Recombinant aav vectors for treating neurodegenerative disorders

Abstract

Provided are recombinant adeno-associated virus (rAAV) vectors comprising one or two of (a) to (c): (a) a nucleotide sequence encoding an aromatic L-amino acid decarboxylase (AADC); (b) a nucleotide sequence encoding a glucocerebrosidase (GBA1); and (c) a nucleotide sequence encoding a neurotrophic factor (NTF) such as a brain dopamine neurotrophic factor (CDNF) or a glial cell-derived neurotrophic factor (GDNF), for use in the treatment of neurodegenerative disorders, in particular Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher's disease (GD), and other proteinopathies. Also provided herein are viral particles comprising the rAAV vectors, pharmaceutical compositions comprising the viral particles, and uses thereof.

Description

[0001] This application is a divisional application of Chinese patent application filed on April 19, 2023, with application number 202380034877.X and invention title "Recombinant AAV Vector for Treating Neurodegenerative Disorders". Technical Field

[0002] This disclosure relates to the technical field of gene therapy. Specifically, this disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising one or both of (a) to (c): (a) a nucleotide sequence encoding an 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-derived neurotrophic factor (GDNF), said recombinant adeno-associated virus (rAAV) vector for the treatment of neurodegenerative disorders, particularly Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD), and other protein disorders. This document also provides viral particles comprising said rAAV vectors, pharmaceutical compositions comprising said viral particles, and uses thereof.

[0003] sequence list

[0004] This disclosure includes a sequence list that is part of this disclosure. Background Technology

[0005] Neurodegenerative diseases (NDs) occur when nerve cells in the brain or peripheral nervous system gradually lose function and eventually die, leading to a loss of mobility, coordination, strength, sensation, and cognition. Due to their slow and progressive nature and the lack of suitable treatments to slow progression or cure them, NDs are associated with enormous socioeconomic, personal, and personal costs and suffering. Aging is a major risk factor for most NDs. With increasing life expectancy, this means that more people will be affected by NDs in the coming decades, imposing a significant economic burden on our societies and posing a major global public health challenge.

[0006] Parkinson's disease (PD), caused by degeneration of dopaminergic transmission in the midbrain and characterized by both motor symptoms (e.g., tremor and rigidity) and nonmotor symptoms (e.g., intellectual disability and depression), affects almost 0.3% of the general population and about 1% of people over 60 years of age (de Lau, LM and MM Breteler, Epidemiology of Parkinson's disease. Lancet Neurol, 2006. 5(6): 525-35).

[0007] Most cases of Parkinson's disease (PD) are sporadic and caused by unknown factors. Only a small number of cases have a family history. Currently, six specific human genomic loci have been identified containing genes whose mutations may lead to rare familial forms of PD (Klein, C. and A. Westenberger, Genetics of Parkinson's disease. Cold Spring Harb Perspect Med, 2012. 2(1): a008888). It is generally believed that the etiology of PD is caused by a complex interaction between intrinsic factors (such as genetics) and extrinsic environmental factors. The pathological changes and their relationship with PD symptoms have been well established (Kouli, A., KM Torsney and WL Kuan, Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects, TB Stoker and JC Greenland eds. 2018: Brisbane (AU)). The pathological features of Parkinson's disease (PD) lie in the degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the loss of the dopaminergic pathway from the SNc to the striatum (putamen and caudate nucleus), leading to decreased dopamine levels in the striatum and consequently impaired motor function. Based on this knowledge, several drugs have been developed using different approaches to restore dopamine levels.

[0008] Another pathological marker of PD is the formation of Lewy bodies (LB), or so-called "Lewy pathology," in the brain of patients. LB or Lewy neurites containing alpha-synuclein (α-syn), induce cytotoxicity in dopaminergic neurons and other neuronal subtypes, and are one of the major pathogenic mechanisms of PD (Teil, M. et al., Targeting alpha-synuclein for PD Therapeutics: A Pursuit on All Fronts. Biomolecules, 2020. 10(3)). Various strategies targeting Lewy pathology are currently under investigation, including stabilizing alpha-syn physiological conformation, reducing alpha-syn expression, inhibiting alpha-syn aggregation, and increasing alpha-syn clearance. Furthermore, Lewy pathology is not limited to dopaminergic circuits but extends to other brain regions via intercellular propagation of alpha-syn aggregates, which may be one of the mechanisms leading to nonmotor PD symptoms. Therefore, theoretically, therapeutic treatments that address alpha-syn aggregation may benefit both motor and nonmotor function in PD patients.

[0009] Among currently available treatments, L-DOPA (levodopa) is effective in treating motor symptoms in some patients with Parkinson's disease (PD). However, L-DOPA has limited efficacy in treating non-motor symptoms or in patients with advanced PD. Furthermore, serious side effects have been shown when L-DOPA is taken at high doses (Zahoor, I., A. Shafi, and E. Haq, Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects, TB Stoker and JC Greenland eds. 2018: Brisbane (AU)). Other treatments (such as deep brain stimulation) only improve symptoms but do not stop 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, TB Stoker and JC Greenland eds. 2018: Brisbane (AU)). Therefore, there is an urgent need for better treatments for PD.

[0010] Currently, biological PD therapies under investigation can be divided into three types. 1) Repairing neurotransmitter imbalances by regulating neuronal signaling. For example, gene therapy that delivers AADC (an enzyme involved in dopamine synthesis) via viral vectors (e.g., AAV2-AADC) (CN 107106689A, in...). https: / / clinicaltrials.gov / ct2 / show The related clinical trials ( / NCT03065192?term=AADC&cond=PD&draw=2&rank=8) and lentivirus-based gene therapy (ProSavin) have shown promising results in early clinical trials, in which patients experienced slight improvements in motor function. 2) Improving neuronal survival by expressing neurotrophic factors and regenerative factors. For example, neurotrophic factor (GDNF) (in the clinical trial at https: / / clinicaltrials.gov / ct2 / show / NCT04167540?term=GDNF&cond=PD&draw=2&rank=1) and neuronal rank protein (NRTN) (in the clinical trial at https: / / clinicaltrials.gov / ct2 / show / NCT00985517?term=Neurturin&draw=2&rank=1) benefit the survival of neurons in the dopaminergic brain. 3) By targeting disease-related genes or gene mutations, such as α-synuclein (SNCA), glucocerebrosidase (GBA1) (in a clinical trial at https: / / clinicaltrials.gov / ct2 / show / NCT04127578?term=GBA1&cond=Parkinson+ Disease&draw=2&rank=1), and leucine-rich repeat kinase 2 (LRRK2). Several gene therapies based on antisense oligonucleotides (ASO), CRISPR, and AAV, aimed at editing genes and / or regulating gene expression, are currently in early-stage clinical trials.

[0011] Therefore, there remains an unmet need for PD treatments that can restore dopamine levels in the striatum, clear α-synuclein, and maintain dopaminergic transmission in the patient's brain.

[0012] AAV-mediated gene replacement therapy may also have the potential to treat other neurodegenerative disorders or neurodevelopmental diseases, including but not limited to multiple system atrophy (MSA), Lewy body dementia (LBD), Alzheimer's disease with amygdala-restricted Lewy body (AD / ALB), Gaucher disease, AADC deficiency, etc.

[0013] MSA is a rapidly progressive, sporadic, adult-onset neurodegenerative disorder. Clinical symptoms of MSA include Parkinson's syndrome (bradykinesia, rigidity, and postural instability, similar to Parkinson's disease); cerebellar syndrome; and autonomic failure due to brainstem degeneration. Based on the primary symptoms, two clinical subtypes, MSA-P (Parkinson's syndrome) and MSA-C (cerebellar syndrome), are defined (Monzio Compagnoni, G. and A. Di Fonzo, Understanding the pathogenesis of multiple system atrophy: state of the art and future perspectives. Acta Neuropathol Commun, 2019. 7(1): p. 113). The main pathological feature of MSA is widespread loss of neurons and oligodendrocytes and glial proliferation in multiple brain regions.

[0014] The etiology of MSA is unclear, but the main pathogenic mechanism is the presence of argyrophilic glial cytoplasmic inclusions (GCIs) (primarily in oligodendrocytes). GCIs are mainly composed of loosely packed filaments of α-synuclein, phosphorylated and ubiquitinated at residue Ser129. Based on its pathology and symptoms, currently available MSA treatments and therapies under development are designed to target the loss of dopamine transmission, neuronal loss, and α-synuclein aggregation, which is quite similar to the approaches used to treat PD.

[0015] Epidemiological studies of MSA have shown a prevalence ranging from 3.4 to 4.9 per 100,000 people, increasing to 7.8 per 100,000 people in those over 40 years of age. Currently available treatments only alleviate symptoms. For example, dopamine derivatives (e.g., Medopa or Duopa) are used to reduce PD-like symptoms. However, these drugs are less effective in treating MSA-P patients than they are in treating PD patients. Furthermore, some MSA-P patients respond only to higher doses of these drugs, and they often become less effective over time. Therefore, enhancing dopamine production via AADC delivery may not be as beneficial to MSA patients as it has been shown in PD patients. Additional disease-modifying therapies that can delay disease progression are needed. Such therapies may potentially target three aspects of the MSA pathogenesis: (1) α-synuclein aggregation, (2) cellular dysfunction and loss, and (3) neuroinflammation. Several active clinical-stage candidates exist, including immunogenic peptides of α-synuclein (PD01 and PD03, NCT02270489), α-synuclein aggregation inhibitors (Anle138b, NCT04208152; ATH434, NCT05109091), antisense oligonucleotides (ASO) targeting α-synuclein (BIIB101, NCT04165486), and neuroprotective factors like GDNF delivered by AAV (AAV2-GDNF, NCT04680065). All of the above-mentioned therapies under development target a single pathology or symptom. For complex ND like MSA, simultaneously targeting multiple pathogenic pathologies may potentially provide better therapeutic effects or even disease-modifying effects, thereby not only alleviating symptoms but also slowing disease progression.

[0016] Gaucher disease (GD) is an autosomal recessive genetic disorder primarily caused by loss-of-function mutations in the GBA1 gene. GD is classified into three types based on the absence (type 1) or presence and severity (nGD, type 2, and type 3) of central nervous system (CNS) damage (Bennett, LL, and C. Fellner, Pharmacotherapy of Gaucher Disease: Current and Future Options. PT, 2018. 43(5): 274–309). Enzyme replacement therapies have been developed that are effective only for type 1 GD because the recombinant enzyme cannot cross the blood-brain barrier (BBB). CNS-loving AAVs expressing GBA1 are a promising approach for treating nGD. A clinical trial of an active agent sponsored by Prevail Therapeutics (NCT04411654) delivered a codon-optimized GBA1 transgene via intracerebellomedullary cistern (ICM) injection of rAAV9.

[0017] AADC deficiency (AADCD) is a rare autosomal recessive genetic disorder primarily caused by loss-of-function mutations in the AADC gene. Abnormalities in the AADC protein lead to a severe deficiency of some of the most important neurotransmitters for brain function, such as serotonin, dopamine, norepinephrine, and epinephrine. rAAV9, expressing functional wild-type AADC, has been developed for the treatment of this disease via a PTC therapeutic agent called Upstaza™ (eladocagene exuparvovec), which has been approved by the EMEA (European Agency for the Evaluation of Medicinal Products) and the UK for the treatment of patients older than 18 months.

[0018] Based on the above evidence, disease ND is a complex, multifaceted, and debilitating disease with a significant impact on global public health. There is an urgent need to develop innovative treatments that modify the disease to slow its progression and alleviate patient suffering. Summary of the Invention

[0019] To develop better therapies for treating various diseases (ND), the inventors have 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, the inventors have developed rAAV vectors that tandemly express two target genes (GOIs) to develop therapies that synergistically target multiple disease pathologies for greater therapeutic efficacy.

[0020] Therefore, in a first aspect, this application provides a nucleotide sequence encoding one of the GOIs listed above (particularly AADC, GBA1, CDNF, and GDNF), wherein the nucleotide sequence is a codon-optimized sequence compared to the wild-type sequence of the GOI.

[0021] In a second aspect, this application provides a nucleic acid construct comprising a nucleotide sequence of the first aspect operatively linked to a promoter.

[0022] In a third aspect, this application provides a nucleic acid construct comprising two nucleotide sequences operatively linked to a promoter, wherein the first and second nucleotide sequences encode two of (a), (b), and (c): (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 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 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, wherein the nucleotide region encoding the signal peptide remains unchanged. In another embodiment, the nucleotide sequence encoding CDNF or GDNF has a reduced number of CpG sites compared to the wild-type coding sequence. In another embodiment, the nucleotide sequence encoding CDNF or GDNF does not contain any CpG islands. In a specific implementation, the nucleotide sequence encoding CDNF or GDNF is the nucleotide sequence of the first aspect.

[0023] In a preferred embodiment, the nucleic acid construct of the third aspect includes a linker sequence between the GOI of the first nucleotide sequence and the second nucleotide sequence. In a specific embodiment, the linker sequence is a 2A peptide. In a specific embodiment, the linker sequence is an IRES.

[0024] In a fourth aspect, this application relates to an 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 vector is AAV9.

[0025] In a fifth aspect, this application relates to a composition, such as a pharmaceutical composition, comprising the rAAV carrier of the fourth aspect and a pharmaceutically acceptable excipient.

[0026] In a sixth aspect, this application relates to a viral particle comprising the rAAV vector described in the fourth aspect.

[0027] In a seventh aspect, the present invention application relates to a method for treating or preventing neurodegenerative disorders (NDs) (e.g., Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD), and other protein diseases) in a subject of need, the method comprising administering the rAAV carrier of the fourth aspect to the subject.

[0028] In an eighth aspect, this invention application relates to the use of the rAAV carrier described in the fourth aspect in the treatment or prevention of neurodegenerative disorders or in the manufacture of medicaments for the treatment or prevention of neurodegenerative disorders, wherein the neurodegenerative disorders are particularly Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD), and other protein disorders.

