A recombinant adeno-associated viral vector and uses thereof

Abstract

This invention belongs to the field of molecular biology and discloses a recombinant adeno-associated virus (AAV) vector and its applications. The recombinant AAV vector comprises an AAV capsid and a non-self-complementary single-stranded genome; the single-stranded genome contains an EF1α promoter and a gene encoding a motor neuron survival protein. By employing the EF1α promoter to regulate SMN1 gene expression, this invention prevents persistently high expression of the SMN gene after SMA patients have developed, thus avoiding delayed motor neuron degeneration caused by persistent SMN overexpression. Therefore, the recombinant AAV vector provided by this invention is significantly more effective in treating SMA than recombinant AAV vectors containing other promoters: it prolongs mouse survival time, increases mouse body weight, and improves the development of motor neurons and skeletal muscle-related tissues. It can be used to prevent, alleviate, or treat neurological diseases.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a recombinant adeno-associated virus vector and its applications. Background Technology

[0002] Adeno-associated virus (AAV) is a species of parvovirus. The AAV genome consists of a linear single-stranded DNA molecule approximately 4.7 kb in length, with two major open reading frames encoding non-structural replication and structural cap (capsid) proteins. Flanking the AAV coding region are two cis-acting inverted terminal repeats (ITRs), approximately 145 nucleotides in length, which act as primers during the initiation of DNA replication. In addition to their role in DNA replication, ITR sequences have been shown to play a role in viral integration, encapsulating viral nucleic acids into mature viral particles, and other processes. The cis-acting sequences guiding viral DNA replication (rep), encapsulation / packaging, and integration into the host cell chromosome are contained within the ITRs. The cap gene, expressed from the p40 promoter, encapsulates three capsid proteins: VP1, VP2, and VP3. These capsid proteins are encoded by the cap gene, and depending on the cap gene, AAV exhibits multiple serotypes and provides different tissue predispositions. AAV has multiple serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. Among them, AAV9 and its mutants AAVPHP.eb and AAVPHP.b are used in cell delivery targeting the central nervous system for the treatment of neurodegenerative diseases.

[0003] AAV vectors have great potential for in vivo delivery of genetic material because (i) they can infect (transduce) a variety of non-dividing and dividing cell types, including myofibrils and neurons; (ii) they do not contain viral structural genes, thus eliminating the host cell’s natural response to viral infection, such as interferon-mediated responses; (iii) wild-type viruses have never been associated with any pathology in humans; (iv) unlike wild-type AAVs that can integrate into the host cell genome, replication-defective AAV vectors are usually present in free form, thus limiting the risk of insertional mutations or oncogene activation; and (v) compared to other vector systems, AAV vectors do not elicit a significant immune response, thus enabling long-term expression of therapeutic transgenes (provided their gene products are not rejected).

[0004] Spinal muscular atrophy (SMA) is a neurogenetic disorder caused by a deletion or mutation in the survival motor neuron 1 gene (SMN1) on chromosome 5q13. This leads to decreased SMN protein levels and selective motor neuron dysfunction. SMA is an autosomal recessive early childhood disease with an incidence of 1 in 10,000. Humans also carry another gene almost identical to SMN1, called SMN2. Both SMN1 and SMN2 genes express SMN protein, but SMN2 produces far less functional full-length protein than SMN1 (10–15%). Although SMN2 cannot completely compensate for the deletion of the SMN1 gene, patients with mild SMA usually have a high SMN2 copy number. Two copies of SMN2 can predict type I SMA with 97%, three copies with 83% predict type II SMA with 83%, and four copies with 84% predict type III SMA. Since these percentages do not reflect the potential impact of gene-modifying mutations, they may underestimate the relationship between copy number (in the absence of genetically modified genes) and clinical phenotype.

[0005] Type I SMA is a leading cause of infant mortality due to genetic disorders. The severity and clinical prognosis depend on the copy number of SMN2. In its most common and severe form (Type I), hypotonia and progressive weakness are noticed in the first few months after birth, diagnosed around 6 months, and often result in death from respiratory failure around 2 years of age. Motor neuron loss in Type I SMA is severe early in life (and may even begin prenatally), and patients never achieve an independent sitting posture. Type I SMA patients typically have 1 or 2 copies of the SMN2 gene. In contrast, Type II SMA appears within the first 18 months, and children with this condition can sit independently but cannot walk. Type II SMA patients typically have 3 copies of the SMN2 gene. Type III SMA patients acquire the ability to walk independently. The motor neurons in Type II and Type III SMA patients appear to adapt and compensate during development and persist into adulthood. Type III SMA patients typically have 3 or 4 copies of the SMN2 gene. The clinical course of Type II and Type III SMA is relatively stable. Furthermore, research indicates that the difference in outcomes is related to the number of SMN2 copies, which enables motor neurons to adapt and compensate during childhood and persist into adulthood. This differs from type I SMA, which suffers from severe motor neuron loss early in life (and may even begin prenatally, particularly in type I SMA patients presenting within three months of birth). Overexpression of SMN has proven well-tolerated in mice and non-human primates, while in humans, high SMN2 copy numbers do not pose a risk (as seen in type II, III, and IV patients, who have higher SMN2 copy numbers). To date, advances in SMA treatment research, particularly for type II and III SMA, have primarily focused on increasing SMN levels using small molecules. These drugs include deacetylase inhibitors such as valproic acid, sodium butyrate, phenyl butyrate, and triamcinolone axetine. These drugs activate the SMN2 promoter, leading to an increase in full-length SMN protein in animal models of SMA, with the aim of altering the disease phenotype to a milder profile seen in type III SMA patients.

