Inhibitory nucleic acid targeting alpha synuclein expression

Abstract

The present invention features a polynucleotide construct comprising a sequence that targets an alpha-synuclein mRNA. Constructs comprising and / or encoding sequences targeting alpha-synuclein mRNA may be used, for example, to inhibit expression of alpha-synuclein mRNA and / or to treat synucleinopathies diseases or disorders.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480078599.2 (22) Application Date 2024.10.18 (30) Priority Data 63 / 591,879 2023.10.20 US 63 / 565,101 2024.03.14 US 63 / 696,066 2024.09.18 US (85) PCT International Application Entering National Phase Date 2026.06.11 (86) PCT International Application Application Data PCT / US2024 / 051907 2024.10.18 (87) PCT International Application Publication Data WO2025 / 085713 EN 2025.04.24 (71) Applicant: Spark Medical, Inc. Address: Pennsylvania, USA (72) Inventors: P. Gonzalez-Allegre, S. Basu, M.G. Bifferi, V. Singh, Li Juan, C. Kali, F. Carnin, E. Ramsburg (74) Patent Agency: Beijing Kunrui Law Firm, 11494 Patent Attorney: Feng Xinqin (51) Int.Cl. C12N 15 / 113 (2006.01) C12N 15 / 11 (2006.01) (54) Invention Title: Inhibitory Nucleic Acid Targeting α-Synuclein Expression (57) Abstract: The present invention is characterized by a polynucleotide construct comprising a sequence targeting α-synuclein mRNA. Constructs comprising a sequence targeting α-synuclein mRNA and / or a coding sequence can be used, for example, to inhibit α-synuclein mRNA expression and / or to treat synucleinosis diseases or conditions. Claims (5 pages), Description (106 pages), Sequence Listing (electronic publication), Figures (29 pages), CN 122374456 A, 2026.07.10, CN 1 22 37 44 56 A. 1. An RNA polynucleotide comprising a targeting RNA sequence having at least 90% sequence identity with a sequence of any one of SEQ ID NO: 36, 38, 32-35, 37, or 39-62. 2. The RNA polynucleotide of claim 1, wherein the targeting RNA sequence comprises a sequence selected from the group consisting of: a) a sequence differing from SEQ ID NO: 34 by 0, 1, 2, or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 108; b) a sequence differing from SEQ ID NO: 36 by 0, 1, 2, or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 109; c) a sequence differing from SEQ ID NO: 34 by 0, 1, 2, or 3 nucleotides;NO: 37 has a sequence that differs from SEQ ID NO: 38 by 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 110; d) has a sequence that differs from SEQ ID NO: 38 by 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 111; e) has a sequence that differs from SEQ ID NO: 39 by 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 112; f) has a sequence that differs from SEQ ID NO: 41 by 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 113; and g) has a sequence that differs from SEQ ID NO: 42 by 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 114. 3. The RNA polynucleotide of claim 1, wherein the target RNA sequence comprises the sequence of SEQ ID NO: 36 or SEQ ID NO: 38. 4. The RNA polynucleotide of claim 3, wherein the targeting RNA sequence comprises the sequence of SEQ ID NO: 95 or SEQ ID NO: 97. 5. The RNA polynucleotide of any one of claims 1 to 4, wherein the RNA polynucleotide further comprises a second RNA sequence, wherein the second RNA sequence is substantially complementary to the targeting RNA sequence. 6. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide is a pre-miRNA, wherein the pre-miRNA comprises a pre-miRNA scaffold, an embedded guide sequence, and an embedded passenger sequence, wherein the guide sequence comprises the targeting sequence and the passenger sequence comprises the second sequence. 7. The RNA polynucleotide of claim 6, wherein the RNA polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 122-128 and 207-224. 8. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 123 or 125. 9. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide is shRNA, wherein the shRNA comprises an shRNA scaffold, an embedded guide sequence, and an embedded passenger sequence, wherein the guide sequenceThe target sequence is included and the passenger sequence includes the second sequence. 10. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide is pri-miRNA, wherein the pri-miRNA includes a pri-miRNA scaffold, an embedded guide sequence and an embedded passenger sequence, wherein the guide sequence includes the target sequence and the passenger sequence includes the second sequence. 11. The RNA polynucleotide of claim 10, wherein the scaffold is miR-1, miR-26, miR16-1, miR-30, miR-33, miR101, miR-64, miR-122, miR-125, miR-135, miR-155, enhanced miR-155 (eSIBR), or miR-451 scaffold. 12. The RNA polynucleotide of claim 10, wherein the scaffold is an S155e scaffold, an S26, an S126 scaffold, an S33 scaffold, or an S155 scaffold. 13. The RNA polynucleotide of claim 10, wherein the RNA polynucleotide comprises a sequence having at least 90% sequence identity with any of SEQ ID NO: 115-121, 207-224, or the RNA version of SEQ ID NO: 243-260. 14. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises a sequence of SEQ ID NO: 116 or SEQ ID NO: 118. 15. An optionally modified repressive RNA comprising (a) a guide strand capable of hybridizing to a target sequence of any of SEQ ID NO: 129-135; and (b) a substantially complementary passenger sequence; wherein one or more nucleotides of the guide strand and the passenger strand are optionally modified RNA. 16. A polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1 to 14. 17. An expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1 to 14, said nucleic acid sequence being operatively linked to one or more expression control elements. 18. The expression cassette of claim 17, wherein the expression cassette comprises an upstream promoter and a downstream polyadenylation signal, said upstream promoter and said downstream polyadenylation signal being operatively linked to said nucleic acid sequence encoding said RNA polynucleotide. 19. The expression cassette of claim 18, wherein the expression cassette is 5' to 3'The expression cassette comprises a promoter or promoter / enhancer, the nucleic acid sequence encoding the RNA polynucleotide, and a polyadenylation signal, wherein the promoter or promoter / enhancer and the polyadenylation signal are operatively linked to the nucleic acid sequence encoding the RNA polynucleotide. 20. The expression cassette of claim 18 or 19, wherein the promoter provides CNS expression. 21. The expression cassette of claim 18 or 19, wherein the promoter is an EF-1α promoter. 22. The expression cassette of any one of claims 17 to 21, wherein the expression cassette is DNA. 23. The expression cassette of claim 22, wherein the expression cassette comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 168-174. 24. The expression cassette of claim 23, wherein the expression cassette comprises the sequence of SEQ ID NO: 169 or 171. 25. The expression cassette according to any one of claims 17 to 24, further comprising a nucleic acid sequence encoding glial cell-derived neurotrophic factor (GDFN), said nucleic acid sequence being operatively linked to said promoter or operatively linked to a second promoter. 26. A recombinant viral vector nucleic acid comprising the expression cassette according to any one of claims 17 to 25, and 5' and 3' viral elements providing viral packaging and / or replication. 27. The recombinant viral vector nucleic acid according to claim 26, wherein said recombinant viral vector nucleic acid is recombinant DNA and comprises an adeno-associated virus (AAV) inverted repeat sequence (ITR) flanking the 5' end of said recombinant viral vector nucleic acid and an AAV ITR flanking the 3' end of said recombinant viral vector nucleic acid. 28. The recombinant viral vector nucleic acid of claim 27, wherein the 5' ITR and 3' ITR are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, or AAV3B. 29. The recombinant viral vector nucleic acid of claim 27, wherein the recombinant viral vector nucleic acid is self-complementary. 30. The recombinant viral vector nucleic acid of claim 27, wherein the 5' ITR comprises a sequence having at least 95% sequence identity with SEQ ID NO: 182, and the 3' ITR comprises a sequence having at least 95% sequence identity with SEQ ID NO: 182.NO: 183 A sequence having at least 95% sequence identity. 31. The recombinant viral vector nucleic acid of claim 27 or 30, comprising a sequence having at least 90% sequence identity with any of the sequences in SEQ ID NO: 175-181. 32. The recombinant viral vector nucleic acid of claim 27, comprising a sequence having at least 95% sequence identity with any of SEQ ID NO: 176 or 178. 33. A delivery medium comprising a viral vector or a non-viral vector and an RNA polynucleotide of any one of claims 1 to 14, a repressive RNA of claim 15, a polynucleotide of claim 16, an expression cassette of any one of claims 17 to 25, or a recombinant viral vector nucleic acid of any one of claims 26 to 32. 34. The delivery medium of claim 33, wherein the delivery medium is the viral vector. 35. The delivery medium of claim 34, wherein the viral vector is a recombinant AAV, a recombinant lentiviral vector, or a recombinant adenovirus vector. 36. The delivery medium of claim 35, wherein the viral vector is a recombinant AAV, the recombinant AAV comprising the recombinant viral vector nucleic acid of any one of claims 26 to 32 and a capsid. 37. The delivery medium of claim 36, wherein the recombinant AAV vector comprises a capsid comprising VP1, VP2, or VP3 having at least 90% sequence identity with any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10; or VP1 of SEQ ID NO: 164 or SEQ ID NO: 167. 38. The delivery medium of claim 36, wherein the capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10; or the capsid comprises SEQ ID NO: 164 or SEQ ID NO: 164.39. The delivery medium of claim 36, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166. 40. The delivery medium of claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequences of SEQ ID NO: 137 and 151. 41. The delivery medium of claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 158. 42. The delivery medium of claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 169. 43. The delivery medium of claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 176. 44. The delivery medium of claim 33, wherein the delivery medium is a nanoparticle selected from the group consisting of: lipid nanoparticles (LNP), polymer nanoparticles, lipid polymer nanoparticles (LPNP), protein- or peptide-based nanoparticles, DNA dendritic polymers or DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. 45. The delivery medium of claim 44, wherein the delivery medium is an LNP or LPNP. 46. ​​A recombinant gland-associated vector comprising rAAV nucleic acid encapsulated in a capsid, wherein the rAAV nucleic acid comprises (a) a 5' ITR of SEQ ID NO: 182; (b) an EF1a promoter of SEQ ID NO: 184; (c) a pri-miRNA coding sequence comprising the sequence of SEQ ID NO: 158; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 185; and (e) a 3' ITR of SEQ ID NO: 183, wherein (c) is operatively linked to (b) and (d). 47. The recombinant AAV vector of claim 46, comprising a sequence at least 95% identical to that of SEQ ID NO: 169. 48. The recombinant AAV vector according to claim 46 or 47, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166.49. A pharmaceutical composition comprising: an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery medium according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48; and a pharmaceutically acceptable carrier. 50. A method for reducing or inhibiting α-synuclein aggregation in cells or a subject, the method comprising administering to the cells or the subject an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery medium according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48, or a pharmaceutical composition according to claim 49. 51. A method of treating a subject with a synucleinosis disease or condition, the method comprising administering to the subject an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery medium according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48, or a pharmaceutical composition according to claim 49. 52. The method of claim 51, wherein the synucleinosis disease or condition is Lewy body disease or multiple system atrophy. 53. The method of claim 51, wherein the synucleinosis disease or condition is infantile axonal dystrophy, atypical axonal dystrophy, adult paroxysmal dystonia-Parkinson's disease, autosomal recessive early-onset Parkinson's disease, POLG-related neurodegeneration, Niemann-Pick disease C1, or Clabbit disease. 54. The method of claim 51, wherein the synucleinopathy disease or condition is Parkinson's disease. 55. A method of treating a disease or condition associated with low dopamine, the method comprising administering to the subject any one of claims 1 to 14.RNA polynucleotide, the repressive RNA according to claim 15, the polynucleotide according to claim 16, the delivery cassette according to any one of claims 17 to 25, the recombinant viral vector nucleic acid according to any one of claims 26 to 32, the delivery medium according to any one of claims 33 to 45, the rAAV vector according to any one of claims 46 to 48, or the pharmaceutical composition according to claim 46. 56. The method according to any one of claims 50 to 55, wherein administration comprises direct intracranial, intracisional, or intraventricular administration. 57. The method according to any one of claims 50 to 56, wherein the initial administration is outside the CNS. 58. The method according to any one of claims 50 to 57, wherein the subject is human. 59. The method according to any one of claims 50 to 58, wherein the method reduces α-synuclein levels in the transduced brain region by about 25% to about 70% relative to baseline. 60. The method according to claim 59, wherein the transduced brain region is the substantia nigra. 61. An AAV vector genomic plasmid comprising recombinant viral nucleic acid according to any one of claims 26 to 32. 62. The AAV genomic plasmid of claim 61, wherein the plasmid lacks the rep and cap genes. 63. A method of producing an rAAV vector, the method comprising the step of culturing an rAAV packaging cell line containing rAAV helper viral activity, wherein the genome of the producing cell comprises nucleic acid, a rep gene, and a cap gene according to any one of claims 26 to 32, wherein the rAAV vector is produced. 64. A method of producing an rAAV vector, the method comprising the step of culturing rAAV-permitting cells containing an rAAV genomic plasmid according to claim 62, wherein the rAAV-permitting cells further comprise (a) rep and cap genes provided as part of the cell genome and / or provided by one or more separate plasmids, and (b) helper viral activity provided by the cell genome and / or provided by one or more separate plasmids. 65. The method of claim 64, wherein the rAAV-allowed cell is a packaging cell, wherein the genome of the packaging cell comprises a cap gene and a rep gene. 66. The method of claim 64, wherein (a) the rep gene, the cap gene, and the...The helper activity is provided in a single plasmid, or (b) the rep gene and the cap gene are provided in a rep / cap plasmid, and the helper activity is provided by a helper plasmid. 67. A method for obtaining an rAAV vector, the method comprising the steps of: (a) producing the rAAV vector using the method according to any one of claims 63 to 66, and (b) purifying the rAAV vector. Claims 5 / 5 Page 6 CN 122374456 A Repressive Nucleic Acid Targeting α-Syntocin Expression

[0001] Cross-Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 591,879, filed October 20, 2023; U.S. Provisional Patent Application No. 63 / 565,101, filed March 14, 2024; and U.S. Provisional Patent Application No. 63 / 696,066, filed September 18, 2024, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Reference to the Electronically Filed Sequence Listing

[0004] The electronic sequence listing (065830.21WO1; size: 338,301 bytes; and creation date: October 11, 2024) is incorporated herein by reference in its entirety. Background Art

[0005] Synucleinopathy is a neurodegenerative disease or condition characterized by neurons and / or glial inclusions. Pathologically, synucleinopathy can be divided into two major disease groups: Lewy body diseases or conditions and multiple system atrophy (MSA). Lewy body diseases and conditions are characterized by aggregated α-synuclein and include Parkinson's disease, Parkinson's dementia, Lewy body dementia, infantile axonal dystrophy, atypical axonal dystrophy, adult paroxysmal dystonia-Parkinson's disease, autosomal recessive early-onset Parkinson's disease, POLG-related neurodegeneration, Niemann-Pick disease C1, and Clab disease. (Koga et al., Molecular Neurodegeneration (2021) 16:83.)

[0006] Parkinson's disease is an age-related progressive neurodegenerative disease. Parkinson's disease is characterized by the abnormal accumulation of misfolded α-synuclein aggregates in various regions of the brain. The loss of dopaminergic neurons in the substantia nigra is a pathological marker of Parkinson's disease. (Lee et al. Neuroimmunol. Neuroinflammation (2021) 8:222-44; and Koga et al. Molecular Neurodegeneration (2021) 16:83.)

[0007] Various symptomatic treatments for Parkinson's disease, such as dopamine replacement therapy, have been used to treat motor symptoms associated with Parkinson's disease. In the early stages of the disease, motor symptoms can usually be well controlled with symptomatic medications. As Parkinson's disease progresses, such treatments become less effective and are associated with debilitating side effects, such as movement disorders, leading to a decline in function and quality of life. (Poewe et al., Nat Rev Dis Primers 3, 17013 (2017.)

[0008] Different repressive RNAs have been proposed to reduce α-synuclein expression. (See, for example, McCormack et al., PLoS One. Aug 11, 2010; 5(8):e12122; Kim et al., Front. Neurol. Jan 31, 2017; 8:13; Alarcón-Arís et al., Mol. Ther. Feb 7, 2018; 26(2):550-567; Bohn and Sapru, U.S. Patent No. 8,389,487; and Menon et al., Brain Communications, Vol. 3, No. 4, 2021, fcab247, hypetext: / / doi.org / 10.1093 / braincomms / fcab247.) Summary of the Invention

[0009] The invention is characterized by a polynucleotide construct comprising a sequence targeting α-synuclein mRNA. The polynucleotide construct comprises a repressive RNA polynucleotide containing a sequence targeting α-synuclein mRNA, such as pri-microRNA (pri-miRNA), pre-microRNA (pre-miRNA), short hairpin RNA (shRNA), microRNA (miRNA), and optionally modified miRNA. Constructs comprising the sequence targeting α-synuclein mRNA and / or the coding sequence described in this specification (page 1 / 106, CN 122374456 A) can be used, for example, in methods for inhibiting α-synuclein expression and / or treating synucleinosis diseases or conditions.

[0010] The α-synuclein mRNA “targeting” sequence is capable of hybridizing with substantially complementary α-synuclein mRNA target sequences. The α-synuclein mRNA target sequences described herein include sequences of any one of SEQ ID NO: 129-135 and SEQ ID NO: 32-62. The complementary sequence of any of them.

[0011] The reference to "inhibitory" RNA polynucleotide indicates that the polynucleotide contains a targetA sequence of mRNA that can inhibit mRNA activity. Inhibition of mRNA activity causes a decrease in protein expression of the targeted mRNA. Repressive RNAs include, for example, miRNAs and miRNA precursors, such as pre-miRNA, sh-RNA, and pri-miRNA.

[0012] Therefore, a first aspect of the invention describes an RNA polynucleotide comprising an RNA sequence targeting α-synuclein mRNA. The RNA polynucleotide comprises a target RNA sequence having at least 90% sequence identity with the sequence of any of SEQ ID NO: 32-62.

[0013] A second aspect of the invention relates to an optionally modified repressive RNA duplex comprising (a) a guide strand capable of hybridizing to a target sequence of any of SEQ ID NO: 129-135; and (b) a substantially complementary passenger sequence; wherein one or more nucleotides of the guide strand and the passenger strand are optionally modified RNA.

[0014] A third aspect of the invention relates to a polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide encoding an α-synuclein mRNA targeting sequence.

[0015] A fourth aspect of the invention relates to an expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide encoding an α-synuclein mRNA targeting sequence and one or more expression control elements operatively linked to the encoding nucleic acid sequence.

[0016] A fifth aspect of the invention relates to a recombinant viral vector nucleic acid comprising (a) an expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide encoding an α-synuclein mRNA targeting sequence and one or more expression control elements operatively linked to the encoding nucleic acid sequence, and (b) 5' and / or 3' viral elements providing viral packaging and / or replication.

[0017] A sixth aspect of the invention relates to a delivery medium comprising a viral or non-viral vector and (a) an RNA polynucleotide comprising a sequence targeting α-synuclein mRNA, (b) an optionally modified repressive RNA duplex targeting α-synuclein mRNA, or (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding an RNA polynucleotide encoding a targeting sequence of α-synuclein mRNA.

[0018] A seventh aspect of the invention relates to a pharmaceutical composition comprising (a) an RNA polynucleotide comprising a sequence targeting α-synuclein mRNA, (b) an optionally modified repressive RNA duplex targeting α-synuclein mRNA, and (c) a polynucleotide encoding α-synuclein mRNA.The eighth aspect of the invention relates to a method of reducing α-synuclein expression in a subject and / or treating synucleinosis disease or condition, comprising administering: (a) an RNA polynucleotide containing a sequence targeting α-synuclein mRNA, (b) an optionally modified repressive RNA duplex targeting α-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid containing a sequence encoding a target sequence of α-synuclein mRNA, or (d) a delivery medium containing (a), (b), or (c); and a pharmaceutically acceptable carrier.

[0020] A ninth aspect of the invention relates to a method of treating a disease or condition associated with low dopamine, the method comprising administering: (a) an RNA polynucleotide comprising a sequence targeting α-synuclein mRNA, (b) optionally modified repressive RNA duplex targeting α-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding the α-synuclein mRNA target sequence, or (d) a delivery medium comprising (a), (b), or (c); and a pharmaceutically acceptable carrier.

