Recombinant AAV vectors for the treatment of neurodegenerative diseases

Abstract

An object is to provide a recombinant AAV vector for treating neurodegenerative diseases. 【Solution】A recombinant adeno-associated virus (rAAV) vector is provided that contains one or two of (a) to (c): (a) a nucleotide sequence encoding aromatic L-amino acid decarboxylase (AADC), (b) a nucleotide sequence encoding glucocerebrosidase (GBA1), and (c) a nucleotide sequence encoding a neurotrophic factor (NTF), such as cerebral dopamine neurotrophic factor (CDNF) or glial cell-derived neurotrophic factor (GDNF), which is a nucleotide sequence for treating neurodegenerative diseases, particularly Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher's disease (GD), and other proteinopathies. Also provided herein are viral particles containing the rAAV vector, pharmaceutical compositions containing the viral particles, and their uses.

Description

[Technical Field]

[0001] The present disclosure relates to the technical field of gene therapy. Specifically, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising one or two of (a) to (c): (a) a nucleotide sequence encoding aromatic L-amino acid decarboxylase (AADC), (b) a nucleotide sequence encoding glucocerebrosidase (GBA1), and (c) a nucleotide sequence encoding a neurotrophic factor (NTF), such as brain dopamine neurotrophic factor (CDNF) or glial cell line-derived neurotrophic factor (GDNF), for treating neurodegenerative diseases, particularly Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD), and other protein disorders. Also provided herein are viral particles comprising the rAAV vector, pharmaceutical compositions comprising the viral particles, and uses thereof. [Sequence table]

[0002] This disclosure includes a sequence listing as part of the disclosure. [Background technology]

[0003] Neurodegenerative diseases (NDs) are caused by the gradual loss of function and eventual death of nerve cells in the brain and peripheral nervous system, resulting in a decline in motor skills, coordination, muscle strength, sensory, and cognitive function. NDs are slowly progressive diseases, and there are no adequate treatments to slow or cure the disease, resulting in significant socioeconomic and personal costs and suffering. Aging is the major risk factor for most NDs. As people live longer, the number of people affected by NDs will increase in the coming decades, placing a huge economic burden on society and posing an immense global public health challenge.

[0004] Parkinson's disease (PD), caused by degeneration of midbrain dopamine transmission and characterized by both motor (e.g., tremor, rigidity) and non-motor (e.g., mental retardation, depression) symptoms, affects approximately 0.3% of the general population and approximately 1% of those aged 60 years or older (de Lau, LM and MMBreteler, Epidemiology of Parkinson's Disease, Lancet Neurol, 2006; 5(6): 525-35).

[0005] Most PD cases are sporadic and of unknown cause. A minority of cases have a family history. Six specific human genomic loci have now been identified that contain genes whose mutations may lead to the development of rare familial forms of PD (Klein, C. and A. Westenberger, Genetics of Parkinson's Disease, Cold Spring Harb Perspect Med, 2012, 2(1): p. a008888) The etiology of PD is widely accepted to result from a complex interplay between endogenous factors, such as genetics, and exogenous environmental factors. The relationship between pathological changes and PD symptoms is well established. (Kouli, A., KM Torsney, and WL Kuan, Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis of Parkinson's Disease: Pathogenesis and Clinical Aspects, TBStoker and JC Greenland, editors, 2018: Brisbane, Australia) The pathological hallmark of PD is the degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the loss of dopaminergic pathways from the SNc to the striatum (putamen and caudate nucleus), leading to decreased dopamine levels in the striatum and motor dysfunction. Based on this knowledge, several drugs have been developed with different approaches to restore dopamine levels.

[0006] Another pathological feature of PD is the formation of Lewy bodies (LBs) in the brains of patients. This phenomenon is known as "Lewy pathology." α-synuclein (α-syn)-containing LBs and Lewy neurites induce cytotoxicity in DA neurons and other neuronal subtypes, contributing to the pathology of PD (Teil, M., et al.: Targeting α-synuclein for PD Treatment: A Full-Scale Approach. Biomolecules, 2020, 10(3)). Multiple strategies to target Lewy pathology are currently under investigation, including stabilizing the physiological conformation of α-syn, reducing α-syn expression, inhibiting α-syn aggregation, and increasing α-syn clearance. Furthermore, Lewy pathology is not limited to dopaminergic circuits but extends to other brain regions via cell-to-cell propagation of α-syn aggregates, which may be one of the mechanisms underlying non-motor PD symptoms. Therefore, theoretically, therapeutic approaches targeting α-syn aggregation could benefit both motor and non-motor functions in PD patients.

[0007] Among currently available treatments, L-DOPA (levodopa) is effective in treating motor symptoms in some PD patients. However, L-DOPA has limited efficacy in treating non-motor symptoms and in patients with end-stage Parkinson's disease. Furthermore, it has been shown that L-DOPA can cause serious side effects when taken at high doses (Zahoor, I., A. Shafi, E. Haq, "Pharmacological Treatment of Parkinson's Disease," Parkinson's Disease: Pathogenesis and Clinical Aspects, TBStoker and JC Greenland, editors, 2018, Brisbane, Australia). Other treatments, such as deep brain stimulation, only alleviate symptoms but do not halt disease progression (Dallapiazza, RF et al., "Considerations for Patient and Target Selection in Deep Brain Stimulation Surgery for Parkinson's Disease," Parkinson's Disease: Pathogenesis and Clinical Aspects, TBStoker and JC Greenland, editors, 2018, Brisbane, Australia). Therefore, better treatments for PD are urgently needed.

[0008] Currently investigated biological PD treatments can be categorized into three types: 1) by modulating neuronal signaling to restore neurotransmitter imbalance. For example, gene therapy using viral vectors to deliver AADC, an enzyme involved in dopamine synthesis, such as AAV2-AADC (CN107106689A, related clinical trial @ https: / / clinicaltrials.gov / ct2 / show / NCT03065192?term=AADC&cond=PD&draw=2&rank=8) and lentiviral gene therapy (ProSavin), has shown promising results in early-stage clinical trials, resulting in mild improvements in motor function in patients. 2) by enhancing neuronal survival through the expression of neurotrophic and regenerative factors. For example, neurotrophic factor (GDNF) (clinical trial @ https: / / clinicaltrials.gov / ct2 / show / NCT04167540?term=GDNF&cond=PD&draw=2&rank=1) and Neurturin (NRTN) (clinical trial @ https: / / clinicaltrials.gov / ct2 / show / NCT04167540?term=GDNF&cond=PD&draw=2&rank=1) trial @ https: / / clinicaltrials.gov / ct2 / show / NCT00985517?term=Neurturin&draw=2&rank=1) is beneficial for the survival of dopaminergic midbrain neurons. 3) Examples include alpha synaptic nuclear protein (SNCA), glucocerebrosidase (GBA1) (clinical trial @ https: / / clinicaltrials.gov / ct2 / show / NCT04127578?term=GBA1&cond=Parkinson+Disease&draw=2&rank=1), and leucine-rich repeat kinase 2 (LRRK2). Several gene therapy approaches using antisense oligonucleotides (ASOs), CRISPR, and AAV for gene editing and gene expression control are currently in early-stage clinical trials.

[0009] Therefore, there is an unmet need for a treatment for PD that can restore striatal dopamine levels, remove α-synthesis, and preserve dopaminergic transmission in the brain of patients. There remains a great need.

[0010] AAV-mediated recombinant therapies also have potential for the treatment of other neurodegenerative and neurodevelopmental diseases, including but not limited to multiple system atrophy (MSA), dementia with Lewy bodies (LBD), Alzheimer's disease with amygdala-restricted Lewy bodies (AD / ALB), Gaucher disease, and AADC deficiency.

[0011] MSA is a rapidly progressive, sporadic, adult-onset neurodegenerative disease. Clinical manifestations of MSA include parkinsonism (bradycardia, rigidity, and postural instability resembling Parkinson's disease), cerebellar syndrome, and autonomic dysfunction due to brainstem nuclei degeneration. Based on the primary symptomatology, two clinical subtypes have been defined: MSA-P (parkinsonism) and MSA-C (cerebellar syndrome) (Monzio Compagnoni, G. and A. Di Fonzo, "Understanding the pathogenesis of multiple system atrophy: current state of the art and future perspectives"). Acta Neuropathol Commun, 2019, 7(1): p. 113). The primary pathological feature of MSA is widespread neuronal and oligodendrocyte loss and gliosis in multiple brain regions.

[0012] Although the cause of MSA remains unknown, the primary pathogenic mechanism of MSA is the presence of argyrophilic fibrillar glial cytoplasmic inclusions (GCIs), primarily present in oligodendrocytes. GCIs are primarily composed of loosely packed filaments of ubiquitinated α-synuclein protein, phosphorylated at Ser129. Based on its pathology and symptoms, currently available and developing treatments for MSA target the loss of dopamine transmission, neuronal loss, and α-synuclein aggregation, which are very similar to therapeutic approaches for PD.

[0013] Epidemiological studies of MSA have shown that the prevalence rate ranges from 3.4 to 4.9 cases per 100,000 people, increasing to 7.8 cases per 100,000 people in those aged 40 years or older. Currently available treatments only alleviate symptoms. For example, dopamine derivatives (e.g., Medopa and Duopa) are used to reduce PD-like symptoms. However, these medications are not as effective in PD patients as they are in MSA-P patients. Furthermore, some MSA-P patients benefit only from high doses of these medications, and their effectiveness usually decreases over time. Therefore, enhancing dopamine production through the administration of AADCs may not be as effective in MSA patients as it is in PD patients. Additional disease-modifying therapies that can slow disease progression are needed. Such therapies have the potential to target three aspects of MSA pathology: (1) α-synuclein aggregation, (2) impaired and lost cellular function, and (3) neuroinflammation. Several promising clinical candidate drugs include immunogenic peptides of α-synuclein (PD01 and PD03, NCT02270489), α-synuclein aggregation inhibitors (Anle138b, NCT04208152, ATH434, NCT05109091), antisense oligonucleotides (ASOs) against α-synuclein (BIIB101, NCT04165486), and neuroprotective factors such as GDNF delivered by AAV (AAV2-GDNF, NCT04680065). All of the above therapies under development target a single pathology or symptom. For complex NDs such as MSA, simultaneously targeting multiple disease-causing pathologies may offer better therapeutic and disease-modifying effects, not only alleviating symptoms but also slowing disease progression.

[0014] Gaucher disease (GD) is an autosomal recessive disorder caused primarily by loss-of-function mutations in the GBA1 gene. GD is classified into three types based on the presence or absence of central nervous system (CNS) involvement (type 1) or the presence and severity of involvement (nGD, types 2 and 3) (Bennett, LL and C. Fellner, Pharmacotherapy for Gaucher Disease: Current and Future Options, PT, 2018. 43(5):274-309). Enzyme replacement therapy has been developed, but is limited to type 1 GD because recombinant enzymes cannot cross the blood-brain barrier (BBB). CNS-directed AAVs expressing GBA1 may be a promising approach for treating nGD. An active clinical trial (NCT04411654) supported by Prevail Therapeutics is administering a codon-optimized GBA1 transgene via intracranial (ICM) injection of rAAV9.

[0015] AADC deficiency (AADCD) is a rare autosomal recessive disorder caused primarily by loss-of-function mutations in the AADC gene. Abnormalities in the AADC protein result in severe deficiencies of serotonin, dopamine, norepinephrine, and epinephrine, neurotransmitters that are crucial for brain function. rAAV9 expressing functional wild-type AADC is being developed by PTC therapeutics as a treatment for this disease, and is being marketed as Upstaza. TM (eladocagene exparvovec) and is approved by the EMEA (European Medicines Agency) and the UK for the treatment of patients aged 18 months and over.

[0016] Taken together, this evidence demonstrates that ND is a complex, multifaceted, and debilitating disease with a significant impact on global public health. There is an urgent need to develop innovative disease-modifying treatments to slow disease progression and alleviate patient suffering. Summary of the Invention [Problem to be solved by the invention]

[0017] To develop better therapeutic approaches for various NDs, we modified the nucleotide sequences encoding AADC, GBA1, and NTFs such as CDNF and GDNF to optimize their expression when delivered to the human brain via rAAV vectors. Furthermore, we developed an rAAV vector that simultaneously expresses two genes of interest (GOIs), enabling us to synergistically target multiple disease pathologies and achieve greater therapeutic efficacy. [Means for solving the problem]

[0018] Thus, in a first aspect, the present application provides a nucleotide sequence encoding one of the above GOIs, specifically AADC, GBA1, CDNF, and GDNF, which nucleotide sequence is codon-optimized compared to the wild-type sequence of the GOI.

[0019] In a second aspect, the present application provides a nucleic acid construct comprising the nucleotide sequence of the first aspect operably linked to a promoter.

[0020] In a third aspect, the present application provides a nucleic acid construct comprising two nucleotide sequences operably linked to a promoter, wherein the first nucleotide sequence and the second nucleotide sequence encode two of (a), (b), and (c), respectively: (a) AADC, (b) GBA1, and (c) GDNF or CDNF. In a preferred embodiment, the nucleotide sequence encoding AADC is a codon-optimized sequence. In a specific embodiment, the nucleotide sequence encoding AADC is the nucleotide sequence of the first aspect. In another preferred embodiment, the nucleotide sequence encoding GBA1 is a codon-optimized sequence. In a specific embodiment, the nucleotide sequence encoding GBA1 is the nucleotide sequence of the first aspect. In another preferred embodiment, the nucleotide sequence encoding CDNF or GDNF is a partially codon-optimized sequence in which the nucleotide region encoding the signal peptide is unchanged. In a further embodiment, the nucleotide sequence encoding CDNF or GDNF has a reduced number of CpG sites compared to the wild-type coding sequence. In a further embodiment, the nucleotide sequence encoding CDNF or GDNF does not contain a CpG island. In a particular embodiment, the nucleotide sequence encoding CDNF or GDNF is the nucleotide sequence of the first aspect.

