Guide RNA (Ribonucleic Acid) molecule, vector and application of guide RNA molecule and vector in medicine for preventing and treating ATTR (Attenuated Transcription Tumor

Abstract

The invention discloses a guide RNA (Ribonucleic Acid) molecule, a vector and application of the guide RNA molecule and the vector in a medicine for preventing and treating ATTR (Attenuated Transporter Receptor), and relates to the technical field of gene therapy. The guide RNA is set according to the coding sequence of the TTR gene, the guide RNA for gene editing is screened, it is found that the sequence similar to the guide sequence of the guide RNA has high TTR gene editing efficiency, the expression level of TTR in the liver of an experimental mouse and the level of TTR in blood circulation can be effectively reduced by constructing a gene editing carrier, and the TTR gene editing efficiency is improved. And the deposition of TTR between important organs and tissues can be effectively improved. Therefore, the guide RNA provided by the invention has a good application prospect in prevention and / or treatment of ATTR.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy technology, and in particular to a guide RNA molecule, a vector and applications thereof in drugs for preventing and treating ATTR. Background Art

[0002] Amyloid transthyretin (ATTR) is a human disease caused by the deposition of transthyretin (TTR). TTR is a homotetrameric protein in the human body that is mainly synthesized in the liver and choroid plexus of the brain and secreted into plasma and cerebrospinal fluid. Under physiological conditions, TTR can transport thyroxine and retinol to tissues and organs throughout the body, thereby supporting physiological functions such as cell metabolism and growth. Aging or TTR gene mutations can cause TTR protein monomers to misfold and aggregate to form amyloid fibers, which are systematically deposited in a variety of important tissues and organs, causing tissue damage or organ failure. ATTR patients have poor quality of life and low survival rate, with an average life expectancy of 7-12 years after the disease is diagnosed. Patients with ATTR involving the heart generally have a median survival of only 35 months, which seriously endangers human life and health.

[0003] Liver transplantation was the first proposed treatment for ATTR, but studies have found that liver transplantation cannot stop the deposition of amyloid TTR in tissues. Molecular drugs targeting different processes of amyloidosis have also been used to treat ATTR, such as the tetramer stabilizer tafamidis, the small interfering RNA drug patisiran, and the antisense nucleotide drug inotersen. In addition, monoclonal antibody drugs that target the removal of TTR amyloid protein (such as N1006) and gene editing drugs that target editing of the TTR gene to reduce TTR protein levels (such as NTLA-2001) have also shown certain therapeutic effects in clinical trials.

[0004] However, these treatments rely on long-term medication or long-term administration of drugs to patients, which leads to poor patient compliance and may increase the risk of infection in patients. In addition, the applicability of drugs that are approved or in clinical research is limited. For example, patisiran is approved for neurological ATTR and cannot alleviate cardiac damage in patients with cardiac ATTR; tafamids can only effectively treat ATTR caused by some gene mutations. Some mutant TTRs (such as TTRF44S) can cause gastrointestinal involvement in patients and significantly affect the quality of life of patients, but all small molecule drugs currently used in clinical practice have not been observed to improve ATTR gastrointestinal symptoms. Therefore, long-acting and widely used ATTR therapies are urgently needed to be developed and applied.

[0005] Invention WO202400385A1 discloses a method for treating ATTR by intravenously administering LNP-encapsulated RNA containing SaCas9 protein and sgRNA to target the liver and edit the TTR gene. However, LNP can cause severe inflammatory reactions and may cause side effects in the subject. In addition, since LNP-mediated RNP delivery cannot be effective for a long time, it may cause the gene editing efficiency to be too low and fail to produce effective therapeutic effects.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a guide RNA molecule, a vector and the use thereof in drugs for preventing and treating ATTR to solve the above-mentioned technical problems.

[0008] In the first aspect, the present invention provides a guide RNA molecule, which includes a guide sequence, wherein the guide sequence comprises: a. a sequence selected from SEQ ID NO:40-47; b. at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of a sequence selected from SEQ ID NO:40-47; or c. a sequence that is at least 90% identical to a sequence selected from SEQ ID NO:40-47.

[0009] In a second aspect, the present invention also provides a vector comprising a nucleic acid encoding the above-mentioned guide RNA molecule.

[0010] In a third aspect, the present invention also provides a composition comprising the above-mentioned guide RNA molecule or the above-mentioned vector.

[0011] In a fourth aspect, the present invention further provides a recombinant cell, which comprises the above-mentioned vector or the above-mentioned composition.