[0029] The nucleic acid constructs of this invention comprise a promoter, a linker, and a combination of one or two therapeutic protein-coding sequences specifically optimized for rAAV vectors to provide the desired expression levels of the therapeutic protein (e.g., one or two of AADC, GBA1, and CDNF / GDNF). Particularly for rAAV vectors containing two GOIs, such rAAV vectors of this invention can achieve co-expression of two therapeutic proteins at desired levels, which will work synergistically as novel therapies to more effectively treat anticipated neurodegenerative diseases, thereby achieving better therapeutic outcomes. Attached Figure Description

[0030] Figure 1 A schematic diagram of an exemplary construct containing an AADC wild-type coding sequence and a GBA1 wild-type coding sequence linked by a P2A peptide is shown.

[0031] Figure 2 A- Figure 2 B shows representative images of Western blot (WB) results of AADC and GBA1 protein expression, alone or in combination, in HEK293 cells. (A) Cells were transfected with constructs containing the coding sequences of AADC, GBA1, or AADC-P2A-GBA1 (the AADC coding sequence linked to the GBA1 coding sequence by a P2A linker). (B) Cells were transfected with constructs containing the AADC and GBA1 coding sequences in different sequences. GpA: GBA1-P2A-AADC; ApG: AADC-P2A-GBA1.

[0032] Figure 3Representative WB images are shown to evaluate the cutting efficiency of different connectors used when expressing both AADC and GBA in the same construct.

[0033] Figure 4 A schematic diagram is shown of a construct containing wild-type AADC (AADC-WT) and optimized GBA1 via a P2A linker, or optimized AADC and wild-type GBA1 via a P2A linker, under the control of a CBh heterozygous promoter consisting of a CMV enhancer, a chicken-β-actin promoter, and a heterozygous intron.

[0034] Figure 5 The expression levels of AADC protein in different candidate constructs in HEK293 cells are shown, as determined by ELISA.

[0035] Figure 6 The expression levels of GBA1 protein in different candidate constructs in HEK293 cells are shown, as determined by ELISA.

[0036] Figure 7 The expression levels of AADC protein in different candidate constructs in HEK293 cells are shown, as determined by Western blotting.

[0037] Figure 8 As shown Figure 7 The normalized AADC protein expression levels shown are expressed as the mean ± SEM from three independent Western blot (WB) experiments.

[0038] Figure 9 The expression levels of GBA1 protein in HEK293 cells by different candidate constructs are shown, as determined by Western blotting.

[0039] Figure 10 As shown Figure 9 The normalized GBA1 protein expression levels shown are expressed as the mean ± SEM from three independent Western blot (WB) experiments.

[0040] Figure 11 The catalytic activity of AADC in HEK293 cells transfected with different candidate constructs is shown.

[0041] Figure 12 The catalytic activity of the GBA1 protein expressed in HEK293 cells transfected with different candidate constructs is shown.

[0042] Figure 13 The expression levels of AADC and GBA1 proteins in HEK293 cells transfected with different combinations of constructs are shown.

[0043] Figure 14 The relative activities of GBA1 protein in HEK293 cells transfected with different combinations of constructs are shown.

[0044] Figure 15 The expression of AADC and GBA1 proteins in HEK293 cells transduced with different candidate rAAV vectors is shown.

[0045] Figure 16 Representative Western blot images show the expression levels of AADC protein in HEK293 cells transfected with different candidate constructs.

[0046] Figure 17 A- Figure 17 B shows a schematic diagram of a wild-type AADC (AADC-WT) connected to an optimized CDNF (A) or GDNF (B) via a P2A connector under the control of the CBh promoter.

[0047] Figure 18 A- Figure 18 B shows the expression levels of CDNF protein in cell lysates and supernatants of HEK293 cells transfected with different candidate constructs, as determined by Western blotting. (A) Western blotting image; (B) Normalized data.

[0048] Figure 19 A- Figure 19 B shows the expression levels of CDNF protein in cell lysates and supernatants of U87 cells transfected with different candidate constructs, as determined by Western blotting (WB). (A) WB image; (B) Normalized data.

[0049] Figure 20 A- Figure 20 B shows the expression levels of CDNF protein in cell lysates and supernatants of HEK293 cells (A) or U87 cells (B) transfected with different candidate constructs, as determined by ELISA.

[0050] Figure 21 The protective effect of CDNF protein in the supernatant of HEK293 cells transfected with different candidate constructs treated with 1.5 mM MPP+ or 40 nM rotenone is shown, as determined by CCK-8 assay.

[0051] Figure 22 The expression levels of GDNF protein in cell lysates and supernatants of HEK293 cells transfected with different candidate constructs are shown, as determined by Western blotting.

[0052] Figure 23The expression levels of GDNF protein in cell lysates and supernatants of HEK293 cells transfected with different candidate constructs are shown, as determined by ELISA.

[0053] Figure 24 The protective effect of GDNF protein in the supernatant of HEK293 cells transfected with different candidate constructs treated with 1.5 mM MPP+ is shown, as determined by CCK-8 assay.

[0054] Figure 25 The expression levels of CDNF (left panel) or GDNF (right panel) proteins in the supernatant of HEK293 cells transfected with the indicated constructs are shown.

[0055] Figure 26 The activity of AADC protein in cell lysates of HEK293 cells transfected with the indicated candidate construct is shown.

[0056] Figure 27 The protective effect of CDNF in the supernatant of HEK293 cells transfected with different candidate constructs treated with 1.5 mM MPP+ or 40 nM rotenone is shown, as determined by CCK-8 and LDH assays.

[0057] Figure 28 The protective effect of GDNF protein in the supernatant of HEK293 cells transfected with different candidate constructs treated with 1.5 mM MPP+ or 40 nM rotenone is shown, as determined by CCK-8 and LDH assays.

[0058] Figure 29 The expression levels of AADC and NTF (CDNF or GDNF) proteins in cell lysates of U87-AAVR cells transduced with the indicated rAAV9 vector are shown, as well as the NTF level in the cell supernatant.

[0059] Figure 30 The relative activities of AADC protein from HEK293 and U87-AAVR cells transduced with the indicated rAAV9 vector are shown.

[0060] Figure 31 The number of contralateral rotations obtained from study subjects in a mouse 6-OHDA-induced PD model after administration of EF1α-A11-GDNF MN and baseline rAAV is shown.

[0061] Figure 32 The net number of rotations obtained from study subjects in a mouse 6-OHDA-induced PD model after administration of EF1α-A11-GDNF MN and baseline rAAV is shown.

[0062] Figure 33 A- Figure 33 B shows a schematic diagram of an optimized GBA1 construct linked to an 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.

[0063] Figure 34 The following figures show the movement speed and distance measurements in PD mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid (MPTP / P) after administration of CBh-A11G11 and baseline A10-vy rAAV, respectively. Before: Data collected before MPTP modeling; After MPTP / P: Data collected after MPTP / P induction and before AAV treatment; After AAV: Data collected after AAV treatment.

[0064] Figure 35 The upright behavior (standing by hind limbs) obtained from subjects in a PD model of α-synuclein A53T transgenic mice after administration of EtI7-A11G11 (EtI7) and CBh-A11G11 (CBh)rAAV is shown. Low: 4E+9 vg, High: 4E+10 vg.

[0065] Figure 36 Representative Western blotting images of AADC and GC enzyme expression in tissue samples collected from subjects in a PD model of α-synuclein A53T transgenic mice after administration of EtI7-A11G11 and CBh-A11G11 rAAV are shown. Low: 4E+9 vg, High: 4E+10 vg.

[0066] Figure 37 A- Figure 37 B shows representative images (A) and normalized data (B) of WB in tissue samples collected from a transgenic α-synuclein A53T mouse PD model after administration of EtI7-A11G11 and CBh-A11G11 rAAV. Low: 4E+9 vg, High: 4E+10 vg.

[0067] Figure 38 A- Figure 38 B shows a schematic diagram of a construct containing different promoters and codon-optimized GBA1.

[0068] Figure 39 The survival curves (vitality) of CBE-induced GD mice after administration of G10-p, CAG-G11 and EF1α-G11 rAAV are shown.

[0069] Figure 40 A- Figure 40 B shows the distance traveled (A) in the open field experiment and the drop latency (B) in the rotundus experiment in CBE-induced GD mice after administration of different doses of G10-p, CAG-G11 and EF1α-G11 rAAV.

[0070] Figure 41 The activity of GC enzymes in striatal samples collected from CBE-induced GD mice after administration of different doses of G10-p, CAG-G11 and EF1α-G11 rAAV is shown.

[0071] Figure 42 Representative Western blot images are shown, illustrating the protein expression of AADC and CDNF in cell lysates of HEK293 cells transfected with the indicated candidate construct, and the level of CDNF protein in the cell supernatant.

[0072] Figure 43 The net number of rotations (amphetamine-induced ipsilateral rotations) in 6-OHDA-induced PD model mice after administration of the indicated doses of CAG-A11-GDNF MN, EF1α-A11-GDNF MN and baseline A10-vyrAAV is shown.

[0073] Figure 44 A- Figure 44 B shows the protein expression levels of AADC (A) and GDNF (B) in striatal samples collected from 6-OHDA-induced PD model mice after administration of indicated amounts of EF1α-A11-GDNF MN and baseline A10-vy rAAV, as determined by ELISA.

[0074] Figure 45 Representative images of tyrosine hydroxylase (TH) levels in substantia nigra samples collected from 6-OHDA-induced PD model mice after administration of indicated amounts of EF1α-A11-GDNF MN and baseline A10-vy rAAV, as determined by immunohistochemistry (IHC), are shown. Blank: Healthy controls.

[0075] Figure 46 The number of Δ rotations (L-DOPA-induced contralateral rotations) obtained from 6-OHDA-induced PD model rats after administration of the indicated dose of EF1α-A11-GDNF MN and baseline A10-vy rAAV is shown. Blank represents healthy controls.

[0076] Figure 47Representative Western blot images are shown, illustrating the protein expression of GC enzymes and GDNF in cell lysates of U87-MG-AAVR cells treated with different candidate rAAV vectors, as well as the level of GDNF protein in the cell supernatant.

[0077] Figure 48 Representative Western blot images showing the protein levels of high molecular weight α-syn (HMW) in SH-SY5Y-AAVR-A53T cells treated with different candidate rAAV vectors are presented.

[0078] Figure 49 Representative Western blot images are shown, illustrating the protein expression of GC enzymes and GDNF in cell lysates of HEK293 cells transfected with different candidate constructs, as well as the level of GDNF protein in the cell supernatant. Detailed Implementation

[0079] Unless explicitly defined elsewhere in this document, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0080] As used herein (including the appended claims), unless the context clearly indicates otherwise, singular terms such as “a”, “an” and “the” include their corresponding plural indicators.

[0081] In the context of this disclosure, unless otherwise indicated, the word “comprise” and its variations such as “comprises” and “comprising” will be understood to imply the inclusion of said elements, such as amino acid sequences, nucleotide sequences, properties, steps, or groups thereof, but not to exclude any other elements, such as amino acid sequences, nucleotide sequences, properties, and steps. When used herein, the term “comprise” or any variation thereof may be replaced by the terms “containing,” “including,” or sometimes “having,” or equivalent variations thereof. In some embodiments, the word “comprise” also includes the case of “consisting of.”

[0082] As used herein, the term "gene" refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) that encodes RNA transcripts and its corresponding nucleotide sequence. As used herein, the term "genomic DNA" may include intermediate non-coding regions and regulatory regions, and may include both 5' and 3' ends. In some cases, the term includes transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region will contain an "open reading frame" encoding a polypeptide. In some cases, "gene" contains only the coding sequence necessary to encode a polypeptide (e.g., "open reading frame" or "coding region"). In some cases, the term "gene" includes not only transcribed sequences but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or a natural gene. A gene may refer to an "exogenous gene" or a non-natural gene. A non-natural gene may refer to a gene that is not normally found in a host organism but is introduced into the host organism through gene transfer. Non-natural genes can also refer to genes that are not located at their natural position in the genome of an organism. Non-natural genes can also refer to naturally occurring nucleic acids containing mutations, insertions, and / or deletions (e.g., non-natural sequences), such as codon-optimized nucleotide sequences. In the context of this application, unless otherwise indicated, “GOI” specifically refers to a CDS region, i.e., a sequence encoding amino acids in a protein.

[0083] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably herein and refer to aggregates of nucleotides of any length. Polynucleotides can be exogenous or endogenous. Polynucleotides can exist in cell-free environments. Polynucleotides can be genes or segments thereof. Polynucleotides can be DNA. Polynucleotides can be RNA. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Polynucleotides can contain one or more analogs (e.g., modified backbones, sugars, or nucleobases).

[0084] As used herein, a “cassette” or “expression cassette” refers to a DNA component contained in a vector (e.g., a plasmid vector or a viral vector) and consisting of one or more gene components under the control of regulatory sequences to be expressed in host cells transduced by the vector.

[0085] As used in this article, "operably linked" is used to describe a way in which two or more components (particularly nucleotide sequences) are linked such that each component can perform its specified function.

[0086] "AAV" refers to adeno-associated virus.

[0087] "AADC" refers to aromatic L-amino acid decarboxylase. AADC is also known as DOPA decarboxylase (DDC).

[0088] "GBA1" refers to the β-glucosidase-linked glucocerebroside lipase that cleaves glucocerebroside. In some cases, "GBA1" may be used interchangeably with "GBA", "GCB" or "GLUC".

[0089] "NTF" refers to neurotrophic factors, a group of supporting proteins that promote neuronal development and maintenance. Both CDNF and GDNF belong to the neurotrophic factor family.

[0090] "CpG islands" refer to regions in the genome rich in CpG sites. "CpG sites" are two consecutive nucleotides consisting of cytosine (C) and guanine (G) in the 5' to 3' direction.

[0091] "2A peptide" refers to a group of short (18-22 amino acids) self-cleaving peptides derived from viruses. During translation, 2A peptides undergo ribosome jumping, causing them to separate from downstream proteins at the 2A sequence terminus.

[0092] “IRES” refers to the internal ribosome entry site.

[0093] "Protein disease" refers to a neurodegenerative disorder in which the accumulation of structurally abnormal proteins (such as α-synuclein) leads to the formation of aggregates or inclusions in the axons or oligodendrocytes of neurons.

[0094] In the context of this application, "subject" refers to an animal, preferably a mammal, such as a primate, like a cynomolgus monkey, and preferably a higher primate, such as a human. Unless otherwise stated, in the context of this application, the term "subject" is used interchangeably with the terms "patient" or "individual".

