AADC, GDNF POLYNUCLEOTIDES AND THEIR USE TO TREAT PARKINSON'S DISEASE

Abstract

The Abstract provides AADC, GDNF polynucleotides and their use for treating Parkinson's disease. Specifically, the Abstract provides an AAV-vectored gene delivery system that delivers aromatic L-amino acid decarboxylase (AADC) and glial cell line-derived neurotrophic factor (GDNF) to specific regions of the brain, and methods and uses for treating neurodegenerative diseases (e.g., Parkinson's disease).

Description

[Technical field]

[0001] This disclosure claims priority to a Chinese patent application filed on November 29, 2021 (application number CN202111434384.4).

[0002] The present disclosure relates to the field of gene therapy, and in particular to a pharmaceutical composition and method for gene therapy for treating Parkinson's disease. Specifically, the present disclosure provides an AAV-vectored gene delivery system that delivers aromatic L-amino acid decarboxylase (AADC) and glial cell line-derived neurotrophic factor (GDNF) to specific regions of the brain to treat Parkinson's disease. [Background technology]

[0003] Parkinson's disease (PD) is a progressive neurodegenerative disorder of the central nervous system (CNS), mainly affecting dopaminergic (DA) neurons in the substantia nigra (SN). Clinically, PD is characterized by resting tremor, bradykinesia, myotonia, and postural gait disorder, and can cause significant disability within 10-15 years of onset, severely affecting the quality of life of patients. Although the etiology of PD remains unclear, previous studies have shown that many factors may be involved, including aging, genetic factors, environmental toxins, infections, oxidative stress, and free radical formation. The main motor symptoms of PD are caused by the death of substantia nigra cells in the midbrain, resulting in a lack of dopamine in the relevant brain regions of patients.

[0004] Aromatic L-amino acid decarboxylase (AADC) is a homodimeric pyridoxal phosphate-dependent enzyme responsible for the synthesis of dopamine and serotonin and can catalyze the decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA or levodopa) to dopamine. AADC deficiency results in serotonin and catecholamine deficiency, reduced neurotransmitter DOPA, and causes severe motor and autonomic dysfunction (Nutt et al. 2020, Pons et al. 2004).

[0005] Glial cell line-derived neurotrophic factor (GDNF) is expressed in various brain regions of the central nervous system, and its relatively certain cellular sources include type I astrocytes, neurons in the substantia nigra-striatum and basal forebrain. Significant GDNF mRNA expression is observed in the basal ganglia, olfactory tubercle and other DA neuron projection regions, some motor-related neural structures such as the substantia innominata, cerebellar Purkinje cells and trigeminal motor nucleus, and some sensory-related structures such as the thalamus, trigeminal sensory nucleus, spinal dorsal horn, dorsal root ganglion and locus coeruleus (Golden et al. 1998). GDNF belongs to the group of neurotrophic factors (NTFs), and has a high affinity for DA neurons and is a highly specific neurotrophic factor for DA neurons. The mode of action of GDNF is mainly target-derived, but it may also have paracrine and autocrine modes of action, with stronger efficacy and broader neurotrophic effect (Lin et al. 1993). Studies have shown that GDNF has obvious trophic, survival and differentiation-promoting effects on cultured embryonic midbrain DAergic neurons in vitro, increases the size of neuronal cell bodies and extends axons (Lin et al. 1993), and also has protective and restorative effects on the substantia nigra and striatal DAergic systems in vivo (Wang et al. 2002). Treating mice with MPTP or rats with 6-hydroxydopa (6-OHDA) and injecting GDNF into the substantia nigra or striatum before or after treatment reduces the damage to DAergic neurons caused by MPTP or 6-OHDA, prevents the degeneration of DAergic neurons, induces the generation of new processes from remaining DAergic neurons, restores their DA levels and the density of DAergic nerve fibers, and significantly improves the animals' motor behavior (Kearns et al. 1997; D. Kirik et al. 2001; D. Kirik, Rosenblad, and Bjorklund 2000; Deniz Kirik, Georgievska, and Bjorklund 2004; Wang et al. 2002).

[0006] Recombinant adeno-associated virus (rAAV) is derived from nonpathogenic wild-type adeno-associated virus and has the characteristics of high safety, a wide range of host cells (dividing and non-dividing cells), low immunogenicity, and long time to express exogenous genes in vivo. It is considered one of the most promising gene delivery vectors and is widely used in gene therapy and vaccine research around the world (Snyder 1999; Xiao, Lentz, and Samulski 2012).

[0007] Currently, the most successful commercially available drug for treating PD is the dopamine drug levodopamine (L-DOPA). However, as the treatment period continues, the benefits of dopamine therapy are gradually lost due to the progressive death of dopaminergic neurons and the loss of AADC activity, which causes a dose-dependent increase in the long-term drug. Meanwhile, systemic administration of high doses of dopamine is accompanied by strong side effects, such as immediate side effects (gastrointestinal side effects, cardiovascular side effects, sleep disorders and worsening of psychiatric symptoms, etc.) and remote side effects (dyskinesia, motor fluctuations, morning stiffness, delayed onset of action, etc.). Therefore, it is an urgent task to provide a safe and long-lasting effective therapeutic drug. The present disclosure provides an improved polynucleotide construct that utilizes AAV and its derivative vectors to deliver genes such as AADC, GDNF, etc., packaged in AAV capsids to specific regions of the brain (e.g., striatum) for the treatment of PD by gene therapy. Summary of the Invention

[0008] The present disclosure provides polynucleotides expressing AADC and / or GDNF proteins, recombinant adeno-associated virus (rAAV) particles, associated expression cassettes, plasmids, vectors, host cells, pharmaceutical compositions, and methods and related pharmaceutical uses thereof for treating, alleviating or preventing neurodegenerative diseases (e.g., Parkinson's disease).

[0009] AADC and / or GDNF Polynucleotide Sequences The present disclosure provides a nucleic acid molecule comprising a polynucleotide encoding an AADC protein (the coding amino acid sequence of the AADC protein is, for example, set forth in SEQ ID NO:1), wherein the polynucleotide has at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% identity to SEQ ID NO:3 (e.g., at least 80.83%, at least 81.21%, at least 79.18%, at least 79.63% or more identity), or at least 95% identity to any one of SEQ ID NOs:4-7. In some embodiments, the coding polynucleotide of the AADC protein comprises a polynucleotide sequence set forth in any one of SEQ ID NOs:3-7.

[0010] The present disclosure provides a nucleic acid molecule comprising a polynucleotide encoding a GDNF protein (the coding amino acid sequence of the AADC protein is, for example, set forth in SEQ ID NO: 2), wherein the polynucleotide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95% identity to SEQ ID NO: 8 (e.g., at least 75.13%, at least 73.99%, at least 77.52%, at least 77.83% or more identity), or at least 95% identity to any one of SEQ ID NOs: 9-12. In some embodiments, the coding polynucleotide of the GDNF protein comprises a polynucleotide sequence set forth in any one of SEQ ID NOs: 8-12.

[0011] In some embodiments of the present disclosure, the AADC protein, GDNF protein includes analogs thereof.

[0012] The present disclosure provides a nucleic acid molecule comprising a first polynucleotide comprising a polynucleotide encoding an AADC protein (the coding amino acid sequence of the AADC protein is, for example, shown in SEQ ID NO: 1) and a second polynucleotide comprising a polynucleotide encoding a GDNF protein (the coding amino acid sequence of the AADC protein is, for example, shown in SEQ ID NO: 2). In some embodiments, the first polynucleotide and the second polynucleotide are operably linked, and the first polynucleotide and the second polynucleotide may be in the same polynucleotide, the same plasmid, or the same expression cassette. In other embodiments, the first polynucleotide and the second polynucleotide are two polynucleotides that exist independently of each other, or two different plasmids that exist independently of each other, or two different expression cassettes that exist independently of each other.

[0013] In some embodiments, the amino acid sequence of the AADC protein comprises or is represented by SEQ ID NO:1 and the amino acid sequence of the GDNF protein comprises or is represented by SEQ ID NO:2.

[0014] In some embodiments, the first polynucleotide sequence encoding the AADC protein and / or the second polynucleotide sequence encoding the GDNF protein are codon-optimized. For example, the codon-optimized polynucleotide sequence encoding the AADC protein has at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% identity to SEQ ID NO:3, and the codon-optimized polynucleotide sequence encoding the GDNF protein has at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity to SEQ ID NO:8.

[0015] In some embodiments, the polynucleotide sequence encoding the AADC protein comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 4 to 7, and / or the polynucleotide sequence encoding the GDNF protein comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 9 to 12. In some specific embodiments, the polynucleotide sequence encoding the AADC is a polynucleotide sequence comprising or represented by any one of SEQ ID NOs: 3 to 7, and the polynucleotide sequence encoding the GDNF is a polynucleotide sequence comprising or represented by any one of SEQ ID NOs: 8 to 12.

[0016] In this disclosure, "at least 95% identity" is meant to encompass at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity.

[0017] The codon-optimized polynucleotide expressing the AADC protein (e.g., any one of SEQ ID NOs: 4 to 7) increases the expression level of the AADC protein compared to a wild-type or non-codon-optimized polynucleotide (e.g., SEQ ID NO: 3). The codon-optimized polynucleotide expressing the GDNF protein (e.g., any one of SEQ ID NOs: 9 to 12) increases the expression level of the GDNF protein compared to a wild-type or non-codon-optimized polynucleotide (e.g., SEQ ID NO: 8). The increase is at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, or at least several times (including 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the protein expression level compared to a wild-type or non-codon-optimized polynucleotide.

[0018] In some embodiments, the nucleic acid molecule provided in the present disclosure further comprises the same expression control sequence operably linked to the first polynucleotide and the second polynucleotide, or two identical or different expression control sequences operably linked to the first polynucleotide and the second polynucleotide, respectively, wherein the expression control sequence comprises (c) a promoter and / or (d) an enhancer.

[0019] In some embodiments, the nucleic acid molecule provided herein comprises: (a) a 5' inverted terminal repeat (5'ITR); (b) a 3' inverted terminal repeat (3'ITR); (e) an intron; (f) a post-transcriptional regulatory element; (g) a polyadenylation signal (polyA); (h) Multiple cloning site (MCS) and The present invention further includes any one or any combination of the following:

[0020] In some specific embodiments, any combination of (a)-(h) can fulfill the function of expressing the target gene (AADC and / or GDNF) (e.g., in the subject's brain (e.g., substantia nigra, striatum)).

[0021] In some specific embodiments, any one or any combination of the polynucleotides (a) to (h) above is operably linked to a polynucleotide encoding the AADC protein and / or a polynucleotide encoding the GDNF protein.

[0022] In some specific embodiments, the 5'ITR and / or 3'ITR are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ or AAV-DJ8, e.g., AAV2, AAV9.

[0023] In some specific embodiments, the promoter is selected from CMV, CAG, CBh, EFS, EF1 (e.g., EF-1α), PGK, SV40, Ubi, RSV, or any combination thereof.

[0024] In some specific embodiments, the enhancer is selected from a Ubi, CMV, RSV enhancer, or any combination thereof.

[0025] In some specific embodiments, the intron is selected from MVM, SV40, β Globin, EF1 (eg, EF-1α), a hybrid intron, or any combination thereof.

[0026] In some specific embodiments, the polyA is selected from PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA, or any combination thereof.

[0027] In some specific embodiments, the post-transcriptional regulatory element is selected from a WPRE, an HPRE, or a combination thereof.

[0028] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an AADC protein, a CMV enhancer (e.g., CMV enhancer 1) and a CBA promoter, and optionally, the first polynucleotide further comprises a hybrid intron, polyA, wherein the polyA is selected from SV40 polyA and hGH polyA, and / or the nucleic acid molecule comprises a polynucleotide encoding a GDNF protein, a CMV enhancer (e.g., CMV enhancer 2) and a CMV promoter, and optionally, the second polynucleotide further comprises a β-globin intron, polyA, wherein the polyA is selected from PA75 polyA and SV40 polyA, wherein the CMV enhancer 1 and CMV enhancer 2 are both CMV enhancers, and the two may be the same or different.

[0029] In some embodiments, AADC and GDNF in the above polynucleotide can be expressed individually using different expression cassettes, or can be shared by linking them to a single expression cassette via a linker (e.g., a P2A sequence).

[0030] The first polynucleotide and the second polynucleotide are operably linked so that the AADC protein and the GDNF protein are expressed at the same target administration site in the subject, thereby avoiding the influence of the mismatch in the administration site of the two on the therapeutic effect of the combination of the two, and reducing side effects caused by leakage of one protein to another site. Referring to the examples of the present disclosure, it can be seen that a remarkable therapeutic effect can be achieved by administering the first polynucleotide and the second polynucleotide to a disease model mouse after operably linking them.

[0031] In some embodiments, the first polynucleotide and the second polynucleotide are linked via a third polynucleotide, and for example, the third polynucleotide encodes an amino acid sequence having a linker function. The order of linkage may be the first polynucleotide-the third polynucleotide-the second polynucleotide from the 5' end to the 3' end, or the second polynucleotide-the third polynucleotide-the first polynucleotide from the 5' end to the 3' end. In some specific embodiments, the amino acid sequence encoded by the third polynucleotide is represented by any one of SEQ ID NOs: 43 to 46, and for example, the sequence of the third polynucleotide is represented by any one of SEQ ID NOs: 47 to 50, or has at least 80%, at least 90%, at least 95%, or at least 98% identity thereto.

[0032] In some embodiments, the polynucleotide encoding the AADC protein and the polynucleotide encoding the GDNF protein are regulated by the same regulatory element or by two different regulatory elements, including, but not limited to, 5'ITR, 3'ITR, promoter, enhancer, intron, polyA, post-transcriptional regulatory element, etc.

[0033] In some embodiments, the nucleic acid molecule comprises from the 5' to the 3' end:

[0034] a) comprising a CMV enhancer, a CBA promoter, a first polynucleotide encoding an AADC protein, a second polynucleotide encoding a GDNF protein, and polyA; In some alternative embodiments, the method further comprises comprising: In some alternative embodiments, the nucleic acid further comprises a WPRE sequence between the second polynucleotide encoding the GDNF protein and the polyA.

[0035] In some specific embodiments, the nucleic acid molecule comprises a CMV enhancer, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, a second polynucleotide encoding a GDNF protein, a WPRE sequence and polyA.

[0036] b) comprising a CMV enhancer, a CBA promoter, a second polynucleotide encoding a GDNF protein, a first polynucleotide encoding an AADC protein, and polyA; In some alternative embodiments, the method further comprises comprising: In some alternative embodiments, the nucleic acid further comprises a WPRE sequence between the first polynucleotide encoding the AADC protein and the polyA.

[0037] In some specific embodiments, the nucleic acid molecule comprises a CMV enhancer, a CBA promoter, a hybrid intron, a second polynucleotide encoding a GDNF protein, a first polynucleotide encoding an AADC protein, a WPRE sequence and polyA.

[0038] c) comprising a CMV enhancer, a CBA promoter, a first polynucleotide encoding an AADC protein, polyA, a CMV enhancer, a CMV promoter, a second polynucleotide encoding a GDNF protein, and polyA; In some alternative embodiments, the method further comprises comprising: In some alternative embodiments, the method further comprises comprising the step of: In some alternative embodiments, the nucleic acid further comprises a WPRE sequence between the second polynucleotide encoding the GDNF protein and the polyA.

[0039] In some specific embodiments, the nucleic acid molecule is CMV enhancer 1, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, polyA, a CMV enhancer 2, a CMV promoter, a second polynucleotide encoding a GDNF protein, and polyA, or CMV enhancer 1, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, polyA, a CMV enhancer 2, a CMV promoter, a second polynucleotide encoding a GDNF protein, a WPRE sequence and polyA, or It comprises CMV enhancer 1, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, polyA, CMV enhancer 2, a CMV promoter, a β-globin intron, a second polynucleotide encoding a GDNF protein, and polyA.

[0040] d) comprising a CMV enhancer, a CMV promoter, a second polynucleotide encoding a GDNF protein, polyA, a CMV enhancer, a CBA promoter, a first polynucleotide encoding an AADC protein, and polyA; In some alternative embodiments, the method further comprises comprising the step of: In some alternative embodiments, the method further comprises comprising: In some alternative embodiments, the method further comprises comprising the steps of: providing a first polynucleotide encoding an AADC protein and a polyA sequence, the first polynucleotide encoding the AADC protein being separated from the polyA sequence by a WPRE sequence; In some specific embodiments, the nucleic acid molecule is CMV enhancer 2, CMV promoter, β globulin intron, a second polynucleotide encoding a GDNF protein, polyA, CMV enhancer 1, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, and polyA, CMV enhancer 2, a CMV promoter, a second polynucleotide encoding a GDNF protein, polyA, a CMV enhancer 1, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, a WPRE sequence and polyA, or It comprises CMV enhancer 2, a CMV promoter, an MVM intron, a second polynucleotide encoding a GDNF protein, polyA, CMV enhancer 1, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, a WPRE sequence and polyA.

[0041] e) comprising a CMV enhancer, a CBA promoter, a first polynucleotide encoding an AADC protein, a third polynucleotide, a second polynucleotide encoding a GDNF protein, and polyA; In some alternative embodiments, the method further comprises comprising: In some alternative embodiments, the method further comprises comprising the steps of: (a) providing a second polynucleotide encoding a GDNF protein and a WPRE sequence between the second polynucleotide and the polyA; In some specific embodiments, the nucleic acid molecule comprises a CMV enhancer, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, a third polynucleotide, a second polynucleotide encoding a GDNF protein, a WPRE sequence and polyA.