[0021] In a preferred embodiment, the nucleic acid construct of the third aspect comprises a linker sequence between the GOI of the first nucleotide sequence and the second nucleotide sequence. In certain embodiments, the linker sequence is a 2A peptide. In certain embodiments, the linker sequence is an IRES.

[0022] In a fourth aspect, the present application relates to a rAAV vector comprising the nucleotide sequence of the first aspect, or the nucleic acid construct of the second or third aspect. In a preferred embodiment, the AAV is AAV9.

[0023] In a fifth aspect, the present application relates to a composition, e.g., a pharmaceutical composition, comprising the rAAV vector of the fourth aspect and a pharmaceutically acceptable excipient.

[0024] In a sixth aspect, the present application relates to a viral particle comprising the rAAV vector of the fourth aspect.

[0025] In a seventh aspect, the present application relates to a method of treating or preventing a neurodegenerative disease (ND), such as Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD), and other proteinopathies, in a subject in need thereof, comprising administering to the subject an rAAV vector of the fourth aspect of the present application.

[0026] In an eighth aspect, the present application relates to the use of the rAAV vector of the fourth aspect in the treatment or prevention of a neurodegenerative disease, or in the manufacture of a medicament for the treatment or prevention of a neurodegenerative disease, in particular Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD), and other proteinopathies.

[0027] The nucleic acid constructs of the present application are comprised of a combination of a promoter, a linker, and one or two therapeutic protein coding sequences specifically optimized for use in rAAV vectors to provide desired levels of expression of one or two therapeutic proteins, e.g., AADC, GBA1, and CDNF / GDNF. In particular, in the case of rAAV vectors containing two GOIs, such rAAV vectors of the present invention can co-express both therapeutic proteins at desired levels, which, in combination, act synergistically as a novel therapeutic method to more effectively treat the targeted neurodegenerative disease and achieve better therapeutic outcomes. [Brief explanation of the drawings]

[0028] FIG. 1 shows a schematic diagram of an exemplary construct containing the AADC wild-type coding sequence and the GBA1 wild-type coding sequence linked by a P2A peptide. Figure 2A-B shows representative images of Western blot (WB) results for the expression of AADC and GBA1 proteins, alone or in combination, in HEK293 cells. (A) Cells were transfected with constructs containing the coding sequences of AADC, GBA1, or AADC-P2A-GBA1 (in which the AADC coding sequence was linked to the GBA1 coding sequence with a P2A linker). (B) Cells were transfected with constructs containing the AADC and GBA1 coding sequences in various orders: GpA: GBA1-P2A-AADC; ApG: AADC-P2A-GBA1. Figure 3 shows representative images of WB showing an evaluation of the cleavage efficiency of various linkers used when expressing both AADC and GBA within the same construct. Figure 4 shows the expression of wild-type AADC (AADC-WT) and optimized GBA1 via a P2A linker, or optimized AADC and wild-type GBA1 (GBA1-WT) via a P2A linker, under the control of the CBh hybrid promoter, which is composed of a CMV enhancer, a chicken β-actin promoter, and a hybrid intron. FIG. 1 is a schematic diagram of the construct. FIG. 5 shows AADC protein expression levels of various candidate constructs in HEK293 cells as measured by ELISA. FIG. 6 shows the GBA1 protein expression levels of various candidate constructs in HEK293 cells as measured by ELISA. FIG. 7 shows the AADC protein expression levels of various candidate constructs in HEK293 cells as measured by WB. FIG. 8 shows the normalized AADC protein expression levels shown in FIG. 7 as the mean±SEM from three independent WB experiments. FIG. 9 shows the GBA1 protein expression levels in HEK293 cells expressed by various candidate constructs, as measured by WB. FIG. 10 shows normalized GBA1 protein expression levels expressed as mean±SEM from three independent WB experiments as shown in FIG. FIG. 11 shows the catalytic activity of AADC in HEK293 cells transfected with various candidate constructs. FIG. 12 shows the catalytic activity of GBA1 protein expressed in HEK293 cells transfected with various candidate constructs. FIG. 13 shows the expression levels of AADC and GBA1 proteins in HEK293 cells transfected with various combination constructs. FIG. 14 shows the relative activity of GBA1 protein in HEK293 cells transfected with various combination constructs. FIG. 15 shows the expression of AADC and GBA1 proteins in HEK293 cells transduced with various candidate rAAV vectors. FIG. 16 shows representative WB images showing AADC protein expression levels in HEK293 cells transfected with various candidate constructs. 17A-B show schematics of constructs containing wild-type AADC (AADC-WT) linked to optimized CDNF (A) or GDNF (B) via a P2A linker under the control of the CBh promoter. Figures 18A-B show CDNF protein expression levels in cell lysates and culture supernatants of HEK293 cells transfected with various candidate constructs, as measured by WB (A) WB images, (B) normalized data (A). Figures 19A-B show CDNF protein expression levels in cell lysates and culture supernatants of U87 cells transfected with various candidate constructs, as measured by WB. (A) WB images, (B) normalized data. Figures 20A-B show CDNF protein expression levels in cell lysates and culture supernatants of HEK293 cells (A) or U87 cells (B) transfected with various candidate constructs, as measured by ELISA. FIG. 21 shows the protective effect of CDNF protein in the culture supernatant of HEK293 cells transfected with various candidate constructs treated with 1.5 mM MPP+ or 40 nM rotenone, as measured by CCK-8 assay. FIG. 22 shows GDNF protein expression levels in HEK293 cells transfected with various candidate constructs as measured by WB. FIG. 23 shows the GDNF protein expression levels measured by ELISA in cell lysates and culture supernatants of HEK293 cells transfected with various candidate constructs. FIG. 24 shows the protective effect of GDNF protein in the culture supernatant of HEK293 cells transfected with various candidate constructs and treated with 1.5 mM MPP+, as measured by CCK-8 assay. FIG. 25 shows CDNF (left panel) or GDNF (right panel) protein expression levels in culture supernatants of HEK293 cells transfected with the indicated constructs. . FIG. 26 shows AADC protein activity in cell lysates of HEK293 cells transfected with the indicated candidate constructs. FIG. 27 shows the protective effect of CDNF protein in the culture supernatant of HEK293 cells transfected with various candidate constructs treated with 1.5 mM MPP+ or 40 nM rotenone, as measured by CCK-8 and LDH assays. FIG. 28 shows the protective effect of GDNF protein in the culture supernatant of HEK293 cells transfected with various candidate constructs treated with 1.5 mM MPP+ or 40 nM rotenone, as measured by CCK-8 and LDH assays. Figure 29 shows AADC and NTF (CDNF or GDNF) protein expression levels in cell lysates and NTF levels in culture supernatants of U87-AAVR cells transduced with the indicated rAAV9 vectors. FIG. 30 shows the relative activity of AADC protein from HEK293 cells and U87-AAVR cells transduced with the indicated rAAV9 vectors. FIG. 31 shows the number of contralateral rotations collected from subjects in a mouse 6-OHDA-induced PD model after administration of EF1α-A11-GDNF MN and benchmark rAAV. FIG. 32 shows the net number of rotations collected from subjects in a mouse 6-OHDA-induced PD model after administration of EF1α-A11-GDNF MN and benchmark rAAV. Figures 33A-B are schematic diagrams of constructs containing optimized GBA1 linked to optimized CDNF (A) or GDNF (B) via a P2A linker under the control of a promoter (CAG or MBP promoter). "OP" refers to the optimized nucleotide sequence. Figure 34 shows measurements of locomotion speed and distance in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid (MPTP / P)-induced PD mice after administration of CBh-A11G11 and benchmark A10-vy rAAV, respectively. Pre: data collected before MPTP modeling; Post MPTP / P: data collected after MPTP / P induction but before AAV treatment; Post AAV: data collected after AAV treatment. Figure 35 shows rearing behavior (hindlimb rearing) collected from subjects in the α-synuclein A53T transgenic mouse model of PD after administration of EtI7-A11G11 (EtI7) and CBh-A11G11 (CBh) rAAV. Low: 4E+9 vg, High: 4E+10 vg. Figure 36 shows representative images of WB showing AADC and GCase expression in tissue samples taken from subjects in the α-synuclein A53T transgenic mouse model of PD after administration of EtI7-A11G11 and CBh-A11G11 rAAV. Low: 4E+9 vg, High: 4E+10 vg. Figures 37A-B show representative images (A) and normalized data (B) of WB showing levels of phosphorylated and total α-synuclein in tissue samples taken from the α-synuclein A53T transgenic mouse model of PD after administration of EtI7-A11G11 and CBh-A11G11 rAAV. Low: 4E+9 vg, High: 4E+10vg Figures 38A-B are schematic diagrams of constructs containing different promoters and codon-optimized GBA1. FIG. 39 shows the survival curves (survival rates) of CBE-induced GD mice after administration of G10-p, CAG-G11, and EF1α-G11 rAAV. Figures 40A-B show the locomotion in the open field test of CBE-induced GD mice after administration of different doses of G10-p, CAG-G11, and EF1α-G11 rAAV, respectively. The distance (A) and the latency to fall (B) in the rotarod test are shown. FIG. 41 shows GCase activity in striatal samples collected from CBE-induced GD mice after administration of different doses of G10-p, CAG-G11, and EF1α-G11 rAAV. FIG. 42 shows representative WB images showing AADC and CDNF protein expression in cell lysates and CDNF protein levels in culture supernatants in HEK293 cells transfected with the indicated candidate constructs. Figure 43 shows the net number of rotations (amphetamine-induced ipsilateral number of rotations) in 6-OHDA-induced PD model mice after administration of the indicated doses of CAG-A11-GDNF MN, EF1α-A11-GDNF MN, and benchmark A10-vy rAAV. Figures 44A-B show AADC (A) and GDNF (B) protein expression levels in striatal samples taken from 6-OHDA-induced PD model mice after administration of the indicated amounts of EF1α-A11-GDNF MN and benchmark A10-vy rAAV, as determined by ELISA. Figure 45 shows representative images showing tyrosine hydroxylase (TH) levels in substantia nigra samples taken from 6-OHDA-induced PD model mice after administration of the indicated amounts of EF1α-A11-GDNF MN and benchmark A10-vy rAAVs, as determined by immunohistochemistry (IHC). Blank: healthy subjects. Figure 46 shows the delta rotations (L-DOPA-induced contralateral rotations) collected from 6-OHDA-induced PD model rats after administration of the indicated doses of EF1α-A11-GDNF MN and benchmark A10-vy rAAV. Blank represents healthy subjects. Figure 47 shows representative WB images showing GCase and GDNF protein expression in cell lysates and GDNF protein levels in culture supernatants of U87-MG-AAVR cells treated with various candidate rAAV vectors. Figure 48 shows representative WB images showing high molecular weight α-syn (HMW) protein levels in SH-SY5Y-AAVR-A53T cells treated with various candidate rAAV vectors. FIG. 49 shows representative WB images showing GCase and GDNF protein expression in cell lysates and GDNF protein levels in culture supernatants of HEK293 cells transfected with various candidate constructs. DETAILED DESCRIPTION OF THE INVENTION

[0029] Unless otherwise defined elsewhere herein, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0030] As used in this specification, including the appended claims, the singular forms of words such as "indefinite article," "definite article," and the like include the corresponding plural forms unless the context clearly dictates otherwise.

[0031] In the context of the present disclosure, unless specifically stated otherwise, the term "comprising" and variations thereof, such as "consisting of" and "consisting of," are understood to mean the inclusion of the stated elements, e.g., amino acid sequences, nucleotide sequences, properties, steps, or groups thereof, but not the exclusion of other elements, e.g., amino acid sequences, nucleotide sequences, properties, and steps. As used herein, the term "consisting of" or variations thereof can be substituted with the terms "including," "including," or in some cases, "having" or equivalent variations. In certain embodiments, the term "comprising" also includes the scenario of "consisting of."

[0032] As used herein, the term "gene" refers to a nucleic acid (DNA, e.g., genomic DNA or The term "genomic DNA" refers to the nucleotide sequences encoding and corresponding RNA transcripts (e.g., cDNA or cDNA). As used herein, the term "genomic DNA" may include regulatory regions as well as intervening non-coding regions, and may include both the 5' and 3' ends. In some cases, the term includes transcribed sequences, such as 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region includes an "open reading frame" that encodes a polypeptide. In some cases, a "gene" consists only of the coding sequence (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions, such as upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or native gene. A gene may refer to a "foreign gene" or non-native gene. A non-native gene refers to a gene not normally present in the host organism but introduced into the host organism by gene transfer. A non-native gene may refer to a gene that does not exist in its natural location in the genome of an organism. A non-native gene may also refer to a naturally occurring nucleic acid that contains mutations, insertions, and / or deletions (such as a non-native sequence), e.g., a codon-optimized nucleotide sequence. In the context of this application, "GOI" refers to a CDS region, i.e., a sequence that codes for amino acids in a protein, unless otherwise specified.

[0033] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein to refer to a multimeric polymer of nucleotides of any length. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or a fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide can contain one or more analogs (e.g., altered backbones, sugars, or nucleobases).