[0095] AADC

[0096] This disclosure provides rAAV vectors that deliver either a single aromatic L-amino acid decarboxylase (AADC) gene or a combination with another target gene, such as GBA1, CDNF, or GDNF. The rAAV vectors delivering AADC can be used to treat disease-related disorders, such as PD or AADCD.

[0097] The biosynthetic pathway of dopamine requires both tyrosine hydroxylase (TH) to convert tyrosine into 1-3,4-hydroxyphenylalanine (L-Dopa) and aromatic L-amino acid decarboxylase (AADC) to decarboxylate L-Dopa to produce dopamine.

[0098] It is known that AADC levels decline with the progression of PD, and that L-DOPA administered to patients cannot be effectively converted to dopamine at the axonal terminals in the striatum. This necessitates more frequent dosing and increased doses of L-DOPA to achieve adequate clinical response. However, increasing the dose of L-DOPA may lead to undesirable side effects such as L-Dopa-induced motor dyskinesia (LID).

[0099] The potential of putaminal AADC delivery of AAV in the treatment of Parkinson's disease has been reported and clinically evaluated (Bankiewicz, 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). Considering that restoring AADC levels will "rescue" and enhance the therapeutic effect of L-DOPA, and that dopamine levels are the most critical factor in restoring motor function, including AADC in candidate constructs to salvage dopamine levels would be particularly beneficial for patients with AADC deficiency.

[0100] AADC deficiency is caused by loss-of-function mutations in the AADC gene. The rAAV described in this invention comprises AADC as a sole GOI or a combination of AADC and another GOI to compensate for the molecular defects leading to the disease.

[0101] In a preferred embodiment, the nucleotide sequence encoding AADC can be optimized for expression in the rAAV construct. Optimization can be codon optimization.

[0102] In one embodiment, the rAAV of this invention comprises a nucleotide sequence encoding an AADC, wherein the nucleotide sequence encoding the AADC comprises or is composed of any one of SEQ ID NO: 1-9, 11, and 46, preferably any one of SEQ ID NO: 1-9 and 46 (optimized sequence), and more preferably a nucleotide sequence or a combination thereof as shown in SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 46. In some embodiments, the rAAV comprising a nucleotide sequence encoding an AADC as a unique GOI can be used to treat PD or AADCD.

[0103] In some embodiments, the rAAV of this application comprises a combined construct containing both AADC and GBA1, for example, for treating PD. In one embodiment, the combined construct comprises a coding sequence for AADC and a coding sequence for GBA1. In some embodiments, the coding sequence for GBA1 is arranged upstream of the 5' of the coding sequence for AADC. In a more preferred embodiment, the coding sequence for AADC is arranged upstream of the 5' of the coding sequence for GBA1. Preferably, the two coding sequences are arranged within the same frame and are under the control of the same promoter. Preferably, the nucleotide sequence encoding GBA1 comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO: 12-20, 45, or 47. Preferably, the nucleotide sequence encoding AADC comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO: 1-9 and 46.

[0104] In one embodiment, the combined AADC and GBA1 construct comprises the coding sequence of AADC as shown in SEQ ID NO: 46 and the coding sequence of GBA1 as shown in SEQ ID NO: 45. In another embodiment, the combined AADC and GBA1 construct from 5' to 3' comprises: a promoter sequence, which is a truncated variant of the EF1α promoter, such as the sequence (EFIt7) shown in SEQ ID NO: 56; the coding sequence of AADC as shown in SEQ ID NO: 46; the coding sequence of GBA1 as shown in SEQ ID NO: 45; and a polyA sequence, such as the hGH polyA sequence. In yet another embodiment, the combined AADC and GBA1 construct from 5' to 3' comprises: a promoter sequence (CBh promoter) as shown in SEQ ID NO: 60; the coding sequence of AADC as shown in SEQ ID NO: 46; the coding sequence of GBA1 as shown in SEQ ID NO: 45; and a polyA sequence, such as the hGH polyA sequence.

[0105] In some embodiments, the rAAV of this invention comprises a combined construct containing both an AADC and an NTF selected from GDNF or CDNF, for example, for treating AADCD. In one embodiment, the combined construct comprises a coding sequence for an AADC and a coding sequence for GDNF or CDNF. In some embodiments, the coding sequence for GDNF or CDNF is arranged upstream of the 5' of the coding sequence for the AADC. In a more preferred embodiment, the coding sequence for the AADC is arranged upstream of the 5' of the coding sequence for GDNF or CDNF. Preferably, the two coding sequences are arranged within the same frame and are under the control of the same promoter. Preferably, the nucleotide sequence encoding the AADC comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO: 1-9 and 46. Preferably, the nucleotide sequence encoding the GDNF comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO: 27-29. Preferably, the nucleotide sequence encoding the CDNF comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO: 23-25.

[0106] In some embodiments, the rAAV of this application comprises a combined construct containing a coding sequence of AADC and a coding sequence of CDNF, for example, for treating AADCD. In one specific embodiment, the combined construct of AADC and CDNF comprises a coding sequence of AADC as shown in SEQ ID NO: 3 or SEQ ID NO: 46 and a coding sequence of CDNF (CDNF-MN) as shown in SEQ ID NO: 25. In one specific embodiment, the combined construct of AADC and CDNF from 5' to 3' comprises: a CAG promoter sequence (CAG) as shown in SEQ ID NO: 58; a coding sequence of AADC as shown in SEQ ID NO: 3; and a coding sequence of CDNF (CDNF-MN) as shown in SEQ ID NO: 25. In one specific embodiment, the combined AADC and CDNF construct from 5' to 3' comprises: the EF1α promoter sequence (EF1α) as shown in SEQ ID NO: 57; the coding sequence of AADC as shown in SEQ ID NO: 46; and the coding sequence of CDNF (CDNF-MN) as shown in SEQ ID NO: 25. In another specific embodiment, the combined AADC and CDNF construct from 5' to 3' comprises: the EF1α promoter sequence (EF1α) as shown in SEQ ID NO: 57; the coding sequence of CDNF (CDNF-MN) as shown in SEQ ID NO: 25; and the coding sequence of AADC as shown in SEQ ID NO: 46.

[0107] In some preferred embodiments, the rAAV of this invention comprises a combined construct containing a coding sequence of AADC and a coding sequence of GDNF, for example, for treating AADCD. In one specific embodiment, the combined construct of AADC and GDNF comprises a coding sequence of AADC as shown in SEQ ID NO: 46 and a coding sequence of GDNF (GDNF-MN) as shown in SEQ ID NO: 29. In one specific embodiment, the combined construct of AADC and GDNF comprises, from 5' to 3': an EF1α promoter sequence (EF1α) as shown in SEQ ID NO: 57; a coding sequence of AADC as shown in SEQ ID NO: 46; and a coding sequence of GDNF (GDNF-MN) as shown in SEQ ID NO: 29.

[0108] GBA1

[0109] This disclosure provides rAAV vectors that deliver either the glucocerebrosidase 1 (GBA1) gene alone or in combination with another gene (e.g., ADCC, CDNF, or GDNF). rAAV vectors delivering GBA1 can be used to treat disease-related disorders (NDs), including Gaucher disease (GD), Parkinson's disease (PD), and multiple system atrophy (MSA). For example, rAAV vectors delivering GBA1 alone can be used to treat Gaucher disease type 2 and 3 (GD) or PD. For example, rAAV vectors delivering both GBA1 and an NTF (such as GDNF) can be used to treat MSA.

[0110] In a preferred embodiment, the nucleotide sequence encoding GBA1 can be optimized for expression via the rAAV construct. Optimization can be codon optimization.

[0111] In one embodiment, the rAAV of this application comprises a nucleotide sequence encoding GBA1, wherein the nucleotide sequence encoding GBA1 comprises or is composed of any one of SEQ ID NO: 12-20, 22, 45 and 47, preferably any one of SEQ ID NO: 12-20, 45 or 47 (codon-optimized sequence), more preferably a nucleotide sequence or a combination thereof as shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 45 or SEQ ID NO: 47.

[0112] In some embodiments, the rAAV of this application comprises a nucleotide sequence encoding GBA1, for example, for the treatment of GD or PD. Preferably, the nucleotide sequence encoding GBA1 comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO: 12-20, 45, or 47. In one specific embodiment, the rAAV comprises the coding sequence for GBA1 as shown in SEQ ID NO: 45. In one specific embodiment, the rAAV comprises a construct from 5' to 3' comprising: a CAG promoter sequence (CAG) as shown in SEQ ID NO: 58; a coding sequence for GBA1 as shown in SEQ ID NO: 45; and a polyA sequence, such as an hGH polyA sequence. In one specific embodiment, the rAAV comprises a construct from 5' to 3' comprising: an EF1α promoter sequence (EF1α) as shown in SEQ ID NO: 57; a coding sequence for GBA1 as shown in SEQ ID NO: 45; optionally, a WPRE; and a polyA sequence, such as an hGH polyA sequence.

[0113] In some embodiments, the rAAV of this invention comprises a combined construct containing both GBA1 and an NTF selected from GDNF or CDNF, for example, for the treatment of MSA. In some embodiments, the coding sequence of GBA1 is arranged 5' upstream of the coding sequence of the NTF. In some embodiments, the coding sequence of the NTF is arranged 5' upstream of the coding sequence of GBA1. Preferably, the two coding sequences are arranged within a frame and are under the control of the same promoter.

[0114] In some embodiments, the rAAV of this application comprises a combined construct containing both GBA1 and GDNF, for example, for the treatment of MSA. Preferably, the nucleotide sequence encoding GBA1 comprises or consists of the nucleotide sequences shown in any one of SEQ ID NO: 12-20, 45, or 47. Preferably, the nucleotide sequence encoding GDNF comprises or consists of the nucleotide sequences shown in any one of SEQ ID NO: 27-29. In a specific embodiment, the combined construct of GBA1 and GDNF comprises the coding sequence for GBA1 as shown in SEQ ID NO: 45 or SEQ ID NO: 47 and the coding sequence for GDNF as shown in any one of SEQ ID NO: 27-29. In one specific embodiment, the combined construct of GBA1 and GDNF from 5' to 3' includes: a CAG promoter sequence (CAG) as shown in SEQ ID NO: 58 or an MBP promoter sequence (MBP) as shown in SEQ ID NO: 59; a coding sequence of GBA1 as shown in SEQ ID NO: 45 or SEQ ID NO: 47; a coding sequence of GDNF (GDNF-MN) as shown in SEQ ID NO: 29; optionally WPRE; and a polyA sequence, such as SV40 polyA or bGH polyA. In one specific embodiment, the combined construct of GBA1 and GDNF from 5' to 3' includes: a CAG promoter sequence (CAG) as shown in SEQ ID NO: 58 or an MBP promoter sequence (MBP) as shown in SEQ ID NO: 59; a coding sequence of GDNF (GDNF-MN) as shown in SEQ ID NO: 29; a coding sequence of GBA1 as shown in SEQ ID NO: 45 or SEQ ID NO: 47; optionally WPRE; and a polyA sequence, such as SV40 polyA or bGH polyA.

[0115] Neurotrophic factors: CDNF and GDNF

[0116] This disclosure provides rAAV vectors that deliver a single neurotrophic factor (specifically CDNF or GDNF) or a combination thereof with another gene (e.g., ADCC or GBA1). rAAV vectors delivering neurotrophic factors can be used to treat neurotrophic disorders (ND).

[0117] The neuroprotective effects of neurotrophic factors have been well established. Both CDNF and GDNF proteins have been studied within the framework of Parkinson's disease (Nasrolahi, A. et al., Neurotrophic factors hold promise for the future of Parkinson's disease treatment: is there a light at the end of the tunnel? Rev Neurosci, 2018. 29(5): 475-489).

[0118] In one implementation, AADC is co-expressed with CDNF or GDNF via an rAAV vector. This is the first example of AADC and neurotrophic factors being co-expressed on the same AAV vector and demonstrating significant efficacy in a mouse PD model.

[0119] In another implementation, GBA1 is co-expressed with GDNF or CDNF via an rAAV vector.

[0120] In a preferred embodiment, the nucleotide sequence encoding CDNF or GDNF can be optimized for expression via an rAAV construct. Optimization can be codon optimization. Codon optimization can be limited to the coding region of the mature protein of GDNF or CDNF without altering the nucleotide sequence of the signal peptide corresponding to CDNF or GDNF, or the nucleotide sequence corresponding to the propeptide GDNF. 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.

[0121] In one embodiment, the rAAV of this application comprises a nucleotide sequence encoding CDNF, wherein the nucleotide sequence encoding CDNF comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NO: 23-25.

[0122] In one embodiment, the rAAV of this application comprises a nucleotide sequence encoding GDNF, wherein the nucleotide sequence encoding GDNF comprises or consists of any of the nucleotide sequences shown in SEQ ID NO: 27-29.

[0123] Expression Box

[0124] The term “expression cassette” in this document refers to a DNA component contained in a vector (e.g., an rAAV vector) and consisting of one or more (especially one or two) GOIs selected from the AADC, GBA1, and NTF (CDNF or GDNF) genes under the control of regulatory sequences, which are to be expressed in host cells transduced by the vector.

[0125] In the context of this application, the term "combinatorial construct" refers to a construct containing two GOIs, particularly two GOIs selected from the AADC, GBA1, and NTF (CDNF or GDNF) genes. In a preferred embodiment, the two gene coding sequences in the combinatorial construct are controlled by the same promoter.

[0126] In one embodiment, the expression box of this invention is characterized by expressing a GOI, preferably expressing a codon-optimized GOI sequence, particularly those as described in this disclosure.

[0127] In one embodiment, the expression cassette of this invention is characterized by co-expressing two GOIs separated by a connector sequence. In another embodiment, one or both GOIs are expressed via codon-optimized sequences (particularly those described in this disclosure). For example, the two GOIs may be selected from combinations of: AADC + GBA1, AADC + CDNF, AADC + GDNF, GBA1 + GDNF, and GBA1 + CDNF.

[0128] By optimizing the cDNA sequences (codons) of the AADC, GBA1, CDNF, and GDNF genes, their regulatory sequences, and optionally adapters, the expression cassettes of this disclosure, inserted into an AAV vector, can achieve higher and more consistent protein expression or co-expression in neuronal cells, either in vitro or in vivo. For example, the expression cassettes of this disclosure have shown superior performance in the expression of one or more GOIs in human cell lines with neuronal identity (such as U87-MG or SH-SY5Y). For example, the expression cassettes of this disclosure have shown superior performance in the expression of one or more GOIs in neuronal cells in the striatum, substantia nigra, or other CNS regions in vivo.