[0042] f) comprising a CMV enhancer, a CBA promoter, a second polynucleotide encoding a GDNF protein, a third polynucleotide, a first polynucleotide encoding an AADC protein, and polyA; In some alternative embodiments, the method further comprises comprising: In some alternative embodiments, the method further comprises comprising the steps of: providing a first polynucleotide encoding an AADC protein and a polyA sequence, the first polynucleotide encoding the AADC protein being separated from the polyA sequence by a WPRE sequence; In some specific embodiments, the nucleic acid molecule comprises a CMV enhancer, a CBA promoter, a hybrid intron, a second polynucleotide encoding a GDNF protein, a third polynucleotide, a first polynucleotide encoding an AADC protein, a WPRE sequence and polyA.

[0043] g) comprising a CMV enhancer, a CBA promoter, a first polynucleotide or a second polynucleotide, and polyA; In some alternative embodiments, the method further comprises a hybrid intron between the CBA promoter and the first polynucleotide, or a hybrid intron between the CBA and the second polynucleotide; In some alternative embodiments, the nucleic acid sequence further comprises a WPRE sequence between the first polynucleotide and the polyA, or a WPRE sequence between the second polynucleotide and the polyA, In some specific embodiments, the nucleic acid molecule comprises a CMV enhancer, a CBA promoter, a hybrid intron, a first polynucleotide and a polyA, A CMV enhancer, a CBA promoter, a hybrid intron, a second polynucleotide and polyA, A CMV enhancer, a CBA promoter, a hybrid intron, a first polynucleotide, a WPRE sequence and polyA, or It comprises a CMV enhancer, a CBA promoter, a hybrid intron, a second polynucleotide, a WPRE sequence and polyA.

[0044] In some embodiments, the polyA is selected from hGH polyA, PA75 polyA, or SV40 polyA.

[0045] In some embodiments, a polynucleotide is provided, the nucleic acid molecule comprising, from the 5' end to the 3' end: (1) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-third polynucleotide-second polynucleotide encoding GDNF-WPRE-SV40 polyA; (2) CMV enhancer-CBA promoter-hybrid intron-second polynucleotide encoding GDNF-third polynucleotide-first polynucleotide encoding AADC-WPRE-SV40 polyA; (3) a first polynucleotide encoding a CMV enhancer-CBA promoter-hybrid intron-AADC-SV40 polyA-CMV enhancer-CMV promoter-a second polynucleotide encoding GDNF-PA75 polyA; (4) a first polynucleotide encoding a CMV enhancer-CBA promoter-hybrid intron-AADC-SV40 polyA-CMV enhancer-CMV promoter-a second polynucleotide encoding GDNF-WPRE-PA75 polyA; (5) a first polynucleotide encoding a CMV enhancer-CBA promoter-hybrid intron-AADC-SV40 polyA-CMV enhancer-CMV promoter-β globulin intron-a second polynucleotide encoding GDNF-PA75 polyA; (6) CMV enhancer-CMV promoter-β globulin intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-SV40 poly A; (7) CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-WPRE-SV40 poly A, or (8) CMV enhancer-CMV promoter-MVM intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-WPRE-SV40 poly A; Includes.

[0046] In some embodiments, a polynucleotide is provided that comprises, in the 5' to 3' direction, the following: 1) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide or GDNF coding polynucleotide, hGH polyA, 3'ITR, 2) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide or GDNF coding polynucleotide, SV40 polyA, 3'ITR, 3) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide or GDNF coding polynucleotide, WPRE, SV40 polyA, 3'ITR, 4) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide, P2A, GDNF coding polynucleotide, WPRE, SV40 polyA, 3'ITR, 5) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, GDNF coding polynucleotide, P2A, AADC coding polynucleotide, WPRE, SV40 polyA, 3'ITR, 6) 5'ITR, MCS, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide, SV40 polyA, CMV enhancer 2, CMV promoter, GDNF coding polynucleotide, PA75 polyA, 3'ITR, 7) 5'ITR, MCS, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide, SV40 polyA, CMV enhancer 2, CMV promoter, GDNF coding polynucleotide, WPRE, PA75 polyA, 3'ITR, 8) 5'ITR, MCS, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide, SV40 polyA, CMV enhancer 2, CMV promoter, β-globulin intron, GDNF coding polynucleotide, PA75 polyA, 3'ITR, 9) 5'ITR, CMV enhancer 2, CMV promoter, β-globulin intron, GDNF coding polynucleotide, PA75 polyA, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide, SV40 polyA, 3'ITR, 10) 5'ITR, CMV enhancer 2, CMV promoter, GDNF coding polynucleotide, PA75 polyA, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide, WPRE, SV40 polyA, 3'ITR, 11) 5'ITR, CMV enhancer 2, CMV promoter, MVM intron, GDNF coding polynucleotide, PA75 polyA, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide, WPRE, SV40 polyA, 3'ITR, 12) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC coding polynucleotide, P2A, GDNF coding polynucleotide, WPRE, SV40 polyA, 3'ITR, and 13) 5'ITR, CMV enhancer 2, CMV promoter, β-globin intron, GDNF-encoding polynucleotide, PA75 polyA, CMV enhancer 1, CBA promoter, hybrid intron, AADC-encoding polynucleotide, SV40 polyA, 3'ITR.

[0047] In some specific embodiments, The CMV enhancer 1 is a sequence comprising or set forth in SEQ ID NO: 27, The CMV enhancer 2 comprises or is a sequence represented by SEQ ID NO: 37, The CBA promoter comprises or is a sequence as set forth in SEQ ID NO: 28, The CMV promoter comprises or is a sequence as set forth in SEQ ID NO: 35, The β-globin intron comprises or is a sequence as set forth in SEQ ID NO: 38, The hybrid intron comprises or has the sequence set forth in SEQ ID NO: 29, The MVM intron comprises or is a sequence as set forth in SEQ ID NO: 39, The PA75 polyA comprises or is a sequence represented by SEQ ID NO: 36, The SV40 polyA comprises or is a sequence represented by SEQ ID NO: 32, The hGH polyA comprises or is a sequence as set forth in SEQ ID NO: 30, The HPRE comprises or is a sequence as set forth in SEQ ID NO: 33, The WPRE is a sequence comprising or set forth in SEQ ID NO: 34, the 5'ITR comprises or is a sequence as set forth in SEQ ID NO: 26, the 3'ITR comprises or is a sequence as set forth in SEQ ID NO: 31, The MCS comprises or is a sequence as set forth in SEQ ID NO: 40, and / or Said P2A is a sequence comprising or represented by SEQ ID NO:33.

[0048] In some embodiments, a nucleic acid molecule is provided comprising a polynucleotide having at least 95% identity to a sequence set forth in any one of SEQ ID NOs: 13 to 25. In some embodiments, the polynucleotide comprises a sequence set forth in any one of SEQ ID NOs: 13 to 25.

[0049] In some embodiments, the AADC protein (e.g., the AADC protein set forth in SEQ ID NO:1) comprises a variant thereof (e.g., the variant has at least 60%, at least 70%, at least 80%, at least 90, at least 95% or more identity to SEQ ID NO:1, NP_000781.2, NP_001076440.2, NP_001229815.2, NP_001229816.2, NP_001229817.2, NP_001229818.2, NP_001229819.2) or a fragment thereof, and the variant or fragment is an AAD It has the same or similar biological activity or function as the GDNF protein, and the GDNF protein (e.g., the GDNF protein shown in SEQ ID NO: 2) includes its mutants (the mutants have, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more identity to SEQ ID NO: 2, NP_001177397.1, NP_001177398.1, NP_001265027.1, NP_954701.1) or fragments, and the mutants or fragments have the same or similar biological activity or function as the GDNF protein.

[0050] In some embodiments, the polynucleotides include precursor molecules that are processed within a cell. The AADC and / or GDNF polynucleotides or their processed forms may be encoded and delivered to a cell in a plasmid, vector, genome or other polynucleotide expression vector.

[0051] In some embodiments, the vector contains an intact replicon so that it can replicate in a cell, for example after transfection, infection or transformation of the cell. In some embodiments, the vector is or is derived from a retrovirus, adenovirus, herpesvirus, baculovirus, papillomavirus, or a modified or altered version of the above. In some embodiments, methods of delivering the vector include, but are not limited to, direct delivery of naked DNA, or delivery by cationic or anionic liposomes, by complexation with cationic polymers, by complexation with proteins or polypeptides.

[0052] In some embodiments, the polynucleotide is designed as a component of an AAV vector genome (or viral genome) and packaged into a rAAV particle, which is processed intracellularly to produce AADC and / or GDNF protein, which may be a wild-type protein or a mutant thereof.

[0053] In some embodiments, the polynucleotide may be the payload of an rAAV particle.

[0054] AAV capsids and AAV particles Any one of the above polynucleotides of the present disclosure can be packaged into the AAV capsid described below to form an AAV particle.

[0055] Does the above AAV contain or originate from the AAV serotype, PHP.B, PHP.A, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV AV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AA V11、AAV12、AAV16.3、AAV24.1、AAV27.3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV 42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44. 4.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61 4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.55 7、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAV C2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh. .65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVc y.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AA Vhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu. 34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.4 6、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.68、AAVhu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVhu. Vrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh. h.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh. 51、AAVrh.52、AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.88 A586R mutation、AAVrh8R R533A mutant、AAAV、BAAV、ヤギAAV、ウシAAV、ヒツジAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr 1.35、AAVhEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr2. r3.1、AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV -LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK16、AAV-LK17、AAV-LK18、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAVShuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAVShuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAVSM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENNAAV 10, Japanese AAV serotype 10, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAVCBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAVCBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAVCBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAVCHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAVCKd-4AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAVCSp-8、AAV CSp-8.10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / , HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, PHP.B (AAV-PHP.B), PHP.A (AAV.PHP.A), G2B-26, G2B-13, TH1.1-32, TH1.1-35, AAVPHP.B2, AAVPHP.B3, AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3, AAVG2B4, AAVG2B5, AAVDJ8 and variants thereof, including but not limited to these.

[0056] In some embodiments, the AAV capsid may be modified or mutated, e.g., includes one or more of the following mutations: Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F, e.g., includes one or more of the following mutations: F129L, D418E, K531E, L584F, V598A, and H642N, e.g., the AAV capsid of AAV2. At least one of the Tyr residues at positions 252, 272, 444, 500, 700, 704, and 730 is substituted with, for example, a Phe residue; for example, the AAV2 capsid includes an N587A, E548A, or N708A mutation; for example, the AAV9 capsid includes a T446F mutation; for example, the AAV capsid includes a V708K mutation; for example, the above AAV capsid is produced by an AAV9 capsid library having mutations in amino acids at positions 390 to 627 (VP1 numbering).

[0057] The present disclosure relates to WO2018232055 (e.g., Table 1 thereof), WO2005033321, WO2015168666, WO2015121501, WO2015038958, WO2016065001, WO2016130589, WO2016049230, WO2016134375, WO2017100671, WO2017083722, WO2017015102, WO2017058892, WO2017066764, US9546112, US7198951, US9233131, US6156303, US96 24274, US9475845, US8734809, US20130224836, US20140359799, US20150315612, US20150376240, US20150159173, US20150376607, US20150238550, US20160369298, US20160361439, US20170145405, and N Pulicerla et al. (Molecular Therapy 19(6):1070-1078 (2011)). In some specific embodiments, the AAV capsid used in the present disclosure is the AAV2 and AAV9 capsid in the above prior art.

[0058] In some embodiments, the AAV capsid is engineered, e.g., a hybrid AAV capsid derived from two or more parent serotypes. For example, the AAV capsid may be AAV2G9, which contains sequences from AAV2 and AAV9, incorporating all of the sequences of AAV2G9 in US20160017005.

[0059] In some embodiments, the AAV capsid is an AAV2 or AAV9 capsid comprising, for example, the amino acid sequence set forth in SEQ ID NO: 41 or 42.

[0060] In some embodiments, other than the encoding heterologous payload, the AAV particles may comprise all or a portion of any naturally occurring and / or recombinant AAV capsid nucleotide sequence or mutant vector genome.

[0061] In some embodiments, the AAV particles may be replication-deficient (e.g., lacking sequences in the vector genome encoding functional Rep and Cap proteins). In some specific embodiments, the replication-deficient AAV particles may lack most or all of the parent coding sequences and have essentially only one or two AAV ITR sequences and a polynucleotide of interest for delivery to a cell, tissue, organ, or organism.

[0062] In some embodiments, the AAV particles may be recombinant AAV (rAAV) particles.

[0063] In some embodiments, the AAV particles may be selected from single-stranded AAV particles (e.g., ssAAV), self-complementary AAV particles (e.g., scAAV), which achieve rapid expression in cells by skipping second strand synthesis.

[0064] In some embodiments, the AAV particles are packaged with a polynucleotide having at least 95% identity to any one of SEQ ID NOs: 13-25.

[0065] In some embodiments, the AAV particles are packaged with a polynucleotide comprising a sequence set forth in any one of SEQ ID NOs: 13 to 25.

[0066] Regulatory elements The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure contains at least one regulatory element to enable replication, transcription and translation of the target gene (e.g., AADC, GDNF) in the payload.

[0067] In some embodiments, the regulatory elements include, but are not limited to, sequences for transcription initiation and / or termination, promoter and / or enhancer sequences, efficient RNA processing signals (e.g., splicing and polyadenylation signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak consensus sequences), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, introns, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences, and upstream enhancers (USEs).

[0068] In some embodiments, the regulatory elements (e.g., promoters) described below drive the payload to be expressed in a target tissue (e.g., the brain (e.g., striatum, substantia nigra) of a subject) for a period of time, such as 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 years or more. Also, for example, 1 to 2 weeks, 1 to 3 weeks, 1 to 4 weeks, 1 to 2 months, 1 to 4 months, 1 to 6 months, 2 to 6 months, 3 to 6 months, 3 to 9 months, 4 to 8 months, 6 to 12 months, 1 to 2 years, 1 to 5 years, 2 to 5 years, 3 to 6 years, 3 to 8 years, 4 to 8 years, 5 to 10 years, 10 to 20 years, 10 to 30 years, 20 to 40 years, 20 to 50 years, or the entire lifetime of the subject.

[0069] inverted terminal repeat (ITR) The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure comprises at least one ITR, e.g., having two ITRs flanking the payload at the 5' and 3' ends, respectively.

[0070] In some embodiments, the ITRs have an origin of replication function.

[0071] In some embodiments, the ITRs contain complementary, symmetrically alignable sequence regions.

[0072] In some embodiments, the ITRs may consist of naturally occurring or recombinantly derived polynucleotide sequences.

[0073] In some embodiments, the ITRs may be derived from the same or different serotype as the capsid. In some embodiments, the 5'ITR and 3'ITR may be derived from the same serotype or from different serotypes. For example, the 5'ITR and 3'ITR are both derived from AAV2 or both derived from AAV9. Also, for example, the 5'ITR comprises the sequence set forth in SEQ ID NO:26 and / or the 3'ITR comprises the nucleotide sequence set forth in SEQ ID NO:31.

[0074] In some embodiments, the length of each ITR may be about 100 to about 150 nucleotides, e.g., 100 to 105 nucleotides, 106 to 110 nucleotides, 111 to 115 nucleotides, 116 to 120 nucleotides, 121 to 125 nucleotides, 126 to 130 nucleotides, 131 to 135 nucleotides, 136 to 140 nucleotides, 141 to 145 nucleotides, or 146 to 150 nucleotides. In one embodiment, the length of the ITR is 140 to 142 nucleotides, e.g., 141 nucleotides. Non-limiting examples of ITR lengths are 102, 140, 141, 142, 145 nucleotides in length, and lengths with at least 95% identity thereto.

[0075] promoter The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure comprises at least one promoter, including, but not limited to, a species-specific, inducible, tissue-specific or cell cycle-specific promoter.

[0076] In some embodiments, the promoter drives expression of a protein or polypeptide (e.g., AADC and / or GDNF) encoded in the vector genome payload of the AAV particle.

[0077] In some embodiments, the promoter is target tissue specific or tropic, for example a promoter capable of expressing the payload in neural tissue.

[0078] In some embodiments, the promoter may be a viral promoter, a plant promoter, a mammalian promoter, or a human promoter.

[0079] In some embodiments, the promoter includes, but is not limited to, a CMV, CBA (including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, RSV, EFS, EF1, GUSB (hGBp), UCOE (promoter of HNRPA2B1-CBX3), NSE, Synapsin, MeCP2, MeP418, MeP426, VMD2, MRHO, TRE, Ac5, Polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, Ubi, GFAP, or PKG promoter, and may be selected from a neurofilament light chain (NFL) promoter, a neurofilament heavy chain (NFH) promoter, a SCN8A promoter, a frataxin (FXN) promoter (also referred to as a FRDA promoter), a H1 promoter, a RNA pol III promoter (e.g., U6 or H1), or a small nuclear RNA (ULB or ULA) promoter. In some embodiments, the promoter is a liver or skeletal muscle promoter, liver promoters such as human alpha-1-antitrypsin (hAAT) and thyroxine-binding globulin (TBG), and skeletal muscle promoters such as desmin, MCK, or synthetic C5-12.

