Recombinant AAV vectors for treatment of neurodegenerative disorders

Abstract

There is provided 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 for 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), for use in the treatment of neurodegenerative disorders, in particular Parkinson's disease (PD), Multi-System Atrophy (MSA), Gaucher's disease (GD) and other proteinopathies. Also provided herein are viral particles comprising the rAAV vector, pharmaceutical compositions comprising the viral particles, and uses thereof.

Description

Technical Field

[0001] The present disclosure relates to the technical field of gene therapy. In particular, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector 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), and the recombinant adeno-associated virus (rAAV) vector is used to treat neurodegenerative disorders, particularly Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD) and other protein diseases. Also provided herein are virus particles comprising the rAAV vector, pharmaceutical compositions comprising the virus particles and uses thereof.

[0002] Sequence Listing

[0003] This disclosure includes a Sequence Listing as a part of this disclosure. Background Art

[0004] Neurodegenerative diseases (NDs) occur when nerve cells in the brain or peripheral nervous system gradually lose function and eventually die, leading to loss of mobility, coordination, strength, sensation, and cognition. Due to their slow and progressive nature and the lack of suitable treatments to slow disease progression or cure, NDs are associated with enormous socioeconomic and personal costs and suffering. Aging is the main risk factor for most NDs. As people live longer, this means that more people will be afflicted with NDs in the coming decades, imposing a huge economic burden on our societies and posing a huge global public health challenge.

[0005] Parkinson's disease (PD), caused by the degeneration of midbrain dopaminergic transmission and characterized by both motor symptoms (e.g., tremor and rigidity) and non-motor symptoms (e.g., mental retardation and depression), affects almost 0.3% of the general population and about 1% of people over 60 years old (de Lau, LM and MM Breteler, Epidemiology of Parkinson's disease. Lancet Neurol, 2006. 5(6): p. 525-35).

[0006] Most PD cases are sporadic and caused by unknown factors. Only a few cases have a family history. Currently, six specific human genomic loci have been identified, which contain genes whose mutations may lead to the onset of rare familial forms of PD (Klein, C. and A. Westenberger, Genetics of Parkinson's disease. Cold Spring Harb Perspect Med, 2012.2 (1): p. a008888). It is generally believed that the etiology of PD is caused by a complex interaction between intrinsic factors (like genetics) and extrinsic environmental factors. Pathological changes and their relationship with PD symptoms have long been established (Kouli, A., KMTorsney and WLKuan, Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects, TB Stoker and JC Greenland, ed. 2018: Brisbane (AU)). The pathological characteristics of PD are the degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the loss of dopaminergic pathways from the SNc to the striatum (putamen and caudate nucleus), resulting in reduced dopamine levels in the striatum and, consequently, impaired motor function. Based on this knowledge, several drugs have been developed through different approaches to restore dopamine levels.

[0007] Another pathological hallmark of PD is the formation of Lewy bodies (LBs) in the patient's brain, or so-called "Lewy pathology". LBs or Lewy neurites containing α-synuclein (α-syn) induce cytotoxicity in DA neurons and other neuronal subtypes, and are one of the main PD pathogenesis (Teil, M. et al., Targeting alpha-synuclein for PD Therapeutics: A Pursuit on All Fronts. Biomolecules, 2020.10 (3)). A variety of strategies targeting Lewy pathology are currently being studied, including stabilizing α-syn physiological conformation, reducing α-syn expression, inhibiting α-syn aggregation, and increasing α-syn clearance. In addition, Lewy pathology is not limited to dopaminergic circuits, but extends to other brain regions via intercellular propagation of α-syn aggregates, which may be one of the mechanisms leading to non-motor PD symptoms. Therefore, in theory, therapeutic treatments that address α-syn aggregation may benefit both motor and non-motor functions in PD patients.

[0008] Among the currently available treatments, L-DOPA (levodopa) is effective in treating the motor symptoms of some PD patients. However, L-DOPA has limited effects in treating non-motor symptoms or advanced PD patients. In addition, it has been shown that L-DOPA may cause serious side effects when taken in high doses (Zahoor, I., A. Shafi and E. Haq, Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects, TBStoker and JCGreenland edited 2018: Brisbane (AU)). Other treatments (such as deep brain stimulation) can only improve symptoms, but cannot prevent the progression of the disease (Dallapiazza, RF et al., Considerations for Patient and Target Selection in Deep Brain Stimulation Surgery for Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects, TBStoker and JCGreenland edited 2018: Brisbane (AU)). Therefore, there is an urgent need for better treatments for PD.

[0009] Biological PD treatments currently under investigation can be divided into three types. 1) Repairing neurotransmitter imbalance 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 / NCT03065192?term=AA DC&cond=PD&draw=2&rank=8) and lentiviral-based gene therapy (ProSavin) have shown promising results in early clinical trials, in which patients' motor function has been slightly improved. 2) Improve neuronal survival by expressing neurotrophic factors and regenerative factors. For example, neurotrophic factor (GDNF) (clinical trial at https: / / clinicaltrials.gov / ct2 / show / NCT04167540?term=GDNF&cond=PD&draw=2&rank=1) and neuro-rank protein (NRTN) (clinical trial at https: / / clinicaltrials.gov / ct2 / show / NCT00985517?term=Neurturin&draw=2&rank=1) are beneficial to the survival of neurons in the dopaminergic midbrain. 3) By targeting disease-associated genes or gene mutations, such as alpha-synuclein (SNCA), glucocerebrosidase (GBA1) (clinical trials 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 (ASOs), CRISPR, and AAV that aim to edit genes and / or regulate gene expression are currently in early clinical trials.

[0010] Therefore, there remains an unmet need for treatments for PD that can restore dopamine levels in the striatum, clear α-syn, and preserve dopaminergic transmission in the patient's brain.

[0011] 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 bodies (AD / ALB), Gaucher disease, AADC deficiency, etc.).

[0012] MSA is a rapidly progressive, sporadic, adult-onset neurodegenerative disorder. Clinical symptoms of MSA include parkinsonism (bradykinesia, rigidity, and postural instability, which are similar to Parkinson's disease); cerebellar syndrome and autonomic failure caused by brainstem nuclear degeneration. Based on the main symptoms, two clinical subtypes, MSA-P (parkinsonism) 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 features of MSA are widespread neuronal and oligodendrocyte loss and gliosis in multiple brain regions.

[0013] The etiology of MSA is still unclear, but the main pathogenic mechanism of MSA is the presence of argyrophilic filamentous glial cytoplasmic inclusions (GCIs), mainly in oligodendrocytes. GCIs are mainly composed of loosely packed filaments of α-synuclein that are 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 aggregation of α-synuclein, which is quite similar to the methods used to treat PD.

[0014] Epidemiological studies of MSA have shown a prevalence ranging from 3.4 to 4.9 / 100,000 people, with a prevalence increasing to 7.8 / 100,000 people in people over 40 years of age. Currently available treatments can only alleviate symptoms. For example, dopamine derivatives (e.g., Medopa or Duopa) are used to reduce PD-like symptoms. However, these drugs are not as effective in treating MSA-P patients as they are in treating PD patients. In addition, some MSA-P patients only respond to higher doses of these drugs, and they generally become less effective over time. Therefore, enhancing dopamine production via delivery of AADC may not benefit MSA patients as much as it has been shown for PD patients. Additional disease-modifying therapies that can delay disease progression are needed. Such therapies may potentially target three aspects of MSA pathogenesis: (1) α-synuclein aggregation, (2) cellular dysfunction and loss, and (3) neuroinflammation. There are several active clinical stage candidates, including immunogenic peptides of α-synuclein (PD01 and PD03, NCT02270489), α-synuclein aggregation inhibitors (Anle138b, NCT04208152; ATH434, NCT05109091), antisense oligonucleotides (ASOs) against α-synuclein (BIIB101, NCT04165486) and neuroprotective factors like GDNF delivered by AAV (AAV2-GDNF, NCT04680065). All of the above therapies being developed 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 its symptoms but also slowing down disease progression.

[0015] Gaucher disease (GD) is an autosomal recessive genetic disorder mainly caused by loss-of-function mutations in the GBA1 gene. GD is divided 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): Pages 274-309). Enzyme replacement therapy has been developed that is only effective for the treatment of type 1 GD because the recombinant enzyme cannot cross the blood-brain barrier (BBB). CNS-tropic AAV expressing GBA1 would be a promising approach for the treatment of nGD. An active agent clinical trial (NCT04411654) sponsored by Prevail Therapeutics delivers a codon-optimized GBA1 transgene via intra-cerebellomedullary cisterna magna (ICM) injection of rAAV9.

[0016] AADC deficiency (AADCD) is a rare autosomal recessive disorder caused primarily by loss-of-function mutations in the AADC gene. Abnormalities in the AADC protein result in severe deficiencies in some of the most important neurotransmitters for brain function, such as serotonin, dopamine, norepinephrine, and epinephrine. rAAV9 expressing functional wild-type AADC was developed for use in PTC therapeutics, called Upstaza TM (eladocagene exuparvovec) to treat this disease, Upstaza TM It has been approved by the EMEA (European Agency for the Evaluation of Medicinal Products) and the UK for the treatment of patients aged 18 months and above.

[0017] Taken together, the above evidence indicates that ND is a complex, multifaceted and debilitating disease with a huge impact on global public health. There is an urgent need to develop innovative disease-modifying treatments to slow disease progression and alleviate patient suffering. Summary of the invention

[0018] In order to develop better therapies for treating various NDs, 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. In addition, the inventors have developed rAAV vectors that express two genes of interest (GOIs) in tandem to develop therapies that synergistically target multiple disease pathologies to achieve greater therapeutic effects.

[0019] Therefore, in a first aspect, the present application provides a nucleotide sequence encoding one of the above-listed GOIs (particularly AADC, GBA1, CDNF and GDNF), wherein the nucleotide sequence is a codon-optimized sequence compared to the wild-type sequence of the GOI.

[0020] In a second aspect, the present application provides a nucleic acid construct comprising the nucleotide sequence of the first aspect operably linked to a promoter.

[0021] In a third aspect, the present application provides a nucleic acid construct comprising two nucleotide sequences operably linked to a promoter, wherein the first nucleotide sequence and the second nucleotide sequence encode two of (a), (b) and (c), respectively: (a) AADC, (b) GBA1 and (c) GDNF or CDNF. In a preferred embodiment, the nucleotide sequence encoding AADC is a codon-optimized sequence. In a specific embodiment, the nucleotide 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 is 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 embodiment, said nucleotide sequence encoding CDNF or GDNF is the nucleotide sequence of the first aspect.

[0022] In a preferred embodiment, the nucleic acid construct of the third aspect comprises a linker sequence between the GOI of the first nucleotide sequence and the second nucleotide sequence. In a specific embodiment, the linker sequence is a 2A peptide. In a specific embodiment, the linker sequence is an IRES.

[0023] In a fourth aspect, the present invention relates to a rAAV vector, the 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.

[0024] In a fifth aspect, the present application relates to a composition, such as a pharmaceutical composition, comprising the rAAV vector of the fourth aspect and a pharmaceutically acceptable excipient.

[0025] In a sixth aspect, the present application relates to a virus particle, wherein the virus particle comprises the rAAV vector of the fourth aspect.

[0026] In a seventh aspect, the present application relates to a method for treating or preventing a neurodegenerative disorder (ND) (e.g., Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD) and other proteinopathies) in a subject in need thereof, the method comprising administering the rAAV vector of the fourth aspect to the subject.

[0027] In an eighth aspect, the present application relates to the use of the rAAV vector of the fourth aspect for treating or preventing neurodegenerative disorders or for manufacturing a medicament for treating or preventing neurodegenerative disorders, wherein the neurodegenerative disorders are particularly Parkinson's disease (PD), multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD) and other proteinopathies.