[0021] Other aspects of the invention include (a) RNA polynucleotides comprising a sequence targeting α-synuclein mRNA, (b) optionally modified repressive RNA duplexes targeting α-synuclein mRNA, (c) polynucleotides, expression cassettes, or recombinant viral nucleic acids comprising a sequence encoding a targeting sequence of α-synuclein mRNA, or (d) delivery media comprising (a), (b), or (c); and pharmaceutically acceptable carriers for use in medicine, reducing α-synuclein, or treating synucleinosis diseases or conditions; and use of (a), (b), (c), or (d) in the preparation of medicaments (e.g., for use in medicine, reducing α-synuclein, or treating synucleinosis diseases or conditions).

[0022] Other features and advantages of the invention will be apparent from the additional description provided herein (including various examples). The examples provided illustrate different components and methods useful in carrying out the invention. Such examples do not limit the claimed invention. Based on this disclosure, those skilled in the art can identify and employ other components and methods useful for carrying out the present invention. Brief Description of the Drawings

[0023] Figure 1 illustrates different small interfering RNAs (siRNAs).The ability to reduce the expression of the human gene encoding α-synuclein (SCNA) in HEK293 cells. siRNA was provided at concentrations of 1 nM, 10 nM, or 100 nM. GAPDH refers to glyceraldehyde-3-phosphate dehydrogenase.

[0024] Figures 2A, 2B, and 2C show the survival percentage of HEK293 cells after administration of different amounts of siRNA. Figure 2A shows the results using 1 nM. Figure 2B shows the results using 10 nM. Figure 2C shows the results using 100 nM. GAPDH refers to glyceraldehyde-3-phosphate dehydrogenase.

[0025] Figure 3 shows the ability of different siRNAs to inhibit the expression of the α-synuclein-GFP fusion protein in HEK293 cells. The activities plotted are normalized to the initial control of green fluorescent protein (GFP) / focal adhesion protein. The provided control is siGLO, which is a scrambling control (a non-targeted control with red fluorescence); mismatch 11 (a non-targeted control); and initial conditions.

[0026] Figures 4A and 4B show the effects of different siRNAs targeting SNCA expression on related genes in HEK293 cells. Figure 4A shows the expression of SNCB (a gene encoding β-synuclein) at siRNA concentrations of 1 nM, 10 nM, or 100 nM. Figure 4B shows the expression of SNCG (a gene encoding γ-synuclein) at siRNA concentrations of 1 nM, 10 nM, or 100 nM. GAPDH refers to glyceraldehyde-3-phosphate dehydrogenase.

[0027] Figure 5 shows the ability of different siRNAs at different concentrations to inhibit expression in SH-SY5Y cells.

[0028] Figure 6 is a graph showing the differential expression (DE) of genes produced by different siRNAs.

[0029] Figure 7 shows the ability of different pre-miRNAs to reduce normalized GFP signal (as a measure of SCNA expression) compared to pCC41. Pre-miRNAs are indicated by miR tags. Statistical analysis was performed using two-factor ANOVA and Dunnett multiple comparison analysis.

[0030] Figures 8A and 8B show the ability of different rAAV vectors containing nucleic acids encoding pre-miRNAs to inhibit α-synuclein expression and α-synuclein aggregation at two different multiplicity of infection. Figure 8A shows human α-synuclein normalized to AAV-α-synuclein (AAV-α-syn). Figure 8B shows human α-synuclein normalized to AAV-α-synuclein.pS129 intensity. pS129 intensity is a measure of α-synuclein aggregation. Statistical analysis was performed using a two-factor ANOVA against AAV-αSyn.

[0031] Figures 9A through 9D show the ability of different rAAV vectors containing nucleic acids encoding pre-miRNA to inhibit α-synuclein expression. Figure 9A shows the fold change relative to human α-synuclein + mis11. Figure 9B shows the inhibition of human α-synuclein production. Figure 9C shows the reduction of mutant α-synuclein (A53T). Figure 9D shows the effect on mouse α-synuclein expression. Statistical analysis was performed using a one-factor ANOVA and Dunnett multiple comparisons.

[0032] Figures 10A through 10D show the miRNA biodistribution and inhibition of mouse α-synuclein. Figure 10A shows the biodistribution in the substantia nigra (SN) and striatum (Str). Figure 10B shows mouse SNCA (mSCNA) expression in the SN and Str. Figure 10C shows mSCNA expression in the substantia nigra. Figure 10D shows mSCNA expression in the striatum.

[0033] Figure 11 shows the guide chain and passenger chain levels from the rAAV vector.

[0034] Figure 12 shows the effect of different doses on the body weight of mice treated with αSyn. Mice were administered a dose of 7.5e9 vg / SN of human αSyn (AAV1 / 2-A53TαSyn) to induce α-synuclein-mediated neuronal loss. Treated animals received doses of rAAV-miR5 of 5e7, 5e8, or 5e9 vector genomes (vg). Controls were administered a diluent or miss11 (missense construct) at a dose of 5e9 vg + αSyn. Statistical analysis: Ordinary one-factor ANOVA and Tukey post-hoc analysis.

[0035] Figures 13A and 13B show the distribution of rAVV-miR5 in the substantia nigra. Treated animals received 5e7, 5e8, or 5e8 vector genome (vg) doses of rAAV-miR5. The control was administered a diluent or miss11 (missense construct) at 5e9 vg + αSyn. The presence of vectors in the substantia nigra was assessed using specific primers designed based on BgH and RbG polyA sequences. The AAV1 / 2-A53TαSyn vector has BgH polyA; rAAV-miR5 and rAAV-CAG-mis11 have RbG polyA. Figure 13A showsThe measurement results of BgH polyA. Figure 13B shows the measurement results of RbG polyA.

[0036] Figure 14 shows the ability of rAAV-miR5 to knock down αSyn as measured by qRT-PCR. The treated animals received 5e7, 5e8, or 5e8 vector genome (vg) doses of rAAV-miR5. The control was administered diluent or miss11 (missense construct) at 5e9 vg + αSyn. Statistical analysis was performed by ordinary one-factor Anova and Tukey multiple comparison tests. **p<0.01, *p<0.05

[0037] Figure 15 shows the ability of rAAV-miR5 to knock down αSyn in the cortex as measured by JESS assay. The treated animals received 5e7, 5e8, or 5e8 vector genome (vg) doses of rAAV-miR5. Diluent or miss11 (missense construct) was administered to the control at 5e9 vg + αSyn. Ordinary 1-factor ANOVA Tukey multiple comparisons. ***p<0.001. Comparison with negative control + AST α-Syn.

[0038] Figure 16 shows the ability of rAAV-miR5 to inhibit dopamine loss induced by A53T α-Syn. Treatment animals received 5e7, 5e8, or 5e8 vector genome (vg) doses of rAAV-miR5. Diluent or miss11 (missense construct) was administered to the control at 5e9 vg + αSyn. Data represent absolute numbers. Ordinary 1-factor ANOVA Tukey post-hoc test was performed on the logarithmic scale values ​​of the data points. Comparison with negative control + AST α-Syn. ****p<0.0001, ***p<0.001, *p<0.05.

[0039] Figure 17 illustrates the ability of rAAV-miR5 to inhibit dopamine loss induced by A53T α-Syn. Treatment animals received doses of rAAV-miR5 vector genome (vg) at doses of 5e7, 5e8, or 5e9. Controls were administered diluent or miss11 (missense construct) with 5e9 vg + α Syn. Ordinary 1-factor ANOVA Tukey multiple comparisons. Comparison with negative control + AST αSyn. ****p<0.0001, **p<0.01, *p<0.05.

[0040] Figure 18 illustrates the microRNA treatment determined by small RNA sequencing after rAAV-miR5 administration. G / P refers to the guide / passenger ratio.

[0041] Figure 19 summarizes the small RNAs.Sequencing results. The endogenous miRNA mechanism is not affected by the rAAV-miR5 overexpression specification, page 4 / 106, 10 CN 122374456 A. Treatment animals received doses of rAAV-miR5 vector genome (vg) of 5e7, 5e8, or 5e9. At the highest dose of 5e9 vg, exogenous mature miRNAs accounted for 1% of the total endogenous miRNA library. Medium and low doses of 5e8 and 5e7 vg resulted in exogenous miRNAs < 0.1% of the total endogenous miRNA library. No miRNAs were found in the diluent-treated group and the negative control group.

[0042] Figure 20 shows the ability of rAAV-miR5 to significantly reduce human SNCA mRNA levels at a dose of 5e9 vg / SN compared to the diluent-injected group in SNCA-OVX female mice, as measured by RT-qPCR of human SNCA mRNA in SN. Statistical comparisons between groups were assessed using a one-way ANOVA and Tukey's multiple comparison test. Data were plotted as individual (symbols) and group means ± SEM (bars).

[0043] Figures 21A and 21B show the biodistribution of the vector genome and miR5 expression in the substantia nigra of African green monkeys (“AGM”) two months after rAAV-miR5 administration. The X-axis plots the three test doses of rAAV-miR5 (3e8 vg per substantia nigra, 3e9 vg per substantia nigra, and 3e10 vg per substantia nigra), and the Y-axis plots the copy number (CN) / μg DNA of the vector genome (Figure 21A) or the CN / μg RNA of miR5 miRNA (Figure 21B). miRNA expression analysis shows the dose-dependent distribution of rAAV-miR5 in the substantia nigra.

[0044] Figure 21C shows the relative expression of SNCA mRNA in the AGM dose groups. The X-axis depicts two control groups (diluent and mis11) and three doses of rAAV-miR5 administered (3e8 vg per substantia nigra, 3e9 vg per substantia nigra, and 3e10 vg per substantia nigra). The Y-axis depicts the fold change relative to the diluent group. For all groups, n=2 AGMs. Mis11 refers to a non-targeted miRNA expressed under the synthetic EF-1α promoter.

[0045] Figure 21D shows an immunohistochemical (IHC) analysis of cytoplasmic α-synuclein (also referred to herein as α-Syn) intensity in tyrosine hydroxylase (TH)-positive dopaminergic (DA) neurons in the substantia nigra, indicating rAAV-miR5-mediated cytoplasmic α-SynThe dose-dependent effect of the protein decreased. Two AGMs were analyzed per group (n=2 / group), and each data point represents the analyzed DA neurons (2000-2500 total cells / group) from slices containing SNs. The X-axis plots group 1 (diluent), group 2 (mis11), group 3 (rAAV-miR5 applied at 3e8 vg per substantia nigra), group 4 (rAAV-miR5 applied at 3e9 vg per substantia nigra), and group 5 (rAAV-miR5 applied at 3e10 vg per substantia nigra). The Y-axis plots α-Syn intensity. Each data point represents the mean for each monkey, with two tissue perforation samples collected for analysis. Error bars represent SEM.

[0046] Figures 22A, 22B, and 22C provide graphical representations of the biodistribution analysis of genome copy number (CN) (reported as CN per microgram of genomic DNA) at various doses of 3e10 vg / SN (Figure 21A), 3e9 vg / SN (Figure 21B), and 3e8 vg / SN (Figure 21C). CN was determined using primer probes targeting the unique sequence of rabbit globulin PolyA. The X-axis depicts the basal ganglia and adjacent brain structures, while the Y-axis depicts CN / μg DNA. A dose-dependent increase in rAAV-miR5 was observed in the substantia nigra, and relatively low to moderate distribution was found in brain regions with adjacent anatomical connections to the basal ganglia structures, such as the hypothalamus and globus pallidus. Biodistribution analysis of other brain regions of the basal ganglia and cortical structures showed that rAAV-miR5 distribution was predominantly contained within the target regions. Two AGMs were included in each group (n = 2 / group), and each data point represents the mean per animal (2 punctures / SN).

[0047] Figure 22D depicts a profile of global SNCA mRNA knockdown generated by rAAV-miR5 in key brain regions. RT-qPCR analysis of SNCA mRNA showed a dose-dependent reduction in SNCA mRNA in the SN at 2 months post-necropsy compared to the control group (including diluent-treated animals and the miR11 control group to account for basal variability in endogenous SNCA levels) at a dose of 3e10 vg / SN. Consistent with biodistribution and miRNA expression analyses, the knockdown of target gene expression was contained in multiple parts of the SN and anatomically connected basal ganglia structures. Two AGMs were included in each group (n = 2 / group), and each data point represents the mean per puncture per animal. Specification 5 / 106 pages 11 CN 122374456 A Detailed Embodiments

[0048] The present invention is characterized by including a targetRNA polynucleotide constructs containing the sequence of α-synuclein mRNA and the sequence encoding nucleic acid. The RNA polynucleotide constructs include repressive RNA polynucleotides containing sequences targeting α-synuclein mRNA, such as pri-miRNA, pre-miRNA, shRNA, miRNA, and optionally modified miRNA.

[0049] In some embodiments, the repressive RNA polynucleotide is a repressive RNA duplex. The term "repressive RNA duplex" refers to a double-stranded RNA containing a guide strand and a passenger strand targeting a target region of RNA. The passenger strand and guide strand are sufficiently complementary to hybridize under physiological conditions. The guide strand associated with an RNA-induced silencing complex (RISC) can inhibit the activity of the target RNA. For example, in the case of an mRNA target, protein expression resulting from the target mRNA can be inhibited.

[0050] Repressive RNA duplexes such as miRNAs can be produced, for example, by gene expression producing pri-miRNA, pre-miRNA, or shRNA followed by biosynthesis; and by chemical synthesis.

[0051] The pri-miRNA scaffold can be embedded with guide and passenger sequences to generate pri-miRNA. The guide strand can be selected to target different types of RNA sequences or regions, such as 5' UTR, 3' UTR, or mRNA. For a given guide sequence, different passenger sequences can be embedded into the pri-miRNA scaffold. The passenger strand can be optimized for a specific scaffold and provide sufficient complementarity with the guide strand to hybridize under physiological conditions.

[0052] Constructs containing sequences targeting α-synuclein mRNA can be used, for example, to generate repressive RNA, wherein the repressive RNA can inhibit α-synuclein expression and / or treat synucleinopathy diseases or conditions. For example, inhibition of α-synuclein expression can be performed for therapeutic and research purposes. Research objectives include examining the effects of inhibiting α-synuclein expression in animal models.

[0053] The term "subject" refers to mammals, such as humans; non-human primates, such as apes, gibbons, gorillas, chimpanzees, orangutans, or macaques; domesticated animals, such as dogs and cats; farm animals, such as poultry, ducks, horses, cattle, goats, sheep, and pigs; and laboratory animals, such as mice, rats, rabbits, or guinea pigs. Humans are preferred as subjects.

[0054] In some embodiments, transgenic mice expressing human A53T mutant α-synuclein are used to evaluate repressive RNAs targeting α-synuclein mRNA. (See, for example, Menon et al., Brain Communications, Vol. 3, ...)Issue 4, 2021, fcab247, hypetext: / / doi.org / 10.1093 / braincomms / fcab247, which is hereby incorporated herein by reference in its entirety.)

[0055] References to “identical percentage,” “percentage of identity,” and similar terms refer to two sequences that have the greatest alignment in a particular region. The region provided refers to the indicated reference sequence. For example, a sequence that is “identical” or “identical” to the target sequence can be calculated by determining the number of identical nucleotides in the sequence aligned to provide the greatest identity and dividing by the total number of nucleotides in the target sequence and multiplying by 100. Differences between aligned sequences may include one or more deletions, substitutions, and / or additions.

[0056] References to indicated percentages of identity with one or more reference sequences or percentages of identical with one or more reference sequences, and similar language provided throughout the specification indicating percentages of identity with one or more reference sequences or percentages of identical with one or more reference sequences, provide indicated percentages or percentage ranges independently of each mentioned sequence. When determining identity or equal percentage, unless otherwise stated (e.g., the molecule is referred to as DNA or RNA), RNA and the corresponding DNA are considered to be identical. The corresponding RNA for DNA is provided with uracil instead of thymine and a ribose backbone instead of a deoxyribose backbone.

[0057] Unless otherwise stated, the terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to different forms of nucleic acids, oligonucleotides (including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). When discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing sequences in the 5' to 3' orientation. Specification 6 / 106 pages 12 CN 122374456 A

[0058] In some embodiments, nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Unless the context otherwise indicates, nucleic acids may be single-stranded, double-stranded, or triple-stranded, linear or circular, and may be of any length.

[0059] According to some embodiments, the polynucleotide is a single-stranded (ssDNA) or double-stranded DNA (dsDNA) molecule. According to some embodiments, the dsDNA molecule is a microsphere, nanoparticle, open linear double-stranded DNA, or closed-end linear double-stranded DNA (CELiD / ceDNA / doggybone DNA). According to some embodiments, the ssDNA molecule is a closed circular or open linear DNA.

[0060] “Transgenic” refers to a nucleic acid that is intended to be or has been introduced into a cell and is operatively linked to a promoter. TransgenicBecause it includes nucleic acids encoding heterologous polynucleotide sequences and / or heterologous promoters.

[0061] In some embodiments, the polynucleotide construct encoding a polynucleotide is “CpG-reduced” or “CpG-depleted”. In some embodiments, the “CpG-reduced” or “CpG-depleted” region is a region outside the pri-miRNA or pre-miRNA sequence. “CpG-reduced” or “CpG-depleted” means (i) a nucleotide sequence in which one or more of the CpG dinucleotides (or motifs) have been removed from a reference nucleic acid sequence; and / or (ii) the percentage of CpG in the polynucleotide is 0% to 10%. In various embodiments, the CpG percentage is 0-5%, 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0062] In some embodiments, CpG motifs are reduced in the 5' and / or 3' untranslated region (UTR), filler sequence, promoter, enhancer, polyadenylation signal, 5' and / or 3' ITR and / or intron.

[0063] Unless otherwise expressly stated, the singular forms “an,” “a,” and “the” include a plural referent.

[0064] The connecting term “and / or” between a plurality of enumerated elements includes both single options and combined options. For example, in the case where two elements are connected by “and / or,” the first option refers to the applicability of the first option without the second option, the second option refers to the applicability of the second option without the first option, and the third option refers to the applicability of the first and second options together. Any of these options is understood to fall within the scope of this meaning and thus satisfies the requirement of the term “and / or.” The parallel applicability of more than one option is also understood to fall within the meaning of the term “and / or.”

[0065] Unless the context otherwise requires, the terms “or” and “and” as used have the same meaning as “and / or” when applied to different alternative members of the list.

[0066] References to terms such as “including,” “for example,” “e.g.,” “like,” and “such as” followed by different members or instances are open-ended descriptions, where the listed members or instances are illustrative and other members or instances may be provided or used.

[0067] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably to refer to amino acid sequences without regard to function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acids. The provided amino acids include naturally occurring amino acids and amino acids provided through cellular modifications.

[0068] The terms “comprise” and “comprises” as used with respect to an element or group of elements are also used.References to variations of “comprising” are open-ended and do not exclude other unlisted elements or method steps. Terms such as “comprising,” “containing,” and “characterized in” are synonyms for “comprising.” In the various aspects and embodiments described herein, references to open-ended terms such as “comprising” may be replaced by “consisting of” or “substantially consisting of.”

[0069] The reference to “consisting of” excludes any element, step, or component not specified in the listed claim elements that is relevant to the claimed invention.

[0070] The reference to the term “substantially consisting of” limits the scope of the claim to specific materials or steps that do not substantially affect the basic and novel features of the claimed invention.

[0071] The term “about” refers to a value within 10% of the base parameter (i.e., plus or minus 10%). For example, “about 1:10” includes 1.1:10.1 or 0.9:9.9 on page 7 / 106 of the specification, CN 122374456 A, and “about 5 hours” includes 4.5 hours or 5.5 hours. The term “about” at the beginning of a series of values ​​modifies each of these values ​​by 10%. In some embodiments, the term “about” refers to a value within 10% of the base parameter.

