Recombinant expression system for expressing human GLB1 protein, protein expression cassette, recombinant adeno-associated virus vector and application of recombinant adeno-associated virus vector in prevention and treatment of GM1 ganglioside storage disease

Abstract

The invention belongs to the technical field of gene therapy, and particularly relates to a recombinant expression system for expressing human GLB1 protein, a protein expression cassette, a recombinant adeno-associated virus vector and application of the recombinant adeno-associated virus vector in prevention and treatment of GM1 ganglioside storage diseases. In order to solve the problem that in an existing GLB1 gene expression system, due to the fact that a transcriptional regulation element combination is not systematically optimized, the expression efficiency in a central nervous system is insufficient, the codon-optimized human GLB1 gene and various different promoter-intron combinations are subjected to systematic pairing and functional verification; it is found that a recombinant expression system formed by a CB7 promoter-chimeric intron 1-codon optimized human GLB1 gene (SEQ ID NO: 2) is packaged into a recombinant adeno-associated virus AAV9.CB7. HGLB1co.bGH, the effect of treating the GM1 ganglioside storage disease is most remarkable, and the GM1 ganglioside storage disease is expected to be efficiently and safely prevented and radically treated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of gene therapy, and particularly relates to a recombinant expression system for expressing human GLB1 protein, a protein expression cassette, a recombinant adeno-associated virus vector and application thereof in preventing and treating GM1 gangliosidosis. BACKGROUND

[0002] GM1 gangliosidosis is an autosomal recessive lethal lysosomal storage disease caused by pathogenic mutations in the GLB1 gene. The mutations lead to a significant decrease or complete loss of the activity of the lysosomal hydrolase, beta-galactosidase (β-gal), which results in the inability to effectively degrade the substrate GM1 ganglioside, leading to abnormal accumulation in the lysosomes of neurons and other cells, ultimately triggering progressive and irreversible neurodegenerative changes. According to the age of onset and clinical features, the disease can be divided into three subtypes: type I (infantile type), type II (late infantile / adolescent type), and type III (adult / chronic type). Infantile patients usually develop the disease within a few months after birth, with rapid disease progression, and most die before the age of 2 to 3. Currently, there is no approved cure for this disease worldwide.

[0003] Existing treatment methods mainly focus on symptomatic support, such as using antiepileptic drugs to control seizures, delaying motor function decline through rehabilitation training, and providing respiratory and nutritional support. However, these methods can only temporarily alleviate some clinical symptoms and cannot fundamentally block disease progression. In the exploratory treatment aspect, enzyme replacement therapy has limited efficacy on the central nervous system due to the difficulty of exogenous enzymes to penetrate the blood-brain barrier; small molecule drugs (such as miglustat) usually rely on residual enzyme activity in patients, so they are essentially ineffective for infantile patients with complete loss of enzyme activity, and also have the problem of narrow therapeutic window.

[0004] For central nervous system diseases, direct drug delivery to the lesion is considered the most efficient treatment strategy. To achieve this goal, a delivery method that can bypass the blood-brain barrier is needed to effectively deliver therapeutic vectors to the central nervous system (CNS). Common central nervous system delivery routes include intrathecal injection, intracerebroventricular injection, and intracerebral injection. These methods can achieve efficient distribution and transduction of viral vectors in brain tissue or spinal cord, thereby overcoming the low efficiency of traditional intravenous injection delivery. Although different delivery routes differ in anatomical location and initial distribution, they all aim to achieve efficient transduction of exogenous genes in the central nervous system.

[0005] Adeno-associated virus (AAV)-based gene therapy provides new hope for GM1 gangliosidosis. AAV vectors have the advantages of high safety and long-term stable expression of exogenous genes. However, the application of this technology to the treatment of GM1 gangliosidosis still faces multiple challenges: first, the expression level of exogenous GLB1 gene in the central nervous system is usually insufficient, making it difficult to produce sufficient enzyme activity to remove accumulated substrates; second, the blood-brain barrier limits the effective delivery of intravenously injected vectors to brain tissue; in addition, most existing treatment options begin to intervene after the onset of clinical symptoms, at which point the nerve damage is often irreversible.

[0006] The analysis of existing patent documents further reveals the limitations of current technical solutions: for example, patent CN116033915A uses AAVrh.10 vectors to deliver GLB1 genes, which uses the CAG promoter and the natural GLB1 sequence, and there is room for further improvement in expression efficiency, and its treatment plan is mainly for individuals who have already developed symptoms, and does not involve preventive treatment in the neonatal period; patent CN116926047A mentions codon-optimized GLB1 sequences or individual variants, but does not explore the synergistic expression effects that may be brought about by combining optimized sequences with different transcription regulatory elements (such as promoters and introns). As known to those skilled in the art, promoters determine the expression intensity and tissue specificity of exogenous genes, and introns can significantly improve mRNA processing and stability; however, in the specific context of GM1 treatment, it is still unknown and difficult to predict which promoter-intron combination can efficiently synergize with the optimized GLB1 gene to achieve efficient and persistent enzyme expression in the central nervous system. Simply adding known optimized elements cannot ensure ideal efficacy, and may even lead to decreased expression efficiency due to compatibility issues between elements, making it difficult to achieve effective clearance of GM1 deposits throughout the brain.

[0007] Therefore, there is an urgent need in the art to develop a GLB1 gene expression system that has been systematically optimized and functionally verified to address the core problem of insufficient expression efficiency in the central nervous system due to the lack of systematic optimization of transcription regulatory element combinations in existing GLB1 gene expression systems. SUMMARY

[0008] To address the core problem of insufficient expression efficiency in the central nervous system due to the lack of systematic optimization of transcription regulatory element combinations in existing GLB1 gene expression systems, the present invention successfully identifies several specific promoter-intron combinations by systematically pairing and functionally verifying codon-optimized human GLB1 genes with a variety of different promoter-intron combinations. Compared with the prior art, the present invention presents the following significant advantages: 1. Better timing of treatment: By intervening in the neonatal period or early in the disease, and using direct delivery to the central nervous system (such as intrathecal injection, brain parenchymal injection or intracerebroventricular injection), it is possible to achieve whole-brain range of gene transduction, so as to implement intervention before or at an early stage of neurodegeneration, and achieve ideal prevention and treatment effect.

[0009] 2. High efficiency of whole-brain expression: By using optimized GLB1 gene and suitable AAV serotypes, combined with direct delivery to the central nervous system, efficient distribution and transduction of viral vectors in the brain is achieved.

[0010] 3. Direct and effective delivery to the central nervous system: Compared with intravenous injection, direct delivery to the central nervous system (such as intrathecal injection, brain parenchymal injection or intracerebroventricular injection) can achieve more efficient delivery to the central nervous system and effectively bypass the blood-brain barrier. The embodiments of the present application prove that even a single intracerebroventricular injection in the neonatal period can achieve widespread and uniform distribution of viruses in the brain, which strongly proves the overall effectiveness of the central nervous system delivery strategy.

[0011] The specific combination of transcription regulatory elements obtained by systematic screening effectively overcomes the key bottlenecks of existing GM1 gene therapy, such as insufficient central expression efficiency, incomplete pathological improvement and limited behavioral recovery, and achieves comprehensive reversal from the molecular, cellular to behavioral levels, providing a highly efficient, safe and durable breakthrough treatment strategy for GM1 gangliosidosis.

[0012] To achieve the above application purposes, the technical solutions adopted by the present application are as follows: In a first aspect, the present application provides a recombinant expression system for expressing human GLB1 protein, which comprises: a codon-optimized human GLB1 coding gene, a promoter driving the expression of the gene, and a chimeric intron located downstream of the promoter; the promoter is selected from at least one of CB7, CBh or UbC; the nucleotide sequence of the codon-optimized human GLB1 coding gene is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0013] Most preferably, the nucleotide sequence of the codon-optimized human GLB1 coding gene is shown in SEQ ID NO: 2.

