression levels of mice in each group were comparable.

[0325] (3) Administration: The drug was injected into the tail vein at a dose of 1 mpk. The day of administration was designated as day 0.

[0326] (4) Regularly collect blood and detect the TTR protein expression in the serum to determine the knockdown level of TTR protein.

[0327] (5) Collection of samples from mice. 28 days after administration, the mice were killed and their livers were collected, quickly frozen in liquid nitrogen, and stored at -80°C.

[0328] (6) Determination of liver editing efficiency. Liver genomic DNA was extracted, the target gene region was amplified by PCR, and the PCR products were subjected to high-throughput sequencing to determine the editing efficiency.

[0329] The results, as shown in Figure 10, show that combinations of sgTTR-0 and SpCas9 mRNA at varying mass ratios all achieved a certain degree of editing efficiency on the TTR gene. The optimal editing efficiency was achieved at gRNA to SpCas9 mRNA ratios of 1:1 and 1:4. This ratio, starting at 1:8, reduced editing efficiency to a certain extent, and at a ratio of 1:32, the TTR editing efficiency was essentially halved.

[0330] 4.3 Off-target rate detection of sgTTR-0 and SpCas9 mRNA combinations at different mass ratios

[0331] To further optimize the editing efficiency of the gene editing system of this application, the ratio of gRNA and Cas9 mRNA in LNP liposomes was optimized based on the off-target efficiency. The specific experimental steps are as follows:

[0332] (1) Cell transfection. sgTTR-0, SpCas9-WT mRNA and double-stranded oligodeoxynucleotides (dsODN) tags were transfected into HepG2 cells simultaneously by electroporation. The total transfection volume was kept at 1 μg, and sgTTR-0 and SpCas9 mRNA were transfected into HepG2 cells at different mass ratios.

[0333] (2) Genomic DNA extraction: 72 hours after transfection, genomic DNA of cells was extracted.

[0334] (3) ODN integration rate detection: T7E1 enzyme digestion was used to detect DNA double-strand cutting efficiency, and Ndel enzyme digestion was used to detect dsODN tag insertion efficiency. Subsequent detection was performed after the Ndel / T7E1 integration efficiency reached 30%.

[0335] (4) Take appropriate quality genomic DNA to construct a library and sequence it using the MGI-2000 system;

[0336] (5) Bioinformatics analysis.

[0337] The results are shown in Figure 11, indicating that the gRNA to mRNA mass ratio of 1:1 is safer.

[0338] By using the transthyretin gene (TTR) as a target through Examples 1-4, and by optimizing the gRNA, Cas9, and LNP delivery system, as well as the ratio of gRNA to Cas9 mRNA, a CRISPR-Cas9 cleavage enzyme system suitable for TTR gene editing with lower off-target efficiency was obtained. In particular, the aforementioned examples screened the low off-target Cas9 enzyme mutant SpCas9-Mut5 through a large number of experiments. While achieving a highly efficient TTR gene knockout effect, it also achieved the effect of reducing off-target effects to the greatest extent, reduced the side effects of the application of the system, and achieved unexpected technical effects.

[0339] Example 5: Verification of the editing efficiency of SpCas9 mutants in different cells

[0340] We further verified whether SpCas9-Mut5 editing efficiency was superior to wild-type Cas9 and other mutants in HEK293T and HepG2 cells. The specific methods are as follows:

[0341] (1) sgTTR-0 was combined with different Cas9 mutants (SpCas9-Mut1 to Mut7) to transfect HEK293T cells and HepG2 cells;

[0342] (2) After 72 h, cells were harvested and genomic DNA was extracted;

[0343] (3) Amplify a DNA fragment of approximately 200 bp near the sgTTR-0 target site and perform amplicon sequencing analysis;

[0344] (4) At the same time, the culture supernatant was collected and the TTR protein content in the cell supernatant was detected by ELISA.

[0345] The results are shown in Figure 12.

[0346] The results in Figure A show that in HEK293T cells, under the guidance of sgTTR-0, mut1-7 showed a high editing efficiency (genomic level) for the TTR gene, ranging from 40% to 60%, which is basically equivalent to the editing efficiency of the control group HF1 (about 55%).