[0028] The nucleic acid construct of the present invention comprises a promoter, a linker and a combination of one or two therapeutic protein coding sequences optimized specifically for rAAV vectors, so as to provide the desired expression level of the therapeutic protein (e.g., one or both of AADC, GBA1 and CDNF / GDNF). In particular, for the rAAV vector comprising two GOIs, the rAAV vector of the present invention can achieve the co-expression of two therapeutic proteins at a desired level, which will work synergistically as a new therapy to more effectively treat the expected neurodegenerative disease and achieve a better treatment outcome. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of an exemplary construct containing the AADC wild-type coding sequence and the GBA1 wild-type coding sequence linked by the P2A peptide is shown.

[0030] 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 (AADC coding sequence linked to the GBA1 coding sequence with a P2A linker). (B) Cells were transfected with constructs containing the AADC and GBA1 coding sequences in different orders. GpA: GBA1-P2A-AADC; ApG: AADC-P2A-GBA1.

[0031] Figure 3 Shown are representative images of WB showing the evaluation of the cleavage efficiency of different linkers used when both AADC and GBA were expressed in the same construct.

[0032] Figure 4 Schematic diagram of constructs containing wild-type AADC (AADC-WT) and optimized GBA1 via a P2A linker or optimized AADC and wild-type GBA1 via a P2A linker (GBA1-WT) under the control of a CBh hybrid promoter consisting of a CMV enhancer, a chicken-β-actin promoter, and a hybrid intron.

[0033] Figure 5 Shown are the AADC protein expression levels of different candidate constructs in HEK293 cells, as determined by ELISA.

[0034] Figure 6 Shown are the GBA1 protein expression levels of different candidate constructs in HEK293 cells, as determined by ELISA.

[0035] Figure 7 Shown are the AADC protein expression levels of different candidate constructs in HEK293 cells, as determined by WB.

[0036] Figure 8 It shows that Figure 7 Normalized AADC protein expression levels shown are presented as mean ± SEM from three independent WB experiments.

[0037] Fig. 9 Shown are the expression levels of GBA1 protein expressed by different candidate constructs in HEK293 cells, as determined by WB.

[0038] Fig.10 It shows that Fig. 9 Normalized GBA1 protein expression levels shown are presented as mean ± SEM from three independent WB experiments.

[0039] Fig.11 Shown is the catalytic activity of AADC in HEK293 cells transfected with different candidate constructs.

[0040] Fig.12 Shown is the catalytic activity of GBA1 protein expressed in HEK293 cells transfected with different candidate constructs.

[0041] Fig.13 Shown are the expression levels of both AADC and GBA1 proteins in HEK293 cells transfected with different combination constructs.

[0042] Fig.14 Shown are the relative activities of GBA1 protein in HEK293 cells transfected with different combinations of constructs.

[0043] Fig.15 Shown is the expression of AADC and GBA1 proteins in HEK293 cells transduced with different candidate rAAV vectors.

[0044] Fig.16 Shown are representative WB images demonstrating AADC protein expression levels in HEK293 cells transfected with different candidate constructs.

[0045] Figure 17A-17B Schematic representation of constructs containing wild-type AADC (AADC-WT) linked to optimized CDNF (A) or GDNF (B) via a P2A linker under the control of the CBh promoter is shown.

[0046] Figure 18A-18B Shown are CDNF protein expression levels in cell lysates and supernatants of HEK293 cells transfected with different candidate constructs, as determined by WB. (A) WB images; (B) normalized data.

[0047] Figure 19A-19B Shown are CDNF protein expression levels in cell lysates and supernatants of U87 cells transfected with different candidate constructs, as determined by WB. (A) WB images; (B) normalized data.

[0048] Figure 20A-20B Shown are CDNF protein expression levels in cell lysates and supernatants of HEK293 cells (A) or U87 cells (B) transfected with different candidate constructs, as determined by ELISA.

[0049] Fig.21 Shown is the protective effect of CDNF protein in supernatants of HEK293 cells transfected with different candidate constructs treated with 1.5 mM MPP+ or 40 nM rotenone, as determined by CCK-8 assay.

[0050] Fig. 22 Shown are GDNF protein expression levels in cell lysates and supernatants of HEK293 cells transfected with different candidate constructs, as determined by WB.

[0051] Fig.23 Shown are GDNF protein expression levels in cell lysates and supernatants of HEK293 cells transfected with different candidate constructs, as determined by ELISA.

[0052] Fig.24 Shown is the protective effect of GDNF protein in the supernatant of HEK293 cells transfected with different candidate constructs treated with 1.5 mM MPP+, as determined by CCK-8 assay.

[0053] Fig.25 Shown are CDNF (left panel) or GDNF (right panel) protein expression levels in supernatants of HEK293 cells transfected with the indicated constructs.

[0054] Fig.26Shown are AADC protein activities in cell lysates of HEK293 cells transfected with the indicated candidate constructs.

[0055] Fig. 27 Shown is the protective effect of CDNF in supernatants of HEK293 cells transfected with different candidate constructs treated with 1.5 mM MPP+ or 40 nM rotenone, as determined by CCK-8 assay and LDH assay.

[0056] Fig.28 Shown is the protective effect of GDNF protein in supernatants of HEK293 cells transfected with different candidate constructs treated with 1.5 mM MPP+ or 40 nM rotenone as determined by CCK-8 assay and LDH assay.

[0057] Fig.29 Shown are AADC and NTF (CDNF or GDNF) protein expression levels in cell lysates of U87-AAVR cells transduced with the indicated rAAV9 vectors, and NTF levels in the supernatants of the cells.

[0058] Fig.30 Shown are the relative activities of AADC proteins from HEK293 and U87-AAVR cells transduced with the indicated rAAV9 vectors.

[0059] Fig.31 Shown are the number of contralateral rotations obtained from study subjects in the mouse 6-OHDA-induced PD model following administration of EF1α-A11-GDNF MNs and baseline rAAV.

[0060] Fig.32 Shown are the net number of rotations obtained from study subjects in the mouse 6-OHDA-induced PD model following administration of EF1α-A11-GDNF MNs and baseline rAAV.

[0061] Figure 33A-Figure 33B Schematic representation of constructs containing optimized GBA1 linked to optimized CDNF (A) or GDNF (B) via a P2A linker under the control of a promoter (CAG or MBP promoter) is shown. "OP" refers to the optimized nucleotide sequence.

[0062] Fig.34The results of movement speed and distance measurements of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid (MPTP / P)-induced PD mice after rAAV administration with CBh-A11G11 and benchmark A10-vy, respectively, are shown. Before: data collected before MPTP modeling; After MPTP / P: data collected after induction by MPTP / P and before AAV treatment; After AAV: data collected after AAV treatment.

[0063] Fig.35 Shown are upright behaviors (by rearing on hind limbs) obtained from study subjects of the α-synuclein A53T transgenic mouse model of PD following administration of EtI7-A11G11 (EtI7) and CBh-A11G11 (CBh) rAAV. Low: 4E+9 vg, High: 4E+10 vg.

[0064] Fig.36 Representative images of WB showing the expression of AADC and GC enzymes in tissue samples collected from study subjects of the α-synuclein A53T transgenic mouse PD model following administration of EtI7-A11G11 and CBh-A11G11 rAAV are shown. Low: 4E+9 vg, High: 4E+10 vg.

[0065] Fig.37 A- Fig.37 B shows representative images (A) and normalized data (B) of WB showing the levels of phosphorylated and total α-synuclein in tissue samples collected from the α-synuclein A53T transgenic mouse PD model after administration of EtI7-A11G11 and CBh-A11G11 rAAV. Low: 4E+9 vg, High: 4E+10 vg.

[0066] Figure 38A-Figure 38B Schematic representation of constructs containing different promoters and codon-optimized GBA1 is shown.

[0067] Fig.39 Shown are the survival curves (vitality) of CBE-induced GD mice after administration of G10-p, CAG-G11, and EF1α-G11 rAAV, respectively.

[0068] Fig.40 A- Fig.40 B shows the moving distance in the open field test (A) and the latency to drop the rod in the rotarod test (B) of CBE-induced GD mice after administration of G10-p, CAG-G11 and EF1α-G11 rAAV at different doses.

[0069] Fig.41Shown are GC enzyme activities in striatal samples collected from CBE-induced GD mice after administration of G10-p, CAG-G11, and EF1α-G11 rAAV at different doses, respectively.

[0070] Fig.42 Shown are representative WB images showing protein expression of AADC and CDNF in cell lysates of HEK293 cells transfected with the indicated candidate constructs and CDNF protein levels in the supernatants of the cells.

[0071] Fig.43 Shown are the net rotation times (amphetamine-induced ipsilateral rotations) of 6-OHDA-induced PD model mice following dosing with the indicated doses of CAG-A11-GDNF MNs, EF1α-A11-GDNF MNs, and baseline A10-vyrAAV.

[0072] Fig.44 A- Fig.44 B shows AADC (A) and GDNF (B) protein expression levels in striatal samples collected from 6-OHDA-induced PD model mice after dosing with the indicated amounts of EF1α-A11-GDNF MNs and benchmark A10-vy rAAV, as determined by ELISA.

[0073] Fig.45 Shown are representative images showing tyrosine hydroxylase (TH) levels in substantia nigra samples collected from 6-OHDA-induced PD model mice following administration of the indicated amounts of EF1α-A11-GDNF MNs and baseline A10-vy rAAV, as determined by immunohistochemistry (IHC). Blank: healthy control.

[0074] Fig.46 The number of delta rotations (L-DOPA-induced contralateral rotations) obtained from 6-OHDA-induced PD model rats after administration of the indicated doses of EF1α-A11-GDNF MN and baseline A10-vy rAAV are shown. Blank represents healthy control.

[0075] Fig.47 Representative WB images showing protein expression of GCase and GDNF in cell lysates of U87-MG-AAVR cells treated with different candidate rAAV vectors and GDNF protein levels in the supernatants of the cells are shown.

[0076] Fig.48 Representative WB images showing the protein levels of high molecular weight α-syn (HMW) of SH-SY5Y-AAVR-A53T cells treated with different candidate rAAV vectors are shown.

[0077] Fig.49 Representative WB images showing protein expression of GCase and GDNF in cell lysates of HEK293 cells transfected with different candidate constructs and GDNF protein levels in the supernatants of the cells are shown. DETAILED DESCRIPTION

[0078] Unless specifically defined elsewhere in this document, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0079] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0080] In the context of the present disclosure, unless otherwise indicated, the word "comprise" and its variants such as "comprises" and "comprising" will be understood to imply the inclusion of the elements, such as amino acid sequences, nucleotide sequences, characteristics, steps or groups thereof, but not to exclude any other elements, such as amino acid sequences, nucleotide sequences, characteristics and steps. When used herein, the term "comprises" or any variants thereof may be replaced with the terms "containing", "including" or sometimes "having" or their equivalent variants. In certain embodiments, the word "comprising" also includes the situation of "consisting of".

[0081] As used herein, the term "gene" refers to nucleic acid (e.g., DNA, such as genomic DNA and cDNA) encoding RNA transcripts and their corresponding nucleotide sequences. As used herein, the term for genomic DNA may include non-coding regions and regulatory regions in between, 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, a "gene" only includes the coding sequence (e.g., "open reading frame" or "coding region") necessary for encoding a polypeptide. 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. Gene may refer to "endogenous genes" or natural genes. Gene may refer to "foreign genes" or non-natural genes. Non-natural genes may refer to genes that are not usually found in host organisms but are introduced into host organisms by gene transfer. A non-natural gene may also refer to a gene that is not located in its natural position in the genome of an organism. A non-natural gene may also refer to a naturally occurring nucleic acid containing a mutation, insertion and / or deletion (e.g., a non-natural sequence), e.g., a codon-optimized nucleotide sequence. In the context of the present application, unless otherwise indicated, "GOI" specifically refers to a CDS region, i.e., a sequence encoding amino acids in a protein.

[0082] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides of any length. Polynucleotides may be exogenous or endogenous to a cell. Polynucleotides may be present in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Polynucleotides may contain one or more analogs (e.g., altered backbones, sugars, or nucleobases).