[0072] Unless the context clearly indicates otherwise, all numerical values ​​or ranges include integers within such ranges and values ​​or fractions of integers within such ranges. Therefore, for demonstration purposes, mentioning a reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, and 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., as well as 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc., and mentions numerical ranges, such as "1-4" including 1, 2, 3, 4, and 1.1, 1.2, 1.3, 1.4, etc. As a further demonstration, "1 to 4 weeks" includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0073] Furthermore, the reference to numerical ranges, such as "0.01 to 10," includes 0.011, 0.012, 0.013, etc., and 9.5, 9.6, 9.7, 9.8, 9.9, etc. For example, a dose of approximately "0.01 mg / kg to approximately 10 mg / kg" of the subject's body weight includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., and 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, etc.mg / kg, 9.9 mg / kg, etc.

[0074] References having an integer greater than (greater than) or less than include numbers greater than or less than the reference number, respectively. Thus, for example, references to more than 2 include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more; and application “twice or more times” includes 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times or more.

[0075] Various references, including articles and patent publications, are cited or described in the background and throughout the specification. Each of these references is incorporated herein by reference in its entirety. No reference is considered prior art for any invention disclosed or claimed. In some cases, a particular reference is indicated to be incorporated herein by reference for prominent inclusion.

[0076] The definitions provided herein, including those in this section, and other sections of this application, apply throughout this application.

[0077] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0078] This description has been divided into different sections and paragraphs, and examples of various embodiments are provided. These separations should not be construed as separating the substance of a paragraph or section or embodiment from the substance of other paragraphs or sections or embodiments. The description provided has a wide application and includes all combinations of various sections, paragraphs, and sentences that can be contemplated. The discussion of any embodiment is intended to be exemplary only and is not intended to imply that the scope of this disclosure (including the claims (unless otherwise specified in the claims)) is limited to these examples.

[0079] This invention is generally disclosed using affirmative language to describe numerous embodiments of the invention. The invention also specifically includes embodiments in which particular subject matter such as substances or materials, method steps and conditions, schemes, or procedures are all or partly excluded. For example, in some embodiments of the invention, materials and / or method steps are excluded. Therefore, even though the invention is not generally described herein with respect to anything not included in the invention, embodiments not expressly excluded in the invention are disclosed herein.

[0080] I. RNA Polynucleotides Targeting α-Synuclein mRNA

[0081] Different α-synuclein mRNA target regions are provided by, for example, the inverse complementary sequences of any of SEQ ID NO: 129-135 and any of SEQ ID NO: 32-62. RNA polynucleotides targeting specific target regions contain sequences substantially complementary to the target region. The substantially complementary sequence contains a region of at least 10 nucleotides, which are at least 70% complementary, at least 80% complementary, at least 90% complementary, or 100% complementary.Complementarity. The degree of complementarity is determined based on alignment to the maximum complementarity sequence as specified in the specification, page 8 / 106, CN 122374456 A. Differences between complementary sequences include additions, deletions, and non-complementary bases. Preferably, the basic complementary sequences can hybridize with each other under physiological conditions.

[0082] In some embodiments, the basic complementary sequence comprises a region of 10-25 nucleotides, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides that are fully complementary, at least 70%, at least 80%, or at least 90% complementary when aligned to maximum complementarity.

[0083] In some embodiments, the basic complementary sequence comprises a region of 10-25 nucleotides, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides that are fully complementary (except for differences of 1, 2, or 3 nucleotides) when aligned to maximum complementarity.

[0084] In some embodiments, the construct containing the α-synuclein mRNA targeting sequence is a repressive RNA duplex, pri-miRNA, pre-miRNA, or shRNA. The α-synuclein mRNA targeting sequence present in these constructs provides a guide strand sequence capable of hybridizing with α-synuclein mRNA. Such constructs also include a passenger sequence that is substantially complementary to the guide sequence. For example, the degree of complementarity between the guide and passenger sequences can vary to allow for sufficient complementarity for hybridization and use in a particular scaffold. In some embodiments, when the alignment reaches maximum complementarity, the leader and passenger strands are perfectly complementary, or have 1, 2, 3, or 4 nucleotide mismatches relative to perfect complementarity. Each mismatch can independently be a non-complementary base, an addition, and / or a deletion.

[0085] In some embodiments, the RNA polynucleotide comprises a targeting sequence comprising: (1) a region that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the RNA sequence of any of SEQ ID NO: 32-62; (2) a sequence that differs from any of SEQ ID NO: 32-62 by 0, 1, 2, or 3 nucleotides; or (4) a region that is identical to the RNA sequence of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 9 ...The difference between any of SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 100 lies in the sequence of 0, 1, 2, or 3 nucleotides. In some embodiments, the nucleotide difference is substitution.

[0086] Different combinations of guide sequences and seed sequences are provided in Table 1.

[0087] Table 1

[0088]

[0089] In some embodiments, the target RNA sequence comprises:

[0090] a) a sequence differing from SEQ ID NO: 34 in that it consists of 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 108; Specification 9 / 106 pages 15 CN 122374456 A

[0091] b) a sequence differing from SEQ ID NO: 36 in that it consists of 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 109;

[0092] c) a sequence differing from SEQ ID NO: 37 in that it consists of 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 110;

[0093] d) a sequence differing from SEQ ID NO: 38 in that it consists of 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises SEQ ID NO: 111

[0094] e) differs from SEQ ID NO: 39 in that it is a sequence of 0, 1, 2 or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 112;

[0095] f) differs from SEQ ID NO: 41 in that it is a sequence of 0, 1, 2 or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 113; and

[0096] g) differs from SEQ ID NO: 42 in that it is a sequence of 0, 1, 2 or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 114. In some embodiments, for (a)-(g), any nucleotide difference (if present) is a substitution.

[0097] In some embodiments, the guide sequence comprises, consists of or is substantially composed of the target sequence, and the RNA polynucleotide further comprises a passenger sequence substantially complementary to the guide.

[0098] In some embodiments, the passenger sequence and guide sequence are provided in the combination shown in Table 2, wherein the guide strand comprisesThe following, substantially composed of, or composed of: the indicated target sequence, and the passenger sequence comprises, substantially composed of, or composed of: the indicated sequence or a sequence differing from the indicated sequence by 1, 2, or 3 nucleotide substitutions.

[0099] Table 2

[0100]

[0101] In some embodiments, the combinations of passenger and guide strands provided in Table 2 are present in pri-miRNA, pre-miRNA, shRNA, or repressive RNA duplexes.

[0102] 1A Repressive RNA Duplexes

[0103] Repressive RNA duplexes can be combined with RNA-induced silencing complexes (RISC) to inhibit mRNA activity. References to RISC include associated proteins, such as core argonaute (AGO) proteins. Without being limited by any particular theory, the guide strand is loaded onto the RISC and hybridized with the target mRNA, wherein hybridization is initiated in a seed region located from n-2 to approximately n-8 at the 5' end of the guide strand. Specification 10 / 106 pages 16 CN 122374456 A

[0104] In some embodiments, the seed region is either completely complementary to its mRNA target or differs from complete complementarity by one nucleotide.

[0105] Preferably, the passenger strand is discarded and degraded by RISC. In different embodiments, the ratio of the guide strand to the passenger strand is approximately 25:1, approximately 15:1, or approximately 10:1.

[0106] Depending on the degree of complementarity and the location of the non-complementary regions, RISC associated with the guide strand can cause mRNA cleavage or translational repression. (See, for example, Lam et al., Mol. Ther. Nucleic Acids. (2015) 4(9):e252 and Kobayashi et al., ACS Omega. (2022) 7(2):2398-2410, each of which is hereby incorporated herein by reference in its entirety.)

[0107] In some embodiments, the length of the guide strand and the passenger strand is independently 18 to 25 nucleotides and each contains a 3' overhang of 1 to 5 nucleotides. In further embodiments, each overhang is 1, 2, 3 or 4 nucleotides. In further embodiments, each guide strand and passenger strand is independently 20, 21, 22 or 23 nucleotides; and each overhang is 2 or 3 nucleotides. In further embodiments, each guide strand and passenger strand is 21, 22 or 23 nucleotides and each overhang is 2 nucleotides.

[0108] IB miRNA

[0109] Using such asPrecursor constructs of pri-miRNA, pre-miRNA, and shRNA facilitate the delivery of repressive RNA duplexes to subjects. These different constructs contain stem-loop structures, guide strand sequences, and passenger strand sequences; and utilize intracellular miRNA biogenesis mechanisms to form repressive RNA duplexes. For example, such constructs can be provided using transgenes encoding repressive RNA.

[0110] The generation and processing of naturally occurring miRNAs involves pri-miRNA transcription from the miRNA gene and cleavage of the 3' and 5' regions mediated by drosha-DGCR8, thereby forming pre-miRNA. The resulting pre-miRNA contains a stem with nucleotide overhangs at the 3' end and a apical stem-loop. (See, for example, Jin et al., Mol. Cell. May 7, 2020;78(3):423-433, which is hereby incorporated herein by reference in its entirety.)

[0111] The pri-miRNA comprises a 5' flanking sequence, a guide / passenger sequence, a apical loop, a guide / passenger sequence, and a 3' flanking sequence. The guide strand sequence may be either 5' or 3' of the loop. When the guide strand sequence is 5' of the loop, the passenger strand sequence is 3' of the apical loop; and when the guide strand sequence is 3' of the loop, the passenger strand sequence is 5' of the loop.

[0112] The 5' and 3' flanking regions form part of the stem region and further comprise a single-stranded region. The length of the flanking regions may vary independently. In some embodiments, the 5' and 3' pri-miRNA flanking regions are each at least 20 nucleotides, at least 50 nucleotides, or at least 100 nucleotides.

[0113] A pri-miRNA scaffold refers to a pri-miRNA region that does not include the guide and passenger sequences. Scaffolds can be used as delivery mediators for the biogenesis of different repressive RNA duplexes, wherein the guide and passenger sequences are embedded in the scaffold.

[0114] Scaffolds capable of incorporating guide and passenger sequences can be based on naturally occurring pri-miRNAs, modifications of naturally occurring miRNAs, or artificially designed with consideration of pri-miRNA characteristics. (See, for example, Xie et al., Mol. Ther. (2020) 28(2):422-430; Fowler et al., Nucleic Acids Research (2016), 44(5): e48; Roden et al., (2017) Genome Res. 27(3):374-384; Jin et al., (2020) Mol. Cell. 7;78(3): 423-433; and Fang...)

[0115] The design considerations for pri-miRNA scaffolds include stem length, loop size, and the presence of specific motifs that may enhance biogenesis and / or repressive RNA. Pri-miRNA stems may contain various structures such as G-U or U-G kinetics, single-base mismatches, protrusions, and multi-base mismatches.

[0116] In some embodiments, the pri-miRNA scaffold contains a UG motif in the 5' arm at position -14 or -13 relative to the drosha 5' cleavage site; and / or a CNNC in the 3' arm at position +14 to +18 relative to the drosha 3' cleavage site.

[0117] In some embodiments, the pri-miRNA scaffold is a miR-1, miR-26, miR16-1, miR-30, miR-33, miR101, miR-64, miR-122, miR-125, miR-135, miR-155, enhanced miR-155 (eSIBR), or miR-451 scaffold. (US Patent No. 10,457,940; Miniarova et al., Mol. Ther. Nucleic Acids (2016) March 22; 5(3):e297; Fowler et al., Nucleic Acids Research (2016), 44(5):e48; Xie et al., Molecular Therapy (2020) 28:2 422-430; Fang and Bartel Molecular Cell (2015) 60, 131-145; Roden et al. (2017) Genome Res. 27, 374–384; and Calloni and Bonatto, Human Gene Therapy Methods (2015) 25(5):162-174.)

[0118] In some embodiments, the pri-miRNA contains a mismatched GHG motif in the 3' arm of the stem.

[0119] In some embodiments, the pri-miRNA stem length is 33, 34, 35, 36, 37, 38, or 39 nucleotides.

[0120] In some embodiments, the pri-miRNA stem length is...34, 35, or 36 nucleotides.

[0121] In some embodiments, the pri-miRNA apical loop is 3 to 23 nucleotides. In further embodiments, the loop is 10 to 23 nucleotides. In some embodiments, the guide strand and passenger strand do not extend into the loop. In some embodiments, the guide and / or passenger strands extend into the loop.

[0122] In some embodiments, the pri-mRNA contains a 5', 7-methylguanylic acid (m7G) cap.

[0123] In some embodiments, the pri-miRNA contains, consists of, or is substantially composed of the sequence of any of SEQ ID NO: 115-121; or a sequence differing from any of SEQ ID NO: 115-121 by 0, 1, 2, 3, or 4 nucleotides. Nucleotide differences can be introduced at different positions, taking into account the guide provided herein, for example, different pri-miRNA scaffold characteristics, guide-target complementarity, passenger-guide strand complementarity, and adjustments to a particular scaffold.

[0124] The pre-miRNA produced by drosha cleavage of the pre-miRNA is exported from the nucleus to the cytoplasm, where it is cleaved by a dicer to produce a repressive RNA duplex. The term "pre-miRNA" refers to an RNA polynucleotide, such as those containing the guide strand and passenger strand described herein, which can be cleaved by a dicer to produce a repressive RNA duplex.

[0125] In some embodiments, the repressive RNA duplex produced by the dicer comprises a guide strand and a passenger strand, each approximately 22 nucleotides in length, wherein each strand contains a 2-base nucleotide overhang at each 3' end. (Fang and Bartel (2015) Molecular Cell, 60:131-145.)

[0126] In some embodiments, the pre-miRNA comprises, consists of, or is substantially composed of: any of the sequences in SEQ ID NO: 122-128; or a sequence differing from any of the sequences in SEQ ID NO: 122-128 by 0, 1, 2, 3, or 4 nucleotides. Nucleotide differences, such as guide sequences, loop sequences, and / or passenger sequences, may be introduced at different positions.

[0127] In some embodiments, the pre-miRNA is introduced into cells using a transgene containing a sequence encoding the pre-miRNA sequence. Biogenesis of the pre-miRNA causes the production of a repressive RNA duplex. The expression cassette containing the transgene may be introduced into cells using a viral or non-viral delivery medium.

[0128] In some embodiments, a transgene containing a sequence encoding the pre-miRNA sequence is introduced into cells using a transgene containing a sequence encoding the pre-miRNA sequence.Transgenic shRNA sequences introduce shRNA into cells. shRNA is structurally and functionally similar to pre-miRNA. shRNA contains a double-stranded RNA containing substantially complementary arms and loops, wherein the shRNA can be exported from the nucleus to the cytoplasm and cleaved by dicer to form a repressive RNA double-stranded RNA. (Aguiar et al., Transl. Neurodegener. (2017) Nov 27; 6:30.)

[0129] IC scaffold

[0130] In some embodiments, the pri-miRNA scaffold is an S155, S155e, S26, S126, or S33 scaffold. The S155 scaffold corresponds to the miR155 scaffold and is used for miR3, miR5, miR6, miR7, miR8, miR10, and miR11 as described in the examples below. The S155e stent corresponds to the enhanced miR155 stent. (See Fowler et al., Nucleic Acids Research (2016), 44(5):e48). The S33 stent corresponds to the stent present in miR33. (See Xie et al., Molecular Therapy (2020) 28:2 422-430 2020.) The S126 stent is an artificial stent produced based on the miR26 stent. Different stents can be used to incorporate different guide chains and passenger chains.

[0131] In some embodiments, the S126 scaffold can be used to incorporate different guide chains and passenger chains, wherein the scaffold comprises SEQ ID NO: 186

[0132] NO1 NO2 NO3 NO4 NO5 NO6 NO7 NO8 NO9 N10 N11 N12 N13 N14 N15 N16 N17 N18 N19 N20 N21 UGUGCAGGUCCCAN22 N23 N24 N25 N26 N27 N28 N29 N30 N31 N32 N33 N34 N35 N36 N37 N38 N39 N40 N41 N42 CG, wherein NO1 to N42 are ribonucleotides, NO1 is complementary to N42, NO2 is not complementary to N41, NO3-N10 are complementary to N33-N40, and N11 is not complementary to N32. The DNA is complementary, and N12-N21 is complementary to N22-N31. In some embodiments, the passenger strand is provided by N01-N21U, wherein the target sequence can be inserted into N01-N21; and the guide strand is provided by N23-N42G. The corresponding DNA can be used to encode RNA sequences.

[0133] In some embodiments, the RNA sequence containing SEQ ID NO: 186 has the following structure:

[0134]

[0135] In different embodiments, the RNA sequence containing SEQ ID NO: 186 may be cleaved by Dicer; or may be cleaved by Drosha and Dicer.

[0136] In some embodiments for an RNA sequence comprising SEQ ID NO: 186, the RNA sequence further comprises a 5' flanking region and a 3' flanking region, wherein the polynucleotide comprises the RNA sequence of SEQ ID NO: 187:

[0137] GUGGCCGN01N02N03N04N05N06N07N08N09N10N11N12N13N14N15N16N17N18N19N20N21UGUGCAGGUCCC AN22N23N24N25N26N27N28N29N30N31N32N33N34N35N36N37N38N39N40N41N42CGGGGACGC, wherein N01 to N42 are ribonucleotides, N01 and N42 are complementary, and N02 Not complementary to N41, N03-N10 are complementary to N33-N40, N11 is not complementary to N32, and N12-N21 are complementary to N22-N31. The corresponding DNA can be used to encode RNA sequences.

[0138] In some embodiments, the repressive RNA polynucleotide targeting α-synuclein is embedded in an S126 scaffold containing SEQ ID NO: 186 or 187, wherein (1) the guide strand contains the sequence of SEQ ID NO: 95 and the passenger strand contains a sequence selected from the group consisting of SEQ ID NO: 189-197; or (2) the guide strand contains the sequence of SEQ ID NO: 97 and the passenger strand contains a sequence selected from the group consisting of SEQ ID NO: 198-206. Tables 3 and 4 show passenger combinations for the guide strands of SEQ ID NO: 95 and SEQ ID NO: 97. The nucleotides shown in bold are located in protrusions formed by non-complementary nucleotides.

[0139] Table 3 Specification 13 / 106 pages 19 CN 122374456 A

[0140]

[0141] Table 4

[0142]

[0143] In some embodiments, the passenger strand and guide strand combinations provided in Tables 3 and 4 are present in pri-miRNA, pre-miRNA, shRNA, or repressive RNA duplexes. The S126 scaffold contains the sequence combinations of Table 3.Examples of pre-miRNAs are provided by SEQ ID NO: 207-215, and examples of pre-miRNAs containing the sequence combinations of Table 4 in the S126 scaffold are provided by SEQ ID NO: 216-224. Longer examples of pre-miRNAs containing the sequence combinations of Table 3 in the S126 scaffold are provided by the RNA version (ribosomal backbone, with U replacing T) of SEQ ID NO: 243-251. Longer examples of pre-miRNAs containing the sequence combinations of Table 4 in the S126 scaffold are provided by the RNA version (ribosomal backbone, with U replacing T) of SEQ ID NO: 252-260.

[0144] ID Modified Repressive RNA

[0145] An alternative to providing repressive RNA duplexes via transgenic expression and biogenesis is direct administration of repressive RNA duplexes. (Instructions for Use 14 / 106 pages, 20 CN 122374456 A) Modified RNA can be used to facilitate direct administration. Modified repressive RNA duplexes can be generated, for example, by modifying one or more nucleotides of the repressive RNA duplex. The repressive RNA duplex can be modified to, for example, improve pharmacokinetics, enhance activity, inhibit innate immune activation, improve targeting, and reduce off-target toxicity.

[0146] The repressive RNA can be modified at various locations such as the 5' end, 3' end, sugar moiety, phosphate group, and nucleobase. Various modifications can be made to allow or facilitate Watson-Crick hybridization to target and inhibit RNA activity. References describing different modifications and modification patterns include Hu et al., Sig Transduct Target Ther., (2020) 5, 101; Varley and Desauniers (2021) RSC Adv., 11, 2415; Alterman et al., Nature Biotechnology (2019) 37:884-894; International Patent Publication No. WO 2021 / 252649; and US Patent Publication No. 2022 / 125823; each of these references is hereby incorporated herein by reference in its entirety.