[0080] In some embodiments, the AAV vector genome of the present disclosure comprises two promoters, e.g., the CMV and CBA promoters, whose sequences are set forth in SEQ ID NOs: 35 and 28, respectively, or, e.g., the EF1α promoter and the CMV promoter, or any two of the above promoters in combination.

[0081] In some embodiments, the promoter is a tissue specific expression element that can limit expression to certain cell types, including, but not limited to, a muscle specific promoter, a B cell promoter, a monocyte promoter, a leukocyte promoter, a macrophage promoter, a pancreatic acinar cell promoter, an endothelial cell promoter, a lung tissue promoter, an astrocyte promoter, or a nervous system promoter, which can be used to limit expression to neurons, astrocytes, or oligodendrocytes.

[0082] In some embodiments, the promoter is selected from the group consisting of neuronal tissue-specific expression element, neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methylated CpG binding protein 2 (MeCP2), Ca 2+ / Calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), β-globin minigene nβ2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoters. Non-limiting examples of tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoters. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include the myelin basic protein (MBP) promoter.

[0083] In some embodiments, the promoter is a truncation or mutant of the above promoters and is less than 1 kb in length, for example, 200-300, 200-400, 300-400, 200-500, 200-600 or more in length.

[0084] In some embodiments, the promoter may be a combination of two or more components of the same or different initiating or parent promoters, for example, CMV and CBA.

[0085] Enhancer The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure can comprise at least one enhancer, including, but not limited to, a species-specific, inducible, tissue-specific or cell cycle-specific enhancer.

[0086] In some embodiments, the enhancer is target tissue specific or tropic, for example an enhancer that can regulate expression of the payload in neural tissue.

[0087] In some embodiments, the enhancer may be or be derived from a viral enhancer, a plant enhancer, a mammalian enhancer, or a human enhancer.

[0088] In some embodiments, the enhancer can be located upstream or downstream of the promoter and operably linked to the promoter, and when the enhancer is present, expression of the target gene (e.g., AADC and / or GDNF) is enhanced by at least 20%, at least 50%, at least 80%, at least 100%, or at least several fold (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 fold or more) compared to when the enhancer is not present.

[0089] In some embodiments, the enhancer includes, but is not limited to, EF-la, Ubc, humanb-actin, CAG, TRE, Ac5, Polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, Ubi, and alpha-1-antitrypsin (hAAT), or a variant or fragment of the enhancer. In some embodiments, the enhancer is selected from IRBP, RSV, or CMV enhancers, or a variant or fragment thereof.

[0090] In some embodiments, the enhancer comprises or is an enhancer set forth in SEQ ID NO: 27 or 37, or a variant or fragment thereof.

[0091] Introns Any one of the above polynucleotides or vector genomes (or viral genomes) of the AAV particle of the present disclosure may include an intron or a portion thereof, In some embodiments, the introns include, but are not limited to, MVM (67-97 bp), intron 1 of F.IX cleavage (approximately 300 bp), β-globulin SD / immunoglobulin heavy chain splicing acceptor (approximately 250 bp), adenovirus splicing donor / immunoglobulin splicing acceptor (approximately 500 bp), SV40 late splicing donor / splice acceptor (19S / 16S) (180 bp), and hybrid adenovirus splicing donor / IgG splicing acceptor (approximately 230 bp), hemoglobin intron, hybrid introns, β-globin intron, or variants or fragments of the above introns.

[0092] In some embodiments, the length of the intron is 100 to 800 nucleotides, for example, about 100, about 200, about 300, about 400, about 500, about 600 nucleotides, or, for example, 200 to 250, 200 to 300, 100 to 200, 300 to 500, 500 to 600, 400 to 600, or 100 to 200 nucleotides.

[0093] In some embodiments, the intron in the technical solution of the present disclosure is selected from a hybrid intron and a β-globin intron, the sequences of which are shown in SEQ ID NO: 29 and SEQ ID NO: 38, respectively.

[0094] Untranslated Regions (UTRs) The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure may include an untranslated region (UTR) selected from 5'UTR and / or 3'UTR to adjust (e.g., increase, decrease or reduce) polynucleotide stability and protein production. Typically, the 5'UTR starts at the transcription start site and ends at the start codon, and the 3'UTR is immediately after the stop codon to the transcription termination signal. The above UTRs may be wild type, mutants thereof, or artificial UTRs.

[0095] In some embodiments, the 5'UTR comprises a Kozak sequence. In other embodiments, the 5'UTR does not comprise a Kozak sequence.

[0096] In some embodiments, the 3'UTR is AU-rich. In some embodiments, the 3'UTR is selected from type I AREs, which contain multiple interspersed copies of AUUUA motifs in a U-rich region, such as, but not limited to, c-Myc and MyoD, type II AREs, which have two or more overlapping UUAUUUA(U / A)(U / A) nonamers, such as, but not limited to, GM-CSF and TNF-a, and type III AREs, such as, but not limited to, c-Jun and myogenic proteins.

[0097] In some embodiments, the 3'UTR may contain an oligo(dT) sequence to template the addition of a polyadenylation sequence (PolyA).

[0098] Polyadenylation sequence (PolyA) The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure may comprise a sequence encoding PolyA, which may be wild-type, a mutant thereof, or a modified PolyA such that protein translation occurs.

[0099] In some embodiments, the PolyA encoding sequence is between the 3' end of the payload coding sequence and the 5' end of the 3' ITR.

[0100] In some embodiments, the length of the sequence encoding the PolyA is 0 to 500 nucleotides, for example, about 75, about 100, about 110, about 120, about 150, about 160, about 200, or about 300 nucleotides, or, for example, 50 to 100, 50 to 150, 50 to 160, 50 to 200, 60 to 100, 60 to 150, 60 to 160, 60 to 200, 70 to 100, 70 to 150, 70 to 160, 70 to 200, 80 to 100, 80 to 150, 80 to 160, 80 to 200, 90 to 100, 90 to 150, 90 to 160, or 90 to 200.

[0101] In some embodiments, the coding PolyA can code for a continuous or discontinuous PolyA. When coding for a discontinuous PolyA, the coding sequence can be interrupted or isolated by other nucleotides. For example, the polyA has at least two 60 adenylic acid fragments, and the at least two 60 adenylic acid fragments are isolated by a sequence comprising 10-90 nucleotides.

[0102] In some embodiments, the sequence encoding PolyA or polyA in the technical solution of the present disclosure is selected from β-globulin polyA, SV40 polyA, bGH polyA, PA75 polyA, MeCP2 polyA, RDH1 polyA, BGH polyA, SPA49 polyA, sNRP-TK65 polyA, sNRP polyA, TK65 polyA, or a variant or fragment of the above PolyA.

[0103] In some embodiments, the polyA is as shown in SEQ ID NO: 32 and SEQ ID NO: 36 in the technical solution of the present disclosure, or a variant or fragment thereof.

[0104] Herein, polyA or its coding sequence in WO2016005324, WO2016005004, WO2016091391, WO2019036513, and WO2020074642 are all incorporated, and all of them can be used in the technical proposals of the present disclosure.

[0105] Filler Arrangement The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure may include filler sequences such that the length of the vector genome is an optimal size for packaging, for example, the length of the vector genome is about 2.3 kb, about 4.6 kb, about 4.7 kb, or about 5.1 kb.

[0106] In some embodiments, the vector genome is a single-stranded or double-stranded genome and the packaged AAV particles are ssAAV or scAAV.

[0107] In some embodiments, a vector genome may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) filler sequences.

[0108] In some embodiments, the filler sequence may be located between or within multiple regulatory elements, for example, between two introns, 3' of the 5'ITR sequence, 5' of the 5'ITR sequence, 5' of the 3'ITR sequence, 3' of the 3'ITR sequence, and before or after regions such as promoters, introns, enhancers, polyA, polyclonal site (MCS) regions, exons, etc.

[0109] Methods for producing or preparing rAAV particles The present disclosure provides methods for producing, preparing and / or modifying rAAV particles, which incorporate all of the methods for producing, preparing and / or modifying rAAV particles in WO200028004, WO200123001, WO2004112727, WO 2005005610, WO2005072364, WO2013123503, WO2015191508 and US20130195801. The rAAV particles can have properties that enhance delivery efficiency, can be efficiently packaged, and can successfully infect target cells (e.g., mammalian or human cells) with high frequency and minimal toxicity.

[0110] In some embodiments, a method for producing rAAV particles is provided that includes packaging any of the polynucleotides or vector genomes (or viral genomes) or vectors of the present disclosure into an AAV capsid. In some specific embodiments, the method includes 1) co-transfecting a baculovirus vector and a viral construct vector and / or an AAV payload construct vector into competent cells, 2) isolating and transfecting the resulting viral construct expression vector and the AAV payload construct expression vector into viral replicating cells, respectively, 3) isolating and purifying the resulting viral construct particles comprising the payload and the viral construct expression vector or the AAV payload construct expression vector, 4) co-transfecting both the AAV payload and the viral construct particles comprising the viral construct expression vector or the AAV payload construct expression vector into viral replicating cells, and 5) collecting and purifying the AAV particles comprising the viral genome.

[0111] In some embodiments, a method for producing rAAV particles is provided, comprising: 1) co-transfecting any of the polynucleotides or vector genomes (or viral genomes) or vectors of the present disclosure, and constructs expressing Rep and Cap genes and auxiliary constructs (to achieve auxiliary functions) simultaneously into mammalian cells (e.g., HEK293 cells); 2) collecting and purifying rAAV particles containing the viral genome; Includes.

[0112] In some embodiments, the viral genome of the rAAV particle optionally encodes a selectable marker, which may include a cell surface marker, e.g., any protein expressed on the cell surface, including, but not limited to, receptor CD markers, lectins, integrins, or truncated forms thereof.

[0113] In some embodiments, an AAV production system for producing rAAV particles of the present disclosure is provided, wherein the production system comprises: 1) a polynucleotide sequence encoding an AAV capsid; and 2) any polynucleotide or vector genome (or viral genome) or vector of the present disclosure; 3) sufficient AAV rep and accessory functions to permit packaging of the polynucleotide or vector genome (or viral genome) or vector in (b) into an AAV capsid; Includes.

[0114] In the above embodiments, the AAV capsid is selected from any AAV capsid of the present disclosure, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, or AAV-DJ8.

[0115] In some specific embodiments, sufficient AAV rep and accessory functions are provided by a packaging cell, which may contain three plasmids: pHelper, pRC9, and pGOI.

[0116] In some embodiments, the Rep gene encodes nonstructural proteins that regulate functions such as replication of the AAV genome and may be selected from Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are typically transcribed from the p5 promoter, whereas Rep52 and Rep40 are typically transcribed from the p19 promoter. The Cap gene encodes the structural proteins VP1, VP2, and / or VP3 that assemble to form the viral capsid. The Cap gene is typically transcribed from the p40 promoter.

[0117] In some embodiments, rAAV particles produced by the AAV production system are provided.

[0118] cell The present disclosure provides a cell capable of packaging AAV, which may be any cell suitable for the production of heterologous proteins.

[0119] In some embodiments, the cell is a mammalian cell, including, but not limited to, HEK293, HELA, CHO, NSO, SP2 / 0, PER.C6, VERO, RD, BHK, HT 1080, A549, COS-7, ARPE-19, and MRC-5.

[0120] In some embodiments, the cells are insect cells and may be derived from a mosquito cell line, such as a fall armyworm, a Drosophila cell line, or an Aedesalbopictus derived cell line, including, but not limited to, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, Ha2302, Hz2E5, HighFive (Invitrogen, CA, USA), AO38, and BM-N.

[0121] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising a prophylactically or therapeutically effective amount of an active ingredient (e.g., any of the polynucleotides disclosed herein (including AADC polynucleotides and / or GDNF polynucleotides) or rAAV particles) and one or more pharma- ceutically acceptable excipients.

[0122] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of polynucleotide (including AADC polynucleotide and / or GDNF polynucleotide) or rAAV particles per unit dose. In some other specific embodiments, the pharmaceutical composition may contain 0.1 to 10 x 10 13 AADC gene copy amount and / or 0.1 to 10 × 10 13 Includes GDNF gene copy amount.

[0123] In some embodiments, the pharmaceutical composition may include a formulation, including, but not limited to, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, and combinations thereof. The formulation may be prepared by any method known or hereafter developed in the art of pharmacology.

[0124] In some embodiments, the rAAV particles may be transfected into cells for transfer or implantation into a subject.

[0125] In some embodiments, the pharmaceutical composition comprises an rAAV particle encapsulated with a polynucleotide, the polynucleotide having at least 95% identity to a sequence set forth in any one of SEQ ID NOs: 13-25, for example, the polynucleotide comprises a sequence set forth in any one of SEQ ID NOs: 13-25.

[0126] Methods for Treating Diseases and Pharmaceutical Uses The present disclosure provides methods or uses of any of the polynucleotides (including AADC polynucleotides and / or GDNF polynucleotides), rAAV particles, and pharmaceutical compositions of the present disclosure for treating, alleviating, or ameliorating a disease or condition.

[0127] The present disclosure provides methods or uses of any of the above polynucleotides, rAAV particles, pharmaceutical compositions in combination with levodopa to treat, alleviate, or ameliorate the above neurological diseases or conditions.

[0128] In some embodiments, any of the polynucleotides, rAAV particles, and pharmaceutical compositions disclosed herein are provided for administration in combination with levodopa.

[0129] In some embodiments, the present disclosure provides levodopa administered in combination with any of the polynucleotides, rAAV particles, and pharmaceutical compositions disclosed herein.

[0130] In some embodiments, the disease or condition is caused by or associated with a deficiency in an AADC protein, and / or the disease or condition is caused by or associated with a deficiency in a GDNF protein.

[0131] In some embodiments, the disease or condition is a neurological disease or condition, such as a central nervous system disease or condition. The central nervous system disease or condition may be a disease or condition affecting any part of the brain (including the hemispheres, diencephalon, thalamus, and cerebellum) or the spinal cord. The neurological disease or condition includes, but is not limited to, diseases or conditions characterized by neuromuscular diseases, lysosomal diseases, trauma, bone marrow injury, pain (including neuropathic pain), nervous system cancers, demyelinating diseases, nervous system autoimmune diseases, neurotoxic syndromes, sleeping sickness, inherited brain diseases, and developmental CNS pathologies. The polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure may alleviate or reduce symptoms due to abnormalities in the levels and / or function of gene products (e.g., protein deficiencies or defects) in a subject in need thereof, or otherwise benefit CNS pathologies in a subject in need thereof.

[0132] In some embodiments, the disease or condition is a neurological disease or condition, including, but not limited to, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob Disease (CJD), Huntington's disease (HD), Friedreich's ataxia (FA), Parkinson's disease (PD), multiple system atrophy (MSA), spinal muscular atrophy (SMA), multiple sclerosis (MS), primary progressive aphasia, progressive supranuclear palsy (PSP), dementia, brain cancer, neurodegenerative diseases, encephalitis, epilepsy, inherited brain diseases causing neurodegeneration, retinitis pigmentosa (RP), head and brain malformations, hydrocephalus, stroke, prion diseases, infantile neuronal ceroid lipofuscinosis (INCL), such as a pediatric neurodegenerative disease caused by a deficiency of lysosomal palmitoyl protein thioesterase-1 (PPT1).

[0133] In some embodiments, the disease or condition is a disease associated with impaired growth and development of the central nervous system, i.e., a neurodevelopmental disorder. In some embodiments, such a neurodevelopmental disorder is caused by a genetic mutation, and includes, but is not limited to, fragile X syndrome (caused by a mutation in the FMR1 gene), Down syndrome (caused by trisomy 21), Rett syndrome, Williams syndrome, Angelman syndrome, Smith-Magenis syndrome, ATR-X syndrome, Barth syndrome, immune dysfunction and / or infections of infancy, such as Sydenham chorea, schizophrenia, congenital toxoplasmosis, congenital rubella syndrome, metabolic diseases (e.g., diabetes mellitus and phenylketonuria), nutritional deficiencies and / or brain trauma, autism and autism spectrum disorders.

[0134] In some embodiments, the disease or condition is a tumor in the central nervous system, including, but not limited to, acoustic neuroma, astrocytoma (grades I, II, III, and IV), chordoma, CNS lymphoma, craniopharyngioma, glioma (e.g., brain stem glioma, ependymoma, optic nerve glioma, subependymoma), medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal tumor (PNET), and schwannoma.

[0135] In some embodiments, the disease or condition is a functional neurological disorder having motor and / or sensory symptoms with a neurological origin in the central nervous system, including, but not limited to, chronic pain, epileptic seizures, speech disorders, involuntary movements, and sleep disorders.

[0136] In some embodiments, the disease or condition is a white matter disorder (a group of disorders that affect nerve fibers in the central nervous system), including, but not limited to, Pelizaeus-Merzbacher disease, hypomyelination with basal ganglia and cerebellar atrophy, Aicardi-Goutieres syndrome, Megalencephalic leukoencephalopathy with subcortical cysts, congenital muscular dystrophy, myotonic dystrophy, Wilson disease, Lowe syndrome, PIBD or Tay syndrome, Cockayne disease, cerebrotendinous xanthomatosis, Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, Zellweger-like syndrome, pseudo-Zellweger-like syndrome, pseudo-neonatal adrenoleukodystrophy, bifunctional protein deficiency, X-linked adrenoleukodystrophy and adrenomyeloneuropathy and Refsum disease.