[0034] As used herein, a "cassette" or "expression cassette" refers to a DNA component comprised of one or more genes contained in a vector (e.g., a plasmid vector or a viral vector) and under the control of regulatory sequences that are expressed in a host cell transduced by the vector.

[0035] As used herein, the term "operably linked" is used to indicate that two or more components, particularly nucleotide sequences, are joined in such a way that each component can perform its designated function.

[0036] "AAV" refers to adeno-associated virus.

[0037] "AADC" refers to aromatic L-amino acid decarboxylase. AADC is also known as DOPA decarboxylase (DDC).

[0038] "GBA1" refers to glucocerebrosidase, which cleaves the β-glucosidic bond of glucocerebroside. In some cases, "GBA1" is referred to interchangeably as "GBA," "GCB," or "GLUC."

[0039] "NTF" stands for neurotrophic factor, a group of supportive proteins that promote the development and maintenance of neurons. Both CDNF and GDNF belong to the neurotrophic factor family.

[0040] "CpG island" refers to a region in the genome that is rich in CpG sites. The term "CpG site" refers to two consecutive nucleotides in the 5' to 3' direction, each consisting of a cytosine (C) and a guanine (G).

[0041] "2A peptides" refer to a group of short (18-22 amino acids) self-cleaving peptides derived from viruses. 2A peptides cause ribosomal skipping during translation, creating a separation between the end of the 2A sequence and the downstream protein.

[0042] "IRES" refers to an internal ribosome entry site.

[0043] "Proteopathies" refer to neurodegenerative diseases in which structurally abnormal proteins, such as alpha-synuclein, accumulate and form aggregates or inclusions within the axons of neurons or oligodendrocytes.

[0044] In the context of this application, a "subject" refers to an animal, preferably a mammal, such as a primate, e.g., a cynomolgus monkey, preferably a higher primate, e.g., a human. Unless otherwise specified, in the context of this application, the term "subject" is interchangeable with the term "patient" or "individual."

[0045] AADC The present disclosure provides rAAV vectors that deliver the aromatic L-amino acid decarboxylase (AADC) gene alone or in combination with another gene of interest, such as GBA1, CDNF, or GDNF. The rAAV vectors that deliver AADC can be used to treat NDs, such as PD and AADCD.

[0046] The dopamine biosynthetic pathway requires both tyrosine hydroxylase (TH), which converts tyrosine to l-3,4-hydroxyphenylalanine (L-DOPA), and aromatic L-amino acid decarboxylase (AADC), which decarboxylates L-DOPA to produce dopamine.

[0047] As PD progresses, AADC levels decline, and L-DOPA ingested by patients is not efficiently converted to dopamine at the axon terminals in the striatum. Therefore, to achieve adequate clinical efficacy, L-DOPA must be administered more frequently and at higher doses. However, increasing the dose of L-DOPA can cause undesirable side effects, such as L-dopa-induced dyskinesia (LID).

[0048] The potential for intraputaminar AADC delivery by AAV in the treatment of PD has been reported and clinically evaluated (Bankiewiecz KS, et al., Long-term clinical improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC). Mol Ther, 2006; 14(4): 564-70; Christine, CW, et al., "Magnetic resonance imaging-guided phase 1 trial of putaminal AADC gene therapy for Parkinson's disease," Ann Neurol, 2019, 85(5): 704-714) Restoring AADC levels would "rescue" and enhance the therapeutic effects of L-DOPA, and given that dopamine levels are the most important factor for the recovery of motor function, AADC is among the candidate components for restoring dopamine levels, which would be particularly beneficial for patients with AADC deficiency.

[0049] AADC deficiency results from loss-of-function mutations in the AADC gene. The present rAAV contains AADC as the sole GOI or in combination with other GOIs, thereby compensating for the molecular defect that leads to the disease.

[0050] In a preferred embodiment, the nucleotide sequence encoding AADC can be optimized for expression in a rAAV construct. The optimization can be codon optimization. good.

[0051] In one embodiment, the rAAV of the present application comprises a nucleotide sequence encoding AADC, which nucleotide sequence comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 9, 11, and 46, preferably SEQ ID NOs: 1 to 9 and 46 (optimized sequences), more preferably the nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 46. In some embodiments, an rAAV comprising a nucleotide sequence encoding AADC as its sole GOI can be used to treat PD or AADCD.

[0052] In some embodiments, the rAAV of the present application comprises a combination construct containing both AADC and GBA1, for use, for example, in treating PD. In one embodiment, the combination construct comprises a coding sequence for AADC and a coding sequence for GBA1. In some embodiments, the coding sequence for GBA1 is located 5' upstream of the coding sequence for AADC. In some embodiments, the coding sequence for AADC is located 5' upstream of the coding sequence for GBA1. Preferably, the two coding sequences are located in frame and under the control of the same promoter. Preferably, the nucleotide sequence encoding GBA1 comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 12 to 20, 45, or 47. Preferably, the nucleotide sequence encoding AADC comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 9, or 46.

[0053] In a specific embodiment, the AADC and GBA1 combination construct is composed of the AADC coding sequence represented by SEQ ID NO: 46 and the GBA1 sequence represented by SEQ ID NO: 45. In a specific embodiment, the AADC and GBA1 combination construct comprises, from 5' to 3', a promoter sequence that is a truncated mutant of the EF1α promoter, for example, the AADC coding sequence represented by SEQ ID NO: 56 (EFIt7), the GBA1 coding sequence represented by SEQ ID NO: 46, and a Poly sequence such as hGH poly A represented by SEQ ID NO: 45. In a specific embodiment, the AADC and GBA1 combination construct comprises, from 5' to 3', a promoter sequence represented by SEQ ID NO: 60 (Cbh promoter), the GBA1 coding sequence represented by SEQ ID NO: 46, and a Poly sequence such as hGH poly A represented by SEQ ID NO: 45.

[0054] In some embodiments, the rAAV of the present application includes a combination construct containing both AADC and an NTF selected from GDNF or CDNF, for example, for use in treating AADCD. In one embodiment, the combination construct includes a coding sequence for AADC and a coding sequence for either GDNF or CDNF. In some embodiments, the coding sequence for GDNF or CDNF is located 5' upstream of the coding sequence for AADC. In some embodiments, the coding sequence for AADC is located 5' upstream of the coding sequence for GDNF or CDNF. Preferably, the two coding sequences are located in frame and under the control of the same promoter. Preferably, the nucleotide sequence encoding AADC comprises or consists of the coding sequence set forth in any of SEQ ID NOs: 1 to 9 or 46. Preferably, the nucleotide sequence encoding GDNF comprises or consists of the coding sequence set forth in any of SEQ ID NOs: 27 to 29. Preferably, the nucleotide sequence encoding CDNF comprises or consists of the coding sequence set forth in any of SEQ ID NOs: 23 to 25.

[0055] In some embodiments, the rAAV of the present application is used in, for example, the treatment of AADCD. and a combination construct comprising the coding sequence for AADC and the coding sequence for CDNF. In a specific embodiment, the combination construct of AADC and CDNF comprises the coding sequence for AADC represented by SEQ ID NO: 3 or SEQ ID NO: 46, and the coding sequence for CDNF represented by SEQ ID NO: 25 (CDNF-MN). In a specific embodiment, the combination construct of AADC and CDNF comprises, from 5' to 3', a CAG promoter sequence represented by SEQ ID NO: 58 (CAG), the coding sequence for AADC represented by SEQ ID NO: 3, and the coding sequence for CDNF represented by SEQ ID NO: 25 (CDNF-MN). In a specific embodiment, the combination construct of AADC and CDNF comprises, from 5' to 3', an EF1α promoter sequence represented by SEQ ID NO: 57 (EF1α), the coding sequence for AADC represented by SEQ ID NO: 46, and the coding sequence for CDNF represented by SEQ ID NO: 25 (CDNF-MN). In one specific embodiment, the AADC and CDNF combination construct comprises, from 5' to 3', the EF1α promoter sequence represented by SEQ ID NO: 57 (EF1α), the CDNF coding sequence represented by SEQ ID NO: 25 (CDNF-MN), and the AADC coding sequence represented by SEQ ID NO: 46 (CDNF-MN).

[0056] In some embodiments, the rAAV of the present application comprises a combination construct comprising a coding sequence for AADC and a coding sequence for GDNF, e.g., for use in treating AADCD. In one specific embodiment, the AADC and GDNF combination construct comprises the coding sequence for AADC represented by SEQ ID NO:46 and the coding sequence for GDNF represented by SEQ ID NO:29 (GDNF-MN). In one specific embodiment, the AADC and GDNF combination construct comprises, from 5' to 3', an EF1α promoter sequence represented by SEQ ID NO:57 (EF1α), the coding sequence for AADC represented by SEQ ID NO:46; and the coding sequence for GDNF represented by SEQ ID NO:29 (GDNF-MN).

[0057] GBA1 The present disclosure provides rAAV vectors that deliver the glucocerebrosidase 1 (GBA1) gene alone or in combination with other genes, such as ADCC, CDNF, or GDNF. rAAV vectors that deliver GBA1 can be used to treat NDs such as Gaucher disease (GD), Parkinson's disease (PD), and multiple system atrophy (MSA). For example, rAAV vectors that deliver only GBA1 can be used to treat types 2 and 3 of Gaucher disease (GD) or PD. For example, rAAV vectors that deliver both GBA1 and an NTF (such as GDNF) can be used to treat MSA.

[0058] In a preferred embodiment, the nucleotide sequence encoding GBA1 can be optimized for expression in an rAAV construct. The optimization can be codon optimization.

[0059] In one embodiment, the rAAV of the present application comprises or consists of a nucleotide sequence encoding GBA1 as set forth in SEQ ID NOs: 12 to 20, 22, 45, and 47, preferably SEQ ID NOs: 12 to 20, 45, or 47 (codon-optimized sequences), more preferably SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 45, or SEQ ID NO: 47.

[0060] In some embodiments, the rAAV of the present application comprises a nucleotide sequence encoding GBA1, e.g., for use in treating GD or PD. Preferably, the nucleotide sequence encoding GBA1 comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 12 to 20, 45, or 47. In one particular embodiment, the rAAV consists of the GBA1 sequence set forth in SEQ ID NO: 45. In one particular embodiment, the rAAV comprises, from 5' to 3', the GBA1 promoter sequence set forth in SEQ ID NO: 58 (CAG) and the GBA1 promoter sequence set forth in SEQ ID NO: 45. In one specific embodiment, the rAAV comprises, from 5' to 3', a construct of a GBA1 promoter sequence represented by SEQ ID NO: 57 (EF1α), a GBA1 coding sequence represented by SEQ ID NO: 45, optionally a WPRE; and a poly A sequence, such as an hGH poly A sequence.

[0061] In some embodiments, the rAAV of the present application comprises a combination construct containing both GBA1 and an NTF selected from GDNF or CDNF, for example, for use in treating MSA. In some embodiments, the coding sequence for GBA1 is located 5' upstream of the coding sequence for the NTF. In some embodiments, the coding sequence for the NTF is located 5' upstream of the coding sequence for GBA1. Preferably, the two coding sequences are located in frame and under the control of the same promoter.

[0062] In some embodiments, the rAAV of the present application comprises a combination construct comprising both GBA1 and GDNF, e.g., for use in treating MSA. Preferably, the nucleotide sequence encoding GBA1 comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 12 to 20, 45, or 47. Preferably, the nucleotide sequence encoding GDNF comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 27 to 29. In one specific embodiment, the combination construct of both GBA1 and GDNF comprises the coding sequence for GBA1 set forth in SEQ ID NO: 45 or SEQ ID NO: 47 and the coding sequence for GDNF set forth in any of SEQ ID NOs: 27 to 29. In a specific embodiment, the combined construct of GBA1 and GDNF comprises, from 5' to 3', the CAG promoter sequence (CAG) represented by SEQ ID NO: 58 or the MBP promoter sequence (MBP) represented by SEQ ID NO: 59, the GBA1 coding sequence represented by SEQ ID NO: 45 or SEQ ID NO: 47, the GDNF coding sequence (GDNF-MN) represented by SEQ ID NO: 29 (GDNF-MN), optionally a WPRE, and a polyA sequence such as SV40 polyA or bGH polyA. In a specific embodiment, the combined construct of GBA1 and GDNF comprises, from 5' to 3', the CAG promoter sequence (CAG) represented by SEQ ID NO: 58 or the MBP promoter sequence represented by SEQ ID NO: 59 (MBP), the GDNF coding sequence (GDNF-MN) represented by SEQ ID NO: 29, the GBA1 coding sequence represented by SEQ ID NO: 45 or SEQ ID NO: 47, optionally a WPRE, and a polyA sequence such as SV40 polyA or bGH polyA sequence.

[0063] Neurotrophic factors: CDNF and GDNF The present disclosure provides rAAV vectors that deliver neurotrophic factors, specifically CDNF or GDNF, alone or in combination with other genes, such as ADCC or GBA1. The rAAV vectors that deliver neurotrophic factors can be used to treat ND.

[0064] The neuroprotective role of neurotrophic factors is well established. Both CDNF and GDNF proteins have been studied in the context of PD (Nasrolahi, A et al., "Neurotrophic Factors Hold Promise for the Future of Parkinson's Disease Treatment: Is There Light at the End of the Tunnel?" Rev Neurosci, 2018, 29(5): 475-489).

[0065] In one embodiment, AADC is co-expressed with CDNF or GDNF, respectively, by an rAAV vector. This is the first time that AADC and a neurotrophic factor have been co-expressed in the same AAV vector and shown significant efficacy in a mouse PD model.