[0147] In some embodiments, the repressive RNA comprises one or more modifications selected from: 5' phosphate mimics (e.g., 5-(E)-vinyl phosphate, 5'-methylene phosphate, 5'-(R)-methyl phosphate, 5'-(S)-methyl phosphate, 5'(R)-MeOCH3Phosphate esters, 5'-(S)-methyl-F phosphate esters, 5-deoxy-5'-morpholino-2'O-methyluridine and thiophosphate esters; internal phosphate modifications (e.g., Rp thiophosphate esters, Sp thiophosphate esters, dithiophosphate esters, methoxyphosphate esters, phenethyl phosphate esters, 2'-5' phosphate bonds and amide bonds); one or more nucleobase modifications (e.g., 5'-nitroindole, pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2'-deoxyuridine, N-ethylpiperidine 7'-EAA triazole-modified adenine, N-ethylpiperidine 6'-triazole-modified adenine, 6'-phenylpyrrolocytosine or 2',4'-difluorotoluyl ribonucleotides); sugar modifications (e.g., 2'-O-methyl, 2'-O-methoxyethyl, 2'-deoxy- 2'-Fluoro, 2'-O-(p-oxazol-2-yl)benzyl, (2'S)-2-deoxy-2'-C-methyl, (2'R)-2-deoxy-2'-C-methyl, 2'-arose-fluorine, 2'-O-benzyl, 2'-O-methyl-4-pyridine, locked nucleic acid, (s)-cET-BNA, tricyclic DNA, PMO, unlocked nucleic acid, glycol nucleic acid, atroitol and nucleic acid; and conjugation groups (e.g., lipophilic groups, hydrophilic groups, cholesterol, GalNAc, docosahexaenoic acid or docosahexaenoic acid having a phosphocholine head group).

[0148] In some embodiments, at least 50%, at least 60%, at least 70%, at least 90% or 100% of the nucleotides are modified.

[0149] In some embodiments, the repressive RNA duplex is a modified divalent repressive RNA duplex. In a further embodiment, the modified divalent repressive RNA duplex is completely chemically stable and comprises one or more of 2'-OMe, 2'-F, thiophosphate, phosphodiester, and 5'-vinylphosphonate. (See, for example, Alterman et al., Nature Biotechnology (2019) 37:884-894, which is hereby incorporated herein by reference in its entirety.)

[0150] The modified RNA can be produced using various techniques, such as stepwise synthesis of RNA and / or modified RNA sequences. The resulting RNA sequence itself can be prepared, for example, by stepwise synthesis or using a nucleic acid encoding a nucleic acid.

[0151] II. Nucleic Acid Encoding an RNA Polynucleotide Containing a Target Sequence

[0152] An RNA polynucleotide containing a target sequence can be produced from a polynucleotide using a nucleic acid sequence encoding an RNA polynucleotide. With respect to the RNA polynucleotide, the nucleic acid encoding sequence provides the same sequence as the corresponding RNA polynucleotide, wherein if the encoding nucleic acid is DNA, then the DNAThe nucleotides will have thymine instead of uracil and deoxyribose instead of ribose. The RNA polynucleotide is generated from a template strand complementary to the coding strand. For example, the coding strand may be provided with and / or used to generate the template strand.

[0153] The polynucleotide containing the nucleic acid sequence encoding the RNA polynucleotide may contain additional components, such as those that promote the generation of the RNA polynucleotide, promote the delivery of the polynucleotide as a viral vector, and / or provide additional activity. For example, additional activity may be provided by encoding a protein and / or encoding a sequence that provides one or more additional repressive RNAs.

[0154] The sequence providing additional repressive RNA may be the same or different and may be directed to the same or different targets. Examples of different conformations include two or more pri-miRNAs encoding the same sequence; two or more pri-mRNAs encoding the same guide strand but different scaffolds; and / or two or more pri-miRNAs encoding different guide strands. Different guide chains can lead to the same target, such as the same mRNA; or different targets, such as different mRNAs. In some embodiments, the polynucleotide encodes 1, 2, 3, 4, or 5 pri-miRNAs, each of which can be the same or different.

[0155] The same type of coding conformation providing pri-mRNA can be used for the polynucleotide encoding shRNA. In some embodiments, the polynucleotide encodes 1, 2, 3, 4, or 5 shRNAs, each of which can be the same or different.

[0156] An expression cassette is used to facilitate the production of RNA polynucleotides containing a target sequence. The expression cassette contains a nucleic acid sequence encoding an RNA polynucleotide and one or more expression control elements operatively linked to the nucleic acid sequence encoding the RNA polynucleotide. The expression cassette can include, for example, the same or different nucleic acid sequences encoding different RNA polynucleotides (e.g., different pri-miRNAs or shRNAs), wherein the different polynucleotides can be operatively linked to the same or different expression elements. For example, the same promoter can be linked to a nucleic acid sequence providing multiple pri-miRNAs, or two or more pri-miRNAs can be operatively linked to different promoters.

[0157] In some embodiments, the polynucleotide (1) encoding the RNA polynucleotide comprises a sequence of at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, or at least 82% of any of the sequences in SEQ ID NO: 136-142.(1) 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical sequences; (2) containing at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any of SEQ ID NO: 136-142; and / or (3) containing sequences that differ from SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142 by 0, 1, 2, or 3 nucleotides.

[0158] Different combinations of target sequences and seed DNA are provided in Table 5.

[0159] Table 5

[0160]

[0161] In some embodiments, the DNA encoding the target sequence comprises:

[0162] a) a sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 136, wherein the target sequence comprises the sequence of SEQ ID NO: 143; Specification 16 / 106 pages 22 CN 122374456 A

[0163] b) a sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 137, wherein the target sequence comprises the sequence of SEQ ID NO: 144;

[0164] c) a sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 138, wherein the target sequence comprises the sequence of SEQ ID NO: 145;

[0165] d) a sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 139, wherein the target sequence comprises SEQ ID NO: 145; The sequence of SEQ ID NO: 146;

[0166] e) differs from SEQ ID NO: 140 in that it is a sequence of 0, 1, 2, or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 147;

[0167] f) differs from SEQ ID NO: 141 in that it is a sequence of 0, 1, 2, or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 148; and

[0168] g) differs from SEQ ID NO: 142 in that it is a sequence of 0, 1, 2, or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 149. In some embodiments, for(a)–(g), any nucleotide difference (if present) is a substitution.

[0169] In some embodiments, the target sequence is a guide strand sequence, and the polynucleotide further comprises a sequence encoding a passenger sequence substantially complementary to the target sequence.

[0170] In some embodiments, the passenger-encoding sequence and guide sequence are provided in the combination shown in Tables 6–8, wherein the guide strand sequence comprises, substantially consists of, or consists of the indicated sequence; and the passenger-encoding sequence comprises, substantially consists of, or consists of the indicated sequence or a sequence differing from the indicated sequence by 1, 2, or 3 nucleotides.

[0171] Table 6

[0172]

[0173] Table 7 Specification 17 / 106 pages 23 CN 122374456 A

[0174]

[0175] Table 8

[0176]

[0177] In some embodiments, the combination encoding the passenger strand and the guide strand is provided as in Table 6, 7 or 8, and the polynucleotide encodes pri-miRNA, pre-miRNA or shRNA.

[0178] In some embodiments, the polynucleotide encoding the RNA polynucleotide (1) comprises at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of any of SEQ ID NO: 157-163; (2) comprises at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any of SEQ ID NO: 157-163; and / or (3) comprises the same sequence as SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, or SEQ ID NO: 163. The difference lies in the sequence of 0, 1, 2 or 3 nucleotides.

[0179] In some embodiments, the polynucleotide comprises or encodes (1) at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least...(1) 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical sequence; (2) at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any one of SEQ ID NO: 225-260; and / or (2) comprising the same sequence as SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240 ... The difference between SEQ ID NO: 241 and any of SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259 and SEQ ID NO: 260 lies in the sequence of 0, 1, 2 or 3 nucleotides.

[0180] In some embodiments, the polynucleotide comprises a sequence encoding a pre-miRNA or pri-miRNA, wherein the polynucleotide comprises the guide / passenger combination provided in Table 6, and (1) comprises a sequence that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of any of SEQ ID NO: 157-163; and / or (2)Contains sequences that differ from SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, or SEQ ID NO: 163 in that they are 0, 1, 2, or 3 nucleotide sequences; wherein the sequences (including percentage identity or difference) are based on combinations of DNA encoding guide / passenger / pre-miRNAs: SEQ ID NO: 136 / 150 / 157; SEQ ID NO: 137 / 151 / 158; SEQ ID NO: 138 / 152 / 159; SEQ ID NO: 139 / 153 / 160; SEQ ID NO: 140 / 154 / 161; SEQ ID NO: 141 / 155 / 162; and SEQ ID NO: 142 / 156 / 163.

[0181] In some embodiments, the polynucleotide comprises a sequence encoding a pre-miRNA or pri-miRNA, wherein the polynucleotide comprises the guide / passenger combination provided in Table 7, and (1) comprises at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of any of SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, and SEQ ID NO: 233. The difference between any of them lies in the sequence of 0, 1, 2, or 3 nucleotides; wherein the sequence (including percentage identity or difference) is based on a combination of DNA encoding the guide / passenger / pre-miRNA: SEQ ID NO: 137 / 261 / 225; SEQ ID NO: 137 / 262 / 226; SEQ ID NO: 137 / 263 / 227; SEQ ID NO: 137 / 264 / 228; SEQ ID NO: 137 / 265 / 229; SEQ ID NO: 137 / 266 / 230; SEQ ID NO:137 / 267 / 231; SEQ ID NO: 137 / 268 / 232; and SEQ ID NO: 137 / 269 / 233.

[0182] In some embodiments, the polynucleotide encodes a pre-miRNA or pri-miRNA polynucleotide, wherein the polynucleotide comprises the guide / passenger combination provided in Table 8, and (1) comprises a sequence that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NO: 234-242; and / or (2) comprises a sequence that is identical to SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239 ...9, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 239, SEQ ID The difference between any of SEQ ID NO: 240, SEQ ID NO: 241, and SEQ ID NO: 242 lies in a sequence of 0, 1, 2, or 3 nucleotides; wherein the sequence (including percentage identity or difference) is based on a combination of DNA encoding a guide / passenger / pre-miRNA from the following: SEQ ID NO: 139 / 270 / 234; SEQ ID NO: 139 / 271 / 235; SEQ ID NO: 139 / 272 / 236; SEQ ID NO: 139 / 273 / 237; SEQ ID NO: 139 / 274 / 238; SEQ ID NO: 139 / 275 / 239; SEQ ID NO: 139 / 276 / 240; SEQ ID NO: 139 / 277 / 241; and SEQ ID NO: 139 / 278 / 242.

[0183] In some embodiments, the polynucleotide comprises a sequence encoding a repressive RNA that targets α-synuclein mRNA, and further comprises a nucleic acid sequence encoding glial cell-derived neurotrophic factor (GDNF). References to GDNF include full-length or mature GDNF and its variants. GDNF is a potent neurotrophic factor for dopamine neurons. The amino acid sequence of full-length GDNF is provided by SEQ ID NO:188, wherein amino acids 1-19 provide the signal sequence. (See, for example, hypertext)Transfer protocol / / www.uniprot.org / uniprotkb / P39905 / entry and hypertext transfer protocol: / / www.ncbi.nlm.nih.gov / protein / NP_000505.1). In some embodiments, the GDNF comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with amino acids 20-211 of SEQ ID NO: 188 (full length) or SEQ ID NO: 188 (mature). In further embodiments, the mature GDNF further comprises a heterologous signal sequence. For example, examples of different signal sequences are provided in International Patent Publications WO2022155665 and WO2022165027, both of which are incorporated herein by reference in their entirety.

[0184] II.A. Expression cassette element

[0185] The expression cassette comprises a nucleic acid sequence encoding a polynucleotide operatively linked to an expression control element. The expression cassette described herein comprises a nucleotide sequence of an RNA polynucleotide encoding a target α-synuclein mRNA and one or more regulatory sequences, and may also comprise additional sequences such as filler sequences and additional coding sequences such as additional RNA polynucleotides and / or polypeptide sequences.

[0186] An “expression control element” affects the expression of a sequence operatively linked to it. Protein expression control elements can affect, for example, transcription, translation, splicing, and information stability. Expression control elements affecting RNA polynucleotide production can affect, for example, transcriptional levels.

[0187] Expression control elements are typically located at the 5' (“upstream”) or 3' (“downstream”) of the transcribed nucleic acid. Expression control elements may also be located within the transcript encoding the protein (e.g., in an intron). Expression control elements may be located adjacent to or at a distance from the transcribed sequence. One or more expression control elements may be present. Examples of expression control elements include promoters, enhancers, introns, polyadenylation signals, Kozak sequences, post-transcriptional regulatory elements, and termination sequences.

[0188] A promoter is a DNA region that initiates transcription. Generally, the transcribed nucleic acid is located at the 3' end of the promoter sequence. In some embodiments, the promoter sequence is linked to an enhancer. An enhancer is a DNA region that increases transcription by the promoter. An enhancer may be adjacent to the promoter or may be located distal to the promoter. Typically, an enhancer is located upstream of the promoter, but it may be located downstream of the promoter sequence or within the promoter sequence.

[0189] Expression control elements, such as promoters and enhancers, can be selected to preferentially drive expression in specific cell or tissue types. Expression control elements are typically active in specific cells, tissues, or organs because they are recognized by transcription activators or other transcriptional regulators, thereby providing activity in the cell, tissue, or organ type. (See, for example, Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th ed., Vol. II, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al., (2010) Current protocols in molecular biology, John Wiley & Sons, New York.)

[0190] Incorporation of tissue-specific regulatory elements into expression constructs provides at least partial tissue tropism for the expression of encoded RNA polynucleotides. References to promoters or enhancers that are specific to a particular cell type or tissue indicate that the promoter or enhancer provides higher levels of expression and / or secretion in the indicated cell or tissue type. Examples of CNS-specific promoters include: neuron-specific promoters such as NSE (neuron-specific enolase), synaptic protein or NeuN, platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), methyl-CpG binding protein 2 (MeCP2), Ca2 / calmodulin-dependent protein kinase II (CaMKII), metabolite glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), β-globin microgene nβ2, proenkephalin (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters; astrocyte-specific promoters such as glial fibrillary acidic protein (GFAP) and EAAT2 promoters; oligodendrocyte-specific promoters such as myelin basic protein (MBP) / myelin-associated glycoprotein and oligodendrocyte transcription factor 2 promoters; and neuron / hypothalamic-specific promoters such as promelanocyte-stimulating hormone (POMC) promoters. (See, for example, U.S. Patent Publication No. 2021 / 214749 and Adeno-Associated Virus Vectors (2019), Castle, ed., 1st edition, Springer, New York.)NY.; Both are hereby incorporated herein by reference in their entirety.)

[0191] Expression control elements also include ubiquitous or mixed promoters and promoter / enhancers capable of driving polynucleotide expression in many different cell types. Such elements include cytomegalovirus (CMV) immediate early promoter / enhancer sequences, Rous sarcoma virus (RSV) promoter / enhancer sequences, phosphoglycerate kinase (PKG) promoters, CAG (a complex of CMV enhancer, chicken β-actin promoter (CBA), and rabbit β-globulin intron) (see, for example, Boshart et al., (1985) Cell, 41:521-530), SV40 promoters, dihydrofolate reductase promoters, and cytoplasmic β-actin promoters.

[0192] Other promoters include superoxide dismutase 1 (SOD1) promoter, U6 promoter, H1, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, SFFV promoter, rat insulin promoter, TBG promoter, desmin promoter and similar muscle-specific promoters, synthetic promoters, hybrid promoters and promoters with multi-tissue specificity.

[0193] In some embodiments, the promoter is an EF-1α promoter (see, for example, Wang et al., J. Cell Mol. Med. (2017) 21 (11): 3044-3054, which is hereby incorporated herein by reference in its entirety) and / or comprises, consists of or substantially consists of a sequence having at least 95%, at least 97%, at least 99% or 100% identity with SEQ ID NO: 184.

[0194] Expression control elements can also influence expression in a manner that can be modulated by increasing or decreasing the expression of a signal or stimulus. A modifiable element that increases the expression of transcribed nucleic acids in response to a signal or stimulus is also called an “inducible element” (i.e., signal-induced). Typically, the amount of increase or decrease conferred by such an element is proportional to the amount of signal or stimulus present. Specific examples include the zinc-inducible sheep metallothionein (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (International Patent Publication No. WO1998 / 10088); the tetracycline-inhibitory system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen et al., Science 268: 1766-1769 (1995)); see also Harvey et al.,Curr. Opin. Chem. Biol. 2:512-518 (1998)); RU486 inducible system (Wang et al., Nat. Specification 21 / 106 pages 27 CN 122374456 A Biotech. 15:239-243 (1997) and Wang et al., Gene Ther. 4:432-441 (1997)); and rapamycin inducible system (Magari et al., J. Clin. Invest. 100:2865-2872 (1997)); and Rivera et al., Nat. Medicine.2:1028-1032 (1996)). Other examples of adjustable control elements include those regulated by specific physiological states such as temperature, acute phase, or development.

[0195] In some embodiments, the expression cassette further comprises one or more introns. A variety of different introns may be used. Examples of introns that can be used include rabbit β-globin introns with splice donors / acceptors, SV40 introns with splice donors / acceptors, human β-globin introns, intron 2 of the human hemoglobin β gene, hFIX int1 (intron 1 of the human coagulation factor IX gene), CBA-rHHB (a synthetic intron derived from the fusion of intron 1 of the chicken β-actin gene and intron 2 of rabbit hemoglobin β), CBA (intron 1 of the chicken β-actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synth (a synthetic intron derived from different parts of the human coagulation factor IX gene and present in pLIVE vectors, Mirus Bio, Madison, WI), synthetic introns of human hemoglobin subunit β (HBB2) and optimized HBB2; and chimeric introns, such as those derived from first human β-globin introns. The intron is composed of a 5'-splicing donor of the globin intron and a branch of the intron located between the leader sequence and the body of the immunoglobulin gene heavy chain variable region, and a 3'-receptor site. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197; Ronzitti et al. Mol. Ther. Methods Clin Dev. (2016) July 20; 3:16049; and HBB-IGG introns provided by the pCMVNT™ vector.)

[0196] In some embodiments, the nucleic acid encoding an RNA polynucleotide containing a target sequence, pri-miRNA, or shRNA is localized within the intron.

[0197] In some embodiments, the expression cassette contains post-transcriptional regulatory elements. Post-translational regulatory elements, such as the Woodchuck post-transcriptional regulatory element (WPRE) and the hepatitis B regulatory element, can increase gene expression. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197.)

[0198] Polyadenylation signaling sequences provide for the formation of a polyA tail, which promotes nuclear export, translation and / or mRNA stability, and may also be involved in transcription termination. Examples of polyadenylation signaling sequences include the SV40 late polyadenylation signal, bovine growth hormone polyA (bGHpA) signal sequence, synthetic polyA, mouse β-globin pA, rabbit β-globin pA (RbG), and H4-based pA (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197).

[0199] In some embodiments, the expression cassette contains a Kozak concordant sequence or a variant thereof. The Kozak concordant sequence plays a role in translation initiation. For example, the Kozak concordant sequence and variants are provided in McClements et al., (2021) Molecular Vision, 27, 233–242.

[0200] In some embodiments, the expression cassette contains an upstream promoter and a downstream polyadenylation signal operatively linked to a nucleic acid sequence encoding an RNA polynucleotide.

[0201] In some embodiments, the expression cassette contains, along the 5' to 3' axis, a promoter or promoter / enhancer, an intron, a nucleic acid sequence encoding an RNA polynucleotide, and a polyadenylation signal operatively linked to the nucleic acid sequence encoding an RNA polynucleotide.