[0137] In some embodiments, the disease or condition may be a lysosomal storage disease (LSD) caused by the inability of cells in the central nervous system to break down metabolic end products, Gaucher disease (due to mutations in the β-glucocerebrosidase (GBA) gene), GM1 / GM2 gangliosidosis, mucopolysaccharidosis conditions, Pompe disease, neuronal ceroid lipofuscinosis, including, but not limited to, Niemann-Pick disease, metachromatic leukodystrophy (MLD), globoid-cellleukodystrophy (GLD), and Fabry disease.

[0138] In some embodiments, the disease or condition is Friedreich's ataxia, amyotrophic lateral sclerosis (ALS), Huntington's disease, or spinal muscular atrophy (SMA).

[0139] In some embodiments, the present disclosure provides a method or use of any of the polynucleotides, rAAV particles, and pharmaceutical compositions disclosed herein for treating, alleviating, or preventing Parkinson's disease or symptoms.

[0140] In some embodiments, before administration of the polynucleotides, rAAV particles, and pharmaceutical compositions described herein, subjects with Parkinson's disease already exhibit motor symptoms such as tremor and altered movement. Non-limiting examples of tremor include mild unilateral or bilateral tremor, moderate bilateral or midline tremor, or refractory tremor. Non-limiting examples of altered movement include mild bradykinesia, moderate bradykinesia, severe bradykinesia, and early morning bradykinesia.

[0141] In some embodiments, a subject with Parkinson's disease may have an alteration in balance, including, but not limited to, impaired balance, impaired righting reflex, obvious balance problems, or falls.

[0142] In some embodiments, a subject with Parkinson's disease may have non-motor symptom changes, and as a non-limiting example, the subject may have mild to moderate cognitive impairment before the compositions described herein are administered, and as another non-limiting example, a subject with Parkinson's disease may have overt cognitive impairment, e.g., dementia, which may also include behavioral disturbances such as hallucinations.

[0143] In some embodiments, a subject with Parkinson's disease may have mild to moderate disabling motor fluctuations in response to one or more dopaminergic agents.

[0144] In some embodiments, prior to administration of the compositions described herein, a subject with Parkinson's disease may have medically refractory motor fluctuations consisting of "wearing off" and / or levodopa-induced ataxia that result in obvious disability.

[0145] In some embodiments, the subject may have mild symptoms associated with Parkinson's disease, including, but not limited to, no cognitive impairment (diagnosed within the past 5 years), a satisfactory response with limited variability to one or more dopamine agonists, mild unilateral or bilateral tremor, little or no impact on quality of life, and / or no balance disorder.

[0146] In some embodiments, the subject may have moderate symptoms associated with Parkinson's disease, including, but not limited to, mild to moderate cognitive impairment, early signs of balance problems and righting reflexes, mild to moderately disabling motor fluctuations (diagnosed within the past 5-10 years) in response to one or more dopamine agonists, moderate bilateral or midline tremor, moderate bradykinesia, and / or some limitation in activities of daily living.

[0147] In some embodiments, the subject may have late symptoms associated with Parkinson's disease, including, but not limited to, having been diagnosed with Parkinson's disease for 10 years or more, moderate refractory motor fluctuations gradually disappearing and / or levodopa-induced dyskinesia, induction of significant physical disability, refractory tremor, evident balance complaints and / or falls, evident cognitive impairment (e.g., dementia with or without behavioral disturbances), severe bradykinesia, evident reduced quality of life due to the disease and / or early morning movement disorders.

[0148] In some embodiments, a subject with Parkinson's disease is receiving levodopa (e.g., DUOPA TM ) was used in combination with the polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure.TM ) alone can achieve success. TM may have achieved no success or limited success using it alone.

[0149] In some embodiments, the subject's UPDRS-3 (or UPDRS-III) drug score is assessed prior to administration of a polynucleotide, rAAV particle, or pharmaceutical composition of the present disclosure. In some embodiments, the subject's UPDRS-3 (or UPDRS-III) drug score is reduced after administration of a polynucleotide, rAAV particle, or pharmaceutical composition of the present disclosure.

[0150] In some embodiments, after administration of a polynucleotide, rAAV particle, pharmaceutical composition of the present disclosure, the subject has improved motor function and / or a reduction in the amount of levodopa medication the subject requires to manage their symptoms.

[0151] In some embodiments, there is provided a method or use of any of the polynucleotides, rAAV particles, and pharmaceutical compositions disclosed herein for the treatment of diseases or conditions associated with circadian rhythms and sleep-wake cycles, including, but not limited to, sleeping sickness (e.g., insomnia), depression, bipolar disorder, seasonal affective disorder, obesity, and diabetes.

[0152] In some embodiments, the polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure may be administered by any delivery route that results in a therapeutically effective result, including, but not limited to, enteral, parenteral, epidural, oral, transdermal, intracerebral, intraventricular (including intraventricular), epidermal, intradermal, subcutaneous, intranasal, intravenous, intravenous, intraarterial, intramuscular, intraosseous infusion (including entry into bone marrow), intrathecal (including entry into the spinal canal), intraparenchymal (entry into brain tissue), intraperitoneal (infusion or injection into the peritoneal cavity), transdermal (diffusion through intact skin and used for systemic distribution), transmucosal, transtracheal (through the tracheal wall), etc. For example, oral, intranasal, subcutaneous, intramuscular, intravascular (e.g., intravenous), intrathecal, intracerebral, and / or intraventricular administration.

[0153] In some embodiments, the polynucleotides, rAAV particles, pharmaceutical compositions of the present disclosure are delivered or administered by injection into the CSF route. For example, they are delivered or administered to cells of the central nervous system (e.g., parenchyma). For example, they are delivered or administered intrathecally and intraventricularly. For example, they are delivered or administered into the substantia nigra or striatum, including the substantia nigra pars compacta (SNpc) and the ventral covering area (VTA), or optionally into the striatum (entering the caudate nucleus and shell) or the subthalamic nucleus (STN). For example, they are delivered or administered to DRG nociceptive neurons, cerebellar-medullary cisterns and transmitting spinal motor neurons and / or astrocytes. The delivery or administration may be by injection or direct infusion.

[0154] In some embodiments, the polynucleotide, rAAV particle, pharmaceutical composition is administered so as to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

[0155] In some embodiments, the polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure may be administered via a single site or multiple sites (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 sites).

[0156] In some embodiments, the polynucleotides, rAAV particles, pharmaceutical compositions of the present disclosure may be administered in a single dose. Single dose intravenous delivery can provide sustained relief to subjects suffering from central nervous system disorders (e.g., Parkinson's disease) and / or associated symptoms. Relief may last for minutes, days, weeks, months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months), or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 years or more).

[0157] The polynucleotides include any AADC polynucleotide and / or GDNF polynucleotide of the present disclosure.

[0158] Reagent kits and equipment The present disclosure provides reagent kits and devices comprising any of the polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure.

[0159] In some embodiments, the reagent kit includes a container device, typically including at least one vial, test tube, flask, bottle, syringe, or other container device into which the polynucleotide, rAAV particles, pharmaceutical composition, or other components may be placed. The reagent kit may also include second, third, or other separate containers into which separate components may be placed, typically including, for example, sterile, pharma- ceutically acceptable buffers and / or other diluents. In some embodiments, the reagent kit includes a pharmaceutical package insert.

[0160] In some embodiments, the polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure can be combined with, coated on, or embedded in a device. The device can include, but is not limited to, a stent, a pump, and / or other implantable therapeutic device. Furthermore, the polynucleotides, rAAV particles, and pharmaceutical compositions can be delivered to a subject using, for example, but not limited to, a compression device, such that the subject is less likely to develop deep vein thrombosis (DVT).

[0161] In some embodiments, the polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure may be delivered by devices including, but not limited to, stents, tubes (including catheters, pipes, and pipettes), and particle guns. The polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure may be administered to a subject by a delivery system that integrates imaging and image-guided therapy, including, but not limited to, lasers, MRgFUS, endoscopes, and robotic surgery devices. The polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure may be administered to a subject by an automated delivery system, including, but not limited to, any of the devices described herein in US5865744, in which a computer adjusts the administration of a probe to a depth appropriate for a particular subject based on images collected by the delivery system. The polynucleotides, rAAV particles, and pharmaceutical compositions of the present disclosure may be delivered to a target point of a subject by an instrument, an enhanced delivery device, or an MIR-guided device, including, but not limited to, any of the methods, systems, and / or computer programs described herein in US8340743.

[0162] definition In order that this disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art.

[0163] Unless the context clearly indicates otherwise, throughout the specification and claims, the words "comprise," "having," "containing," and the like, are to be understood to have an inclusive meaning, i.e., "including but not limited to," rather than an exclusive or exhaustive meaning.

[0164] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0165] "Aromatic L-amino acid decarboxylase", "aromatic amino acid dopa decarboxylase" or "AADC (Amino Acid Decorboxylase)" is a homodimeric pyridoxal phosphate-dependent enzyme responsible for the synthesis of dopamine and serotonin. AADC catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA or levodopa) to dopamine, L-5-hydroxytryptophan to serotonin, and L-tryptophan to tryptamine. AADC proteins and their encoding polynucleotides in this disclosure encompass wild-type AADC proteins, variants thereof, and associated encoding polynucleotides. Exemplary AADC protein sequences and their encoding polynucleotide sequences are set forth in SEQ ID NOs: 1 and 3 in this disclosure. The wild-type AADC protein may be any naturally occurring isotype or variant derived from the dopa decarboxylase (DDC) gene, and multiple alternatively spliced ​​transcript variants encoding different isotypes of AADC have been identified. Specifically, the DDC gene produces seven transcript variants encoding six different isoforms. DDC transcript variants 1 and 2 both encode isoform 1 of AADC. In some embodiments, the AADC polynucleotide encodes DDC transcript variant 2, thereby encoding naturally occurring isoform 1 of AADC (NCBI database: NP_000781.1). The present disclosure also incorporates all of the AADCs in WO2018232055A as exemplary AADCs. An "AADC polynucleotide" is any polynucleotide polymer that encodes an AADC protein and expresses such an AADC protein in a cell, tissue, organ, or organism when in a vector, plasmid, or translatable construct.

[0166] "Glial Cell Line-Derived Neurotrophic Factor" or "GDNF" is a neurotrophic factor that supports the development and survival of peripheral sympathetic, parasympathetic, enteric and sensory neurons, as well as midbrain dopamine and motor neurons, and the GDNF protein and its coding polynucleotide in the present disclosure encompasses wild-type GDNF protein, its variants and related coding polynucleotides. It is known that there are two GDNF splice variants, pre-(α)pro-GDNF (conventionally referred to as GDNFα) and pre-(β)pro-GDNF (conventionally referred to as GDNFβ), which are produced by different splicing of GDNF mRNA (Suter-Crazzolara and Unsicker, 1994). The mature GDNF proteins produced by the two splice variants are likely to be the same, with mature GDNF consisting of 134 amino acids, containing two putative N-glycosylation sites and seven conserved cysteines that are in the same relative spacing from other members of the TGF-β protein family, and the biologically active mature GDNF dimer being formed by a covalent disulfide bond between the unpaired cysteines in the monomer (Lin et al., 1993; Eigenbrot and Gerber, 1997; Chang et al., 2002). Exemplary GDNF protein sequences and their encoding polynucleotide sequences are shown in SEQ ID NOs: 2 and 8 of the present disclosure. The present disclosure also incorporates the (α)pro-GDNF protein in US6362319, the truncated forms of GDNF in US6184200, and the GDNF in WO2009053536A, all as exemplary GDNFs. A "GDNF polynucleotide" is any polynucleotide polymer that encodes a GDNF protein and, when in a vector, plasmid, or translatable construct, expresses such a GDNF protein in a cell, tissue, organ, or organism.

[0167] "Adeno-associated virus (AAV)" includes "recombinant adeno-associated virus (rAAV)," which is a replication-deficient, non-enveloped virus that is a member of the Parvoviridae family of the Dependovirus genus. An "AAV particle" or "rAAV particle" consists of at least two components, including a capsid and a polynucleotide encapsidated therein. AAV particles may be derived from any serotype described in this disclosure or known in the art, including combinations of serotypes (i.e., "pseudotyped" AAV) or various genomes (e.g., single-stranded or self-complementary). Additionally, AAV particles may be replication-deficient and / or targeted. AAV of the present disclosure may include variants that have sequences with significant homology at the amino acid level (capsid) with the polynucleotide (genome or capsid), such that constructs are produced that are generally physical and functional equivalents, replicate by similar mechanisms, and are assembled by similar mechanisms.

[0168] "Polypeptide," "protein," or "protein" can be used interchangeably and refer to a polymer of amino acid residues and apply to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly covers conservatively modified variants thereof.

[0169] A "polypeptide variant" or "protein variant" refers to a molecule whose amino acid sequence differs slightly from a native or starting sequence. The variant may have substitutions, deletions and / or insertions at certain positions in the amino acid sequence. A "native" or "starting" sequence should not be confused with a wild-type sequence. As used in this disclosure, a native or starting sequence is a relative term that refers to the original molecule to which it can be compared. A "native" or "starting" sequence or molecule may refer to a wild-type (a sequence found in nature), but is not necessarily a wild-type sequence. Generally, a variant has at least about 70%, preferably at least about 80%, more preferably at least about 90% identity to a native sequence.

[0170] An "analog" is a polypeptide variant that contains one or more amino acid modifications (e.g., substitution, addition, or deletion of an amino acid residue) while still maintaining the properties of the parent polypeptide. Analogs can be obtained by conservative substitution of the parent polypeptide, a method of conservative substitution commonly used in the art.

[0171] "Conservative substitution" means substitution of another amino acid residue with similar properties to the original amino acid residue in a protein or polypeptide. For example, lysine, arginine and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine ​​and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have non-polar side chains. Furthermore, tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Therefore, it is clear to those skilled in the art that even if amino acid residues in the above-mentioned group showing similar properties are substituted, it does not show a specific change in properties.

[0172] "Linker" or "joint" refers to a linking unit that links two polypeptide fragments or their encoding polynucleotides, and generally has a certain degree of flexibility, and the use of the joint does not cause the original function of the protein domain to be lost. The linker may be a peptide linker or its encoding polynucleotide, and includes one or more amino acids (typically, about 1 to 30, 2 to 24, or 3 to 15 amino acids) or their encoding polynucleotide. The linkers used in the present disclosure may be the same or different.

[0173] The term "fusion" or "linkage" refers to the covalent linkage of elements (e.g., the coding polynucleotides of two target genes or the proteins or polypeptides encoded thereby) directly or via one or more linkers. When the linker is a peptide linker, the covalent bond is a peptide bond.

[0174] "Polynucleotide", "polynucleotide" may be used interchangeably and refers to a deoxyribonucleotide or ribonucleotide polymer that may assume a linear or cyclic conformation and may assume a single-stranded or double-stranded form. In the present disclosure, the term should not be interpreted as limiting in length. The term can encompass known analogs of natural nucleotides and nucleotides modified in the base, sugar and / or phosphate moieties (e.g., phosphorothioate backbones). Generally, an analog of a particular nucleotide has the same base pairing specificity, e.g., an analog of A will pair with a T base. A polynucleotide is "effectively linked" when it is placed in a functional relationship with another polynucleotide sequence. For example, an expression control sequence (e.g., a promoter or enhancer) is effectively linked to a target gene when the expression control sequence affects the transcription of the target gene.

[0175] "RNA" or "ribopolynucleotide" refers to a polymer of ribonucleotides. "DNA" or "deoxyribopolynucleotide" refers to a polymer of deoxyribonucleotides. DNA and RNA may be naturally synthesized (e.g., by DNA replication and DNA transcription, respectively) or chemically synthesized. DNA and RNA may be single-stranded (i.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" refers to a single-stranded RNA of an amino acid sequence that codes for one or more polypeptide chains.

[0176] "Operably linked" refers to a functional linkage between two or more molecules, constructs, transcripts, entities, moieties, etc., and includes both cases where an expression control sequence is adjacent to a target gene and cases where an expression control sequence acts in trans or at a remote location to control expression of the target gene.

[0177] "A polynucleotide is operably linked to an expression control sequence" means that the polynucleotide is expressed under the control of said expression control sequence. Two or more polynucleotides may be expressed under the control of the same expression control sequence, or may be expressed under the control of two identical or different expression control sequences.

[0178] As used herein, "expression control sequence" encompasses any polynucleotide that controls the expression of a coding sequence, including, but not limited to, promoters, enhancers, and the like.

[0179] "Sequence" refers to an amino acid sequence or a polynucleotide sequence of any length, which, if a polynucleotide sequence, may be DNA or RNA, may be linear, circular or branched, and may be single- or double-stranded.

