# US 2025/0051750 A1 · Aadc/gdnf polynucleotide, and use thereof in treating parkinson's disease

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- Number: US 2025/0051750 A1
- Document ID: US20250051750A1
- Status: Published application
- Kind: A1
- Application number: 18/714,057
- Filed: 2022-11-29
- Published: 2025-02-13
- Earliest claimed: 2021-11-29
- Assignees: Shanghai Regenelead Therapies Co., Ltd.
- Applicants: Shanghai Regenelead Therapies Co., Ltd.
- Inventors: Jun Jiang; Mingyue Wu; Wei Ning; Cheng Liao
- Web page: https://patentgrep.com/patent/US20250051750A1

## CPC

- C07K 14/48: Nerve growth factor [NGF]
- C12N 9/88: Lyases (4.)
- A61K 38/185: {Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 25/16: Anti-Parkinson drugs
- C07K 14/475: Growth factors; Growth regulators
- C12N 15/86: Viral vectors
- C12Y 401/01015: Glutamate decarboxylase (4.1.1.15)
- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- C12N 2800/40: Systems of functionally co-operating vectors
- C12N 2830/42: being an intron or intervening sequence for splicing and/or stability of RNA
- C12N 2830/48: regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
- C12N 2830/50: regulating RNA stability, not being an intron, e.g. poly A signal
- C12Y 401/01028: Aromatic-L-amino-acid decarboxylase (4.1.1.28), i.e. tryptophane-decarboxylase
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES

## Abstract

Provided are an aromatic L-amino acid decarboxylase (AADC)/glial cell line-derived neurotrophic factor (GDNF) polynucleotide, and a use thereof in treating Parkinson's disease. Specifically, provided are a method for and a use in treating neurodegenerative diseases (such as Parkinson's disease) by delivering AADC and a GDNF to specific areas of the brain by a gene delivery system using AAV as a vector.

## Claims

### Claim 1

A nucleic acid molecule, comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes aromatic L-amino acid decarboxylase (AADC) protein, and the second polynucleotide encodes glial cell line-derived neurotrophic factor (GDNF) protein.

### Claim 2 (depends on 1)

The nucleic acid molecule according to claim 1, wherein 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.

### Claim 3 (depends on 1)

The nucleic acid molecule according to claim 1, wherein the first polynucleotide comprises a sequence having at least 75% identity to SEQ ID NO: 3 or a sequence having at least 80% identity to any one of SEQ ID NOs: 4-7, and/or the second polynucleotide comprises a sequence having at least 70% identity to SEQ ID NO: 8 or a sequence having at least 80% identity to any one of SEQ ID NOs: 9-12.

### Claim 4 (depends on 1)

The nucleic acid molecule according to claim 1, wherein the first polynucleotide and the second polynucleotide are linked by a third polynucleotide, the third polynucleotide encodes an amino acid sequence with linker function;
and/or,
the nucleic acid molecule 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; the expression control sequence comprises a promoter and/or an enhancer.

### Claim 5

(canceled)

### Claim 6 (depends on 1)

The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule further comprises 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).

### Claim 7 (depends on 6)

The nucleic acid molecule according to claim 6, wherein:
the 5′ ITR and/or 3′ ITR are/is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, 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 the group consisting of Ubi, CMV and RSV enhancers, or any combination thereof,
the intron is selected from the group consisting of MVM, SV40, β globin, EF-1α and hybrid introns, or any combinations thereof;
the polyA is selected from the group consisting of PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA, or any combination thereof, and/or
the post-transcriptional regulatory element is selected from the group consisting of WPRE, HPRE, or a combination thereof.