[0014] Preferably, the promoter is a CB7 promoter.

[0015] Most preferably, the nucleotide sequence of the CB7 promoter is shown in SEQ ID NO: 9.

[0016] Preferably, the chimeric intron is selected from at least one of hybrid intron, SV40 intron, chimeric intron 1 or chimeric intron 2.

[0017] More preferably, the nucleotide sequence of the hybrid intron is shown as SEQ ID NO: 6.

[0018] More preferably, the nucleotide sequence of the SV40 intron is shown as SEQ ID NO: 8.

[0019] More preferably, the nucleotide sequence of the chimeric intron 1 is shown as SEQ ID NO: 10.

[0020] More preferably, the nucleotide sequence of the chimeric intron 2 is shown as SEQ ID NO: 12.

[0021] Most preferably, the chimeric intron is selected from chimeric intron 1, the nucleotide sequence of which is shown as SEQ ID NO: 10.

[0022] In a second aspect, the present application provides a human GLB1 protein expression cassette, which contains the above-mentioned recombinant expression system for expressing human GLB1 protein.

[0023] Preferably, the expression cassette comprises, from 5' end to 3' end, a promoter, an intron, a target gene and a polyA signal sequence; wherein the target gene is a codon-optimized human GLB1 coding gene; the nucleotide sequence of the target gene is shown as SEQ ID NO: 2.

[0024] Preferably, the promoter is CB7 promoter, the nucleotide sequence of which is shown as SEQ ID NO: 9.

[0025] Preferably, the chimeric intron is chimeric intron 1, the nucleotide sequence of which is shown as SEQ ID NO: 10.

[0026] In a third aspect, the present application provides a recombinant adeno-associated virus vector, which contains the above-mentioned human GLB1 protein expression cassette.

[0027] Preferably, the recombinant adeno-associated virus vector is selected from at least one of the following serotypes or variants thereof: AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh74.

[0028] Most preferably, the recombinant adeno-associated virus vector is AAV9.

[0029] Preferably, the recombinant adeno-associated virus vector is prepared by co-transfecting host cells with three plasmids: the REP and CAP protein expression plasmid pAAV2 / 9 of AAV, the helper plasmid pAdΔF6, and the cis-plasmid of the recombinant adeno-associated virus vector containing the human GLB1 protein expression cassette described above, using a transfection reagent.

[0030] Preferably, the recombinant adeno-associated virus vector contains a recombinant adeno-associated virus, and the nucleotide sequence of the recombinant adeno-associated virus is selected from at least one of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0031] Preferably, the vector is suitable for administration by central nervous system delivery, including but not limited to intrathecal injection, brain parenchyma injection, or intracerebroventricular injection.

[0032] In a fourth aspect, the present application provides a pharmaceutical composition containing the recombinant adeno-associated virus vector described above.

[0033] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

[0034] Preferably, the pharmaceutical composition is suitable for administration by central nervous system delivery, including but not limited to intrathecal injection, brain parenchyma injection, or intracerebroventricular injection.

[0035] In a fifth aspect, the present application provides use of the recombinant expression system expressing human GLB1 protein, the human GLB1 protein expression cassette, the recombinant adeno-associated virus vector, or the pharmaceutical composition described above in the preparation of a medicament for preventing and / or treating GM1 gangliosidosis.

[0036] Preferably, in the use, the prepared medicament is administered by central nervous system delivery, including intrathecal injection, brain parenchyma injection, or intracerebroventricular injection.

[0037] Preferably, the GM1 gangliosidosis is type I, type II, and / or type III.

[0038] Beneficial effects: The present application successfully identifies several specific promoter-intron combinations of the recombinant expression system for expressing human GLB1 protein by systematically pairing and functionally verifying the codon-optimized human GLB1 gene with various different promoter-intron combinations. Through experimental verification, the codon-optimized human GLB1 gene hGLB1co2 (SEQ ID NO: 2) can significantly improve the expression and secretion levels of GLB1 enzyme in HEK293 cells. The hGLB1co2 gene combined with the promoter CB7, CBh or UbC and the corresponding intron combination exhibits a synergistic effect in the treatment of GM1 gangliosidosis, especially the specific combination of CB7 promoter-chimeric intron 1-codon-optimized human GLB1 gene (SEQ ID NO: 2), which is packaged and prepared into recombinant adeno-associated virus AAV9.CB7.hGLB1co.bGH, and the effect is most significant in the treatment of GM1 gangliosidosis, as follows: 1. Significant and synergistic improvement of gene expression efficiency The recombinant adeno-associated virus AAV9.CB7.hGLB1co.bGH of the specific combination of the present application achieves a leap in GLB1 enzyme activity in vivo and in vitro, and the effect is much better than that of other combinations. In vitro experiments, the supernatant enzyme activity of the codon-optimized sequence hGLB1co2 is 3.2 times that of the wild type, specifically 115.83 ± 12.35 vs 36.11 ± 1.66 μmol / (h·mL). In vivo experiments, the enzyme activity of this combination in the cerebral cortex region reaches 9.85 times that of the wild type, which is significantly higher than that of the CBh promoter combination (4.78 times) and the hSyn promoter combination (3.81 times), and it is confirmed by X-gal staining that it achieves the most extensive and highest intensity expression in the whole brain.

[0039] 2. Deep reversal of central nervous system pathology and neuroinflammation Through ultra-high efficiency enzyme expression, the recombinant adeno-associated virus AAV9.CB7.hGLB1co.bGH of the present application achieves deep pathological improvement from molecular accumulation to cell structure: Effective clearance of GM1 deposition: In key brain regions such as the cortex, thalamus and hippocampus, the amount of GM1 deposition returns to the level comparable to that of wild-type mice, and the GM1 content in the cortex is 98% of that of wild-type mice.

[0040] Neuronal structure repair and protection: Transmission electron microscopy shows that typical pathological phenomena have basically disappeared; the neuron number maintenance rate in the hippocampus reaches 96% of that of wild-type mice, which is significantly higher than that of the untreated group (73%).

[0041] Inhibition of neuroinflammatory system: The level of pro-inflammatory factor IL-1β returns to the normal range, and the degree of microglial cell activation is significantly reduced (p<0.001).

[0042] 3. Full recovery of behavioral function Under long-term (32 weeks) intervention, the recombinant adeno-associated virus AAV9.CB7.hGLB1co.bGH of the present application achieved near full recovery of motor and cognitive functions: Motor function: In the rotarod test, the average fall time was 163 seconds, which was not statistically different from the wild type of 160 seconds.

[0043] Cognitive function: In the DMP dry maze experiment, the escape time was 109 seconds, which was restored to a level comparable to the wild type of 105 seconds.

[0044] 4. Synchronous improvement of systemic and peripheral metabolism Through central delivery, the combination unexpectedly achieved recovery of enzyme activity and correction of metabolic disorders in peripheral tissues: Peripheral enzyme activity: In the heart and liver, it reached 13.24 times and 2.81 times the wild type enzyme activity, respectively.

[0045] Metabolite clearance: Urinary GAG levels decreased to 49% of the wild type level (199.19 ± 47.90 μg / mg); serum AST levels returned to the normal range.