[0202] In some embodiments, the expression cassette further contains a sequence encoding glial cell-derived neurotrophic factor (GDFN). The GDFN coding sequence can be operatively linked to a promoter encoding an RNA polynucleotide containing an α-synuclein targeting sequence, or operatively linked to a separate promoter. In a further embodiment, the expression cassette contains a nucleic acid encoding GDNF operatively linked to a promoter encoding an RNA polynucleotide containing an α-synuclein targeting sequence, wherein the GDNF coding sequence is upstream of the RNA polynucleotide coding sequence. Specification 22 / 106 pages 28 CN 122374456 A

[0203] In some embodiments, the expression cassette further contains a miRNA target sequence, which in a further embodiment is incorporated into the 3' UTR of the expression cassette. The miRNA target sequence is formed by the presence of specific cells or tissues.miRNAs recognize and thus cause the degradation of mRNA transcripts. Based on the presence of certain miRNAs in specific cells, incorporating miRNA target sequences can be used to reduce expression in certain cell or tissue types. Multiple tandem repeats of miRNA target sequences can be used to increase degradation. (Geisle et al., (2016) World Journal of Experimental Medicine 6(2): 37–54.)

[0204] In some embodiments, expression cassettes excluding sequences encoding pri-miRNA or shRNA contain any of 0–5, 0–10, 0–15, 0–50, or 0–100 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 CpGs; and / or contain 0% to 5%, 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0% CpGs.

[0205] II.B. Recombinant Viral Vector Nucleic Acid

[0206] The polynucleotide recombinant viral nucleic acid contains 5' and / or 3' viral elements that provide viral packaging and replication, and may provide additional activities such as promoter activity, genome integration, or add-on tandem coupling. The 5' and 3' elements are typically located at or near the 5' and 3' ends of the recombinant viral nucleic acid and may be a naturally occurring sequence or a modified form of a naturally occurring sequence. Examples of 5' and 3' elements include adenovirus ITRs, adeno-associated virus ITRs and packaging sequences; and retrovirus 5' and 3' long terminal repeats (LTRs) and packaging sequences. (Naso et al., (2017) BioDrugs, 31(4), 317–334; Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: 53 (2021); and Liu and Seol (2020) BMB Reports; 53(11):565–575.)

[0207] The term “recombinant” as a modifier for nucleic acid or vector refers to a combination of elements that do not exist in nature. For example, a recombinant viral vector nucleic acid provides 5' and / or 3' viral elements and an expression cassette containing one or more elements not naturally associated with the 5' and / or 3' viral elements. Similarly, viral vectors (such as rAAV vectors) may contain a naturally occurring or modified capsid that encapsulates the recombinant viral vector nucleic acid.

[0208] Polynucleotides, expression cassettes, and viral vector nucleic acids are compatible with specific viral vectors. For example, rAAVs containing ssDNA or dsDNA can be generated, adenoviral vectors containing dsDNA can be generated, and retroviral vectors containing ssRNA can be generated.

[0209] In some embodiments, the viral vector nucleic acid outside the pri-miRNA or shRNA coding region contains any of 0-5, 0-10, 0-15, 0-50, 0-100, or 0 to 150 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 CpGs; and / or 0%. Up to 10%, 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0% CpG, about 6%, about 7%, about 8%, about 9%, or about 10% CpG.

[0210] III. Viral Vectors

[0211] In some embodiments, the gene delivery medium is a viral vector. A viral vector contains a protein capsid encapsulating recombinant viral nucleic acid and can deliver the nucleic acid to cells or tissues. Depending on the specific vector, the viral vector may further contain a viral envelope. Examples of viral vectors that can be used include adenovirus vectors, rAAV, retroviral vectors, and herpes simplex virus vectors.

[0212] Different serotypes exist in different types of viruses. Different serotypes can provide different activities, such as cell or tissue tropism and the likelihood of generating a host immune response. The term “serotype” broadly refers to serologically different viruses as well as non-serologically different viruses that can be within subgroups or variants of a given serotype. Serological uniqueness can be determined based on the lack of cross-reactivity between antibodies of one capsid and antibodies of another. Such cross-reactivity differences are usually due to differences in capsid protein sequence / antigenic determinants (e.g., due to differences in VP1, VP2 and / or VP3 sequences of AAV serotypes).

[0213] As more naturally occurring viral isolates are discovered or capsid mutants are generated, serological differences may or may not exist with any currently existing serotype. Therefore, in the case of a new virus that does not have serological differences, the new virus will be a subgroup or variant of the corresponding serotype.

[0214] III.A. Adenovirus Vector

[0215] Adenovirus is a non-enveloped double-stranded DNA.Virus. The recombinant adenovirus vector contains recombinant adenovirus nucleic acid lacking one or more proteins involved in viral replication and further contains an adenovirus capsid. Recombinant adenovirus vectors containing varying amounts of adenovirus DNA can be produced. The Ad genome is flanked by hairpin-like inverted terminal repeats (ITRs) with terminal lengths ranging from 30 bp to 371 bp. The ITRs act as self-initiating structures that facilitate primase-independent DNA replication. The 5' and 3' inverted repeat sequences do not need to be exact inverted repeat sequences. Viral genome packaging requires a packaging signal located on the left arm of the genome. (Liu and Seol (2020) BMB Reports; 53(11):565–575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)

[0216] In some embodiments, the recombinant adenovirus vector is a third-generation vector, which is also referred to as “gutless” or “helper-dependent”. Enterofree vectors can be generated from recombinant adenovirus nucleic acid, in which all or substantially all of the viral sequence is absent except for the ITR and packaging signals. Enterofree adenovirus vectors are high-capacity vectors capable of holding up to about 36 kb of DNA inserts. Preferred recombinant adenovirus nucleic acid is about 27 kb to about 37 kb. Filler sequences can be added to the recombinant adenovirus nucleic acid to increase the nucleic acid size and capsid incorporation. Preferred filler sequences avoid coding sequences, repetitive sequences, recombinant sequences, and immunogenic sequences. (Liu and Seol (2020) BMB Reports; 53(11):565-575; Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53; and Sandig et al., PNAS (2000) 97(3): 1002-1007, each of which is hereby incorporated herein by reference in its entirety.)

[0217] In some embodiments, recombinant adenovirus vectors may be generated based on rare human or chimpanzee serotypes. The use of chimpanzee and rare human serotypes may help reduce the host immune response to the recombinant adenovirus vector due to pre-existing immunity. (Guo et al., (2018) Human vaccines & immunotherapeutics, 14 (7):1679–1685 and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)

[0218] Adenoviral vectors can be produced by supplying trans vectors with, for example, appropriate helper viruses or plasmids and cell lines to produce the viral proteins required. (Liu and Seol)(2020) BMB Reports; 53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53. )

[0219] III.B. AAV Vector

[0220] The recombinant adeno-associated virus (referred to herein as “rAAV”) vector is based on adeno-associated virus. Adeno-associated virus is a single-stranded DNA virus containing a 4.7-kb genome flanked by 145-nt ITRs at both ends of the genome. ITR activity is important for self-initiation and packaging and can also provide additional activities such as promoter activity. The sizes of the rAAV 5' and 3' ITRs may differ, and the 5' and 3' inverted repeat sequences do not need to be exact inverted repeat sequences.

[0221] The rAAV vector contains recombinant AAV nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks a sequence encoding one or more AAV proteins involved in viral replication. In some embodiments, the rAAV vector contains AAV 5' and / or 3' ITRs and a DNA insert. In some embodiments, the rAAV nucleic acid contains a 5' ITR and / or a 3' ITR, independently selected from the 5' and 3' ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3B ITRs. In a further embodiment, both the 5' and 3' ITRs are present, and both ITRs are derived from the same serotype genome.

[0222] In a further embodiment, the 5' ITR comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 182, and the 3' ITR independently comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 183.

[0223] In some embodiments, the rAAV nucleic acid is at least about 2.5 kb. If desired, a filler sequence can be used to increase the rAAV nucleic acid size and packaging efficiency. In various embodiments, the rAAV nucleic acid including the filler is 4–5.2 kb, 3.0–5.5 kb, 4.0–5.0 kb, 4.3–4.8 kb, about 4.2 kb, about 4.3 kb, about4.4 kb, about 4.5 kb, about 4.6 kb, or about 4.7 kb. Preferred filler sequences circumvent coding sequences, repetitive sequences, recombinant sequences, and immunogenic sequences.

[0224] In some embodiments, rAAV is a self-complementary adeno-associated virus vector (scAAV) or a short hairpin adeno-associated virus vector (shAAV). scAAV and shAAV provide double-stranded recombinant adeno-associated virus nucleic acid that can be incorporated into an AAV capsid. scAAV and shAAV contain an inverse dimer repeat sequence that provides intramolecular double-stranded DNA. scAAV can be generated by mutating the terminal unwinding site of the ITR so that Rep cannot cleave the terminal unwinding site. shAAV can be generated using a short hairpin. scAAV and shAAV are double-stranded DNA, which provides an advantage in circumventing the DNA synthesis steps required for single-stranded rAAV nucleic acid to enter the cell. A potential disadvantage of scAAV and shAAV is that the size of the DNA insert that can be incorporated is reduced by about half compared to single-stranded rAAV nucleic acid. (US Patent No. 10,457,940; Xie et al., Mol Ther. (2017) June 7; 25(6):1363-1374; and McCarty Mol. Ther. (2008) 16(10): 1648-1656; each of which is hereby incorporated herein by reference in its entirety.)

[0225] Naturally occurring AAV capsids contain viral proteins VP1, VP2, and VP3 in a ratio of about 1:1:10. AAV vectors can be generated in which all three viral proteins are based on a specific serotype, or one, two, or all three viral proteins are based on different serotypes.

[0226] Recombinant AAV capsids and nucleic acids can be based on the same serotype (or subgroup or variant), or they can be different from each other. In some embodiments, the rAAV nucleic acid has the same serotype genome (e.g., ITR) as the encapsulating capsid protein.

[0227] In different embodiments, the rAAV capsid comprises a protein having a sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.9%, or 100% identical to the following: AAV1, AAV2,VP1, VP2, or VP3 of any of AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10; or VP1 of SEQ ID NO: 164 or SEQ ID NO: 167.

[0228] In some embodiments, the AAV capsid comprises VP1, VP2, and VP3, each independently having at least 80%, at least 90%, at least 95%, or 100% sequence identity with any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10; or VP1 of SEQ ID NO: 164 or SEQ ID NO: 167; and variants thereof (e.g., capsid variants, such as amino acid insertions, additions, substitutions, and deletions). (See, for example, U.S. Patent Nos. 9,909,142 and 9,840,719 disclosing RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6; U.S. Patent Specification 25 / 106 pages 31 CN 122374456 A Publication No. 2013 / 0059732 and U.S. Patent No. 9,169,299 disclosing LK01, LK02 and LK03; and U.S. Patent No. 11,110,153, the disclosures of which are incorporated herein by reference in their entirety.)

[0229] In some embodiments, the capsid comprises VP1 having the sequence of SEQ ID NO: 164, VP2 having the sequence of SEQ ID NO: 165 and VP3 having the sequence of SEQ ID NO: 166.

[0230] In some embodiments, the AAV capsid can cross the blood-brain barrier and provide CNS expression. Examples of such AAV capsids and designs of AAV capsids capable of providing CNS expression are provided in Chen et al., (2021) J. Control. Release 333, 129-138 (e.g., AAV9, AAV-PHP⋅B, AAV-PHP).In .eB, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAV1 / rh.10), U.S. Patent No. 9,585,971, and Goertsen et al., (2022) Nat. Neurosci. 25, 106–115 (2022).

[0231] The AAV genome contains two major genes: rep and cap. Transcription from the rep gene is initiated by two different promoters, resulting in non-structural proteins named Rep78, Rep68, Rep52, and Rep40. The rep proteins play a role in genome replication and / or encapsulation. The cap gene encodes structural proteins (VP1, VP2, and VP3) that make up the capsid; non-structural assembly activator proteins (APP) that perform functions related to capsid assembly; and membrane-associated accessory proteins that may be associated with production phases of the replication cycle. (Maurer and Weitzman (2020) Hum. Gene Ther. 31(9–10): 499–511, which are hereby incorporated herein by reference in their entirety.)

[0232] AAV requires helper viral function to complete its replication cycle. Helper viral function can be supplied by different viruses in permissive cell lines. Permissive cell lines are cell lines capable of supporting viral replication. Examples of helper viruses for AAV include adenoviruses, HSV-1, HPV-16, and HboV1, which can be used in combination with, for example, primate permissive cells; and baculoviruses, which can be used in combination with, for example, insect permissive cells such as sf9. (Maurer and Weitzman Hum. Gene Ther. (2020) 31(9–10):499–511 and Meier et al., (2020) Viruses 19;12(6): 662, both of which are incorporated herein by reference in their entirety.)

[0233] Recombinant AAV can be produced by supplying the viral proteins required for trans vector production using, for example, suitable helper viruses or plasmids and cell lines. In some embodiments, rAAV is produced using an rAAV vector genomic plasmid. The plasmid contains the portion of the rAAV nucleic acid that is ultimately packaged or encapsulated to form a viral (e.g., rAAV) vector. The “plasmid backbone” contains elements important for replication and the production of recombinant viruses. The plasmid backbone itself is not packaged or encapsulated into viral particles, except for possible 3' ITR and / or 5' ITR cloning remnants.

[0234] The vector genomic plasmid may contain regions such as origin of replication and optional markers. Other sites that may be present include cloning sites.

[0235] Recombinant AAV can be derived from different types of cell lines, includingHeLa, A549, BHK, Vero, and HEK293 or derivatives thereof are used. In some embodiments, HEK293 cells (ATCC CRL1573) are used. Other host cell lines suitable for the production of rAAV vectors are described, for example, in Robert et al., (2017) Biotechnol. J. (2017) 12 (3), 1600193; and international application PCT / US2017 / 024951, the disclosure of which is incorporated herein by reference in its entirety.

[0236] Recombinant AAVs can be cultured under a variety of different conditions suitable for providing cell growth and gene expression. References describing rAAV fabrication include Clément and Grieger (2016) Mol. Ther. Methods Clin. Dev. 16;3:16002; Robert et al. (2017) Biotechnol. J. 12(3), 1600193; and Adeno-Associated Virus Vectors (2019), Castle. ed., 1st edition, Springer, New York, NY.; each of which is hereby incorporated herein by reference in its entirety. )

[0237] In some embodiments, AAV helper functionality is introduced into host cells by transfecting host cells with AAV helper constructs before or simultaneously with transfection of AAV expression vectors. Host cells with AAV helper functionality may be referred to as “helper cells” or “packaging helper cells”. AAV helper constructs are sometimes used to provide at least transient expression of the AAV rep and / or cap genes to supplement the missing AAV function necessary for productive AAV transduction. AAV helper constructs typically lack the AAV ITR and are neither self-replicating nor self-packaging. These constructs can be, for example, in the form of plasmids, phages, transposons, granules, viruses, or virions. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both the rep and cap expression products. Many other vectors encoding the rep and / or cap expression products are known. For example, recombinant AAVs can be generated, as described, for example, in U.S. Patent No. 9,408,904; and International Applications PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are incorporated herein by reference in their entirety.

[0238] In some embodiments, the rAAV vector is produced from rAAV production cells containing rAAV helper viral activity. The genome of the rAAV production cell contains rAAV nucleic acid, the rep gene, and the cap gene.

[0239] In some embodiments, the rAAV vector is produced by culturing rAAV-permitted cells containing an AAV genome plasmid, wherein the rAAV-permitted cells further contain the rep and cap genes as part of the cell genome and / or provided by one or more separate plasmids; and helper viral activity as part of the cell genome and / or provided by one or more separate plasmids. In further embodiments, (a) the rAAV-permitted cell line is a packaging cell, wherein the genome of the packaging cell contains the cap gene and the rep gene; (b) the rep gene, the cap gene, and the helper activity are provided by the same plasmid; or (c) the rep gene and the cap gene are provided by a rep / cap plasmid, and the helper activity is provided by a helper plasmid.

[0240] In some embodiments involving the use of HSV helper functions, the helper functions are provided by genes encoding at least UL5, UL8, UL52, and ICP8.

[0241] In some embodiments involving the use of adenovirus helper functions, the helper functions are provided by genes encoding at least E1A, E1B19K, E1B55K, E2A, E4orf6, and VA RNA. In some embodiments, the E1, E2A, and VR RNA functions are provided by helper plasmids, while additional helper functions are provided by the host strain.

[0242] In some embodiments, the rAAV vector is obtained by generating and purifying rAAV using the methods described herein. Purification of rAAV can be performed using techniques such as gradient-based purification, column-based purification, and combination methods. (See, for example, Ayuso et al., Curr Gene Ther. (2010) 10(6):423-36, which is hereby incorporated herein by reference in its entirety.)

[0243] III.C. Retroviral Vectors

[0244] Retroviruses are enveloped single-stranded RNA viruses containing 5' and 3' LTRs, and signal packaging sequences located just outside the LTRs. Different types of retroviral vectors may contain varying amounts of viral genome. In some embodiments, the retroviral vector is an HIV-based lentiviral vector that retains all cis-acting sequences required for viral RNA packaging, reverse transcription, and proviral DNA integration, while removing all HIV protein-coding genes. Lentiviral vectors have packaging capacities up to approximately 9 kb. If desired, filler sequences can be used to increase rAAV nucleic acid size and packaging efficiency.Rate. Lentiviral vectors can be produced by supplying the required viral proteins to the trans vector using appropriate plasmids and cell lines. (Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)

[0245] III.D. Other parvovirus vectors

[0246] In some embodiments, the vector is based on parvoviruses other than AAV, such as Erythroparvovirus, Protoparvovirus, or Tetraparvovirus. Members of Erythroparvovirus (e.g., human parvovirus B19) typically have a genome size of about 5.6 kb, and therefore recombinant vectors based on Erythroparvovirus are expected to accommodate larger recombinant payloads than AAV vectors. The genome size of the protoparvirus or Tetraparvovirus is approximately 5.3 kb, and therefore, recombinant vectors based on these viruses are also expected to accommodate larger recombinant payloads than AAV vectors.

[0247] Examples of the erythroparvovirus capsid are disclosed in International Patent Publications WO2023 / 220043, WO2023 / 220035 and WO2023 / 220040, all of which are hereby incorporated herein by reference in their entirety.

[0248] Examples of the protoparvirus and Tetraparvovirus capsid are disclosed in International Patent Publication WO2022 / 140683, all of which are hereby incorporated herein by reference in their entirety.

[0249] IV. Non-viral vectors

[0250] In some embodiments, the gene delivery medium is a non-viral vector. Preferred non-viral vectors are nanoparticles. A wide variety of nanoparticles can be used, including lipid nanoparticles (LNPs), polymer nanoparticles, lipid polymer nanoparticles (LPNPs), protein and peptide-based nanoparticles, DNA dendritic polymers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. (See, for example, Riley and Vermerris Nanomaterials (2017) 201, 7, 94; Thomas et al., Molecules (2019) , 24, 3744; Bochicchio et al., (2021) , 13, 198; Munagala et al., Cancer Letters (2021) , 505 , 58; Fu et al., NanoImpact 20 ,100261; Neshat et al. (2020) Current Opin. Biotechnol. 66:1-10; Ouranidis et al., (2022) Biomedicines, 10, 50; and Qin et al., Signal Transduct Target Ther. (2022) May 21;7(1):166, each of which is hereby incorporated herein by reference in its entirety. )

[0251] If desired, nanoparticles may be used to target cell types using, for example, targeting ligands that recognize target cell receptors. Examples of targeting ligands include carbohydrates (e.g., galactose, mannose, glucose, and galactomannan), endogenous ligands (e.g., folic acid and transferrin), antibodies and proteins / peptides (e.g., RGD, epidermal growth factor, and low-density lipoprotein) and peptides. (e.g., Teo et al., Advanced Drug Delivery Reviews (2016), 98, 41.)

[0252] Nanoparticles can be used to deliver repressive RNA or encoding polynucleotide constructs to cells. In various embodiments, nanoparticles can deliver additional therapeutic compounds; and one or more additional compounds are provided in different nanoparticles. The compounds mentioned include small molecules and macromolecules (e.g., therapeutic proteins and antibodies).