[0083] As used herein, "cassette" or "expression cassette" refers to a DNA component contained in a vector (e.g., a plasmid vector or a viral vector) and consists of one or more genes under the control of regulatory sequences to be expressed in a host cell transduced by the vector.

[0084] As used herein, "operably linked" is used to describe that two or more components (especially nucleotide sequences) are linked in a manner such that each component can perform its designated function.

[0085] "AAV" refers to adeno-associated virus.

[0086] "AADC" refers to aromatic L-amino acid decarboxylase. AADC is also known as DOPA decarboxylase (DDC).

[0087] "GBA1" refers to glucocerebrosidase that cleaves the β-glucosidic linkage of glucocerebroside. In some cases, "GBA1" may be referred to interchangeably as "GBA," "GCB," or "GLUC."

[0088] "NTF" refers to neurotrophic factors, which are a group of support proteins that promote the development and maintenance of neurons. Both CDNF and GDNF belong to the neurotrophic factors.

[0089] "CpG island" refers to a region in the genome that is rich in CpG sites. "CpG site" refers to two consecutive nucleotides consisting of cytosine (C) and guanine (G) in the 5' to 3' direction.

[0090] "2A peptides" refers to a group of short (18-22 amino acids) self-cleaving peptides derived from viruses. 2A peptides produce ribosome skipping during translation, resulting in separation between the end of the 2A sequence and the downstream protein.

[0091] "IRES" refers to internal ribosome entry site.

[0092] "Proteinopathy" refers to a neurodegenerative disorder in which structurally abnormal proteins (eg, α-synuclein) accumulate, resulting in the formation of aggregates or inclusions in axons of neurons or in oligodendrocytes.

[0093] In the context of the present application, "subject" refers to an animal, preferably a mammal, such as a primate, such as a cynomolgus monkey, preferably a higher primate, such as a human. Unless otherwise indicated, in the context of the present application, the term "subject" is interchangeable with the term "patient" or "individual".

[0094] AADC

[0095] The present disclosure provides rAAV vectors that deliver the aromatic L-amino acid decarboxylase (AADC) gene alone or in combination with another gene of interest (eg, GBA1, CDNF, or GDNF). rAAV vectors that deliver AADC can be used to treat ND, such as PD or AADCD.

[0096] The biosynthetic pathway for dopamine requires both tyrosine hydroxylase (TH), which converts tyrosine to 1-3,4-hydroxyphenylalanine (L-Dopa), and aromatic L-amino acid decarboxylase (AADC), which decarboxylates L-Dopa to produce dopamine.

[0097] It is known that AADC levels decline as PD progresses, and L-DOPA taken by patients cannot be effectively converted into dopamine at axon terminals in the striatum. This requires more frequent and increased doses of L-DOPA to achieve an adequate clinical response. However, increasing the dose of L-DOPA may bring undesirable side effects, such as L-Dopa-induced dyskinesia (LID).

[0098] The potential of AAV intra-putamen AADC delivery in the treatment of PD 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): p. 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): p. 704-714). Considering that restoring AADC levels will "rescue" and enhance the therapeutic effect of L-DOPA, and dopamine levels are the most critical factor in restoring motor function, AADC is included in the candidate construct to rescue dopamine levels, which will be particularly beneficial for AADC-deficient patients.

[0099] AADC deficiency is caused by loss-of-function mutations in the AADC gene. The rAAV of the present invention comprises AADC as the only GOI or a combination of AADC and another GOI to compensate for the molecular defect that causes the disease.

[0100] In a preferred embodiment, the nucleotide sequence encoding AADC can be optimized for expression in a rAAV construct. The optimization can be codon optimization.

[0101] In one embodiment, the rAAV of the present application comprises a nucleotide sequence encoding AADC, and the nucleotide sequence encoding AADC comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 1-9, 11 and 46, preferably any one of SEQ ID NOs: 1-9 and 46 (optimized sequence), and more preferably a nucleotide sequence 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 AADC as the only GOI can be used to treat PD or AADCD.

[0102] In some embodiments, the rAAV of the present application comprises a combined construct containing both AADC and GBA1, for example, for the treatment of PD. In one embodiment, the combined construct comprises the coding sequence of AADC and the coding sequence of GBA1. In some embodiments, the coding sequence of GBA1 is arranged 5' upstream of the coding sequence of AADC. In a more preferred embodiment, the coding sequence of AADC is arranged 5' upstream of the coding sequence of GBA1. Preferably, the two coding sequences are arranged in frame and under the control of the same promoter. Preferably, the nucleotide sequence encoding GBA1 comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NO: 12-20, 45 or 47. Preferably, the nucleotide sequence encoding AADC comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NO: 1-9 and 46.

[0103] In a specific embodiment, the combined construct of AADC and GBA1 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 a specific embodiment, the combined construct of AADC and GBA1 comprises, from 5' to 3': a promoter sequence that is a truncated variant of the EF1α promoter, such as the sequence (EFIt7) as 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 poly A sequence, such as the hGH poly A sequence. In a specific embodiment, the combined construct of AADC and GBA1 comprises, from 5' to 3': 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 poly A sequence, such as the hGH poly A sequence.

[0104] In some embodiments, the rAAV of the present application comprises a combination construct containing both AADC and NTF selected from GDNF or CDNF, for example, for the treatment of AADCD. In one embodiment, the combination construct comprises the coding sequence of AADC and the coding sequence of GDNF or CDNF. In some embodiments, the coding sequence of GDNF or CDNF is arranged 5'upstream of the coding sequence of AADC. In a more preferred embodiment, the coding sequence of AADC is arranged 5'upstream of the coding sequence of GDNF or CDNF. Preferably, the two coding sequences are arranged in frame and under the control of the same promoter. Preferably, the nucleotide sequence encoding AADC comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 1-9 and 46. Preferably, the nucleotide sequence encoding GDNF comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 27-29. Preferably, the nucleotide sequence encoding CDNF comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 23-25.

[0105] In some embodiments, the rAAV of the present application comprises a combined construct containing a coding sequence of AADC and a coding sequence of CDNF, for example, for the treatment of AADCD. In a 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 as shown in SEQ ID NO:25 (CDNF-MN). In a specific embodiment, the combined construct of AADC and CDNF comprises from 5' to 3': 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 as shown in SEQ ID NO:25 (CDNF-MN). In a specific embodiment, the combined construct of AADC and CDNF 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 CDNF as shown in SEQ ID NO:25 (CDNF-MN). In a specific embodiment, the combined construct of AADC and CDNF comprises from 5' to 3': 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.

[0106] In some preferred embodiments, the rAAV of the present application comprises a combined construct containing a coding sequence of AADC and a coding sequence of GDNF, for example, for the treatment of AADCD. In a 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 as shown in SEQ ID NO:29 (GDNF-MN). In a 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 as shown in SEQ ID NO:29 (GDNF-MN).

[0107] GBA1

[0108] The present disclosure provides rAAV vectors that deliver the glucocerebrosidase 1 (GBA1) gene alone or in combination with another gene (e.g., ADCC, CDNF, or GDNF). The rAAV vectors that deliver GBA1 can be used to treat ND, including Gaucher disease (GD), Parkinson's disease (PD), and multiple system atrophy (MSA). For example, the rAAV vectors that deliver GBA1 alone can be used to treat Gaucher disease (GD) or PD type 2 and 3. For example, the rAAV vectors that deliver both GBA1 and NTF (such as GDNF) can be used to treat MSA.

[0109] In a preferred embodiment, the nucleotide sequence encoding GBA1 may be optimized for expression by a rAAV construct. The optimization may be codon optimization.

[0110] In one embodiment, the rAAV of the present application comprises a nucleotide sequence encoding GBA1, and the nucleotide sequence encoding GBA1 comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 12-20, 22, 45 and 47, preferably any one of SEQ ID NOs: 12-20, 45 or 47 (codon-optimized sequences), and more preferably a nucleotide sequence as shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 45 or SEQ ID NO: 47.

[0111] In some embodiments, the rAAV of the present 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 a nucleotide sequence as shown in any one of SEQ ID NOs: 12-20, 45 or 47. In a specific embodiment, the rAAV comprises a coding sequence for GBA1 as shown in SEQ ID NO: 45. In a specific embodiment, the rAAV comprises a construct comprising, from 5' to 3': 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 poly A sequence, such as a hGH poly A sequence. In a specific embodiment, the rAAV comprises a construct comprising, from 5' to 3': 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 WPRE; and a poly A sequence, such as a hGH poly A sequence.

[0112] In some embodiments, the rAAV of the present application comprises a combined construct containing both GBA1 and 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 NTF. In some embodiments, the coding sequence of NTF is arranged 5' upstream of the coding sequence of GBA1. Preferably, the two coding sequences are arranged in frame and under the control of the same promoter.

[0113] In some embodiments, the rAAV of the present 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 a nucleotide sequence as shown in any one of SEQ ID NOs: 12-20, 45 or 47. Preferably, the nucleotide sequence encoding GDNF comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 27-29. In a specific embodiment, the combined construct of GBA1 and GDNF comprises a coding sequence of GBA1 as shown in SEQ ID NO: 45 or SEQ ID NO: 47 and a coding sequence of GDNF as shown in any one of SEQ ID NOs: 27-29. In a specific embodiment, the combined construct of GBA1 and GDNF comprises, from 5' to 3': a CAG promoter sequence (CAG) as shown in SEQ ID NO:58 or a 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 as shown in SEQ ID NO:29 (GDNF-MN); optionally WPRE; and a poly A sequence, such as SV40 poly A or bGH poly A. In a specific embodiment, the combined construct of GBA1 and GDNF comprises, from 5' to 3': a CAG promoter sequence (CAG) as shown in SEQ ID NO:58 or a 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 poly A sequence, such as SV40 poly A or bGH poly A.

[0114] Neurotrophic factors: CDNF and GDNF

[0115] The present disclosure provides rAAV vectors that deliver a neurotrophic factor (particularly CDNF or GDNF) alone or in combination with another gene (eg, ADCC or GBA1). rAAV vectors that deliver neurotrophic factors can be used to treat ND.

[0116] The neuroprotective effects of neurotrophic factors have been well established. Both CDNF and GDNF proteins have been studied in the framework of PD (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): p. 475-489).

[0117] In one embodiment, AADC is co-expressed with CDNF or GDNF, respectively, by rAAV vectors. This is the first example of AADC and neurotrophic factors co-expressed in the same AAV vector and showing significant efficacy in a mouse PD model.

[0118] In another embodiment, GBA1 is co-expressed with GDNF or CDNF via a rAAV vector.

[0119] In a preferred embodiment, the nucleotide sequence encoding CDNF or GDNF can be optimized for expression by rAAV constructs. The optimization can be codon optimization. Codon optimization can be limited to the coding region of the mature protein of GDNF or CDNF without changing the nucleotide sequence corresponding to the signal peptide of 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.

[0120] In one embodiment, the rAAV of the present application comprises a nucleotide sequence encoding CDNF, and the nucleotide sequence encoding CDNF comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 23-25.

[0121] In one embodiment, the rAAV of the present application comprises a nucleotide sequence encoding GDNF, and the nucleotide sequence encoding GDNF comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 27-29.

[0122] Expression cassette

[0123] The term "expression cassette" herein refers to a DNA component which is contained in a vector (e.g., a rAAV vector) and consists of one or more (particularly one or two) GOIs selected from AADC, GBA1 and NTF (CDNF or GDNF) genes under the control of regulatory sequences to be expressed in a host cell transduced by the vector.

[0124] In the context of the present application, the term "combination construct" refers to a construct comprising two GOIs, in particular two GOIs selected from AADC, GBA1 and NTF (CDNF or GDNF) genes. In a preferred embodiment, the two gene coding sequences in the combination construct are under the control of the same promoter.

[0125] In one embodiment, the expression cassette of the present application is characterized by the expression of a GOI, preferably a codon-optimized GOI sequence, in particular those as described in the present disclosure.