### Claim 8 (depends on 7)

The nucleic acid molecule according to claim 7, wherein the nucleic acid molecule comprises, from the 5′ end to the 3′ end:
a) the CMV enhancer, the CBA promoter, the first polynucleotide, the second polynucleotide, and the polyA;
a-2) the CMV enhancer, the CBA promoter, the first polynucleotide, the second polynucleotide, and the polyA; wherein the hybrid intron is further comprised between the CBA promoter and the first polynucleotide;
a-3) the CMV enhancer, the CBA promoter, the first polynucleotide, the second polynucleotide, and the polyA; wherein the WPRE sequence is further comprised between the second polynucleotide and the polyA;
b) the CMV enhancer, the CBA promoter, the second polynucleotide, the first polynucleotide, and the polyA;
b-2) the CMV enhancer, the CBA promoter, the second polynucleotide, the first polynucleotide, and the polyA; wherein the hybrid intron is further comprised between the CBA promoter and the second polynucleotide;
b-3) the CMV enhancer, the CBA promoter, the second polynucleotide, the first polynucleotide, and the polyA; wherein the WPRE sequence is further comprised between the first polynucleotide and the polyA;
c) the CMV enhancer, the CBA promoter, the first polynucleotide, the polyA, the CMV enhancer, the CMV promoter, the second polynucleotide, and the polyA;
c-2) the CMV enhancer, the CBA promoter, the first polynucleotide, the polyA, the CMV enhancer, the CMV promoter, the second polynucleotide, and the polyA: wherein the hybrid intron is further comprised between the CBA promoter and the first polynucleotide;
c-3) the CMV enhancer, the CBA promoter, the first polynucleotide, the polyA, the CMV enhancer, the CMV promoter, the second polynucleotide, and the polyA: wherein the β globin intron is further comprised between the CMV promoter and the second polynucleotide;
c-4) the CMV enhancer, the CBA promoter, the first polynucleotide, the polyA, the CMV enhancer, the CMV promoter, the second polynucleotide, and the polyA: wherein the WPRE sequence is further comprised between the second polynucleotide and the polyA;
d) the CMV enhancer, the CMV promoter, the second polynucleotide, the polyA, the CMV enhancer, the CBA promoter, the first polynucleotide, and the polyA;
d-2) the CMV enhancer, the CMV promoter, the second polynucleotide, the polyA, the CMV enhancer, the CBA promoter, the first polynucleotide, and the polyA: wherein, the R globin intron or the MVM intron is further comprised between the CMV promoter and the second polynucleotide;
d-3) the CMV enhancer, the CMV promoter, the second polynucleotide, the polyA, the CMV enhancer, the CBA promoter, the first polynucleotide, and the polyA: wherein, the hybrid intron is further comprised between the CBA promoter and the first polynucleotide;
d-4) the CMV enhancer, the CMV promoter, the second polynucleotide, the polyA, the CMV enhancer, the CBA promoter, the first polynucleotide, and the polyA: wherein, the WPRE sequence is further comprised between the first polynucleotide and the polyA;
e) the CMV enhancer, the CBA promoter, the first polynucleotide, the third polynucleotide, the second polynucleotide, and the polyA;
e-2) the CMV enhancer, the CBA promoter, the first polynucleotide, the third polynucleotide, the second polynucleotide, and the polyA; wherein, the hybrid intron is further comprised between the CBA promoter and the first polynucleotide;
e-3) the CMV enhancer, the CBA promoter, the first polynucleotide, the third polynucleotide, the second polynucleotide, and the polyA: wherein, the WPRE sequence is further comprised between the second polynucleotide and the polyA; or,
f) the CMV enhancer, the CBA promoter, the second polynucleotide, the third polynucleotide, the first polynucleotide, and the polyA;
f-2) the CMV enhancer, the CBA promoter, the second polynucleotide, the third polynucleotide, the first polynucleotide, and the polyA; wherein, the hybrid intron is further comprised between the CBA promoter and the second polynucleotide;
f-3) the CMV enhancer, the CBA promoter, the second polynucleotide, the third polynucleotide, the first polynucleotide, and the polyA; wherein, the WPRE sequence is further comprised between the first polynucleotide and the polyA.

### Claim 9 (depends on 8)

The nucleic acid molecule according to claim 8, wherein:
the CMV enhancer comprises the 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 set forth in SEQ ID NO: 36;
the SV40 polyA comprises the sequence set forth in SEQ ID NO: 32;
the hGH polyA comprises the sequence set forth in SEQ ID NO: 30;
the WPRE sequence is set forth 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.