[0066] In another embodiment, GBA1 is co-expressed with CDNF or GDNF, respectively, by an rAAV vector.

[0067] In a preferred embodiment, the nucleotide sequence encoding CDNF or GDNF can be optimized for expression by an rAAV construct. The optimization can be codon optimization. Codon optimization can be limited to the coding region of the mature GDNF or CDNF protein, without altering the nucleotide sequence corresponding to the CDNF or GDNF signal peptide or the nucleotide sequence corresponding to the GDNF prepeptide. In a preferred embodiment, the codon-optimized CDNF or GDNF coding sequence has a reduced number of CpG islands compared to the wild-type coding sequence.

[0068] In one embodiment, the rAAV of the present application comprises a nucleotide sequence encoding CDNF that comprises or consists of a coding sequence set forth in any of SEQ ID NOs: 23-25.

[0069] In one embodiment, the rAAV of the present application comprises or consists of a coding sequence set forth by any of SEQ ID NOs:27 to 29.

[0070] Expression cassette As used herein, the term "expression cassette" refers to a DNA component comprised of one or more, specifically one or two GOIs selected from AADC, GBA1, and NTF (CDNF or GDNF) genes, contained in a vector (e.g., an rAAV vector) and under the control of regulatory sequences that are expressed in a host cell transduced by the vector.

[0071] The term "combination construct" in the context of this application refers to a construct comprising two GOIs, specifically two GOIs selected from AADC, GBA1, and NTF (CDNF or GDNF) genes. In a preferred embodiment, the two coding sequences are arranged in frame and under the control of the same promoter.

[0072] In one embodiment, the expression cassette of the present application features the expression of one GOI, preferably the expression of a codon-optimized sequence of the GOI, particularly as described in this disclosure.

[0073] In one embodiment, the expression cassette of the present application features the co-expression of two GOIs separated by a linker sequence. In a further embodiment, one or two of the GOIs are expressed via optimized sequences, particularly as described in this disclosure. For example, the two GOIs can be selected from the following combinations: AADC+GBA1, AADC+CDNF, AADC+GDNF, GBA1+GDNF, and GBA1+CDNF.

[0074] The expression cassette of the present disclosure inserted into the AAV vector can achieve higher and more stable protein expression or co-expression in neurons in vitro or in vivo by optimizing the cDNA sequences (codons) of the AADC gene, GBA1 gene, CDNF gene, and GDNF gene, their regulatory sequences, and optionally linkers. For example, the expression cassette of the present disclosure shows better performance in expressing GOIs in human cell lines with characteristic properties of neurons, such as U87-MG or SH-SY5Y. For example, the expression cassette of the present disclosure shows better performance in expressing GOIs in neurons in the striatum, substantia nigra, or other central nervous system regions in vivo.

[0075] In one embodiment, the present disclosure provides, as an essential part of an expression cassette, a group of wild-type or codon-optimized nucleotide sequences encoding an AADC protein, and a second protein selected from the group consisting of a GBA1 protein, a CDNF protein, and a GDNF protein, specifically a human AADC protein having the amino acid sequence set forth in SEQ ID NO: 31, a human AADC protein having the amino acid sequence set forth in SEQ ID NO: 32, and a human AADC protein having the amino acid sequence set forth in SEQ ID NO: 33. the human GBA1 protein, the human CDNF protein having the amino acid sequence shown in SEQ ID NO: 33, or the nucleotide sequence shown in SEQ ID NO: 34, or consisting of said nucleotide sequence.

[0076] In one embodiment, the present disclosure provides, as an essential part of an expression cassette, first comprising or consisting of a group of wild-type or codon-optimized nucleotide sequences encoding GBA1 protein and a second protein selected from the group consisting of CDNF protein and GDNF protein, specifically a human GBA1 protein having the amino acid sequence set forth in SEQ ID NO: 32, a human CDNF protein having the amino acid sequence set forth in SEQ ID NO: 33, or a nucleotide sequence set forth in SEQ ID NO: 34.

[0077] By "isolated nucleic acid" is meant DNA or RNA in which the isolated polynucleotide has been removed from all or part of a naturally occurring polynucleotide, or DNA or RNA associated with a polynucleotide with which it is not naturally associated. An isolated nucleic acid molecule "comprising" a particular nucleotide sequence may include, in addition to the designated sequence, operably linked regulatory sequences that control expression of the coding region of the described nucleic acid sequence. Those skilled in the art will understand that due to codon degeneracy, any particular amino acid sequence may be encoded by several different nucleotide sequences.

[0078] As used herein, a "codon-optimized coding sequence" refers to a nucleotide sequence encoding a protein, such as an AADC protein, a GBA1 protein, a CDNF protein, or a GDNF protein, that has been modified from a wild-type coding sequence to take codon bias into account. Optimization may be achieved by reducing sequence complexity, adjusting GC content, adjusting codon usage, and avoiding rare codons. A codon-optimized coding sequence typically improves the translation efficiency of a gene of interest (GOI) and enhances protein expression. Tools incorporating algorithms for designing codon-optimized coding sequences (e.g., JCat) are readily available to those skilled in the art. In a preferred embodiment, the codons in the AADC coding sequence of the present application have a codon adaptation index (CAI) greater than 0.8. CAI is a measure of codon bias. Those skilled in the art will understand that the actual effectiveness of a sequence generated by running an algorithm still needs to be verified experimentally.

[0079] In a preferred embodiment, a human The codon-optimized coding sequence for the AADC protein comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 1 to 10 and 46; the codon-optimized coding sequence for the human GBA1 protein comprises or consists of the nucleotide sequence of SEQ ID NOs: 12 to 21, 45 and 47; the codon-optimized coding sequence for the human CDNF protein comprises or consists of the nucleotide sequence of SEQ ID NOs: 23-25; and the codon-optimized coding sequence for the human GDNF protein comprises a nucleotide sequence selected from SEQ ID NOs: 27-29.

[0080] Furthermore, the expression cassette can contain one or more regulatory sequences in addition to the coding sequence. The regulatory sequence can be selected from one or more of a promoter, an enhancer, a polyadenylation sequence, and a translation termination signal. The specific combination of the regulatory sequences disclosed herein can achieve unexpected effects in improving the expression efficiency of the coding sequence.

[0081] "Promoter" refers to a DNA sequence that allows for the initiation of transcription of a downstream gene under the control of said promoter. Promoters include, but are not limited to, constitutive promoters, cell type-specific promoters, tissue-specific promoters, and developmental stage-specific promoters. A promoter may be a gene's naturally occurring promoter, a promoter specific to a gene, or a promoter specific to a gene. The promoter may be a modified version of the original promoter, or a synthetic promoter.

[0082] In a preferred embodiment, the promoter of the present disclosure may be a constitutive promoter. In a preferred embodiment, the promoter may be a CBh promoter, an EF1α promoter, a CAG promoter, a MBP promoter (myelin basic protein promoter), or a promoter derived therefrom.

[0083] An "enhancer" is a regulatory DNA sequence that, together with a promoter, can enhance the transcription of a GOI in a rAAV. In a preferred embodiment, the expression cassette of the present application includes an enhancer. More preferably, the enhancer may be, for example, a CMV enhancer in the CBh promoter.

[0084] In some embodiments, intron sequences that function as enhancers can be included, for example, intron sequences derived from an intron of the GOI can be included in the expression cassette.

[0085] In some cases, a promoter together with enhancer and / or intron sequences is collectively referred to as a "promoter" or a "promoter element." In a preferred embodiment, the promoter is a CBh promoter. In another preferred embodiment, the promoter is composed of an EFS promoter and an intron sequence.

[0086] Preferably, the total length of the intron sequence is about 200 bp or less, about 250 bp or less, about 300 bp or less, about 350 bp or less, or about 400 bp or less.

[0087] For example, the intron sequences of the present disclosure are derived from a gene of interest, e.g., the intron sequences are composed of one or more fragments derived from one or more intron regions of the gene of interest.

[0088] In preferred embodiments, the promoter or promoter / intron element is no more than 1000 bp, 900 bp, 850 bp, 800 bp, 700 bp, 600 bp, 500 bp, or 400 bp in length due to the limited packaging capacity of AAV.

[0089] In some cases, if the intron sequence is derived from an intron region of a gene of interest, it can be inserted into the coding sequence (e.g., a codon-optimized coding sequence) at a position corresponding to its naturally occurring position in the gene, e.g., between two exons, rather than being located 5' upstream of the coding sequence to constitute a promoter / intron element.

[0090] The Kozak consensus sequence (Kozak sequence), named after the scientist who discovered it, is a nucleic acid sequence motif present in most eukaryotic mRNA transcripts and serves as a protein translation initiation site. The Kozak sequence ensures accurate and efficient protein translation.

[0091] In one specific embodiment, the expression cassette comprises a CMV enhancer, a chicken β-actin promoter, a first coding sequence of a first GOI, a linker, a second coding sequence of a second GOI, and SV40 Poly A, wherein the first coding sequence and the second coding sequence are independently selected from those disclosed herein and encode any of the following combinations of two GOIs: :AADC+GBA1, AADC+CDNF, AADC+GDNF, GBA1+GDNF and GBA1+CDNF.

[0092] Linker sequence In one aspect, the present application provides linker sequences that produce high efficiency and fidelity when used to link two coding sequences of the present application that are co-expressed by one rAAV vector of the present application.

[0093] As an example of a linker sequence, a sequence encoding a 2A peptide (such as P2A, F2A, or E2A) or an IRES (the full-length version is referred to herein as the "ECMV IRES" and the shorter, truncated version as the "mini-IRES") can be used to link two coding sequences of the present application. The position of the GOI relative to the linker sequence can be adjusted to achieve the desired performance in protein expression and function. In a preferred embodiment, a P2A linker sequence is used between two GOIs within the rAAV.

[0094] In certain embodiments, the linker sequence of the present application comprises or consists of the nucleotide sequence set forth by SEQ ID NO: 35, 37, 39, 41, 43 or 44.

[0095] promoter The rAAV vectors of the present application may contain promoters conventionally used in rAAV vectors (including native promoters, mutants or hybrids thereof).

[0096] In some embodiments, the promoter can be a truncated variant of a wild-type promoter, for example, the promoter can be a truncated version of the EF1α promoter having a nucleotide sequence set forth in any one of SEQ ID NOs: 50 to 56, as shown in Table 2 of Example 6.

[0097] In some embodiments, the promoters of the present application may also be used in conjunction with enhancers, which may or may not be native to the promoter. For example, the CBh promoter is a hybrid of the CMV enhancer and the chicken β-actin (CBA) promoter. In this case, reference to a promoter refers to the inclusion of an enhancer, as would be understood by one of skill in the art. In one embodiment, the rAAV vectors of the present application comprise or consist of a CBh promoter, e.g., the nucleotide sequence set forth by SEQ ID NO:60.

[0098] In another embodiment, an EF1α promoter, such as an EF1α promoter having the nucleotide sequence set forth in SEQ ID NO: 57, is used to drive co-expression of AADC and CDNF, or co-expression of AADC and GDNF. In another embodiment, a truncated EF1α promoter, such as a truncated version of the EF1α promoter having the nucleotide sequence set forth in any one of SEQ ID NOs: 50 to 56, is used to promote co-expression of AADC and GBA1. In another aspect, a CAG promoter having the nucleotide sequence set forth in SEQ ID NO: 58 or an MBP promoter having the nucleotide sequence set forth in SEQ ID NO: 59 (or a truncation thereof) is used to drive co-expression of GBA1 and GDNF.

[0099] Poly A signal The rAAV vectors of the present application also contain a polyadenylation signal (Poly A).

[0100] For example, poly A sequences that can be used in the present application include SV40 poly A, human growth hormone (hGH) poly A, and bovine growth hormone (bGH) poly A.

[0101] In one embodiment, the expression cassette of the present disclosure comprises hGHPoly A. In one embodiment, the expression cassette of the present disclosure comprises hGHPoly A where the promoter is an EF1α promoter or a variant thereof.

[0102] In some embodiments, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is placed downstream of the GOI and proximal to the polyadenylation signal.

[0103] AAV vector serotype In a preferred embodiment, the rAAV of the present application is an AAV9 vector.

[0104] Previous reports have shown that intracerebral injection of AAV9 showed better distribution at the target site than other serotypes, including AAV1, AAV2, AAV5, and AAV8 (Watakabe, 2014). A et al., Comparative analysis of adeno-associated virus vector serotypes 1, 2, 5, 8, and 9 in marmoset, mouse, and macaque cerebral cortex. Neurosci Res, 2015, 93: 144-57. Because the human striatum is a relatively large brain region, molecular engineering of wild-type AAV capsids is necessary to cover a wider range of genes delivered by AAV.

[0105] The inventors have identified a novel AAV serotype that exhibits significantly improved tissue tropism for the putamen, a target region of interest for AAV-based gene therapy in the treatment of PD, compared to currently known AAVs. Thus, in another preferred embodiment, the present rAAV utilizes a novel AAV capsid.

[0106] Exemplary Constructs In one embodiment, the present application provides an expression cassette comprising the following sequence from 5' to 3': (a) 5′ITR; (b) promoter; (c) optional intron sequences; (d) Coding sequence of the GOI (e) a polyA, and (f) 3′ ITR, However, the GOI is selected from AADC, GBA1, CDNF, or GDNF, and the coding sequence is a codon-optimized sequence.