[0012] In a fifth aspect, the present invention further provides a virus particle, which comprises the above-mentioned vector or the above-mentioned composition and a capsid.

[0013] In a sixth aspect, the present invention also provides the use of the above-mentioned guide RNA molecule, the above-mentioned vector, the above-mentioned composition, the above-mentioned recombinant cell or the above-mentioned virus particle in the preparation of a drug for preventing and / or treating ATTR.

[0014] In a seventh aspect, the present invention further provides a pharmaceutical composition, which includes: the above-mentioned guide RNA molecule, the above-mentioned vector, the above-mentioned composition, the recombinant cell or the above-mentioned virus particle.

[0015] The present invention has the following beneficial effects:

[0016] The present invention sets a guide RNA for the coding sequence of the TTR gene, screens the guide RNA for gene editing, and finds that the sequence with a certain similarity to SEQ ID NO: 40-47 has a higher TTR gene editing efficiency. By constructing a gene editing vector, the expression level of TTR in the liver of experimental mice and the level of TTR in the blood circulation can be effectively reduced, and the deposition of TTR in important organs and tissues can be effectively improved. Therefore, the guide RNA provided by the present invention has a good application prospect in the prevention and / or treatment of ATTR. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 This is the structure diagram of the sgRNA targeting region designed in the CRISPR / SaCas9 gene editing system;

[0019] Figure 2 Schematic diagram of the expression vector structure of CRISPR / SaCas9;

[0020] Figure 3 This is a graph showing the gene editing efficiency test results for the CRISPR / SaCas9 gene editing system of gRNA1-4;

[0021] Figure 4 This is a diagram of the structure of the sgRNA targeting region designed in the CRISPR / SpCas9 gene editing system;

[0022] Figure 5 Schematic diagram of the expression vector structure of CRISPR / SpCas9;

[0023] Figure 6 This is a graph showing the gene editing efficiency test results for the CRISPR / SpCas9 gene editing system of gRNA1-6;

[0024] Figure 7 Schematic diagram of the rAAV vector structure of ssAAV8-TTR-SaCRISPR-U6-sgRNA;

[0025] Figure 8 Schematic diagram of the scAAV8-U6-sgRNA and ssAAV8-TTR-SpCas9 vector structures;

[0026] Fig. 9The results of the editing efficiency test of the human TTR (F44S) gene after administration of the SpCas9 group and the SaCas9 group (A) and the statistical results of the quantitative gene editing efficiency by next-generation sequencing (B);

[0027] Fig.10 The results of immunohistochemistry and immunoblotting to detect and quantify the expression level of TTR (F44S) gene in the liver tissue of mice three months after injection;

[0028] Fig.11 This is a statistical result diagram of the ELISA quantification of the level of TTRF44S in the blood circulation of the SpCas9 group and the SaCas9 group after administration;

[0029] Fig.12 Micrographs (A) and statistical results (B) of immunohistochemical detection and quantification of TTRF44S deposition levels in the heart, eyes, brain, cerebellum, spinal cord, stomach, small intestine, kidney and fat of mice three months after injection;

[0030] Fig.13 The results of the efficiency test of rAAV editing TTR (V122I) gene in mouse liver tissue three months after injection (A), the expression level test of TTRV122I (B) and the statistical results (C), and the statistical results of the level of TTRV122I in the blood circulation (D);

[0031] Fig.14 This is the off-target effect detection result diagram of the SpCas9 group and the SaCas9 group. DETAILED DESCRIPTION

[0032] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0033] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0034] As used herein, the term "guide RNA (gRNA)" refers to a combination of CRISPR RNA (crRNA) and tracrRNA (trRNA). "Guide RNA" can be used interchangeably with "guide". CrRNA and trRNA can be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" can refer to each type, i.e., sgRNA or dgRNA; "sgRNA" of this application may also be referred to as "gRNA" for short. TrRNA can be a naturally occurring sequence, or the trRNA sequence can have modifications or variations compared to a naturally occurring sequence.