[0046] 5. Optimization of treatment strategy and long-term safety The present application achieves early intervention and efficient transduction by using direct delivery to the central nervous system in the neonatal period, such as intracerebroventricular injection. The 32-week long-term experiment confirmed that this program effectively reversed the pathology of the central nervous system while not causing significant toxic reactions, and the therapeutic effect was stable and lasting, showing excellent biological safety and treatment persistence. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The structure diagram of various rAAV expression plasmids constructed in Example 1 of the present application.

[0048] Figure 2 The comparison of enzyme activity of wild type and codon optimized human GLB1 in HEK293 cells in Example 1 of the present application: (A) Cell culture supernatant enzyme activity; (B) Cell lysate enzyme activity.

[0049] Figure 3 The virus structure diagram of AAV9.CB7.hGLB1co.bGH, AAV9.CBh.hGLB1co.bGH, AAV9.UbC.hGLB1co.bGH and AAV9.hSyn.hGLB1co.bGH in Example 2 of the present application.

[0050] Figure 4Long-term recovery of GLB1 enzyme activity in serum of GM1 model mice after AAV9 gene therapy in Example 3 of the present application.

[0051] Figure 5 Recovery effect of GLB1 enzyme activity in peripheral tissues of GM1 model mice after AAV9 gene therapy in Example 3 of the present application. (A-E) are the enzyme activity determination results of heart, liver, spleen, lung and kidney, respectively.

[0052] Figure 6 Recovery and distribution of GLB1 enzyme activity in brain tissues of GM1 model mice after AAV9 gene therapy in Example 3 of the present application: (A) X-gal staining of whole brain shows enzyme activity distribution; (B-H) are the enzyme activity quantitative analysis of olfactory bulb, cortex, spinal cord, striatum, thalamus and brainstem, hippocampus and cerebellum, respectively.

[0053] Figure 7 Clearance effect of GM1 deposition in brain tissues of GM1 model mice by AAV9 gene therapy in Example 4 of the present application: (A-C) LC-MS / MS quantitative analysis of GM1 content in cortex, cerebellum and spinal cord; (D-F) semi-quantitative analysis of GM1 immunofluorescence in cortex, thalamus and hippocampus; (G) representative images of GM1 immunofluorescence in the above brain regions.

[0054] Figure 8 Improvement effect of AAV9 gene therapy on neuron pathology of GM1 model mice in Example 5 of the present application: (A) representative image of cortical neuron transmission electron microscope; (B) representative image of brain tissue NeuN immunofluorescence; (C) quantitative analysis of NeuN immunofluorescence intensity in hippocampus.

[0055] Figure 9 Improvement effect of AAV9 gene therapy on neuroinflammation of GM1 model mice in Example 6 of the present application: (A-C) quantitative analysis of IBA1 and IL-1β immunofluorescence intensity in cortex, thalamus and hippocampus; (D) representative images of IBA1 and IL-1β immunofluorescence in the above brain regions.

[0056] Figure 10 Improvement effect of AAV9 gene therapy on motor function of GM1 model mice in Example 7 of the present application: (A) schematic diagram of rotating rod experiment device; (B) statistical results of fall latency of mice in each group on the rotating rod.

[0057] Figure 11 Improvement effect of AAV9 gene therapy on cognitive function of GM1 model mice in Example 8 of the present application: (A) schematic diagram of delayed matching place (DMP) dry maze device; (B) average escape time of mice in each group in the maze; (C) maze movement trajectory and heat map of a representative mouse.

[0058] Figure 12 Effects of AAV9 gene therapy on body weight gain in GM1 model mice in Example 9 of the present application: (A) body weight change curve of female mice; (B) body weight change curve of male mice.

[0059] Figure 13 Effects of AAV9 gene therapy on the level of glucosaminoglycan (GAG) in the urine of GM1 model mice in Example 10 of the present application.

[0060] Figure 14 Distribution of AAV9 viral vectors in various tissues of GM1 model mice in Example 11 of the present application: (A) liver; (B) cerebral cortex; (C) cerebellum; (D) spinal cord.

[0061] Figure 15 Effects of AAV9 gene therapy on serum biochemical indicators in GM1 model mice in Example 11 of the present application: (A) serum ALT level; (B) serum AST level.

[0062] Figure 16 Results of histopathological evaluation (H&E staining) of GM1 model mice after AAV9 gene therapy in Example 11 of the present application.

[0063] Statistical Notes: All statistical analyses were performed using GraphPad Prism 10 software. Data are presented as mean ± standard deviation (SD) or mean ± standard error (SEM). Dunnett's test was used for comparison between groups. p values are as follows: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; # p<0.05; ## p<0.01; ### p<0.001; #### p<0.0001.

[0064] Pathological Notes: Figure 8 A, N normal nucleus; N* abnormal nucleus; mit normal mitochondria; mit* abnormal mitochondria; RER rough endoplasmic reticulum; LF lipofuscin; AL autolysosome; SL secondary lysosome; * mild expansion of perinuclear space. Scale bar: 20 μm. DETAILED DESCRIPTION

[0065] In order to make the technical problems, technical solutions and beneficial effects to be solved in the present application more clear and explicit, the present application will be further described in detail below in combination with embodiments. Unless otherwise defined, all technical terms used in this text have the same meaning as understood by those of ordinary skill in the art.

[0066] In some embodiments of the present application, the human GLB1 gene is codon-optimized to obtain multiple optimized sequences with high expression potential, named hGLB1col (SEQ ID NO: 1), hGLB1co2 (SEQ ID NO: 2) and hGLB1co3 (SEQ ID NO: 3). Each GLB1 gene sequence is cloned into a backbone plasmid vector to obtain a plasmid expressing human GLB1 protein, and the expression of GLB1 enzyme activity is determined.

[0067] In some preferred embodiments of the present application, it is found that the codon-optimized human GLB1 gene hGLB1co2 (SEQ ID NO: 2) can significantly improve the expression and secretion level of GLB1 enzyme in HEK293 cells.

[0068] In some embodiments of the present application, the optimized human GLB1 gene hGLB1co2 (SEQ ID NO: 2) is systematically paired and functionally verified with different promoter-intron combinations. For this purpose, a variety of expression vectors are constructed, which respectively contain the codon-optimized GLB1 gene (SEQ ID NO: 2) and are combined with promoters selected from CBh, CB7, UbC, hSyn and their corresponding introns to package and produce recombinant adeno-associated virus.

[0069] In some embodiments of the present application, the recombinant adeno-associated virus vector is selected from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 or AAVrh74.

[0070] In some preferred embodiments of the present application, the AAV9 viral vector with good neural tropism is selected.

[0071] In some more preferred embodiments of the present application, the optimized human GLB1 gene hGLB1co2 (SEQ ID NO: 2) is combined with the promoters CBh, CB7, UbC, hSyn and their corresponding introns, respectively, and AAV9 is used as the recombinant adeno-associated virus vector to package and produce recombinant AAV9 virus, and the obtained viruses are named as: AAV9.CB7.hGLB1co.bGH, AAV9.CBh.hGLB1co.bGH, AAV9.UbC.hGLB1co.bGH and AAV9.hSyn.hGLB1co.bGH.

[0072] In one embodiment of the present application, in a neonatal mouse model of GM1 disease, through central nervous system delivery of AAV9 vectors loaded with different combinations of GLB1 gene expressing hGLB1co2 (SEQ ID NO: 2), it is confirmed through rigorous in vivo experiments that, although all treatment groups can improve GLB1 activity in the brain to some extent, a specific combination, especially the combination of CB7 promoter-chimeric intron 1 and the codon-optimized GLB1 gene of the present application (denoted as AAV9.CB7.hGLB1co.bGH), shows better GLB1 enzyme activity and more significant pathological improvement in long-term in vivo experiments. This preferred combination not only achieves a wide and high-intensity distribution of GLB1 enzyme activity in the whole brain (confirmed by X-gal staining), for example, the enzyme activity in the cerebral cortex region is increased by nearly 10 times the wild-type level, but also induces the most significant pathological improvement and functional recovery, which is specifically manifested as: GM1 deposits are effectively cleared, neuronal ultrastructure is well repaired, microglial cell activation is effectively inhibited, and motor and cognitive functions are restored to near normal levels.