[0347] The results in Figure B show that in HepG2 cells, under the guidance of sgTTR-0, the editing efficiency of mut1-7 on the TTR gene (genomic level) is basically equivalent to the editing efficiency of the control group HF1 (about 66%).

[0348] The results in Figure C show that in HepG2 cells, under the guidance of sgTTR-0, mut1-7 has a strong inhibitory effect on TTR protein (protein level), and its knockdown level of TTR protein (downregulation of approximately 60%) is higher than that of the control SpCas9-HF1 (downregulation of approximately 49.5%).

[0349] Example 6 Detection of SpCas9 mutant off-target rates at high-frequency off-target sites

[0350] After using GUIDE-Seq technology to predict the high-frequency off-target sites of SpCas9-WT, it was found that the off-target rate of SpCas9-Mut5 was lower among the four major high-frequency off-target sites. The specific method is as follows:

[0351] (1) sgTTR-0 was combined with different Cas9 mutants to transfect HepG2 cells;

[0352] (2) After 72 hours, cells were harvested and genomes were extracted;

[0353] (3) Amplify a DNA fragment of approximately 200 bp near the high-frequency off-target site;

[0354] (4) Perform amplicon sequencing on the amplified DNA fragments.

[0355] The editing efficiency of the wild-type SpCas9 and its mutants at four different off-target sites (off-target site 1 / 2 / 3 / 4) was analyzed. The results are shown in FIG13 . Specifically,

[0356] The results in Figure A show that under the guidance of sgTTR-0, spCas9-WT had an off-target editing efficiency of nearly 20% at off-target site 1, while mut2 had an off-target editing efficiency of approximately 4%. The remaining Cas9 mutants (including mut5) reduced off-target editing at this site to a level equivalent to that of the ctrl group (<1%). In terms of editing efficiency at this off-target site, mut5 showed a significant difference from WT (P<0.0001), mut5 showed a significant difference from mut2 (P=0.0015), and there was no statistical difference between mut5 and mut1 / 3 / 4 / 6 / 7.

[0357] The results in Figure B show that under the guidance of sgTTR-0, Cas9-WT had an off-target editing efficiency of over 10% at off-target site 2, mut2 had an off-target editing efficiency of nearly 10%, and mut4 had an off-target editing efficiency of approximately 2%. The remaining Cas9 mutants (including mut5) reduced off-target editing at this site to a level equivalent to that of the ctrl group (<0.5%). In terms of editing at this off-target site, mut5 showed significant differences from WT (P<0.0001), mut5 showed significant differences from mut2 (P<0.0001), mut5 showed significant differences from mut4 (P<0.0001), and mut5 showed significant differences from mut1 / 3 / 6 / 7.

[0358] The results in Figure C show that under the guidance of sgTTR-0, Cas9-WT had an off-target editing efficiency of nearly 10% at off-target site 3, and mut2 had an off-target editing efficiency of nearly 5%. The other Cas9 mutants (including mut5) reduced the off-target editing efficiency at this site to the level of the ctrl group (approximately 1%). In terms of editing efficiency at this off-target site, mut5 showed a significant difference from WT (P<0.0001), mut5 showed a significant difference from mut2 (P=0.0015), and there was no statistical difference between mut5 and mut1 / 3 / 4 / 6 / 7.

[0359] The results in Figure D show that under the guidance of sgTTR-0, Cas9-WT measured an off-target editing efficiency of approximately 7% at the off-target site 4 site, and mut5 significantly reduced the editing of this off-target site (reduced to approximately 4%, p value = 0.003).

[0360] Combined with the results of Example 3 (e.g., Figure 8), it can be seen that SpCas9-Mut5 has fewer off-target sites and a lower overall off-target rate compared to wild-type Cas9 and other mutants, and also has a lower off-target rate at each high-frequency off-target site than the wild type or other mutants.