[0180] "Homology", "identity" or "sequence identity" refers to the overall correlation between polymer (e.g., oligonucleotide, polynucleotide or polypeptide) molecules. For example, two polynucleotide sequences can be aligned to calculate the percentage of identity between them. Because of gaps and penalties introduced in the calculation, the identity value may vary, but an optimal method can be selected (e.g., gaps can be introduced in one or both of the two sequences to be aligned to achieve optimal alignment, and different sequences can be ignored to achieve comparison, one of the two sequences being the target sequence to be aligned and the other being the reference sequence). In some embodiments, the length of the target sequence to be aligned may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence, and then the nucleotides at the corresponding nucleotide positions are compared. If the position in the aligned target sequence is occupied by the same nucleotide as the corresponding position in the reference sequence, then the two are considered to be identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences and determination of the percentage of identity between two sequences can be accomplished by a mathematical algorithm.For example, the percentage of identity between two polynucleotide sequences can be measured by methods such as those described in Computational Molecular Biology, edited by Lesk, A.M., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, D.W., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, edited by Griffin, A.M., and Griffin, H.G., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, edited by Gribskov, M. and Devereux, J., M Stockton Press, New York, 1991, all of which are incorporated herein in their entireties. For example, the percentage of identity between two nucleotide sequences can be measured by the Meyers and Miller (CABIOS, 1989, 4:11-17) algorithm incorporated into the ALIGN program (version 2.0) using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage of identity between two nucleotide sequences can be measured by the GAP program in the GCG software package using the NWSgapdna.CMP matrix. In general, methods for measuring the percentage of identity between sequences include, but are not limited to, those described in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073.

[0181] "Conserved" refers to a nucleotide or amino acid residue of a polynucleotide or polypeptide sequence that is not changed at the same position in two or more sequences being compared. A relatively conserved nucleotide or amino acid is more related to the conserved nucleotide or amino acid in the sequence than the nucleotide or amino acid that appears at other positions in the sequence. In some embodiments, two or more sequences are "fully conserved" when they are 100% identical, "highly conserved" when they are at least 70%, 75%, 80%, 90%, 95% or more identical, and "conserved" when they are at least 30%, 40%, 50%, 60% or more identical. Sequence conservation may apply to the entire length of an oligonucleotide, polynucleotide or polypeptide, or to a portion or region thereof. Conserved sequences may be discontinuous, and one of skill in the art will understand how to achieve alignment when there are gaps in the contiguous alignment between sequences, and how to align corresponding residues when the presence of insertions or deletions is not permitted.

[0182] "Gene expression" refers to the conversion of information contained in a gene into a gene product, e.g., a polynucleotide undergoes a process of successful transcription and often translation to produce a protein or peptide. A gene product may be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by mRNA translation. Gene products further include RNA modified by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, e.g., methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation. "Modulation" of gene expression refers to a change in gene activity, and modulation of expression may include, but is not limited to, gene activation, gene optimization, and gene suppression. Genome editing (e.g., truncation, alteration, inactivation, random mutation) can be used to modulate expression. Reference to measuring "gene expression" should be understood to mean that what is measured can be the transcribed polynucleotide product, such as RNA or mRNA, or the translated amino acid product, such as a polypeptide or peptide. Methods for measuring the amount or levels of RNA, mRNA, polypeptides, and peptides are known in the art.

[0183] "Transfection" refers to a method for introducing an exogenous polynucleotide into a cell. Transfection methods include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures.

[0184] A "payload" refers to one or more polynucleotides or polynucleotide regions encoded by or in a viral genome, or the expression product of such polynucleotides or polynucleotide regions, e.g., a transgene, a polynucleotide encoding a polypeptide, or a regulatory polynucleotide. In some embodiments of the present disclosure, the payload encodes or expresses a target gene, such as AADC and / or GDNF. A "payload construct" is one or more polynucleotide regions that encode or comprise a payload flanked on one or both sides by inverted terminal repeat (ITR) sequences, the payload construct being a template that replicates in a viral producer cell to produce a viral genome. A "payload construct vector" is a vector that encodes or comprises a payload construct and regulatory regions used for replication and expression in bacteria, cells. A "payload construct expression vector" is a vector that encodes or comprises a payload construct and further comprises one or more polynucleotide regions that encode or comprise components for expression of the virus in a viral replicating cell.

[0185] A "vector" is a construct that can be transported, transferred or otherwise used as a heterologous molecule, for example, a construct that can deliver and express one or more target genes in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors bound to cationic polymers, and DNA or RNA expression vectors encapsulated in liposomes. In some embodiments, the vector may be recombinantly produced, may be produced based on, or may include, an AAV parent or reference sequence. The parent or reference AAV sequence may serve as the first or subsequent sequence of an engineered vector. The parent or reference AAV sequence may include a polynucleotide sequence that expresses a target gene (encoding a target protein, polypeptide or part thereof), a polynucleotide sequence of a regulatory element (e.g., promoter, enhancer), and the sequence may be wild type or modified or mutated.

[0186] A "vector genome" comprises a polynucleotide encoding at least one inverted terminal repeat (ITR), at least one regulatory sequence, and at least one payload. A vector genome is derived by replicating a payload construct from a payload construct expression vector. A vector genome encodes at least one copy of a payload construct.

[0187] A "viral construct vector" is a vector that contains one or more polynucleotide regions that encode or include Rep and / or Cap proteins. A "viral construct expression vector" is a vector that contains one or more polynucleotide regions that encode or include Rep and / or Cap proteins, and further contains one or more polynucleotide regions that encode or include components for expression of the virus in a viral replicating cell.

[0188] "Modified" refers to an altered state or structure of a molecule or entity compared to a parent or reference molecule or entity, and may be in a variety of ways, including chemical, structural, and functional modifications. In some embodiments, polynucleotides, polypeptides, vectors, constructs, capsids, etc. of the present disclosure are modified by introducing non-natural amino acids or non-natural nucleotides.

[0189] A "host cell" includes any cell or cell culture that may be or has been a recipient of a vector for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, and due to natural, accidental, or deliberate mutations, the progeny are not necessarily completely identical (in morphology or genomic DNA complement) to the original parent cell. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide according to the present disclosure. "Cells," "cell lines," and "cell cultures" may be used interchangeably, and any such designation includes the progeny. It should also be understood that due to deliberate or unintentional mutations, any progeny may not be precisely identical in DNA content. Mutant progeny that have the same function or biological activity as screened from the originally transformed cell are included. Host cells may include microbial (e.g., bacterial), plant, or animal cells. Bacteria susceptible to transformation include members of the enterobacteriaceae, such as strains of Escherichia coli and Salmonella, Bacillaceae, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), NS0 cells, and 293 cells.

[0190] A "pharmaceutical composition" refers to a mixture containing one or more of the rAAVs described herein and other chemical components, as well as other components such as physiologically / pharmaceutical acceptable vectors and excipients, to facilitate administration to a living organism and to contribute to the absorption of the active ingredients to further exert biological activity.

[0191] A "pharmaceutically acceptable vector" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows that ingredient to retain its biological activity while being non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical vector, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various wetting agents.

[0192] "Giving", "administration" and "treatment", when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refer to contact of an exogenous agent, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid, e.g., therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Giving", "administration" and "treatment" also refer to treating, e.g., cells, ex vivo and in vitro, with a reagent, diagnostic, binding composition, or through another cell. When applied to a human, veterinary or research subject, refer to therapeutic treatment, preventative or prophylactic measures, research and diagnostic uses.

[0193] "Treatment" refers to providing a subject with an internal or external therapeutic agent, such as any one of the polynucleotides disclosed herein or its encoded polypeptide, rAAV containing said polynucleotide, or related pharmaceutical composition, as a therapeutic agent, where the subject suffers from, is at risk of suffering from, or is prone to suffer from, one or more neurodegenerative diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is provided to the subject or population being treated in an amount that effectively relieves one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively relieves any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on several factors, such as the disease state, age and weight of the subject, and the ability of the drug to produce the required therapeutic effect in the subject. Whether the disease symptoms have been reduced can be evaluated by any clinical detection method commonly used by a physician or other professional health care provider to evaluate the severity or progression of the condition. An embodiment of the present disclosure (e.g., a method of treatment or product) may be ineffective in alleviating a target disease symptom in a subject, but should alleviate the target disease symptom in a statistically significant number of subjects, as determined by any statistical testing method known in the art, such as, for example, Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0194] "Co-administration" means administration of two or more agents (e.g., rAAV particles) to a subject simultaneously or at intervals such that there is overlap in the effects of each agent on a patient and / or subject exposed to both at the same time. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minutes of each other, or within about 24 hours, 12 hours, 6 hours, 3 hours of at least one dose of one or more other agents.

[0195] "Delivery" refers to the act or manner of delivering a compound, such as a parvovirus, e.g., AAV and / or AAV compound, substance, entity, moiety, cargo or payload, to a target. Such a target may be a cell, tissue, organ, organism or system.

[0196] "Amelioration" means reducing the severity of at least one indicator of a condition or disease. For example, in the case of a neurodegenerative disease, amelioration includes a reduction in neuronal loss.

[0197] An "effective amount" includes an amount that cures, alleviates, or improves one or more symptoms of a medical condition or the condition itself such that a beneficial or desired clinical outcome is achieved. An "effective amount" is determined by the circumstances of its application, and the effective amount used in a subject can vary depending on factors such as the condition being treated, the overall health of the subject, the method, route and dose of administration, and the severity of side effects. For example, when administering a pharmaceutical composition to treat Parkinson's disease, an effective amount is an amount sufficient to achieve treatment of Parkinson's disease, as defined in the present disclosure, as compared to the response obtained without administration of the pharmaceutical composition.

[0198] "Encapsulating" means wrapping, surrounding, or enveloping, for example, wrapping, surrounding, or enveloping a vector with an AAV capsid.

[0199] "Delivery" refers to the act or manner of delivering a compound, such as a parvovirus, e.g., AAV and / or AAV compound, substance, entity, moiety, cargo or payload, to a target. Such a target may be a cell, tissue, organ, organism or system (whether biological or manufactured). "Delivery agent" refers to any agent or substance that at least partially facilitates in vivo and / or in vitro delivery of a polynucleotide and / or one or more substances (including but not limited to compounds and / or compositions of the invention, e.g., viral particles or expression vectors) to a target cell.

[0200] "Optionally" and "optionally" mean that the subsequently described event or circumstance may, but need not, occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0201] By "subject" or "patient" is meant mammals, particularly primates, especially humans.

[0202] "About" or "approximately" means that a numerical value is within an acceptable error range of a specific value as determined by one of ordinary skill in the art, as determined by how the numerical portion is measured (i.e., the limitations of the measurement system). For example, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" or "essentially including" may mean a variation of at most 20%, for example, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 0.5% and 1%, and in this disclosure, any instance where the term "about" precedes a number or range of values ​​includes the specified number of embodiments. Unless otherwise stated, when a specific value appears in this application and the claims, the meaning of "about" or "essentially including" should be assumed to be within an acceptable error range of the specific value. [Brief description of the drawings]

[0203] [Figure 1] FIG. 1 is a schematic diagram of the structure of a polynucleotide expressing AADC and / or GDNF according to the present disclosure. [Figure 2A] The plasmid was transfected into SHSY5Y cells, and the L-DOPA concentration was then detected by HPLC after treatment with L-DOPA. [Figure 2B] The plasmid was transfected into SHSY5Y cells and then the concentration of GDNF was detected by ELISA. [Figure 3A] Amount of AADC protein expressed by each AAV plasmid after codon optimization of AADC. [Figure 3B] Quantity of GDNF protein expressed by each AAV plasmid after codon optimization of GDNF. [Figure 4A]Immunofluorescence signals of mouse striatal AADC after transfection with AAV2 and AAV9 serotypes. [Figure 4B] Immunofluorescence coverage of mouse striatal AADC after transfection with AAV2 and AAV9 serotypes. [Figure 5A] Expression levels of striatal AADC after injection of AAV2-02A and AAV9-02A. [Figure 5B] Expression levels of striatal GDNF after injection of AAV2-02A and AAV9-02A. [Figure 6A] This shows the response of mice to L-DOPA (counterclockwise) after injection of HRPDAAV02-AI (i.e., 02AI, the same below) and HRPDAAV03-DI (i.e., 03DI, the same below). [Figure 6B] Response to L-DOPA in mice (contralateral forelimb utilization rate) after drug injection of HRPDAAV02-AI and HRPDAAV03-DI. [Figure 7A] This shows the response of mice to L-DOPA after injection of HRPDAAV02-AI and HRPDAAV03-DI drugs (DOPA concentration on the non-administered side). [Figure 7B] This shows the response of mice to L-DOPA (DOPA concentration on the administration side) after injection of HRPDAAV02-AI and HRPDAAV03-DI drugs. [Figure 7C] This shows the response of mice to L-DOPA (DOPA concentration in blood) after injection of HRPDAAV02-AI and HRPDAAV03-DI drugs. [Figure 7D] Protection of mouse dopamine neurons after drug injection of HRPDAAV02-AI and HRPDAAV03-DI, and statistics on immunofluorescence signal density of dopamine nerve terminals. [Figure 8A] This shows the response of animals in each group to apomorphine after the establishment of the 6-OHDA model. [Figure 8B]Responses of rats to L-DOPA (rotation test) after injection of HRPDAAV02-AI and HRPDAAV03-DI drugs. [Figure 8C] The detection results of the rat model established in the cylinder test experiment. [Figure 8D] Response status of rats to L-DOPA (contralateral forelimb utilization rate) after drug injection of HRPDAAV02-IA and HRPDAAV03-DI. [Figure 8E] These are the results of detecting the metabolic capacity of the striatum for L-DOPA after administration of HRPDAAV02-IA and HRPDAAV03-DI (DOPA ratio in the affected side / healthy side). [Figure 8F] Protective effect on DA neurons after administration of HRPDAAV-03DI. In this disclosure, **** represents p<0.0001, *** represents p between 0.0001 and 0.001, both of which indicate highly statistically significant differences, ** represents p between 0.001 and 0.01, which indicates highly statistically significant differences, and * represents p between 0.01 and 0.05, which indicates statistically significant differences. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0204] The present disclosure will be further described below with reference to examples, but these examples are not intended to limit the scope of the present disclosure.

[0205] Experimental methods for which no specific conditions are specified in the Examples or Test Examples of this disclosure generally follow conventional conditions or conditions recommended by the manufacturers of materials or products. Reference is made to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Methods in Molecular Biology, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which no specific source is specified are conventional reagents that are commercially available.

[0206] Example 1. Design of AADC and GDNF polynucleotide structures In this embodiment, sequences were designed and engineered for the coding polynucleotides of AADC and GDNF for cell delivery or in vivo delivery in humans and animals. The above sequences can be inserted into plasmids, lentiviruses, adenoviruses, mRNA-LNPs, and adeno-associated viruses (AAV) vectors, for example, recombinant adeno-associated viruses (rAAV) for in vivo and in vitro delivery. The specific sequences between the ITRs in the above rAAV plasmid constructs are shown in Table 2, and the enhancer used may include, for example, a CMV enhancer, and the promoter may include, for example, a CMV promoter or a CBA promoter. None of the sequences in Table 2 were codon-optimized. The structural diagram is shown in FIG. 1.

[0207] [Table 1]

[0208] Example 2. Verification of the efficiency of transformation of AADC to L-DOPA and expression of GDNF by in vitro expression of AADC / GDNF polynucleotides DOPA is mainly produced in the substantia nigra region of the midbrain and its signaling pathway is as follows:

[0209] [ka] SHSY5Y cells are a type of human neuroblastoma cell line. In this example, a rAAV vector plasmid containing AADC / GDNF was transfected into SHSY5Y cells (ATCC), levodopa (L-DOPA) was added to the medium, and the supernatant was collected.

[0210] 1. The dopamine (DOPA) concentration was detected by HPLC to observe the transformation efficiency of the rAAV vector to transform L-DOPA into DOPA.

[0211] The method was as follows: on day 0, 2E+5 to 6E+5 SHSY5Y cells were seeded in each well of a 6-well plate so that the cell density was 60% to 80% at the time of transfection 24 h later. On day 1, the transfection systems of HRPDAAV01-A to HRPDAAV03-F containing tube A (2.5 μg of rAAV plasmid, 250 μL of Opti-MEM (GIBCO, 31985088), 5 μL of Lipofectamine 3000 (Thermo Fisher Scientific, L3000001)) and tube B (250 μL of Opti-MEM, 7.5 μL of Lipofectamine 3000) were prepared, respectively. After preparing tube A and tube B, they were mixed uniformly and left to stand for 2 to 3 min, and the solution in tube A was slowly added to tube B, gently pipetted, mixed uniformly, and left to stand for 10 to 15 min. Before transfection, 1.5 mL of cells (DMEM / F12 (GIBCO, 31331093) + 15% FBS (GIBCO, 26140079)) was replaced, and the prepared transfection system was added dropwise. 6 h after transfection, 3 mL of medium (DMEM / F12 + 15% FBS) was replaced. 24 h after transfection, 3 mL of medium (DMEM / F12 + 15% FBS) containing L-DOPA (Sigma-Aldrich, D9628) (2 mMol) was replaced. 48 h after transfection, the supernatant was taken and the concentration of DOPA was detected by HPLC to detect the expression and function of AADC.

