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 of this invention comprises an AAV capsid and sequence elements, including an EFS promoter sequence and a sequence encoding a motor neuron survival protein. The recombinant AAV vector provided by this invention can avoid the toxicity caused by overexpression of SMN1, stably maintain the SMN1 protein level in vivo within the therapeutic window, prolong the survival time of SMA mice, increase mouse body weight, improve the development of motor neurons and skeletal muscle-related tissues, and restore neuronal physiological function. This invention employs a gene therapy strategy and can be used to prevent, alleviate, or treat neurological diseases.

Description

Technical Field

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

[0002] Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder caused by the deletion or mutation of the survival motor neuron 1 gene (SMN1). Its main clinical features are muscle weakness and atrophy resulting from the degeneration of the anterior motor neurons in the spinal cord. SMA frequently affects the respiratory system, causing pathophysiological changes that lead to a range of respiratory problems such as hypoventilation, weakened cough, and sputum blockage. Respiratory failure is the most common cause of death in children with SMA. The incidence of this disease in surviving newborns in European and American populations is approximately 1 in 10,000, ranking first among fatal genetic diseases in children under two years of age.

[0003] The SMN2 gene, located on the centromere side, is highly homologous to the SMN1 gene on the telomere side, differing only in five single nucleotides at their respective 3' ends. While SMN2 gene deletion is not pathogenic, it is a modifier of the SMN1 gene and plays a dose-compensating role in the presence of SMN1 deletion. The SMN2 copy number is correlated with the severity of the SMA phenotype. Based on age of onset, severity of muscle weakness, and achieved motor function, SMA can be classified into four common clinical types: SMA-I, SMA-II, SMA-III, and SMA-IV. Type I SMA, also known as Werdnig-Hoffman disease (infantile type), accounts for approximately 45% of all SMA cases. Onset occurs within 6 months of birth, presenting with rapidly progressing, symmetrical limb weakness, with maximum motor ability not reaching independent sitting. Most children die from respiratory failure within 2 years of age. The most common SMN2 copy number in children with type I SMA is 2 copies. Type II SMA, also known as Dubowitz's disease, is the intermediate type, accounting for approximately 30%–40% of cases. Patients typically develop the disease between 6 and 18 months of age, with a slower progression than type I. Maximum motor ability is achieved at sitting independently, but not standing or walking independently. Most patients live into adulthood. Type II predominantly has 3 copies of SMN2. Type III, also known as Kugelberg-Welander disease, is the juvenile type, accounting for approximately 20% of cases. Most patients develop the disease after 18 months of age, with normal early motor development and the ability to walk independently. With age, proximal muscle weakness develops, eventually leading to partial loss of independent walking ability. Life expectancy is not shortened or only slightly reduced. Type III predominantly has 3 or 4 copies of SMN2. Type IV, the late-onset type, is the adult type. Patients have normal early motor development, developing the disease in adulthood, with proximal limb weakness and mild motor impairment. Disease progression is slow, and life expectancy is generally unaffected. Type IV predominantly has 4 copies of SMN2.

[0004] Currently, treatment strategies for SMN-dependent diseases mainly include SMN1 gene replacement therapy, enhancing SMN2 gene promoter activity, and increasing the expression of the full-length SMN2 gene transcript. Drugs that have entered clinical trials include phenylbutyric acid, valproic acid, and hydroxyurea, but have not yet produced sufficient clinical benefits. Nusinersen (SPINRAZA), an antisense oligonucleotide (ASO) drug approved by the FDA, aims to increase the production of the full-length SMN protein by regulating the splicing of the SMN2 gene, thereby compensating for potential genetic defects. Clinical studies show promise in improving movement disorders; however, this treatment requires indefinite administration via intrathecal injection, necessitates a long induction period before effectiveness, and has safety considerations requiring clinical monitoring. Zolgensma, approved by the FDA, delivers its complementary double-stranded SMN1 expression sequence into the body via an AAV9 vector and has achieved therapeutic effects under the regulation of the cytomegalovirus (CMV) immediate / early enhancer and the chicken β-actin (CB) promoter, but based on current clinical data, its efficacy in treating SMA patients needs further improvement.