[0361] Example 7: Testing the editing effect of Cas9-mut5 at more sites in the genome

[0362] 7.1 Editing Efficiency Detection

[0363] To investigate whether the editing efficiency of Cas9-mut5 at sites other than TTR is comparable to that of wild-type Cas9 and reported high-efficiency, low-off-target editors, we selected four gRNAs (sgRNA-1 / 2 / 3 / 4) and combined them with Cas9WT / HF1 / HiFiCas9 / Mut5 mRNA to transfect HepG2 cells. After 72 hours, the cells were harvested and DNA fragments of approximately 200 bp near the corresponding target sites were amplified and analyzed by amplicon sequencing. The results are shown in Figure 14. Specifically:

[0364] A shows that under the guidance of sgRNA-1 (SEQ ID NO. 25, target site TRAC), the editing efficiency of Cas9-mut5 is comparable to that of Cas9WT / HF1 / HiFiCas9, and their on-target editing efficiency is around 60%.

[0365] Figure B shows that under the guidance of sgRNA-2 (SEQ ID NO. 26, target gene is TRAC), the editing efficiency of Cas9-mut5 is comparable to that of Cas9WT / HF1 / HiFiCas9, and their on-target editing efficiencies are all around 55%.

[0366] C shows that under the guidance of sgRNA-3 (SEQ ID NO. 27, target gene is TRAC), the editing efficiency of Cas9-mut5 is comparable to that of Cas9WT / HF1 / HiFiCas9, and their on-target editing efficiencies are all around 60%.

[0367] D shows that under the guidance of sgRNA-4 (SEQ ID NO.28, target gene is REGNASE-1), the editing efficiency of Cas9-mut5 is comparable to that of Cas9WT / HF1 / HiFiCas9, and their on-target editing efficiency is around 40%.

[0368] The above results indicate that Mut5's editing ability at sites other than TTR is not inferior to WT or reported high-efficiency, low-off-target editing tools.

[0369] 7.2 Off-target rate detection

[0370] To investigate the off-target effects of Cas9-mut5 when combined with additional gRNAs, HEK293T cells were transfected with Cas9WT / HF1 / HiFiCas9 / mut5, respectively, along with EMX1 and VEGFA3 gRNAs. The control group was transfected with gRNA only, and cells were harvested 72 hours later. A DNA fragment approximately 200 bp near the target site was amplified and analyzed by next-generation sequencing. The results are shown in Figure 15, where:

[0371] The results in Figure A show that under the guidance of EMX1gRNA, the on-target editing efficiency of WT / HF1 / HiFiCas9 / mut5 for the EMX1 gene exceeded 70%, and the on-target editing efficiency of SpCas9-Mut5 was better than that of SpCas9-WT; at the same time, like other high-efficiency and low-off-target editors, mut5 had a significantly reduced off-target editing efficiency at the Sp-Cas9-WT high-frequency off-target site OT1 (from 18.5% to 0.9%, P<0.0001), almost dropping to the level of the ctrl group, and SpCas9-Mut5 had the most significant effect in reducing the off-target rate.

[0372] The results in Figure B show that under the guidance of VEGFA3gRNA, the on-target editing efficiency of SpCas9-Mut5 for the VEGFA3 gene exceeds 70%, which is better than SpCas9-WT; at the same time, the off-target rate of SpCas9-Mut5 at the SpCas9-WT high-frequency off-target site OT2 is lower than that of SpCas9-WT and other mutants, and it can significantly reduce the off-target editing of the SpCas9-WT high-frequency off-target site OT1 (from 11.2% to 6.0%, P < 0.0001).

[0373] Overall, the off-target rate of Mut5 in genes other than TTR is relatively lower, and its gene editing efficiency is often better than SpCas9-WT and other low-off-target Cas9 variants.

[0374] The relevant sequences of this application are shown in the following table:

[0375] Sequence Listing

[0376] Note: When SEQ ID NO.1-SEQ ID NO.9 are used as mRNA sequences, all thymidine (T) represents uridine (U). In the examples, when the sequences of SEQ ID NO.1-SEQ ID NO.9 are used for mRNA, all U represents 1-methylpseudouridine.