[0129] In one embodiment, as an essential part of the expression cassette, this disclosure first provides a set of wild-type or codon-optimized nucleotide sequences encoding an AADC protein and a second protein selected from GBA1, CDNF, and GDNF proteins, specifically, human AADC protein having the amino acid sequence shown in SEQ ID NO: 31, human GBA1 protein having the amino acid sequence shown in SEQ ID NO: 32, human CDNF protein having the amino acid sequence shown in SEQ ID NO: 33, and human GDNF protein having the amino acid sequence shown in SEQ ID NO: 34.

[0130] In one embodiment, as an essential part of the expression cassette, this disclosure first provides a set of wild-type or codon-optimized nucleotide sequences encoding a GBA1 protein and a second protein selected from CDNF and GDNF proteins, specifically, a human GBA1 protein having the amino acid sequence shown in SEQ ID NO: 32, a human CDNF protein having the amino acid sequence shown in SEQ ID NO: 33, and a human GDNF protein having the amino acid sequence shown in SEQ ID NO: 34.

[0131] "Isolated nucleic acid" means DNA or RNA extracted from all or part of a polynucleotide, wherein the isolated polynucleotide is found in nature or linked to a polynucleotide not found in nature. Isolated nucleic acid molecules "containing" a specific nucleotide sequence may, in addition to the specified sequence, include operablely linked regulatory sequences that control the expression of the coding region of said nucleic acid sequence. Due to codon degeneracy, those skilled in the art will understand that any particular amino acid sequence can be encoded by several different nucleotide sequences.

[0132] The term "codon-optimized coding sequence" as used herein refers to a nucleotide sequence encoding a protein (such as AADC, GBA1, CDNF, or GDNF) modified from its wild-type coding sequence to adapt to codon bias. Optimization can be achieved by reducing sequence complexity, adjusting GC content, adjusting codon usage, and / or avoiding rare codons. Codon-optimized coding sequences typically exhibit increased translation efficiency of the target gene (GOI), leading to higher protein expression. Tools with embedding 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 of the AADC coding sequence of this application have a codon fitness index (CAI) greater than 0.8. CAI is a measure of codon bias. Those skilled in the art will understand that the actual efficiency of any sequence generated by running the algorithm still needs to be verified experimentally.

[0133] In a preferred embodiment, the codon-optimized coding sequence of the human AADC protein comprises or consists of nucleotide sequences selected from or composed of SEQ ID NO: 1-10 and 46; the codon-optimized coding sequence of the human GBA1 protein comprises or consists of nucleotide sequences selected from or composed of SEQ ID NO: 12-21, 45 and 47; the codon-optimized coding sequence of the human CDNF protein comprises or consists of nucleotide sequences selected from or composed of SEQ ID NO: 23-25; and the codon-optimized coding sequence of the human GDNF protein comprises or consists of nucleotide sequences selected from or composed of SEQ ID NO: 27-29.

[0134] In addition to the coding sequence, the expression cassette may also contain one or more regulatory sequences. The regulatory sequences may be selected from one or more of the following: promoters, enhancers, polyadenylation sequences, and translation termination signals. Specific combinations of the regulatory sequences in this disclosure can achieve unexpected effects in improving the expression efficiency of the coding sequence.

[0135] A "promoter" is a DNA sequence that can initiate the transcription of downstream genes under the control of the promoter. Promoters include, but are not limited to, constitutive promoters, cell type-specific promoters, tissue-specific promoters, and developmental stage-specific promoters. Promoters can be naturally occurring promoters of genes, modified forms of naturally occurring promoters, or synthetic promoters.

[0136] In a preferred embodiment, the promoter of this disclosure may be a constitutive promoter. In a preferred embodiment, the promoter may be a CBh promoter, an EF1α promoter, a CAG promoter, an MBP promoter (myelin basic protein promoter), or a promoter derived therefrom.

[0137] An enhancer is a regulatory DNA sequence that, together with a promoter, can enhance the transcription of GOI in rAAV. In a preferred embodiment, the expression cassette of this application contains an enhancer. More preferably, the enhancer may be a CMV enhancer, for example, in the CBh promoter.

[0138] In some implementations, intron sequences that act as enhancers may be included. For example, an intron sequence derived from GOI may be included in the expression cassette.

[0139] In some cases, the promoter, along with the enhancer and / or intron sequence, is collectively referred to as a "promoter" or "promoter element". In a preferred embodiment, the promoter is a CBh promoter. In another preferred embodiment, the promoter consists of an EFS promoter and an intron sequence.

[0140] Preferably, the total length of the intron sequence is about or less than 200 bp, about or less than 250 bp, about or less than 300 bp, about or less than 350 bp, or about or less than 400 bp.

[0141] For example, the intron sequences in this disclosure are derived from the target gene. For example, the intron sequences consist of one or more segments derived from one or more intron regions of the target gene.

[0142] In a preferred embodiment, due to the limited packaging capacity of AAV, the length of the promoter or promoter / included element does not exceed 1000 bp, 900 bp, 850 bp, 800 bp, 700 bp, 600 bp, 500 bp, or 400 bp.

[0143] In some cases, when the intron sequence originates from the intron region of the target gene, the intron sequence can be inserted into the coding sequence (e.g., a codon-optimized coding sequence) at a position corresponding to its location in the gene in nature, for example, between two exons rather than at the 5' upstream of the coding sequence, and constitute a promoter / intron element.

[0144] The Kozak concordant sequence (Kozak sequence) (named after the scientist who discovered it) is a nucleic acid sequence motif present in the mRNA transcripts of most eukaryotes, serving as a protein translation initiation site. The Kozak sequence ensures that proteins are translated accurately and efficiently.

[0145] 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 an SV40 polyA, wherein the first and second coding sequences 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.

[0146] Connector sequence

[0147] In one aspect, this application provides a connector sequence that, when used to connect two coding sequences of this application to be co-expressed by an rAAV vector of this application, produces high efficiency and fidelity.

[0148] As examples of adapter sequences, sequences encoding 2A peptides (such as P2A, F2A, or E2A) or IRES (referred to herein as “ECMV IRES” in full-length form and “miniature IRES” in shorter truncated form) can be used to link the two coding sequences of this invention. The position of one or more GOIs relative to the adapter sequence can be modulated to achieve desired performance in protein expression and function. In a preferred embodiment, a P2A adapter sequence is used between the two GOIs in rAAV.

[0149] In specific embodiments, the adapter sequence of this invention application comprises or consists of nucleotide sequences as shown in SEQ ID NO: 35, 37, 39, 41, 43 or 44.

[0150] promoter

[0151] The rAAV vector of this invention may contain promoters conventionally used in rAAV vectors, including naturally occurring promoters, their variants, or hybrids.

[0152] In some implementations, the promoter may be a truncated variant of a wild-type promoter. For example, the promoter may be a truncated form of the EF1α promoter as shown in Table 2 of Example 6, having a nucleotide sequence as shown in any of SEQ ID NO: 50-56.

[0153] In some embodiments, the promoter of this application may also be used in conjunction with an enhancer, which may be natural or non-natural with respect 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 the promoter implies the inclusion of an enhancer, which will be understood by those skilled in the art. In one embodiment, the rAAV vector of this application comprises the CBh promoter, for example, the CBh promoter having the nucleotide sequence shown in SEQ ID NO: 60.

[0154] In another embodiment, to drive the co-expression of AADC and CDNF or AADC and GDNF, an EF1α promoter is used, for example, an EF1α promoter having the nucleotide sequence shown in SEQ ID NO: 57. In another embodiment, to drive the co-expression of AADC and GBA1, a truncated EF1α promoter is used, for example, a truncated EF1α promoter having the nucleotide sequence shown in any one of SEQ ID NO: 50-56. On the other hand, to drive the co-expression of GBA1 and GDNF, a CAG promoter having the nucleotide sequence shown in SEQ ID NO: 58 or an MBP promoter (or a truncated version thereof) having the nucleotide sequence shown in SEQ ID NO: 59 is used.

[0155] Poly-A signal

[0156] The rAAV vector of this invention application also contains a polyadenylation signal (polyA).

[0157] For example, the polyA sequences that can be used in this invention application include SV40 polyA, human growth hormone (hGH) polyA, and bovine growth hormone (bGH) polyA.

[0158] In one embodiment, the expression box of this disclosure contains hGH polyA. In one embodiment, the expression box of this disclosure contains hGH polyA when the promoter is an EF1α promoter or a variant thereof.

[0159] In some implementations, the marmot hepatitis virus post-transcriptional regulatory element (WPRE) is positioned downstream of the GOI and close to the polyadenylation signal.

[0160] AAV vector serotype

[0161] In a preferred embodiment, the rAAV of this invention is an AAV9 carrier.

[0162] Based on previous reports, intraplasmic injection of AAV9 showed superior dispersion at target sites compared to other serotypes, including AAV1, AAV2, AAV5, and AAV8 (Watakabe, A. et al., Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8, and 9 in marmoset, mouse, and macaquecerebral cortex. Neurosci Res, 2015. 93: 144–57). Since the human striatum is a relatively large brain region, molecular engineering of the wild-type AAV capsid is necessary to achieve broader coverage of genes delivered by AAVs.

[0163] The inventors have identified novel AAV serotypes that exhibit significantly improved tissue tropism compared to currently known AAVs targeting the capsid (the intended target region for AAV-based gene therapy in the treatment of PD). Therefore, in another preferred embodiment, the rAAV of this application utilizes the novel AAV capsid.

[0164] Exemplary builder

[0165] In one embodiment, this application provides an expression box, the expression box comprising, from 5' to 3':

[0166] (a) 5' ITR;

[0167] (b) Promoter;

[0168] (c) Optionally containing subsequences;

[0169] (d) The encoding sequence of GOI;

[0170] (e) Poly A; and

[0171] (f)3' ITR,

[0172] The GOI is selected from AADC, GBA1, CDNF or GDNF, and the encoded sequence is a codon-optimized sequence.

[0173] In another embodiment, this application provides an expression box, the expression box comprising from 5' to 3':

[0174] (g)5' ITR;

[0175] (h) promoter;

[0176] (i) Optionally containing subsequences;

[0177] (j) The encoding sequence of the first GOI;

[0178] (k) Connector sequence;

[0179] (l) The encoding sequence of the second GOI;

[0180] (m) Poly A; and

[0181] (n)3' ITR,

[0182] The first and second coding sequences are independently selected from wild-type coding sequences or codon-optimized sequences (e.g., those described in this disclosure) and encode any of the following combinations of two GOIs: AADC + GBA1, AADC + CDNF, AADC + GDNF, GBA1 + GDNF, and GBA1 + CDNF, wherein the two GOIs can be arranged in any order.

[0183] In the constructs indicated in the two paragraphs above, each element may be independently selected from those described in this disclosure. For example, the first nucleotide sequence may be an AADC coding sequence as shown in any one of SEQ ID NO: 1-9, 11, and 46; a GBA1 coding sequence as shown in any one of SEQ ID NO: 12-20, 22, 45, and 47; or a CDNF or GDNF coding sequence as shown in any one of SEQ ID NO: 23-30. For example, the second nucleotide sequence may be an AADC coding sequence as shown in any one of SEQ ID NO: 1-9, 11, and 46; a GBA1 coding sequence as shown in any one of SEQ ID NO: 12-20, 22, 45, and 47; or a CDNF or GDNF coding sequence as shown in any one of SEQ ID NO: 23-30. Preferably, the first nucleotide sequence may be an AADC coding sequence as shown in SEQ ID NO: 3 or 46; or a GBA1 coding sequence as shown in any one of SEQ ID NO: 16, 45, and 47. Alternatively or additionally, in a preferred embodiment, the second nucleotide sequence may be a CDNF coding sequence as shown in SEQ ID NO: 25 or a GDNF coding sequence as shown in SEQ ID NO: 29. In some embodiments, the first and second nucleotide sequences are linked by a P2A adapter sequence.

[0184] Pharmaceutical Composition

[0185] The term "pharmaceutical composition" refers to a composition suitable for delivery to a subject. The pharmaceutical compositions of this disclosure comprise isolated nucleic acids, rAAV vectors, or viral particles of this disclosure, and pharmaceutically acceptable excipients. 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.

[0186] delivery method

[0187] When applied to subjects (e.g., animals, including humans) or to cells, tissues, organs, or biological fluids, the terms “administration,” “administering,” “treating,” and “treatment” as used herein refer to contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and a subject, cell, tissue, organ, or biological fluid. Cellular treatment encompasses contact between the reagent and cells, as well as contact between the reagent and a fluid, wherein the fluid is in contact with cells. The terms “administration” and “treatment” also include in vitro and ex vivo treatment of, for example, cells by means of a reagent, diagnostic agent, conjugated compound, or another cell.

[0188] In a preferred embodiment, the rAAV carrier of this invention can be delivered via intravenous, intraventricular, intrathecal, or striatal administration. In a specific embodiment, the rAAV carrier is delivered via the striatal route. In another specific embodiment, the rAAV carrier is delivered via the intraventricular route. In the most preferred embodiment, treatment or administration is performed via the intraventricular (ICV) (e.g., via ICV injection).

[0189] The rAAV vector can be administered via single or multiple doses. In a specific implementation, the rAAV vector is administered via a single injection.

[0190] The dosage of rAAV carrier injection can vary based on the route of administration. For example, given the motor problems in PD patients caused by the loss of dopaminergic transmission in the striatum, intraparenchymal / intramural injections typically require delivering a specific volume of rAAV to cover as much of the striatum as possible to achieve adequate therapeutic effect. The dosage can also vary based on the subject's weight. Therefore, the dosage range can cover an area of ​​1.5 × 10⁻⁶. 10 - 1.5×10 14 The range of vg / kg is wide.

[0191] Therapeutic uses

[0192] The terms “treat,” “treating,” or “treatment” include curing or at least alleviating the symptoms of neurodegenerative disorders such as PD, MSA, GD, AADCD, or other protein diseases.