[0126] In one embodiment, the expression cassette of the present application is characterized by co-expression of two GOIs separated by a linker sequence. In other embodiments, one or both GOIs are expressed via codon-optimized sequences, in particular those as described in the present disclosure. For example, the two GOIs can be selected from the following combinations: AADC+GBA1, AADC+CDNF, AADC+GDNF, GBA1+GDNF and GBA1+CDNF.

[0127] By optimizing the cDNA sequences (codons) of the AADC gene, the GBA1 gene, the CDNF gene, and the GDNF gene, their regulatory sequences, and optionally the linker, the expression cassette of the present disclosure inserted into the AAV vector can achieve higher and more consistent protein expression or co-expression in neuronal cells in vitro or in vivo. For example, the expression cassette of the present disclosure shows better 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 cassette of the present disclosure shows better performance in the expression of one or more GOIs in neuronal cells in the striatum, substantia nigra, or other CNS regions in vivo.

[0128] In one embodiment, as an essential part of the expression cassette, the present disclosure first provides a set of wild-type or codon-optimized nucleotide sequences, which encode an AADC protein and a second protein selected from a GBA1 protein, a CDNF protein and a GDNF protein, in particular, a human AADC protein having an amino acid sequence as shown in SEQ ID NO: 31, a human GBA1 protein having an amino acid sequence as shown in SEQ ID NO: 32, a human CDNF protein having an amino acid sequence as shown in SEQ ID NO: 33, and a human GDNF protein having an amino acid sequence as shown in SEQ ID NO: 34.

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

[0130] "Isolated nucleic acid" means DNA or RNA removed from all or part of a polynucleotide, wherein the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. An isolated nucleic acid molecule "comprising" a particular nucleotide sequence may include, in addition to the specified sequence, operably linked regulatory sequences that control the expression of the coding region of the nucleic acid sequence. Due to codon degeneracy, those skilled in the art understand that any particular amino acid sequence may be encoded by several different nucleotide sequences.

[0131] The "codon-optimized coding sequence" herein refers to a nucleotide sequence encoding a protein (such as an AADC protein, a GBA1 protein, a CDNF protein, or a GDNF protein), which is 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 generally show increased translation efficiency of the gene of interest (GOI), resulting in higher protein expression. Tools (e.g., JCat) with embedded algorithms for designing codon-optimized coding sequences are readily available to those skilled in the art. In a preferred embodiment, the codons of the AADC coding sequence of the present invention application have a codon adaptation index (CAI) greater than 0.8. CAI is a measure of codon bias. Those skilled in the art will understand that the actual efficiency of any sequence generated by running the algorithm still needs to be verified by experiment.

[0132] In a preferred embodiment, the codon-optimized coding sequence of the human AADC protein comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 1-10 and 46; the codon-optimized coding sequence of the human GBA1 protein comprises or consists of a nucleotide sequence of SEQ ID NOs: 12-21, 45 and 47; the codon-optimized coding sequence of the human CDNF protein comprises or consists of a nucleotide sequence of SEQ ID NOs: 23-25; the codon-optimized coding sequence of the human GDNF protein comprises or consists of a nucleotide sequence of SEQ ID NOs: 27-29.

[0133] In addition, the expression cassette may also contain one or more regulatory sequences in addition to the coding sequence. The regulatory sequence may be selected from one or more of the following: a promoter, an enhancer, a polyadenylation sequence, and a translation termination signal. Specific combinations of regulatory sequences of the present disclosure may achieve unexpected results in improving the expression efficiency of the coding sequence.

[0134] "Promoter" refers to a DNA sequence that can initiate transcription of a downstream gene 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. The promoter can be a naturally occurring promoter of a gene, a modified form of a naturally occurring promoter, or a synthetic promoter.

[0135] In a preferred embodiment, the promoter of the present disclosure may be a constitutive promoter. In a preferred embodiment, the promoter may be a CBh promoter, an EF1α promoter, a CAG promoter, an MBP promoter (myelin basic protein promoter) or a promoter derived therefrom.

[0136] An "enhancer" is a regulatory DNA sequence that can enhance the transcription of a GOI in rAAV together with a promoter. In a preferred embodiment, the expression cassette of the present application comprises an enhancer. More preferably, the enhancer can be a CMV enhancer, for example in a CBh promoter.

[0137] In some embodiments, intron sequences that act as enhancers may be included. For example, intron sequences derived from introns of a GOI may be included in the expression cassette.

[0138] In some cases, the promoter together 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.

[0139] 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, about or less than 400 bp.

[0140] For example, the intron sequence of the present disclosure is derived from a target gene. For example, the intron sequence is composed of one or more fragments derived from one or more intron regions of a target gene.

[0141] In preferred embodiments, due to the limited packaging capacity of AAV, the length of the promoter or promoter / intron element does not exceed 1000 bp, does not exceed 900 bp, does not exceed 850 bp, does not exceed 800 bp, does not exceed 700 bp, does not exceed 600 bp, does not exceed 500 bp or does not exceed 400 bp.

[0142] In some cases, when the intron sequence is derived from an intron region of a gene of interest, the intron sequence can be inserted into a coding sequence (e.g., a codon-optimized coding sequence) at a position corresponding to its position in the gene in nature, for example, between two exons rather than at a position 5' upstream of the coding sequence, and constitute a promoter / intron element.

[0143] The Kozak consensus sequence (Kozak sequence), named after the scientist who discovered it, is a nucleic acid sequence motif present in most eukaryotic mRNA transcripts that functions as a protein translation start site. The Kozak sequence ensures that proteins are accurately and efficiently translated.

[0144] In a specific embodiment, the expression cassette comprises a CMV enhancer, a chicken β-actin promoter, a first coding sequence of a first GOI, a linker, a second coding sequence of a second GOI, and SV40 poly A, wherein the first coding sequence and the second coding sequence are independently selected from those disclosed herein and encode any of the following combinations of two GOIs: AADC+GBA1, AADC+CDNF, AADC+GDNF, GBA1+GDNF, and GBA1+CDNF.

[0145] Adapter sequence

[0146] In one aspect, the present application provides a linker sequence that produces high efficiency and fidelity when used to connect two coding sequences of the present application to be co-expressed by an rAAV vector of the present application.

[0147] As examples of linker sequences, sequences encoding 2A peptides (such as P2A, F2A or E2A) or IRES (the full-length form is referred to herein as "ECMV IRES", and the shorter truncated form is referred to herein as "mini-IRES") can be used to connect the two coding sequences of the present application. The position of one or more GOIs relative to the linker sequence can be adjusted to achieve the desired performance in terms of protein expression and function. In a preferred embodiment, a P2A linker sequence is used between two GOIs in rAAV.

[0148] In a specific embodiment, the linker sequence of the present application comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 35, 37, 39, 41, 43 or 44.

[0149] Promoter

[0150] The rAAV vector of the present invention may comprise a promoter conventionally used in rAAV vectors, including a naturally occurring promoter, a variant thereof, or a hybrid thereof.

[0151] In some embodiments, the promoter may be a truncated variant of a wild-type promoter. For example, the promoter may be a truncated version of the EF1α promoter as shown in Table 2 of Example 6, having a nucleotide sequence as shown in any one of SEQ ID NOs: 50-56.

[0152] In some embodiments, the promoter of the present invention application can also be used with an enhancer, which may be natural or not natural to the promoter. For example, the CBh promoter is a hybrid of a CMV enhancer and a chicken beta-actin (CBA) promoter. In this case, mentioning a promoter means including an enhancer, which can be understood by those skilled in the art. In one embodiment, the rAAV vector of the present invention application includes a CBh promoter, for example, a CBh promoter having a nucleotide sequence as shown in SEQ ID NO:60.

[0153] In another embodiment, to drive the co-expression of AADC and CDNF or the co-expression of AADC and GDNF, an EF1α promoter is used, for example, an EF1α promoter having a nucleotide sequence as 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 a nucleotide sequence as shown in any one of SEQ ID NOs: 50-56. On the other hand, to drive the co-expression of GBA1 and GDNF, a CAG promoter having a nucleotide sequence as shown in SEQ ID NO: 58 or an MBP promoter having a nucleotide sequence as shown in SEQ ID NO: 59 (or a truncation thereof) is used.

[0154] Poly A signal

[0155] The rAAV vector of the present invention also contains a polyadenylation signal (poly A).

[0156] For example, poly A sequences that can be used in the present application include SV40 poly A, human growth hormone (hGH) poly A, and bovine growth hormone (bGH) poly A.

[0157] In one embodiment, the expression cassette of the present disclosure comprises hGH poly A. In one embodiment, when the promoter is the EF1α promoter or a variant thereof, the expression cassette of the present disclosure comprises hGH poly A.

[0158] In some embodiments, a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) is placed downstream of the GOI and proximal to the polyadenylation signal.

[0159] AAV vector serotype

[0160] In a preferred embodiment, the rAAV of the present invention is an AAV9 vector.

[0161] Based on previous reports, the dispersion of intraparenchymal injected AAV9 at the target site is superior 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 macaque cerebral cortex. Neurosci Res, 2015. 93: p. 144-57). Since the human striatum is a relatively large brain region, in order to achieve a wider coverage of genes delivered by AAV, molecular engineering of wild-type AAV capsids is necessary.

[0162] The inventors have identified new AAV serotypes that have significantly improved tissue tropism compared to currently known AAVs for the putamen, the intended target area for AAV-based gene therapy in treating PD. Therefore, in another preferred embodiment, the rAAV of the present application uses a new AAV capsid.

[0163] Exemplary constructs

[0164] In one embodiment, the present application provides an expression cassette, the expression cassette comprising from 5' to 3':

[0165] (a) 5'ITR;

[0166] (b) promoter;

[0167] (c) optionally intron sequences;

[0168] (d) the coding sequence of GOI;

[0169] (e) poly A; and

[0170] (f) 3'ITR,

[0171] The GOI is selected from AADC, GBA1, CDNF or GDNF, and the coding sequence is a codon-optimized sequence.

[0172] In another embodiment, the present application provides an expression cassette, the expression cassette comprising from 5' to 3':

[0173] (g) 5'ITR;

[0174] (h) promoter;

[0175] (i) optionally an intron sequence;

[0176] (j) a coding sequence of a first GOI;

[0177] (k) a linker sequence;

[0178] (1) a coding sequence of a second GOI;

[0179] (m) poly A; and

[0180] (n) 3'ITR,

[0181] wherein the first coding sequence and the second coding sequence are independently selected from a wild-type coding sequence or a codon-optimized sequence (e.g., as described in the present 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 may be arranged in any order.

[0182] In the constructs indicated in the above two paragraphs, each element can be independently selected from those described in the present disclosure. For example, the first nucleotide sequence can be an AADC coding sequence as shown in any one of SEQ ID NOs: 1-9, 11 and 46; a GBA1 coding sequence as shown in any one of SEQ ID NOs: 12-20, 22, 45 and 47; or a CDNF or GDNF coding sequence as shown in any one of SEQ ID NOs: 23-30. For example, the second nucleotide sequence can be an AADC coding sequence as shown in any one of SEQ ID NOs: 1-9, 11 and 46; a GBA1 coding sequence as shown in any one of SEQ ID NOs: 12-20, 22, 45 and 47; or a CDNF or GDNF coding sequence as shown in any one of SEQ ID NOs: 23-30. Preferably, the first nucleotide sequence can be an AADC coding sequence as shown in SEQ ID NOs: 3 or 46; or a GBA1 coding sequence as shown in any one of SEQ ID NOs: 16, 45 and 47. Additionally or alternatively, in preferred embodiments, 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 nucleotide sequence and the second nucleotide sequence are linked by a P2A linker sequence.

[0183] Pharmaceutical composition

[0184] The term "pharmaceutical composition" refers to a composition suitable for delivery to a subject. The pharmaceutical composition of the present disclosure comprises an isolated nucleic acid, a rAAV vector or viral particle of the present disclosure, and a pharmaceutically acceptable excipient. Conventional pharmaceutically acceptable excipients are known in the art and can be solid or liquid excipients. In one embodiment, the pharmaceutical composition can be a liquid for injection.