[0377] When SEQ ID NOs. 10-30 are used as gRNA sequences, thymidine (T) represents uridine (U). When gRNA sequences comprising or as shown in SEQ ID NOs. 10-19 are used in the Examples, the sequences of SEQ ID NOs. 10-19 all have the following modifications: 2'-O-methylation of the first to third nucleotides and the last one to three nucleotides from the 5' end, and phosphorothioate modification of the first to third internucleotide linkages and the last one to three internucleotide linkages from the 5' end.

[0378] The above describes the implementation methods of the present application. However, the present application is not limited to the above implementation methods. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A gene editing composition comprising: 1) Cas9 nuclease or an enzymatically active fragment thereof, or a nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof; and 2) a guide RNA (gRNA) targeting a transthyretin (TTR) gene or a nucleic acid molecule encoding the gRNA, wherein the Cas9 nuclease comprises a mutation at one or more amino acid residue positions selected from the group consisting of K526, N692, Q695, H698, N497, Y450, Q926, K377, E387, D397, R400, D406, A421, L423, R424, Q426, Y430, K442, P449, V452, A456, R457, W464, M465, K466, 68. E470, T474, P475, W476, F478, K484, S487, A488, T496, F498, L502, N504, K506, P509, F518, N522, E523, L540, S541, I5 48. D550, F553, V561, K562, E573, A589, L598, D605, L607, N609, N612, E617, D618, D628, R629, R635, K637, L651, K652, R65 4. T657, G658, L666, K673, S675, I679, L680, L683, N690, R691, F693, S701, F704, Q712, G715, Q716, H723, I724, L727, I73 3. L738, Q739, N803, Q805, Q807, K810, Y812, D829, N831, R832, ​​S834, D835, Q844, S845, K848, R859, K862, R864, K866, K890 , T893, Q894, D898, N899, K902, K913, K918, Q920, T924, R925, T928, K929, H930, S960, K961, S964, K968, R976, H982, H983, Y1013, K1031, T1033, SI106, K1107, S1109, Y1237, Y1242, K1244 and K1246, wherein the amino acid residue positions correspond to the amino acid numbers in the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence SEQ ID NO:38 or are defined with reference to the amino acid numbering in the Streptococcus pyogenes Cas9 (SpCas9) protein amino acid sequence SEQ ID NO:

38.

2. The gene editing composition according to claim 1, wherein the Cas9 nuclease comprises or is selected from one or more mutations of the group consisting of K526X, Q695X, H698X and R691X relative to SEQ ID NO. 38, wherein each X is independently selected from the group consisting of glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), aspartic acid (D), glutamate (E), asparagine (N), glutamine (Q), serine (S), threonine (T), lysine (K), arginine (R), phenylalanine (F) or tyrosine (Y).

3. The gene editing composition according to claim 2, wherein each X is independently selected from any one of the following: glycine (G), alanine (A), aspartic acid (D) and glutamic acid (E).

4. The gene editing composition according to claim 3, wherein the Cas9 nuclease comprises or is a mutation combination selected from any one of the following groups relative to the mutation of SEQ ID NO.38: K526A+R691A+Q695A+H698A, K526A+R691A+N692A+Q695A+H698A, K526G+R691G+Q695G+H698G, K526D+R691A+Q695A+H698A, K526A+R691D+Q695A+H698A, K526A+R691A+Q695A+H698D, K526E+R691A+Q695A+H698A, K526A+R691E+Q695A+H698A and K526A+R691A+Q695A+H698E.

5. The gene editing composition according to any one of claims 1 to 4, wherein the Cas9 nuclease or its enzymatically active fragment further comprises a nuclear localization signal located at the N-terminus and / or the C-terminus.

6. The gene editing composition according to claim 5, wherein: The amino acid sequence of the nuclear localization signal is as shown in SEQ ID NO.39 or SEQ ID NO.40, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO.39 or SEQ ID NO.40; Optionally, the amino acid sequence of the nuclear localization signal at the N-terminus is as shown in SEQ ID NO.40, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO.40; and / or the amino acid sequence of the nuclear localization signal peptide at the C-terminus is as shown in SEQ ID NO.39, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO.

39.