[0073] In addition, in a neonatal mouse model, through central nervous system delivery of AAV9.CB7.hGLB1co.bGH vectors, not only GLB1 enzyme activity in brain tissue is significantly restored, but also GLB1 enzyme activity in serum and peripheral tissues is restored, and the expressed enzyme activity can be maintained for a long time, showing good stability and persistence.

[0074] In some embodiments of the present application, the central nervous system delivery method is selected from at least one of intracerebroventricular injection, intrathecal injection or brain parenchyma injection.

[0075] In some preferred embodiments of the present application, the central nervous system delivery method is selected from intracerebroventricular injection.

[0076] Based on the teachings of the present application, the general principles of central nervous system delivery disclosed in this specification, and the efficient transduction and expression effects of viral vectors in brain tissue demonstrated by the following specific examples, other central nervous system delivery methods, such as intrathecal injection or brain parenchyma injection, are also suitable for delivering the recombinant viral vectors of the present application to achieve the purpose of treating GM1 gangliosidosis. The "intracerebroventricular injection" used in the examples should be regarded as a successful example of all central nervous system delivery methods, rather than the exclusive and only choice.

[0077] Specific embodiments will be illustrated below to explain the scheme of the present application. Those skilled in the art will understand that the following embodiments are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product manual is used. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0078] Reagents used in the following tests (1) Experimental animals: GM1 gangliosidosis model mice (GLB1 - / - ), genotype GLB1 G455R / G455R , C57BL / 6N genetic background, 1-2 days old, both male and female. Littermates wild-type C57BL / 6N mice and heterozygotes (GLB1 + / - ) mice were used as controls. All mice were raised in the animal house of the Sichuan University Animal Experiment Center and fed with standard feed. The animal experiment program was reviewed and approved by the Sichuan University West China Hospital Experimental Animal Ethics Committee.

[0079] (2) Cell lines: HEK293 cells (human embryonic kidney cell line) were purchased from ATCC, USA. Cell culture used DMEM high glucose medium (Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin double antibody, cultured at 37℃, 5% CO2.

[0080] (3) Main reagents: In-Fusion HD Cloning Kit was purchased from Clontech Laboratories Company; TransIT-X2 transfection reagent was purchased from MirusBio Company; 4-methyl umbelliferyl-β-D-galactose (4-MU-β-D-Gal) was purchased from Macklin Company; 4-methyl umbelliferyl (4-MU) standard was purchased from Sigma-Aldrich Company; BCA protein quantification kit was purchased from Thermo Fisher Scientific Company; creatinine quantification kit was purchased from Sigma-Aldrich Company; Glucosaminoglycan (GAG) detection kit was purchased from Biocolor Company; tissue genomic DNA extraction kit was purchased from Tiangen Biotech Co., Ltd.; 4% paraformaldehyde was purchased from Biosharp Company; enzyme activity reaction buffer was purchased from Feijie Biotech; Artificial cerebrospinal fluid (aCSF) was prepared according to the method described in the literature (Hinderer et al., Hum Gene Ther, 2020), and its composition was as follows: 1 mM phosphate buffer (pH 7.2), 150 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, 0.8 mM MgCl2, and 0.001% Pluronic F-68.

[0081] (4) Antibodies: Anti-ganglioside GM1 antibody (Bioryt, orb312249) was used at a concentration of 1:100; anti-NeuN antibody (Servicebio, GB11138-50) was used at a concentration of 1:200; anti-IL-1β antibody (Abcam, ab9722) was used at a concentration of 1:100; anti-IBA1 antibody (Wako, 016-26721) was used at a concentration of 1:100; CY3-labeled goat anti-rabbit IgG (Servicebio, GB21303) was used at a concentration of 1:300; Alexa Fluor 488-labeled goat anti-mouse IgG (Servicebio, GB25301) was used at a concentration of 1:400.

[0082] (5) Staining reagents: X-gal (5-bromo-4-chloro-3-indole-β-D-galactoside) staining solution was prepared according to the method described in the literature (Hocquemiller et al., Molecular ther, 2022), and its composition is as follows: 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 1 mg / mL X-gal.

[0083] The detection methods used in the following tests (1) Histopathological examination: hematoxylin-eosin (H&E) staining, GM1 immunofluorescence staining, NeuN immunofluorescence staining, IBA1 and IL-1β immunofluorescence staining; (2) Enzyme activity detection: GLB1 enzyme activity was measured using the 4-MU-β-D-Gal fluorescent substrate method and X-gal substrate enzyme activity staining. (3) Biochemical index detection: urine GAG ​​content measurement, serum ALT / AST level detection; (4) Molecular biological detection: extraction of tissue genomic DNA and detection of viral copy number; (5) Behavioral tests: rotating rod test, DMP dry maze test; (6) Ultrastructural observation: Transmission electron microscopy (TEM) analysis.

[0084] Example 1: Construction and in vitro verification of human GLB1 codon-optimized, expression plasmid vectors In this example, human GLB1 gene was first codon-optimized to obtain three optimized nucleotide sequences, named hGLB1col (SEQ ID NO: 1), hGLB1co2 (SEQ ID NO: 2) and hGLB1co3 (SEQ ID NO: 3), respectively. The wild-type hGLB1wt (SEQ ID NO: 4) and the above three codon-optimized sequences (hGLB1col-co3) were obtained by the method of whole gene synthesis. Then, each GLB1 gene sequence was cloned into a backbone plasmid vector using In-Fusion ligation technology. The backbone plasmid vector contains 5’ AAV2 ITR (SEQ ID NO: 17), CBh promoter (SEQ ID NO: 5), hybrid intron (SEQ ID NO: 6), bGH polyA signal sequence (SEQ ID NO: 18) and 3’ AAV2 ITR (SEQ ID NO: 19). Finally, the following plasmids were constructed: pAAV.CBh.hGLB1wt.bGH expressing wild-type human GLB1 protein (SEQ ID NO: 20), and pAAV.CBh.hGLB1col.bGH, pAAV.CBh.hGLB1co2.bGH and pAAV.CBh.hGLB1co3.bGH expressing codon-optimized human GLB1 protein (SEQ ID NO: 21).

[0085] Each of the above constructed plasmids was taken 500 ng and transfected into HEK293 cells plated in a 24-well plate using TransIT-X2 transfection reagent. After 48 hours of transfection, the cell culture supernatant was collected, and the cell lysate was obtained by lysing the cells. The total protein in the cell lysate was quantified by BCA method. The GLB1 enzyme activity was determined as follows: 10 μL of cell supernatant or cell lysate containing 500 ng of total protein (made up to 30 μL with the corresponding buffer) was mixed with 30 μL of 4-MU-β-D-Gal substrate solution (0.75 μmol / mL, dissolved in pH 4.5 buffer), and incubated at 37°C for 30 minutes in the dark. Then, 30 μL of termination reaction solution was taken into a 96-well plate, 150 μL of sodium carbonate solution (pH 10.7) was added to terminate the reaction, and the fluorescence intensity was detected using a multifunctional enzyme marker. The enzyme activity was calculated according to the 4-MU standard curve, and the enzyme activity in the cell supernatant was expressed as μmol / (h·mL), and the enzyme activity in the cell lysate was expressed as μmol / (h·mg). The experimental results are shown in Table 1. Figure 2 (n=3): Cell supernatant enzyme activity [pmol / (h mL)]: pAAV.CBh.hGLB1wt.bGH: 36.11 ± 1.66; pAAV.CBh.hGLB1col.bGH: 82.87 ± 6.7; pAAV.CBh.hGLB1co2.bGH: 115.83 ± 12.35; pAAV.CBh.hGLB1co3.bGH: 102.89 ± 0.88.