[0107] In another embodiment, the present application provides an expression cassette comprising the following sequence from 5' to 3': (h) 5′ITR; (h) promoter; (i) optional intron sequences; (j) coding sequence of the GOI; (k) linker sequence; (l) coding sequence of the second GOI; (m)a polyA, and (n) 3' ITR, In this case, the first coding sequence and the second coding sequence are independently selected from wild-type or codon-optimized sequences, e.g., as described in this disclosure, and encode any of the following combinations of two GOIs: AADC+GBA1, AADC+CDNF, AADC+GDNF, GBA1+GDNF, GBA1+CDNF, where the two GOIs can be arranged in any order.

[0108] In the constructs described in the above two paragraphs, each element can be independently selected from those described in the present disclosure. For example, the first nucleotide sequence can be an AADC coding sequence represented by any one of SEQ ID NOs: 1 to 9, 11, and 46, a GBA1 coding sequence represented by any one of SEQ ID NOs: 12 to 20, 22, 45, and 47, or a CDNF or GDNF coding sequence represented by any one of SEQ ID NOs: 23 to 30. For example, the second nucleotide sequence can be an AADC coding sequence represented by any one of SEQ ID NOs: 1 to 9, 11, and 46, a GBA1 coding sequence represented by any one of SEQ ID NOs: 12 to 20, 22, 45, and 47, or a CDNF or GDNF coding sequence represented by any one of SEQ ID NOs: 23 to 30. Preferably, the first nucleotide sequence can be an AADC coding sequence such as that represented by SEQ ID NO: 3 or 46; or a GBA1 coding sequence such as that represented by any one of SEQ ID NOs: 16, 45, and 47. Additionally or alternatively, in preferred embodiments, the second nucleotide sequence may be the CDNF coding sequence set forth in SEQ ID NO: 25, or the nucleotide sequence set forth by SEQ ID NO: 29. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are linked by a P2A linker sequence.

[0109] Pharmaceutical Composition The term "pharmaceutical composition" refers to a composition suitable for delivery to a subject. The pharmaceutical composition of the present application comprises an isolated nucleic acid of the present disclosure, an rAAV vector or viral particle, and a pharmaceutically acceptable excipient. Conventional pharmaceutically acceptable excipients are known in the art and may be solid or liquid excipients. In one embodiment, the pharmaceutical composition may be a liquid for injection.

[0110] Administration method As used herein, the terms "administration," "administering," "treating," and "treatment," when applied to a subject, e.g., an animal, including a human, or a cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with the subject, cell, tissue, organ, or biological fluid. Treatment of a cell includes contact of a reagent with the cell, and contact of a reagent with a fluid. The terms "administration" and "treatment" also include in vitro and ex vivo treatments of cells, e.g., with reagents, diagnostics, binding compounds, or other cells.

[0111] In a preferred embodiment, the rAAV vector of the present application can be delivered by intravenous, intracerebroventricular, intrathecal, or intrastriatal administration. In a specific embodiment, the rAAV vector is delivered via the intrastriatal route. In a specific embodiment, the rAAV vector is delivered via the intracerebroventricular route. In a most preferred embodiment, the treatment or administration is performed intracerebroventricularly (ICV), such as by ICV injection.

[0112] The rAAV vector can be administered in a single dose or multiple doses. In certain embodiments, the rAAV vector is administered in a single injection.

[0113] The dosage of rAAV vector injection can vary depending on the route of administration. For example, considering that the loss of dopamine transmission in the striatum induces movement disorders in Parkinson's disease patients, intraparenchymal / intrastriatal injection usually requires the delivery of a certain amount of rAAV to cover the striatum as much as possible to achieve a sufficient therapeutic effect. The dosage can also be varied depending on the subject's weight. Therefore, the dosage range is 1.5 × 10 10 to 1.5 x 10 14 This falls within a wide range covering vg / kg.

[0114] therapeutic use The terms "treat," "treatment," or "treatment" connote curing or at least alleviating the symptoms of a neurodegenerative disease such as PD, MSA, GD, AADCD, or other proteinopathy.

[0115] Patients suffering from any of these neurodegenerative diseases can be diagnosed by a well-trained neurologist according to clinical diagnostic criteria (Postuma, R.B. et al., Clinical Diagnostic Criteria for Parkinson's Disease (MDS) in Mov Disord, 2015; 30(12): 1591-601; Palma, J.A., L. Norcliffe-Kaufmann, and H. Kaufmann, Diagnosis of Multiple System Atrophy in Auton Neurosci, 2018; 211: 15-25) based on genetic background, medical history, symptoms and signs, and results of neurological and physical examinations.

[0116] Viral vectors expressing one of the aforementioned genes or co-expressing two of the aforementioned genes can be used to treat subjects with the aforementioned neurodegenerative diseases. The subject may be a clinically diagnosed early-stage PD patient with one or more mutations in the AADC gene, GBA1 gene, or other PD genetic risk genes, such as SNCA. The subject may also be a clinically diagnosed late-stage PD patient, with or without one or more mutations in the AADC gene, GBA1 gene, or other PD genetic risk genes, as described above. The subject may have previously been treated with or is currently being treated with a dopamine derivative, such as medpa, with or without L-DOPA-induced dyskinesia. The subject may also be resistant to currently available treatments.

[0117] Subjects may be patients with clinically diagnosed GD type 2 or type 3, or GD type 1 with central nervous system symptoms. Subjects may also be patients with clinically diagnosed MSA who exhibit at least one feature suggestive of parkinsonism (rigid tremor or bradykinesia with postural instability), cerebellar syndrome (gait ataxia with cerebellar dysarthria, limb ataxia, or cerebellar oculomotor disorder), or autonomic dysfunction, with or without a mutation in the GBA1 gene or reduced activity of the GBA1 protein.

[0118] Subjects may also be clinically diagnosed MSA patients who are resistant to currently available treatments such as dopamine derivatives.

[0119] Example In order to facilitate understanding and utilization of the present invention, the advantages of the present invention will be explained in more detail with reference to examples and the accompanying drawings. It should be understood, however, that the following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention, the scope of which is defined by the claims.

[0120] Example 1 Determination of the optimal linker for joining the AADC and GBA1 coding sequences In this example, the performance of different linker sequences, specifically E2A (SEQ ID NO:35), F2A (SEQ ID NO:37), T2A (SEQ ID NO:39), P2A (SEQ ID NO:41), mini-IRES (SEQ ID NO:43), and ECMV IRES (SEQ ID NO:44), was tested in constructs containing the wild-type coding sequences of both AADC and GBA1.

[0121] To construct an AAV vector containing both AADC and GBA1, the wild-type coding sequences of AADC (SEQ ID NO: 11) and GBA1 (SEQ ID NO: 22) were linked in the order of AADC, P2A, and GBA1 via a self-cleaving P2A sequence and placed under the control of the CBh promoter (SEQ ID NO: 60) (Figure 1). The GBA1 coding sequence was inserted upstream of the AADC coding sequence. Another construct was also prepared. Both constructs were introduced into a plasmid. The resulting plasmids were designated CBh-AADC-P2A-GBA1-WPRE-SV40pA ("AADC-P2A-GBA" or "ApG") and CBh-GBA1-P2A-AADC-WPRE-SV40pA ("GBA-P2A-AADC" or "GpA") and transfected into HEK293 cells (Procell, CL-0001). Plasmids containing the AADC or GBA1 coding sequence alone ("AADC," "GBA") or together with the P2A coding sequence ("AADC-P2A," "P2A-GBA") were also constructed and transfected into the same cells for comparison. Plasmids expressing GFP were used as controls.

[0122] Western blots were performed to detect the expression of AADC and GBA1 induced by each construct. Specifically, 72 hours after transfection, cells were collected in lysis buffer (RIPA buffer, Thermo Fisher Scientific 89901) and denatured in 5x SDS-PAGE Sample Loading Buffer (Beyotime, P0015L) at 95°C for 15 minutes. Cells were separated on a 10% SDS-PAGE gel (Sangon, #C631100) and blotted onto a 0.22 μm PVDF membrane (Merck Millipore). Protein expression levels of AADC-WT, GBA1-WT, and the housekeeping gene GAPDH were detected with antibodies against human AADC (Millipore, #AB1569), GBA1 (Sigma, #G4171), and GAPDH (CST, #2118), respectively. Long-exposure and normal-exposure WBs were performed.

[0123] As shown in Figure 2A, the molecular weight of the GBA1 protein expressed from the AADC-P2A-GBA1 (ApG) plasmid was almost the same as that of GBA1 expressed from a GBA1-only plasmid, but the ApG-derived AADC was slightly larger than that of AADC expressed from a AADC-only plasmid due to the P2A fragment attached to it. This result indicates that the self-cleaving 2A peptide functions in this setting.

[0124] Contrary to expectations, when the GBA1 gene was placed in front of (5' upstream of) the AADC gene, the expression of both genes was significantly reduced after transfection into HEK293 cells (see Fig. 2B).

[0125] Additional linkers, including E2A, F2A, T2A, P2A, mini-IRES, and ECMV IRES, were evaluated using the same experimental setup as P2A, and the results showed that all linkers evaluated, except for the mini-IRES, purified both AADC and GBA1 proteins at the expected molecular sizes.

[0126] Furthermore, to confirm the presence of incompletely cleaved fusion proteins (AADC + GBA1), we performed tests with extended exposure times. For the E2A and F2A constructs, clear positive bands were detected with both the AADC and GBA1 antibodies (see Figure 3). The cleavage efficiency of various linkers, from highest to lowest, was ECMV IRES, P2A, T2A, E2A, and F2A. When the full-length ECMV IRES was used as a linker, constructs were produced that exceeded the packaging capacity of rAAV. Therefore, P2A was selected as the linker used in this invention.

[0127] Example 2 Codon optimization to improve protein expression of AADC and GBA1 In this example, nine sequences containing more frequent codons were generated for each of AADC (referred to as A1 to A9, SEQ ID NOs: 1 to 9) and GBA1 (referred to as G1 to G9, SEQ ID NOs: 12 to 20). All of these sequences have a codon adaptation index (CAI) greater than 0.85, calculated using an online tool (https: / / www.genscript.com / tools / rare-codon-analysis). The AADC and GBA1 coding sequences disclosed in previous patent application publications were synthesized and used as references, and designated A10 (SEQ ID NO: 10, CN107106689A, Voyager Therapeutics Inc.) and G10 (SEQ ID NO: 21, WO2020210698A1, Prevail Therapeutics, Inc.), respectively. A10 encodes the M17V mutant AADC protein.

[0128] By combining one of the codon-optimized AADC coding sequences (A1 to A10) with the GBA1-WT (G0) sequence, or by combining the AADC-WT (A0) sequence with one of the codon-optimized GBA1 coding sequences (G1 to G10) (see Table 1), 20 candidate constructs with the configurations shown in Figure 4 were refined, with P2A used as a linker between the two coding sequences. Vectors containing G0 (wild-type GBA1 alone), A0 (wild-type AADC alone), and G0A0 were used as controls.

[0129] [Table 1]

[0130] The vectors were transfected into HEK293 cells using Lipofectamine 3000 Transfection Reagent (Invitrogen, #L3000008) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were washed once with 1x PBS and harvested in RIPA buffer (Thermo, 89901). AADC and GBA1 protein levels in cell lysates were measured by sandwich ELISA as described below.

[0131] ELISA for detecting AADC and GBA1 protein levels 1. On day 1, pre-coat a 96-well plate with 100 μL of coating buffer at 4°C. Overnight, they were coated with the following capture antibodies: human DOPA decarboxylase (DDC) monoclonal antibody (Sinobiological, 10560-R003, 2 μg / mL) for AADC samples and GBA1 antibody (Abcam, ab55080, 1 μg / mL) for GBA1 samples.

[0132] 2. The next day, the sample wells were washed three times with washing buffer. 300 μM of blocking buffer (containing 2% BSA) was added and left at room temperature for approximately 2 hours, after which three additional washing steps were performed.

[0133] 3. 100 μL assay buffer containing the following detection antibodies was added to each well: anti-DDC monoclonal antibody (HRP) (Sinobiological, 10560-R040, 1:1000 dilution) for AADC protein detection, and polyclonal antibody (Abcam, ab96246) for GBA1 antibody detection. After incubation at room temperature for approximately 1 hour, the sample wells were washed three times with wash buffer.

[0134] 4. To detect GBA1 protein expression, an HRP-conjugated antibody (goat anti-rabbit IgG-Fc secondary antibody (HRP) (SinoBiological, SSA003)) was applied, followed by a washing procedure.

[0135] 5. The sample wells were then incubated with 100 μL TMB solution (Solarbio, #PR1200) for approximately 20 minutes, after which the reaction was stopped by adding 100 μL stop solution (Solarbio, #C1058).

[0136] 6. The absorbance of each sample (excitation wavelength 450 nm, emission wavelength 630 nm) was detected using a fluorometer (SPECTRAmax Gemini XPS, Molecular Devices, San Jose, CA, USA), and the results are shown in Figures 5 and 6.

[0137] As shown in Figure 5, A3 and A9 were the top two candidates among the AADC coding sequences evaluated. A10 expressed the AADC mutant protein and therefore showed the highest AADC protein expression level. As shown in Figure 6, G4 and G5 were the top two candidates among the GBA1 coding sequences evaluated. Furthermore, both A0G4 and A0G5 expressed relatively higher GBA1 protein levels than the reference A0G10 construct, which contains the conventional reference GBA1 coding sequence G10.

[0138] Western blot Western blot (WB) assays were also performed to detect protein expression. Again, candidate vectors (except G1, G2, G7, A1, A2, and A7) were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Invitrogen, #L3000008).