[0006] Currently, drugs in clinical trials include phenylbutyric acid, valproic acid, and hydroxyurea, but none have produced sufficient clinical benefits. Nusinersen (SPINRAZA), an antisense oligonucleotide (ASO) drug approved by the FDA, aims to increase the production of full-length SMN protein by regulating the splicing of the SMN2 gene, thereby compensating for potential genetic defects. Clinical studies show promise in improving motor disorders; however, this treatment requires indefinite intrathecal administration, a long induction period before it takes effect, and safety considerations necessitate clinical monitoring. Zolgensma, also approved by the FDA, delivers its complementary double-stranded SMN1 expression sequence into the body via an AAV9 vector and achieves therapeutic effects under the regulation of cytomegalovirus (CMV) immediate / early enhancers and chicken β-actin (CB) promoters; however, current clinical data suggest that its efficacy in treating SMA patients needs further improvement. Summary of the Invention

[0007] The first aspect of the present invention is to provide a recombinant adeno-associated virus vector.

[0008] A second aspect of the present invention is to provide a pharmaceutical composition.

[0009] A third aspect of the present invention is to provide the application of the recombinant adeno-associated virus vector of the first aspect and the pharmaceutical composition of the second aspect.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a recombinant adeno-associated virus (AAV) vector comprising an AAV capsid and a non-self-complementary single-stranded genome; said single-stranded genome comprising an EF1α promoter and a gene encoding a motor neuron survival (SMN) protein.

[0012] Preferably, the adeno-associated virus (AAV) capsid is one of AAVPHP.eb, AAVPHP.b, and AAV9 capsid; more preferably, it is the AAV9 capsid.

[0013] Preferably, the EF1α promoter is any one of (a1) to (a3):

[0014] (a1) A promoter having the nucleotide sequence shown in SEQ ID NO.1;

[0015] (a2) The promoter shown in SEQ ID NO.1 is a nucleotide sequence that still has the function of the EF1α promoter after substitution, deletion or addition of one or more bases;

[0016] (a3) A promoter shown in a nucleotide sequence having 80%, 85%, 90%, 95%, 98% or 99% or more homology with the nucleotide sequence shown in SEQ ID NO.1 and having EF1α promoter function.

[0017] Preferably, the motor neuron survival (SMN) protein is the human motor neuron survival (SMN) protein.

[0018] Preferably, the motor neuron survival (SMN) protein is any one of (b1) to (b3):

[0019] (b1) A protein having the amino acid sequence shown in SEQ ID NO.2;

[0020] (b2) The protein shown in SEQ ID NO.2 that still has the function of a motor neuron survival protein after one or more amino acid sequences have been substituted, deleted or added;

[0021] (b3) Proteins having an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% or more homologous to the amino acid sequence shown in SEQ ID NO.2 and having the function of motor neuron survival protein;

[0022] Among them, SEQ ID NO.2 is: MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSP ICEVANNIEQNAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN.

[0023] More preferably, the gene encoding the survival motor neuron (SMN) protein is the human SMN1 gene (NM_000344).

[0024] Preferably, the single-stranded genome further comprises: WPRE.

[0025] Preferably, the single-stranded genome further comprises at least one of the following: a Kozak sequence, polyadenylated nucleotide (polyA), and AAV inverted terminal repeat (ITR) sequences.

[0026] More preferably, the single-stranded genome also includes a Kozak sequence, polyadenylated acid (polyA), WPRE, and AAV inverted terminal repeat (ITR) sequences.

[0027] Preferably, the AAV inverted terminal repeat (ITR) sequence comprises AAV 5'-ITR and AAV 3'-ITR.

[0028] Preferably, the polyadenylated acid is BGH polyadenylated acid (polyA).

[0029] Preferably, a recombinant adeno-associated virus (AAV) vector comprises an AAV capsid and a non-self-complementary single-stranded genome; said single-stranded genome comprises an AAV 5'-ITR, an EF1α promoter, a Kozak sequence, a gene encoding a survival motor neuron (SMN) protein, polyadenylate (polyA), WPRE, and AAV 3'-ITR.

[0030] A second aspect of the present invention provides a pharmaceutical composition comprising a recombinant adeno-associated virus vector of the first aspect of the present invention and a pharmaceutically acceptable carrier / excipient.

[0031] A third aspect of the present invention provides the use of any one of (1) to (2) in the preparation of a medicament for the prevention, alleviation or treatment of nervous system diseases:

[0032] (1) The recombinant adeno-associated virus vector of the first aspect of the present invention;

[0033] (2) The pharmaceutical composition of the second aspect of the present invention.

[0034] Preferably, the neurological disease is a motor neuron disease involving motor function.

[0035] Preferably, the motor neuron disease is selected from at least one of spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), bulbar muscular atrophy, spinocerebellar ataxia, primary lateral sclerosis (PLS), and traumatic spinal cord injury; more preferably, spinal muscular atrophy (SMA).

[0036] Preferably, the spinal muscular atrophy (SMA) includes type I spinal muscular atrophy (SMA), type II spinal muscular atrophy (SMA), type III spinal muscular atrophy (SMA), and type IV spinal muscular atrophy (SMA).

[0037] Preferably, the drug is administered by injection.

[0038] Preferably, the injection is at least one of intravenous injection, intraventricular injection, and intrathecal injection; more preferably, it is intraventricular injection.