[0193] Subjects with any of these neurodegenerative disorders can be diagnosed by a well-trained neurologist based on genetic background, medical history, symptoms and signs, and the results of neurological and physical examinations, according to clinical diagnostic criteria (Postuma, RB et al., MDS clinical diagnosticcriteria for Parkinson's disease. Mov Disord, 2015. 30(12): 1591-601; Palma, JA, L. Norcliffe-Kaufmann and H. Kaufmann, Diagnosis of multiple systematrophy. Auton Neurosci, 2018. 211: 15-25).

[0194] Subjects with the aforementioned neurodegenerative disorders can be treated using viral vectors expressing one or both of the aforementioned genes. Subjects may be clinically diagnosed early-stage PD patients with one or more mutations in the AADC gene, GBA1 gene, or other PD genetic risk genes (like SNCA). Subjects may also be clinically diagnosed late-stage PD patients without mutations in the AADC gene, GBA1 gene, or other PD genetic risk genes, or with one or more mutations. Subjects may be those previously treated with or currently being treated with dopamine derivatives (like Medopa), with or without L-DOPA-induced motor dysfunction. Subjects may also be those resistant to currently available treatments.

[0195] Subjects may be clinically diagnosed patients with GD, classified as type 2 or 3, or type 1 with central nervous system symptoms. Subjects may also be clinically diagnosed patients with MSA who have shown Parkinson's disease (bradykinesia with tonic tremor or postural instability), cerebellar syndrome (gait ataxia with cerebellar dysarthria, limb ataxia, or cerebellar oculomotor dysfunction), or at least one feature suggesting autonomic dysfunction, with or without GBA1 gene mutation or reduced GBA1 protein activity.

[0196] Subjects can also be clinically diagnosed MSA patients who are resistant to currently available treatments (such as dopamine derivatives).

[0197] This application also relates to the following:

[0198] 1. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a first protein, the first nucleotide sequence being operatively linked to a second nucleotide sequence encoding a second protein, wherein the first protein and the second protein are selected from combinations (a) to (i):

[0199] (a) The first protein is human AADC, and the second protein is human GBA1;

[0200] (b) The first protein is human AADC, and the second protein is human CDNF;

[0201] (c) The first protein is human AADC, and the second protein is human GDNF;

[0202] (d) The first protein is human GBA1, and the second protein is human CDNF;

[0203] (e) The first protein is human GBA1, and the second protein is human GDNF;

[0204] (f) The first protein is human CDNF, and the second protein is human AADC;

[0205] (g) The first protein is human CDNF, and the second protein is human GBA1;

[0206] (h) The first protein is human GDNF, and the second protein is human AADC;

[0207] (i) The first protein is human GDNF and the second protein is human AADC.

[0208] 2. The isolated nucleic acid molecule according to claim 1, wherein the first nucleotide sequence is upstream of the second nucleotide sequence at the 5'.

[0209] 3. The isolated nucleic acid molecule according to claim 1 or 2, characterized in that:

[0210] (i) The polypeptide sequence of AADC contains or is an amino acid sequence as shown in SEQ ID NO: 31;

[0211] (ii) The polypeptide sequence of GBA1 contains or is an amino acid sequence as shown in SEQ ID NO: 32;

[0212] (iii) The polypeptide sequence of CDNF contains or is an amino acid sequence as shown in SEQ ID NO: 33;

[0213] (iv) The polypeptide sequence of GDNF contains or is an amino acid sequence as shown in SEQ ID NO: 34.

[0214] 4. The isolated nucleic acid molecule according to any one of items 1-3, wherein the first nucleotide sequence and / or the second nucleotide sequence is a wild-type coding sequence or a codon-optimized coding sequence.

[0215] 5. The isolated nucleic acid molecule according to any one of claims 1-4, wherein when the first protein is human ADCC, the first nucleotide sequence comprises or is selected from the nucleotide sequences shown in any one of SEQ ID NO: 1-9, 11 or 46.

[0216] 6. The isolated nucleic acid molecule according to claim 5, wherein the nucleotide sequence encoding human AADC is a codon-optimized coding sequence of AADC having the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 46.

[0217] 7. The isolated nucleic acid molecule according to any one of claims 1-6, wherein the first protein or the second protein is human GBA1, wherein the first nucleotide sequence or the second nucleotide sequence comprises or is selected from the nucleotide sequences shown in any one of SEQ ID NO: 12-20, 22, 45 or 47.

[0218] 8. The isolated nucleic acid molecule according to claim 7, wherein the nucleotide sequence encoding human GBA1 is a codon-optimized coding sequence of GBA1 having the nucleotide sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 45 or SEQ ID NO: 47.

[0219] 9. The isolated nucleic acid molecule according to any one of claims 1-8, wherein the second protein is human CDNF, wherein the second nucleotide sequence comprises or is selected from the nucleotide sequences shown in any one of SEQ ID NO: 23-26.

[0220] 10. The isolated nucleic acid molecule according to any one of claims 1-8, wherein the nucleotide sequence encoding human CDNF is a codon-optimized coding sequence for CDNF, and the distinct nucleotides between the codon-optimized coding sequence and the wild-type coding sequence for CDNF as shown in SEQ ID NO: 26 are not present in the region of the signal peptide encoding CDNF.

[0221] 11. The isolated nucleic acid molecule according to claim 10, wherein the nucleotide sequence encoding human CDNF is a codon-optimized coding sequence of CDNF and has fewer CpG sites or no CpG islands compared to the wild-type coding sequence of CDNF as shown in SEQ ID NO: 26.

[0222] 12. The isolated nucleic acid molecule according to claim 11, wherein the nucleotide sequence encoding human CDNF is a codon-optimized coding sequence of CDNF having the nucleotide sequence shown in any one of SEQ ID NO: 23-25.

[0223] 13. The isolated nucleic acid molecule according to claim 12, wherein the nucleotide sequence encoding human CDNF is a codon-optimized coding sequence of CDNF having the nucleotide sequence shown in SEQ ID NO: 25.

[0224] 14. The isolated nucleic acid molecule according to any one of claims 1-8, wherein the second protein is human GDNF, wherein the second nucleotide sequence comprises or is selected from the nucleotide sequences shown in any one of SEQ ID NO: 27-30.

[0225] 15. The isolated nucleic acid molecule according to any one of claims 1-8, wherein the nucleotide sequence encoding human GDNF is a codon-optimized coding sequence for GDNF, and the distinct nucleotides between the codon-optimized coding sequence and the wild-type coding sequence for GDNF as shown in SEQ ID NO: 30 are not present in the region encoding the signal peptide or propeptide of GDNF.

[0226] 16. The isolated nucleic acid molecule according to claim 15, wherein the nucleotide sequence encoding human GDNF is a codon-optimized coding sequence for GDNF and has fewer CpG sites or no CpG islands compared to the wild-type coding sequence for GDNF as shown in SEQ ID NO: 30.

[0227] 17. The isolated nucleic acid molecule according to claim 16, wherein the nucleotide sequence encoding human GDNF is a codon-optimized coding sequence of GDNF having the nucleotide sequence shown in any one of SEQ ID NO: 27-29.

[0228] 18. The isolated nucleic acid molecule according to claim 17, wherein the nucleotide sequence encoding human GDNF is a codon-optimized coding sequence of GDNF having the nucleotide sequence shown in SEQ ID NO: 29.

[0229] 19. The isolated nucleic acid molecule according to any one of claims 1-18, wherein the first nucleotide sequence and the second nucleotide sequence are linked within a frame and are operatively linked to a promoter located 5' upstream of both the first nucleotide sequence and the second nucleotide sequence.

[0230] 20. The isolated nucleic acid molecule according to claim 19, wherein the promoter is selected from the CBh promoter, EF1α promoter, CAG promoter, MBP promoter or variants thereof.

[0231] 21. The isolated nucleic acid molecule according to claim 20, wherein the promoter is a truncated variant of the EF1α promoter having a nucleotide sequence of any one of SEQ ID NO: 50-56.

[0232] 22. The isolated nucleic acid molecule according to any one of claims 1-21, wherein the isolated nucleic acid molecule further comprises a linker sequence between the first nucleotide sequence and the second nucleotide sequence.

[0233] 23. The isolated nucleic acid molecule according to claim 22, wherein the adapter sequence is a coding sequence for a self-cleaving peptide or an internal ribosome entry site.

[0234] 24. The isolated nucleic acid molecule according to item 23, wherein the self-cleaving peptide is a 2A peptide.

[0235] 25. The isolated nucleic acid molecule according to item 24, wherein the 2A peptide is selected from E2A, F2A, T2A, and P2A.

[0236] 26. The isolated nucleic acid molecule according to item 25, wherein the 2A peptide is P2A.

[0237] 27. The isolated nucleic acid molecule according to item 23, wherein the adapter sequence is an ECMV IRES or a miniature IRES.

[0238] 28. The isolated nucleic acid molecule according to any one of items 1-27, wherein the isolated nucleic acid molecule further comprises a polyadenylation signal.

[0239] 29. The isolated nucleic acid molecule according to claim 28, wherein the polyadenylation signal is selected from SV40 polyA, human growth hormone (hGH) polyA and bovine growth hormone (bGH) polyA.

[0240] 30. A codon-optimized sequence of an AADC having a nucleotide sequence as shown in any one of SEQ ID NO: 1-9 or 46.

[0241] 31. A codon-optimized sequence of GBA1 having a nucleotide sequence as shown in any one of SEQ ID NO: 12-20, 45 or 47.

[0242] 32. A codon-optimized sequence of a CDNF having a nucleotide sequence as shown in any one of SEQ ID NO: 23-25.

[0243] 33. A codon-optimized sequence of GDNF, said codon-optimized sequence of GDNF having a nucleotide sequence as shown in any one of SEQ ID NO: 27-29.

[0244] 34. A recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid molecule isolated according to any one of claims 1-29 or a codon-optimized sequence according to any one of claims 30-33.

[0245] 35. The rAAV carrier according to claim 34, wherein the rAAV carrier further comprises an ITR of AAV2.

[0246] 36. A viral particle comprising the rAAV vector according to claim 34 or 35.

[0247] 37. The viral particle according to claim 36, wherein the viral particle comprises a capsid protein of AAV1, AAV2, AAV5, AAV8 or AAV9.

[0248] 38. A pharmaceutical composition comprising an rAAV vector according to claim 34 or 35 or a viral particle according to claim 36 or 37, and a pharmaceutically acceptable excipient.

[0249] 39. The pharmaceutical composition according to claim 38, wherein the pharmaceutical composition is used to treat neurodegenerative disorders.

[0250] 40. The pharmaceutical composition according to claim 39, wherein the neurodegenerative disorder is Parkinson's disease, multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD), or other protein disorders.

[0251] 41. A method for treating or preventing neurodegenerative disorders in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the rAAV vector according to claim 34 or 35, viral particles according to claim 36 or 37, or a pharmaceutical composition according to any one of claims 38-40.

[0252] 42. The method according to claim 41, wherein the neurodegenerative disorder is Parkinson's disease, and the isolated nucleic acid molecule contains a nucleotide sequence encoding a human AADC, preferably a codon-optimized sequence of the AADC according to claim 30, and preferably as the first nucleotide sequence.

[0253] 43. The method according to claim 42, wherein the isolated nucleic acid molecule further comprises a nucleotide sequence encoding GBA1, CDNF or GDNF, preferably as the second nucleotide sequence.

[0254] 44. The method according to claim 41, wherein the neurodegenerative disorder is Parkinson's disease, and the isolated nucleic acid molecule contains a nucleotide sequence encoding human GBA1, preferably a codon-optimized sequence of GBA1 according to claim 31, and preferably as the first nucleotide sequence.

[0255] 45. The method according to claim 44, wherein the isolated nucleic acid molecule further comprises a nucleotide sequence encoding AADC, CDNF or GDNF, preferably as the second nucleotide sequence.

[0256] 46. ​​The method according to claim 41, wherein the neurodegenerative disorder is Gaucher disease, and the isolated nucleic acid molecule contains a nucleotide sequence encoding human GBA1, preferably a codon-optimized sequence of GBA1 according to claim 31.

[0257] 47. The method according to item 46, wherein the Gaucher disease is type 2 or type 3 Gaucher disease.

[0258] 48. The method according to claim 41, wherein the neurodegenerative disorder is multiple system atrophy (MSA), and the isolated nucleic acid molecule contains a nucleotide sequence encoding human GBA1, preferably a codon-optimized sequence of GBA1 according to claim 31.

[0259] 49. The method according to claim 48, wherein the isolated nucleic acid molecule further comprises a nucleotide sequence encoding CDNF or GDNF, preferably GDNF, and preferably as the second nucleotide sequence.

[0260] 50. The method according to claim 41, wherein the neurodegenerative disorder is AADC deficiency (AADCD), and the isolated nucleic acid molecule contains a nucleotide sequence encoding human AADC, preferably a codon-optimized sequence of AADC according to claim 30, and preferably as the first nucleotide sequence.

[0261] 51. The method according to claim 50, wherein the isolated nucleic acid molecule further comprises a nucleotide sequence encoding CDNF or GDNF, preferably GDNF, and preferably as the second nucleotide sequence.

[0262] 52. Use of the rAAV vector according to item 34 or 35 or the viral particles according to item 36 or 37 in the manufacture of a medicament for treating neurodegenerative disorders.

[0263] 53. According to the use described in item 52, the neurodegenerative disorder is Parkinson's disease, multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency, or other protein disorders.

[0264] Example

[0265] To facilitate understanding and utilization of the invention, its advantages will be described in more detail with reference to embodiments and accompanying drawings. However, it should be understood that the following embodiments are intended to illustrate the invention only and are not intended to limit its scope. The scope of the invention should be defined by the claims.

[0266] Example 1. Determination of the optimal connector for linking the coding sequences of AADC and GBA1

[0267] In this embodiment, the performance of different connector sequences (specifically E2A (SEQ ID NO: 35), F2A (SEQ ID NO: 37), T2A (SEQ ID NO: 39), P2A (SEQ ID NO: 41), miniature IRES (SEQ ID NO: 43), and ECMV IRES (SEQ ID NO: 44)) was tested in a construct containing wild-type coding sequences of both AADC and GBA1.