### Claim 10 (depends on 7)

The nucleic acid molecule according to claim 7, wherein the nucleic acid molecule comprises, 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 globin intron-second polynucleotide encoding GDNF-PA75 polyA;
(6) CMV enhancer-CMV promoter-β globin intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-SV40 polyA;
(7) CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-PA75 poly A-CMV Preliminary Amendment Page 6 enhancer-CBA promoter-hybrid intron-first polynucleotide-WPRE-SV40 polyA; or
(8) CMV enhancer-CMV promoter-MVM intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-WPRE-SV40 polyA.

### Claim 11 (depends on 1)

The nucleic acid molecule according to claim 1, comprising the sequence set forth in any one of SEQ ID NOs: 13-25 or a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 13-25.

### Claim 12

A nucleic acid molecule, wherein the nucleic acid molecule comprises
a polynucleotide encoding AADC protein, wherein the nucleic acid molecule comprises the sequence set forth in any one of SEQ ID NOs: 4-7 or a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4-7; or,
a polynucleotide encoding GDNF protein, wherein the nucleic acid molecule comprises the sequence set forth in any one of SEQ ID NOs: 9-12 or a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-12.

### Claim 13

(canceled)

### Claim 14 (depends on 12)

The nucleic acid molecule according to claim 12, further comprising 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);
the 5′ ITR and/or 3′ ITR are/is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, 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 the group consisting of Ubi, CMV and RSV enhancers, or any combination thereof,
the intron is selected from the group consisting of MVM, SV40, β globin, EF-1α and hybrid introns, or any combination thereof,
the polyA is selected from the group consisting of PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA, or any combination thereof, and/or
the post-transcriptional regulatory element is selected from the group consisting of WPRE, HPRE, or a combination thereof.

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 15)

The rAAV particle according to claim 15, wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, AAVrh8, AAVrh10, AAV-DJ, or AAV-DJ8.

### Claim 17 (depends on 1)

A pharmaceutical composition, comprising the nucleic acid molecule according to claim 1, and one or more pharmaceutically acceptable excipients.

### Claim 18 (depends on 1)

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

### Claim 19 (depends on 1)

A cell, comprising or expressing the nucleic acid molecule according to claim 1.

### Claim 20 (depends on 15)

A rAAV production system for producing the recombinant rAAV particle according to claim 15, wherein the production system comprises:
(a) a polynucleotide sequence encoding an amino acid sequence of the AAV capsid;
(b) a nucleic acid molecule or a vector comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes aromatic L-amino acid decarboxylase (AADC) protein, and the second polynucleotide encodes glial cell line-derived neurotrophic factor (GDNF) protein; and
(c) sufficient AAV rep function and helper function to allow packaging of the nucleic acid molecule or the vector into the AAV capsid;
wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, or AAV-DJ8.

### Claim 21

(canceled)

### Claim 22 (depends on 15)

A method for producing the rAAV particle according to claim 15, comprising packaging a nucleic acid molecule into the AAV capsid using a packaging cell, wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, or AAV-DJ8; wherein the nucleic acid molecule comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes aromatic L-amino acid decarboxylase (AADC) protein, and the second polynucleotide encodes glial cell line-derived neurotrophic factor (GDNF) protein.

### Claim 23 (depends on 1)

A method for treating, alleviating, or preventing a central nervous system disease or symptom, comprising administering to the brain of a subject in need thereof an amount, effective in treatment or alleviation, of the nucleic acid molecule according to claim 1 wherein such that the AADC protein and GDNF protein are expressed in the brain of the subject.

### Claim 24 (depends on 15)

A pharmaceutical composition, comprising the rAAV particle according to claim 15 and one or more pharmaceutically acceptable excipients.

### Claim 25 (depends on 15)

A method for treating, alleviating, or preventing a central nervous system disease or symptom, comprising administering to the brain of a subject in need thereof an amount, effective in treatment or alleviation, of the rAAV particle according to claim 15, wherein such that the AADC protein and GDNF protein are expressed in the brain of the subject.

### Claim 26 (depends on 23)

The method according to claim 23, wherein the AADC protein and GDNF protein are expressed in the striatum of the subject; the central nervous system disease or symptom is a neurodegenerative disease or symptom.

### Claim 27 (depends on 26)

The method according to claim 26, wherein neurodegenerative disease or symptom is dyskinesia or sleep disorder or Parkinson's disease.