[0185] Delivery Method

[0186] As used herein, the terms "administration", "administering", "treating", and "treatment" refer to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with a subject, cell, tissue, organ, or biological fluid when applied to a subject (e.g., an animal, including a human) or to a cell, tissue, organ, or biological fluid. Treatment of cells encompasses contact of an agent with a cell and contact of an agent with a fluid, wherein the fluid is in contact with the cell. The terms "administration" and "treatment" also include in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic agent, a binding compound, or by another cell.

[0187] In a preferred embodiment, the rAAV vector of the present application can be delivered via intravenous, intraventricular, intrathecal or intrastriatal administration. In a specific embodiment, the rAAV vector is delivered via an intrastriatal route. In another specific embodiment, the rAAV vector is delivered via an intraventricular route. In a most preferred embodiment, treatment or administration is carried out by intracerebroventricular (ICV) (such as by ICV injection).

[0188] The rAAV vector can be administered via a single dose or multiple doses. In a specific embodiment, the rAAV vector is administered via a single injection.

[0189] The dose of rAAV vector injection can vary based on the route of administration. For example, given that loss of dopaminergic transmission in the striatum leads to motor problems in PD patients, intraparenchymal / intrastriatal injections often require the delivery of a specific volume of rAAV to cover as much of the striatum as possible to achieve adequate therapeutic effect. The dose can also vary based on the subject’s weight. Thus, the dose range can range from 1.5 × 10 10 -1.5×10 14 vg / kg in a wide range.

[0190] Therapeutic Uses

[0191] The terms "treat," "treating," or "treatment" include curing or at least alleviating the symptoms of a neurodegenerative disorder such as PD, MSA, GD, AADCD or other proteinopathy.

[0192] 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 diagnostic criteria for Parkinson's disease. Mov Disord, 2015. 30(12): pp. 1591-601; Palma, JA, L. Norcliffe-Kaufmann and H. Kaufmann, Diagnosis of multiple systematrophy. Auton Neurosci, 2018. 211: pp. 15-25).

[0193] A viral vector expressing one of the aforementioned genes or co-expressing two of the aforementioned genes can be used to treat a subject suffering from the aforementioned neurodegenerative disorder. The subject can be a clinically diagnosed early PD patient with one or more mutations in the AADC gene, GBA1 gene or other PD genetic risk genes (like SNCA). The subject can also be a clinically diagnosed late PD patient with no mutation or one or more mutations in the above-mentioned AADC gene, GBA1 gene or other PD genetic risk genes. The subject can be a subject who has previously been treated with a dopamine derivative (like Medopa) or is being treated with a dopamine derivative, with or without L-DOPA-induced dyskinesia. The subject can also be a subject who is resistant to currently available treatments.

[0194] The subject may be a clinically diagnosed GD patient classified as type 2 or type 3 or as type 1 with central nervous system symptoms. The subject may also be a clinically diagnosed MSA patient who has shown parkinsonism (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 suggestive of autonomic dysfunction, with or without GBA1 gene mutation or decreased GBA1 protein activity.

[0195] The subject may also be a clinically diagnosed MSA patient resistant to currently available treatments like dopamine derivatives.

[0196] Example

[0197] In order to facilitate understanding and utilization of the present invention, the advantages of the present invention will be described in more detail with reference to Examples and drawings. However, it should be understood that the following examples are only intended to illustrate the present invention without any intention to limit the scope of the present invention. The scope of the present invention should be limited by the claims.

[0198] Example 1. Determination of the Optimal Linker for Connecting the Coding Sequences of AADC and GBA1

[0199] In this example, the performance of different linker sequences, specifically E2A (SEQ ID NO:35), F2A (SEQ ID NO:37), T2A (SEQ ID NO:39), P2A (SEQ ID NO:41), mini-IRES (SEQ ID NO:43), and ECMV IRES (SEQ ID NO:44), were tested in constructs containing wild-type coding sequences for both AADC and GBA1.

[0200] 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 via the self-cleaving P2A sequence in the following order: AADC sequence placed under the control of the CBh promoter (SEQ ID NO: 60), followed by P2A, and then GBA1 sequence ( Figure 1 ). Another construct having the GBA1 coding sequence 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 they were transfected into HEK293 cells (Procell, CL-0001). Plasmids containing the coding sequence of AADC or GBA1 alone ("AADC", "GBA") or together with the coding sequence of P2A ("AADC-P2A", "P2A-GBA") were also constructed and transfected into the same cells for comparison. A plasmid expressing GFP was used as a control.

[0201] Western blotting was performed to detect the expression of AADC and GBA1 of each construct. In particular, cells were collected in lysis buffer (RIPA buffer, Thermo fisher 89901) 72 hours after transfection, denatured at 95°C for 15 min in 5×SDS-PAGE sample loading buffer (Beyotime, P0015L), separated in 10% SDS-PAGE gel (Sangon, #C631100), and blotted onto 0.22 μm PVDF membrane (Merck Millipore). The protein expression levels of AADC-WT, GBA1-WT and housekeeping gene GAPDH were detected by antibodies against human AADC (Millipore, #AB1569), GBA1 (Sigma, #G4171) and GAPDH (CST, #2118), respectively. Long-term exposure and normal exposure were performed using WB.

[0202] like Figure 2 As shown in A, the GBA1 protein expressed by the plasmid AADC-P2A-GBA1(ApG) has almost the same molecular weight as that of GBA1 expressed by a plasmid containing only GBA1, while the AADC derived from ApG is slightly larger than that expressed by a plasmid containing only AADC due to the P2A fragment attached thereto. This result indicates that the self-cleaving 2A peptide is functional in this environment.

[0203] Unexpectedly, 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 Figure 2 B).

[0204] Additional linkers including E2A, F2A, T2A, P2A, mini-IRES, and ECMV IRES were evaluated in the same experimental setting as P2A. The results showed that, except for mini-IRES, all linkers evaluated produced both AADC and GBA1 proteins with the expected molecular size.

[0205] Additional tests were performed by extending the exposure time to find out if any fusion protein (AADC+GBA1) was present due to incomplete cleavage. Clear positive bands were detected with both AADC and GBA1 antibodies in the E2A and F2A constructs (see Figure 3 ). The cleavage efficiency of different linkers is ranked from high to low as ECMV IRES, P2A, T2A, E2A, and then F2A. Using the full-length ECMV IRES as a linker will produce a construct that exceeds the packaging capacity of rAAV. Therefore, P2A was selected as the linker for the present invention.

[0206] Example 2. Codon optimization for improving protein expression of AADC and GBA1

[0207] In this example, nine sequences containing higher 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. The codon adaptation index (CAI) of all these sequences was greater than 0.85, as calculated by an online tool (https: / / www.genscript.com / tools / rare-codon-analysis). The AADC coding sequence and GBA1 coding sequence disclosed in the previous patent application publication were synthesized, and they were used as references and named A10 (SEQ ID NO: 10, disclosed in CN 107106689A, Voyager Therapeutics Inc.) and G10 (SEQ ID NO: 21, disclosed in WO 2020210698A1, Prevail Therapeutics, Inc.), respectively. A10 encodes an AADC protein with an M17V mutation.

[0208] Produce a Figure 4 Twenty candidate constructs (see Table 1) of the configurations shown: one of the codon-optimized AADC coding sequences (A1-A10) was combined with the GBA1-WT (G0) sequence, or the AADC-WT (A0) sequence was combined with one of the codon-optimized GBA1 coding sequences (G1-G10), wherein P2A was used as a linker between the two coding sequences. Vectors containing G0 (wild-type GBA1 alone), A0 (wild-type AADC alone), and G0A0 were used as controls.

[0209] Table 1. Candidate recombinant constructs

[0210]

[0211]

[0212] The vectors were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Invitrogen, #L3000008) according to the manufacturer's instructions. 48 hours after 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.

[0213] ELISA for detection of AADC and GBA1 protein levels

[0214] 1. On day 1, a 96-well plate was coated with the following capture antibodies in 100 μL coating buffer overnight at 4°C: 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.

[0215] 2. On the next day, the sample wells were washed three times with washing buffer. 300 μL of blocking buffer (containing 2% BSA) was added and left at room temperature for about 2 h, followed by another three washing steps.

[0216] 3. Add 100 μL of assay buffer containing the following detection antibodies to each well: anti-DDC monoclonal antibody (HRP) for AADC protein detection (Sinobiological, 10560-R040, 1:1000 dilution) and polyclonal anti-GBA antibody for GBA1 protein detection (Abcam, ab96246). After incubation at room temperature for about 1 hour, wash the sample wells 3 times with wash buffer.

[0217] 4. To detect GBA1 protein expression, HRP-conjugated antibody (goat anti-rabbit IgG-Fc secondary antibody (HRP) (SinoBiological, SSA003)) was applied, followed by a washing step.

[0218] 5. The sample wells were then incubated with 100 μL TMB solution (Solarbio, #PR1200) for about 20 min, and the reaction was then terminated by adding 100 μL stop solution (Solarbio, #C1058).

[0219] 6. The absorbance of each sample (at an excitation wavelength of 450 nm and emission at 630 nm) was measured by a fluorimeter (SPECTRAmax Gemini XPS, Molecular Devices, San Jose, CA, USA), and the results are shown in Figure 5 and Figure 6 middle.

[0220] like Figure 5 As shown in Figure 2, A3 and A9 are the top two candidates among the AADC coding sequences evaluated. A10 showed the highest AADC protein expression level because it expressed an AADC mutant protein. Figure 6 As shown, G4 and G5 are the top 2 candidates among the GBA1 coding sequences evaluated. 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.

[0221] Western blotting

[0222] Western blot (WB) assay was also performed to detect protein expression. Again, candidate vectors (except G1, G2, G7, A1, A2 and A7) were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Invitrogen, #L3000008).

[0223] 48 hours after transfection, cells were harvested in RIPA buffer (Thermo, 89901), diluted in 5× loading buffer (Beyotime, P0015L) and boiled (denaturation). All WB samples were separated on SDS polyacrylamide gels (BioRad, 1703932) and transferred to 0.22 μm PVDF membranes (BIO-RAD, 1620177).

[0224] The membrane was incubated with anti-AADC antibody (Millipore, #AB1569) or anti-GBA1 antibody (Sigma, #G4171) or antibody against housekeeping gene GAPDH (CST, #2118) at 4°C overnight, followed by incubation with HRP-linked secondary antibody at room temperature for 2 hours the next day.

[0225] 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 in Figure 7 and Fig. 9 The area of ​​the bands of AADC protein or GBA1 protein was determined by ImageJ 1.53 (NIH) and normalized to the level of GAPDH. The GADPH-normalized values ​​were then normalized to the values ​​of A0 for the AADC group or G0 for the GBA1 group. The normalized data from three independent experiments are presented in Figure 8 and Fig.10 middle.

[0226] like Figure 7-10 As shown in the WB results in , it was found 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, which was consistent with the results from the ELISA assay. A3, A9 (for AADC) and G4, G5 (for GBA1) were selected as codon-optimized sequences for further combinatorial expression construct optimization.

[0227] Example 3. Catalytic activity of co-expressed AADC and GBA1 proteins

[0228] This example is intended to determine whether exogenously expressed AADC and GBA1 proteins have catalytic activity, indicating normal function. In addition, catalytic activity can also be used as an additional evaluation criterion for candidate sequences. The method for examining AADC and GBA1 activity in cell lysates is described below.

[0229] AADC catalytic activity (HPLC analysis)

[0230] The cultures expressing AADC protein (A3G0, A9G0, A10G0, A0G0, A0) and the control samples expressing GFP protein were exposed to 100 μM L-DOPA for 24 h. 100 μL 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 the cells were scraped from the bottom.

[0231] The cell homogenate was centrifuged at 13,000 g for 10 min, and 30 μL of each sample was used for HPLC determination. The samples were subjected to HPLC / ECD to detect the dopamine levels produced by the catalytically active AADC proteins expressed by the different constructs. The relative AADC activity was calculated using a standard curve of dopamine, as shown by the amount of dopamine transferred from L-DOPA in each sample.