[0039] The beneficial effects of this invention are:

[0040] This invention provides a recombinant adeno-associated virus (AAV) vector comprising an AAV capsid and a non-self-complementary single-stranded genome; the single-stranded genome contains an EF1α promoter and a gene encoding a motor neuron survival (SMN) protein; existing gene therapy drugs or strategies for SMA mostly use promoters with broad expression throughout the entire time period, especially those based on CMV (Human Cytomegalovirus Immediate Early Warning System). The enhancer / promoter (the immediate early enhancer promoter of human cytomegalovirus) is derived from the CB series promoter or the CAG series promoter. These promoters can maintain high levels of expression of the human SMN1 gene sequence they control in vivo. This invention uses the EF1α promoter to regulate SMN1 gene expression, so that the SMN gene is no longer persistently highly expressed after the development of SMA patients, avoiding the delayed degenerative lesions of motor neurons caused by persistent overexpression of SMN. As a result, the recombinant adeno-associated virus (AAV) vector provided by this invention has a significantly better therapeutic effect on SMA than recombinant adeno-associated virus (AAV) vectors containing other promoters: prolonging the survival time of mice, increasing the weight of mice, and improving the development of motor neurons and skeletal muscle-related tissues. It can be used to prevent, alleviate or treat neurological diseases.

[0041] This invention enables the efficient delivery of non-self-complementary single-stranded genomes to target organs and tissues by defining specific AAV viral vector serotypes. Attached Figure Description

[0042] Figure 1 This is the pDown-hSMN1[NM_000344] plasmid map.

[0043] Figure 2 This is the pAAV[Exp]-EF1α>hSMN1[NM_000344]:WPRE plasmid map.

[0044] Figure 3 This is the pAAV[Exp]-CAG>hSMN1[NM_000344]]:WPRE plasmid map.

[0045] Figure 4 This is a pDown-EGFP plasmid map.

[0046] Figure 5This is the pAAV[Exp]-EF1α>EGFP:WPRE plasmid map.

[0047] Figure 6 This is the pscAAV[Exp]-CBh>EGFP plasmid map.

[0048] Figure 7 This is the pAAV[Exp]-CAG>EGFP:WPRE plasmid map.

[0049] Figure 8 This is the pAAV[Exp]-EF1α>hSMN1[NM_000344] plasmid map.

[0050] Figure 9 This is the pAAV[Exp]-CAG>hSMN1[NM_000344] plasmid map.

[0051] Figure 10 The images show the results of SMN1 expression in 293T cells transduced with the viruses prepared in Examples 1 and 2: A is a gel electrophoresis result of SMN1 expression in 293T cells transduced with the viruses prepared in Examples 1 and 2; B is a statistical graph of SMN1 expression level in 293T cells transduced with the viruses prepared in Examples 1 and 2 relative to SMN1 expression level in 293T cells without virus transduction; 1 is the SMN1 expression level in 293T cells transduced with the viruses prepared in Example 2; 2 is the SMN1 expression level in 293T cells transduced with the viruses prepared in Example 1; and 3 is the SMN1 expression level in 293T cells without virus transduction.

[0052] Figure 11 The images show the EGFP expression results of the viruses prepared in Examples 4 and 5 in different tissues: A is the EGFP expression result of the virus prepared in Example 4 in different tissues; B is the EGFP expression result of the virus prepared in Example 5 in different tissues; and C is the EGFP expression result of the virus prepared in Example 3 in different tissues.

[0053] Figure 12 The images show the expression results of EGFP in the anterior horn of the spinal cord for the viruses prepared in Examples 3, 4, and 5: A is the expression result of EGFP in the anterior horn of the spinal cord for the viruses prepared in Examples 4 and 5 (scale bar is 50 μm); B is the expression result of EGFP in the anterior horn of the spinal cord for the virus prepared in Example 3 (scale bar is 100 μm).

[0054] Figure 13The images show the expression results of EGFP in the hippocampus of brain tissue by the viruses prepared in Examples 3, 4, and 5: A is the expression result of EGFP in the hippocampus of brain tissue by the viruses prepared in Examples 4 and 5 (scale bar is 50 μm); B is the expression result of EGFP in the hippocampus of brain tissue by the virus prepared in Example 3 (scale bar is 100 μm).

[0055] Figure 14 The images show the expression results of EGFP in the anterior horn of the spinal cord and hippocampus 10 or 40 days after injection of the virus prepared in Example 5: A is the expression result of EGFP in the anterior horn of the spinal cord 10 or 40 days after injection of the virus prepared in Example 5 (scale bar: 100 μm); B is the expression result of EGFP in the hippocampus 10 or 40 days after injection of the virus prepared in Example 5 (scale bar: 100 μm); C is the expression result of EGFP in the anterior horn of the spinal cord 10 or 40 days after injection of the virus prepared in Example 3 (scale bar: 100 μm); D is the expression result of EGFP in the hippocampus 10 or 40 days after injection of the virus prepared in Example 3 (scale bar: 100 μm).

[0056] Figure 15 The graph shows the effect of different injection methods on the survival rate of mice when the viruses prepared in Examples 1, 2, 6, and 7 are injected.

[0057] Figure 16 This is a graph showing the effect of different injection methods on body weight and survival days of the viruses prepared in Examples 1, 2, 6, and 7.