7. The gene editing composition according to any one of claims 1 to 6, wherein the amino acid sequence of the Cas9 nuclease is as shown in SEQ ID NO.36, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.

36.

8. A gene editing composition according to any one of claims 1 to 7, wherein the nucleic acid molecule encoding the Cas9 nuclease or its enzymatically active fragment comprises a 5' non-coding region sequence (5'UTR) and a 3' non-coding region sequence (3'UTR); optionally, the 5'UTR comprises or is the 5'UTR of the tobacco epithelium virus (TEV) gene, and / or the 3'UTR comprises or is the 3'UTR of the human hemoglobin alpha 1 (hHBA1) gene.

9. The gene editing composition according to claim 8, wherein the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO.6 or 7, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence as shown in SEQ ID NO.6 or 7; and / or The nucleotide sequence of the 3'UTR is as shown in SEQ ID NO.9, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence as shown in SEQ ID NO.

9.

10. A gene editing composition according to claim 8 or 9, wherein the nucleic acid molecule encoding the Cas9 nuclease further comprises a polyadenosine tail (Poly (A) tail) sequence or a tailing signal sequence, and preferably the Poly (A) tail sequence comprises or is a nucleic acid sequence as shown in SEQ ID NO.

8.

11. The gene editing composition according to any one of claims 8 to 10, wherein the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof is an mRNA molecule. 12 . The gene editing composition according to claim 11 , wherein the mRNA molecule comprises a 5′ cap structure, and optionally the 5′ cap structure is m7G(5′)ppp(5′)(2′OMeA)pG.

13. A gene editing composition according to any one of claims 1 to 12, wherein the nucleotide sequence of the nucleic acid molecule encoding the Cas9 nuclease is as shown in SEQ ID NO: 2, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:

2.

14. The gene editing composition according to any one of claims 1 to 13, wherein the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof is an mRNA molecule, which contains base-modified uridine (U); optionally, the base-modified U is 1-methylpseudouridine; optionally, each U in the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof is 1-methylpseudouridine.

15. A gene editing composition according to any one of claims 1-14, wherein the gRNA comprises a spacer sequence, and the spacer sequence is as shown in any one of SEQ ID NO.20-24, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NO.20-24.

16. The gene editing composition according to claim 15, wherein the gRNA consists of crRNA and tracrRNA comprising the spacer sequence; optionally, the nucleotide sequence of the crRNA comprises or is the nucleotide sequence shown in SEQ ID NO.18, and the tracrRNA comprises or is the nucleotide sequence shown in SEQ ID NO.

19.

17. The gene editing composition of claim 15, wherein the gRNA is a single guide RNA (sgRNA); optionally, the nucleic acid sequence of the sgRNA is as shown in any one of SEQ ID NOs: 10-16, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 10-16.

18. The gene editing composition of claim 17, wherein the nucleotide sequence of the sgRNA is as shown in SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 16, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO:

16.

19. The gene editing composition according to any one of claims 15 to 18, wherein the sgRNA, crRNA and tracrRNA are chemically modified, preferably the chemical modification comprises or is 2'-O-methylation modification of the 1st to 3 nucleotides and the last 1 to 3 nucleotides from the 5' end of the sgRNA, crRNA or tracrRNA, and phosphorothioate modification of the 1st to 3 nucleotide bonds and the last 1-3 nucleotide bonds from the 5' end.

20. The gene editing composition according to any one of claims 1-19, wherein the mass ratio of the nucleic acid molecule encoding the Cas9 nuclease or an enzymatically active fragment thereof to the gRNA or the nucleic acid molecule encoding the gRNA is about 1:4 to about 4:1, for example about 1:

1.

21. A gene editing composition according to any one of claims 1 to 20, comprising lipid nanoparticles (LNPs), wherein the lipid component of the LNPs comprises or consists of 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% auxiliary lipids and 0.5-15 mol% surfactants; and: The structural lipid is selected from cholesterol and cholesterol derivatives; The cationic lipid is selected from: SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, DlinDMA, preferably SM-102; The helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC; and / or The surfactant is selected from PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, preferably PEG2000-DMG.