[0268] 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 following order via a self-cutting P2A sequence: the AADC sequence under the control of the CBh promoter (SEQ ID NO: 60), followed by the P2A sequence, and then the GBA1 sequence. Figure 1 Another construct with a GBA1 coding sequence located upstream of the AADC coding sequence was also prepared. Both constructs were introduced into plasmids. The resulting plasmids were named 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 either the coding sequences for AADC or GBA1 alone (“AADC”, “GBA”) or together with the coding sequence for P2A (“AADC-P2A”, “P2A-GBA”) were also constructed and transfected into the same cells for comparison. Plasmids expressing GFP were used as controls.

[0269] Western blotting was performed to detect the expression of AADC and GBA1 in each construct. Specifically, 72 hours post-transfection, cells were collected in lysis buffer (RIPA buffer, Thermo Fisher 89901), denatured at 95°C for 15 min in 5×SDS-PAGE sample loading buffer (Beyotime, P0015L), 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 using antibodies against human AADC (Millipore, #AB1569), GBA1 (Sigma, #G4171), and GAPDH (CST, #2118), respectively. Long-term and normal exposures were performed using Western blotting.

[0270] like Figure 2 As shown in Figure A, the GBA1 protein expressed by plasmid AADC-P2A-GBA1 (ApG) has almost the same molecular weight as GBA1 expressed by plasmids containing only GBA1, while the AADC derived from ApG is slightly larger than the AADC expressed by plasmids containing only AADC due to the attached P2A fragment. This result indicates that the self-cleaved 2A peptide is functional in this context.

[0271] Surprisingly, when the GBA1 gene was placed before the AADC gene (5' upstream), the expression of both genes was significantly reduced after transfection into HEK293 cells (see [link]). Figure 2 B).

[0272] Other adaptors, including E2A, F2A, T2A, P2A, miniature IRES, and ECMV IRES, were evaluated in the same experimental environment as P2A. The results showed that, except for miniature IRES, all evaluated adaptors produced both AADC and GBA1 proteins of the expected molecular size.

[0273] Further testing was performed by extending the exposure time to determine if any fusion protein (AADC + GBA1) was present due to incomplete cleavage. Clear positive bands were detected in both the E2A and F2A constructs using both AADC and GBA1 antibodies (see [link to test]). Figure 3 The cutting efficiencies of different connectors, ranked from highest to lowest, are ECMV IRES, P2A, T2A, E2A, and then F2A. Using a full-length ECMV IRES connector will produce a build with a packaging capacity exceeding rAAV. Therefore, P2A was selected as the connector for use in this invention.

[0274] Example 2. Codon optimization for improving protein expression of AADC and GBA1

[0275] In this embodiment, nine sequences containing high-frequency codons were generated for AADC (referred to as A1-A9, SEQ ID NO: 1-9) and GBA1 (referred to as G1-G9, SEQ ID NO: 12-20), respectively. All these sequences had a codon fitness 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 applications were synthesized and used as references, named A10 (SEQ ID NO: 10, disclosed in CN 107106689A, Voyager Therapeutics Inc.) and G10 (SEQ ID NO: 21, disclosed in WO 2020210698A1, PrevailTherapeutics, Inc.), respectively. A10 encodes an M17V-mutant AADC protein.

[0276] Generate in the following ways that have the following characteristics Figure 4The twenty candidate constructs shown (see Table 1) combine one of the codon-optimized AADC coding sequences (A1-A10) with a GBA1-WT (G0) sequence, or combine the AADC-WT (A0) sequence with one of the codon-optimized GBA1 coding sequences (G1-G10), where P2A acts as a connector between the two coding sequences. Vectors containing G0 (wild-type GBA1 alone), A0 (wild-type AADC alone), and G0A0 are used as controls.

[0277] Table 1. Candidate Recombinant Constructs

[0278]

[0279] The vector was transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Invitrogen, #L3000008) according to the manufacturer's instructions. Forty-eight hours post-transfection, cells were washed once with 1× PBS and harvested in RIPA buffer (Thermo, 89901). Protein levels of AADC and GBA1 in cell lysates were determined by sandwich ELISA as described below.

[0280] ELISA for detecting AADC and GBA1 protein levels

[0281] 1. On day 1, coat the 96-well plate overnight at 4°C with the following capture antibodies in 100 μL of coating buffer: 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.

[0282] 2. On the second day, wash the sample wells three times with washing buffer. Add 300 μL of blocking buffer (containing 2% BSA) and incubate at room temperature for about 2 h, followed by three more washing steps.

[0283] 3. Add 100 μL of assay buffer containing the following detection antibodies to each well: anti-DDC monoclonal antibody (HRP) (Sinobiological, 10560-R040, 1:1000 dilution) for AADC protein detection and polyclonal anti-GBA antibody (Abcam, ab96246) for GBA1 protein detection. After incubating at room temperature for approximately 1 h, wash the sample wells three times with washing buffer.

[0284] 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 step.

[0285] 5. The sample wells were then incubated with 100 μL of TMB solution (Solarbio, #PR1200) for about 20 min, and the reaction was terminated by adding 100 μL of stop solution (Solarbio, #C1058).

[0286] 6. The absorbance of each sample (at an excitation wavelength of 450 nm and an emission wavelength of 630 nm) was measured using a fluorometer (SPECTRAmax Gemini XPS, Molecular Devices, San Jose, California, USA), and the results are shown in [Table data would be inserted here]. Figure 5 and Figure 6 middle.

[0287] like Figure 5 As shown, A3 and A9 are the top two candidates among the evaluated AADC coding sequences. A10 exhibits the highest AADC protein expression level because it expresses the AADC mutant protein. Figure 6 As shown, G4 and G5 are the top two candidates among the evaluated GBA1 coding sequences. Furthermore, both A0G4 and A0G5 express relatively higher levels of GBA1 protein compared to the reference A0G10 construct containing the prior art reference GBA1 coding sequence G10.

[0288] Protein blot

[0289] Western blot (WB) assays were also performed to detect protein expression. Next, candidate vectors (except G1, G2, G7, A1, A2, and A7) were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Invitrogen, #L3000008).

[0290] Forty-eight hours post-transfection, cells were harvested in RIPA buffer (Thermo, 89901), diluted in 5× loading buffer (Beyotime, P0015L), and boiled (denaturing). All Western blot samples were separated on an SDS-polyacrylamide gel (BioRad, 1703932) and transferred to a 0.22 μm PVDF membrane (BIO-RAD, 1620177).

[0291] The membrane was incubated overnight at 4°C with anti-AADC antibody (Millipore, #AB1569), anti-GBA1 antibody (Sigma, #G4171), or anti-GAPDH housekeeping gene antibody (CST, #2118), and then incubated at room temperature for 2 hours the next day with HRP-linked secondary antibody.

[0292] The BeyoECL Moon kit (Beyotime, P0018F) was then applied to the membrane. Exposure and image capture were performed using a Tannon 5600 system, and the results are shown below. Figure 7 and Figure 9 In this study, the band areas of AADC or GBA1 proteins were determined using ImageJ 1.53 (NIH) and normalized relative to GAPDH levels. The GAPDH-normalized values ​​were then normalized relative to the A0 value of the AADC group or the G0 value of the GBA1 group. Normalized data from three independent experiments are presented in [the table / image / image]. Figure 8 and Figure 10 middle.

[0293] like Figures 7-10 The Western blot results showed that A3G0 and A9G0 had the highest AADC protein expression among the codon-optimized AADC candidates, and A0G4 and A0G5 were the best candidates among the GBA1 candidates, consistent with the results from ELISA assays. A3, A9 (for AADC) and G4, G5 (for GBA1) were selected as codon-optimized sequences for further optimization of combined expression constructs.

[0294] Example 3. Catalytic activity of co-expressed AADC and GBA1 proteins

[0295] This embodiment aims to determine whether exogenously expressed AADC and GBA1 proteins possess catalytic activity, indicating normal function. Furthermore, catalytic activity can also be used as an additional evaluation criterion for candidate sequences. The method for examining the activity of AADC and GBA1 in cell lysates is described below.

[0296] AADC catalytic activity (HPLC analysis)

[0297] Cultures expressing AADC protein (A3G0, A9G0, A10G0, A0G0, A0) and control samples expressing GFP protein were exposed to 100 μM L-DOPA for 24 h. 100 μL of homogenization buffer (50 mM phosphate buffer, pH 7.4, containing 0.2 mM pyridoxal phosphate and 0.2 mM pargyline) was added to each well, and cells were scraped from the bottom.

[0298] Cell homogenates were centrifuged at 13,000 g for 10 min, and HPLC analysis was performed using 30 μL of each sample. HPLC / ECD was performed on the samples to detect dopamine levels produced by the catalytically active AADC protein expressed by different constructs. Relative AADC activity was calculated using a dopamine standard curve, as shown by the amount of dopamine transferred from L-DOPA in each sample.

[0299] GBA1 activity

[0300] The GBA1 protein activity was tested in cultures expressing GBA1 protein (A0G4, A0G5, A0G10, A0G0, G0) and control samples expressing GFP protein. Recombinant human GBA (rhGBA) was used as a positive control.

[0301] 1. Dilute rhGBA (catalog number 7410-GHB) to 0.2 ng / µL in assay buffer (50 mM sodium citrate, 25 mM sodium cholate, 5 mM MDT, pH 6.0).

[0302] 2. Dilute the substrate (4-methylumbellatus-β-D-glucopyranoside, Sigma, catalog number M3633, 10 mM in DMSO) to 6 mM in assay buffer.

[0303] 3. Load 25 µL of cell lysate (100 ng / µL total protein) or 0.2 ng / µL rhGBA into the wells of a 96-well plate. Initiate the reaction by adding 25 μL of 6 mM substrate. Load 25 μL of assay buffer into the wells of the standard curve.

[0304] 4. Seal the plate and incubate at 37°C for 3 hours.

[0305] 5. After incubation, the reaction was terminated by adding 50 µL of a stop solution (0.5 M glycine, 0.3 M NaOH (approximately pH 10)) to each well.

[0306] 6. In endpoint mode, record readings at excitation and emission wavelengths of 365 nm and 445 nm respectively (top readings).

[0307] 7. Specific activity was calculated as follows: (adjusted fluorescence * 25) / (180 min * 0.0025 mg). Results are shown in... Figure 9 and Figure 10 middle.

[0308] like Figure 11As shown, among all the constructs evaluated, the AADC protein expressed by construct A3G0 exhibited the highest catalytic activity. Figure 12 As shown, the GBA1 protein expressed by the A0G4 construct exhibits the highest catalytic activity.

[0309] Example 4. Identification of the optimal combination of codon-optimized AADC and GBA1 sequences

[0310] To identify the optimal combination of codon-optimized AADC and GBA1 sequences, the AADC and GBA1 coding sequences with better performance, as shown above, were paired together and used to construct recombinant AAV vectors. Specifically, constructs A3G4, A9G4, A3G5, and A9G5 were generated. Furthermore, construct A11G11 was generated, based on codons A3 and G4 with further optimization to completely remove CpG islands from the AADC and GBA1 coding sequences. Two baseline vectors were also constructed: A10-vy (SEQ ID NO: 48, codon A10 containing a promoter and other regulatory elements described in the same patent) and G10-p (SEQ ID NO: 49, codon G10 containing a promoter and other regulatory elements described in the same patent).

[0311] The above vectors, along with A0-P2A, A0G0, A10G0, A10-vy, and G10-p, were transfected into HEK293 cells. AADC and GBA1 protein expression, as well as the catalytic activity of GBA, were measured. It was found that among the evaluated co-constructions, A11G11, A3G5, and A9G4 expressed the highest levels of AADC protein, as determined by Western blotting analysis. Figure 13 For GBA1 protein expression, constructs A3G5 and A11G11 expressed the highest levels of the protein (they are also the most active), followed by construct A9G4. Figure 14 ).

[0312] Example 5. Evaluation of AADC and GBA1 protein expression in AAV9-packaged combinatorial constructs

[0313] The constructs A0G0, A9G4, A11G11, and A10-vy were packaged into AAV cells using serotype AAV9. AAV9-A0G0, A9G4, and A11G11, along with GFP-only control AAV, were administered at MOIs 1e5 and 1e6 into U-87 MG cells stably expressing the AAV receptor to enhance transduction efficiency. Five days post-infection, the expression levels of AADC and GBA1 proteins in these cell samples were determined by Western blotting. Figure 15As shown, cells transduced with A11G11 AAV virus expressed higher levels of AADC and GBA1 proteins compared to A9G4-packaged AAV virus. AADC protein expression was observed to be significantly higher in cells transduced with A10-vy AAV virus, most likely due to the stronger promoter used in the A10-vy construct.

[0314] Example 6. Optimization of the regulating elements used in the combined expression construct

[0315] To further improve protein expression in the combinatorial constructs, the promoters and polyA sequences used were optimized. The CBh promoter in 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. Since EF1α-A11G11-hGH exceeded the packaging capacity of AAV, its length was reduced by truncating a portion of the EF1α promoter, resulting in seven different truncated variants (numbered 5-11) as shown in Table 2.

[0316] The constructs listed in Table 2 were transfected into HEK293 cells along with A3G5, A10-vy, and GFP. AADC protein expression levels were determined by Western blotting. Among the first group of constructs evaluated, EF1α-A11G11-hGH expressed the highest level of AADC protein. Among the constructs with a truncated EF1α promoter (EFStI1-7, SEQ NO: 50-56), construct EFStI2 expressed the highest level of AADC protein. Figure 16 ).

[0317] Table 2. Combinatorial constructs with EF1a and truncated EF1a promoters.

[0318]

[0319] Example 7. In vivo function of AAV candidates expressing optimized AADC and GBA1 sequences in a PD mouse model effect

[0320] The therapeutic effects of AAV expressing optimized AADC and GBA1 were evaluated in a PD mouse model to determine whether candidate AAVs could rescue kinesia in PD animals.

[0321] First, a widely used chemically induced PD mouse model was used. Mice were treated with MPTP / probenecid. The candidate rAAV vector CBh-A11G11 was administered once to the striatum of the PD mouse model at a higher dose of 2E+10 vg / animal and a lower dose of 2E+09 vg / animal (“CBh-A11G11 Low” and “CBh-A11G11 High”, respectively). The rAAV vector A10-vy (2E+09 vg / animal) was injected in a similar manner to the baseline control (“A10-vy”), and a GFP-expressing rAAV vector was used as a negative control (“GFP”). Motor function of the mice was evaluated by measuring movement speed and walking distance before PD modeling (before induction with MPTP / P; “before”) and before and after administration of the corresponding rAAV vectors (“MPTP / P Post” and “AAV Post”, respectively). The “before” behavioral test was performed for 6 days, followed by daily administration of MPTP / probenecid for 35 days. After a 3-day acclimatization period, subjects underwent the same behavioral tests (“post-MPTP / P”) followed by rAAV injection. “Post-AAV” behavioral tests were performed 21 days after rAAV injection. Results are shown in… Figure 34 middle.