[0035] As used herein, "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and is used to guide the guide RNA to the target sequence for binding or modification (e.g., cutting) by an RNA-guided DNA binder. A "guide sequence" may also be referred to as a "guide region," "targeting sequence," "spacer sequence," or "protospacer sequence." The guide sequence may be about 20 base pairs in length, for example, in the case of Streptococcus pyogenes (i.e., SpyCas9) and related Cas9 homologs / orthologs. Shorter or longer sequences may also be used as guide sequences, for example, 14, 15, 16, 17, 18, 19, 21, 22 nucleotides in length. In some embodiments, the guide sequence and the targeting sequence may be 100% complementary or identical to each other in sequence. In other embodiments, the guide sequence and the targeting sequence may contain at least one mismatch. For example, the guide sequence and the targeting sequence can contain 1, 2, 3, 4, 5, or 6 mismatches, wherein the total length of the targeting sequence is at least 15, 16, 17, 18, 19, 20, 21, or more base pairs. In some embodiments, the guide sequence and the targeting sequence can contain 1-4 mismatches, wherein the guide sequence contains at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the targeting sequence can contain 1, 2, 3, or 4 mismatches, wherein the guide sequence contains at least 19 or 20 nucleotides.

[0036] As used herein, the "target sequence" or "targeting sequence" of a gRNA comprises both strands of genomic DNA, i.e., the target sequence and the complementary sequence of the target sequence.

[0037] As used herein, "complementary" means that two nucleic acid sequences can perform base pairing in the direction from 5' to 3', for example, the complementary sequence of the sequence "5'-GAACTGAG-3'" is "5'-CTCAGTTC-3'".

[0038] As used herein, "scaffold" refers to the nucleic acid sequence in the sgRNA other than the guide sequence.

[0039] It should be understood that gRNA, guide sequence, and gRNA scaffold can be an RNA sequence, or a DNA sequence or DNA region corresponding to the RNA sequence.

[0040] As used herein, "ATTR" is a human disease caused by TTR deposition, including wild-type ATTR and hereditary ATTR. Wild-type ATTR is an acquired amyloidosis disease, generally believed to be caused by aging-related TTR protein misfolding. Hereditary ATTR is a genetic disease caused by TTR gene mutations, and typical phenotypes include familial amyloid polyneuropathy, familial amyloid cardiomyopathy, and familial leptomeningeal amyloidosis. A variety of TTR pathogenic mutations have been found, including but not limited to all mutation types published by amyloidosismutations.com (DOI: 10.1002 / humu.22619). The combination of two TTR alleles in a hereditary ATTR patient can be a wild-type TTR gene and a mutant TTR gene; or two mutant TTR alleles, wherein the two mutation types can be the same or different. It should be understood that, unless otherwise specified, ATTR should include wild-type ATTR and hereditary ATTR, and the hereditary ATTR should include ATTR caused by all TTR mutations that have been discovered and may be discovered in the future.

[0041] In the first aspect, the present invention provides a guide RNA molecule, the RNA molecule comprising a guide sequence, the guide sequence comprising: a. a sequence selected from SEQ ID NO:40-47; b. at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of a sequence selected from SEQ ID NO:40-47; or c. a sequence that is at least 90% identical to a sequence selected from SEQ ID NO:40-47.

[0042] SEQ ID NO: 40-41 targets exon 2 of the human TTR protein coding region; SEQ ID NO: 42-47 targets exon 2 of the human TTR protein coding region. SEQ ID NO: 40-41 are two sgRNA sequences targeting human TTR protein screened based on the PAM motif 5'-NNGRRT-3' (N represents any nucleotide, R represents A or G) of SaCas9, and SEQ ID NO: 42-47 are sgRNA sequences targeting six human TTR proteins screened based on the PAM motif 5'-NGG-3' (N represents any nucleotide) of SpCas9.

[0043] The present invention found that sequences with a certain degree of similarity to SEQ ID NO: 40-47 have a higher TTR gene editing efficiency. By constructing a gene editing vector, the expression level of TTR in the liver of experimental mice and the level of TTR in the blood circulation can be effectively reduced, and the deposition of TTR in important organs and tissues can be effectively improved. Therefore, the guide RNA provided by the present invention has a good application prospect in the prevention and / or treatment of ATTR.

[0044] For example, the guide sequence of the guide RNA molecule is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 40-47.

[0045] In a preferred embodiment of the present invention, the guiding sequence comprises: a. a sequence selected from SEQ ID NO:41 or SEQ ID NO:43; b. at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of a sequence selected from SEQ ID NO:41 or SEQ ID NO:43; or c. a sequence that is at least 90% identical to a sequence selected from SEQ ID NO:41 or SEQ ID NO:43.