[0086] Cell lysate enzyme activity [pmol / (h mg)]: pAAV.CBh.hGLB1wt.bGH: 1076.21 ± 35.19; pAAV.CBh.hGLB1col.bGH: 1980.38 ± 94.86; pAAV.CBh.hGLB1co2.bGH: 1774.17 ± 285.69; pAAV.CBh.hGLB1co3.bGH: 1958.35 ± 48.83.

[0087] The above results show that codon optimization can significantly improve the expression and secretion level of GLB1 enzyme in HEK293 cells compared with the wild-type sequence. Comprehensive comparison of enzyme activity in cell supernatant and lysate, hGLB1co2 sequence (SEQ ID NO: 2) exhibits the most optimal comprehensive expression efficiency. Therefore, hGLB1co2 is selected as the optimal sequence and used for subsequent all vector construction. Unless otherwise specified, "hGLB1co" mentioned in the following refers to hGLB1co2 (SEQ ID NO: 2).

[0088] Example 2: Human GLB1 vector optimization and virus preparation Based on the selected codon-optimized sequence hGLB1co (SEQ ID NO: 2), expression plasmids containing different promoters and corresponding introns were further constructed: pAAV.hSyn.hGLB1co.bGH (containing hSyn promoter SEQ ID NO: 7 and SV40 intron SEQ ID NO: 8), pAAV.CB7.hGLB1co.bGH (containing CB7 promoter SEQ ID NO: 9 and chimeric intron 1 SEQ ID NO: 10), pAAV.UbC.hGLB1co.bGH (containing UbC promoter SEQ ID NO: 11 and chimeric intron 2 SEQ ID NO: 12). Subsequently, referring to the three-plasmid co-transfection method of Lock et al. (Hum Gene Ther, 2010), using PEIpro transfection reagent, AAV2 Rep / AAV9 Cap protein expression plasmid (pAAV2 / 9), helper plasmid (pAdAF6) and each AAV packaging cis-plasmid (such as pAAV.CB7.hGLB1co.bGH) were co-transfected into HEK293 cells to package and produce recombinant AAV9 virus. The resulting viruses were named: AAV9.hSyn.hGLB1co.bGH (SEQ ID NO: 16), AAV9.CB7.hGLB1co.bGH (SEQ ID NO: 13), AAV9.UbC.hGLB1co.bGH (SEQ ID NO: 15) and AAV9.CBh.hGLB1co.bGH (SEQ ID NO: 14), and the structural schematic diagram is shown in Figure 3 .

[0089] After transfection for 144 hours, the cell culture supernatant was collected, and then concentrated by tangential flow filtration and purified by iodixanol density gradient ultracentrifugation. The purified virus was desalted using an Amicon Ultra-100K ultrafiltration centrifuge tube and replaced into artificial cerebrospinal fluid (aCSF, composition see reagent part). The final virus stock was evaluated for purity by SDS-PAGE, endotoxin content was detected by limulus reagent method, and absolute quantification of viral genome titer was performed by digital PCR (ddPCR), and the obtained virus was used for subsequent animal experiments.

[0090] Example 3: Long-term AAV9 gene therapy restores GLB1 enzyme activity in serum and peripheral tissues of GM1 disease model The AAV9 viral vectors AAV9.CBh.hGLB1co.bGH, AAV9.CB7.hGLB1co.bGH, AAV9.UbC.hGLB1co.bGH, and AAV9.hSyn.hGLB1co.bGH, carrying different promoters (CBh, CB7, UbC, hSyn) to drive the hGLB1co gene and conjugated with bGH polyA, were administered to 1-2 day old GM1 model mice via central nervous system delivery (in this example, intraventricular injection). The injection dose was 6 × 10⁶ mice per mouse. 10 Genome copy number (GC). Wild-type mice, heterozygous mice, and untreated GM1 model mice were used as control groups.

[0091] Serum GLB1 enzyme activity restored Starting from week 8 of treatment, blood was collected every 4 weeks via the orbital venous plexus, and serum was separated. GLB1 enzyme activity (unit: μmol / (h·mL)) was measured using the method described in Example 1. At week 32 of treatment, the serum GLB1 enzyme activity results for each group are as follows: Figure 4 As shown (n=10 for each group): Untreated group: 5.69 ± 0.22 Heterozygous control group: 26.83 ± 3.02 Wild-type control group: 59.76 ± 3.96 AAV9.CB7.hGLB1co.bGH treatment group: 23.95 ± 3.50 AAV9.CBh.hGLB1co.bGH treatment group: 26.87 ± 3.46 AAV9.UbC.hGLB1co.bGH treatment group: 14.78 ± 2.67 AAV9.hSyn.hGLB1co.bGH treatment group: 7.03 ± 0.69 Statistical analysis showed that serum GLB1 enzyme activity in the AAV9.CBh.hGLB1co.bGH and AAV9.CB7.hGLB1co.bGH treatment groups recovered to the level of the heterozygous control group (p>0.05 compared with the heterozygous group).

[0092] tissue GLB1 enzyme activity analysis At the end of treatment (around week 32), peripheral organs (heart, liver, spleen, lungs, kidneys) and different brain regions of mice were collected, tissue homogenates were prepared, and GLB1 enzyme activity was detected.

[0093] Peripheral tissues ( Figure 5A-E): AAV9.CB7.hGLB1co.bGH treatment group significantly improved enzyme activity in heart, liver tissue ( Figure 5 A and Figure 5 B), reaching supraphysiological levels (13.24-fold and 2.81-fold of wild type, respectively, p<0.0001). AAV9.CBh.hGLB1co.bGH and AAV9.UbC.hGLB1co.bGH treatment groups restored cardiac enzyme activity to wild type levels ( Figure 5 A), and liver enzyme activity to heterozygote levels ( Figure 5 B); enzyme activity in spleen, lung, and kidney tissue was increased but not restored to normal levels ( Figure 5 C-E) Brain tissue: GLB1 enzyme activity was detected in various brain regions including olfactory bulb, cortex, hippocampus, striatum, thalamus, brainstem, cerebellum, and spinal cord ( Figure 6 B-H). All treatment groups significantly improved GLB1 activity in the brain, with AAV9.CB7.hGLB1co.bGH showing superior delivery and expression efficiency in the brain than other treatment groups, achieving the highest GLB1 enzyme activity restoration in multiple brain regions including olfactory bulb, cortex, spinal cord, striatum, thalamus and brainstem, hippocampus, and cerebellum, reaching 5.20-fold, 9.85-fold, 5.77-fold, 7.17-fold, 7.35-fold, 8.39-fold, and 6.34-fold of wild type levels, respectively.

[0094] The fold increase in cortex enzyme activity was as follows ( Figure 6 B): AAV9.CB7.hGLB1co.bGH: 9.85-fold of wild type (p<0.0001, n = 7); AAV9.UbC.hGLB1co.bGH: 9-fold of wild type (p<0.0001, n = 7); AAV9.CBh.hGLB1co.bGH: 4.78-fold of wild type (p<0.001, n = 7); AAV9.hSyn.hGLB1co.bGH: 3.81-fold of wild type (p<0.05, n = 6).