[0212] As for the HPLC process, pre-cooled PBS (1 mg tissue: 10 μL PBS) was added at the time of tissue dissolution, and the sample was thoroughly homogenized with a pre-cooled tissue grinder, after which 100 μL of the homogenate was taken and used for the HPLC experiment. An equal volume of pre-cooled sample treatment solution (0.2 M perchloric acid, 0.2 mM sodium pyrosulfite, 0.01% EDTA 2Na) was added, which also contained 0.6 μM DHBA as an internal standard, and centrifuged at 10,000 g for 20 min in a centrifuge pre-cooled at 4 ° C. The supernatant was taken and pressure-filtered with a 0.22 μm aqueous phase microwell filtration membrane. It was stored at 20 ° C. The sample was detected with an HPLC EDC system. The mobile phase was 16% aqueous methanol (containing 40 mM sodium acetate, 15 mM citric acid, 0.25 mM sodium octanesulfonate, and 0.2 mM 2Na EDTA, pH 4.3). Chromatographic peaks were analyzed using the Eigen Chromatography Data Workstation Software V5.0, and the peak areas were corrected using DHBA as an internal standard. A calibration curve was fitted based on the results of the injection of the standard, and the content of dopamine in the sample was calculated, and the results were expressed in "ng dopamine / mg tissue".

[0213] 2. The GDNF expression efficiency of the rAAV vector was observed by detecting the concentration of GDNF by ELISA.

[0214] The ELISA experiment was performed strictly according to the instruction manual of the reagent kit (RayBiotech, ELH-DDC). The specific experimental procedure was as follows: pre-cooled PBS (1 mg tissue: 10 μL PBS) was added when dissolving the tissue, the sample was thoroughly homogenized with a pre-cooled tissue grinder, and then centrifuged to remove the supernatant for ELISA experiment. 100 μL of standard or sample was added to each well in the reagent kit (RayBiotech, ELH-DDC) and incubated at room temperature for 2.5 h, 100 μL of biotin antibody was added to each well and incubated at room temperature for 1 h, 100 μL of Streptavidin solution was added to each well and incubated at room temperature for 45 min, 100 μL of TMB was added to each well and incubated at room temperature for 30 min. 50 μL of stop solution was added to each well. The spectrum was immediately read at 450 nm.

[0215] The detection results, referring to Figures 2A and 2B, show that HRPDAAV02-A and HRPDAAV03-D are relatively strong in the expression and function of AADC and GDNF.

[0216] Example 3. Codon optimization of AADC and GDNF In this embodiment, four codon-optimized polynucleotide sequences were designed for AADC and GDNF. The wild-type polynucleotide sequence of AADC refers to SEQ ID NO:3, and the codon-optimized sequences AADC01, AADC02, AADC03, and AADC04 refer to SEQ ID NOs:4-7, respectively, and the sequence identities between SEQ ID NOs:4-7 and SEQ ID NO:3 are 80.83%, 81.21%, 79.18%, and 79.63%, respectively. The wild-type polynucleotide sequence of GDNF refers to SEQ ID NO:8, and the codon-optimized sequences GDNF01, GDNF02, GDNF03, and GDNF04 refer to SEQ ID NOs:9-12, respectively, and the sequence identities between SEQ ID NOs:9-12 and SEQ ID NO:8 are 75.13%, 73.99%, 77.52%, and 77.83%, respectively.

[0217] AADC01, AADC02, AADC03, and AADC04 were used to replace the AADC sequence in HRPDAAV02-A, respectively, to obtain HRPDAAV02-A(AADC01), HRPDAAV02-A(AADC02), HRPDAAV02-A(AADC03), and HRPDAAV02-A(AADC04), which correspond to 02A(AADC01), 02A(AADC02), 02A(AADC03), and 02A(AADC04), respectively, in the drawings. GDNF01, GDNF02, GDNF03, and GDNF04 were used to replace the GDNF sequence in HRPDAAV02-A, respectively, to obtain HRPDAAV02-A(GDNF01), HRPDAAV02-A(GDNF02), HRPDAAV02-A(GDNF03), and HRPDAAV02-A(GDNF04), which correspond to 02A(GDNF01), 02A(GDNF02, 02A(GDNF03), and 02A(GDNF04) in the figure, respectively. Then, the screening of Example 2 was repeated to obtain the AADC and GDNF codon-optimized sequences AADC01 and GDNF04 with the strongest expression and function, and the results are shown in Figures 3A and 3B.

[0218] After replacing AADC and GDNF in HRPDAAV02-A and HRPDAAV03-D with AADC01 (SEQ ID NO: 3) and GDNF04 (SEQ ID NO: 12), HRPDAAV02-AI (SEQ ID NO: 24) and HRPDAAV03-DI (SEQ ID NO: 25) were obtained. HRPDAAV02-AI and HRPDAAV03-DI correspond to 02AI and 03DI, respectively, in the figure. The configurations of both are as shown in Table 3.

[0219] [Table 2-1] [Table 2-2]

[0220] Example 4. Screening for AAV serotypes HRPDAAV-02A was packaged in the AAV2 serotype (see SEQ ID NO: 41 for the amino acid sequence) and the AAV9 serotype (see SEQ ID NO: 42 for the amino acid sequence) and purified to obtain AAV2-02A and AAV9-02A.

[0221] The viral packaging process is as follows.

[0222] Packaging cells (viral production cells, Thermo A3152801) were cultured at 0.5 × 10 cells / mL in 1 L of medium (Irvine 91165) in a 3 L shake flask. 6 After inoculation at a density of 10 vc / mL and incubation in a CO2 shaker for 3 days, the cells were diluted in fresh medium to 3.0 × 10 6 Dilute to vc / mL A total of 4 μg of three packaging plasmids (pHelper, pRC9, pGOI) were added to 25 mL of fresh medium and mixed evenly, PEIpro (Polyplus 115-100) was added to another 25 mL of fresh medium and mixed evenly, and the PEIpro dilution was poured into the plasmid dilution, mixed evenly quickly, and left at room temperature for 15 min, after which the transfection complex was added to the packaging cells. After 3 days of incubation in a CO2 shaker, 50 mL of cell lysis solution (0.5 M hepes, 40 mM MgCl2, 10% tween-20) and 200 μL of Benzonase (Merck 1.01697.0010) were added, and the cells were returned to the CO2 shaker for 4 h to lyse the cells, which became the harvest solution.

[0223] The virus purification process is as follows.

[0224] The harvest fluid was filtered through a depth filter (Cobetter) to remove cellular debris, then concentrated 10-fold through a 100 kDa hollow fiber column (Repligen, D06-E100-05-N) and replaced in PBS (5 volumes). The exchanged solution was applied to an affinity chromatography column (Thermo A36652) equilibrated with PBS, rinsed with 5 column volumes of PBS, and then eluted with 100 mM glycine, pH 3.0. The eluate was collected and then an appropriate volume of BTP 1M, pH 10.0 was added as quickly as possible to adjust the pH to about 7.0. The eluate was diluted with 30 volumes of IEX equilibration buffer (25 mM BTP, pH 9.5) and applied to a buffer-equilibrated anion exchange column (BIA 311.5113-2). After rinsing with 10 column volumes of IEX equilibration buffer, NaCl salt gradient elution was performed and the solid peak was collected according to UV 260 / 280. The eluate was concentrated to 1mL using ultrafiltration centrifuge tubes (Merck, UFC810008), replaced with PBS + 0.01% Pluronic F-68, and filtered through a 0.22μm syringe filter to obtain purified virus. AAV2-02A and AAV9-02A were injected stereotaxically into the striatum of rats (SD strain, male, purchased from Shanghai Jieshijie Laboratory Animal Co., Ltd.), and some rat striatum were harvested 4 and 8 weeks after injection to detect the expression of AADC and GDNF by EILSA, and AADC immunofluorescence sections were performed 8 weeks after injection.

[0225] The method is as follows.

[0226] SD male rats were taken and first anesthetized with 50 mg / kg zoletil (ip), the animals were fixed in a stereotaxic apparatus, the skin was incised sagittally along the middle of the skull, the anterior section (Bregma) was exposed, and the right striatal region was positioned at position 1: AP-0.0 mm, ML-2.6 mm, DV-4.7 mm, position 2: AP0.0 mm, ML-2.6 mm, DV-4.7 mm. After opening the skull with a dental engine, a microsyringe was inserted and 2 μL of the test drug (0.2 μL / min) was automatically injected into each site with a micro peristaltic pump. After the injection was completed, the needle was left in place for 10 min, then slowly withdrawn and the wound was sutured. To prevent infection, each rat was subcutaneously injected with 0.3 mL of levofloxacin injection. The animals were kept for 8 weeks after surgery.

[0227] 1) Rats were perfused transcardially with 4% paraformaldehyde, and then the brains were harvested and fixed for more than 24 h. The samples were then transferred to a 30% sucrose solution and thoroughly precipitated and dehydrated. Serial coronal sections of the striatum were made using a freezing microtome to a section thickness of 30 μm, and the sections were placed in a freezing solution and stored in a refrigerator at 4°C. The brain sections were washed three times with PBS, treated with 0.3% TritonX100 for 10 min, blocked in PBS containing 10% goat serum for 1 h, and incubated overnight at 4°C with rabbit anti-AADC antibody (Absin, abs110350) (1:1000) as the primary antibody. The next day, the sections were washed with PBS three times for 5 min each, incubated with donkey anti-rabbit IgG AlexaFluor488 antibody (1:1500) and nuclear dye DAPI (1:15000) at room temperature for 1 h, washed three times with PBS, and mounted with Dako anti-fluorescence attenuating mounting medium. Observed and photographed under an Olympus fluorescent microscope, as shown in Figure 4A and Figure 4B, the staining results showed that the fluorescent area of ​​AADC was obviously larger after injection of AAV9-02A compared with AAV2-02A (P<0.01, AAV2-02A vs AAV9-02A), proving that AAV9 has a higher expression efficiency in the striatum region than AAV2.

[0228] 2) After rats were reared for 4 or 8 weeks after stereotaxic surgery, they were decapitated and the striatal tissue from the injected side was extracted. The tissue was weighed and stored in a -80°C freezer. When dissolving the tissue, pre-cooled PBS (1 mg of tissue: 10 μL of PBS) was added, and the sample was thoroughly homogenized with a pre-cooled tissue grinder. 100 μL of the homogenate was taken and used for HPLC experiments, 40 μL of the homogenate was taken and used for tissue DNA extraction and qPCR experiments, and the remaining homogenate was used for ELISA experiments.

[0229] The concentrations of AADC and GDNF were detected by ELISA. The ELISA experiment was performed strictly according to the instruction manual of the reagent kit (RayBiotech, ELH-DDC), and the specific experimental procedure is as follows:

[0230] The method for detecting AADC was as follows: 100 μL of dissolved standard or sample was added to each well in the reagent kit (RayBiotech, ELH-DDC), incubated at room temperature for 2.5 h, centrifuged and washed, 100 μL of biotin antibody was added to each well, incubated at room temperature for 1 h, centrifuged and washed, 100 μL of Streptavidin solution was added to each well, incubated at room temperature for 45 min, centrifuged and washed, 100 μL of TMB was added to each well, incubated at room temperature for 30 min, centrifuged and washed. 50 μL of stop solution was added to each well. The spectrum was immediately read at 450 nm.

[0231] The method for detecting GDNF was as follows: 100 μL of dissolved standard or sample was added to each well in the reagent kit (R&D, DY212), incubated at room temperature for 2 h, centrifugally dehydrated, washed, 100 μL of detection antibody was added to each well, incubated at room temperature for 2 h, centrifugally dehydrated, washed, 100 μL of Streptavidin-HRP solution was added to each well, incubated at room temperature for 20 min, centrifugally dehydrated, washed, 100 μL of substrate solution was added to each well, incubated at room temperature for 20 min. 50 μL of stop solution was added to each well. The spectrum was immediately read at 450 nm.

[0232] As shown in Figure 5A and Figure 5B, the detection results of target gene expression levels showed that AAV9-02A had obviously higher expression levels of striatal AADC and GDNF than AAV2-02A group, proving that AAV9 had higher transduction efficiency in the striatal region compared with AAV2.

[0233] Example 5. Confirmation of efficacy in MPTP-induced mouse PD model In this embodiment, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was selected to establish a mouse Parkinson's disease model. MPTP is highly lipophilic and can easily pass through the blood-cerebrospinal fluid barrier. Once in the brain, it can be transformed into methyl-phenylpyridine ion (MPP+) under the action of glial monoamine oxidase B. MPP+ is actively taken up into the mitochondria of dopamine (DA)-ergic neurons by DA transporters, causing degeneration and death of DA-ergic neurons, a process that closely mimics the symptoms and onset of PD. MPTP can be administered intraperitoneally to adult male mice for 5 consecutive days to induce apoptosis of DA neurons and cause Parkinson's disease-like symptoms in mice.

[0234] The mice were divided into four groups: a sham-operated control group (i.e., normal control), a model control group (i.e., MPTP model control, hereinafter the same), a 02AI group (i.e., HRPDAAV-02AI), and a 03DI group (i.e., HRPDAAV-03DI). HRPDAAV-02AI and HRPDAAV-03DI, both of which use AAV9 capsids, were injected into the unilateral striatum of 8-week-old male mice. The sham-operated control group and the model control group were subjected to sham surgery. A male C57BL / 6N mouse was anesthetized by intraperitoneal injection of 1% sodium pentobarbital (35-50 mg / kg). After confirming that the mouse was anesthetized, the mouse was fixed in a stereotaxic apparatus (Ruivode, model no. 68018) in a prone position. After the instruments were disinfected as usual, the scalp was incised and the soft tissue and periosteum were bluntly peeled off to expose the right parietal bone. A bone window was opened 0.8 mm anteriorly from the anterior section (AP = +0.8 mm) and 1.8 mm from the midline (ML = 1.8 mm). AAV drugs were injected using a microsyringe (specification: 1 μL, outer diameter: 0.55 mm), the needle advance speed was 1 mm / min, the needle was advanced to 3 mm below the dura (DV = -3 mm) and withdrawn to 2.8 mm (DV = -2.8 mm), and the liquid was injected at a constant rate of 0.2 μL / min, and the needle was left in place for 3 min after the injection was completed, and the needle withdrawal speed was 1 mm / min.

[0235] The mice in the model control group and treatment group were intraperitoneally injected with 30mg / kg MPTP, the injection volume was 10mL / kg, 28 days after the injection into the striatum, and the mice in the sham operation group were intraperitoneally injected with 10mL / kg saline. Five days after the model construction, the mice were allowed to rest for 3 days and behavioral detection was performed.

[0236] 1) Rotation test To confirm the response to L-DOPA after AADC delivered by HRPDAAV-02AI and HRPDAAV-03DI drugs was expressed in the unilateral striatum, a rotation experiment was performed. Levodopa (L-DOPA) (20 mg / kg) + benserazide (6 mg / kg) was intragastrically administered to each group of experimental mice. After levodopa administration, the mice were immediately placed in the test environment to acclimate, and the number of rotations of the mice was recorded 45 min after administration, and the rotations of the healthy side (left rotations) of the mice within 30 min were detected.

[0237] As a result, referring to Figure 6A and Table 4, compared to the sham-operated control group and the model control group, the rotation number was significantly increased after L-DOPA administration in the HRPDAAV-02AI and HRPDAAV-03DI-administered groups (02AI group vs. model control group, p = 0.07, 03DI group vs. model control group, p = 0.01), demonstrating that AADC delivered and expressed by AAV has an excellent response effect to L-DOPA.

[0238] [Table 3]

[0239] 2) Cylinder test A cylinder test experiment was conducted to detect the preference of the mice for using their forelimbs under background conditions. Four days after the last MPTP injection, L-DOPA (10 mg / kg) + benserazide (3 mg / kg) were administered intragastrically to the mice, and the mice were placed in a transparent cylinder (10 cm in diameter, 45 cm in height). The number of times the mice touched the wall with their left and right forelimbs and both forelimbs within 5 min was observed and recorded. A mouse's touching of the wall with one or both forelimbs and then returning to the bottom of the cylinder was recorded as one time (when the mouse first touched the wall with one paw and then the other paw, it was recorded as touching both walls). After each mouse's experiment, the cylinder was wiped with ethyl alcohol. The left forelimb use of the mice was analyzed: left forelimb use rate (%) = (left side + 0.5 both sides) / (right side + left side + both sides) × 100.

[0240] As a result, referring to FIG. 6B and Table 5, after L-DOPA administration, the HRPDAAV-02AI and HRPDAAV-03DI administration groups showed an increase in the contralateral forelimb utilization rate compared to the model control group, with the 02AI group increasing by 20.4% compared to the model control group and the 03DI group increasing by 8.2% compared to the model control group. The responsive effect of AADC delivered and expressed by AAV to L-DOPA was again demonstrated.

[0241] [Table 4]

[0242] 3) Detection of dopamine in the striatum and blood After the behavioral test, mice from each group were given intragastric administration of L-DOPA (20mg / kg, 5mL / kg) and benserazide (5mg / kg, 5mL / kg). The mice were sacrificed 1h after the end of administration, and peripheral blood was collected before sacrifice to detect dopamine in the blood. After sacrifice, the striatum of the healthy and affected sides was collected separately. DOPA was detected by UPLC-MS / MS.

[0243] Referring to FIG. 7A and Table 6-1, in the detection of DOPA in the left striatum (non-administered side), the model control group and the administration group showed a clear decrease in DOPA compared to the sham-operated control group, proving the success of the MPTP model construction.

[0244] [Table 5]

[0245] Referring to Figure 7B and Table 6-2, in the detection of DOPA in the right striatum (administration side), it was found that the HRPDAAV-02AI and HRPDAAV-03DI administration groups showed a clear increase in DOPA compared to the model control group. The DOPA level was restored to the level of the sham-operated control group.