[0046] Compared with SEQ ID NO:40, the above-mentioned sequence of SEQ ID NO:41 has a higher gene editing efficiency; compared with SEQ ID NO:42 and SEQ ID NO:44-47, the above-mentioned sequence of SEQ ID NO:43 has a higher gene editing efficiency.

[0047] In particular, compared with the CRISPR / SpCas9 gene editing system, the CRISPR / SaCas9 gene editing system has lower potential off-target effects. The CRISPR / SaCas9 system can effectively reduce the level of TTR in the blood circulation and the expression level of TTR in mouse liver, and can more significantly improve the deposition of TTR in important organs and tissues.

[0048] In a preferred embodiment of the present invention, the sequence of SEQ ID NO:41 has a better therapeutic effect than the sequence of SEQ ID NO:43, can more effectively reduce the level of TTR in the blood circulation and reduce the expression level of TTR in mouse liver, and can more significantly improve the deposition of TTR between important organs and tissues, and has a lower potential off-target effect.

[0049] In a preferred embodiment of the present invention, the guide RNA molecule further comprises a scaffold sequence or a portion thereof, wherein the scaffold sequence comprises the nucleotide sequence shown in SEQ ID NO: 48 or 49, wherein the scaffold sequence is operably linked to the guide sequence.

[0050] In a preferred embodiment of the present invention, the guide RNA is a dual guide RNA (dgRNA) or a single guide RNA (sgRNA).

[0051] In a second aspect, the present invention also provides a vector comprising a nucleic acid encoding the above-mentioned guide RNA molecule.

[0052] In a preferred embodiment of the present invention, the vector further comprises a gene regulatory sequence and a nuclear localization signal coding sequence;

[0053] In a preferred embodiment of the present invention, the gene regulatory sequence includes: a promoter and a terminator;

[0054] In the preferred embodiment of the present invention, the vector further comprises a coding sequence of a Cas nuclease or a Cas nickase. In some embodiments, the Cas nuclease is a Class 2 Cas nuclease. In some embodiments, the Cas nuclease is Cas9, Cpfl, C2cl, C2c2, and C2c3 or a modified protein thereof. In some embodiments, the Cas nuclease is a Streptococcus pyogenes or Staphylococcus aureus Cas9 nuclease or a modified protein thereof. In some embodiments, the Cas nuclease is from a Type II CRISPR / Cas system. In some embodiments, the Cas nuclease is fused to an exonuclease. In some embodiments, the exonuclease is selected from the group consisting of TREX1, TREX2, and MRE11. In some embodiments, the exonuclease is a truncated exonuclease. In some embodiments, the Cas nuclease is fused to the exonuclease at the C-terminus via a GGGGS linker.

[0055] In a preferred embodiment of the present invention, the Cas nuclease is selected from the SaCas9 protein.

[0056] In a preferred embodiment of the present invention, the promoter is selected from a constitutive promoter, an inducible promoter, a ubiquitous promoter, a tissue-specific promoter, a cell type-specific promoter, or a developmental stage-specific promoter.

[0057] In a preferred embodiment of the present invention, the promoter is a type II promoter or a type III promoter.

[0058] In a preferred embodiment of the present invention, the promoter is selected from at least one of a TTR promoter, an EF promoter and a human U6 promoter.

[0059] In a preferred embodiment of the present invention, the terminator is selected from SV40 polyA, bGH polyA, BETAPolyA or 6XT.

[0060] In a preferred embodiment of the present invention, the vector comprises at least one of a recombinant adeno-associated virus vector, a lentivirus, an adenovirus and a poxvirus.

[0061] The AAV nucleic acid sequence of the recombinant adeno-associated virus is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or a combination thereof.

[0062] The terms AAV vector, AAV particle, AAV vector particle, recombinant AAV particle, recombinant AAV vector particle and rAAV are used interchangeably and, as used herein, refer to AAV capsids with or without a DNA genome comprising ITRs produced by producer cells or packaging cells, respectively.

[0063] In the preferred embodiment of the present invention, the vector also includes a selectable marker gene or a reporter gene. The term "selectable marker" refers to a gene that will help select cells that actively express the nucleic acid sequence. Examples of suitable selectable markers include enzymes that encode resistance to antibiotics (i.e., antibiotic resistance genes), such as kanamycin, neomycin, puromycin, hygromycin, blasticidin or bleomycin.