[0095] Brain tissue GLB1 enzyme activity distribution staining At 32 weeks post-treatment, mouse brain tissue was fixed with 4% paraformaldehyde, dehydrated, OCT-embedded, and snap-frozen to prepare 8 μm thick sagittal frozen sections. After fixation with 0.25% glutaraldehyde and PBS washing, the sections were incubated in X-gal staining solution at 37°C in the dark overnight, then counterstained with nuclear fast red and mounted. The images were collected using a whole-slide scanning system (3DHISTECH, Pannoramic MIDI). The staining results showed that Figure 6A): The AAV9.CB7.hGLB1co.bGH treatment group showed the widest distribution and highest intensity of enzyme activity staining, followed by the AAV9.UbC.hGLB1co.bGH treatment group, AAV9.CBh.hGLB1co.bGH treatment group, and AAV9.hSyn.hGLB1co.bGH treatment group. This result is consistent with the quantitative analysis of enzyme activity in brain tissue. AAV9.CB7.hGLB1co.bGH can achieve efficient GLB1 gene expression in both the central nervous system and peripheral tissues of GM1 disease model mice, especially causing the most significant increase in GLB1 enzyme activity in brain tissue, showing superior therapeutic potential.

[0096] Example 4: Long-term AAV9 gene therapy significantly reduced neuronal GM1 deposition in brain tissue of a GM1 disease model. Brain tissue from mice treated for 32 weeks in Example 3 was paraffin-embedded and sectioned. Semi-quantitative analysis was performed by GM1 immunofluorescence staining, and the GM1 content in the cortex, cerebellum and spinal cord was precisely quantified by LC-MS / MS.

[0097] Immunofluorescence staining analysis GM1 immunofluorescence staining results ( Figure 7 DG showed that, compared with the untreated model group, all AAV9 treatment groups (including AAV9.CBh.hGLB1co.bGH, AAV9.CB7.hGLB1co.bGH, AAV9.UbC.hGLB1co.bGH, and AAV9.hSyn.hGLB1co.bGH) showed significant improvements in key brain regions—the cortex (DG). Figure 7 D) Thalamus ( Figure 7 E) and hippocampus ( Figure 7 GM1 deposits in F) were effectively cleared and restored to normal levels. Specifically, the AAV9.CB7.hGLB1co.bGH treatment group showed an average GM1 fluorescence intensity in the cortical region that was 98% of the wild-type control group.

[0098] Quantitative verification The GM1 content in different brain tissue regions (cortex, cerebellum, spinal cord) was precisely quantified using LC-MS / MS, and the results ( Figure 7 The results (AC) showed that all AAV9 treatment groups significantly reduced GM1 levels in all brain regions, restoring them to the normal range in wild-type or heterozygous mice. This result is corroborated by the semi-quantitative immunofluorescence analysis.

[0099] The above results show that each AAV9-hGLB1 co-vector delivered by intracerebroventricular injection can effectively mediate the expression and secretion of GLB1 enzyme in the brain tissue of GM1 model mice, thereby significantly reducing the abnormal deposition of GM1 in neurons and restoring it to the physiological level. This confirms the effectiveness of AAV9 carrying the codon-optimized human GLB1 gene therapy for GM1 gangliosidosis from the pathological storage level.

[0100] Example 5: Long-term AAV9 gene therapy improves neuronal morphology and maintains neuron number in GM1 disease model The brain tissue of the mice treated for 32 weeks in Example 3 was taken to prepare paraffin sections, and the neuron number was semi-quantitatively analyzed by NeuN immunofluorescence staining, and the ultrastructure of cortical neurons was observed by transmission electron microscopy (TEM).

[0101] Immunofluorescence staining analysis The results of NeuN immunofluorescence staining Figure 8 B-C) show that compared with the untreated model group, the NeuN immunofluorescence signal of each AAV9 treatment group is enhanced in the entire brain sagittal section. Semi-quantitative analysis of the hippocampal region Figure 8 C) shows that the AAV9.CB7.hGLB1co.bGH treatment group has the highest average fluorescence intensity, reaching 96% of the wild-type level, which is significantly higher than the untreated disease group (73% of the wild-type level).

[0102] Transmission electron microscopy ultrastructure analysis The results of TEM observation of cortical neurons Figure 8 A) shows that the untreated group has typical pathological changes such as autophagolysosome (AL), a large amount of lipofuscin (LF), and nuclear membrane swelling (*). Each AAV9 treatment group shows different degrees of ultrastructure improvement. Among them, the AAV9.CB7.hGLB1co.bGH treatment group has a neuronal structure that basically recovers to the level of wild-type mice, and no obvious pathological features are observed; the remaining treatment groups do not observe obvious autophagolysosomes, but still have mitochondrial (mit*) swelling, and the improvement degree is similar to that of the heterozygous mice.

[0103] The above results show that long-term AAV gene therapy can effectively maintain the neuron number in the brain tissue of GM1 model mice and improve the ultrastructure pathology. AAV9.CB7.hGLB1co.bGH shows the best effect in neuroprotection.

[0104] Example 6: Long-term AAV9 gene therapy improves neuroinflammatory response in GM1 disease model The brain tissues of GM1 disease model mice treated for 32 weeks in Example 3 were paraffin-embedded and sectioned, and semi-quantitative analysis was performed by IBA1 (microglia marker) and IL-1β (inflammatory factor) immunofluorescence staining to evaluate the improvement of gene therapy on neuroinflammation.

[0105] IL-1β immunofluorescence staining results Figure 9 D): In the key brain regions of cortex (A), thalamus (B) and hippocampus (C), AAV9.CBh.hGLB1co.bGH, AAV9.CB7.hGLB1co.bGH and AAV9.UbC.hGLB1co.bGH treatment can significantly reduce IL-1β expression, so that it returns to the level without significant difference from normal mice. Figure 9 Figure 9 Figure 9

[0106] IBA1 immunofluorescence staining results: Although the degree of microglial cell activation in the cortex and hippocampus regions of the AAV9.CB7.hGLB1co.bGH treatment group did not completely return to the normal level, it was significantly decreased compared with the untreated disease group (p<0.001). The rest of the treatment groups (AAV9.CBh.hGLB1co.bGH and AAV9.UbC.hGLB1co.bGH) did not show statistically significant improvement in IBA1 expression.

[0107] The above results show that AAV9.CBh.hGLB1co.bGH, AAV9.CB7.hGLB1co.bGH and AAV9.UbC.hGLB1co.bGH can effectively inhibit IL-1β-related inflammatory response in GM1 model brain tissues, among which AAV9.CB7.hGLB1co.bGH shows the most significant effect in reducing microglial cell activation, showing the best potential for improving neuroinflammation.

[0108] Example 7: Long-term AAV9 gene therapy improves motor dysfunction in GM1 disease model The accelerating rotarod test was used to evaluate the motor coordination of mice (instrument schematic diagram see Figure 10 A). Briefly, the mice were first adapted to a rotation speed of 4 revolutions per minute for 3 minutes. In the formal test, the rotation speed increased uniformly from 5 revolutions per minute to 40 revolutions per minute, and the latency of the mouse falling off the rod was recorded, with a single test lasting up to 180 seconds, repeated three times. Tests were performed at weeks 8, 16, 24 and 32 after treatment, and each mouse in each group was tested 3 times, and the average latency was taken for statistical analysis. The results of motor function evaluation at week 32 after treatment are shown in Figure 10 B. Wild type group (n=11): 160 seconds​​​ Heterozygote group (n=8): 143 seconds Disease untreated group (n=9): 91 seconds AAV9.CB7.hGLB1co.bGH group (n=12): 163 seconds AAV9.CBh.hGLB1co.bGH group (n=12): 161 seconds AAV9.UbC.hGLB1co.bGH group (n=12): 155 seconds AAV9.hSyn.hGLB1co.bGH group (n=12): 127 seconds The above results show that AAV9.CB7.hGLB1co.bGH, AAV9.CBh.hGLB1co.bGH and AAV9.UbC.hGLB1co.bGH treatment can effectively correct the motor defects of GM1 model mice, and make their motor coordination ability recover to the wild type level. Although the AAV9.hSyn.hGLB1co.bGH treatment group has improved, it is not completely restored.