[0005] Adeno-associated virus (AAV) is a species of parvovirus with great potential for in vivo genetic material delivery. The AAV vector system is a popular in vivo and in vitro gene delivery system, exhibiting high transduction efficiency across various mammalian cell types. In packaging cells, a DNA fragment located between two inverted repeat sequences (ITRs) is packaged with viral proteins expressed by helper plasmids to form viral particles. When the virus transduces a host cell, the AAV virus carrying exogenous DNA enters the cell, and the linear double-stranded DNA genome exists in the cell nucleus as cell-free DNA. Based on differences in the viral capsid proteins, they are classified into different serotypes. Different serotypes of the virus have different tissue affinities. AAV9, for example, has affinity for tissues such as the myocardium, lungs, retina, and skin. Furthermore, due to its ability to cross the blood-brain barrier, AAV9 has wide applications in neuroscience. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a recombinant adeno-associated virus vector and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a recombinant adeno-associated virus vector comprising an adeno-associated virus capsid and sequence elements, wherein the sequence elements include an EFS promoter sequence and a sequence encoding a motor neuron survival protein.

[0009] The recombinant adeno-associated virus vector provided by this invention can avoid the toxicity caused by the overexpression of SMN1, and stably maintain the SMN1 protein level in vivo within the therapeutic window, thereby prolonging the survival time of SMA mice, increasing mouse weight, and improving the development of motor neurons and skeletal muscle-related tissues.

[0010] As a preferred embodiment of the recombinant adeno-associated virus vector of the present invention, the nucleotide sequence of the EFS promoter is shown in SEQ ID NO.1.

[0011] As a preferred embodiment of the recombinant adeno-associated virus vector of the present invention, the amino acid sequence of the motor neuron survival protein is shown in SEQ ID NO.2.

[0012] In a preferred embodiment of the recombinant adeno-associated virus vector of the present invention, the sequence encoding the motor neuron survival protein is cloned using the primer set shown in SEQ ID NO.4 and SEQ ID NO.5 with hSMN1 cDNA as a template.

[0013] In a preferred embodiment of the recombinant adeno-associated virus vector of the present invention, the gene for the neuronal survival protein is the SMN1 gene, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0014] As a preferred embodiment of the recombinant adeno-associated virus vector of the present invention, it further includes at least one of the WPRE sequence, Kozak sequence, polyadenylated nucleotide sequence, and AAV inverted terminal repeat sequence.

[0015] In a preferred embodiment of the recombinant adeno-associated virus vector of the present invention, the AAV inverted terminal repeat sequence comprises AAV 5'-ITR and AAV 3'-ITR.

[0016] In a preferred embodiment of the recombinant adeno-associated virus vector of the present invention, the adeno-associated virus capsid is any one of AAVPHP.eb, AAVPHP.b, and AAV9 capsid.

[0017] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-described recombinant adeno-associated virus vector.

[0018] Preferably, the pharmaceutical composition further includes any pharmaceutically acceptable carrier and / or excipient.

[0019] Furthermore, the pharmaceutical composition is administered by injection;

[0020] Furthermore, the injection is at least one of intravenous injection, intraventricular injection, and intrathecal injection.

[0021] Thirdly, the present invention applies the recombinant adeno-associated virus vector and the pharmaceutical composition to the preparation of medicaments for the treatment and / or prevention of nervous system diseases.

[0022] Preferably, the neurological disease is a motor neuron disease involving motor function.

[0023] This invention employs a gene therapy strategy, regulating SMN1 gene expression through the EFS promoter to restore neuronal physiological function and significantly prolong the survival time of an SMA mouse model. It can be used to prevent, alleviate, or treat neurological diseases.