[0246] [Table 6]

[0247] Referring to Figure 7C and Table 6-3, it was found that the detection of plasma DOPA did not show significant differences between the groups, demonstrating that the effects of HRPDAAV-02AI and HRPDAAV-03DI were mainly limited to the striatum of the brain and no obvious leakage occurred.

[0248] [Table 7]

[0249] 4) Immunohistochemical detection in brain tissue Each group of mice was perfused transcardially to collect brain tissue for fluorescent immunohistochemical detection. The specific method was as follows: after general observation of the experimental mice, the cardiac tissue was released from the thoracic cavity, an intravenous needle was inserted into the left ventricle and fixed, the right atrial appendage was incised, pre-cooled PBS was perfused into the right atrial appendage to drain out a clear liquid, and then 4% paraformaldehyde was perfused transcardially. The brain tissue was cut out and placed in a fixative (4% paraformaldehyde solution), and the fixed tissue was stained for tyrosine dehydrogenase (TH) using paraffin sections.

[0250] Referring to Figure 7D and Table 7, the fluorescent immunohistochemical analysis revealed that the TH fluorescence intensity in both sides of the sham-operated control group and the model control group was similar, while the TH fluorescence intensity in the right striatum (administered side) was significantly increased compared to the left striatum (non-administered side) in the HRPDAAV-02AI and HRPDAAV-03DI administration groups. It was demonstrated that the HRPDAAV-02AI and HRPDAAV-03DI drugs had excellent protective effects on mouse dopamine neurons.

[0251] [Table 8]

[0252] Example 6. Confirmation of efficacy in 6-OHDA-induced rat PD model In this example, a rat PD model was established using 6-OHDA. 6-OHDA is a neurotoxin and structurally similar to dopamine (DA), so it is often mistakenly taken up into DAergic neurons as a transmitter, and selectively causes the death of DAergic neurons through the formation of hydroxyl radicals, inhibition of mitochondrial oxidative respiratory chain complexes, and interference with ATP synthesis. Due to the reduction in DA content in the striatum of the damaged side, rats exhibit symptoms similar to those of human PD, and the model has the characteristics of being reliable, stable, and irreversible, and its behavioral defects can be intuitively quantitatively analyzed, and it is widely used to evaluate anti-Parkinson's drugs, especially drugs effective against DA and its receptors. Therefore, in this example, the Parkinson's model induced by 6-OHDA injection into the unilateral MFB was used to evaluate the therapeutic effects of Parkinson's disease with HRPDAAV-02AI and HRPDAAV-03DI.

[0253] The method involved dividing SD rats into six experimental groups: a sham-operated control group (i.e., a normal control group), a model control group, a high-dose 02AI group (1E+10vg / dose), a low-dose 02AI group (2E+9vg / dose), a high-dose 03DI group (1E+10vg / dose), and a low-dose 03DI group (2E+9vg / dose), all of which used AAV9 capsids. After anesthetizing the rats with an intraperitoneal injection of 3% sodium pentobarbital (at a dose of 50-60 mg / kg), the rats were fixed in a prone position. After the instruments were disinfected as usual, the scalp was incised, the soft tissue and periosteum were bluntly peeled off to expose the right parietal bone, the connective tissue near the midline was removed, the sagittal suture, anterior term and lambda were determined, and a microsyringe (specifications: 10 μL, outer diameter: 0.5 mm) containing a model construction reagent was fixed to a stereotaxic apparatus to set the coordinates, and the striatum was positioned on the right side of the rat with the anterior term as the stereotaxic center point. One bone window was opened at a position 0.0 mm (AP = +0.0 mm) anterior to the anterior term and 3 mm (ML = 3 mm) away from the midline. At a needle feed rate of 1 mm / min, the needle was advanced to 6 mm (DV=-6 mm) below the dura mater, then withdrawn to 5.8 mm (DV=-5.8 mm), and 2 μL of liquid was injected at a constant rate of 0.6 μL / min. After the injection was completed, the needle was withdrawn to 3.8 mm (DV=-3.8 mm), and 2 μL of liquid was injected at a constant rate of 0.6 μL / min. After the two-point injection was completed, the needle was left in place for 3 min, and the needle withdrawal rate was 2 mm / min. The scalp was sutured and disinfected, and the rat was placed on a 37 °C heating pad until it woke up. After the operation, the rat was given an injection of penicillin sodium (80,000 units / day) and an oral administration of ibuprofen oral solution for 3 consecutive days.

[0254] After 3 weeks of drug injection into the striatum, the model was constructed and anesthetized by intraperitoneal injection of 3% sodium pentobarbital, with an injection dose of 60 mg / kg and an injection volume of 2 mL / kg. The anesthetized rat was transferred to a workbench and fixed in a stereotaxic apparatus in a prone position so that the skull was in a horizontal plane. The rat's eyelid reflex and pain response were observed, and surgery should not be started until the eyelid reflex and pain response of the limbs and tail were absent. A hole was drilled vertically using the smallest drill, taking care not to dislodge or damage the brain tissue. The rat was positioned on the right side (administration side), and one bone window was drilled 2.0 mm (AP = -2.0 mm) posterior to the anterior section and 2 mm (ML = 2 mm) away from the midline. At a needle advance rate of 6 mm / min, the needle was advanced to 8.8 mm (DV=-8.8 mm) below the dura mater and withdrawn to 8.5 mm (-8.5 mm), and 6-OHDA was injected at a constant rate of 0.6 μL / min, and each rat was injected with 4 μL of 6-OHDA (5 μg / μL), and the needle was left in place for 3 min after the completion of the injection, with a needle withdrawal rate of 2 mm / min.The scalp was sutured and disinfected, and the rats were placed on a 37°C heating pad until they woke up, and they were given injections of penicillin sodium (80,000 units / day) and oral administration of ibuprofen oral solution for 3 consecutive days after surgery.

[0255] Three weeks after constructing the model, the rats' response to the apomorphine drug was detected. Apomorphine (0.5 mg / kg) was intraperitoneally injected into the rats, and 10 minutes later, the rats were placed in the test environment and the number of rotations within 30 minutes was recorded.

[0256] Referring to FIG. 8A, the number of rotations in the 6-OHDA model rats was significantly increased compared to the sham-operated control group, proving that the model experiment was successful.

[0257] 1) Rotation test To detect the response of exogenously expressed AADC in the striatum to exogenous L-DOPA, we detected the rotations of the healthy side (left rotations) of rats that had been intragastrically administered L-DOPA. Rats were orally administered L-DOPA (20 mg / kg, 5 mL / kg) + benserazide (5 mg / kg, 5 mL / kg), and 45 min later, the rats were placed in the test environment and the number of rotations within 30 min was recorded.

[0258] Referring to Figure 8B, the model control group did not show a significant increase in rotation number compared to the sham-operated control group, but the high and low dose groups of HRPDAAV-02AI and HRPDAAV-03DI showed a significant increase compared to the model control group, indicating that AADC delivered and expressed in the striatum by AAV has an excellent response effect to exogenous L-DOPA drug.

[0259] 2) Cylinder test A cylinder test experiment was conducted to detect the rats' forelimb preference under background conditions. Each group of rats was placed in a transparent cylinder with a diameter of 20 cm, and the number of times the rats touched the wall with their left and right forelimbs and both forelimbs within 10 min was observed and recorded. A rat's hindlimb was used as a fulcrum, and the number of times it touched the wall with one or both forelimbs and then returned to the bottom of the cylinder was recorded as one time (when a rat first touched the wall with one paw and then the other paw, it was recorded as a bilateral wall touch). The left forelimb usage rate of the rats was calculated: left forelimb usage rate (%) = (left side + 0.5 both sides) / (right side + left side + both sides) × 100.

[0260] Referring to Figure 8C, the model mice and the treatment group both showed a decreased left forelimb utilization rate compared to the blank control group, again confirming the validity of the model.

[0261] To detect the utilization of L-DOPA in rats treated with AAV drugs, L-DOPA (5 mg / kg, 5 mL / kg) and benserazide (2.5 mg / kg, 5 mL / kg) were orally administered intragastrically to the rats. After 30 min, the rats were placed in a transparent cylinder with a diameter of 20 cm, and the number of times the rats touched the wall with their left and right forelimbs and both forelimbs within 10 min was observed and recorded.

[0262] Referring to Figure 8D, the model control group supplemented with L-DOPA did not show a significant improvement in the left forelimb utilization rate, but the HRPDAAV-02AI and HRPDAAV-03DI groups showed a clear improvement in the left forelimb utilization rate compared to the model control group, proving that the rats after administration showed a clear improvement in utilization rate to L-DOPA. It was again proven that AADC delivered and expressed in the striatum by AAV has an excellent response effect to exogenous L-DOPA drug.

[0263] 3) Detection of the striatal metabolic capacity for L-DOPA after administration of HRPDAAV-02AI and HRPDAAV-03DI After the behavioral test, rats from each group were given L-DOPA (20mg / kg, 5mL / kg) and benserazide (5mg / kg, 5mL / kg) intragastrically. The rats were sacrificed 1h after the end of the administration. After sacrifice, the striatum of the healthy and affected sides was collected. DOPA was detected by UPLC-MS / MS.

[0264] Referring to Figure 8E and Table 8, the DOPA ratio of the affected side / healthy side was detected, and the model control group and the administration group were compared, and the DOPA of the affected side of the model control group was obviously decreased. The construction of the 6-OHDA model was proved to be successful.

[0265] [Table 9]

[0266] In the detection of DOPA in the striatum of the administered side (affected side), it was found that DOPA was significantly improved in both the HRPDAAV-02AI and HRPDAAV-03DI administered groups compared to the model control group. Compared to the model control group, the DOPA ratio of the affected side / healthy side was significantly improved in each administered group. It was demonstrated that the striatum's metabolic ability to metabolize L-DOPA drugs was significantly improved after administration of HRPDAAV-02AI and HRPDAAV-03DI.

[0267] 4) Detection of protective effects on DA neurons after administration of HRPDAAV-03DI Each group of rats was perfused transcardially to obtain brain tissue for fluorescent immunohistochemical detection. The specific method was as follows: after general observation of the experimental rats, the cardiac tissue was released from the thoracic cavity, an intravenous needle was inserted into the left ventricle and fixed, the right atrial appendage was incised, pre-cooled PBS was perfused into the right atrial appendage to allow a clear liquid to flow out, and then 4% paraformaldehyde was perfused transcardially. The brain tissue was cut out and placed in a fixative (4% paraformaldehyde solution), and the fixed tissue was stained for tyrosine dehydrogenase (TH) using paraffin sections.

[0268] Referring to FIG. 8F, the ratio of TH positive cells in the affected side / contralateral side was analyzed by fluorescent immunohistochemistry. The TH cell survival rate in the affected side of the experimental model control group was less than 15% compared to the sham-operated group, demonstrating the success of the 6-OHDA model construction.

[0269] Meanwhile, the TH fluorescence intensity ratio of the affected side to the healthy side in the low-dose HRPDAAV-03DI group was significantly improved compared to the model control group, proving that the HRPDAAV-03DI drug had an excellent protective effect on rat dopamine neurons and improved the survival rate of TH cells.

[0270] The sequences used in this disclosure are as follows:

[0271] >AADC amino acid sequence (SEQ ID NO:1) MNASEFRRRGKEMVDYVANYMEGIEGRQVYPDVEPGYLRPLIPAAAPQEPDTFEDIINDVEKIIMPGVTHWHSPYFFAYFPTASSYPAMLADMLCGAIGCIGFSWAASPACTELETVMMD WLGKMLELPKAFLNEKAGEGGGVIQGSASEATLVALLAARTKVIHRLQAASPELTQAAIMEKLVAYSSDQAHSSVERAGLIGGVKLKAIPSDGNFAMRASALQEALERDKAAGLIPFFMV ATLGTTTCCSFDNLLEVGPICNKEDIWLHVDAAYAGSAFICPEFRHLLNGVEFADSFNFNPHKWLLVNFDCSAMWVKKRTDLTGAFRLDPTYLKHSHQDSGLITDYRHWQIPLGRRFRSL KMWFVFRMYGVKGLQAYIRKHVQLSHEFESLVRQDPRFEICVEVILGLVCFRLKGSNKVNEALLQRINSAKKIHLVPCHLRDKFVLRFAICSRTVESAHVQRAWEHIKELAADVLRAERE

[0272] > Amino acid sequence of GDNF (SEQ ID NO:2) MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI

[0273] >AADC wild type DNA (SEQ ID NO:3)

[0274] >AADC01 DNA (SEQ ID NO:4)

[0275] >AADC02 DNA (SEQ ID NO:5)

[0276] >AADC03 DNA (SEQ ID NO:6)

[0277] >AADC04 DNA (SEQ ID NO:7)

[0278] >Wild-type GDNF DNA (SEQ ID NO: 8) atgaagttatgggatgtcgtggctgtctgcctggtgctgctccacaccgcgtccgccttcccgctgcccgccggtaagaggcctcccgaggcgcccgccgaagaccgctccctcggccgccgccgcgcgcccttcgcgctgagcagtgactcaaatatgccagaggattatcctgatcagttcgatgatgtcatggattttattcaagccaccattaaaagactgaaaaggtcaccagataaacaaatggcagtgcttcctagaagagagcggaatcggcaggctgcagctgccaacccagagaattccagaggaaaaggtcggagaggccagaggggcaaaaaccggggttgtgtcttaactgcaatacatttaaatgtcactgacttgggtctgggctatgaaaccaaggaggaactgatttttaggtactgcagcggctcttgcgatgcagctgagacaacgtacgacaaaatattgaaaaacttatccagaaatagaaggctggtgagtgacaaagtagggcaggcatgttgcagacccatcgcctttgatgatgacctgtcgtttttagatgataacctggtttaccatattctaagaaagcattccgctaaaaggtgtggatgtatctga

[0279] >GDNF01 DNA (SEQ ID NO: 9) atgaagctgtgggatgtggtggccgtgtgcctggtgctgctgcatacagctagcgcctttcccctgccagctggcaagagaccacctgaggctcctgccgaggacagatctctgggcaggagacgggcccctttcgctctgtctagcgattccaacatgcccgaggactatccagaccagttcgatgacgtgatggacttcatccaggctaccatcaagaggctgaagagaagcccagacaagcagatggctgtgctgcctagacgcgagaggaatagacaggccgctgctgccaatcctgagaacagcaggggcaagggccggaggggccagcgcggcaagaatagaggctgcgtgctgaccgctatccacctgaacgtgacagacctgggcctgggctacgagaccaaggaggagctgatcttccgctattgttccggctcttgtgatgccgctgagaccacatatgacaagatcctgaagaacctgagcagaaacaggagactggtgtctgacaaggtgggccaggcctgttgcagaccaatcgccttcgatgatgacctgagctttctggacgataacctggtgtatcacatcctgagaaagcactctgccaagagatgtggctgcatctga

[0280] >GDNF02 DNA (SEQ ID NO: 10) atgaagctgtgggatgtggtggccgtgtgcctggtgctgctgcatacagcttctgcctttcctctgcccgctggcaagaggcctccagaggccccagctgaggacagatccctgggcagaaggagagccccttttgctctgtccagcgactctaacatgcccgaggattatcccgaccagttcgacgatgtgatggacttcatccaggctaccatcaagaggctgaagagaagcccagataagcagatggctgtgctgccacgcagagagaggaacagacaggccgctgctgctaaccctgagaactctcggggcaagggcagaaggggccagagaggcaagaatcgcggctgcgtgctgacagctatccacctgaacgtgacagatctgggcctgggctacgagacaaaggaggagctgatcttcagatactgttctggcagctgtgatgctgccgagacaacctacgacaagatcctgaagaacctgagcagaaatcggaggctggtgtccgataaggtgggccaggcctgttgcagacctatcgctttcgacgacgatctgagcttcctggacgataacctggtgtaccacatcctgcggaagcattctgccaagagatgcggctgtatctga

[0281] >GDNF03 DNA(SEQ ID NO: 11) atgaagctgtgggatgtggtggccgtgtgcctggtgctgctgcatacagcttctgccttccctctgccagctggcaagagaccacctgaggcccccgctgaggatagaagcctgggccgcagaagggctccttttgccctgagctccgactctaatatgcctgaggattacccagaccagttcgatgacgtgatggactttatccaggccacaatcaagcgcctgaagaggtctccagacaagcagatggctgtgctgcctaggagagagagaaaccggcaggctgccgctgctaatccagagaactctaggggcaagggcaggagaggccagaggggcaagaatcgcggctgcgtgctgaccgctatccatctgaatgtgaccgatctgggcctgggctacgagacaaaggaggagctgatcttcaggtactgcagcggctcttgtgacgccgctgagaccacatacgacaagatcctgaagaacctgagcagaaaccgcagactggtgtctgataaggtgggccaggcttgttgcaggcccatcgcttttgatgacgatctgtctttcctggacgataatctggtgtaccacatcctgcggaagcactctgctaagcggtgtggctgcatctga

[0282] >GDNF04 DNA (SEQ ID NO: 12) atgaagctgtgggatgtggtggccgtgtgcctggtgctgctgcacaccgcttctgccttcccactgcctgccggcaagagacctcccgaggcccctgccgaggacagaagcctgggcaggcggagagccccatttgctctgtctagcgattccaacatgcctgaggattaccccgatcagttcgatgacgtgatggatttcatccaggccaccatcaagagactgaagagatctcctgacaagcagatggctgtgctgcctagaagggagagaaacaggcaggccgctgctgccaatccagagaactccaggggcaagggcagaaggggccagcgcggcaagaatagaggctgcgtgctgacagccatccacctgaacgtgaccgacctgggcctgggctacgagaccaaggaggagctgatcttcaggtactgtagcggctcctgtgatgctgccgagaccacatacgacaagatcctgaagaacctgtccaggaacagaaggctggtgtctgacaaggtgggccaggcttgctgtaggccaatcgctttcgacgacgatctgtcctttctggatgacaacctggtgtaccacatcctgaggaagcattccgctaagagatgtggctgcatctga