[0139] Forty-eight hours after transfection, cells were harvested with RIPA buffer (Thermo, 89901), diluted, and boiled (denatured) in 5x loading buffer (Beyotime, P0015L). All WB samples were separated on SDS-polyacrylamide gels (BioRad, 1703932) and transferred to 0.22 μm PVDF membranes (BIO-RAD, 1620177).

[0140] The membrane was incubated overnight at 4°C with anti-AADC antibody (Millipore, #AB1569) or anti-GBA1 antibody (Sigma, #G4171), or an antibody against the housekeeping gene GAPDH (CST, #2118), and the next day was incubated with HRP-conjugated secondary antibody at room temperature for 2 hours.

[0141] The membrane was coated with the BeyoECL Moon kit (Beyotime, P0018F). Exposure and imaging were performed using a Tannon 5600 system, and the results are shown in Figures 7 and 9. The area of the AADC or GBA1 protein band was determined using ImageJ 1.53 (NIH) and normalized to GAPDH levels. The GADPH normalized values were normalized to A0 for the AADC group and G0 for the GBA1 group. Normalized data obtained from three independent experiments are shown in Figures 8 and 10.

[0142] As shown in the WB results in Figures 7 to 10, among the codon-optimized AADC candidates, A3G0 and A9G0 showed the highest AADC protein expression, and among the GBA1 candidates, A0G4 and A0G5 were the best, consistent with the results of the ELISA assay. A3 and A9 (for AADC) and G4 and G5 (for GBA1) were selected as codon-optimized sequences for further optimization of the combinatorial expression construct.

[0143] Example 3 Catalytic activity of co-expressed AADC and GBA1 proteins In this example, we aimed to determine whether exogenously expressed AADC and GBA1 proteins are catalytically active and functional. Furthermore, catalytic activity can be used as an additional evaluation criterion for candidate sequences. The method for examining AADC and GBA1 activity in cell lysates is as follows.

[0144] AADC catalyst activity (HPLC analysis) AADC protein-expressing cultures (A3G0, A9G0, A10G0, A0G0, A0) and GFP protein-expressing control samples were exposed to 100 μM L-DOPA for 24 h. The cultures were homogenized in 100 μM homogenization buffer (50 mM phosphate buffer, pH 7.4, 0.2 μM). A solution containing 0.2 mM pyridoxyl phosphate and 0.2 mM pargyline was added to each well, and the cells were scraped off the bottom.

[0145] The cell homogenates were centrifuged at 13,000 g for 10 minutes, and 30 μL of each sample was used for HPLC assays. Samples were subjected to HPLC / ECD to detect dopamine levels purified from catalytically active AADC protein expressed by various constructs. A dopamine standard curve was used to calculate relative AADC activity, which is expressed as the amount of dopamine transferred from L-DOPA in each sample.

[0146] GBA1 activity GBA1 protein activity was examined in the GBA1 protein-expressing cultures (A0G4, A0G5, A0G10, A0G0, and G0) and GFP protein-expressing control samples. Recombinant human GBA (rhGBA) was used as a positive control.

[0147] 1. rhGBA (Cat. No. 7410-GHB) was dissolved in assay buffer (50 mM The solution was diluted to 0.2 ng / μL in 25 mM sodium citrate, 25 mM sodium cholate, 5 mM DTT, pH 6.0.

[0148] 2. Substrate (4-methylumbelliferyl-beta-D-glucopyranoside, Sigma, Cat. No. M3633, 10 mM in DMSO) was diluted to 6 mM in assay buffer.

[0149] 3.25 μL of cell lysate (100 ng / μL total protein) or 0.2 ng / μL rhGBA was loaded into wells of a 96-well plate. 25 μL of 6 mM The reaction was initiated by adding substrate. 25 μL of assay buffer was loaded into the standard curve wells.

[0150] 4. The plate was sealed and incubated at 37°C for 3 hours.

[0151] 5. After incubation, the reaction was stopped by adding 50 μL of stop solution (0.5 M glycine, 0.3 M NaOH (approximately pH 10)) to each well.

[0152] 6. Measurements were recorded in endpoint mode at an excitation wavelength of 365 nm and an emission wavelength of 445 nm (top lead), respectively.

[0153] 7. Specific activity was calculated as follows: (adjusted fluorescence * 25) / (180 min * 0.0025 mg). The results are shown in Figures 9 and 10.

[0154] As shown in Figure 11, the AADC protein expressed by construct A3G0 exhibited the highest catalytic activity of all the constructs evaluated, and as shown in Figure 12, the GBA1 protein expressed by construct A0G4 exhibited the highest catalytic activity.

[0155] Example 4 Identification of the best combination of codon-optimized AADC and GBA1 sequences To identify the best combination of codon-optimized AADC and GBA1 sequences, the best-performing AADC and GBA1 coding sequences were paired and used to construct recombinant AAV vectors. Specifically, constructs A3G4, A9G4, A3G5, and A9G5 were generated. Furthermore, to completely remove the CpG islands from the AADC and GBA1 coding sequences, a further optimized construct, A11G11, was generated based on the A3 and G4 codons. Two benchmark vectors were also constructed: A10-vy (SEQ ID NO: 48, codon A10, including the promoter and other regulatory elements described in the same patent) and G10-p (SEQ ID NO: 49, codon G10, including the promoter and other regulatory elements described in the same patent).

[0156] The above vectors and A0-P2A, A0G0, A10G0, A10-vy, and G10-p were transfected into HEK293 cells. AADC and GBA1 protein expression and GBA catalytic activity were measured. Among the combination constructs evaluated, A11G11, A3G5, and A9G4 were found to express the highest levels of AADC protein as determined by WB analysis (Figure 13). For GBA1 protein expression, constructs A3G5 and A11G11 expressed the highest levels of protein and were the most active, followed by construct A9G4 (Figure 14).

[0157] Example 5 Evaluation of AADC and GBA1 protein expression of AAV9 packaging combination constructs Constructs A0G0, A9G4, A11G11, and A10-vy were packaged into AAVs using the AAV9 serotype. AAV9-A0G0, A9G4, and A11G11 were administered at MOIs of 1e5 and 1e6, along with a control AAV expressing only GFP, into U-87MG cells stably expressing the AAV receptor to increase transduction efficiency. Five days after infection, AADC and GBA1 protein expression levels in these cell samples were determined by WB analysis. As shown in Figure 15, cells transduced with the A11G11 AAV virus expressed higher levels of AADC and GBA1 proteins than A9G4-packaged AAV viruses. AADC protein expression was observed to be significantly higher in cells transduced with the A10-vy AAV virus, most likely due to the strong promoter used in the A10-vy construct.

[0158] Example 6 Optimization of control elements used in combinatorial expression constructs To further improve protein expression of the combination construct, the promoter and P The polyA sequence was also optimized. The CBh promoter of A11G11 was replaced with the EF1α promoter (SEQ ID NO: 57), and the SV40 polyA tail was replaced with bovine growth hormone polyA (bGH) or human growth hormone polyA (hGH) as shown in Table 2. Because EF1α-A11G11-hGH exceeded the packaging capacity of AAV, the length was shortened by truncating part of the EF1α promoter, resulting in seven truncations (Nos. 5-11) as shown in Table 2.

[0159] The constructs in Table 2, A3G5, A10-vy, and GFP were transfected into HEK293 cells. AADC protein expression levels were determined by WB analysis. Among the constructs initially evaluated, EF1α-A11G11-hGH expressed the highest level of AADC protein. Among the constructs with truncated EF1α promoter (EFStI1-7, SEQ ID NOs: 50-56), construct EFStI2 expressed the highest level of AADC protein (Figure 16).

[0160] [Table 2]

[0161] Example 7 In vivo efficacy of AAV candidates expressing optimized AADC and GBA1 sequences in a PD mouse model The therapeutic efficacy of the optimized AADC- and GBA1-expressing AAVs was evaluated in a PD mouse model to determine whether the candidate AAVs could restore motor deficits in PD animals.

[0162] We first used a widely used chemical-induced PD mouse model. Mice were treated with MPTP / probenecid. The candidate rAAV vector CBh-A11G11 was injected into the striatum of PD model mice at a high dose of 2E+10 vg / mouse and a low dose of 2E+09 vg / mouse ("CBh-A11G11 Low" and "CBh-A11G11 High"). The rAAV vector A10-vy (2E+09 vg / mouse) was injected in the same manner as a benchmark control ("A10-vy"), and a GFP-expressing rAAV vector was used as a negative control ("GFP"). The mice's motor function was assessed by measuring their movement speed and distance traveled before PD modeling (before MPTP / P induction; "Pre"), and before and after administration of each rAAV vector ("Post MPTP / P" and "Post AAV", respectively). "Pre" behavioral testing was performed for 6 days, followed by daily administration of MPTP / probenecid for 35 days. After 3 days of acclimation, subjects underwent the same behavioral testing ("post-MPTP / P") followed by rAAV injection. 21 days after rAAV injection, "post-AAV" behavioral testing was performed. The results are shown in Figure 34.

[0163] As shown in Figure 34, the movement speed of mice decreased after MPTP / probenecid administration. Mice treated with A10-vy (N = 3) showed a smaller decrease in speed than the other mice. All mice, except for the A10-vy-treated mice, showed a reduction in movement distance. After administration of the corresponding rAAV, negative control mice (N = 5) showed a sustained decline in motor function, with a continuous decrease in movement speed and distance. Meanwhile, mice treated with both low (N = 5) and high (N = 7) doses of CBh-A11G11-SV40 showed a recovery in movement speed and distance, although the high dose showed slightly better results than the low dose. These results suggest that CBh-A11G11-SV40 exerts a significant effect in restoring the deficits in movement speed and distance in the MPTP / probenecid-induced PD mouse model. Since the damage to the locomotion ability of A10-vy-treated mice was less severe than that of other mice, CBh-A11G11-SV40 was considered to have achieved a superior effect compared with the benchmark in restoring the deficits in locomotion speed and distance in the MPTP / probenecid-induced PD mouse model.

[0164] Next, we validated our results using a transgenic PD mouse model. To recapitulate the pathology of PD, we genetically engineered mice to overexpress mutant α-synuclein (point mutation A53T). Two types of rAAV, EFSIt7-A11G11-hGH (EtI7) and CBh-A11G11-SV40 (CBh), were produced based on the A11G11 construct using different promoters and poly A tails. rAAV was administered at a low dose of 4E+9. Mice were administered intrastriatal injections of 4E+10 vg and a high dose of 4E+10 vg. Untreated wild-type mice and vehicle (PBS)-treated A53T mice were included as controls. Motor function was assessed by measuring the time to rear up (hindlimb rearing), and the results are shown in Figure 35.

[0165] As shown in Figure 35, mice treated with vehicle (N = 6) performed fewer vertical movements than WT (N = 8) mice, indicating impaired motor function. Mice treated with both high (N = 7) and low (N = 7) doses of EFSIt7-A11G11-hGH (EtI7) performed better than Veh, with the high dose showing significantly better results. Mice treated with low doses of CBh-A11G11-SV40 (CBh) (N = 7) also showed increased time spent on vertical movement compared to WT and vehicle-A11G11-based rAAV. The increased number of vertical movements indicates recovery of motor function.

[0166] Brain tissue samples were collected from A53T mice and protein expression was determined. The expression of AADC and GCase proteins, which are involved in PD pathology, was determined by WB. As shown in Figure 36, AADC and GCase expression was observed in all treatment groups. Furthermore, the levels of phosphorylated α-synuclein and total α-synuclein in the striatum were measured. The level of phosphorylated α-synuclein was considered a biomarker of PD pathology. As shown in Figures 37A and 37B, both candidate rAAV vectors dose-dependently reduced the level of phosphorylated α-synuclein, indicating an improvement in PD pathology. The illustrative bands in Figure 37A were from two representative subjects in each group. The relative phosphorylated α-synuclein levels shown in Figure 37B were averaged across all subjects in the indicated group.

[0167] Example 8 In vivo therapeutic efficacy of AAV candidates expressing optimized GBA1 sequences in a mouse model of GD The therapeutic efficacy of AAV expressing the optimized GBA1 was tested in a GD mouse model.

[0168] As shown in Figure 38, two candidate vectors expressing optimized GBA1 (G11) were constructed, each containing or consisting of the nucleotide sequence indicated by 38. The first candidate vector was designed to contain a CAG promoter, a G11 codon, and hGH Poly A ("CAG"). The second candidate vector was designed to contain an EF1α promoter, a G11 codon, a WPRE, and hGH poly A ("EF1α"). Both vectors were packaged as rAAV in AAV9. The candidate rAAV vectors were injected into the lateral ventricles of chemically induced and genetically engineered GD mouse models to evaluate their efficacy.

[0169] The chemical-induced mouse model was the CBE-induced GD mouse model. GCase activity was inhibited by conduritol B epoxide (CBE), a specific irreversible GCase inhibitor that mimics loss-of-function mutations in the GBA1 gene. On postnatal day 2 (P2), mice were administered PBS, AAV9-GFP (negative control), or three different doses of candidate rAAV vectors (low dose, 2.75E+09vg; medium dose, 8.8E+09vg; high dose, 2.8E+10vg per animal) via ICV injection. G10-p was used as a reference. The disease model was initiated at P8 to allow sufficient time for gene expression. Mice were administered 37.5 mg / kg CBE via IP injection once daily. Lifespan assessment was performed until P28.