[0024] Furthermore, the motor neuron diseases include spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), bulbar muscular atrophy, spinocerebellar ataxia, primary lateral sclerosis, and traumatic spinal cord injury.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The recombinant adeno-associated virus vector of this invention can avoid the toxicity caused by SMN1 overexpression, stably maintaining the SMN1 protein level in vivo within the therapeutic window, thus prolonging the survival time of SMA mice, increasing mouse weight, and improving the development of motor neurons and skeletal muscle-related tissues. This invention employs a gene therapy strategy, regulating SMN1 gene expression through the EFS promoter to restore neuronal physiological function and significantly prolong the survival time of mouse models. Compared to existing SMA gene therapy strategies based on AAV vectors, especially the FDA-approved Zolgensma, the unique SMN1 expression cassette design of this invention can avoid the safety risks associated with exogenous gene overexpression while treating SMA, and avoid delayed motor neuron degeneration caused by persistent SMN overexpression. This is especially true for CB series or CAG series promoters derived from the CMV (Human cytomegalovirus immediate early enhancer / promoter) promoter, which allow the human SMN1 gene sequence they control to maintain a high level of expression in vivo. The recombinant adeno-associated virus (AAV) vector provided by this invention is significantly more effective in treating SMA than recombinant AAV vectors containing other promoters. It can be used to prevent, alleviate, or treat neurological diseases. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure of the pDown-hSMN1[NM-000344] plasmid.

[0028] Figure 2Schematic diagram of the structure of pAAV[Exp]-EFS>hSMN1[NM_000344.4]:WPRE plasmid.

[0029] Figure 3 This is a schematic diagram of the pDown-EGFP plasmid structure.

[0030] Figure 4 This is a schematic diagram of the pAAV[Exp]-EFS>EGFP plasmid structure.

[0031] Figure 5 The image shows the expression of EGFP after transfection with the pAAV[Exp]-EFS>EGFP plasmid (100×). The image also shows the EGFP expression results 48 hours after transfection of 293T cells with the plasmid prepared in Example 2. The white light exposure interval is 10ms, and the green fluorescence exposure interval is 100ms.

[0032] Figure 6 The expression of SMN1 in 293T cells transduced with the AAV9 (pAAV[Exp]-EFS>EGFP) vector (100×). Immunofluorescence staining results of SMN1 in 293T cells 48 hours after transduction with the AAV9 vector prepared in Example 1.

[0033] Figure 7 The image shows the expression of EGFP in cortical neurons after transduction with the AAV9 (pAAV[Exp]-EFS>EGFP) vector (200×). The image shows the EGFP expression results 72 hours after transduction with the AAV9 vector prepared in Example 2 into mouse embryo-derived cortical neurons. The exposure time between white light and green fluorescence is 100 ms. The red boxes mark some neurons expressing EGFP.

[0034] Figure 8 The expression of EGFP in spinal motor neurons after transduction with AAV9(pAAV[Exp]-EFS>EGFP) and AAV9(pAAV[Exp]-CAG>EGFP) vectors (200×). The exposure time between white light and green fluorescence was 100 ms. Red arrows mark non-motor neurons that partially express EGFP.

[0035] Figure 9Survival curves of newborn mice after injection treatment. Survival curves of the ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) vector prepared in Example 1 after intraventricular injection (ICV) or intravenous injection (IV) into SMA mice. WT = wild-type mice, US = untreated SMA mouse model, ICV = Intracerebroventricular, IV = Intravenous. # indicates that only one mouse survived at that time point.

[0036] Figure 10 The figures show the changes in mouse body weight during treatment. The changes in body weight of mice in each group after the ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) vector prepared in Example 1 was injected into SMA mice via ICV or IV. WT = wild-type mouse, US = untreated SMA mouse model, ICV = intracerebroventricular, IV = intravenous.