[0058] Figure 17The graph shows the SMN1 expression levels in different SMA strain mice after injection with the virus prepared in Example 1: A is a gel electrophoresis image of SMN1 expression in different SMA strain mice after injection with the virus prepared in Example 1; B is a statistical result graph of SMN1 expression in different SMA strain mice after injection with the virus prepared in Example 1: 1 is brain tissue of SMN2+ / +, SMNΔ7+ / +, and smn+ / + mice that were not injected with the virus; 2 is brain tissue of SMN2+ / +, SMNΔ7+ / +, and smn- / - mice that were not injected with the virus; 3 is brain tissue of SMN2+ / +, SMNΔ7+ / +, and smn- / - mice that were intravenously injected with the virus prepared in Example 1. 4 is brain tissue of SMN2+ / +, SMNΔ7+ / +, smn- / - mice injected intravenously with the virus prepared in Example 1; 5 is spinal cord tissue of SMN2+ / +, SMNΔ7+ / +, smn+ / + mice that were not injected with the virus; 6 is spinal cord tissue of SMN2+ / +, SMNΔ7+ / +, smn- / - mice that were not injected with the virus; 7 is spinal cord tissue of SMN2+ / +, SMNΔ7+ / +, smn- / - mice injected intravenously with the virus prepared in Example 1; 8 is spinal cord tissue of SMN2+ / +, SMNΔ7+ / +, smn- / - mice injected intravenously with the virus prepared in Example 1.

[0059] Figure 18 The following are graphs showing the SMN1 expression levels in different SMA strains of mice after injection with the virus prepared in Example 6: 1 is the brain tissue of SMN2+ / +, SMNΔ7+ / +, and smn- / - mice that were not injected with the virus; 2 is the brain tissue of SMN2+ / +, SMNΔ7+ / +, and smn+ / - mice that were not injected with the virus; 3 is the brain tissue of SMN2+ / +, SMNΔ7+ / +, and smn- / - mice injected intraventricularly with the virus; 4 is the spinal cord tissue of SMN2+ / +, SMNΔ7+ / +, and smn- / - mice that were not injected with the virus; 5 is the spinal cord tissue of SMN2+ / +, SMNΔ7+ / +, and smn+ / - mice that were not injected with the virus; 6 is the spinal cord tissue of SMN2+ / +, SMNΔ7+ / +, and smn- / - mice injected intraventricularly with the virus.

[0060] Figure 19 This is a graph showing the effect of brain injection of ssAAV9 (EF1α>SMN1:WPRE) on the balance ability of mice.

[0061] Figure 20 This is a graph showing the effect of brain injection of ssAAV9 (EF1α>SMN1:WPRE) on the flipping ability of mice. Detailed Implementation

[0062] The present invention will be further described in detail below through specific embodiments.

[0063] Unless otherwise specified, the raw materials used in this embodiment were prepared by conventional means or purchased through commercial channels.

[0064] Example 1: Preparation of virus ssAAV9 (EF1α>SMN1:WPRE) with EF1α promoter

[0065] 1. Plasmid vector construction

[0066] (1) Construction of SMN1 gene vector

[0067] 1) Construction of the pDown-hSMN1 vector: Using pDown-AarI-ccdB-cmR-AarI (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB210730-1146dss) as the backbone, the hSMN1 [NM-000344] fragment was cloned into the pDown-AarI-ccdB-cmR-AarI vector between the AarI restriction sites using the Golden Gate reaction to obtain the pDown-hSMN1 vector; specifically: using hSMN1 [NM-000344] cDNA as a template, two primers were designed (AarI-hSMN1-F:atcgCACCTGCATCGGGGTTTAATTTAAGGAATGTGAGCACCTTC (SEQ ID NO.3) and AarI-hSMN1-R:atcgCACCTGCATCGGGCTgccaccATGGCGATGAGCAGCGGCG (SEQ ID NO.3)). NO.4)) PCR amplification yielded a sequence fragment containing the hSMN1 coding region; vector construction was performed using Golden Gate technology, and the AarI reaction system is shown in Table 1 and the reaction procedure in Table 2. The Golden Gate reaction product was converted to VB UltraStable TM Competent cells were selected, and single clones were subjected to PCR and sequencing verification to obtain the pDown-hSMN1[NM_000344] vector (e.g. Figure 1 (As shown).

[0068] Table 1 AarI Reaction System

[0069]

[0070] Table 2 AarI Reaction Procedure

[0071]

[0072] 2) Construction of pAAV[Exp]-EF1α>hSMN1[NM-000344]:WPRE: The introductory clones pUp-EF1α (pUp-EF1α purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB210730-1149xuq), pDown-hSMN1[NM_000344], and the target vector pAAV.Des2d-WPRE (px601 / 5'ITR modified, purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB180718-1039jtn(Bacteria)) were subjected to a Gateway LR recombination reaction. The LR reaction product was converted to VBUltraStable TM Competent cells were selected, and single clones were subjected to PCR and sequencing verification to obtain the pAAV[Exp]-EF1α>hSMN1[NM-000344]:WPRE vector (plasmid map as shown). Figure 2 (As shown in Table 3). The reaction system and reaction conditions are shown in Table 3.