[0232] GBA1 activity

[0233] Cultures expressing GBA1 protein (A0G4, A0G5, A0G10, A0G0, G0) and control samples expressing GFP protein were tested for GBA1 protein activity. Recombinant human GBA (rhGBA) was used as a positive control.

[0234] 1. Dilute rhGBA (Cat. No. 7410-GHB) to 0.2 ng / μL in assay buffer (50 mM sodium citrate, 25 mM sodium cholate, 5 mM DTT, pH 6.0).

[0235] 2. Dilute substrate (4-methylumbelliferyl-β-D-glucopyranoside, Sigma, Cat. No. M3633, 10 mM in DMSO) to 6 mM in assay buffer.

[0236] 3. Load 25 μL of cell lysate (total protein 100 ng / μL) or 0.2 ng / μL rhGBA into the wells of a 96-well plate. Start the reaction by adding 25 μL of 6 mM substrate. Load 25 μL of assay buffer into the standard curve wells.

[0237] 4. Seal the plate and incubate at 37°C for 3 hours.

[0238] 5. After incubation, stop the reaction by adding 50 μL of stop solution (0.5 M glycine, 0.3 M NaOH (approximately pH 10)) to each well.

[0239] 6. In endpoint mode, record readings at excitation and emission wavelengths of 365 nm and 445 nm respectively (upper reading).

[0240] 7. Calculate the specific activity as follows: (adjusted fluorescence * 25) / (180 min * 0.0025 mg). The results are shown in Fig. 9 and Fig.10 middle.

[0241] like Fig.11 As shown, among all constructs evaluated, the AADC protein expressed by construct A3G0 exhibited the highest catalytic activity. Fig.12 As shown, the GBA1 protein expressed from construct A0G4 exhibited the highest catalytic activity.

[0242] Example 4. Identification of the Optimal Combination of Codon-Optimized AADC and GBA1 Sequences

[0243] In order to identify the best combination of codon-optimized AADC and GBA1 sequences, the coding sequences of AADC and GBA1 with better performance as shown above were paired together and used to construct recombinant AAV vectors. In particular, constructs A3G4, A9G4, A3G5 and A9G5 were generated. In addition, construct A11G11 was generated, which is based on codons of A3 and G4 with further optimization to completely remove CpG islands from AADC and GBA1 coding sequences. Two benchmark vectors, A10-vy (SEQ ID NO: 48, codon A10 containing promoter and other regulatory elements described in the same patent) and G10-p (SEQ ID NO: 49, codon G10 containing promoter and other regulatory elements described in the same patent) were also constructed.

[0244] The above vectors were transfected into HEK293 cells along with A0-P2A, A0G0, A10G0, A10-vy and G10-p. AADC and GBA1 protein expression and the catalytic activity of GBA were determined. It was found that among the combinatorial constructs evaluated, A11G11, A3G5 and A9G4 expressed the highest levels of AADC protein, as determined by WB analysis ( Fig.13 For GBA1 protein expression, constructs A3G5 and A11G11 expressed the highest levels of protein (they were also the most active), followed by construct A9G4 ( Fig.14 ).

[0245] Example 5. Evaluation of AADC and GBA1 protein expression of AAV9-packaged combinatorial constructs

[0246] Constructs A0G0, A9G4, A11G11 and A10-vy were packaged into AAV, using serotype AAV9. AAV9-A0G0, A9G4 and A11G11 were administered at MOI 1e5 and 1e6 to U-87MG cells stably expressing the AAV receptor to increase transduction efficiency, along with a control AAV expressing only GFP. Five days after infection, AADC and GBA1 protein expression levels in these cell samples were determined by WB analysis. Fig.15 As shown, cells transduced with A11G11 AAV virus expressed higher levels of AADC and GBA1 proteins compared to A9G4 packaged AAV virus. It was observed that AADC protein expression was significantly higher in cells transduced with A10-vy AAV virus, most likely due to the stronger promoter used in the A10-vy construct.

[0247] Example 6. Optimization of regulatory elements used in combinatorial expression constructs

[0248] In order to further improve the protein expression of the combined construct, the promoter and poly A sequences used were also optimized. The CBh promoter in A11G11 was replaced with the EF1α promoter (SEQ ID NO: 57), and the SV40 poly A tail was replaced with bovine growth hormone poly A (bGH) or human growth hormone poly A (hGH), as shown in Table 2. Since EF1α-A11G11-hGH exceeded the packaging capacity of AAV, the length of the EF1α promoter was reduced by truncating a portion of the promoter, resulting in seven different truncations (numbered 5-11) as shown in Table 2.

[0249] The constructs in Table 2 were transfected into HEK293 cells along with A3G5, A10-vy and GFP. The AADC protein expression level was determined by WB analysis. In the first group of constructs evaluated, EF1α-A11G11-hGH expressed the highest level of AADC protein. In the constructs with truncated EF1α promoter (EFStI1-7, SEQ NO: 50-56), construct EFStI2 expressed the highest level of AADC protein ( Fig.16 ).

[0250] Table 2. Combination constructs with EF1a and truncated EF1a promoters.

[0251]

[0252]

[0253] Example 7. In vivo efficacy of AAV candidates expressing optimized AADC and GBA1 sequences in a PD mouse model

[0254] The therapeutic efficacy of AAV expressing optimized AADC and GBA1 was evaluated in a PD mouse model to determine whether the candidate AAV could rescue the motor deficits of PD animals.

[0255] First, a widely used chemically induced PD mouse model was used. The mice were treated with MPTP / probenecid. The candidate rAAV vector CBh-A11G11 was administered to the striatum of the PD mouse model at a higher dose of 2E+10vg / animal and a lower dose of 2E+09vg / animal (respectively "CBh-A11G11 low" and "CBh-A11G11 high") once. The rAAV vector A10-vy (2E+09vg / animal) was injected in a similar manner to the baseline control ("A10-vy"), and a rAAV vector expressing GFP was used as a negative control ("GFP"). The motor function of mice was evaluated by measuring the movement speed and walking distance before PD modeling (before induction with MPTP / P; "before") and before and after the application of the corresponding rAAV vector (respectively "MPTP / P after" and "AAV after") . The "before" behavioral test was performed for 6 days, and then MPTP / probenecid was applied every day for 35 days. After 3 days of acclimatization, subjects underwent the same behavioral test ("MPTP / P post") followed by rAAV injection. 21 days after rAAV injection, "AAV post" behavioral test was performed. Results are shown in Fig.34 middle.

[0256] like Fig.34 As shown, the movement speed of mice decreased after using MPTP / probenecid. Compared with other mice, mice to be treated with A10-vy (N=3) showed less severe speed loss. All mice (except those to be treated with A10-vy) showed a shortened movement distance. After treatment with the corresponding rAAV, the motor function of mice (N=5) as negative controls showed a continuous decline, manifested as a continuously reduced movement speed and distance. In contrast, mice treated with low doses (N=5) and high doses (N=7) of CBh-A11G11-SV40 showed recovery of movement speed and distance, with higher doses showing slightly better results compared to lower doses. These results show that in the MPTP / probenecid-induced PD mouse model, CBh-A11G11-SV40 has achieved significant results in rescuing insufficient movement speed and walking distance. Given that the impairment of mobility in mice treated with A10-vy was less severe than in other mice, CBh-A11G11-SV40 showed superior effects in rescuing deficits in locomotor speed and walking distance compared with baseline in the MPTP / probenecid-induced PD mouse model.

[0257] The transgenic PD mouse model was then used to verify the results. Mice were genetically engineered to overexpress mutant α-synuclein (point mutation A53T) to recapitulate PD pathology. Two rAAVs, EFSIt7-A11G11-hGH (EtI7) and CBh-A11G11-SV40 (CBh), were produced based on the construct A11G11 using different promoters and poly A tails. rAAV was administered to mice at a low dose of 4E+9vg and a high dose of 4E+10vg by intrastriatal injection. Wild-type mice without any treatment and A53T mice treated with vehicle (PBS) were included as controls. Motor function was determined by measuring the number of vertical movements (standing upright by the hind limbs), and the results are shown in Fig.35 middle.

[0258] like Fig.35 As shown, mice treated with the vehicle (N=6) showed fewer vertical movements compared to WT mice (N=8), indicating impaired motor function. Mice treated with high doses (N=7) and low doses (N=7) of EFSIt7-A11G11-hGH (EtI7) showed better performance than mice treated with the vehicle, with the high dose showing significantly increased results. Mice treated with low doses of CBh-A11G11-SV40 (CBh) (N=7) also showed increased vertical movement times compared to both WT and vehicle A11G11-based rAAV. An increase in the number of vertical movements indicates recovery of motor function.

[0259] Brain tissue samples of A53T mice were collected for determination of protein expression. The expression of AADC and GC enzyme proteins in the striatum was determined by WB. Fig.36 As shown, all treatment groups showed expression of AADC and GC enzymes. In addition, 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. Fig.37 A and Fig.37 As shown in B, both candidate rAAV vectors reduced phosphorylated α-synuclein levels in a dose-dependent manner, indicating improvement in PD pathology. Fig.37 The exemplary bands in A are from 2 representative subjects in each group. Fig.37 The relative phosphorylated α-synuclein levels shown in B are the means of all subjects in the indicated groups.

[0260] Example 8. In vivo therapeutic efficacy of AAV candidates expressing optimized GBA1 sequences in a GD mouse model The therapeutic efficacy of AAV expressing optimized GBA1 was tested in a GD mouse model.

[0261] Two candidate vectors expressing optimized GBA1 (G11) were constructed, as shown in Figure 38. The first candidate vector was designed to contain the CAG promoter, the G11 codon, and the hGH poly A ("CAG"). The second candidate vector was designed to contain the EF1α promoter, the G11 codon, WPRE, and the hGH poly A ("EF1α"). Both vectors were packaged into AAV9 as rAAV. The candidate rAAV vectors were injected into the lateral ventricle of the chemically induced GD mouse model and the genetically engineered GD mouse model to evaluate their effects.

[0262] The chemically induced mouse model is a CBE-induced GD mouse model. GC enzyme activity is inhibited by cyclohexene tetraol β-epoxide (CBE), a specific irreversible GC enzyme inhibitor that mimics a loss-of-function mutation in the GBA1 gene. On postnatal day 2 (P2), mice were treated with PBS, AAV9-GFP (negative control) or three different doses of candidate rAAV vectors (low, 2.75E+09vg; medium, 8.8E+09vg; high, 2.8E+10vg, per animal) via intracerebroventricular injection. G10-p was used as a reference. In order to allow enough time for gene expression, disease modeling was started at P8. 37.5mg / kg CBE was injected into mice via intraperitoneal injection once a day. In-life evaluation was performed until P28.

[0263] Fig.39 The viability of GD mice is shown until P28. As can be seen, the survival percentage of the PBS group and the negative control group dropped 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 increased the viability of GD mice.

[0264] Fig.40 The changes in motor function of the studied animals are shown. Both candidate vectors rescued the hypokinesia and ataxia caused by long-term CBE administration. Fig.40 A and Fig.40 As shown in B, CAG increased the distance moved in the open field test at all doses and enhanced motor coordination in the rotarod at the middle and high doses, while EF1α increased the distance moved at the middle and high doses but improved motor coordination only at the high dose.

[0265] At postnatal day 30, brain samples were collected for determination of protein activity. Brain samples were homogenized in lysis buffer (N-PER neuronal protein extraction reagent, Thermo Scientific #87792) containing protease inhibitors (Roche #11697498001) to produce lysate samples. GC enzyme activity in these lysate samples was determined by GBA1 activity assay as described above. GC enzyme activity was found to be dose-dependently increased in all candidate vector treatment groups ( Fig.41 ).

[0266] Example 9. Codon-optimized human CDNF sequence for AADC combinatorial constructs

[0267] In this example, an AAV vector was designed to co-express AADC and a gene encoding NTF (particularly CDNF). P2A was used as a linker sequence between the two coding sequences.