[0037] Figure 11 The image shows the results of the righting reflex test. The ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) vector prepared in Example 1 was injected into SMA mice via ICV or IV for 12 and 30 days, and the results of the righting reflex test were obtained in each group of mice. WT = wild-type mice, US = untreated SMA mouse model, ICV = Intracerebroventricular; IV = Intravenous. # indicates that only one mouse survived in the group at the time of testing; * indicates that all mice in the group died at the time of testing, and no data was available. Scoring criteria: 6 points: 0-4s, 5 points: 5-9s, 4 points: 10-14s, 3 points: 15-19s, 2 points: 20-24s, 1 point: 25-30s, 0 points: >30s.

[0038] Figure 12 Figure 1 shows the swimming test results. The swimming test results of mice in each group 60 days after ICV or IV injection of the ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) vector prepared in Example 1 into SMA mice. WT = wild-type mice, US = Untreated SMA mouse model, ICV = Intracerebroventricular, IV = Intravenous. # indicates that only one mouse survived in this group at the test time.

[0039] Figure 13 This image shows the results of the balance beam experiment. The ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) vector prepared in Example 1 was injected into SMA mice via ICV or IV for 60 days, resulting in the balance beam experiment results for each group of mice. WT = wild-type mouse, US = untreated SMA mouse model, ICV = Intracerebroventricular, IV = Intravenous. # indicates that only one mouse in that group survived at the detection time.

[0040] Figure 14 To investigate the effect of different vectors on the survival curves of SMA, the survival time curves of ssAAV9(pAAV[Exp]-EFS>hSMN1:WPRE) and Zolgensma, ssAAV9(pAAV[Exp]-EF1A>hSMN1:WPRE), ssAAV9(pAAV[Exp]-EF1A>hSMN1:WPRE), and ssAAV9(pAAV[Exp]-CMV>hSMN1:WPRE) vectors prepared in Example 1 after intraventricular injection (ICV) into SMA mice were presented. Detailed Implementation

[0041] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0042] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0043] Example 1: Preparation of ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) vector

[0044] (1) Construction of SMN1 gene vector

[0045] 1) Constructing the pDown-hSMN1 vector

[0046] 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 via the Golden Gate reaction to obtain the pDown-hSMN1 vector;

[0047] Specifically, using hSMN1[NM-000344]cDNA as a template, two primers were designed, and PCR amplification was performed to obtain a sequence fragment containing the hSMN1 coding region. The vector was constructed using Golden Gate technology; the AarI reaction system is shown in Table 1, and the reaction procedure is shown in Table 2. The Golden Gate reaction product was then transformed into VBUltraStable. TM Competent cells were selected, and single clones were subjected to PCR and sequencing verification as shown in SEQ ID NO.3. This yielded the pDown-hSMN1[NM-000344] vector (e.g., ...). Figure 1 (As shown).

[0048] The primer sequences are:

[0049] AarI-hSMN1-F:atcgCACCTGCATCGGGGTTTAATTTAAGGAATGTGAGCACCTTC;

[0050] AarI-hSMN1-R: atcgCACCTGCATCGGGCTgccaccATGGCGATGAGCAGCGGCG.

[0051] Table 1 AarI Reaction System

[0052] Components Added amount AarI 1 μL (2 U / μL) T7 DNA ligase 1μL 10×AarI buffer 2μL ATP (10mM) 1μL DTT (10mM) 4μL AarI-hSMN1-AarI 1 μL (140 ng / μL) pDown-AarI-ccdB-cmR-AarI 1uL (20ng / uL) water Up to 20uL

[0053] Table 2 AarI Reaction Procedure

[0054]

[0055] 2) Construct pAAV[Exp]-EFS>hSMN1[NM-000344]

[0056] The introductory clones pUp-EFS (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.) and pDown-hSMN1[NM-000344] were subjected to a Gateway LR recombination reaction with the target vector pAAV.Des2d-WPRE (px601 / 5'ITR modified) (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.). The LR reaction product was then converted to VB UltraStable. TM Competent cells were selected, and single clones were subjected to PCR and sequencing verification to obtain the pAAV[Exp]-EFS>hSMN1[NM-000344]:WPRE vector. The reaction system and reaction conditions are shown in Table 3.