[0073] Table 3 Reaction system and reaction conditions

[0074]

[0075] (2) Virus production

[0076]

[0077] HEK293T cells were cultured in 10% FBS medium (DMEM (Gibco, 11965-02), 10% fetal bovine serum (FBS, Gibco), 2mM glutamine (Gibco), 1% penicillin / streptomycin (Thermo Fisher Scientific), and 0.1mM non-essential amino acids (Gibco)) at 5% CO2 and 37°C. After transfecting the 293T cells with the three-plasmid system using calcium phosphate transfection, the medium was changed 6 hours later. 48 hours after transfection, the flask walls were tapped to detach most of the cells. Repeatedly pipet and agitate the cells to detach any remaining cells from the cell wall. Collect the cells and supernatant together and transfer to a sterile 50 mL centrifuge tube. Centrifuge at 4°C and 4000 rpm for 20 min, discarding the supernatant. Resuspend the cell pellet in AAV lysis buffer (500 μL / 25 mL of collected cells), then freeze-thaw three times in liquid nitrogen and a 37°C water bath, shaking vigorously during thawing. Incubate the suspension overnight at 37°C and observe the cell lysis status the next day. After confirming successful cell lysis, add 2 mM MgCl2 and 62.5 U / mL Benzonase, mix thoroughly by pipetting, and incubate at 37°C for 45 min to digest the cell genome. Centrifuge the suspension at 4°C and 4000 rpm for 20 min, collecting the supernatant. Then, purify the AAV using density gradient ultracentrifugation. Dilute the bottom layer with PBS and concentrate using ultrafiltration. The concentrated AAV virus was titered using qPCR, and the resulting AAV virus was named ssAAV9(EF1α-SMN1:WPRE).

[0078] Example 2: Preparation of virus ssAAV9 (CAG>SMN1:WPRE) with CAG promoter

[0079] The preparation method in this embodiment is the same as that in Example 1, except that:

[0080] In step 2), the initiator clone was replaced with pUp-CAG (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB210730-1148mga); resulting in pAAV[Exp]-CAG>hSMN1[NM_000344]]:WPRE (plasmid map as shown). Figure 3 As shown), ssAAV9(CAG-SMN1:WPRE) was obtained through viral packaging.

[0081] Example 3: Preparation of virus ssAAV9 (EF1α>EGFP:WPRE) with EF1α promoter

[0082] The preparation method in this embodiment is the same as that in Example 1, except that:

[0083] In step 1), the template is EGFP cDNA (SEQ ID NO. 5), and the primers are AarI-EGFP-F: atcgCACCTGCATCGGGGTTTACTTGTACAGCTCGTCCATGC (SEQ ID NO. 6) and AarI-EGFP-R: atcgCACCTGCATCGGGCTgccaccATGGTGAGCAAGGGCGAGGA (SEQ ID NO. 7), resulting in the pDown-EGFP vector (plasmid map as shown). Figure 4 (as shown);

[0084] Step 2) Obtain the pAAV[Exp]-EF1α>EGFP:WPRE vector (plasmid map as shown) Figure 5 As shown), ssAA V9 (EF1α>EGFP:WPRE) was obtained through viral packaging.

[0085] Example 4: Preparation of scAAV9 virus with CBh promoter (CBh>EGFP)

[0086] The preparation method in this embodiment is the same as that in Example 1, except that:

[0087] In step 1), the template is EGFP cDNA (SEQ ID NO. 5), and the primers are AarI-EGFP-F: atcgCACCTGCATCGGGGTTTACTTGTACAGCTCGTCCATGC (SEQ ID NO. 6) and AarI-EGFP-R: atcgCACCTGCATCGGGCTgccaccATGGTGAGCAAGGGCGAGGA (SEQ ID NO. 7), resulting in the pDown-EGFP vector (plasmid map as shown). Figure 4 (as shown);

[0088] In step 2), the initiator clone was replaced by pUp-EF1α with pUp-CBh (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB210730-1150ugd); the target vector was changed from pAAV.Des2d-WPRE (px601 / 5'ITR modified, purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB180718-1039jtn (Bacteria)) to pscAAV.Des2d (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB190301-1011vej), resulting in the pscAAV[Exp]-CBh>EGFP vector (plasmid map as shown). Figure 6As shown), scAAV9 (CBh>EGFP) was obtained through viral packaging.

[0089] Example 5: Preparation of virus ssAAV9 (CAG>EGFP:WPRE) with CAG promoter

[0090] The preparation method in this embodiment is the same as that in Example 1, except that:

[0091] In step 1), the template is EGFP cDNA (sequence: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA, SEQ ID NO.5), the primers are AarI-EGFP-F: atcgCACCTGCATCGGGGTTTACTTGTACAGCTCGTCCATGC (SEQID NO.6) and AarI-EGFP-R: atcgCACCTGCATCGGGCTgccaccATGGTGAGCAAGGGCGAGGA (SEQ IDNO.7), obtaining the pDown-EGFP vector (the plasmid map is as Figure 4 shown);

[0092] In step 2), the initiator clone was replaced with pUp-CAG (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB210730-1148mga); resulting in pAAV[Exp]-CAG>EGFP:WPRE (plasmid map as shown). Figure 7 As shown), ssAAV9(CAG>EGFP:WPRE) was obtained through viral packaging.

[0093] Example 6: Preparation of virus ssAAV9 (EF1α>SMN1) with EF1α promoter

[0094] The preparation method in this embodiment is the same as that in Example 1, except that:

[0095] In step 2), the target vector was changed from pAAV.Des2d-WPRE(px601 / 5'ITR modified, purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB180718-1039jtn (Bacteria)) to pAAV.Des2d(px601-5'ITR modified) (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB180605-1173cfq) to obtain pAAV[Exp]-EF1α>hSMN1[NM_000344] (plasmid map as shown). Figure 8 As shown), ssAAV9 (EF1α>SMN1) was obtained through viral packaging.