[0064] In addition, the selectable marker gene also comprises an amplifiable selectable marker gene. Mentioning " amplifiable selectable marker gene " as used herein refers to the gene that allows to increase this gene under suitable growth conditions. The amplifiable selectable marker gene can respond to the shortage of inhibitor or essential metabolite by amplification to increase the expression product (that is, by the expression of the protein encoded by the amplifiable selectable marker gene). In one embodiment, the amplifiable selectable marker gene can be characterized as being able to complement with an auxotrophic host.

[0065] An example of a reporter gene is a fluorescent protein, such as green fluorescent protein (GFP), red fluorescent protein (RFP), or blue fluorescent protein (BFP).

[0066] In a preferred embodiment of the present invention, the nuclear localization signal encoding sequence is selected from any one of SEQ ID NOs: 23-24.

[0067] In a third aspect, the present invention also provides a composition comprising the above-mentioned guide RNA molecule or the above-mentioned vector.

[0068] The composition is a gene editing system.

[0069] In a preferred embodiment of the present invention, when the composition is a CRISPR / SpCas9 system, it also includes a recombinant vector encoding SpCas9;

[0070] In a preferred embodiment of the present invention, the recombinant vector encoding SpCas9 further includes a promoter, a nuclear localization signal coding sequence and a terminator.

[0071] In a fourth aspect, the present invention further provides a recombinant cell, which comprises the above-mentioned vector or the above-mentioned composition.

[0072] In a preferred embodiment of the present invention, the recombinant cells include but are not limited to: bacteria, fungi or 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6 cells, 293 series cells, Per6 cells and CHO cells.

[0073] In a preferred embodiment of the present invention, the bacteria include at least one of Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli and Bacillus subtilis.

[0074] In a preferred embodiment of the present invention, the fungus comprises at least one of Trichoderma reesei and yeast.

[0075] The host cells mentioned above include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parental cells in nucleic acid content, but may contain mutations.

[0076] The above-mentioned recombinant cells are prepared by transforming the recombinant expression vector into a host cell (such as a microorganism) by conventional methods in the art; the host microorganism can be any conventional host microorganism in the art, as long as the recombinant expression vector can stably replicate itself and the foreign gene carried can be effectively expressed. The host microorganism includes bacteria or fungi.

[0077] In a fifth aspect, the present invention also provides a virus particle, which comprises the above-mentioned vector or the above-mentioned composition and a capsid. The capsid (CAP) is composed of VP1, VP2, and VP3, which are capsid proteins encoded by the cap gene.

[0078] In a sixth aspect, the present invention also provides the use of the above-mentioned guide RNA molecule, the above-mentioned vector, the above-mentioned composition or the above-mentioned viral particle in the preparation of a drug for preventing and / or treating ATTR.

[0079] In a preferred embodiment of the present invention, ATTR is caused by a gene mutation or a non-gene mutation;

[0080] In a preferred embodiment of the present invention, ATTR is selected from at least one of the following: F44S, V122I, D18G, G53R, Y114C, T49P, T60A, P24S, D38A, L58H, V30M, F64L, G47R, S50I, F33V, A45T, E89K and E89Q.

[0081] The drug also includes pharmaceutically acceptable carriers, including but not limited to fillers, lubricants, disintegrants, binders, glidants, etc.

[0082] In the preferred technical scheme of the present invention, the pharmaceutically acceptable carrier includes but is not limited to polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo or a combination thereof.

[0083] In a seventh aspect, the present invention further provides a pharmaceutical composition, which includes: the above-mentioned guide RNA molecule, the above-mentioned vector, the above-mentioned composition, the above-mentioned recombinant cell or the above-mentioned virus particle.

[0084] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0085] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0086] The inventors screened the sgRNAs of CRISPR / SaCas9 and CRISPR / SpCas9 systems, selected the sgRNA with the highest editing efficiency to construct rAAV vectors, and + / TTR(F44S) Model mice and Ttr + / TTR(V122I) The therapeutic effect of rAAV treatment on TTR was evaluated in model mice.

[0087] Example 1

[0088] Screening sgRNA for CRISPR / SaCas9 gene editing system

[0089] The inventors screened the protospacer sequences of four human TTR protein coding regions based on the PAM motif 5'-NNGRRT-3' (N represents any nucleotide) of SaCas9, and designed sgRNAs for the CRISPR / SaCas9 system, including a guide region and a scaffold sequence. The designed sgRNA targeting region is as follows Figure 1 shown.