[0057] Table 3 Reaction system and reaction conditions

[0058]

[0059] (2) Virus production

[0060] AAV virus packaging was performed by transfecting 293T cells using a three-plasmid system (vector plasmid, packaging plasmid, and helper plasmid). Plasmid 1 (vector plasmid) contained the ITR sequence at the 5' end of the AAV genome and an expression cassette for SMN1. This expression cassette contained the EFS promoter sequence (SEQ ID NO.1), Kozak sequence, SMN1 transgene (NM_000344.4), BGH pA, 3' ITR, kanamycin, and pUC ori (i.e., pAAV[Exp]-EFS>hSMN1[NM-000344.4]:WPRE vector). pAAV2 / 2 (retro) (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB180917-1100ncc) was used as the recombinant AAV helper plasmid (packaging plasmid), which provided the AAV2 Rep gene and AAV9 in a trans configuration. Cap gene; and pHelper helper viral plasmid (helper plasmid, purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., ID: VB180712-1021bcx), containing the helper genes E2a, E4 and VA.

[0061] HEK293T cells were cultured in DMEM medium (Gibco, 11965-02) containing 10% fetal bovine serum (FBS), 2 mM glutamine (Gibco), 1% penicillin / streptomycin (Thermo Fisher Scientific), and 0.1 mM non-essential amino acids (Gibco) at 5% CO2 and 37°C. The medium was changed 6 hours after transfection of 293T cells with the three-plasmid system using calcium phosphate transfection. Forty-eight 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(pAAV[Exp]-EFS>hSMN1:WPRE).

[0062] Example 2: Preparation of ssAAV9 (pAAV[Exp]-EFS>EGFP) vector

[0063] The preparation method of this Example 2 differs from that of Example 1 only in that:

[0064] When constructing pAAV[Exp]-EFS>EGFP, the introductory clones pUp-EFS and pDown-EGFP (e.g.) will be used. Figure 3 The expression cassette (as shown) was used in a Gateway LR recombination reaction with the target vector pAAV.Des2d(px601-5'ITR modified) (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.). The expression cassette contained the EFS promoter sequence, Kozak sequence, EGFP, 3'ITR, kanamycin, and pUC orii (i.e., pAAV[Exp]-EFS>EGFP vector); thus obtaining pAAV[Exp]-Kan-EFS>EGFP (as shown). Figure 4As shown), ssAAV9(pAAV[Exp]-EFS>EGFP) was obtained through viral packaging.

[0065] Example 3: In vitro evaluation experiment of EFS promoter

[0066] 293T cells were administered at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 1 mL per well in 24-well plates. On the day of transduction, cells reached 50%–60% confluence. Before transduction, cells in one well were digested for cell counting. The required plasmid was calculated based on the DNA copy number 2.5E+11. Plasmid transfection was performed according to the Lipo2000 Transfection Reagent instructions and added to 293T cells. A control group (NC) was established by adding buffer without plasmid. After gentle shaking, the cells were returned to a 37°C incubator for 72 hours. EGFP expression was observed under a fluorescence microscope. Results are as follows: Figure 5 As shown, compared with the NC group, EGFP expression was significantly higher in 293T cells after transfection with pAAV[Exp]-EFS>EGFP plasmid. The results suggest that the EFS promoter can initiate the expression of the target gene in mammalian cells.