[0096] Example 7: Preparation of virus ssAAV9 (CAG>SMN1) with CAG promoter

[0097] The preparation method in this embodiment is the same as that in Example 1, except that:

[0098] In step 2), the initiator clone was replaced by pUp-EF1α with pUp-CAG (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB210730-1148mga), and the target vector was changed from pAAV.Des2d-WPRE (px601 / 5'ITR modified, purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB180718-1039jtn (Bacteria)) to pAAV.Des2d (px601-5'ITR modified) (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB180605-1173cfq) to obtain pAAV9[Exp]-CAG>hSMN1[NM_000344] (plasmid map as shown). Figure 9 As shown), ssAAV9 (CAG>SMN1) was obtained through viral packaging.

[0099] Example 8: In vitro evaluation of the function of AAV viral vector expressing SMN1

[0100] The day before transduction, 293T cells were prepared at a rate of 3 × 10⁻⁶. 6 Cells were seeded at a density of 1 mL per well in 12-well plates. On the day of transduction, cells reached 50%–60% confluence. Before transduction, cells from one well were digested and counted. The required AAV virus (prepared in Examples 1 and 2) was calculated based on MOI = 1.0E+05, and the corresponding volume of virus was added to 293T medium. A control group (NC control) containing buffer without virus particles was also included (the composition of this group was identical to the experimental group except for the absence of virus particles). After gentle shaking, the cells were returned to a 37°C incubator for 72 hours. 72 hours after transduction, the cells were collected into 1.5 mL EP tubes, ensuring a cell count of 1 × 10⁶ cells per tube. 7 The cells were centrifuged at 1300 rpm for four minutes, the supernatant was discarded, and the mixture was flash-frozen in liquid nitrogen and stored at -80°C. Total protein was extracted from the cells using RIPA lysis buffer, and SMN1 expression levels were detected by Western blot. The experimental procedures followed standard industry practices, and the above experiments were performed in triplicate. The experimental results are as follows: Figure 10 As shown: The transduction of the virus in Examples 1 and 2 significantly increased the expression level of SMN1 in 293T cells, proving that the viral vector expresses SMN1 normally and has a significant effect on improving the expression level of SMN1.

[0101] Example 9: Evaluation of AAV virus tissue affinity in vivo

[0102] Twelve SMA strain mice (SMN2+ / +, SMNΔ7+ / +, smn+ / -, purchased from Jackson Laboratory, catalog number 005025; SMN2+ / +, SMNΔ7+ / +, smn+ / - mice were bred to produce SMN2+ / +, SMNΔ7+ / +, smn- / - mice and SMN2+ / +, SMNΔ7+ / +, smn+ / + mice, which were used as animal models for designing SMA gene therapy drugs) within 48 hours of birth were used as experimental subjects. The animals were divided into three groups: the NC control group (the group with added buffer containing no virus particles was the same as the experimental group except that it did not contain virus particles), the scAAV9 virus with the CBh promoter prepared in Example 4 (CBh>EGFP), and the ssAAV9 virus with the CAG promoter prepared in Example 5 (CAG>EGFP:WPRE), with 6, 3, and 6 animals respectively. They were injected intravenously into the face, and the injection dose of both viruses was 1E+14vg / kg. Ten or forty days after administration (three mice from each of the NC, CBh, and CAG groups were sacrificed on day 10, and the remaining three mice from the NC and CAG groups were sacrificed on day 40), the mice in the above three groups were deeply anesthetized and sacrificed, and their hearts were perfused with 4% paraformaldehyde (PFA). The mice were dissected, and tissues such as the spine, heart, brain, and kidneys were removed and placed on transparent bags for photographing under blue light. The tissues were then fixed with 4% PFA, dehydrated with 30% sucrose solution, and then frozen sections were prepared to observe the expression of EGFP in different tissues. Nine SMA strain mice (SMN2+ / +, SMNΔ7+ / +, smn+ / -, purchased from Jackson Laboratory, catalog number 005025; SMN2+ / +, SMNΔ7+ / +, smn+ / - mice were bred to obtain SMN2+ / +, SMNΔ7+ / +, smn- / - mice and SMN2+ / +, SMNΔ7+ / +, smn+ / + mice, which were used as animal evaluation models for designing SMA gene drugs) within 48 hours of birth were used as experimental subjects. They were divided into two groups: a control group (the group supplemented with buffer without virus particles was the NC control group (the composition of this group was the same as the experimental group except that it did not contain virus particles) and a group of 6 mice each containing the ssAAV9 virus with the EF1α promoter prepared in Example 3. Both groups were injected intravenously, and the injection dose of both viruses was 1E+14vg / kg.Ten or forty days after drug administration (three mice from each of the NC and EF1α groups were sacrificed on day 10, and the remaining three mice from each of the NC and EF1α groups were sacrificed on day 40), the mice in both groups were euthanized under deep anesthesia, and their hearts were perfused with 4% paraformaldehyde (PFA). The mice were then dissected, and tissues such as the spine, heart, brain, and kidneys were removed and placed in dishes for photographing under blue light. The tissues were then fixed with 4% PFA, dehydrated with 30% sucrose solution, and frozen sections were prepared. The expression of EGFP in different tissues was then observed. The results are as follows. Figure 11 (Elimination after 10 days) As shown: Under laser confocal scanning microscopy, it was observed that, compared with the NC group, different tissues in the scAAV9(CBh>EGFP), ssAAV9(EF1α>EGFP:WPRE), and ssAAV9(CAG>EGFP:WPRE) groups showed different degrees of fluorescence intensity, indicating that all three viruses are distributed and expressed in vivo. To further compare the expression of these two viruses in the anterior horn of the spinal cord and the hippocampus of brain tissue, frozen sections were prepared. Experimental results are as follows: Figure 12 , Figure 13 As shown, fluorescence microscopy revealed that ssAAV9(EF1α>EGFP:WPRE), scAAV9(CBh>EGFP), and ssAAV9(CAG>EGFP:WPRE) were expressed in the anterior horn of the spinal cord and the hippocampus in brain tissue. The same dose of ssAAV9(EF1α>EGFP:WPRE) showed the most prominent expression in the target cells (motor neurons) of the anterior horn of the spinal cord and the hippocampus. Furthermore, from... Figure 14 The results show that the expression of ssAAV9(EF1α>EGFP:WPRE) and ssAAV9(CAG>EGFP:WPRE) in vivo after 40 days was significantly better than that in the 10-day group, indicating that the administration time is also a major factor affecting gene expression in vivo. In conclusion, ssAAV9(CAG>EGFP:WPRE) and ssAAV9(EF1α>EGFP:WPRE), after intravenous injection into different tissues, can be significantly expressed in target cell motor neurons, and the duration of in vivo expression has a significant impact on gene expression levels.