[0170] Figure 39 shows the survival rate of GD mice up to P28. As shown, the survival rates of the PBS and negative control groups rapidly decreased after P22. No animals in the negative control group survived at P25. In contrast, both the candidate vector and the reference G10-p significantly improved the survival rate of GD mice.

[0171] Figure 40 shows the changes in motor function in the study animals. Both candidate vectors reversed the motor dysfunction and motor coordination deficits induced by chronic CBE administration. In Figures 40A and 40B, CAG increased the distance traveled in the open field test at all doses and improved motor coordination in the rotarod test at the medium and high doses, while EF1α increased the distance traveled at the medium and high doses but improved motor coordination only at the high dose.

[0172] At postnatal day 30, brain samples were collected to determine protein activity. Lysate samples were prepared by homogenizing the brain samples in lysis buffer (N-PER Neuronal Protein Extraction Reagent, Thermo Scientific #87792) containing protease inhibitors (Roche #11697498001). GCase activity in these lysate samples was measured using the GBA1 activity assay described above. A dose-dependent increase in GCase activity was observed in all candidate vector-treated groups (Figure 41).

[0173] Example 9 Codon-optimized human CDNF sequences for AADC combination constructs In this example, an AAV vector was designed to co-express AADC and NTF-encoding genes, specifically CDNF, with P2A used as a linker sequence between the two coding sequences.

[0174] To determine the optimal coding sequence for CDNF, the wild-type AADC coding sequence (AADC WT) was ligated via P2A with different codon-optimized CDNF sequences. Codon optimization was performed based on different algorithms.

[0175] Two codon-optimized CDNF sequences with high CAI (>0.85) were obtained and designated CDNF GS and CDNF SA, respectively. Both sequences were codon-optimized except for the sequence fragment encoding the signal peptide (nucleotide positions 1-72 of the nucleotide sequence represented by SEQ ID NO: 26). In CDNF SA, the nine nucleotides encoding the three amino acids immediately following the signal peptide (nucleotide positions 73-81) were unchanged.

[0176] On the other hand, another CDNF candidate sequence, named CDNF manual (CDNF MN), was obtained by manually identifying and reducing the "CG" components in the mature protein-coding sequence (nucleotide positions 82-561) based on the CDNF SA sequence, resulting in the intentional removal of all CpG islands from the CDNF MN sequence.

[0177] For this study, an AAV backbone vector was prepared containing 5′ to 3′, 5′ ITR, CBh promoter, AADC WT coding sequence, P2A sequence, SV40 Poly A, and 3′ ITR.

[0178] The sequences of CDNF GS, CDNF SA, CDNF MN, and CDNF wild-type (CDNF WT) were inserted into the aforementioned backbone vector 3' of the P2A linker sequence (Figures 17A and 17B). The resulting vectors were designated ApC-GS, ApC-SA, ApC-MN, and ApC-WT. A reference vector consisting of CDNF WT Alone (without AADC) was also constructed.

[0179] HEK293 cells were transfected with the five plasmids listed above and one negative control plasmid expressing only GFP using Lipofectamine 3000 transfection reagent (Invitrogen, # L3000008). 72 hours after transfection, the culture supernatant was centrifuged at 2,000 rpm for 10 minutes to remove debris. 40 μL of the culture supernatant and 10 μL of 5x loading buffer (Beyotime, P0015L) were transferred to a new 1.5 mL tube and incubated at 95°C for 5 minutes. The cells were washed once with 1x PBS and harvested with RIPA buffer (Thermo, 89901).

[0180] For WB analysis, samples were diluted, boiled in 5x loading buffer (Beyotime, P0015L), separated on an SDS-polyacrylamide gel (BIO-RAD, 1703932), and transferred to a 0.22 μm PVDF membrane (BIO-RAD, 1620177). The membrane was incubated with antibodies against CDNF protein (Sigma, HPA044587), AADC protein (Merck Millipore, AB1569), and β-tubulin protein (Tubulin, Proteintech, 66240-1-Ig) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies for 2 hours at room temperature the following day. Next, the membrane was coated with the BeyoECL Moon kit (Beyotime, P0018F) (Figure 18A). Exposure and imaging were performed using a Tannon 56 The results were shown in Figure 18B.

[0181] As shown in Figure 18B, among all the constructs evaluated, cells transfected with the ApC-MN construct had the highest levels of CDNF protein secreted into the culture supernatant, which was also confirmed in U-87 MG cells transfected with the same set of plasmids (Figures 19A-B).

[0182] The levels of secreted CDNF protein in the culture supernatants were further measured by sandwich ELISA. Briefly, 96-well microplates (Costar, 42592) were coated overnight at 4°C with CDNF mouse MAb (SinoBiological, 15691-MM1) as a capture antibody. The next day, the plates were washed three times with PBST (phosphate-buffered saline with Tween) and then blocked with 5% nonfat dry milk (Solarbio, D8340) for 2 hours at room temperature. After an additional wash step, CDNF protein standards (SinoBiological, 15691-H08H) or protein samples were added to the plates in duplicate. The samples were then incubated with detection antibody (SinoBiological, 15691-R104) for 2 hours at room temperature and then washed. The samples were then incubated with HRP-conjugated goat anti-rabbit antibody (SinoBiological, HO14SE1801) for 1 hour and washed once. The bound HRP conjugate was detected by adding TMB (Solarbio, PR1200), and the reaction was terminated with stop solution (Solarbio, C1058). The luminescence intensity of each sample (excitation wavelength 450 nm, emission wavelength 630 nm) was measured using a fluorometer (SPECTRAmax Gemini XPS, Molecular Devices, San Jose, CA, USA). The results are shown in Figure 20A-B.

[0183] As shown in Figure 20A-B, both HEK293 and U-87 MG cells transfected with ApC-MN expressed significantly higher levels of CDNF protein, as indicated by the high levels of CDNF secreted into the culture supernatant. Consequently, ApC-MN was selected for further evaluation.

[0184] Example 10 Codon-optimized CDNF protein exhibited significant cytoprotective effects In this example, we investigated the cytoprotective effects of CDNF protein expressed and secreted in cells transfected with the plasmid AADC-CDNF MN (ApC-MN). Two cell-based assays were used to investigate the cytoprotective effects of CDNF: an LDH cytotoxicity assay and a CCK-8 cell viability assay in cells treated with MPP+ or rotenone.

[0185] For the LDH assay, SH-SY5Y cells (National Collection A 96-well assay plate containing MPP (of Authenticated Cell Cultures, SCSP-5014) was pretreated with or without culture supernatant containing secreted CDNF protein collected from previous experiments. To determine background luminescence levels, some wells were left without cells as blank controls. + Plates were treated with rotenone or DMSO solvent control. After incubating the plates at 37°C, LDH detection reagent (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega) was prepared by mixing 12 mL of Assay Buffer with one bottle of Substrate Mix. 45 minutes before adding the CytoTox 96® reagent, 1 / 10 volume of 10x Lysis Solution was added to the positive control (maximum LDH release control) wells. Before reading the plate, 50 μM CytoTox 96® reagent was added to each well. The ELISA kit (Protein™) reagent was added. The plate was covered with foil to protect it from light exposure and incubated at room temperature for 30 minutes. 50 μM of stop solution was then added to each well to stop the reaction. One hour after adding the stop solution, the absorbance at 490 nm was recorded using a plate reader. Percent cytotoxicity = 100 x experimental LDH release (OD490) / maximum LDH release (OD490).

[0186] For the CCK-8 assay, 5 × 10 SH-SY5Y cells in logarithmic growth phase were plated in a 96-well plate. 3The cells were plated at a density of 1000 / mL and cultured in high-glucose / DMEM cell culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C in a cell incubator with 5% CO2 and 95% air for 12 hours. The cells were then treated with or without culture supernatant containing the secreted CDNF collected in the previous experiment. The final concentrations of MPP were 0 to 2 mM. + Alternatively, serum-free medium containing rotenone was used to induce cell apoptosis. After 24 h, cell viability was assessed by adding 10 μM CCK-8 (Cell Counting Kit-8, Dojindo), and after 2 h of incubation, relative cell viability was determined by spectrophotometry at a wavelength of 450 nm.

[0187] As shown in Figure 21, MPP + SH-SY5Y cells treated with the culture supernatant of ApC-MN-transfected cells in the presence of 1.5 mM or rotenone (40 nM) showed higher survival rates than cells treated with the culture supernatant of ApC-WT-transfected cells, indicating that ApC-MN expresses and secretes higher levels of CDNF protein into the culture supernatant and exerts stronger cytoprotective effects than ApC-WT.

[0188] Example 11 Codon-optimized human GDNF sequence for AADC combination constructs In this example, the mature protein coding sequence of human GDNF was optimized by the codon optimization process performed in Example 9 (Figure 17B) to obtain the GDNF-GS sequence (SEQ ID NO: 27), the GDNF-SA sequence (SEQ ID NO: 28), and the GDNF-MN sequence (SEQ ID NO: 29).

[0189] The same in vitro cell experiments as described in Example 10 were performed using HEK293 cells transfected with constructs containing GDNF GS, GDNF SA, and GDNF MN (Figures 22 to 24). Based on the results, the GDNF MN sequence without a CpG island had the highest GDNF expression and protection capacity among all the codon-optimized GDNF sequences evaluated. Therefore, the GDNF MN sequence was selected as the GDNF coding sequence for the AADC combination construct for further evaluation.

[0190] Example 12 Determination of the optimal promoter for use in AADC and CDNF / GDNF combination constructs To determine the optimal promoter elements for use in AADC and CDNF or GDNF combination constructs, eight new combination constructs were prepared, as shown in Table 3. The four CDNF-containing constructs were CAG-A3-P2A-CDNF MN, CAG-A11-P2A-CDNF MN, EF1α-A3-P2A-CDNF MN, and EF1α-A11-P2A-CDNF MN; and the four GDNF-containing constructs were CAG-A3-P2A-GDNF MN, CAG-A11-P2A-GDNF MN, EF1α-A3-P2A-GDNF MN, and EF1α-A11-P2A-GDNF MN. The CBh-AADC WT-P2A-CDNF WT and CBh-AADC WT-P2A-GDNF WT constructs were also prepared and used as controls.

[0191] [Table 3]

[0192] The aforementioned constructs were transfected into HEK293 cells. Both cell lysates and culture supernatants of these samples were collected. Secreted CDNF or GDNF protein levels were measured by ELISA. The results showed that EF1α-A11-P2A-CDNF MN expressed higher levels of CDNF protein than CAG-A3-CDNF MN, while the other two constructs expressed undetectable levels of CDNF protein (Figure 25).

[0193] Of the GDNF-containing constructs, EF1a-A11-GDNF MN expressed and secreted the highest levels of GDNF (FIG. 25).

[0194] The catalytic activity of the AADC proteins expressed by these combination constructs was also measured by HPLC assay of cell lysates, as described in the previous example. Figure 26 shows that the relative AADC activity of EF1α-A11-CDNF MN and EF1α-A11-GDNF MN was significantly higher than that of all other candidate constructs.

[0195] The CDNF codon was placed before the AADC codon and linked with P2A to prepare the construct EF1α-CDNF MN-A11.

[0196] Both EF1α-CDNF MN-A11 (CDNF MN-A11) and EF1α-A11-CDNF MN (A11-CDNF MN) were transfected into HEK293 cells using Lipofectamine 3000. 72 hours after transfection, the culture supernatant and cell lysates were collected. The protein levels of AADC and CDNF in the cell lysates and the CDNF level (secreted CDNF) in the culture supernatant were measured by WB. The protein level of secreted CDNF was found to be much higher in the CDNF MN-A11 group than in the A11-CDNF MN group (Figure 42).

[0197] Example 13: Verification of the cytoprotective effects of CDNF and GDNF proteins derived from candidate constructs To determine the cytoprotective effects of the GDNF constructs identified in Example 12, MPP+ and rotenone assays were performed as described in previous experiments. Culture supernatants from cells transfected with the constructs EF1α-A11-CDNF MN (Figure 27) or EF1α-A11-GDNF MN (Figure 28) improved the viability of SH-SY5Y cells in the presence of MPP+ (1.5 mM) or rotenone (40 nM). LDH assays were also performed to confirm the cytoprotective effects of both candidate constructs.

[0198] Example 14 Evaluation of AADC and CDNF or GDNF protein expression of AAV9 packaging constructs Both the EF1a-A11-CDNF MN and EF1a-A11-GDNF MN combination constructs were packaged into AAV9. The resulting AAV9-EF1α-A11-CDNF MN and AAV9-EF1α-A11-GDNF MN constructs were added to U87-AAVR cells at an MOI of 1e5. At 72 hours post-viral transduction, cells were harvested in lysis buffer. The expressed CDNF and AADC protein levels were measured according to the WB protocol. Meanwhile, culture supernatants from all samples were also harvested and concentrated as described above. The levels of secreted CDNF and GDNF proteins were also detected by WB, and the results are shown in Figure 29.

[0199] The relative activity of the expressed AADC protein in the above cell lysates is shown in FIG.

[0200] Taken together, the data shown in Figures 29 and 30 show that both constructs performed as expected when packaged into AAV9 as rAAV.