[0268] To determine the optimal coding sequence for CDNF, the wild-type AADC coding sequence (AADC WT) was ligated to different codon-optimized CDNF sequences via P2A. Codon optimization was performed based on different algorithms.

[0269] 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 9 nucleotides encoding the three amino acids immediately following the signal peptide (nucleotide positions 73-81) remained unchanged.

[0270] Meanwhile, based on the sequence of CDNF SA, another candidate CDNF sequence called CDNF manual (CDNF MN) was obtained by manually identifying and reducing the "CG" components 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.

[0271] For this study, an AAV backbone vector was prepared containing, from 5' to 3', the 5' ITR, the CBh promoter, the AADC WT coding sequence, the P2A sequence, the SV40 poly A, and the 3' ITR.

[0272] The sequences of CDNF GS, CDNF SA, CDNF MN and CDNF wild type (CDNF WT) were respectively inserted into the 3' position of the P2A linker sequence of the above backbone vector ( Fig.17A and Fig. 17BThe obtained vectors were named ApC-GS, ApC-SA, ApC-MN and ApC-WT. A reference vector containing only CDNF WT (without AADC) was also constructed.

[0273] Using Lipofectamine 3000 transfection reagent (Invitrogen, #L3000008), the above 5 plasmids and a negative control plasmid expressing only GFP were transfected into HEK293 cells. 72 hours after transfection, the supernatant was centrifuged at 2,000 rpm for 10 minutes to remove debris in the culture medium, and 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 min. The cells were washed once with 1×PBS and harvested in RIPA buffer (Thermo, 89901).

[0274] For WB analysis, the samples were diluted and boiled in 5× loading buffer (Beyotime, P0015L), separated on SDS polyacrylamide gel (BIO-RAD, 1703932) and transferred to 0.22 μm PVDF membrane (BIO-RAD, 1620177). The membrane was incubated with antibodies against CDNF protein (Sigma, HPA044587), AADC protein (Merck Millipore, AB1569) and β-tubulin (tubulin, Proteintech, 66240-1-Ig) at 4°C overnight, followed by incubation with HRP-linked secondary antibodies at room temperature for 2 hours the next day. The BeyoECL Moon kit (Beyotime, P0018F) was then applied to the membrane ( Fig.18A ). Exposure and image capture were performed using a Tannon 5600 system. The intensity of the CDNF bands was calculated and normalized to the construct ApC-WT, and the results are shown in Fig.18B middle.

[0275] like Fig.18B As shown, cells transfected with construct ApC-MN showed the highest levels of CDNF protein secreted into the supernatant among all constructs evaluated, which was confirmed in U-87MG cells transfected with the same set of plasmids ( Figure 19A-19B ).

[0276] The level of secreted CDNF protein in the supernatant was further determined by sandwich ELISA method. Briefly, 96-well microplates (Costar, 42592) were coated with CDNF mouse Mab (SinoBiological, 15691-MM1) antibody as capture antibody at 4°C overnight. The next day, the plates were washed 3 times with PBST (phosphate buffered saline with Tween) and then blocked with 5% skim milk powder (Solarbio, D8340) for 2 hours at room temperature. After an additional washing step, CDNF protein standards (SinoBiological, 15691-H08H) or protein samples were added to the plates in duplicate. The samples were incubated with 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 bound HRP conjugate was detected by adding TMB (Solarbio, PR1200), and the reaction was stopped by stop solution (Solarbio, C1058). The absorbance of each sample (at an excitation wavelength of 450 nm and emission at 630 nm) was detected by a fluorometer (SPECTRAmax Gemini XPS, Molecular Devices, San Jose, CA, USA), and the results are shown in Figure 20A-20B middle.

[0277] like Figure 20A-20B As shown, both HEK293 and U-87MG cells transfected with construct ApC-MN expressed significantly higher levels of CDNF protein, as shown by high levels of secreted CDNF in the supernatant. As a result, ApC-MN was selected for further evaluation.

[0278] Example 10. Protein expressed by codon-optimized CDNF exhibits significant cytoprotective effects

[0279] In this example, 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, namely the LDH cytotoxicity assay and the CCK-8 cell viability assay of cells treated with MPP+ or rotenone.

[0280] In the LDH assay, 96-well assay 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. Several wells without cells were left as blank controls to determine background luminescence levels. The cells were then incubated with MPP + While the plates were incubated at 37°C, the LDH detection reagent (CytoTox Non-radioactive cytotoxicity assay, Promega). 45 minutes before the reagent, add 1 / 10 volume of 10× lysis solution to the positive control (maximum LDH release control) wells. Before reading the plate, add 50 μL of CytoTox The 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 by a plate reader. Cytotoxicity percentage = 100 × experimental LDH release (OD490) / maximum LDH release (OD490).

[0281] In the CCK-8 assay, SH-SY5Y cells in the logarithmic growth phase were cultured at 5 × 10 3 The cells were plated at a density of 100 μg / mL in a 96-well plate and cultured in high glucose / DMEM cell culture 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. The cells were then treated with or without supernatant containing secreted CDNF collected from a previous experiment. MPP containing 1 μM MPP was used at a final concentration of 0 to 2 mM. + Cell apoptosis was induced by adding serum-free medium containing 1% or rotenone. After 24 h, cell viability was assessed by adding 10 μL CCK-8 (Cell Counting Kit-8, Dojindo) to the culture medium and after 2 h of incubation, relative cell viability was determined by spectroscopy at a wavelength of 450 nm.

[0282] like Fig.21 As shown, in the presence of MPP +In the presence of either 5-nitropropene (1.5 mM) or rotenone (40 nM), SH-SY5Y cells treated with the supernatant from cells transfected with the construct ApC-MN had a higher survival rate than SH-SY5Y cells treated with the supernatant from cells transfected with the construct ApC-WT, suggesting that the construct ApC-MN expressed and secreted a higher level of CDNF protein into the supernatant than the construct ApC-WT to exert its stronger cytoprotective effect.

[0283] Example 11. Codon-optimized human GDNF sequence for AADC combination constructs

[0284] In this example, the codon optimization process was performed as in Example 9 ( Fig. 17B ) was used 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).

[0285] In vitro cell experiments as described in Example 10 were performed in HEK293 cells transfected with constructs containing GDNF GS, GDNF SA and GDNF MN ( Figure 22-Figure 24 Based on the results, the GDNF MN sequence without CpG islands had the best GDNF expression and protection potential among all the codon-optimized GDNF sequences evaluated. Therefore, the GDNF MN sequence was selected as the GDNF coding sequence for the AADC combination construct for further evaluation.

[0286] Example 12. Determination of the optimal promoter for use in AADC and CDNF / GDNF combination constructs

[0287] To determine the best promoter element to use in the combination constructs of AADC and CDNF or GDNF, eight new combination 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. Constructs CBh-AADC WT-P2A-CDNF WT and CBh-AADC WT-P2A-GDNF WT were generated and used as controls.

[0288] Table 3. Constructs of CDNF / GDNF and AADC combined with different regulatory sequences

[0289]

[0290] *ND: Not Detectable

[0291] The above constructs were transfected into HEK293 cells. Both cell lysates and supernatants of these samples were collected. Secreted protein levels of CDNF or GDNF were determined by ELISA. The results showed that EF1α-A11-P2A-CDNF MNs expressed higher levels of CDNF protein compared to CAG-A3-CDNF MNs, while the other two constructs expressed undetectable levels of CDNF protein ( Fig.25 ).

[0292] For constructs containing GDNF, EF1a-A11-GDNF MNs expressed and secreted the highest levels of GDNF ( Fig.25 ).

[0293] The catalytic activity of the AADC proteins expressed by these combined constructs was also determined by HPLC assay of cell lysates as described in the previous examples. Fig.26 As shown, the relative AADC activities of EF1α-A11-CDNF MNs and EF1α-A11-GDNF MNs were much higher than those of all other candidate constructs.

[0294] The construct EF1α-CDNFMN-A11 was generated by placing the CDNF codon in front of the AADC codon and ligating via P2A.

[0295] Both EF1α-CDNF MN-A11 (CDNF MN-A11) and EF1α-A11-CDNF MN (A11-CDNF MN) were transfected into HEK293 cells by Lipofectamine 3000. 72 hours after transfection, supernatants and cell lysates were collected. Protein levels of AADC and CDNF in cell lysates and CDNF levels in supernatants (secreted CDNF) were determined by WB. It was interesting to find that the protein level of secreted CDNF was much higher in the CDNF MN-A11 group than in A11-CDNF MN ( Fig.42 ).

[0296] Example 13. Validation of the cytoprotective effects of CDNF and GDNF proteins derived from candidate constructs

[0297] MPP+ and rotenone assays as described in previous experiments were performed to determine the cytoprotective effects of the CDNF and GDNF constructs identified in Example 12. Fig. 27) or EF1α-A11-GDNF MN ( Fig.28 ) transfected cells increased 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 the two candidate constructs.

[0298] Example 14. Evaluation of AADC and CDNF or GDNF protein expression in AAV9-packaged combination constructs

[0299] Both EF1a-A11-CDNF MN and EF1a-A11-GDNF MN combination constructs were packaged into AAV9. The obtained AAV9-EF1α-A11-CDNF MN and AAV9-EF1α-A11-GDNF MN were added to U87-AAVR cells at MOI=1e5. 72 hours after viral transduction, the cells were collected in lysis buffer. Following the WB protocol, the expressed protein levels of CDNF and AADC were determined. At the same time, the supernatants of all samples were also collected and concentrated as described above. The levels of secreted CDNF and GDNF proteins were also detected by WB, and the results are shown in Fig.29 middle.

[0300] The relative activities of the expressed AADC proteins in the above cell lysates are shown in Fig.30 middle.

[0301] and Fig.29 and Fig.30 Taken together, the data shown in Figure 2 indicate that both constructs behaved as expected when packaged into AAV9 as rAAV.

[0302] Example 15. AAV candidates expressing optimized AADC in combination with GDNF or CDNF in PD mouse models In vivo therapeutic efficacy

[0303] The therapeutic effect of AAV delivered with optimized AADC+GDNF combination constructs was tested in a unilaterally injured 6-OHDA PD mouse model. Briefly, C57 mice were placed in a stereotaxic frame and anesthetized with isoflurane mixed in oxygen. 1 μL 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, apomorphine (Sigma, PHR2621-500MG) was administered intraperitoneally to mice, 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 of the subjects (coordinates AP = +0.6 mm, ML = -1.8 mm, DV = -3.2 mm) at three doses (low: 1E+12 vg, medium: 3.16E+12 vg, high: 1E+13 vg, in mL). AAV-GFP and two doses of the benchmark AAV-A10-vy (low: 1E+12 vg, high: 1E+13 vg, in mL) were also applied. 21 days after the AAV injection, a significant reduction in contralateral rotations triggered by apomorphine was observed in all study groups treated with the candidate rAAV vector compared to the control group ( Fig.31 and Fig.32 ).like Fig.31 As shown, the three study groups treated with three different doses of EF1α-A11-GDNF MN rAAV performed significantly better than the group treated with the baseline rAAV. More notably, the number of net rotations (ipsilateral-contralateral) in the low- and medium-dose treatment groups were close to zero, indicating that these study subjects had fully recovered their balance motor behavior, while the group receiving the high-dose baseline showed overcompensatory ipsilateral rotations (excess AADC in the treated side) ( Fig.32 ).

[0304] In another in vivo efficacy test in PD mice (Study 2), CAG-A11-GDNF MN rAAV (AAV9) was also tested in a unilaterally injured 6-OHDA PD mouse model. Two different doses (2.0E+09vg and 2.0E+10vg) 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 a benchmark. 21 days after AAV injection, EF1α-A11-GDNF MN at 2E+10vg / animal was found to significantly reduce the ipsilateral rotation triggered by amphetamine (5mg / kg intraperitoneally), indicating that motor function was rescued ( Fig.43 ).