[0253] The preparation of different nanoparticles and the incorporation of nucleic acids and other compounds are well known in the art. For example, examples of publications demonstrating the incorporation of nucleic acids into specific nanoparticles, such as LPNP and LNP, include Teo et al., Advanced Drug Delivery Reviews (2016) 98, 41; Bochicchio et al., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12(1), 65; and Teixeira et al., (2017) Prog. Lipid Res. Oct;68:1–11 (each of which is hereby incorporated herein by reference in its entirety). Factors influencing the incorporation of small molecules into nanoparticles include the presence of hydrophobic and ionizable moieties. (See, for example, Nii and Ishii, International Journal of Pharmaceutics (2005) 298, 198; and Chen et al., Journal of Controlled Release (2018) 286, 46.)

[0254] IV.A. Lipid-based delivery systems

[0255] Lipid-based delivery systems include those using lipids as a component. Examples of lipid-based delivery systems include liposomes, LNPs, micelles, and extracellular vesicles.

[0256] "Lipid nanoparticles" or "LNPs" refer to lipid-based vesicles that can be used to deliver nucleic acid molecules and have a nanoscale size. In different embodiments, the nanoparticles are about 10 nm to about 1000 nm, about 50 nm to about 500 nm, or about 50 nm to about 200 nm. Specification 28 / 106 pages 34 CN 122374456 A

[0257] DNA carries a negative charge. Therefore, it may be advantageous for LNPs to contain cationic lipids (such as, for example, aminolipids). Exemplary amino lipids are described in U.S. Patent Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122, and 7,745,651, and U.S. Patent Publications Nos. 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, and 2012 / 0172411. All of these references, including those in 2010 / 0117125, are incorporated herein in their entirety. In some embodiments, the LNP comprises the aminolipid described in U.S. Patent No. 9,512,073, which is hereby incorporated herein in its entirety.

[0258] In this document, the terms “cationic lipid” and “amino lipid” are used interchangeably to include lipids and their salts having one, two, three or more fatty acid or aliphatic alkyl chains and a pH-titrile amino group (e.g., alkylamino or dialkylamino group). Cationic lipids are generally protonated (i.e., positively charged) at pH below the pKa of the cationic lipid; and are substantially neutral at pH above the pKa. Cationic lipids may also be titrable cationic lipids. In some embodiments, the cationic lipid comprises a protonable tertiary amine (e.g., pH-titrile) group; a C18 alkyl chain, wherein each alkyl chain may independently have one or more double bonds, one or more triple bonds; and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0259] Cationic lipids include 1,2-dilinyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinyloxy-N,N-dimethylaminopropane (DLenDMA), and 1,2-di-γ-- Linaloyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinyl-4-(2-dimethylaminoethyl)-[1,3]-dioxacyclopentane (Dlin-K-C2-DMA, also known as Dlin-C2K-DMA, XTC2 and C2K), 2,2-dilinyl-4-dimethylaminomethyl-[1,3]-dioxacyclopentane (Dlin-K-DMA), dilinylmethyl-3-dimethylaminopropionate (Dlin-M-C2-DMA, also known as MC2), 4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-ester (Dlin-M-C3-DMA, also known as MC3), their salts and mixtures thereof. Other cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinyl-4-(3-dimethylaminopropyl)-[1,3]-dioxacyclopentane (Dlin-K-C3-DMA), 2,2-dilinyl-4-(3-dimethylaminobutyl)-[1,3]-dioxacyclopentane (Dlin-K-C4-DMA), Dlen-C2K-DMA, γ-Dlen-C2K-DMA, and (Dlin-MP-DMA) (also known as 1-B11).

[0260] Further cationic lipids include 2,2-dilininyl-5-dimethylaminomethyl-[1,3]-dioxane (Dlin-K6-DMA), 2,2-dilininyl-4-N-methylpiperazino-[1,3]-dioxane (Dlin-K-MPZ), 1,2-dilininylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP), 1,2-dilininyloxy-3-(dimethylamino)acetoxypropane (Dlin-DAC), 1,2-dilininyloxy-3-morpholinopropane (Dlin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1 2-Dilinoleylthio-3-dimethylaminopropane (Dlin-S-DMA), 1-linoleoyl-2-linolenico-3-dimethylaminopropane (Dlin-2-DMAP), 1,2-dilinolenico-3-trimethylaminopropane chloride (Dlin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (Dlin-TAP.Cl), 1,2-dilinolenico-3-(N-methylpiperazinyl)propane (Dlin-MPZ), 3-(N,N-dilinolenico)-1,2-propanediol(DlinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilininyloxo-3-(2-N,N-dimethylamino)ethoxypropane (Dlin-EG-DMA), N,N-dioleo-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxypropyl-3-yl)-N N-Dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoxy-N-[2(amino-formamido)ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), bis(octadecyl)amidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholesterol-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesterol-5-en-3-β-oxy)-3'-oxaproxy)-3- Dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS) and disubstituted spermine (D2S) or mixtures thereof.

[0261] A variety of commercial cationic lipid formulations can be used, such as LIPOFECTIN® (containing DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE® (containing DOSPA and DOPE, available from GIBCO / BRL).

[0262] Other ionizable lipids that can be used include C12-200, 306Oi10, MC3, cKK-E12, bCKK-E12, lipid 5, lipid 9, ATX-002, ATX-003, and Merck-32. Merck-32 is described in U.S. Patent Application Publication No. 2017 / 0367988.

[0263] In a further embodiment, the cationic lipid may be present in an amount of about 10% to about 85% of the LNP by molar ratio, or about 50% to about 75% of the LNP by molar ratio.

[0264] The LNP may contain neutral lipids. Neutral lipids may be lipid substances present at physiological pH in an uncharged or neutral zwitterionic form. Such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided by factors including particle size and stability. In some embodiments, the neutral lipid component may be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0265] Lipids with various acyl chain groups having different chain lengths and saturations are available, or may be isolated or synthesized. In some embodiments, lipids containing saturated fatty acids with carbon chain lengths in the C14 to C22 range may be used. In some embodiments, lipids containing monounsaturated or diunsaturated fatty acids with carbon chain lengths in the C14 to C22 range may be used. Additionally, lipids containing a mixture of saturated and unsaturated fatty acid chains may be used. Typical neutral lipids include 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-ethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), or phosphatidylcholine. Neutral lipids may also consist of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups such as serine and inositol.

[0266] In a further embodiment providing neutral lipids, the neutral lipids may be present in amounts from about 0.1% by weight to about 99% by weight of the LNP, or from about 5% by weight to about 15% by weight of the LNP, for example, in amounts of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0267] The LNP may contain other components, such as sterols and polyethylene glycol. Sterols may impart fluidity to the LNP. As used herein, "sterol" refers to naturally occurring plant sterols (phytosterols) or animal sterols (animal sterols), as well as non-naturally occurring synthetic sterols. All of these sterols are characterized by their presence in sterols.A hydroxyl group is present at position 3 of ring A. Suitable sterols include those commonly used in the preparation of liposomes, lipid vesicles, or lipid particles, most commonly cholesterol. Phytosterols include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application 30 / 106, page 36, CN 122374456 A, Publication No. 2011 / 0177156. In different embodiments providing sterols, the sterols are present in amounts from about 1% by weight to about 80% by weight of LNP, or from about 10% by weight to about 25% by weight of LNP.

[0268] Polyethylene glycol (PEG) is a water-soluble polymer of ethylene PEG repeating units with terminal hydroxyl groups. PEGs are classified by their molecular weight; for example, PEG 2000 has an average molecular weight of about 2,000 Daltons, and PEG 5000 has an average molecular weight of about 5,000 Daltons. PEGs commercially available from Sigma Chemical Co. and other companies include monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succiniminosuccinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-trifluoromethanesulfonate (MePEG-TRES), and monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0269] In some embodiments involving PEG, the PEG has an average molecular weight of about 550 to about 10,000 Daltons and is optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In further embodiments, the PEG is substituted with a methyl group at the terminal hydroxyl position. In a further embodiment, the PEG has an average molecular weight of about 750 to about 5,000 Daltons, or about 1,000 to about 5,000 Daltons, or about 1,500 to about 3,000 Daltons, or about 2,000 Daltons, or about 750 Daltons.

[0270] PEG-modified lipids include PEG-dialkyloxypropyl conjugates (PEG-DAA) described in U.S. Patent Nos. 8,936,942 and 7,803,397. PEG-modified lipids (or lipid-polyoxyethylene conjugates) may have a variety of “anchoring” lipid moieties to fix the PEG moieties to the surface of lipid vesicles. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-neuro-phosphatidylethanolamine described in U.S. Patent No. 5,820,873, and PEG-neuro-phosphatidylethanolamine.Amide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. In some embodiments, the PEG-modified lipids may be PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, the amount of PEG may be from about 0.1% by weight to about 50% by weight of the LNP, or from about 5% by weight to about 15% by weight of the LNP.

[0271] In a further embodiment regarding the size of the LNP, the size of the LNP ranges from about 10 nm to 500 nm, or from about 50 nm to about 200 nm, or from 75 nm to about 125 nm before encapsulating the nucleic acid.

[0272] In some embodiments of LNPs, Billingsley et al., Nano Lett. 2020, 20, 1578; Billingsley et al., International Patent Publication No. WO 2021 / 077066; or U.S. Patent Application No. 2022 / 396556 (each of which is hereby incorporated herein by reference in its entirety) describe LNPs. These publications describe LNPs containing lipid-anchored PEG, cholesterol, phospholipids, and ionizable lipids. In some embodiments, the LNPs contain a C14-4 polyamine core and / or have a particle size of about 70 nm. C14-4 has the following structure. Specification 31 / 106 pages 37 CN 122374456 A

[0273]

[0274] In some embodiments, the LNP is composed of cationic lipids or lipopeptides described in U.S. Patent No. 10,493,031, U.S. Patent No. 10,682,374, or WO2021 / 077066 (each of which is hereby incorporated herein by reference in its entirety). In some embodiments, the LNP contains cationic lipids, cholesterol-based lipids, and / or one or more PEG-modified lipids. In some embodiments, the LNP contains cKK-E12 (Dong et al., PNAS (2014) 111 (11), 3955):

[0275]

[0276] In some embodiments, the LNP contains a modified form of cKK-E12 referred to herein as “bCKK-E12”, having the following structure:

[0277]

[0278] In some embodiments, the LNP contains lipids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as described in Sabnis et al., Molecular Therapy 2018, 26:6,As described in 1509-1519 (which are hereby incorporated herein by reference in their entirety). In some embodiments, the LNP comprises lipids 5, 8, 9, 10, or 11 as described by Sabnis et al.

[0279] Lipid 5 of Sabnis et al. has the following structure: Specification 32 / 106 page 38 CN 122374456 A

[0280]

[0281] Lipid 9 of Sabnis et al. has the following structure:

[0282]

[0283] Other lipids that can be utilized include Roces et al., Pharmaceutics, 2020, 12, 1095; Jayaraman et al., Angew. Chem. Int. Ed., 2012, 51, 8529-8533; Maier et al., www.moleculartherapy.org, 2013, Vol. 21, No. 8, 1570-1578; Liu et al., Adv. Mater. 2019, 31, 1902575, for example, BAMEA-O16B; Cheng et al., Adv. Mater., 2018, 30, 1805308, e.g., 5A2-SC8; Hajj and Ball, Small, 2019 15, 1805097, e.g., 306Oi10; Du et al., U.S. Patent Application Publication No. 20160376224; and those described in Tanaka et al., Adv. Funct. Mater., 2020, 30, 1910575; each of them is hereby incorporated herein by reference in its entirety.

[0284] In a further embodiment, the nanoparticles are LNPs. In a further embodiment, the LPN, in molar percentage, comprises, is substantially composed of, or is composed of the following components: (1) about 20% to about 65% of one or more cationic lipids, about 1% to about 50% of one or more phospholipids, about 0.1% to about 10% of one or more PEG-conjugated lipids, and 0% to about 70% of cholesterol; and (2) about 20% to about 50% of one or more cationic lipids, about 5% to about 20% of one or more phospholipids, about 0.1% to about 5% of one or more PEG-conjugated lipids, and about 20% to about 60% of cholesterol. In a further embodiment, the phospholipid is a neutral lipid; and the phospholipid is DOPE or DSPC.

[0285] In a further embodiment, the LNP, in molar percentage, comprises, is composed of, or is substantially composed of, the following components: (1) about 20% to about 65% of one or more cationic lipids, about 1% to about 50% of one or more phospholipids, about 0.1% to about 5% of one or more PEG-conjugated lipids, and about 20% to about 60% of cholesterol.It contains, is substantially composed of, or is composed of the following components: (1) approximately 35% cKK-E12; approximately 2.5% C14-PEG2000; approximately 46.5% cholesterol; and approximately 16% DOPE; (2) approximately 35% bCKK-E12; approximately 2.5% C14-PEG2000; approximately 46.5% cholesterol; and approximately 16% DOPE; (3) approximately 50% lipid 9; approximately 1.5% C14-PEG2000; approximately 38.5% cholesterol; and approximately 10% DSPC; (4) approximately 50% lipid 5; approximately 1.5% C14-PEG2000; approximately 38.5% cholesterol; and approximately 10% DSPC; (5) approximately 50% ionizable lipids; and approximately 10% DSPC. DSPC; approximately 37.5% cholesterol; and approximately 2.5% stabilizer (PEG-lipid); or (6) GenVoy-ILM™ LNP (Precision NanoSystems).

[0286] IV.B. Polymer-based Nanoparticles

[0287] Polymer-based delivery systems can be made from a variety of different natural and synthetic materials. DNA and other compounds can be trapped in the polymer matrix of polymer nanoparticles, or can be adsorbed or conjugated on the surface of nanoparticles. Examples of commonly used polymers for nucleic acid delivery include poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly(ethyleneimine) (PEI) and PEI derivatives, chitosan, dendritic polymers, polyanhydrides, polycaprolactone, polymethacrylates, poly-L-lysine, pullulan, dextran, hyaluronic acid, and poly-β-amino esters. (Thomas et al., (2019) Molecules 24, 3744.)

[0288] Polymer-based nanoparticles can have different sizes, ranging from about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, and about 150 nm or smaller.

[0289] IV.C. Lipid Polymer Nanoparticles

[0290] Lipid polymer nanoparticles are hybrid nanoparticles that provide both lipid and polymer components, and can therefore be considered LNPs or LPNPs. LPNP configurations can provide an outer polymer and an inner lipid or an outer lipid and an inner polymer. The presence of two different types of materials helps in designing nanoparticles to provide delayed release of the components.Considering the material to be delivered, different lipid and polymer components can be selected. (See, for example, Teo et al., Advanced Drug Delivery Reviews (2016) 98, 41; Bochicchio et al., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12(1), 65; and Teixeira et al., (2017) Prog. Lipid Res. Oct;68:1-11.)

[0291] IV.D. Protein and Peptide-Based Nanoparticles

[0292] Protein and peptide-based systems can use a variety of different proteins and peptides. Examples of proteins that can be used include gelatin and elastin. Peptide-based systems can employ, for example, CPP.

[0293] CPP is a short peptide (6–30 amino acid residues) that may be able to penetrate intracellularly to deliver therapeutic molecules. Most CPPs are composed primarily of arginine and lysine residues, giving them cationic and hydrophilic properties, but CPPs can also be amphiphilic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., HIV-1 Tat protein) or obtained through synthetic methods (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018;25(1):1996–2006). Examples of CPPs include cationic CPPs (highly positively charged), such as Tat peptide, penetrin, protamine, poly-L-lysine, and polyarginine; amphiphilic CPPs (chimeric or fusion peptides constructed from different sources, containing both positively and negatively charged amino acid sequences), such as transporters, VT5, bacitracin-7 (Bac7), proline-rich peptides (PPR), SAP (VRLPPP)3, TP10, pep-1, and MPG; membrane-loving CPPs (exhibiting both hydrophobic and amphiphilic properties, and containing both large aromatic residues and small residues), such as H625, SPIONs-PEG-CPP, and NP; and hydrophobic CPPs (containing only nonpolar motifs or residues), such as SG3, PFVYLI, pep-7, and fibroblast growth factor.

[0294] Protein and peptide nanoparticles can be provided in different sizes, for example, ranging from about 1 nm to about 1000 nm, from about 10 nm to about 500 nm, from about 50 nm to about 200 nm, from about 100 nm to about 150 nm, or about 150 nm or smaller.

[0295] IV.E. Peptide cage nanoparticles

[0296] Peptide cage-based delivery systems can be produced from protein materials capable of assembling into cage-like structures to form a confined internal environment. Peptide cages can comprise protein shells (e.g., structures with lumens, which are either naturally accessible to the solvent or can be created by varying the solvent concentration, pH, or equilibrium ratio) that assemble to form the protein cage. The monomers of the protein cages can be naturally occurring or variant forms, including amino acid substitutions, insertions, and deletions (e.g., fragments).

[0297] Different types of protein “shells” can be assembled and loaded with different types of materials. Protein cages can be produced using viral capsid proteins (e.g., protein capsids from Cowpea Chlorotic Mottle Virus) and non-viral proteins (e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Publication No. 34 / 106, page 40, CN 122374456 A 20040028694, and U.S. Patent Publication No. 20090035389, each of which is incorporated herein by reference for its characteristics).

[0298] Examples of protein cages derived from non-viral proteins include: eukaryotic or prokaryotic ferritins and deferroferritins, such as 12- and 24-subunit ferritins; and heat shock proteins (HSPs), such as 24-subunit heat shock proteins that form the internal core space, small HSPs of *Methanococcus jannaschii*, dodecimal Dsp HSPs of *E. coli*; and MrgA proteins.

[0299] Protein cages can have different core sizes, such as about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, or about 150 nm or smaller.

[0300] IV.F. Exosomes

[0301] Exosomes are small biomembrane vesicles. Exosomes have been used to deliver a variety of cargoes, including small molecules, peptides, proteins, and nucleic acids. Exosomes typically range in size from about 30 nm to 100 nm and can be absorbed by cells to deliver their cargo (e.g., expression cassettes containing nucleic acids encoding RNA polynucleotides containing α-synuclein targeting sequences). The cargo can associate with exosome surface structures or can be encapsulated within an exosome bilayer.

[0302] Various modifications can be made to exosomes to facilitate cargo delivery and cell targeting. Modifications for facilitating cargo transport include structures for association with the cargo, such as protein scaffolds and polymers. Modifications for cell targeting include target...To ligands and modify surface charge. Publications describing the production, modification and use of exosomes for delivery of various cargoes include Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., NanoImpact 20, 100261 (2020); and Dooley et al., Molecular Therapy 29 (5), 1729 (each of which is hereby incorporated herein by reference in its entirety).

[0303] V. Pharmaceutical Compositions

[0304] Pharmaceutical compositions may be used to facilitate the storage and / or delivery of a medicament administered to a subject. In some embodiments, the pharmaceutical composition comprises: (a) an RNA polynucleotide containing a sequence targeting α-synuclein mRNA, (b) an optionally modified repressive RNA duplex targeting α-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid containing a sequence encoding the α-synuclein mRNA targeting sequence, or (d) a delivery medium comprising (a), (b), or (c); and a pharmaceutically acceptable carrier.

[0305] The reference to “pharmaceutically acceptable” means that the component will not cause substantial undesirable biological effects at the amount used. Pharmaceutically acceptable carriers may contain different components, such as one or more pharmaceutically acceptable excipients, such as salts, sugars, buffers, solvents, preservatives, proteins, and surfactants. A particular excipient may have more than one function. Examples of pharmaceutically acceptable excipients and carriers that can be used as viral vectors are provided, for example, in International Patent Publications WO2021 / 071835 and WO2024 / 138129 (which are hereby incorporated herein by reference in their entirety).

[0306] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery. Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions, or emulsions, the formulations of which are generally sterile and can be isotonic with the blood of the intended recipient. Exemplary examples include water, buffered saline, Hanas solution, Ringer's solution, dextran, fructose, ethanol, animal and vegetable oils, and synthetic oils. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0307] In one embodiment, the pharmaceutical composition contains a formulation capable of being injected into a subject. Examples of injectable formulation components include isotonic, sterile, saline solutions, salts (e.g., monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, and mixtures of such salts), buffered saline solutions, sugars (e.g., dextrose), and water for injection. Pharmaceutical compositions include dry...Dry compositions, such as freeze-dried compositions, allow for the formation of a solution suitable for administration, as per instructions for use, page 35 / 106, 41 CN 122374456 A, upon addition of sterile water or physiological saline.