[0067] Example 4: In vitro evaluation of ssAAV9 (pAAV[Exp]-EFS>EGFP) expression function

[0068] 293T cells were administered at a rate of 5 × 10⁻⁶. 5 Cells were seeded at a density of 1 mL per well in 24-well plates. On the day of transduction, cells reached 50%–60% confluence. Before transduction, cells in one well were digested for cell counting. The required AAV vector was calculated based on MOI = 2.0E+05 and added to the 293T cells. A control group (NC) was established by adding buffer without virus particles. After gentle shaking, the cells were returned to a 37°C incubator for 72 hours. EGFP expression was observed under a fluorescence microscope. The primary antibody Anti-EGFP bound to the EGFP expressed in the cells, amplifying the fluorescence signal. The secondary antibody Goat anti-Rabbit 594 bound to the primary antibody, producing red fluorescence. Figure 6 As shown, compared with the NC group, EGFP expression was observed in 293T cells transfected with ssAAV9(pAAV[Exp]-EFS>EGFP).

[0069] Example 5: EFS promoter regulates the expression of target genes in cortical neurons

[0070] The cerebral cortex of mouse embryos was harvested to obtain cortical neurons. After culturing for 48 hours, ssAAV9 (pAAV[Exp]-EFS>EGFP) was transduced at MOI = 5E+05. EGFP protein expression was observed under a fluorescence microscope 72 hours after transduction. Figure 7 As shown, compared with the NC group, significant EGFP expression was observed in the transduction group; the results suggest that ssAAV9(pAAV[Exp]-EFS>EGFP) can be expressed in cerebral cortical neurons. The EFS promoter can regulate gene expression in cerebral cortical neurons.

[0071] Example 6: Expression of target genes regulated by different promoters in spinal motor neurons

[0072] Spinal cord tissue from mouse embryos was used to obtain spinal motor neurons. After culturing for 48 hours, the neurons were transduced into ssAAV9 (pAAV[Exp]-CAG>EGFP) and ssAAV9 (pAAV[Exp]-EF1A>EGFP) viral vectors at MOI = 2E+05. EGFP protein expression was observed under a fluorescence microscope 72 hours after transduction. Figure 8 As shown, compared with the NC group, significant EGFP expression was observed in the transduced ssAAV9(pAAV[Exp]-EF1A>EGFP) group; ssAAV9(pAAV[Exp]-CAG>EGFP) could not be expressed in motor neuron cells and had poor targeting of motor neurons.

[0073] Example 7: In vivo evaluation of the efficacy of ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE)

[0074] The ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) vector was delivered intravenously (IV) (7.5E+13vg / kg, 1.5E+14vg / kg) and intracerebroventricularly (ICV) (4E+13vg / kg, 7.5E+13vg / kg, 1.5E+14vg / kg) to SMA model mice (SMN2) within 48 hours of birth via intravenous injection and intracerebroventricular injection, respectively. + / + ,SMNΔ7 + / + smn + / - Purchased from Jackson Laboratory, item number 005025, SMN2 + / + SMNΔ7 + / + smn + / - SMN2 is obtained by breeding mice. + / +SMNΔ7 + / + smn - / - The mice used as evaluation mice in this embodiment were injected with the vector into newborn mice, and their growth status, such as weight changes and survival days, were recorded daily.

[0075] Survival curves as follows Figure 9 As shown. The WT (wild-type mice) group consisted of wild-type mice that had not undergone any injection procedures. The US (Untreated SMA mouse model) group had a median survival time of 15 days and could not acquire normal motor abilities. Compared with the US group, the survival rate of mice injected with ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) was significantly improved in all groups. Specifically, in the ICV-4E+13vg / kg group, 11.1% of mice had a survival time extended to 62 days; in the ICV-7.5E+13vg / kg group, 84.6% of mice had a survival time extended to 170 days, and 76.9% had a survival time extended to 185 days, approximately 7 times that of the US group; in the ICV-1.5E+14vg / kg group, 100% of mice had a survival time extended to 111 days, and 83.3% had a survival time extended to 161 days. Compared with the US group, 20% of mice in the IV-7.5E+13vg / kg group survived for up to 99 days; 60% of mice in the IV-1.5E+14vg / kg group survived for 20 days, and no mice survived for more than 30 days. These results suggest that using EFS as a promoter to regulate SMN1 expression can significantly improve the survival time of SMA mice.