[0322] like Figure 34 As shown, the movement speed of mice decreased after MPTP / probenecid treatment. Mice awaiting A10-vy treatment (N = 3) showed less severe speed loss compared to other mice. All mice (except those awaiting A10-vy treatment) showed shortened walking distance. Following treatment with the corresponding rAAV, the motor function of the negative control mice (N = 5) showed a sustained decline, manifested as a continuous decrease in movement speed and distance. In contrast, mice treated with both low (N = 5) and high (N = 7) doses of CBh-A11G11-SV40 showed recovery of movement speed and distance, with the higher dose showing slightly better results compared to the lower dose. These results indicate that CBh-A11G11-SV40 is significantly effective in rescuing insufficient movement speed and walking distance in an MPTP / probenecid-induced PD mouse model. Given that mice treated with A10-vy experienced less severe impairment in mobility compared to other mice, CBh-A11G11-SV40 demonstrated better performance in rescuing insufficient movement speed and distance in MPTP / probenecid-induced PD mouse models compared to baseline.

[0323] The results were then validated using a transgenic PD mouse model. Mice were genetically engineered to overexpress a mutant α-synuclein (point mutation A53T) to reenact PD pathology. Two rAAVs, EFSIT7-A11G11-hGH (EtI7) and CBh-A11G11-SV40 (CBh), were generated based on the construct A11G11 using different promoters and poly-A tails. rAAVs were administered to mice via intrastriatal injection at low doses of 4E+9 vg and high doses of 4E+10 vg. Wild-type mice without any treatment and A53T mice treated with a mediated substance (PBS) served as controls. Motor function was assessed by measuring the number of vertical movements (through hindlimb erection), and the results are shown in [Table data would be inserted here]. Figure 35 middle.

[0324] like Figure 35 As shown, mice treated with the medium (N = 6) exhibited fewer vertical movements compared to WT mice (N = 8), indicating impaired motor function. Mice treated with both high-dose (N = 7) and low-dose (N = 7) EFSIt7-A11G11-hGH (EtI7) showed better performance than the medium-treated mice, with the high dose showing a significant increase. Mice treated with a low dose of CBh-A11G11-SV40 (CBh) (N = 7) also showed an increased number of vertical movements compared to both WT and the medium-based A11G11 rAAV. The increased number of vertical movements indicates a recovery of motor function.

[0325] Brain tissue samples were collected from A53T mice for protein expression determination. The expression of AADC and GC enzyme proteins in the striatum was measured by Western blotting. Figure 36 As shown, all treatment groups exhibited expression of AADC and GC enzymes. Furthermore, the levels of phosphorylated α-synuclein and total α-synuclein in the striatum were measured. The level of phosphorylated α-synuclein is considered a biomarker for PD pathology. Figure 37 A and Figure 37 As shown in B, both candidate rAAV vectors reduced phosphorylated α-synuclein levels in a dose-dependent manner, indicating an improvement in PD pathology. Figure 37 The exemplary bands in A are derived from two representative subjects in each group. For example... Figure 37 The relative phosphorylated α-synuclein levels shown in B are the average of all subjects in the indicated group.

[0326] Example 8. In vivo therapeutic efficacy of an AAV candidate expressing an optimized GBA1 sequence in a GD mouse model.

[0327] The therapeutic effect of AAV expressing optimized GBA1 was tested in a GD mouse model.

[0328] Two candidate vectors expressing the optimized GBA1 (G11) were constructed, such as... Figure 38 As shown. The first candidate vector was designed to contain the CAG promoter, G11 codon, and hGH polyA (“CAG”). The second candidate vector was designed to contain the EF1α promoter, G11 codon, WPRE, and hGH polyA (“EF1α”). Both vectors were packaged into AAV9 as rAAV. The candidate rAAV vectors were injected into the lateral ventricles of chemically induced GD mouse models and genetically engineered GD mouse models to evaluate their effects.

[0329] The chemically induced mouse model was a CBE-induced GD mouse model. GC enzyme activity was inhibited by cyclohexenetetrol β-epoxide (CBE), a specific irreversible GC enzyme inhibitor mimicking the loss-of-function mutation in the GBA1 gene. On day 2 after birth (P2), mice were treated via intracerebroventricular injection with PBS, AAV9-GFP (negative control), or three different doses of the candidate rAAV vector (low, 2.75E+09 vg; medium, 8.8E+09 vg; high, 2.8E+10 vg, per animal). G10-p was used as a reference. To allow sufficient time for gene expression, disease modeling began at P8. Mice were injected with 37.5 mg / kg CBE via intraperitoneal injection once daily. In-life assessments were performed until P28.

[0330] Figure 39 The viability of GD mice is shown up to P28. As can be seen, the survival percentage in both the PBS group and the negative control group decreased rapidly after P22. No animals survived in the negative control group at P25. In contrast, both the candidate vector and the reference G10-p significantly improved the viability of GD mice.

[0331] Figure 40 The study demonstrated changes in motor function in the animals. Both candidate vectors rescued motor insufficiency and ataxia induced by long-term CBE administration. Figure 40 A and Figure 40 As shown in B, CAG increased the distance traveled in the open field experiment at all doses and enhanced motion coordination in the rotarod apparatus at medium and high doses, while EF1α increased the distance traveled at medium and high doses, but only improved motion coordination at high doses.

[0332] Brain samples were collected on day 30 postnatally to determine protein activity. Brain samples were homogenized in lysis buffer (N-PER neuronal protein extraction reagent, Thermo Scientific #87792) containing a protease inhibitor (Roche #11697498001) to produce lysed samples. GC enzyme activity in these lysed samples was determined by GBA1 activity assay as described above. GC enzyme activity was found to increase dose-dependently in all candidate vector treatment groups. Figure 41 ).

[0333] Example 9. Codon-optimized human CDNF sequences for AADC combinatorial constructs

[0334] In this embodiment, an AAV vector is designed to co-express the genes encoding AADC and NTF (specifically CDNF). P2A is used as a linker sequence between the two coding sequences.

[0335] To determine the optimal coding sequence for CDNF, the wild-type AADC coding sequence (AADC WT) is concatenated with different codon-optimized CDNF sequences via P2A. Codon optimization is performed based on different algorithms.

[0336] Two codon-optimized CDNF sequences with high CAI (> 0.85) were obtained and named 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 shown in SEQ ID NO:26). In CDNF SA, the nine nucleotides (nucleotide positions 73-81) encoding the three amino acids immediately following the signal peptide remained unchanged.

[0337] Simultaneously, based on the CDNF SA sequence, another candidate CDNF sequence, known as CDNF manual (CDNF MN), was obtained by manually identifying and reducing the "CG" component in the mature protein coding sequence (nucleotide positions 82 to 561). As a result, all CpG islands were intentionally removed from the CDNF MN sequence.

[0338] For this study, an AAV backbone vector was prepared, which contains a 5' ITR, a CBh promoter, an AADC WT coding sequence, a P2A sequence, an SV40 polyA, and a 3' ITR from 5' to 3'.

[0339] The sequences of CDNF GS, CDNF SA, CDNF MN, and CDNF wild-type (CDNF WT) were inserted at the 3' position of the P2A linker sequence of the aforementioned backbone vector. Figure 17 A and Figure 17B). The obtained vectors were named ApC-GS, ApC-SA, ApC-MN, and ApC-WT. Reference vectors containing only CDNF WT (without AADC) were also constructed.

[0340] Using Lipofectamine 3000 transfection reagent (Invitrogen, # L3000008), the above five plasmids and one negative control plasmid expressing only GFP were transfected into HEK293 cells. 72 hours post-transfection, the supernatant was centrifuged at 2,000 rpm for 10 minutes to remove debris from the culture medium. Then, 40 µL of supernatant and 10 µL of 5× 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 1× PBS and harvested in RIPA buffer (Thermo, 89901).

[0341] For Western blotting analysis, samples were diluted and boiled in 5× 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 overnight at 4°C with antibodies against CDNF protein (Sigma, HPA044587), AADC protein (Merck Millipore, AB1569), and β-tubulin (tubulin, Proteintech, 66240-1-Ig), followed by incubation the next day at room temperature for 2 hours with HRP-linked secondary antibody. The BeyoECL Moon kit (Beyotime, P0018F) was then applied to the membrane. Figure 18 A). Exposure and image capture were performed using a Tannon 5600 system. The intensity of the CDNF bands was calculated and normalized relative to the construct ApC-WT, and the results are shown in... Figure 18 B in.

[0342] like Figure 18 As shown in B, among all the constructs evaluated, cells transfected with construct ApC-MN showed the highest levels of CDNF protein secreted into the supernatant, which was confirmed in U-87 MG cells transfected with plasmids from the same group. Figure 19 A- Figure 19 B).

[0343] The level of secreted CDNF protein in the supernatant was further determined using a sandwich ELISA method. In short, 96-well microplates (Costar, 42592) were coated overnight at 4°C with CDNF mouse Mab (SinoBiological, 15691-MM1) antibody as the capture antibody. The next day, the plates were washed three times with PBST (phosphate-buffered saline with a Tween) and then blocked for 2 hours at room temperature with 5% skim milk powder (Solarbio, D8340). After another washing step, CDNF protein standards (SinoBiological, 15691-H08H) or protein samples were added to the plates in duplicate. The samples were incubated with the detection antibody (SinoBiological, 15691-R104) at room temperature for 2 hours, followed by a washing step. The samples were then incubated with HRP-conjugated goat anti-rabbit antibody (SinoBiological, HO14SE1801) for 1 hour, followed by another washing step. The binding of HRP conjugates was detected by adding TMB (Solarbio, PR1200), and the reaction was terminated by a stop solution (Solarbio, C1058). The absorbance of each sample (at an excitation wavelength of 450 nm and an emission wavelength of 630 nm) was measured using a fluorometer (SPECTRAmax Gemini XPS, Molecular Devices, San Jose, California, USA), and the results are shown below. Figure 20 A- Figure 20 B in.

[0344] like Figure 20 A- Figure 20 As shown in Figure B, both HEK293 and U-87 MG cells transfected with the ApC-MN construct expressed significantly higher levels of CDNF protein, as indicated by the high levels of secreted CDNF in the supernatant. As a result, ApC-MN was selected for further evaluation.

[0345] Example 10. Codon-optimized CDNF-expressed proteins exhibit significant cell-protective effects.

[0346] In this embodiment, the cytoprotective effect of CDNF protein expressed and secreted by cells transfected with plasmid AADC-CDNF MN (ApC-MN) was investigated. The cytoprotective effect of CDNF was examined using two cell-based assays: the LDH cytotoxicity assay for cells treated with MPP+ or rotenone and the CCK-8 cell viability assay.

[0347] In the LDH assay, 96-well plates containing SH-SY5Y cells (National Collection of Authenticated Cell Cultures, SCSP-5014) were pretreated with or without supernatant containing secreted CDNF protein collected from previous experiments. A few cell-free wells were left as blank controls to measure background luminescence levels. The cells were then subjected to MPP... + Alternatively, rotenone or DMSO mediators were used for control treatment. The LDH assay reagent (CytoTox96® non-radioactive cytotoxicity assay, Promega) was prepared by mixing 12 mL of assay buffer with a vial of substrate mixture while the plate was incubated at 37°C. 45 minutes before adding the CytoTox 96® reagent, 1 / 10 volume of 10× lysis solution was added to the positive control (maximum LDH release control) well. Before reading the plate, 50 μL of CytoTox96® reagent was added to each well. The plate was covered with foil to prevent light exposure and incubated at room temperature for 30 min. Then, 50 μL of stop solution was added to each well to terminate the reaction. One hour after adding the stop solution, the absorbance at 490 nm was recorded using a plate reader. Cytotoxicity percentage = 100 × experimental LDH release (OD490) / maximum LDH release (OD490).

[0348] In the CCK-8 assay, SH-SY5Y cells in the logarithmic growth phase were subjected to a concentration of 5 × 10⁻⁶ cells. 3 Cells were seeded at a density of 1 / mL in 96-well plates and cultured in high glucose / DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, and maintained at 37°C for 12 h in a cell culture incubator with 5% CO2 and 95% air. Cells were then treated with or without supernatant containing secreted CDNF collected from previous experiments. MPP was then used to concentrate the medium to a final concentration of 0-2 mM. + Apoptosis was induced in serum-free medium containing rotenone or strychnine. Cell viability was assessed by adding 10 μL CCK-8 (Cell Counting Kit-8, Dojindo) to the medium after 24 h of incubation, and relative cell viability was determined by spectrophotometry at 450 nm after 2 h of incubation.

[0349] like Figure 21 As shown, in the presence of MPP +In the case of either rotenone (1.5 mM) or rotenone (40 nM), SH-SY5Y cells treated with the supernatant of cells transfected with construct ApC-MN had a higher survival rate than those treated with the supernatant of cells transfected with construct ApC-WT. This suggests that construct ApC-MN expresses and secretes higher levels of CDNF protein into the supernatant to exert its stronger cytoprotective effect compared to construct ApC-WT.

[0350] Example 11. Codon-optimized human GDNF sequences for AADC combinatorial constructs

[0351] In this embodiment, the codon optimization process performed as in Embodiment 9 is employed ( Figure 17 B) to optimize the mature protein coding sequence of human GDNF to obtain the GDNF-GS sequence (SEQ ID NO: 27), GDNF-SA sequence (SEQ ID NO: 28) and GDNF-MN sequence (SEQ ID NO: 29).

[0352] In vitro cell experiments as described in Example 10 were performed in HEK293 cells transfected with a construct containing GDNF GS, GDNF SA, and GDNF MN. Figures 22-24 Based on the results, among all the codon-optimized GDNF sequences evaluated, the GDNF MN sequence without CpG islands exhibited the best GDNF expression and protection potential. Therefore, the GDNF MN sequence was selected as the GDNF encoding sequence for the AADC combinatorial construct for further evaluation.