[0109] Example 8: Long-term AAV9 gene therapy improves cognitive dysfunction in GM1 disease model In the GM1 disease model mice treated for 32 weeks in Example 3, the spatial learning and memory ability of the mice was evaluated by DMP (delayed matching place) dry maze experiment (see Figure 11 A) to verify the improvement effect of gene therapy on neurocognitive function. Experimental method: The experiment used a DMP dry maze device, which was a circular platform with a diameter of 122 cm and a thickness of 1.2 cm, with 40 holes and the escape hole position changed daily, and the platform wall was provided with visual cues. During testing, the mice were placed at the edge of the platform and covered with an opaque funnel; after a delay of 30 seconds, a 2 kHz, 85 dB noise was played and the funnel was removed, exposing the mice to 1200 lux of light. The mice needed to find the escape hole on their own within 3 minutes. The whole process was collected by the ANY-Maze system, and the experimental scheme was 4 tests per day for each group, for 4 consecutive days. The average escape time of each group of mice for 4 consecutive days is shown in Figure 11 B, and the maze trajectory and heat map directly show the escape differences of each group (see Figure 11 C), and the escape time on the 4th day is as follows: Wild type group: 105 seconds Heterozygote group: 129 seconds Disease untreated group: 170 seconds AAV9.CB7.hGLB1co.bGH group: 109 seconds AAV9.CBh.hGLB1co.bGH group: 130 seconds AAV9.UbC.hGLB1co.bGH group: 135 seconds AAV9.hSyn.hGLB1co.bGH group: 136 seconds The above results show that all AAV9-hGLB1co treatment groups can improve the cognitive dysfunction of GM1 model mice. Among them, the cognitive function of the AAV9.CB7.hGLB1co.bGH treatment group is restored to the wild type level, and the effect is the most significant.

[0110] Example 9: Long-term AAV9 gene therapy improves weight gain in GM1 disease model The weight changes of the GM1 model mice treated for a long time (32 weeks) in Example 3 were monitored, and the weight was measured every 4 weeks from the 4th week after treatment. The weight gain curve is shown in Figure 12 A (female) and Figure 12 B (male). Taking the 16th week weight as an example: Female: wild type (21.41 ± 0.84 g), heterozygote (22.12 ± 1.52 g), GM1 untreated (23.11 ± 1.51 g). The body weight of each treatment group had no significant difference with the wild type, and the AAV9.CB7.hGLB1co.bGH group was 21.82 ± 0.79 g.

[0111] Male: wild type (26.98 ± 2.15 g), heterozygote (27.46 ± 1.18 g), GM1 untreated (29.69 ± 0.69 g). The body weight of each treatment group had no significant difference with the wild type, and the AAV9.CB7.hGLB1co.bGH group was 25.50 ± 1.22 g.

[0112] The above results show that each AAV9 treatment can restore the weight gain trend of GM1 model mice to normal, which is comparable to wild type mice.

[0113] Example 10: Long-term AAV9 gene therapy reduces GAG deposition levels in urine of GM1 disease model At the 32nd week of treatment, the urine of mice in each group was collected, and the urine glucosaminoglycan (GAG) content standardized by creatinine (unit: μg / mg creatinine) was detected. The urine GAG content detection results are shown in Figure 13 Wild type group (n=12): 403.14 ± 38.66 μg / mg ​Disease-untreated group (n=8): 720.05 ± 133.79 μg / mg (1.78 times that of wild type, p<0.05) AAV9.CB7.hGLB1co.bGH group (n=12): 199.19 ± 47.90 μg / mg (0.49 times that of wild type, p<0.0001 compared with untreated group) AAV9.CBh.hGLB1co.bGH group (n=12): 392.35 ± 64.95 μg / mg AAV9.UbC.hGLB1co.bGH group (n=12): 425.49 ± 54.14 μg / mg AAV9.hSyn.hGLB1co.bGH group (n=8): 412.04 ± 80.65 μg / mg The above results indicate that all AAV9-hGLB1co treatments significantly reduced GAG levels in the urine of GM1 model mice, restoring them to wild-type ranges. The AAV9.CB7.hGLB1co.bGH group showed the most significant GAG clearance effect, with levels lower than the wild-type baseline.

[0114] Example 11: Preliminary safety evaluation of long-term AAV9 gene therapy A comprehensive safety assessment was conducted on GM1 disease model mice treated for 32 weeks as described in Example 3.

[0115] Organizational distribution Genome data were extracted from mouse brain and liver tissues, and the absolute quantification of AAV9 viral genome copy number was performed using TaqMan probe qPCR. Results showed ( Figure 14 AAV9 is widely distributed in brain tissue (including the cortex, cerebellum, and spinal cord) and liver, indicating that the virus is widely distributed and does not cause significant toxicity.

[0116] Physiological index analysis Serum biochemical results showed that there were no significant differences in ALT levels among the groups (including the untreated group, wild-type group, and all treated groups). Figure 15 A). The AST levels are as follows ( Figure 15 B): Wild-type group: 49.96 ± 1.5 (n=9) Heterozygous group: 57.8 ± 3.62 (n=7) Disease untreated group: 79.91 ± 4.37 (n = 9), which is 1.6 times of the wild type group (p < 0.001); AAV9.CB7.hGLB1co.bGH treatment group: 59.5 ± 1.84 (n = 9); AAV9.CBh.hGLB1co.bGH treatment group: 61.22 ± 3.97 (n = 9); AAV9.UbC.hGLB1co.bGH treatment group: 55.56 ± 3.15 (n = 9); AAV9.hSyn.hGLB1co.bGH treatment group: 78.34 ± 10.45 (n = 9).

[0117] The above results show that: AAV9.CB7.hGLB1co.bGH, AAV9.CBh.hGLB1co.bGH and AAV9.UbC.hGLB1co.bGH treatment can significantly reduce the serum AST level of GM1 disease model mice, and restore it to near the wild type or heterozygote range.

[0118] Histopathological analysis (H&E staining) The brain tissue (including cortex, thalamus) and peripheral main organs (heart, liver, spleen, lung, kidney) were paraffin-embedded, sectioned and analyzed by H&E staining. Figure 16 ).

[0119] Brain tissue: The pathological results show that the degree of neuron vacuolization in the cerebral cortex and thalamic regions of all treatment groups of mice is significantly improved compared with the untreated group, and the neuron morphology is basically restored to the wild type mouse level, arranged in order, clear structure, and no obvious pathological phenomenon.

[0120] Peripheral organs: The H&E stained sections of the peripheral main organs (heart, liver, spleen, lung, kidney) were pathologically evaluated. The results show that compared with the disease untreated group with inflammatory cell infiltration, the above organs of the mice in each treatment group have complete morphological structure, and the inflammatory lesions of the liver are significantly improved. In addition, no significant pathological changes related to AAV viral vectors were observed in all treatment groups.

[0121] In summary, the above comprehensive analysis of viral distribution, histopathology and serum biochemical indicators shows that AAV9-hGLB1co given by intracerebral ventricular injection in newborns can effectively improve the neuropathology related to GM1 disease, and does not cause significant treatment-related toxicity in brain tissue and peripheral main organs, showing good biological safety.