[0090] The pre-spacer sequence and sgRNA sequence contained in the designed sgRNA are shown in Table 1:

[0091] Table 1

[0092]

[0093] Clone the above gRNA into the CRISPR / SaCas9 expression vector, the vector structure is as follows Figure 2 (EF promoter sequence is shown in SEQ ID NO.21, NLS1 nuclear localization signal is shown in SEQ ID NO.23, NLS2 nuclear localization signal is shown in SEQ ID NO.24, BGH transcription terminator is shown in SEQ ID NO.27, SaCas9 nucleotide sequence is shown in SEQ ID NO.25, SaCas9 amino acid sequence is shown in SEQ ID NO.26, human U6 promoter sequence is shown in SEQ ID NO.29).

[0094] The above cloned plasmids were transfected into HEK293T cells, and cell samples were collected 48h after transfection. The gene editing efficiency of the above gRNAs was compared by T7E1 restriction analysis. Cells transfected with empty plasmids were used as negative controls (NC). The results showed that compared with the control group, gRNA3 and gRNA4 had obvious restriction bands, and the original bands were significantly faded after T7E1 treatment.

[0095] This indicates that it has a high gene editing efficiency ( Figure 3 , "+" represents T7E1 treatment, "-" represents no T7E1 treatment). Among them, gRNA4 was used to construct rAAV.

[0096] Example 2

[0097] This example screened sgRNAs for the CRISPR / SpCas9 gene editing system

[0098] The inventors screened the protospacer sequences of six human TTR protein coding regions based on the SpCas9 PAM motif 5'-NGG-3' (N represents any nucleotide), and designed sgRNAs for the CRISPR / SpCas9 system, including a guide region and a scaffold. The designed sgRNA targeting region is as follows Figure 4 The amino acid sequence of SpCas9 is shown in SEQ ID NO.31, the nucleotide sequence of SpCas9 is shown in SEQ ID NO.30, and the polyA sequence is shown in SEQ ID NO.28. The pre-spacer sequence and sgRNA sequence included in the designed sgRNA are shown in Table 2:

[0099] Table 2

[0100]

[0101] Clone the above gRNA into the CRISPR / SpCas9 expression vector, the vector structure is as follows Figure 5 shown.

[0102] The above cloned plasmids were transfected into HEK293T cells, and cell samples were collected 48h after transfection. The gene editing efficiency of the above gRNAs was compared by T7E1 restriction analysis. Cells transfected with empty plasmids were used as negative controls (NC). The results showed that compared with the control group, gRNA2 showed the highest gene editing efficiency, and the original band was significantly diluted after T7E1 treatment ( Figure 6 , "+" represents T7E1 treatment, and "-" represents no T7E1 treatment). Therefore, gRNA2 was used to construct rAAV.

[0103] Example 3

[0104] Preparation of rAAV for targeted editing of the human TTR gene.

[0105] In this example, ssAAV8-TTR-SaCRISPR-U6-sgRNA was prepared based on the CRISPR / SaCas9 system to target and edit the human TTR gene. Figure 7 As shown in the figure, sgRNA is driven by the U6 promoter; SaCas9 is expressed by the liver-specific TTR promoter and is introduced into the nucleus through the NLS signal, and then binds to the sgRNA to target and edit the human TTR gene.

[0106] The ssAAV8-TTR-SaCRISPR-U6-sgRNA delivery sequence is shown in SEQ ID NO.32.

[0107] Example 4

[0108] Preparation of rAAV for targeted editing of the human TTR gene.

[0109] In this example, rAAV was prepared based on the CRISPR / SpCas9 system to target and edit the human TTR gene. The rAAV consisted of two rAAVs: scAAV8-U6-sgRNA and ssAAV8-TTR-SpCas9. The structure of the rAAVs is shown in FIG. Figure 8 As shown. Among them, scAAV8-U6-sgRNA can express sgRNA under the drive of U6 promoter ( Figure 8 ssAAV8-TTR-SpCas9 can express SpCas9 under the mediation of the liver-specific TTR promoter and enter the nucleus under the mediation of NLS ( Figure 8 Down).

[0110] The scAAV8-U6-sgRNA delivery sequence is shown in SEQ ID NO.33, and the ssAAV8-TTR-SpCas9 delivery sequence is shown in SEQ ID NO.34.

[0111] Example 5

[0112] Using Ttr + / TTR(F44S) Model mice were used to evaluate the therapeutic effect of rAAV in vivo.