[0076] Weight changes such as Figure 10 As shown in the figure. The body weight of mice was monitored during the injection treatment. The body weight curve of the mice showed that the body weight gradually increased over time after injection of the ssAAV9(pAAV[Exp]-EFS>hSMN1:WPRE) vector.

[0077] Example 8: ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) improves the development of motor neuron-skeletal muscle-related tissues.

[0078] The ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) vector was delivered into SMA model mice via intravenous injection and lateral ventricle injection, respectively. The developmental level of motor neurons and skeletal muscle-related tissues was evaluated using the righting reflex test, swimming test, and balance beam test. The results showed that ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) could prolong the survival time and improve the survival rate of SMA mice while promoting the development of motor neurons and skeletal muscle-related tissues, thus improving the motor ability of SMA mice.

[0079] The results of the righting reflection experiment are as follows Figure 11 As shown. When the test mice are gently placed on their side or back, they immediately return to their normal posture, a reflex known as the righting reflex. Figure 11 As shown in Figure A, 12 days after injection treatment, the righting reflex test score in the WT group was 6.00. Compared with the WT group, the score in the US group was significantly lower, with an average score of 1.00. Compared with the US group, the righting reflex scores of mice in all injection treatment groups were significantly increased, with the most significant increase observed in the lateral ventricle injection medium-dose groups (ICV-4E+13vg / kg group: 4.67±0.67; ICV-7.5E+13vg / kg group: 5.67±0.33; ICV-1.5E+14vg / kg group: 5.00±1.00; IV-7.5E+13vg / kg group: 5.00±0.58; IV-1.5E+14vg / kg group: 0.00±0.00). Figure 11 As shown in Figure B, 30 days after injection treatment, the righting reflex score in the WT group was 6.00. Data were unavailable for the US group and the IV-1.5E+14vg / kg group due to mouse mortality. The righting reflex scores of the remaining treated mice all improved to 6.00.

[0080] The results of the swimming experiment are as follows Figure 12 As shown. Mice were placed in an inescapable, transparent container filled with water, and the time required for the mouse to climb onto the escape platform was recorded. Figure 12 As shown, swimming tests were performed on mice in each group 30 days after injection treatment. In the WT group, 3 mice were able to quickly climb ashore, with an average time of 6.33 ± 88 s. Data were unavailable for the US group and the IFV-1.5E+14 vg / kg group due to mouse mortality. The average time for mice in the ICV-7.5E+13 vg / kg group to climb ashore was 5.67 ± 0.88 s. The average time for mice in the IFV-7.5E+13 vg / kg group to climb ashore was 5.00 s.

[0081] The results of the balance beam experiment are as follows Figure 13As shown. The test assesses an animal's ability to maintain balance while moving along a narrow wooden slat, observing the use of its fore and hind limbs and the number of falls, and assigning a score to the animal. For example... Figure 13 As shown, 60 days after injection treatment, all mice in the WT group were able to turn around without falling. Data were unavailable for the US and IV-1.5E+14vg / kg groups due to mouse mortality. All mice in the ICV-7.5E+13vg / kg group were able to turn around, with an average of 0.67±0.67 falls. Only one mouse survived in the IV-7.5E+13vg / kg group, and was able to turn around during the balance beam test, falling a total of 3 times.

[0082] Example 9: The effect of WPRE regulatory elements on the therapeutic effect of hSMN1

[0083] The effects of ssAAV9(pAAV[Exp]-EF1A>hSMN1:WPRE) and ssAAV9(pAAV[Exp]-EF1A>hSMN1) on the survival of SMA model mice were compared using lateral ventricle injection. Figure 14 As shown, the WPRE regulatory element can significantly prolong the survival time of SMA mice and plays a very important role in the therapeutic effect of AAV9-hSMN1.