22. A Cas9 nuclease or an enzymatically active fragment thereof, comprising or being selected from the group consisting of one or more mutations relative to SEQ ID NO.38: K526X, Q695X, H698X and R691X, wherein each X is independently selected from the group consisting of glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), serine (S), threonine (T), lysine (K), arginine (R), phenylalanine (F) or tyrosine (Y), wherein the amino acid residue positions correspond to the amino acid numbers in the amino acid sequence of Streptococcus pyogenes Cas9 (SpCas9) protein SEQ ID NO: 38 or are defined with reference to the amino acid numbers in the amino acid sequence of Streptococcus pyogenes Cas9 (SpCas9) protein SEQ ID NO:

38.

23. The Cas9 nuclease or its enzymatically active fragment according to claim 22, wherein the Cas9 nuclease comprises or is any combination of mutations selected from the group consisting of K526A+R691A+Q695A+H698A, K526A+R691A+N692A+Q695A+H698A, K526G+R691G+Q695G+H698G, K526D+R691A+Q695A+H698A, K526A+R691D+Q695A+H698A, K526A+R691A+Q695A+H698D, K526E+R691A+Q695A+H698A, K526A+R691E+Q695A+H698A and K526A+R691A+Q695A+H698E.

24. The Cas9 nuclease or enzymatically active fragment thereof according to claim 22 or 23, wherein the Cas9 nuclease is SpCas9, and optionally, the amino acid sequence of the Cas9 nuclease is as shown in SEQ ID NO: 36, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:

36.

25. A gRNA targeting a TTR gene, which is a sgRNA, and whose nucleotide sequence is as shown in any one of SEQ ID NOs: 10-16 or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the nucleotide sequence as shown in any one of SEQ ID NOs: 10-16; or It consists of crRNA and tracrRNA, wherein the crRNA is as shown in SEQ ID NO.18 or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the nucleotide sequence as shown in SEQ ID NO:18, and the tracrRNA is as shown in SEQ ID NO.19 or comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the nucleotide sequence as shown in SEQ ID NO.

19.

26. A composition, a ribonucleoprotein complex or a protein lipid complex comprising the Cas9 nuclease or an enzymatically active fragment thereof according to any one of claims 22-23, or the gRNA according to claim 24.

27. An engineered vector encoding the Cas9 nuclease or enzymatically active fragment thereof according to any one of claims 21-23 and / or the gRNA according to claim 24.

28. A method for editing a transthyretin gene in a host cell, comprising introducing into the host cell the gene editing composition of any one of claims 1-21, the ribonucleoprotein complex or protein lipid complex of claim 26, or the engineered vector of claim 27.

29. A method for preventing or treating a transthyretin gene-related disease or condition, comprising introducing into a subject the gene editing composition of any one of claims 1-21, the ribonucleoprotein complex or protein lipid complex of claim 26, or the engineered vector of claim 27.

30. The method of claim 28 or 29, wherein the TTR-related disease or disorder comprises transthyretin amyloidosis.

31. The method of claim 30, wherein the TTR-related disease or condition comprises cardiomyopathy and neuropathy caused by transthyretin amyloidosis.

32. Use of the gene editing composition of any one of claims 1-21, the ribonucleoprotein complex or protein lipid complex of claim 26, or the engineered vector of claim 27 in the preparation of a drug for editing the transthyretin gene in a host cell.

33. Use of the gene editing composition of any one of claims 1-20, the ribonucleoprotein complex or protein lipid complex of claim 26, or the engineered vector of claim 27 in the preparation of a drug for preventing or treating a transthyretin gene-related disease or condition.

34. The use of claim 32 or 33, wherein the TTR-related disease or disorder comprises transthyretin amyloidosis.

35. The use of claim 34, wherein the TTR-related disease or condition comprises cardiomyopathy and neuropathy caused by transthyretin amyloidosis.

36. A pharmaceutical composition comprising the gene editing composition of any one of claims 1-21, the ribonucleoprotein complex or protein lipid complex of claim 26, or the engineered vector of claim 27, and a pharmaceutically acceptable carrier.

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