[0353] Example 12. Determination of the optimal promoter for use in the AADC and CDNF / GDNF combined construct

[0354] To determine the optimal promoter element for use in the combined AADC with CDNF or GDNF constructs, eight new combined constructs were prepared, as shown in Table 3 below. The four constructs containing CDNF are CAG-A3-P2A-CDNF MN, CAG-A11-P2A-CDNF MN, EF1α-A3-P2A-CDNF MN, and EF1α-A11-P2A-CDNF MN; and the four constructs containing GDNF are CAG-A3-P2A-GDNF MN, CAG-A11-P2A-GDNF MN, EF1α-A3-P2A-GDNF MN, and EF1α-A11-P2A-GDNF MN. The constructs CBh-AADC WT-P2A-CDNF WT and CBh-AADC WT-P2A-GDNF WT were generated and used as controls.

[0355] Table 3. Constructs of CDNF / GDNF and AADC with different regulatory sequence combinations

[0356]

[0357] * ND: Undetectable

[0358] The above constructs were transfected into HEK293 cells. Cell lysates and supernatants from these samples were collected. The protein levels of secreted CDNF or GDNF were measured by ELISA. The results showed that EF1α-A11-P2A-CDNF MN expressed higher levels of CDNF protein compared to CAG-A3-CDNF MN, while the other two constructs expressed undetectable levels of CDNF protein. Figure 25 ).

[0359] For constructs containing GDNF, EF1a-A11-GDNF MN expresses and secretes the highest levels of GDNF. Figure 25 ).

[0360] The catalytic activity of the AADC protein expressed by these combined constructs was also determined by HPLC analysis of cell lysates as described in the previous examples. Figure 26 As shown, the relative AADC activities of EF1α-A11-CDNF MN and EF1α-A11-GDNF MN are significantly higher than those of all other candidate constructs.

[0361] The construct EF1α-CDNFMN-A11 was generated by placing the CDNF codon before the AADC codon and connecting them via P2A.

[0362] 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 post-transfection, supernatants and cell lysates were collected. Western blotting was used to determine the protein levels of AADC and CDNF in the cell lysates and the CDNF level (secreted CDNF) in the supernatant. Interestingly, the protein level of secreted CDNF was found to be significantly higher in the CDNF MN-A11 group compared to that in the A11-CDNF MN group. Figure 42 ).

[0363] Example 13. Validation of the cell-protective effects of CDNF and GDNF proteins derived from candidate constructs

[0364] MPP+ and rotenone assays were performed as described in previous experiments to determine the cell-protective effects of the CDNF and GDNF constructs identified in Example 12. The cytoprotective activity was determined using the construct EF1α-A11-CDNF MN (… Figure 27) or EF1α-A11-GDNF MN ( Figure 28 The supernatant of transfected cells 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.

[0365] Example 14. Evaluation of AADC and CDNF or GDNF protein expression in AAV9-packaged combinatorial constructs

[0366] Both EF1a-A11-CDNF MN and EF1a-A11-GDNF MN constructs were packaged into AAV9 cells. The resulting AAV9-EF1α-A11-CDNF MN and AAV9-EF1α-A11-GDNF MN were added to U87-AAVR cells at an MOI of 1e5. 72 hours after viral transduction, cells were collected in lysis buffer. Protein levels of CDNF and AADC expression were determined according to Western blotting protocol. Simultaneously, the supernatants of all samples were collected and concentrated as described above. The levels of secreted CDNF and GDNF proteins were also detected by Western blotting, and the results are shown in [Figure number missing]. Figure 29 middle.

[0367] The relative activity of the expressed AADC protein in the above cell lysates is shown in Figure 30 middle.

[0368] and Figure 29 and Figure 30 The data shown, when combined, shows that both constructs behave as expected when packaged into AAV9 as rAAV.

[0369] Example 15. Expression of an AAV candidate with an optimized combination of AADC and GDNF or CDNF in a PD mouse model. Internal therapeutic effects

[0370] The therapeutic effect of AAV delivered with an optimized AADC + GDNF combination construct was tested in a unilateral 6-OHDA PD mouse model. Briefly, C57 mice were placed in a stereotactic frame and anesthetized with isoflurane mixed with oxygen. 1 μL of 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 administered apomorphine (Sigma, PHR2621-500 mg) intraperitoneally, and their rotational behavior (rotation to the intact side) was recorded and analyzed. The candidate rAAV vector EF1α-A11-GDNF MN was then injected into the right striatum (coordinates AP = +0.6 mm, ML = -1.8 mm, DV = -3.2 mm) of the subjects at three doses (low: 1E+12 vg, medium: 3.16E+12 vg, high: 1E+13 vg, in mL). AAV-GFP and two doses of baseline AAV-A10-vy (low: 1E+12 vg, high: 1E+13 vg, in mL) were also applied. At 21 days post-AAV injection, a significant reduction in apomorphine-triggered contralateral rotation was observed in all study groups treated with the candidate rAAV vector compared to the control group. Figure 31 and Figure 32 ).like Figure 31 As shown, the three study groups treated with three different doses of EF1α-A11-GDNF MN rAAV significantly outperformed the group treated with baseline rAAV. More notably, the net number of rotations (ipsilateral-contralateral) in the low- and medium-dose treatment groups was close to zero, indicating that these subjects had fully recovered their balance movement behavior, while the high-dose baseline group showed overcompensated ipsilateral rotations (excessive AADC in the treated side). Figure 32 ).

[0371] In another in vivo efficacy test in PD mice (Study 2), CAG-A11-GDNF MN rAAV (AAV9) was also tested in a unilaterally damaged 6-OHDA PD mouse model. Two different doses (2.0E+09 vg and 2.0E+10 vg) of CAG-A11-GDNF MN and EF1α-A11-GDNF MN were delivered to PD mice via intrastriatal injection. AAV-GFP was used as an untreated control, and two doses of AAV2-A10-vy were used as baselines. At 21 days post-AAV injection, EF1α-A11-GDNF MN at 2E+10 vg / animal significantly reduced ipsilateral rotation triggered by amphetamine (5 mg / kg intraperitoneally), indicating salvage of motor function. Figure 43 ).

[0372] Tissue samples were collected for the expression of AADC and NTF proteins and the measurement of dopamine levels. GOI expression was observed in the substantia nigra-striatal pathway. Figure 44 As shown, striatal tissue samples were collected from subjects in the 6-OHDA mouse Study 2 on day 91 after AAV injection. Protein levels of AADC and GDNF in both the EF1α group and the baseline group were determined by Western blotting. Unexpectedly, it was found that EF1α expressed more AADC (AADC protein) in the striatum (injection site) compared to A10-vy. Figure 44 A). Simultaneously, it was confirmed that EF1α expressed GDNF in vivo in a dose-dependent manner (A). Figure 44 B).

[0373] Furthermore, tyrosine hydroxylase (TH) staining showed that, compared with control animals that received only AAV9-GFP, more dopaminergic terminals survived in the nigrostriatal pathway of EF1α-treated animals, confirming the protective effect of the candidate rAAV. Figure 45 ).

[0374] The efficacy of AAV9-EF1α-A11-GDNF MN (EF1α) was also tested in a unilateral 6-OHDA lesion rat model of PD. This model was derived from a previous study (6-OHDA Lesion Models of Parkinson's Disease in the Rat. Animal Models of Movement Disorders: Vol. I, Neuromethods, Vol. 61, DOI 10.1007 / 978-1-61779-298-4_13). In short, 6-OHDA was injected into the right substantia nigra (coordinates AP = -4.4 mm, ML = -1.1 mm, DV = -8 mm). Two weeks after lesion surgery, EF1α was injected at two sites in the right striatum of the subjects (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 doses (low: 4.0E + 9 vg / animal, high: 4E + 10 vg / animal). A baseline dose of AAV2-A10-vy (4E + 10 vg / animal) was also administered. Spontaneous contralateral rotational behavior was recorded 21 days after AAV injection, before and after administration of L-DOPA (5 mg / kg L-DOPA + 2.5 mg / kg benserazide). Δ rotations per minute (back-forward) represents the subject's response to L-DOPA. Figure 46 As shown, EF1α significantly enhanced the response of treated subjects to low-dose L-DOPA, suggesting that this candidate treatment could enhance the bioavailability of dopamine derivatives like L-DOPA in future clinical applications. Furthermore, at the same dosing level (4E + 10 vg), EF1α showed better results compared to A10-vy.

[0375] Example 16. Expression of optimized combinations of GBA1 and CDNF or GBA1 and GDNF in MSA animal models. In vivo therapeutic efficacy of combined AAV candidates

[0376] Generates with the following characteristics Figure 33 A and Figure 33 Candidate constructs with configurations shown in B. These constructs contain a combination of a CAG or MBP promoter, the GBA1 gene (including SEQ ID NO: 45 and 47) and CDNF (SEQ ID NO: 23-25) or GDNF (SEQ ID NO: 27-29).

[0377] CAG-G11-P2A-GDNF MN-SV40 polyA (CAG-G11-GDNF MN), CAG-G12-P2A-GDNF MN-SV40 polyA (CAG-G12-GDNF MN), MBP-G11-P2A-GDNF MN-bGH polyA (MBP-G11-GDNF MN), and CAG-GDNF MN-WPRE-hGH (CAG-GDNF MN) were constructed and packaged into AAVs using serum-type AAV9. These candidate rAAV vectors were applied to U-87 MG-AAVR cells (U-87 MG cells overexpressing AAVR), and cell lysates and supernatants were collected. GC enzyme and GDNF protein levels were measured in these samples. Figure 47 ).

[0378] The efficacy of the aforementioned candidate rAAV vectors was tested in vitro in a cell model used to recreate the pathological features of PD and MSA. This cell model consisted of stable SH-SY5Y cells overexpressing both AAVR and a mutant α-synuclein (point-mutated A53T) protein. Lentiviral vectors containing either the AAVR sequence or the mutant α-synuclein (A53T) sequence were prepared by Azenta Life Science and sequentially added to SH-SY5Y cells. Following antibiotic and GFP-based selection, a stable SH-SY5Y-AAVR-A53T cell line was generated. Figure 48 As shown, the candidate rAAV vector was found to significantly reduce the level of high molecular weight (HMW) α-synuclein (a biomarker of α-syn aggregation) in cell models.

[0379] A CAG-GDNF MN-P2A-G11-SV40 polyA (CAG-GDNF MN-G11) was constructed and transfected into HEK293 cells along with CAG-G11-GDNFMN. Seventy-two hours after transfection, supernatant and cell lysate samples were collected and analyzed. It was found that CAG-GDNF MN-G11 expressed more GDNF than CAG-G11-GDNF MN in both supernatant and cell lysate, and unexpectedly maintained the ability to express GC enzymes. Figure 49 ).

[0380] The therapeutic effects of AAV delivered with optimized GBA1 + CDNF or GBA1 + GDNF combination constructs were then evaluated in a mouse model of MSA. The candidate rAAV vector, injected into the lateral ventricle (icv), rescued motor dysfunction in MSA animals. Tissue samples were collected. Reduced expression of GBA1 and NTF proteins, as well as decreased levels of α-syn and phosphorylated α-syn, were observed in the substantia nigra-striatal pathway. Furthermore, tyrosine hydroxylase (TH) staining revealed increased dopaminergic terminal survival in the striatum of treated animals, confirming the protective effect of the candidate AAV.

[0381] Example 17. In vivo therapeutic efficacy of an optimized GBA1-expressing AAV candidate in a PD mouse model.

[0382] The therapeutic effect of AAV expressing optimized GBA1 was tested in a PD mouse model.

[0383] PD models were established using genetically engineered mice that overexpressed a mutant α-synuclein (point mutation A53T). CAG-G11-hGH poly-A (as in Example 8 and...) were used. Figure 38 (As shown in A) was packaged into AAV9 to generate the rAAV vector. The candidate rAAV vector was injected into the lateral ventricle of an A53T PD mouse model to evaluate its efficacy. rAAV9-CAG-G11-hGH significantly alleviated motor insufficiency in the PD mouse model and reduced the levels of α-syn and phosphorylated α-syn in the substantia nigra-striatal pathway, suggesting its potential for treating PD and other α-syn protein disorders.

Claims

1. A recombinant adeno-associated virus vector comprising an isolated nucleic acid molecule, said isolated nucleic acid molecule comprising a first nucleotide encoding human AADC as shown in SEQ ID NO: 46, a second nucleotide encoding human GDNF as shown in SEQ ID NO: 29, and a 2A peptide; wherein the first nucleotide is upstream of the 5' end of the second nucleotide, and the two are linked by the 2A peptide.

2. The recombinant adeno-associated virus vector of claim 1, wherein the first nucleotide and the second nucleotide are linked within the frame and are operatively linked to a promoter located 5' upstream of both the first nucleotide and the second nucleotide.

3. The recombinant adeno-associated virus vector according to claim 1, wherein the 2A peptide is selected from E2A, F2A, T2A, and P2A.

4. The recombinant adeno-associated virus vector according to claim 3, wherein the 2A peptide is P2A.

5. The recombinant adeno-associated virus vector according to any one of claims 1-4, wherein the isolated nucleic acid molecule further comprises a polyadenylation signal.

6. The recombinant adeno-associated virus vector according to claim 5, wherein the polyadenylation signal is selected from SV40 polyA, human growth hormone polyA, and bovine growth hormone polyA.

7. The recombinant adeno-associated virus vector according to claim 1, wherein the recombinant adeno-associated virus vector further comprises an ITR of AAV2.

8. A viral particle comprising a recombinant adeno-associated virus vector according to any one of claims 1-7.

9. The virus particle according to claim 8, wherein the virus particle comprises a capsid protein of AAV1, AAV2, AAV5, AAV8 or AAV9.

10. A pharmaceutical composition comprising a recombinant adeno-associated virus vector according to any one of claims 1-7 or a virus particle according to claim 8 or 9, and a pharmaceutically acceptable excipient.

11. The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is used to treat neurodegenerative disorders.

12. Use of the recombinant adeno-associated virus vector according to any one of claims 1-7 or the viral particles according to claim 8 or 9 in the preparation of a medicament for treating neurodegenerative disorders, wherein the neurodegenerative disorder is Parkinson's disease.

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