[0305] Tissue samples were collected for the expression of AADC and NTF proteins and the determination of dopamine levels. The expression of GOI was observed in the nigrostriatal pathway. Fig.44 As shown, striatal tissue samples were collected from subjects in 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 WB. It was unexpectedly found that EF1α expressed more AADC in the striatum (injection site) compared with A10-vy ( Fig.44 A). At the same time, it was confirmed that EF1α expressed GDNF in a dose-dependent manner in vivo ( Fig.44 B).

[0306] Furthermore, tyrosine hydroxylase (TH) staining revealed that more dopaminergic terminals survived in the nigrostriatal pathway in EF1α-treated animals compared to control animals that received AAV9-GFP alone, confirming the protective effect of the candidate rAAV ( Fig.45 ).

[0307] The efficacy of AAV9-EF1α-A11-GDNF MN (EF1α) was also tested in a unilaterally injured 6-OHDA PD rat model. The model was generated based on previous studies (6-OHDA Lesion Models of Parkinson'sDisease in the Rat. Animal Models of Movement Disorders: Vol. I, Neuromethods, Vol. 61, DOI 10.1007 / 978-1-61779-298-4_13). Briefly, 6-OHDA was injected into the right substantia nigra (coordinates AP = -4.4 mm, ML = -1.1 mm, DV = -8 mm). Two weeks after lesion surgery, EF1α was then injected into two sites on 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 dose of baseline AAV2-A10-vy (4E+10 vg / animal) was also applied. 21 days after AAV injection, spontaneous contralateral rotation behavior was recorded before and after L-DOPA (5 mg / kg L-DOPA + 2.5 mg / kg benserazide) administration. ΔNumber of rotations per minute (post-pre) represents the subject's response to L-DOPA. As Fig.46As shown, EF1α was found to significantly enhance 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. In addition, at the same dosing level (4E+10vg), EF1α showed better results compared to A10-vy.

[0308] Example 16. Expression of optimized GBA1 and CDNF combination or GBA1 and GDNF in MSA animal model In vivo therapeutic efficacy of combined AAV candidates

[0309] Produce Fig.33A and Fig.33B Candidate constructs with the configuration shown. These constructs contain a combination of CAG or MBP promoter, GBA1 gene (including SEQ ID NOs: 45 and 47) and CDNF (SEQ ID NOs: 23-25) or GDNF (SEQ ID NOs: 27-29).

[0310] CAG-G11-P2A-GDNF MN-SV40 poly A (CAG-G11-GDNF MN), CAG-G12-P2A-GDNF MN-SV40 poly A (CAG-G12-GDNF MN), MBP-G11-P2A-GDNF MN-bGH poly A (MBP-G11-GDNF MN), and CAG-GDNF MN-WPRE-hGH (CAG-GDNF MN) were constructed and packaged into AAV using serotype AAV9. These candidate rAAV vectors were applied to U-87MG-AAVR cells (U-87MG cells overexpressing AAVR), and cell lysate and supernatant samples were collected. GC enzyme and GDNF protein levels were determined in these samples ( Fig.47 ).

[0311] The efficacy of the above-mentioned candidate rAAV vector was tested in vitro in a cell model for recapitulating the pathological features of PD and MSA, which is a stable cell line based on SH-SY5Y cells and overexpressing both AAVR and mutant α-synuclein (point mutation A53T) proteins. Lentiviral vectors with AAVR sequences or mutant α-synuclein (A53T) sequences were prepared by Azenta Life Science and applied to SH-SY5Y cells in sequence. After selection based on antibiotics and GFP, the SH-SY5Y-AAVR-A53T stable cell line was generated. Fig.48 As shown, the candidate rAAV vector was found to significantly reduce the level of high molecular weight (HMW) α-synuclein, a biomarker for α-syn aggregation, in a cell model.

[0312] CAG-GDNF MN-P2A-G11-SV40 poly A (CAG-GDNF MN-G11) was constructed and transfected into HEK293 cells together with CAG-G11-GDNFMN. 72 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 enzyme ( Fig.49 ).

[0313] The therapeutic effect of AAV delivered optimized GBA1+CDNF or GBA1+GDNF combination constructs was then evaluated in a mouse model of MSA. Candidate rAAV vectors after injection into the lateral ventricle (icv) rescued the motor deficits of MSA animals. Tissue samples were collected. Expression of GBA1 and NTF proteins in the nigrostriatal pathway, as well as decreased levels of α-syn and phosphorylated α-syn were observed. In addition, tyrosine hydroxylase (TH) staining showed that more dopaminergic terminals survived in the striatum of treated animals, confirming the protective effect of the candidate AAV.

[0314] Example 17. In vivo therapeutic efficacy of AAV candidates expressing optimized GBA1 in a PD mouse model The therapeutic efficacy of AAV expressing optimized GBA1 was tested in a PD mouse model.

[0315] Mice genetically engineered to overexpress mutant α-synuclein (point mutation A53T) were used as a PD model. CAG-G11-hGH poly A (as described in Examples 8 and Fig.38A The candidate rAAV vector was injected into the lateral ventricle of the A53T PD mouse model to evaluate its effect. rAAV9-CAG-G11-hGH can significantly alleviate the motor deficit in the PD mouse model and reduce the levels of α-syn and phosphorylated α-syn in the nigrostriatal pathway, indicating that it has the potential to treat PD and other α-syn protein diseases.

[0316] Sequence information

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Claims

1. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a first protein, the first nucleotide sequence being operably linked to a second nucleotide sequence encoding a second protein, wherein the first protein and the second protein are selected from the following combinations (a) to (i): (a) the first protein is human AADC, and the second protein is human GBA1; (b) the first protein is human AADC, and the second protein is human CDNF; (c) the first protein is human AADC, and the second protein is human GDNF; (d) the first protein is human GBA1, and the second protein is human CDNF; (e) the first protein is human GBA1, and the second protein is human GDNF; (f) the first protein is human CDNF, and the second protein is human AADC; (g) the first protein is human CDNF, and the second protein is human GBA1; (h) the first protein is human GDNF, and the second protein is human AADC; (i) The first protein is human GDNF, and the second protein is human AADC.

2. The isolated nucleic acid molecule of claim 1, wherein the first nucleotide sequence is 5' upstream of the second nucleotide sequence.

3. The isolated nucleic acid molecule according to claim 1 or 2, characterized in that: (i) the polypeptide sequence of AADC comprises or is the amino acid sequence shown in SEQ ID NO:31; (ii) the polypeptide sequence of GBA1 comprises or is the amino acid sequence shown in SEQ ID NO:32; (iii) the polypeptide sequence of CDNF comprises or is the amino acid sequence shown in SEQ ID NO:33; (iv) The polypeptide sequence of GDNF comprises or is the amino acid sequence shown in SEQ ID NO:

34.

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

5. The isolated nucleic acid molecule according to any one of claims 1 to 4, when the first protein is human ADCC, the first nucleotide sequence comprises or is a nucleotide sequence selected from the nucleotide sequence shown in any one of SEQ ID NOs: 1-9, 11 or 46.

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 a nucleotide sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO:

46.

7. The isolated nucleic acid molecule according to any one of claims 1 to 6, when the first protein or the second protein is human GBA1, wherein the first nucleotide sequence or the second nucleotide sequence comprises or is a nucleotide sequence selected from the nucleotide sequence shown in any one of SEQ ID NOs: 12-20, 22, 45 or 47.

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 a nucleotide sequence as shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:45 or SEQ ID NO:

47.

9. The isolated nucleic acid molecule according to any one of claims 1-8, when the second protein is human CDNF, wherein the second nucleotide sequence comprises or is a nucleotide sequence selected from the nucleotide sequence shown in any one of SEQ ID NOs: 23-26.

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 of CDNF, and the different nucleotides between the codon-optimized coding sequence and the wild-type coding sequence of CDNF as shown in SEQ ID NO: 26 are not present in the region encoding the signal peptide of CDNF.

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 reduced CpG sites or no CpG islands compared to the wild-type coding sequence of CDNF as shown in SEQ ID NO:

26.

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 a nucleotide sequence as shown in any one of SEQ ID NOs: 23-25.

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 a nucleotide sequence as shown in SEQ ID NO:

25.

14. The isolated nucleic acid molecule according to any one of claims 1-8, when the second protein is human GDNF, wherein the second nucleotide sequence comprises or is a nucleotide sequence selected from the nucleotide sequence shown in any one of SEQ ID NOs: 27-30.

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

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

30.

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 a nucleotide sequence as shown in any one of SEQ ID NOs: 27-29.

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 a nucleotide sequence as shown in SEQ ID NO:

29.

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

20. The isolated nucleic acid molecule of claim 19, wherein the promoter is selected from the group consisting of a CBh promoter, an EF1α promoter, a CAG promoter, an MBP promoter, or a variant thereof.

21. The isolated nucleic acid molecule of claim 20, wherein the promoter is a truncated variant of the EF1α promoter having a nucleotide sequence of any one of SEQ ID NOs: 50-56.

22. The isolated nucleic acid molecule of any one of claims 1-21, further comprising a linker sequence between the first nucleotide sequence and the second nucleotide sequence.

23. The isolated nucleic acid molecule of claim 22, wherein the linker sequence is a sequence encoding a self-cleaving peptide or an internal ribosome entry site.

24. The isolated nucleic acid molecule of claim 23, wherein the self-cleaving peptide is a 2A peptide.

25. The isolated nucleic acid molecule of claim 24, wherein the 2A peptide is selected from the group consisting of E2A, F2A, T2A, P2A.

26. The isolated nucleic acid molecule of claim 25, wherein the 2A peptide is P2A.

27. The isolated nucleic acid molecule of claim 23, wherein the linker sequence is ECMV IRES or mini-IRES.

28. The isolated nucleic acid molecule of any one of claims 1-27, further comprising a polyadenylation signal.

29. The isolated nucleic acid molecule of claim 28, wherein the polyadenylation signal is selected from the group consisting of SV40 poly A, human growth hormone (hGH) poly A, and bovine growth hormone (bGH) poly A.

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

31. A codon-optimized sequence of GBA1, the codon-optimized sequence of GBA1 having a nucleotide sequence as shown in any one of SEQ ID NOs: 12-20, 45 or 47.

32. A codon-optimized sequence of CDNF, the codon-optimized sequence of CDNF having a nucleotide sequence as shown in any one of SEQ ID NOs: 23-25.

33. A codon-optimized sequence of GDNF, the codon-optimized sequence of GDNF having a nucleotide sequence as shown in any one of SEQ ID NOs: 27-29.

34. A recombinant adeno-associated viral (rAAV) vector comprising the isolated nucleic acid molecule of any one of claims 1-29 or the codon-optimized sequence of any one of claims 30-33.

35. The rAAV vector of claim 34, further comprising ITRs of AAV2.

36. A viral particle comprising the rAAV vector of claim 34 or claim 35.

37. The viral particle of claim 36, comprising a capsid protein of AAV1, AAV2, AAV5, AAV8 or AAV9.

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

39. The pharmaceutical composition of claim 38, for use in treating a neurodegenerative disorder.

40. The pharmaceutical composition of claim 39, wherein the neurodegenerative disorder is Parkinson's disease, multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency (AADCD), or other proteinopathy.

41. A method of treating or preventing a neurodegenerative disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the rAAV vector of claim 34 or claim 35, the viral particle of claim 36 or claim 37, or the pharmaceutical composition of any one of claims 38-40.

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

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.

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

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.

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

47. The method of claim 46, wherein the Gaucher disease is type 2 or type 3 Gaucher disease.

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

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.

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

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.

52. Use of the rAAV vector of claim 34 or claim 35 or the viral particle of claim 36 or claim 37 in the manufacture of a medicament for treating a neurodegenerative disorder.

53. The use according to claim 52, wherein the neurodegenerative disorder is Parkinson's disease, multiple system atrophy (MSA), Gaucher disease (GD), AADC deficiency or other proteinopathy.

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