[0308] Additionally, suspensions can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or mediators include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, thereby facilitating the preparation of concentrated solutions.

[0309] "Effective amount" or "sufficient amount" refers to an amount that provides the indicated or desired effect. Effective amounts may be administered alone or in combination with one or more other compositions (e.g., additional therapeutic agents or immunosuppressants), treatments, regimens, or treatment regimens in a single or multiple dose; and provide long-term or short-term responses.

[0310] A transgenic pharmaceutical composition comprising an RNA polynucleotide encoding an α-synuclein mRNA may be delivered to a subject to allow the production of the encoded polynucleotide. Delivery can be in vivo or ex vivo. In some embodiments, the pharmaceutical composition contains sufficient genetic material to enable the receptor to produce a therapeutically effective amount in a subject.

[0311] A “therapeutically effective amount” is the amount that elicits a desired or indicated biological or pharmaceutical response in a subject. Therapeutically effective amounts can be determined based on observed symptoms and / or by using biomarkers associated with a specific disease or condition. Various factors can be considered to optimize the selection of a particular effective dose, including the disease to be treated or prevented, the symptoms involved, the targeted disease or condition, safety and efficacy in animal models, the patient’s weight, and the patient’s immune status. The optimal dose used in the formulation will also depend on the route of administration and the severity of the disease or condition and can be assessed based on the patient’s condition. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0312] In some embodiments, the pharmaceutical composition containing the rAAV carrier contains an empty AAV capsid. In some embodiments, in a pharmaceutical composition comprising an rAAV carrier and an empty AAV capsid, the ratio of the empty AAV capsid to the rAAV carrier is within or between about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100. In some embodiments, the ratio of the empty AAV capsid to the rAAV carrier is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0313] Other guidelines and examples of pharmaceutical compositions and delivery systems are provided, for example, in Remington: The Science and Practice of Pharmacy (2020) 23rd edition, University of the Science in Philadelphia, published by Elsevier; The Merck Index (2013) 15th edition, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; and Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th edition, Lippincott Williams & Wilkins, Baltimore, MD.

[0314] VI. Administration and Treatment

[0315] RNA polynucleotides containing an inhibitory sequence targeting α-synuclein can be used in methods of reducing α-synuclein expression and / or treating synucleinopathy diseases or conditions in subjects. For example, such methods may include administering (a) an RNA polynucleotide containing a sequence targeting α-synuclein mRNA, (b) an optionally modified repressive RNA duplex targeting α-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid containing a sequence encoding a targeting sequence of α-synuclein mRNA, or (d) a delivery medium containing (a), (b), or (c); and a pharmaceutically acceptable carrier.

[0316] Treatment may be administered to a subject, preferably a human subject, to provide prophylactic treatment thereby reducing the likelihood or severity of a disease or condition and / or treating a diagnosed disease or condition. Treating a diagnosed disease or condition includes improving one or more symptoms and / or delaying the onset of one or more symptoms. In some embodiments, specific therapeutic agents, routes of administration, and / or pharmaceutical compositions are selected in consideration of the specific disease or condition being treated.

[0317] Subjects with a specific disease or condition, or subjects at increased risk of a specific disease or condition, can be identified, for example, based on symptoms, α-synuclein, biomarkers, and / or genetic markers. For example, subjects can be...Subjects at increased risk of developing synucleinosis or other conditions are treated. Examples of markers include SNCA A53T and SNCA A30P substitutes. (Er et al., Biochem Soc Trans. 27 Feb 2023; 51(1): 245–257, which is hereby incorporated herein by reference in its entirety.)

[0318] In some embodiments, the diagnosis involves α-synuclein seed amplification assay. (Siderowf et al., Lancet Neurol. 2023 May; 22(5): 407–417, which is hereby incorporated herein by reference in its entirety.)

[0319] In some embodiments, the treatment is for Lewy body disease or other conditions. In further embodiments, the Lewy body disease or condition is Parkinson's disease, Parkinson's dementia, Lewy body dementia, infantile axonal dystrophy, atypical axonal dystrophy, adult paroxysmal dystonia-Parkinson's disease, autosomal recessive early-onset Parkinson's disease, POLG-related neurodegeneration, Niemann-Pick disease C1, or Krabby's disease.

[0320] Some embodiments relate to the treatment of Parkinson's disease, Parkinson's dementia, or Lewy body dementia. Symptoms of Parkinson's disease include resting tremor, bradykinesia, gait disturbances, speech difficulties, weak voice, muscle atrophy, instability, and postural deformities. Parkinson's disease may also be accompanied by nonmotor symptoms such as rapid eye movement, sleep behavior disorder (RBD), decreased sense of smell, depression, and autonomic failure. Early symptoms include pain, stiffness or numbness in the limbs, bradykinesia, tremor, and decreased facial expression. Late symptoms include motor fluctuations, motor impairment, gait freezing, and falls. A preliminary diagnosis can be based on an assessment of the patient’s medical history and clinical characteristics of the examination. Response to dopamine drugs can also be used for the diagnosis of Parkinson’s disease over time. Most patients with Parkinson’s disease eventually develop dementia. (Koga et al. Molecular Neurodegeneration (2021) 16:83; and Lee et al. Neuroimmunol Neuroinflammation 2021;8:222-44, both of which are hereby incorporated in their entirety.)

[0321] Symptoms of Lewy body dementia include dementia, cognitive fluctuations, visual hallucinations, rapid eye movement, sleep behavior disorder, and Parkinson’s disease. (Koga et al. Molecular Neurodegeneration (2021) 16:83.)

[0322] In some embodiments, diagnosis, patient monitoring, and / or clinical assessment involve the use of questionnaires, scales, and / or assessment tools used by clinicians in the treatment of movement disorders, such as Parkinson’s disease, and in the assessment of clinical outcomes. Such clinical outcome assessments can be made, for example, by referencing the International Parkinson's and Movement Disorders website.(found at (hypertext transfer protocol: / / www.movementdisorders.org / MDS / MDS-Clinical-Outcome-Assessment.htm).

[0323] In some embodiments, the Movement Disorders Association Parkinson's Disease Comprehensive Assessment Scale (MDS-UPDRS) is used to assess the progression of Parkinson's disease. (Goetz et al., 2023 Mov Disord 2023;38(2):342-7; and Goetz et al., Mov Disord 2008;23(15):2129-70, both of which are incorporated herein by reference in their entirety.)

[0324] In some embodiments, imaging biomarkers may be used to assess the progression of Parkinson's disease. In a further embodiment, imaging biomarkers and techniques for monitoring the pathology and progression of Parkinson's disease include: SV2A and VMAT2 PET for studying changes in the integrity of dopamine terminals in the caudate nucleus and putamen; structural MRI for studying volume loss in the substantia nigra, and quantitative susceptibility mapping for assessing structural changes based on iron accumulation in the substantia nigra; biofluid biomarkers for tracking disease progression, such as total α-Syn, total phosphorylated tau, dopa decarboxylase, and CSF levels of 3,4-dihydroxyphenylacetic acid; and α-Syn protein knockdown can be measured in neuronal-derived exosomes isolated from plasma.

[0325] In another embodiment of disease progression, the Roche Parkinson's Disease mobile application DHT, whose specification 37 / 106 pages 43 CN 122374456 A includes a set of performance outcome (“PerfO”) assessments designed to measure the progression of major motor characteristics in patients with early-stage Parkinson's disease. (Essioux et al., Digital health technologies can reduce sample size and enable shorter proof of concept clinical trial in Parkinson's disease . Presented at the 17th AD / PD 2023 International Conference on Alzheimer's and Parkinson's Diseases and Related Neurological Disorders; March 28-April 1, 2023; Gothenburg, Sweden). DHTThe impairment measurements that form the basis of the core motor symptoms of Parkinson's disease are provided, including bradykinesia of the upper and lower body, gait dysfunction, postural instability, tremor, and speech and voice difficulties. These can be derived from seven motor DHT PerfO tests: hand turning, dexterity, head turning, resting tremor, postural tremor, speech and voice.

[0326] Some embodiments relate to the treatment of MSA. MSA is characterized by a variable combination of autonomic failure, levodopa (L-DOPA) unresponsive Parkinson's disease, cerebellar ataxia, pyramidal signs, and nonmotor symptoms. Nonmotor symptoms may include RBD, sleep apnea, dysphagia, severe dysarthria, and dysarthria. (Koga et al., Molecular Neurodegeneration (2021) 16:83.)

[0327] Administration can be administered via various routes, such as subcutaneous, epidermal, intradermal, intrathecal, intraorbital, intramucosal, intranasal, intraperitoneal, intravenous, intrapleural, intraarterial, intracavitary, oral, via portal vein, intramuscular, intraparenchymal, intracisional, or intraventricular. In some embodiments, administration to the patient is performed via infusion in a drug carrier.

[0328] In some embodiments, administration is targeted at the CNS, for example, intraparenchymal, intracisional, or intraventricular administration.

[0329] In some embodiments, the route of administration and / or carrier targets the caudate nucleus-putamen and / or substantia nigra. In further embodiments, the caudate nucleus-putamen + substantia nigra is targeted for the treatment of MSA; or the substantia nigra is targeted for the treatment of Parkinson's disease.

[0330] In some embodiments, administration is targeted at the CNS, for example, via intraparenchymal, intracisional, or intraventricular administration.

[0331] In some embodiments, CNS administration is performed by direct application to the brain using a needle or catheter. (e.g., International Publication No. WO 2021 / 108809, Cohen-Pferrer et al., Pediatric Neurology 67 (2017) 23-35; and U.S. Patent No. 10369329; each of which is hereby incorporated herein by reference in its entirety.)

[0332] In some embodiments, CNS administration is performed by convection-enhanced delivery. Convection-enhanced delivery involves surgically exposing the brain, placing a catheter directly into the target region, and then infusing the therapeutic agent. (U.S. Patent Publication No. 2022 / 010001; and Debinski et al. (2009) Expert Rev Neurother. 9(10): 1519-27; both of which are hereby incorporated herein by reference in their entirety.)

[0333] In some embodiments, CNS administration is performed via IP to the caudate nucleus and putamen or substantia nigra, using convection-enhanced delivery.Bilateral infusion is used for intraparenchymal administration. In a further embodiment, administration is performed using bilateral stereotactic infusion. In a further embodiment, administration is performed under MRI guidance, wherein rAAV is provided in a formulation containing an imaging agent. Different devices can be used for infusion, including, for example, the ClearPoint® SmartFlow cannula and ClearPoint® Neuro Navigation system via convection-enhanced delivery (CED) under MRI guidance.

[0334] In some embodiments, the presence of a repressive nucleic acid encoded by the transgene is measured in cerebrospinal fluid (CSF) after IP infusion. The presence of the repressive nucleic acid in CSF can be monitored at different time points, such as at baseline and at 3, 6, 12, 18, and 24 months. In a further embodiment, a vector encoding miR5 is administered and the presence of miR5 is measured in cerebrospinal fluid (CSF). For example, measuring the repressive nucleic acid in CSF can be used as a durable marker.

[0335] CNS delivery devices, systems, and techniques also include, for example, those described in U.S. Patent No. 8,128,600, U.S. Patent Publication No. 2020 / 0324089, U.S. Patent No. 1,112,9643, U.S. Patent No. 1,115,4377, U.S. Patent Publication No. 2021 / 0343,397, U.S. Patent Publication No. 2021 / 0282,866, U.S. Patent No. 9,572,928, U.S. Patent No. 8,337,458, U.S. Patent No. 1072,2265, and U.S. Patent Publication No. 2021 / 214,749, each of which is incorporated herein by reference in its entirety.

[0336] CNS administration may also be performed using techniques that facilitate transport across the blood-brain barrier, including disruption of the blood-brain barrier and utilization of blood-brain barrier carriers. (Chen et al., (2021) Journal of Controlled Release 333, 129–138 and Bellettato and Scrapa, Italian Journal of Pediatrics (2018) 44 (Supplement 2): 131; Haumann et al., (2020) CNS Drugs 34, 1121–1131; and Camalleri et al., J. Clin. Neurophysiol. (2020) Mar; 37(2): 104–117, each of which is hereby incorporated in its entirety by reference.) Technologies that facilitate crossing the blood-brain barrier can be used on both viral and nonviral delivery media.

[0337] In some embodiments, CNS delivery is achieved using techniques and / or agents that facilitate crossing the blood-brain barrier.

[0338] In some embodiments, CNS delivery is achieved by using techniques that bypass the blood-brain barrier.

[0339] In some embodiments, CNS delivery is achieved by direct administration to the brain.

[0340] Techniques that facilitate transport across the blood-brain barrier include disrupting the blood-brain barrier, using blood-brain barrier carriers, and using vectors capable of crossing the blood-brain barrier. General techniques for facilitating crossing the blood-brain barrier can be used on both viral and non-viral delivery media. Furthermore, specific delivery media, such as certain AAV serotypes, can facilitate crossing the blood-brain barrier. (Chen et al., (2021) Journal of Controlled Release 333, 129–138; Bellettato and Scrapa, Italian Journal of Pediatrics (2018) 44 (Supplement 2): 131; Haumann et al., (2020) CNS Drugs 34, 1121–1131; Cammalleri et al., J. Clin. Neurophysiol. (2020) Mar; 37(2): 104–117); and Fischell and Fishman, Front. Neurosci., Sep 23, 2021, Vol. 15, Article 747726, doi: 10.3389 / fnins.2021.747726; each of these is hereby incorporated in its entirety by reference.

[0341] In some embodiments, an AAV capsid that facilitates CNS entry is used. Examples of such capsids are provided in Chen et al., (2021) Journal of Controlled Release 333, 129-138 (e.g., AAV9, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAV1 / rh.10), U.S. Patent Nos. 9,585,971, US Patent Publications US2021 / 214749 and US2021 / 107947, U.S. Patent Nos. 11,518,787, and International Patent Publication WO2019 / 158619.

[0342] In some embodiments, the AAV capsid contains inserts that target CNS cells, such as those present in the caudate nucleus and / or putamen and / or substantia nigra and / or ventral tegmentum. Examples of targeted sequences are found in US Patent Publication No. US2021 / 214749 and International Patent Publication No. WO2019 / 158619.Both are provided in [reference to], and are hereby incorporated herein by reference in their entirety. Targeting peptides can be inserted into different AAV capsid sites. In some embodiments, the AAV capsid insertion site is outside the lipase domain of VP1 and outside the assembly activation protein (AAP).

[0343] In some embodiments, the AAV capsid comprises a targeting peptide insert comprising the amino acid sequence described in U.S. Patent Publication No. US2021 / 214749 or International Patent Publication No. WO2019 / 158619. In a further embodiment, the capsid is an AAV2 capsid comprising a peptide insert containing SEQ ID NO: 279, 280, 281, or 282; and the insert is centered at amino acid residue 587 of AAV2 VP1 or the insert is located between N587 and R588 of the AAV2 VP1 capsid protein.

[0344] In some embodiments, CNS delivery involves the use of focused ultrasound and microbubbles. The combination of focused ultrasound and microbubbles can temporarily disrupt the blood-brain barrier, thereby facilitating the entry of therapeutic agents, including transgenic delivery agents. Focused ultrasound can deposit energy into selected areas of human anatomy, and its use can be facilitated by magnetic resonance imaging (MRI). Different types of microbubbles can be used in conjunction with focused ultrasound to temporarily disrupt the blood-brain barrier. (Cammalleri et al., Specification 39 / 106 pp. 45 CN 122374456 AJ Clin Neurophysiol. (2020) Mar;37(2):104-117; Noroozian et al., Methods Mol Biol. (2019) 1950, 177–197; US Patent No. 10,322,178; and International Patent Publication No. WO 2019 / 113538; each of which is hereby incorporated herein by reference in its entirety.)

[0345] Microbubbles are typically injected intravenously. In some embodiments, the microbubbles are Definity™ (Lantheus Medical Imaging, North Billrica, Massachusetts, USA), containing perfluoropropane lipid microspheres (average diameter 1.1–3.3 µm, with a maximum diameter of 20 µm); SonoVue™ (Bracco Imaging, Milan, Italy), a suspension of phospholipid microspheres containing sulfur hexafluoride gas (average diameter approximately 2.5 µm; over 90% of the bubbles are less than 8 µm); and Optison™ (GE Healthcare, Princeton, New Jersey, USA), a sterile, nonpyrogenous suspension of human serum albumin and perfluoropropane microspheres (average diameter 3.0–4.5 µm).µm, of which the maximum diameter is 32 µm). (See Cammalleri et al., J Clin Neurophysiol. (2020) Mar;37(2):104-117, which is hereby incorporated herein by reference in its entirety.)

[0346] In some embodiments, microbubbles are generated and applied as described in U.S. Patent No. 10,322,178, which is hereby incorporated herein by reference in its entirety.

[0347] In some embodiments, the rAAV carrier is selected or engineered to facilitate use with focused ultrasound delivery. (Kofed et al., Journal of Controlled Release 351 (2022) 667–680; Kofeed et al., Molecular Therapy: Methods & Clinical Development, Vol. 27, Dec. 2022, 167–195; U.S. Patent Publication No. 2023 / 0047753; and International Patent Publication No. WO2023 / 0044161; each of these is incorporated herein by reference in its entirety.)

[0348] In some embodiments, the rAAV vector comprises the acoustically targeted peptide described in U.S. Patent Publication No. 2023 / 0047753 and / or International Patent Publication No. WO2023 / 004416.

[0349] In some embodiments, intranasal administration is used to achieve CNS vector delivery. Intranasal delivery bypasses the blood-brain barrier and reduces systemic exposure. In further embodiments, intranasal delivery includes the use of focused ultrasound and microbubbles. It has been noted that the use of a combination of focused ultrasound and microbubbles with intranasal delivery enhances the penetration of therapeutic agents already in the perivascular space across the blood-brain barrier. (Ye et al., The Lancet (2022) October: Vol. 84: 1–14, which is hereby incorporated herein by reference in its entirety.)

[0350] In some embodiments providing CNS expression, the polynucleotide expressing the RNA polynucleotide comprises a PGK promoter, a CBh promoter, and / or an EF1-α promoter.

[0351] The optimal dose can vary depending on various factors, such as the specific treatment and desired endpoint. The dose, amount, frequency, or duration can be increased or decreased proportionally, taking into account adverse side effects, complications, or other risk factors of the treatment or therapy, as well as the condition of the subject.

[0352] A “unit dosage form” refers to a physically separated unit containing a predetermined effective amount of the active ingredient in combination with a pharmaceutically acceptable carrier. Unit dosage forms may be provided, for example, in ampoules and vials, which may include a pharmaceutically acceptable carrier or a composition in a lyophilized or freeze-dried state. In freeze-dried or lyophilized conditions, it can be appliedA sterile liquid carrier is added beforehand. Individual unit dosage forms may be included in multi-dose kits or containers.

[0353] An “effective amount” achieves the intended or indicated effect. For example, an effective amount for treatment reduces one or more adverse symptoms, decreases the likelihood of one or more symptoms associated with the disease or condition, or reduces the progression of the disease or condition. A preferred effective amount for treatment effectively reduces multiple or all adverse symptoms.

[0354] Since α-Syn is a naturally occurring protein, a certain amount of the protein may be important or helpful for certain functions. Although the biological function of α-Syn has not been well characterized, the SNCA gene within it has been shown to develop normally and remain healthy throughout its lifespan in α-Syn-deficient animals with a sex deletion (Chandra et al., Proc Natl Acad Sci USA 2004; 101 (41): 14966-71), although imperceptible learning deficits have been reported in one α-Syn-deficient mouse strain (Kokhan et al., Behav Brain Res 2012; 231 (1): 226-30). In population-based studies, individuals identified as lacking one SNCA allele lacked traits of Parkinson's disease or dementia, contrary to the apparent loss-of-function effect of α-Syn (Blauwendraat et al., UK Biobank. Neurobiol Dis 2021;148:105182; and Burré et al., Mov Disord. 30 June 2024. doi: 10.1002 / mds.29897).