[0122] The following is the sequence information involved in the present application SEQ ID NO: 1: hGLB1co1 (codon-optimized human GLB1 sequence 1) 2034 bp SEQ ID NO: 2: hGLBl co2 (codon-optimized human GLB 1 sequence 2) 2034 bp SEQ ID NO:3: hGLB1 co3 (codon-optimized human GLB1 sequence 3) 2034 bp SEQ ID NO:4: hGLB1 wt (wild-type human GLB1 sequence) 2034 bp SEQ ID NO:5: CBh promoter sequence 565 bp SEQ ID NO:6: Intron sequence / hybrid intron 228 bp SEQ ID NO:7: hSyn promoter sequence 448 bp SEQ ID NO:8: SV40 intron sequence 97 bp SEQ ID NO:9: CB7 promoter sequence 659 bp SEQ ID NO:10: Chimeric intron 1 sequence 1017 bp SEQ ID NO:11: UbC promoter sequence 1212 bp SEQ ID NO:12: Chimeric intron 2 sequence 133 bp SEQ ID NO:13: AAV9.CB7.hGLB1 co.bGH sequence (5' ITR - CB7 - Chimeric intron 1 - hGLB1 co - bGH - 3' ITR) 4479 bp SEQ ID NO: 14: AAV9.CBh.hGLBlco.bGH (5'ITR-CBh-hybrid intron-hGLBlco-bGH-3'ITR) sequence 3509 bp SEQ ID NO: 15: AAV9.UbC.hGLBlco.bGH (5'ITR-UbC-chimeric intron 2- hGLBlco-bGH-3'ITR) sequence 4204 bp SEQ ID NO: 16: AAV9.hSyn.hGLBlco.bGH (5'ITR-hSyn-SV40 Intron-hGLBlco-bGH-3'ITR) sequence 3388 bp SEQ ID NO: 17: 5' AAV2 ITR sequence 168 bp SEQ ID NO: 18: bGH polyA sequence 225 bp SEQ ID NO: 19: 3' AAV2 ITR sequence 168 bp SEQ ID NO: 20: Amino acid sequence of wild-type human GLB1 protein 677 amino acid residues SEQ ID NO: 21: Codon-optimized human GLB1 protein amino acid sequence 677 amino acid residues MPGFLVRILPLLLVLLLLGPTRGLRNATQRMFEIDYSRDSFLKDGQPFRYISGSIHYSRVPRFYWKDRLLKMKMAGLNAIQTYVPWNFHEPWPGQYQFSEDHDVEYFLRLAHELGLLVILRPGPYICAEWEMGGLPAWLLEKESILLRSSDPDYLAAVDKWLGVLLPKMKPLLYQNGGPVITVQVENEYGSYFACDFDYLRFLQKRFRHHLGDDVVLFTTDGAHKTFLKCGALQGLYTTVDFGTGSNITDAFLSQRKCEPKGPLINSEFYTGWLDHWGQPHSTIKTEAVASSLYDILARGASVNLYMFIGGTNFAYWNGANSPYAAQPTSYDYDAPLSEAGDLTEKYFALRNIIQKFEKVPEGPIPPSTPKFAYGKVTLEKLKTVGAALDILCPSGPIKSLYPLTFIQVKQHYGFVLYRTTLPQDCSNPAPLSSPLNGVHDRAYVAVDGIPQGVLERNNVITLNITGKAGATLDLLVENMGRVNYGAYINDFKGLVSNLTLSSNILTDWTIFPLDTEDAVRSHLGGWGHRDSGHHDEAWAHNSSNYTLPAFYMGNFSIPSGIPDLPQDTFIQFPGWTKGQVWINGFNLGRYWPARGPQLTLFVPQHILMTSAPNTITVLELEWAPCSSDDPELCAVTFVDRPVIGSSVTYDHPSKPVEKRLMPPPPQKNKDSWLDHV*.

Claims

1. A recombinant expression system expressing human GLB 1 protein, characterized in that, The system comprises: a codon-optimized human GLB1 coding gene, a promoter driving expression of the gene, and a chimeric intron downstream of the promoter; the promoter is selected from at least one of CB7, CBh, or UbC; the nucleotide sequence of the codon-optimized human GLB1 coding gene is as shown in SEQ ID NO: 2, SEQ ID NO: 1, or SEQ ID NO:

3.

2. The recombinant expression system expressing human GLB1 protein according to claim 1, characterized in that, the nucleotide sequence of the codon-optimized human GLB1 coding gene is as shown in SEQ ID NO:

2.

3. The recombinant expression system expressing human GLB1 protein according to claim 2, characterized in that, the promoter is a CB7 promoter, and the nucleotide sequence of the CB7 promoter is as shown in SEQ ID NO:

9.

4. The recombinant expression system expressing human GLB 1 protein according to any one of claims 1 to 3, characterized in that, the chimeric intron is selected from at least one of a hybrid intron, an SV40 intron, chimeric intron 1, or chimeric intron 2.

5. The recombinant expression system expressing human GLB1 protein according to claim 4, characterized in that, at least one of the following is met: the nucleotide sequence of the hybrid intron is as shown in SEQ ID NO: 6; the nucleotide sequence of the SV40 intron is as shown in SEQ ID NO: 8; the nucleotide sequence of the chimeric intron 1 is as shown in SEQ ID NO: 10; the nucleotide sequence of the chimeric intron 2 is as shown in SEQ ID NO:

12.

6. The recombinant expression system expressing human GLB1 protein according to claim 4, characterized in that, the chimeric intron is selected from chimeric intron 1, and the nucleotide sequence of the chimeric intron 1 is as shown in SEQ ID NO:

10.

7. A human GLB1 protein expression cassette characterized in that, The recombinant expression system for expressing human GLB1 protein according to any one of claims 1-6.

8. The human GLB1 protein expression cassette of claim 7, wherein, The expression cassette comprises, from 5' to 3', a promoter, an intron, a target gene, and a polyA signal sequence; wherein the target gene is a codon-optimized human GLB1 coding gene, and the nucleotide sequence of the target gene is as shown in SEQ ID NO:

2.

9. The human GLB1 protein expression cassette of claim 7 or 8, wherein, The promoter is a CB7 promoter, and the nucleotide sequence of the CB7 promoter is as shown in SEQ ID NO:

9.

10. The human GLB 1 protein expression cassette according to any one of claims 7 to 9, characterized in that, The chimeric intron is chimeric intron 1, and the nucleotide sequence of the chimeric intron 1 is as shown in SEQ ID NO:

10.

11. A recombinant adeno-associated viral vector characterized in that, The human GLB1 protein expression cassette according to any one of claims 7-10.

12. The recombinant adeno-associated viral vector of claim 11, wherein, The recombinant adeno-associated virus vector is selected from at least one of the following serotypes or a variant thereof: AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh74; preferably, the recombinant adeno-associated virus vector is AAV9.

13. A pharmaceutical composition, characterized by, The recombinant adeno-associated virus vector according to claim 11 or 12.

14. Use of the recombinant expression system for expressing human GLB1 protein according to any one of claims 1-6, the human GLB1 protein expression cassette according to any one of claims 7-10, the recombinant adeno-associated virus vector according to any one of claims 11-12, or the pharmaceutical composition according to claim 13 in the preparation of a medicament for preventing and / or treating GM1 gangliosidosis.

15. Use according to claim 14, characterized in that, In the use, the prepared medicament is administered by a central nervous system delivery mode, and the central nervous system delivery mode comprises intrathecal injection, brain parenchyma injection, or intracerebroventricular injection.

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