[0201] Example 15 In vivo therapeutic efficacy of an AAV candidate expressing optimized AADC in combination with GDNF or CDNF in a PD mouse model The therapeutic efficacy of AAV delivering the optimized AADC+GDNF combination construct was tested in a unilaterally lesioned 6-OHDA PD mouse model. Briefly, C57 mice were placed in a stereotaxic frame and anesthetized with isoflurane in oxygen. 1 μM 6-OHDA (3 mg / ml, Sigma, #162957) was injected into the right substantia nigra (coordinates AP = -2.9 mm, ML = -1.1 mm, DV = -4.5 mm). One week after lesion surgery, mice were intraperitoneally administered apomorphine (Sigma, PHR2621-500MG), and rotational behavior (turning toward the uninjured side) was recorded and analyzed. Next, we injected the candidate rAAV vector EF1α-A11-GDNF MN into the right striatum of each subject (coordinates AP = +0.6 mm, ML = -1.8 mm, DV = -3.2 mm) at three doses (low dose: 1E+12 vg, medium dose: 3.16E+12 vg, high dose: 1E+13 vg / mL). Two doses of AAV-GFP and benchmark AAV-A10-vy (low: 1E+12 vg, high: 1E+13 vg mL) were also administered. Twenty-one days after AAV injection, apomorphine-induced contralateral rotation was significantly reduced in all test groups administered the candidate rAAV vectors compared to the control group (Figure 31 and Figure 32). As shown in Figure 31, the three study groups treated with the three different doses of EF1α-A11-GDNF MN rAAV performed significantly better than the study treated with the benchmark rAAV. More notably, the net rotation counts (ipsilateral-contralateral) for the low-dose and mid-dose groups were both close to zero, indicating that these subjects were able to perform balance motor activities. Although complete recovery was observed, overcompensation of ipsilateral rotation (excessive AADC on the treated side) was observed in the high-dose benchmark group (Figure 32).

[0202] In another in vivo efficacy study in PD mice (Study 2), CAG-A11-GDNF MN rAAV (AAV9) was also tested in a unilateral 6-OHDA PD mouse model. Two doses (2.0E+09 vg and 2.0E+10 vg) of CAG-A11-GDNF MN and EF1α-A11-GDNF MN were intrastriatally injected into PD mice. AAV-GFP was used as an untreated control, and two doses of AAV2-A10-vy were used as a benchmark. At 21 days after AAV injection, EF1α-A11-GDNF MN at 2E+10 vg / animal significantly reduced ipsilateral rotations induced by amphetamine (5 mg / kg i.p.), indicating recovery of motor function (Figure 43).

[0203] Tissue samples were collected to measure AADC and NTF protein expression and dopamine levels. GOI expression was observed in the nigrostriatal pathway. As shown in Figure 44, striatal tissue samples were collected from 6-OHDA mice in Study 2 on day 91 after AAV injection. AADC and GDNF protein levels were measured by WB in both the EF1α and benchmark groups. Surprisingly, EF1α expressed more AADC in the striatum (injection site) than A10-vy (Figure 44A). Meanwhile, EF1α was confirmed to express GDNF in vivo in a dose-dependent manner (Figure 44B).

[0204] Furthermore, tyrosine hydroxylase (TH) staining showed that more dopaminergic terminals survived in the nigrostriatal pathway in animals treated with EF1α compared to control animals treated with AAV9-GFP alone, confirming the protective effect of the candidate rAAV (Figure 45).

[0205] The efficacy of AAV9-EF1α-A11-GDNF MN (EF1α) was also tested in a unilateral 6-OHDA PD rat model. The model was generated according to a previous study (6-OHDA Lesion Model of Parkinson's Disease in Rats). Animal Models of Movement Disorders: Volume 1, Neuromethods, Volume 61, DOI 10.1007 / 978-1-61779-298-4_13). Briefly, 6-OHDA was injected into the right substantia nigra (coordinates AP = -4.4 mm, ML = -1.1 mm, DV = -8 mm). Two weeks after the lesion surgery, subjects received two injections of EF1α into two sites in the right striatum (coordinates AP = +1.0 mm, ML = -3.0 mm, DV = -4.5 mm; AP = -0.2 mm, ML = -3.5 mm, DV = -5.0 mm) at two different doses (low dose: 4.0E+9 vg / mouse, high dose: 4E+10 vg / mouse). A single injection of the benchmark AAV2-A10-vy (4E+10 vg / mouse) was also administered. Twenty-one days after AAV injection, spontaneous contralateral rotations were recorded before and after administration of L-DOPA (5 mg / kg L-DOPA + 2.5 mg / kg benserazide). The 1-minute delta rotation time (post-minus pre) represented the subject's response to L-DOPA. As shown in Figure 46, EF1α significantly enhanced the subject's response to low doses of L-DOPA, suggesting that this therapeutic candidate may enhance the bioavailability of dopamine derivatives such as L-DOPA in future clinical applications. Furthermore, EF1α showed better results than A10-vy at the same dose level (4E + 10 vg).

[0206] Example 16 In vivo therapeutic efficacy of AAV candidates combining optimized GBA1 and CDNF or GBA1 and GDNF in MSA animal models Candidate constructs were purified, with the configurations shown in Figures 33A and 33B. These constructs comprise the CAG or MBP promoter, the GBA1 gene (including SEQ ID NOs: 45 and 47) in combination with CDNF (SEQ ID NOs: 23 to 25) or GDNF (SEQ ID NOs: 27 to 29).

[0207] We constructed CAG-G11-P2A-GDNF MN-SV40 Poly A (CAG-G11-GDNF MN), CAG-G12-P2A-GDNF MN-SV40 Poly A (CAG-G12-GDNF MN), MBP-G11-P2A-GDNF MN-bGH Poly A (MBP-G11-GDNF MN), and CAG-GDNF MN-WPRE-hGH (CAG-GDNF MN) vectors and packaged them into AAV serotype AAV9. These candidate rAAV vectors were injected into U-87 MG-AAVR cells (U-87 MG cells overexpressing AAVR), and cell lysates and culture supernatant samples were collected. GCase and GDNF protein levels were measured in these samples (Figure 47).

[0208] The efficacy of the candidate rAAV vectors was tested in vitro using a cellular model recapitulating the pathological characteristics of PD and MSA (a stable cell line based on SH-SY5Y cells overexpressing both AAVR and mutant α-synuclein (point mutation A53T) proteins). Lentiviral vectors carrying either the AAVR sequence or the mutant α-synuclein (A53T) sequence were prepared at Azenta Life Sciences and sequentially administered into SH-SY5Y cells. After antibiotic and GFP-based selection, the SH-SY5Y-AAVR-A53T stable cell line was generated. As shown in Figure 48, the candidate rAAV vectors were found to significantly reduce the levels of high molecular weight (HMW) α-synuclein, a biomarker of α-syn aggregation, in the cellular model.

[0209] CAG-GDNF MN-P2A-G11-SV40 polyA(CAG-GDNF We constructed a CAG-GDNF MN-G11 construct and transfected it into HEK293 cells along with CAG-G11-GDNF MN. Seventy-two hours after transfection, the culture supernatants and cell lysates were collected and analyzed. Compared with CAG-G11-GDNF MN, CAG-GDNF MN-G11 expressed significantly more GDNF in both the culture supernatants and cell lysates, and surprisingly, maintained the ability to express GCase (Figure 49).

[0210] We then evaluated the therapeutic efficacy of AAVs delivering the optimized GBA1 + CDNF or GBA1 + GDNF combination constructs in a mouse model of MSA. Intravenous injection of the candidate rAAV vectors rescued motor deficits in MSA animals. Tissue samples were collected and observed to decrease GBA1 and NTF protein expression, as well as α-synthesis and phosphorylated α-synthesis levels in the nigrostriatal pathway. Furthermore, tyrosine hydroxylase (TH) staining demonstrated that more dopaminergic terminals survived in the striatum of treated animals, confirming the protective effect of the candidate AAVs.

[0211] Example 17 In vivo therapeutic efficacy of AAV candidates expressing optimized GBA1 in PD mouse models The therapeutic efficacy of AAV expressing optimized GBA1 was tested in a PD mouse model.

[0212] Mice genetically engineered to overexpress mutant α-synuclein (point mutation A53T) are used as a PD model. CAG-G11-hGH Poly A is packaged into AAV9 (as shown in Example 8 and Figure 38A) to purify the rAAV vector. Candidate rAAV vectors are injected into the lateral ventricle of the A53T PD mouse model to evaluate their efficacy. rAAV9-CAG-G11-hGH significantly alleviates motor dysfunction in the PD mouse model and reduces the levels of α-synuclein and phosphorylated α-synuclein in the nigrostriatal pathway, potentially demonstrating potential therapeutic potential for the treatment of PD and other α-synuclein protein disorders.

[0213] [Table 4] TIFF2025516136000005.tif253170TIFF2025516136000006.tif253170TIFF2025516136000007.tif253170TIFF2025516136000008.tif253170TIFF2025516136000009.tif253170TIFF2025516136000010.tif253170TIFF2025516136000011.tif253170TIFF2025516136000012.tif253170TIFF2025516136000013.tif253170TIFF2025516136000014.tif253170TIFF2025516136000015.tif253170TIFF2025516136000016.tif253170TIFF2025516136000017.tif253170TIFF2025516136000018.tif253170TIFF2025516136000019.tif253170TIFF2025516136000020.tif253170TIFF2025516136000021.tif253170TIFF2025516136000022.tif253170TIFF2025516136000023.tif253170TIFF2025516136000024.tif253170TIFF2025516136000025.tif253170TIFF2025516136000026.tif253170TIFF2025516136000027.tif253170TIFF2025516136000028.tif253170TIFF2025516136000029.tif253170TIFF2025516136000030.tif253170TIFF2025516136000031.tif253170TIFF2025516136000032.tif253170TIFF2025516136000033.tif253170TIFF2025516136000034.tif253170TIFF2025516136000035.tif253170TIFF2025516136000036.tif253170TIFF2025516136000037.tif253170TIFF2025516136000038.tif253170TIFF2025516136000039.tif253170TIFF2025516136000040.tif253170TIFF2025516136000041.tif253170TIFF2025516136000042.tif253170.

Claims

1. Isolated nucleic acid molecules, A first nucleotide sequence encoding a first protein is operationally linked to a second nucleotide sequence encoding a second protein, and the first and second proteins are selected from the following combinations (a) or (b): (a) The first protein is human AADC and the second protein is human GDNF; or (b) The first protein is human GDNF and the second protein is human AADC, Here, The first nucleotide sequence is located 5' upstream of the second nucleotide sequence. The human AADC is encoded by a nucleotide sequence containing any of the sequence numbers 46, 3, 5, 6, 7, or 9, or consisting of any of the sequence numbers. Isolated nucleic acid molecules.

2. The isolated nucleic acid molecule according to claim 1, having the following characteristics: (i) The polypeptide sequence of AADC includes the amino acid sequence shown by SEQ ID NO: 31, or is an amino acid shown by the SEQ ID NO: (ii) The polypeptide sequence of GDNF includes the amino acid sequence shown by Sequence ID No. 34, or is the amino acid sequence shown by the Sequence ID No.

34.

3. The isolated nucleic acid molecule according to claim 1, wherein the first protein is human AADC.

4. The isolated nucleic acid molecule according to claim 1, wherein the second protein is human GDNF, and the second nucleotide sequence comprises or is a nucleotide sequence selected from the group consisting of nucleotide sequences represented by any one of sequence numbers 27 to 30.

5. The isolated nucleic acid molecule according to claim 1, wherein the nucleotide sequence encoding human GDNF is the codon-optimized coding sequence of GDNF, and in nucleotides not present in the region encoding the signal peptide or prepeptide of GDNF, the codon-optimized coding sequence is different from the wild-type coding sequence of GDNF indicated by SEQ ID NO:

30.

6. The isolated nucleic acid molecule according to claim 5, wherein the nucleotide sequence encoding human GDNF is a codon-optimized coding sequence of GDNF, and has reduced CpG sites or lacks CpG islands compared to the wild-type coding sequence of GDNF shown by Sequence ID No.

30.

7. The isolated nucleic acid molecule according to claim 1, wherein the first nucleotide sequence and the second nucleotide sequence are linked within a frame and operably linked to a promoter located 5' upstream of both the first nucleotide sequence and the second nucleotide sequence.

8. The isolated nucleic acid molecule according to claim 7, wherein the promoter is selected from the CBh promoter, the EF1α promoter, the CAG promoter, the MBP promoter, or variants thereof.

9. The isolated nucleic acid molecule according to claim 8, wherein the promoter is a truncated variant of the EF1α promoter having a nucleotide sequence indicated by any one of sequence numbers 50 to 56.

10. The isolated nucleic acid molecule according to claim 1, further comprising a linker sequence between the first nucleotide sequence and the second nucleotide sequence.

11. The isolated nucleic acid molecule according to claim 10, wherein the linker sequence is a coding sequence for a self-cleaving peptide or an internal ribosome entry site.

12. The isolated nucleic acid molecule according to claim 11, wherein the self-cleaving peptide is a 2A peptide.

13. The isolated nucleic acid molecule according to claim 12, wherein the 2A peptide is selected from the group consisting of E2A, F2A, T2A and P2A.

14. The isolated nucleic acid molecule according to claim 11, wherein the linker sequence is ECMV IRES or mini IRES.

15. Sequence ID: A codon-optimized sequence of AADC having a nucleotide sequence indicated by one of the following: 46, 3, 5, 6, or 9.

16. A recombinant adeno-associated virus (rAAV) vector comprising an isolated nucleic acid molecule according to any one of claims 1 to 14 or the codon-optimized sequence according to claim 15.

17. A virus particle comprising the rAAV vector described in claim 16.

18. The viral particle according to claim 17, comprising the capsid protein of AAV1, AAV2, AAV5, AAV8, or AAV9.

19. A pharmaceutical composition comprising the rAAV vector according to claim 16 and a pharmaceutically acceptable excipient.

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