[0283] >HRPDAAV01-A(SEQ ID NO: 13)

[0284] >HRPDAAV01-B (SEQ ID NO: 14)

[0285] >HRPDAAV01-C (SEQ ID NO: 15)

[0286] >HRPDAAV02-A (SEQ ID NO: 16)

[0287] >HRPDAAV02-B (SEQ ID NO: 17)

[0288] >HRPDAAV03-A (SEQ ID NO: 18)

[0289] >HRPDAAV03-B (SEQ ID NO: 19)

[0290] >HRPDAAV03-C (SEQ ID NO: 20)

[0291] >HRPDAAV03-D (SEQ ID NO: 21)

[0292] >HRPDAAV03-E (SEQ ID NO: 22)

[0293] >HRPDAAV03-F (SEQ ID NO: 23)

[0294] >HRPDAAV02-AI (SEQ ID NO: 24)

[0295] >HRPDAAV03-DI (SEQ ID NO: 25)

[0296] >5’ ITR (SEQ ID NO: 26) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct

[0297] >CMV enhancer 1 (SEQ ID NO: 27) cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg

[0298] >CBA promoter (SEQ ID NO: 28) tcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcg

[0299] >hybrid intron (SEQ ID NO: 29) ggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcag

[0300] >hGH poly(A)(SEQ ID NO: 30) Gggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtcctt

[0301] >3’ ITR(SEQ ID NO: 31) Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg

[0302] >SV40 poly(A) (SEQ ID NO: 32) Taagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagtt

[0303] >HPRE (SEQ ID NO: 33) ataacaggcctattgattggaaagtttgtcaacgaattgtgggtcttttggggtttgctgccccttttacgcaatgtggatatcctgctttaatgcctttatatgcatgtatacaagcaaaacaggcttttactttctcgccaacttacaaggcctttctcagtaaacagtatatgaccctttaccccgttgctcggcaacggcctggtctgtgccaagtgtttgctgacgcaacccccactggttggggcttggccataggccatcagcgcatgcgtggaacctttgtgtctcctctgccgatccatactgcggaactcctagccgcttgttttgctcgcagcaggtctggagcaaacctcatcgggaccgacaattctgtcgtactctcccgcaagtatacatcgtttccatggctgctaggctgtgctgccaactggatcctgcgcgggacgtcctttgtttacgtcccgtcggcgctgaatcccgcggacgacccctcccggggccgcttggggctctaccgcccgcttctccgtctgccgtaccgtccgaccacggggcgcacctctctttacgcggactccccgtctgtgccttctcatctgccggaccgtgtgcacttcgcttcacctctgcacgtcgcatggaggccaccgtgaacgcccaccggaacctgcccaaggtcttgcataagaggactcttggactttcagcaatgtcatc

[0304] >WPRE (Accession No. 34) Aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgc

[0305] >CMV promoter (Accession No. 35) Gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagct

[0306] >PA75 polyA (Accession No. 36)

[0307] >CMV enhancer 2 (SEQ ID NO: 37) Cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgtcaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccact tggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg

[0308] >β-globin intron (SEQ ID NO:38) Gtgagtttggggacccttgattgttctttctttttcgstattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaagatgtcccttgtatcaccatggaccctcatgataattttgttt ctttcactttctactctgttgacaaccattgtctcctcttttttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttc tctaatcacttttttttcaaggcaatcagggtatattatattgtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggctggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttcttttatggttacaatgatatacactgtttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttttttcctacag

[0309] MVM intron aagaggtaagggtttaaggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttgg

[0310] MCS atcgatatccgttaca

[0311] AAV2

[0312] >AAV9 capsid (SEQ ID NO: 42)

[0313] >T2A amino acid sequence (SEQ ID NO:43) EGRGSLLTCGDVEENPGP

[0314] >P2A amino acid sequence (SEQ ID NO:44) ATNFSLLKQAGDVEENPGP

[0315] >E2A amino acid sequence (SEQ ID NO:45) QCTNYALLKLAGDVESNPGP

[0316] >F2A amino acid sequence (SEQ ID NO:46) VKQTLNFDLLKLAGDVESNPGP

[0317] >T2A nucleotide sequence (SEQ ID NO: 47) gagggcagaggcagtctgctgacatgcggtgacgtggaagagaatcccggccct

[0318] >P2A nucleotide sequence (SEQ ID NO:48) gccaccaacttctccctgctgaagcaggccggcgacgtggaggagaacccccggcccc

[0319] >E2A nucleotide sequence (SEQ ID NO: 49) cagtgcaccaactacgccctgctgaagctggccggcgatgtggagagcaaccccgggccc

[0320] >F2A nucleotide sequence (SEQ ID NO:50) Gtgaaacagactttgaattttgaccttctcaagttggcgggagacgtggagtccaaccctggacct

Claims

1. A nucleic acid molecule comprising a first polynucleotide encoding an aromatic L-amino acid decarboxylase (AADC) protein and a second polynucleotide encoding a glial cell line-derived neurotrophic factor (GDNF) protein; Nucleic acid molecule.

2. the AADC protein comprises the amino acid sequence set forth in SEQ ID NO: 1, and / or the GDNF protein comprises the amino acid sequence set forth in SEQ ID NO: 2; The nucleic acid molecule of claim 1.

3. the first polynucleotide comprises a sequence having at least 75% identity to SEQ ID NO: 3 or a sequence having at least 95% identity to any one of SEQ ID NOs: 4 to 7, and / or the second polynucleotide comprises a sequence having at least 70% identity to SEQ ID NO: 8 or a sequence having at least 95% identity to any one of SEQ ID NOs: 9 to 12; Preferably, the first polynucleotide comprises a sequence set forth in any one of SEQ ID NOs: 3 to 7, Preferably, the second polynucleotide comprises a sequence set forth in any one of SEQ ID NOs: 8 to 12. A nucleic acid molecule according to claim 1 or 2.

4. the first polynucleotide and the second polynucleotide are linked via a third polynucleotide; Preferably, the third polynucleotide encodes an amino acid sequence having a linker function, more preferably, the amino acid sequence encoded by the third polynucleotide is set forth in any one of SEQ ID NOs: 43 to 46, and most preferably, the sequence of the third polynucleotide is set forth in any one of SEQ ID NOs: 47 to 50 or has at least 95% identity thereto. The nucleic acid molecule of claim 1.

5. The nucleic acid molecule further comprises identical expression control sequences operably linked to a first polynucleotide and a second polynucleotide, or two identical or different expression control sequences operably linked to the first polynucleotide and the second polynucleotide, respectively; the expression control sequence comprises a promoter and / or an enhancer; Preferably, the promoter is selected from the group consisting of CMV, CAG, CBA, CBh, EFS, EF1, PGK, SV40, Ubi, RSV promoters or any combination thereof; and / or The enhancer is selected from a Ubi, CMV, RSV enhancer, or any combination thereof; The nucleic acid molecule of claim 1.

6. Further comprising any one or any combination of a 5' inverted terminal repeat (5' ITR), a 3' inverted terminal repeat (3' ITR), an intron, a post-transcriptional regulatory element, a polyadenylation signal (polyA), and a multiple cloning site (MCS); The nucleic acid molecule of claim 1.

7. the 5' ITR and / or 3' ITR is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhlO, AAV-DJ or AAV-DJ8, preferably AAV2 or AAV9; the intron is selected from MVM, SV40, β-globulin, EF-1α, hybrid introns, or any combination thereof; the polyA is selected from PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA or any combination thereof; and / or The post-transcriptional regulatory element is selected from WPRE, HPRE, or a combination thereof; The nucleic acid molecule of claim 6.

8. The nucleic acid molecule comprises, from the 5' end to the 3' end: a) comprising a CMV enhancer, a CBA promoter, a first polynucleotide, a second polynucleotide and polyA, optionally further comprising a hybrid intron between the CBA promoter and the first polynucleotide, and optionally further comprising a WPRE sequence between the second polynucleotide and polyA; b) comprising a CMV enhancer, a CBA promoter, a second polynucleotide, a first polynucleotide and polyA, optionally further comprising a hybrid intron between the CBA promoter and the second polynucleotide, and optionally further comprising a WPRE sequence between the first polynucleotide and polyA; c) comprising a CMV enhancer, a CBA promoter, a first polynucleotide, polyA, a CMV enhancer, a CMV promoter, a second polynucleotide and polyA, optionally further comprising a hybrid intron between the CBA promoter and the first polynucleotide, optionally further comprising a β-globin intron between the CMV promoter and the second polynucleotide, and optionally further comprising a WPRE sequence between the second polynucleotide and polyA; d) comprising a CMV enhancer, a CMV promoter, a second polynucleotide, polyA, a CMV enhancer, a CBA promoter, a first polynucleotide and polyA, optionally further comprising a β-globin intron or an MVM intron between the CMV promoter and the second polynucleotide, optionally further comprising a hybrid intron between the CBA promoter and the first polynucleotide, and optionally further comprising a WPRE sequence between the first polynucleotide and polyA; e) comprising a CMV enhancer, a CBA promoter, a first polynucleotide, a third polynucleotide, a second polynucleotide and polyA, optionally further comprising a hybrid intron between the CBA promoter and the first polynucleotide, and optionally further comprising a WPRE sequence between the second polynucleotide and polyA; or f) comprising a CMV enhancer, a CBA promoter, a second polynucleotide, a third polynucleotide, the first polynucleotide and polyA, optionally further comprising a hybrid intron between the CBA promoter and the second polynucleotide, and optionally further comprising a WPRE sequence between the first polynucleotide and polyA; Preferably, the polyA is selected from hGH polyA, PA75 polyA, or SV40 polyA. The nucleic acid molecule of claim 1.

9. The CMV enhancer comprises a sequence set forth in SEQ ID NO: 27 or 37; The CBA promoter comprises the sequence set forth in SEQ ID NO: 28, The CMV promoter comprises the sequence set forth in SEQ ID NO: 35, The β-globin intron comprises the sequence set forth in SEQ ID NO: 38, The hybrid intron comprises the sequence set forth in SEQ ID NO: 29, The MVM intron comprises the sequence set forth in SEQ ID NO: 39, The PA75 polyA comprises the sequence shown in SEQ ID NO: 36, The SV40 polyA comprises the sequence shown in SEQ ID NO: 32, the hGH polyA comprises the sequence set forth in SEQ ID NO: 30; The WPRE sequence is shown in SEQ ID NO: 34, the 5' ITR comprises the sequence set forth in SEQ ID NO: 26, and / or The 3' ITR comprises the sequence set forth in SEQ ID NO:

31. A nucleic acid molecule according to any one of claims 5 to 8.

10. From the 5' end to the 3' end, (1) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-third polynucleotide-second polynucleotide encoding GDNF-WPRE-SV40 polyA; (2) CMV enhancer-CBA promoter-hybrid intron-second polynucleotide encoding GDNF-third polynucleotide-first polynucleotide-WPRE-SV40 polyA; (3) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-SV40 polyA-CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-PA75 polyA; (4) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-SV40 polyA-CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-WPRE-PA75 polyA; (5) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-SV40 polyA-CMV enhancer-CMV promoter-β globulin intron-second polynucleotide encoding GDNF-PA75 polyA; (6) CMV enhancer-CMV promoter-β globulin intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-SV40 poly A; (7) CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-WPRE-SV40 poly A, or (8) A CMV enhancer-CMV promoter-MVM intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-WPRE-SV40 poly A; The nucleic acid molecule of claim 1.

11. A sequence having at least 95% sequence identity to any one of SEQ ID NOs: 13 to 25, or any one of SEQ ID NOs: 13 to 25. The nucleic acid molecule of claim 1.

12. A nucleic acid molecule comprising a polynucleotide encoding an AADC protein, the nucleic acid molecule comprising a sequence set forth in any one of SEQ ID NOs: 4 to 7, or a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 4 to 7. Nucleic acid molecule.

13. A nucleic acid molecule comprising a polynucleotide encoding a GDNF protein, the nucleic acid molecule comprising a sequence set forth in any one of SEQ ID NOs: 9 to 12, or a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 9 to 12. Nucleic acid molecule.

14. The nucleic acid molecule further comprises a 5' ITR, a 3' ITR, a promoter, an enhancer, an intron, a post-transcriptional regulatory element, a polyadenylation signal (polyA), and a multiple cloning site (MCS); Preferably, the 5' ITR and / or 3' ITR is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhlO, AAV-DJ or AAV-DJ8; The promoter is selected from the group consisting of CMV, CAG, CBA, CBh, EFS, EF1, PGK, SV40, Ubi, and RSV promoters, or any combination thereof; the enhancer is selected from a Ubi, CMV, RSV enhancer, or any combination thereof; the intron is selected from MVM, SV40, β-globulin, EF-1α, hybrid introns, or any combination thereof; the polyA is selected from PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA or any combination thereof; and / or The post-transcriptional regulatory element is selected from WPRE, HPRE, or a combination thereof; More preferably, The CMV enhancer comprises a sequence set forth in SEQ ID NO: 27 or 37; The CBA promoter comprises the sequence set forth in SEQ ID NO: 28, The CMV promoter comprises the sequence set forth in SEQ ID NO: 35, The β-globin intron comprises the sequence set forth in SEQ ID NO: 38, The hybrid intron comprises the sequence set forth in SEQ ID NO: 29, The MVM intron comprises the sequence set forth in SEQ ID NO: 39, The PA75 polyA comprises the sequence shown in SEQ ID NO: 36, The SV40 polyA comprises the sequence shown in SEQ ID NO: 32, the hGH polyA comprises the sequence set forth in SEQ ID NO: 30; The WPRE sequence is shown in SEQ ID NO: 34, the 5' ITR comprises the sequence set forth in SEQ ID NO: 26, and / or the 3' ITR comprises the sequence set forth in SEQ ID NO: 31; Preferably, the nucleic acid molecule comprises, from the 5' end to the 3' end: g) a CMV enhancer, a CBA promoter, a first polynucleotide or a second polynucleotide and polyA, optionally further comprising a hybrid intron between the CBA promoter and the first polynucleotide, or further comprising a hybrid intron between the CBA and the second polynucleotide, and optionally further comprising a WPRE sequence between the first polynucleotide and polyA, or further comprising a WPRE sequence between the second polynucleotide and polyA; A nucleic acid molecule according to claim 12 or 13.

15. A recombinant adeno-associated virus (rAAV) particle comprising the nucleic acid molecule of claim 1 and an AAV capsid.

16. The AAV capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, AAVrh8, AAVrhlO, AAV-DJ or AAV-DJ8, preferably AAV2 or AAV9, more preferably AAV9; rAAV particles according to claim 15.

17. A pharmaceutical composition comprising the nucleic acid molecule of claim 1 or the rAAV particle of claim 15 or 16, and one or more pharmaceutically acceptable excipients. Pharmaceutical compositions.

18. A vector comprising the nucleic acid molecule of claim 1, preferably selected from a plasmid, a lentivirus, an adenovirus, an mRNA-LNP, an adeno-associated virus (AAV) vector, and a recombinant adeno-associated virus (rAAV) vector. vector.

19. A cell that contains or expresses the nucleic acid molecule of claim 1 or the vector of claim 18, or that packages and produces the recombinant rAAV particle of claim 15 or 16. cell.

20. 17. An rAAV production system for producing recombinant rAAV particles according to claim 15 or 16, comprising: (a) a polynucleotide sequence encoding the amino acid sequence of an AAV capsid; (b) a nucleic acid molecule according to claim 1 or a vector according to claim 18; (c) sufficient AAV rep and accessory functions to allow packaging of the nucleic acid molecule of claim 1 or the vector of claim 18 into the AAV capsid; the AAV capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV-DJ or AAV-DJ8, preferably AAV2 or AAV9, more preferably AAV9; rAAV production system.

21. Sufficient AAV rep and accessory functions are provided by a packaging cell, which preferably contains three plasmids: pHelper, pRC9, and pGOI.

21. The rAAV production system of claim 20.

22. A method for producing an rAAV particle according to claim 15 or 16, comprising packaging the polynucleotide of claim 1 or the vector of claim 18 into an AAV capsid by a packaging cell, wherein the AAV capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV-DJ or AAV-DJ8, preferably AAV2 or AAV9, more preferably AAV9. method.

23. A pharmaceutical composition for use in a method for treating, alleviating or preventing a central nervous system disease or symptom, comprising the polynucleotide of claim 1, the rAAV particle of claim 15 or 16 or the vector of claim 18, said method comprising administering a therapeutically or alleviatively effective amount of said pharmaceutical composition into the brain of a subject in need thereof, thereby causing expression of AADC protein and GDNF protein in the brain of said subject; Preferably, the AADC protein and the GDNF protein are expressed in the striatum of the subject; Preferably, the central nervous system disease or condition is a neurodegenerative disease or condition, more preferably a movement disorder, a sleep disorder, most preferably Parkinson's disease. Pharmaceutical compositions.

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