[0103] Example 10: Evaluation of the efficacy of AAV virus in vivo

[0104] In the following examples, the viruses prepared using the vectors were administered via both intravenous injection and lateral ventricle injection. The following viruses were prepared at a ratio of 1 x 102: ssAAV9 (CAG>SMN1:WPRE) with CAG promoter prepared in Example 2, ssAAV9 (EF1α>SMN1:WPRE) with EF1α promoter prepared in Example 1, ssAAV9 (EF1α>SMN1) with EF1α promoter prepared in Example 6, and ssAAV9 (CAG>SMN1) with CAG promoter prepared in Example 7. 14 A dose of vg / kg was injected into newborn SMA model mice (SMN2+ / +, SMNΔ7+ / +, smn+ / -, purchased from Jackson Laboratory, catalog number 005025; SMN2+ / +, SMNΔ7+ / +, smn+ / - mice were bred to obtain SMN2+ / +, SMNΔ7+ / +, smn- / - mice, used as evaluation mice in this example) within 48 hours of birth. Following the viral injection, daily changes in body weight, motor function, and survival time were recorded (details for each treatment are as follows). Figure 16 As shown), the results are as follows: Figure 15 , 16As shown: Compared with the NC control group (which received no injections and died within 10–14 days without acquiring normal motor function), the ssAAV9(EF1α>SMN1), ssAAV9(CAG>SMN1), ssAAV9(CAG>SMN1:WPRE), and ssAAV9(EF1α>SMN1:WPRE) groups showed significantly improved body weight and survival rate. Among these, intraventricular injection of ssAAV9(CAG>SMN1) resulted in the longest survival time of mice, up to 30 days; intraventricular injection of ssAAV9(EF1α>SMN1:WPRE) resulted in normal survival of mice up to the statistical time. Mice were still surviving for more than 7 months (216 days), and the survival time of these mice was about 20 times that of the NC group. Intraventricular injection of ssAAV9 (EF1α>SMN1) could extend the survival time of mice to 145 days. It can be seen that the recombinant adeno-associated virus using EF1α as the promoter is more effective than CAG, the recombinant adeno-associated virus containing WPRE is more effective than the one without WPRE, and intraventricular injection of recombinant adeno-associated virus is more effective than intravenous injection. Furthermore, the parameters of the mice's motor ability were analyzed: body weight and rolling ability (the time it took for the mice to roll their four paws onto the worktable was recorded by placing the mice supine and recording the video, while the weight of the mice was measured).The ability of mice to flip over was quantified using fractions: 6 represents 0–5s, 5 represents 5–9s, 4 represents 10–14s, 3 represents 15–19s, 2 represents 20–24s, 1 represents 25–30s, and >30s was 0; NC represented untreated model mice (SMN2+ / +, SMNΔ7+ / +, smn- / - mice, without any injections, 3 mice per treatment). Balance ability was also assessed (under rest, the experimental animals were placed on a balance beam (approximately 25cm long, 2cm wide, and 40cm above the ground), and the time taken to turn around on the balance beam was recorded (maximum 60s), including the number of times the mouse fell and whether it could turn over; NC represented untreated model mice (SMN2+ / +, SMNΔ7+ / +, smn- / - mice, without any injections, 3 mice per treatment), etc., compared with the NC control group (untreated mice). Compared to any other injection procedure, the body weight of mice injected intravenously and intraventricularly showed little change, while the body weight of mice injected intravenously (EF1α>SMN1:WPRE) and intraventricularly (CAG>SMN1) increased. The body weight of mice injected intraventricularly (EF1α>SMN1:WPRE) was significantly higher than that of mice injected intravenously (EF1α-SMN1:WPRE). The body weight of mice injected intraventricularly (EF1α>SMN1) was significantly higher than that of mice injected intraventricularly (CAG>SMN1). The body weight of mice injected intraventricularly (EF1α>SMN1:WPRE) was significantly higher than that of mice injected intraventricularly (EF1α>SMN1). Figure 16 It is evident that recombinant adeno-associated virus (AAV) using EF1α as the promoter is superior to CAG, recombinant AAV containing WPRE is superior to that without WPRE, and intraventricular injection of recombinant AAV is superior to intravenous injection; in the balance beam experiment, compared with the NC control group, mice injected intraventricularly with ssAAV9 (EF1α>SMN1:WPRE) experienced significantly fewer falls and were able to turn on the balance beam. Figure 19 In the EF1α flipping experiment, compared with the NC control group, mice injected intracerebroventricularly with ssAAV9 (EF1α>SMN1:WPRE) showed significantly improved flipping ability, which increased over time, while the NC control group mice died after 18 days. Figure 20 This indicates that intraventricular injection of ssAAV9(EF1α>SMN1:WPRE) is more effective in treating SMA model mice.