[0084] Example 10: Comparison of in vivo efficacy of AAV9-hSMN1

[0085] The effects of ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) on the survival of SMA model mice were compared using intraventricular injection. The effects were compared with Zolgensma, ssAAV9 (pAAV[Exp]-EF1A>hSMN1:WPRE), ssAAV9 (pAAV[Exp]-EF1A>hSMN1), and ssAAV9 (pAAV[Exp]-CMV>hSMN1:WPRE). AAV9 vectors were delivered to SMA model mice within 48 hours of birth at doses ranging from 7.0E+13 to 1.0+13 vg / kg, and the survival days were recorded. Figure 14As shown, ssAAV9(pAAV[Exp]-EFS>hSMN1:WPRE) and Zolgensma, ssAAV9(pAAV[Exp]-EF1A>hSMN1:WPRE), ssAAV9(pAAV[Exp]-EF1A>hSMN1), and ssAAV9(pAAV[Exp]-CMV>hSMN1:WPRE) all significantly improved the survival time of mice. Among them, the survival rate of mice injected with ssAAV9(pAAV[Exp]-EFS>hSMN1:WPRE) vector was higher than that of mice injected with Zolgensma, ssAAV9(pAAV[Exp]-EF1A>hSMN1:WPRE), ssAAV9(pAAV[Exp]-EF1A>hSMN1), and ssAAV9(pAAV[Exp]-CMV>hSMN1:WPRE) vectors.

[0086] The recombinant adeno-associated virus vector ssAAV9 (pAAV[Exp]-EFS>hSMN1:WPRE) provided by this invention can avoid the toxicity caused by SMN1 overexpression, stably maintain the SMN1 protein level in vivo within the therapeutic window, prolong the survival time of mice, increase mouse body weight, and improve the development of motor neurons and skeletal muscle-related tissues. This invention employs a gene therapy strategy, regulating SMN1 gene expression through the EFS promoter to restore neuronal physiological function and significantly prolong the survival time of mouse models. Compared to existing AAV vector-based SMA gene therapy strategies, especially the FDA-approved Zolgensma, the unique SMN1 expression cassette design of this invention can avoid the safety risks associated with exogenous gene overexpression while treating SMA, and prevent delayed motor neuron degeneration caused by persistent SMN overexpression. This is particularly true for CB-series or CAG-series promoters derived from the CMV (Human Cytomegalovirus Immediate Early Enhancer / Promoter) promoter, which allow the human SMN1 gene sequence they control to maintain high levels of expression in vivo. This can be used to prevent, alleviate, or treat neurological diseases.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A recombinant adeno-associated virus vector, comprising an adeno-associated virus capsid and sequence elements, characterized in that, The sequence elements sequentially include AAV 5'-ITR, EFS promoter sequence, Kozak sequence, sequence encoding motor neuron survival protein, WPRE sequence, polyadenylate sequence, and AAV 3'-ITR; The nucleotide sequence of the EFS promoter is shown in SEQ ID NO.1; The amino acid sequence of the motor neuron survival protein is shown in SEQ ID NO.2; The adeno-associated virus capsid is an AAV9 capsid.

2. The recombinant adeno-associated virus vector according to claim 1, characterized in that, The sequence encoding the motor neuron survival protein was cloned using the primer set shown in SEQ ID NO.4 and SEQ ID NO.5 with hSMN1 cDNA as a template.

3. The recombinant adeno-associated virus vector according to claim 1, characterized in that, The gene for the neuronal survival protein is the SMN1 gene, and its nucleotide sequence is shown in SEQ ID NO.

3.

4. A pharmaceutical composition, characterized in that, Includes the recombinant adeno-associated virus vector as described in any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is administered by injection.

6. The use of the recombinant adeno-associated virus vector according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 4 or 5, in the preparation of a medicament for treating spinal muscular atrophy.

Images