[0113] The wild-type coding sequence of human TTR is shown in SEQ ID NO: 35, the full-length amino acid sequence of human TTR is shown in SEQ ID NO: 36, the partial amino acid sequence of human TTR is shown in SEQ ID NO: 37, and the human TTR F44S The amino acid sequence is shown in SEQ ID NO: 38.

[0114] Humanized male C57BL / 6 mouse Ttr + / TTR(F44S) (Customized mice) were grouped according to Table 3 and injected with ssAAV8-TTR-SaCas9-U6-sgRNA, scAAV8-U6-sgRNA / ssAAV8-TTR-SpCas9 or PBS, respectively, to evaluate the effects of these rAAVs in treating ATTR.

[0115] Table 3

[0116]

[0117] The liver tissue of mice was taken three months after injection, and the efficiency of rAAV editing of TTR (F44S) gene was detected by T7E1 enzyme digestion analysis and next-generation sequencing. The results of T7E1 enzyme digestion analysis showed that compared with the PBS group, both the SpCas9 group and the SaCas9 group had enzyme digestion bands, indicating that both the SpCas9 group and the SaCas9 group could effectively edit the human TTR (F44S) gene ( Fig. 9 In the A, "+" represents T7E1 treatment, and "-" represents no T7E1 treatment). The results of the quantitative gene editing efficiency of the second-generation sequencing showed that the editing efficiency of the SpCas9 group was about 15%, while the editing efficiency of the SaCas9 group was about 41%, which was significantly higher than that of the SpCas9 group ( Fig. 9 (B).

[0118] The expression level of TTR(F44S) gene in the liver tissue of mice three months after injection was detected and quantified by immunohistochemistry. The results showed that compared with the PBS group, the expression level of TTR(F44S) gene in the liver of the SpCas9 group was significantly higher than that in the PBS group. F44S There was no significant change in the expression level, but TTR F44S The expression level was significantly downregulated ( Fig.10The expression of TTR(F44S) gene in the liver tissue of mice three months after injection was detected and quantified by western blotting, and it was also found that the expression level of TTRF44S in the liver of the SaCas9 group was significantly downregulated ( Fig.10 C and D in ).

[0119] Blood samples were collected from mice every two weeks after injection and the circulating TTR was quantified by ELISA. F44S The results showed that both the SpCas9 group and the SaCas9 group could effectively reduce the level of TTR in the blood circulation. F44S level, and the SaCas9 group showed better therapeutic effect ( Fig.11 ).

[0120] Immunohistochemistry was used to detect and quantify TTR in the heart, eyes, brain, cerebellum, spinal cord, stomach, small intestine, kidney and fat of mice three months after injection. F44S The results showed that SaCas9 administration could significantly alleviate TTR F44S Deposits in the heart, cerebellum, spinal cord, stomach, small intestine, kidneys, and fat ( Fig.12 ).

[0121] Example 6

[0122] Using Ttr + / TTR(V122I) Model mice to evaluate the therapeutic effect of rAAV in vivo

[0123] Human TTR V122I The amino acid sequence of is shown in SEQ ID NO: 39. Humanized male C57BL / 6 mouse Ttr + / TTR(V122I) (Customized mice) were grouped according to Table 4 and injected with ssAAV8-TTR-SaCRISPR or PBS, respectively, to evaluate the effect of ssAAV8-TTR-SaCas9-U6-sgRNA in treating ATTR.

[0124] Table 4

[0125]

[0126] The genome was extracted from the liver tissue of mice three months after injection, and the efficiency of rAAV editing of the TTR (V122I) gene was detected by next-generation sequencing. The results showed that the editing efficiency of the SaCas9 group was about 48% ( Fig.13 Middle A) Western blotting was used to detect TTR in the liver tissue of mice three months after injection V122I The expression level of TTR in the liver of the SaCas9 group was significantly higher than that in the PBS group. V122I The expression level of Fig.13B and C). Blood samples were collected from mice every two weeks after injection and circulating TTR was quantified by ELISA. V122I The results showed that the administration of SaCas9 group can effectively reduce the level of TTR in blood circulation V122I Level ( Fig.13 (middle D).

[0127] Example 7

[0128] Assessing the safety of rAAV-mediated TTR-targeted editing

[0129] The rAAV genome plasmids in Examples 3-4 were transfected into HEK293T cells. Cell samples were collected 48 h after transfection and the genome was extracted. Potential off-target regions were amplified by PCR (Tables 5 and 6), and the off-target editing efficiency of CRISPR / SpCas9 and CRISPR / SaCas9 was detected by next-generation sequencing technology.