[0105] Meanwhile, from the perspective of animal physiology, the lateral ventricle and the spinal sheath are both cavity structures in the cerebrospinal fluid circulation system. In principle, the effect of injection through the spinal sheath is similar to that of injection through the lateral ventricle. The drug can quickly reach all parts of the central nervous system through the cerebrospinal fluid circulation. However, it is easier to perform spinal sheath injection on patients in clinical practice.

[0106] Example 11: Evaluation of SMN expression in vivo

[0107] SMA strain mice (SMN2+ / +, SMNΔ7+ / +, smn+ / -, purchased from Jackson Laboratory, catalog number 005025; SMN2+ / +, SMNΔ7+ / +, smn+ / - mice were bred to obtain SMN2+ / +, SMNΔ7+ / +, smn- / - mice and SMN2+ / +, SMNΔ7+ / +, smn+ / + mice) were subjected to the following treatments: ① SMN2+ / +, SMNΔ7+ / +, smn+ / + mice were not injected; ② SMN2+ / +, SMNΔ7+ / +, smn- / - mice were not injected. ③ Intravenous injection of SMN2+ / +, SMNΔ7+ / +, smn- / -; ④ Intraventricular injection of SMN2+ / +, SMNΔ7+ / +, smn- / -; The mice receiving the injections used a virus with the EF1α promoter prepared in Example 1; the injection dose was 1.0E+14vg / kg, and the treatment lasted for 10 days; at two weeks of age, brain and spinal cord tissues were collected from the mice, and total protein was extracted using RIPA lysis buffer. The SMN1 expression level was detected by Western blot. The experimental procedures were the same as standard industry procedures, and the above experiments were performed in parallel three times. The experimental results are as follows. Figure 17 As shown: In the absence of injection, the expression of SMN1 protein in the brain and spinal cord of SMN2+ / +, SMNΔ7+ / +, smn- / - mice was lower than that in SMN2+ / +, SMNΔ7+ / +, smn+ / + mice; intravenous injection of the virus with the EF1α promoter prepared in Example 1 slightly upregulated the expression of SMN1 in the spinal cord of SMN2+ / +, SMNΔ7+ / +, smn- / - mice, while no significant change was observed in the expression of SMN1 in the brain tissue; intraventricular injection of the virus with the EF1α promoter prepared in Example 1 significantly increased the expression level of SMN1 in the brain and spinal cord of SMN2+ / +, SMNΔ7+ / +, smn- / - mice.

[0108] SMA strain mice (SMN2+ / +, SMNΔ7+ / +, smn+ / -, purchased from Jackson Laboratory, catalog number 005025; SMN2+ / +, SMNΔ7+ / +, smn+ / - mice were bred to obtain SMN2+ / +, SMNΔ7+ / +, smn- / - mice and SMN2+ / +, SMNΔ7+ / +, smn+ / + mice) were treated as follows: ① SMN2+ / +, SMNΔ7+ / +, smn+ / - mice were not injected; ② SMN2+ / +, SMNΔ7+ / + ③ SMN2+ / +, SMNΔ7+ / +, SMN- / - injected into the ventricle; the corresponding virus for the mice injected was ssAAV9 (EF1α>SMN1) virus with the EF1α promoter prepared in Example 6; the injection dose was 1.0E+14vg / kg, and the treatment lasted for 10 days; when the mice were two weeks old, brain and spinal cord tissues were taken from the mice, and total protein was extracted using RIPA lysis buffer. The expression level of SMN1 was detected by Western blot experiment. The experimental operation was the same as the industry standard operation, and the above experiment was performed in parallel 3 times. The experimental results are as follows. Figure 18 As shown: In the absence of injection, the expression of SMN1 protein in the brain and spinal cord of SMN2+ / +, SMNΔ7+ / +, smn- / - mice was lower than that in SMN2+ / +, SMNΔ7+ / +, smn+ / - mice; intraventricular injection of the virus with the EF1α promoter prepared in Example 6 significantly upregulated the expression of SMN1 in the brain and spinal cord of SMN2+ / +, SMNΔ7+ / +, smn- / - mice.

[0109] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A recombinant adeno-associated virus vector comprising an adeno-associated virus capsid and a non-self-complementary single-stranded genome; said single-stranded genome comprising an AAV 5'-ITR, an EF1α promoter, a Kozak sequence, a gene encoding a motor neuron survival protein, polyadenylates, WPRE, and an AAV 3'-ITR; The EF1α promoter is a promoter having the nucleotide sequence shown in SEQ ID NO.1; The motor neuron survival protein is a protein having the amino acid sequence shown in SEQ ID NO.2; The adeno-associated virus capsid is an AAV9 capsid.

2. A pharmaceutical composition comprising the recombinant adeno-associated virus vector of claim 1 and a pharmaceutically acceptable carrier / excipient.

3. The use of any one of (1) to (2) in the preparation of drugs for alleviating or treating spinal muscular atrophy: (1) The recombinant adeno-associated virus vector according to claim 1; (2) The pharmaceutical composition according to claim 2.

4. The application according to claim 3, characterized in that: The drug is administered by injection.

5. The application according to claim 4, characterized in that: The injection is at least one of intravenous injection, intraventricular injection, and intrathecal injection.

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