[0130] Table 5: Potential off-target sites of the CRISPR / SpCas9 system

[0131]

[0132] Table 6: Potential off-target sites of the CRISPR / SaCas9 system

[0133]

[0134] The regions with mutations of no more than 4 nt from the targeted region were defined as potential off-target sites. The results showed that compared with the PBS transfection group, no obvious off-target effect was observed in the SaCas9 group, while significant off-target editing was observed in the SpCas9 group ( Fig.14 ).

[0135] In summary, the gene editing vector targeting TTR delivered by rAAV described in the present invention can effectively reduce the expression level of TTR in the liver of experimental mice, the level of TTR in the blood circulation, and can effectively improve the deposition of TTR in important organs and tissues. Compared with the CRISPR / SpCas9 gene editing system, the CRISPR / SaCas9 gene editing system has lower potential off-target effects.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A guide RNA molecule, characterized in that The guide RNA molecule includes a guide sequence, which comprises: a. a sequence selected from SEQ ID NO:40-47; b. at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of a sequence selected from SEQ ID NO:40-47; or c. a sequence that is at least 90% identical to a sequence selected from SEQ ID NO:40-47.

2. The guide RNA molecule according to claim 1, characterized in that The guide sequence comprises: a. a sequence selected from SEQ ID NO:41 or SEQ ID NO:43; b. at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of a sequence selected from SEQ ID NO:41 or SEQ ID NO:43; or c. a sequence that is at least 90% identical to a sequence selected from SEQ ID NO:41 or SEQ ID NO:

43.

3. The guide RNA molecule according to claim 1, characterized in that The guide RNA molecule further comprises a scaffold sequence or a portion thereof, wherein the scaffold sequence comprises the nucleotide sequence shown in SEQ ID NO: 48 or 49, wherein the scaffold sequence is operably linked to the guide sequence; Preferably, the guide RNA is a dual guide RNA (dgRNA) or a single guide RNA (sgRNA).

4. A carrier, characterized in that It comprises a nucleic acid encoding the guide RNA molecule according to any one of claims 1 to 3.

5. The carrier according to claim 4, characterized in that The vector also includes a gene regulatory sequence and a nuclear localization signal coding sequence; Preferably, the gene regulatory sequence includes: a promoter and a terminator; Preferably, the vector further comprises a coding sequence for Cas nuclease or Cas nickase; Preferably, the Cas nuclease is selected from SaCas9 protein; Preferably, the promoter is selected from a constitutive promoter, an inducible promoter, a ubiquitous promoter, a tissue-specific promoter, a cell type-specific promoter, or a developmental stage-specific promoter; Preferably, the promoter is selected from at least one of a TTR promoter, an EF promoter and a human U6 promoter; Preferably, the vector comprises a recombinant adeno-associated virus vector, a lentivirus, an adenovirus or a poxvirus; Preferably, the nuclear localization signal encoding sequence is selected from any one of SEQ ID NOs: 23-24.

6. A composition, characterized in that It comprises the guide RNA molecule according to any one of claims 1 to 3 or the vector according to any one of claims 4 to 5; Preferably, when the composition is a CRISPR / SpCas9 system, it further comprises a recombinant vector encoding SpCas9; Preferably, the recombinant vector encoding SpCas9 further comprises a promoter, a nuclear localization signal coding sequence and a terminator.

7. A recombinant cell, characterized in that It comprises the carrier according to any one of claims 4 to 5 or the composition according to claim 6.

8. A virus particle, characterized in that It comprises the vector according to any one of claims 4 to 5 or the composition according to claim 6 and a capsid.

9. Use of the guide RNA molecule according to any one of claims 1 to 3, the vector according to any one of claims 4 to 5, the composition according to claim 6, the recombinant cell according to claim 7 or the viral particle according to claim 8 in the preparation of a drug for preventing and / or treating ATTR; Preferably, the ATTR is caused by a gene mutation or a non-gene mutation; Preferably, the ATTR is selected from at least one of the following: F44S, V122I, D18G, G53R, Y114C, T49P, T60A, P24S, D38A, L58H, V30M, F64L, G47R, S50I, F33V, A45T, E89K and E89Q.

10. A pharmaceutical composition, characterized in that It includes: The guide RNA molecule according to any one of claims 1 to 3, the vector according to any one of claims 4 to 5, the composition according to claim 6, the recombinant cell according to claim 7, or the viral particle according to claim 8.

Images