[0355] Nonclinical studies in rodents and NHPs have shown that reductions in SNCA mRNA (up to 90%) and α-Syn protein (up to 70%) are well tolerated over 12 months and do not lead to dopaminergic neurodegeneration or dysfunction in ascending dopaminergic projections (Zharikov et al., Neurobiol Dis 2019; 125:146-53; and Zharikov et al., J Clin Invest 2015; 125(7):2721-35). In NHPs, repetitive intrathecal delivery of antisense oligonucleotides (ASOs) causes up to 70% knockdown of SNCA transcripts and α-Syn protein in the brain with no adverse effects up to 90 days (Cole et al., JCI Insight 2021; 6(5): e135633). Immunotherapy aimed at reducing α-Syn levels in the brain...The therapy has shown a safety profile supporting further development (Jankovic et al., JAMA Neurol 2018; 75 (10): 1206–14; and Pagano N Engl J Med 2022; 387 (5): 421–32.), indicating that the reduction of α-Syn in adults may be tolerable.

[0356] In some embodiments, a pharmaceutical composition comprising a viral or non-viral vector is administered to the subject at a dose suitable for reducing α-synuclein or α-synuclein expression. In various embodiments, in the transduced brain region (e.g., substantia nigra), α-synuclein is reduced by at least 15%, at least 20%, at least 50%, at least 60%, or at least 70% relative to baseline levels; and / or α-synuclein or α-synuclein expression is reduced by at least 20%, at least 50%, at least 60%, at least 70%, or about 30% to about 70%, or about 30% to about 60%, or about 25% to about 70%.

[0357] In various embodiments, suitable doses are about 0.01 mg / kg to about 10 mg / kg of the carrier per kg of subject body weight, about 0.01 mg / kg to about 0.1 mg / kg of the carrier per kg of subject body weight, about 0.1 mg / kg to about 1.0 mg / kg of the carrier per kg of subject body weight, or about 1.0 mg / kg to about 10 mg / kg of the carrier per kg of subject body weight.

[0358] Typically, the dose range of the rAAV vector is at least 1 x 10⁸ vector genomes per kilogram of subject body weight (vg / kg) or higher, for example, 1 x 10⁹ vector genomes per kilogram of subject body weight (vg / kg), 1 x 10¹⁰ vector genomes per kilogram of subject body weight (vg / kg), 1 x 10¹¹ vector genomes per kilogram of subject body weight (vg / kg), 1 x 10¹² vector genomes per kilogram of subject body weight (vg / kg), 1 x 10¹³ vector genomes per kilogram of subject body weight (vg / kg) or 1 x 10¹⁴ vector genomes per kilogram of subject body weight (vg / kg) or higher, to achieve therapeutic effects. In different embodiments, the rAAV dose is approximately 5 x 10¹¹ rAAV vg / kg or greater than approximately 5 x 10¹¹ rAAV vg / kg; approximately 1 x 10¹² rAAV vg / kg or greater than approximately 1 x 10¹² rAAV vg / kg; approximately 2 x 10¹² rAAV vg / kg or greater than approximately 2 x 10¹² rAAV vg / kg; approximately 3 x 10¹² rAAV vg / kg or greater than approximately 1 x 10¹² rAAV vg / kg;3 x 10¹² rAAV vg / kg; approximately 4 x 10¹² rAAV vg / kg or greater; approximately 5 x 10¹² rAAV vg / kg or greater; approximately 1 x 10¹³ rAAV vg / kg or greater; approximately 2 x 10¹³ rAAV vg / kg or greater; approximately 3 x 10¹³ rAAV vg / kg or greater; approximately 4 x 10¹³ rAAV vg / kg or greater; approximately 5 x 10¹³ rAAV vg / kg or greater; approximately 6 x 10¹³ rAAV vg / kg; rAAV vg / kg or greater than approximately 6 x 10¹³ rAAV vg / kg.

[0359] Examples of dose ranges for rAAV vg / kg include the range of about 5 x 10¹¹ to about 6 x 10¹³ rAAV vg / kg as described in the dosage instructions (page 41 / 106, CN 122374456 A); the range of about 5 x 10¹¹ to about 5.5 x 10¹¹ rAAV vg / kg; the range of about 5.5 x 10¹¹ to about 6 x 10¹¹ rAAV vg / kg; the range of about 6 x 10¹¹ to about 6.5 x 10¹¹ rAAV vg / kg; the range of about 6.5 x 10¹¹ to about 7 x 10¹¹ rAAV vg / kg; the range of about 7 x 10¹¹ to about 7.5 x 10¹¹ rAAV vg / kg; and the range of about 7.5 x 10¹¹ to about 8 x 10¹¹ rAAV vg / kg. The dosage range is approximately 8 x 10¹¹ to approximately 8.5 x 10¹¹ rAAV vg / kg; the dosage range is approximately 8.5 x 10¹¹ to approximately 9 x 10¹¹ rAAV vg / kg; the dosage range is approximately 9 x 10¹¹ to approximately 9.5 x 10¹¹ rAAV vg / kg; the dosage range is approximately 9.5 x 10¹¹ to approximately 1 x 10¹² rAAV vg / kg; the dosage range is approximately 1 x 10¹² to approximately 1.5 x 10¹² rAAV vg / kg; the dosage range is approximately 1.5 x 10¹² to approximately 2 x 10¹² rAAV vg / kg; the dosage range is approximately 2 x 10¹² to approximately 2.5 x 10¹² rAAV vg / kg; the dosage range is approximately 2.5 x 10¹² to approximately 3 x 10¹² rAAV vg / kg; approximatelyDosage range from 3x10¹² to approximately 3.5x10¹² rAAV vg / kg; dosage range from approximately 3.5x10¹² to approximately 4x10¹² rAAV vg / kg; dosage range from approximately 4x10¹² to approximately 4.5x10¹² rAAV vg / kg; dosage range from approximately 4.5x10¹² to approximately 5x10¹² rAAV vg / kg; dosage range from approximately 5x10¹² to approximately 5.5x10¹² rAAV vg / kg; dosage range from approximately 5.5x10¹² to approximately 6x10¹² rAAV vg / kg; dosage range from approximately 6x10¹² to approximately 6.5x10¹² rAAV vg / kg; dosage range from approximately 6.5x10¹² to approximately 7x10¹² rAAV vg / kg; dosage range from ...3x10¹² to approximately 4x10¹² rAAV vg / kg; dosage range from approximately 4x10¹² to approximately 4.5x10¹² rAAV vg / kg; dosage range from approximately 4.5x10¹² to approximately 5x10¹² rAAV vg / kg; dosage range from approximately 5x10¹² to approximately 5.5x10¹² rAAV vg / kg; dosage range from approximately 5x10¹² to approximately 6x10¹² rAAV vg / kg; dosage range from approximately 6x10¹² to approximately 6.5x10¹² rAAV vg / kg; dosage range from approximately 6.5x10¹² to approximately 7x10¹² rAAV vg / kg; dosage range from approximately 7x Dosage range from approximately 7.5 x 10¹² rAAV vg / kg; from approximately 7.5 x 10¹² to approximately 8 x 10¹² rAAV vg / kg; from approximately 8 x 10¹² to approximately 8.5 x 10¹² rAAV vg / kg; from approximately 8.5 x 10¹² to approximately 9 x 10¹² rAAV vg / kg; from approximately 9 x 10¹² to approximately 9.5 x 10¹² rAAV vg / kg; from approximately 9.5 x 10¹² to approximately 1 x 10¹³ rAAV vg / kg; from approximately 1 x 10¹³ to approximately 1.5 x 10¹³ rAAV vg / kg; from approximately 1.5 x 10¹³ to approximately 2 x 10¹³ rAAV vg / kg; from approximately 2 x 10¹³ to approximately 2.5 x 10¹³ rAAV vg / kg. Dosage ranges from approximately 2.5 x 10¹³ to approximately 3 x 10¹³ rAAV vg / kg; from approximately 3 x 10¹³ to approximately 3.5 x 10¹³ rAAV vg / kg; from approximately 3.5 x 10¹³ to approximately 4 x 10¹³ rAAV vg / kg; from approximately 4 x 10¹³ to approximately 4.5 x 10¹³ rAAV vg / kg; from approximately 4.5 x 10¹³ to approximately 5 x 10¹³ rAAV vg / kg; from approximately 5 x 10¹³ to approximately 5.5 x 10¹³ rAAV vg / kg; from approximately 5.5 x 10¹³ to approximately 6 x 10¹³ rAAV vg / kg; from approximately 6 x 10¹³ to approximately 1 x 10¹⁴ rAAV vg / kg.

[0360] In some embodiments, rAAV vg / kg is approximately 5 x 10¹¹ vg / kg, approximatelyAdministered at doses of 6 x 10¹¹ vg / kg, approximately 7 x 10¹¹ vg / kg, approximately 8 x 10¹¹ vg / kg, approximately 9 x 10¹¹ vg / kg, approximately 1 x 10¹² vg / kg, approximately 2 x 10¹² vg / kg, approximately 3 x 10¹² vg / kg, approximately 4 x 10¹² vg / kg, approximately 5 x 10¹² vg / kg, approximately 6 x 10¹² vg / kg, approximately 7 x 10¹² vg / kg, approximately 8 x 10¹² vg / kg, approximately 9 x 10¹² vg / kg, approximately 1 x 10¹³ vg / kg, approximately 2 x 10¹³ vg / kg, approximately 3 x 10¹³ vg / kg, approximately 4 x 10¹³ vg / kg, approximately 5 x 10¹³ vg / kg, or approximately 6 x 10¹³ vg / kg.

[0361] In some embodiments, the dosages and dosage ranges of other viral vectors are as provided herein with respect to rAAV. For example, in some embodiments, the dosages and dosage ranges of recombinant adenovirus vectors, recombinant retroviral vectors (e.g., lentiviruses), and recombinant herpes simplex virus vectors are the same as those shown above with respect to rAAV.

[0362] In some embodiments, the provided vector genome dosage is based on a target region, such as the caudate nucleus, putamen, and / or substantia nigra (SN). In further embodiments, the provided dosage is 3e8 vg / target brain region (TBR) to 3e11 vg / TBR, about 3e8 vg / TBR, about 3e9 vg / TBR, about 3e10 vg / TBR, or about 3e11 vg / TBR; or 3e8 vg / SN to 3e11 vg / SN, about 3e8 vg / SN, about 3e9 vg / SN, about 3e10 vg / SN, or about 3e11 vg / SN.

[0363] In some embodiments, the polynucleotide constructs, viral vectors, and nonviral vectors described herein are administered in combination with additional compounds or treatments targeting a specific disease or condition; and / or in combination with compounds that reduce the immune response to the polynucleotide, delivery medium, and / or generated proteins. Additional compounds or treatments may be provided in various ways, such as by administration alone; and may be administered or performed substantially simultaneously with or after administration of the polynucleotide constructs, viral vectors, and nonviral vectors described herein, as per page 42 / 106 of the specification 48 CN 122374456 A.

[0364] In some embodiments, the administration of the polynucleotide constructs, viral vectors, and nonviral vectors described herein is combined with one or more additional treatments targeting synucleinopathy diseases or conditions. Treatments for Parkinson's disease include, for example, dopaminergic drugs (e.g., levodopa); monoamine oxidase inhibitors and antagonists; and glial cell line-derived neurotrophic factors.(GDNF). (Lee et al. Neuroimmunol Neuroinflammation 2021;8:222-44, which is hereby incorporated herein by reference in its entirety.)

[0365] In some embodiments, (a) treatment is performed in combination with one or more additional compounds for the treatment of Parkinson's disease, the compounds being selected from dopamine decarboxylase inhibitors / dopamine precursors (e.g., carbidopa-levodopa); catechol-O-methyltransferase inhibitors that inhibit the breakdown of levodopa (e.g., entacapone, tocapone, or octopcapone); dopamine agonists (e.g., pramipexole, ropinilol, apomorphine, or rotigotine); monoamine oxidase B inhibitors that inhibit the breakdown of dopamine (e.g., selegiline, rasagiline, or safinamide); mixed mechanism inhibitors, including N-methyl-D-aspartate antagonism (e.g., amantadine and memantine); adenosine 2A Antagonists (e.g., itratheline); and / or anticholinergics (e.g., trihexyphenidyl and benzalkonium chloride) (see, for example, hypertexttransferprotocol / / www.apdaparkinson.org / what-is-parkinsons / treatment-medication / medication); and / or (b) the patient has been or is currently undergoing treatment with (a). In a further embodiment, the patient has been or is currently receiving treatment with (a) and has a reduced responsiveness to treatment involving (a).

[0366] In some embodiments, (a) the treatment is performed in combination with one or more additional compounds selected from cholinesterase inhibitors (e.g., rivastigmine, donepezil, and galantamine), N-methyl-D-aspartate antagonists (e.g., memantine and amantadine), dopamine decarboxylase inhibitors / dopamine precursors (e.g., carbidopa-levodopa), and antipsychotics (e.g., clozapine and quetiapine), and / or (b) the patient has been or is undergoing treatment with (a). In further embodiments, the patient has been or is receiving treatment with (a) and has reduced responsiveness to treatment involving (a).

[0367] In some embodiments, multiple system atrophy is treated in combination with one or more additional compounds or other treatments targeting specific symptoms. Examples of such additional compounds and treatments include medications that raise blood pressure (e.g., fludrocortisone, midodrine, pyridostigmine, and drocedopa); medications that relieve Parkinson's-like symptoms (e.g., levodopa and carbidopa); and medications that treat erectile dysfunction (e.g., sildenafil). (See, for example, hypertext transfer)protocol / / www.mayoclinic.org / diseases-conditions / multiple-system-atrophy / diagnosis-treatment / drc-20356157, which is hereby incorporated herein by reference.)

[0368] In some embodiments, an RNA polynucleotide comprising a repressive sequence targeting α-synuclein is used in combination with GDNF. In further embodiments, GDNF or a nucleic acid encoding GDNF is administered. The nucleic acid encoding GDNF may be provided on the same or different polynucleotide as the polynucleotide encoding the repressive sequence targeting α-synuclein.

[0369] In some embodiments, the administration of the polynucleotide constructs, viral vectors and non-viral vectors described herein is combined with immunosuppressants or regimens. Such agents and regimens may be used as needed to achieve immune tolerance or to mitigate the immune response to the provided polynucleotide or delivery medium. Examples of immunosuppressants and regimens include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, ImmTOR® (synthetic vaccine particle (SVP)-rapamycin (rapamycin encapsulated in biodegradable nanoparticles)), ImmTOR-ILTM (ImmTOR with a Treg-selective IL-2 agonist), B cell depletion, immunoadsorption, and plasma clearance.

[0370] In some embodiments, the polynucleotide construct, viral vector, or non-viral vector is administered with one or more immunosuppressant formulations, wherein one or more immunosuppressants are administered substantially simultaneously with or after the administration of the polynucleotide construct, viral vector, or non-viral vector. In some embodiments, one or more immunosuppressants are administered concurrently with the polynucleotide construct, viral vector, or non-viral vector. In some embodiments, one or more immunosuppressants are administered 1-12, 12-24, or 24-48 hours before administration of the polynucleotide construct, viral vector, or non-viral vector; or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50 days, or more than 50 days after administration of the polynucleotide construct, viral vector, or non-viral vector. In some embodiments, one or more immunosuppressants are administered 1-12, 12-24, or 24-48 hours after administration of the polynucleotide construct, viral vector, or non-viral vector; or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50 days, or more than 50 days after administration.

[0371] In some embodiments, the immunosuppressant is an anti-inflammatory agent. In some embodiments, the immunosuppressant is a steroid, such as a corticosteroid. In some embodiments, the immunosuppressants are prednisone, prednisolone, calcineurin inhibitors (e.g., cyclosporine, tacrolimus), MMF (mycophenolic acid, e.g., CellCept®, Myfortic®), CD52 inhibitors (e.g., alemtuzumab), CTLA4-Ig (e.g., abatacept, beraccept), anti-CD3 mAb, anti-LFA-1 mAb (e.g., elfazumab), anti-CD40 mAb (e.g., ASKP1240), anti-CD22 mAb (e.g., epazolizumab), anti-CD20 mAb (e.g., rituximab, oliguria, ovalitumumab, vitazolizumab), proteasome inhibitors (e.g., bortezomib), TACI-Ig (e.g., acecept), anti-C5 mAb (e.g., tocilizumab), mycophenolic acid esters, azathioprine, sirolimus, everolimus, TNFR-Ig, and anti-TNF. mAbs, tofacitinib, anti-IL-2R (e.g., baribizumab), anti-IL-17 mAbs (e.g., secukinumab), anti-IL-6 mAbs (e.g., anti-IL-6 antibody cilucurumab, anti-IL-6 receptor antibody tocilizumab (Actemra®)), IL-10 inhibitors, TGF-β inhibitors, B cell-targeting antibodies (e.g., rituximab), mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin), synthetic vaccine particles (SVP™)-rapamycin (rapamycin encapsulated in biodegradable nanoparticles), intravenous gamma globulin (IVIG), omalizumab, methotrexate, tyrosine kinase inhibitors (e.g., ibrutinib), cyclophosphamide, fingolimod, B cell activating factor (BAFF) inhibitors (e.g., anti-BAFF mAbs, such as belimumab), proliferation-inducing ligand (APRIL) inhibitors, anti-IL-1b mAbs (e.g., kanamycin (Haris®)). C3a inhibitors, Tregitope (see, for example, U.S. Patent No. 10,213,496), or combinations and / or derivatives thereof.

[0372] Strategies to reduce (overcome), avoid, or mitigate the effects of humoral immunity on viral vectors (such as rAAV vectors) include administering high vector doses; using empty AAV capsids as bait to adsorb anti-AAV antibodies; administering immunosuppressive drugs to reduce, decrease, suppress, prevent, or eradicate humoral immune responses to rAAV; altering the rAAV capsid serotype or engineering the rAAV capsid to make it less sensitive to neutralizing antibodies; and using plasma exchange circulation to adsorb anti-AAV immunoglobulins, thereby reducing anti-AAV.Antibody titer; and the use of delivery techniques, such as balloon catheters, followed by saline flushing. For example, such strategies are described in Mingozzi et al., (2013) Blood, 122:23-36. Similar techniques and strategies can be used for other types of viral vectors.

[0373] Empty capsids used as decoy probes, provided in the same or separate formulation as the rAAV vector, can be provided at a different ratio than the viral vector. In some embodiments, the decoy probes are provided simultaneously with rAAV. The amount of empty capsids applied can be calibrated based on the amount (titer) of antibodies generated in a particular subject. In some embodiments, the ratio of empty AAV capsid to rAAV vector is within or between about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100. In certain aspects, the applied ratio of empty AAV capsid to rAAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Preferably, the serotype of the empty capsid is the same as that of the rAAV serotype.

[0374] In some embodiments, a method bypassing blood flow and viral antibodies is used to deliver the viral vector. Examples of such techniques include delivery to the liver via the hepatic artery; and delivery to the liver via endoscopic retrograde cholangiopancreatography (ERCP). Other specification 44 / 106 pages 50 CN 122374456 A Catheter systems, such as the submandibular gland duct, can also be used as an entry point for delivering viral vectors to a subject who has or has pre-existing anti-antibodies against the viral vector.

[0375] Additional strategies for reducing humoral immunity to rAAV (which can be applied to other viral vectors) include methods for removing, depleting, capturing, and / or inactivating AAV antibodies, commonly referred to as ablation, and more particularly as plasma exchange involving blood products. Ablation or plasma exchange is a procedure in which the plasma of a human subject is circulated ex vivo (in vitro) through a device that alters the plasma by adding, removing, and / or replacing components before the plasma is returned to the patient. Plasma exchange can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from blood products (e.g., plasma). This procedure can be used to deplete, capture, inactivate, reduce, or remove AAV-bound immunoglobulins (antibodies), thereby lowering the titer of AAV antibodies in treated subjects that may contribute to rAAV neutralization. An example is the use of a device comprising an AAV capsid affinity matrix column, through which blood products (e.g., plasma) are passed, thereby producing different isotypes.Binding of AAV antibodies. (See, for example, Bertin et al., 2020, Sci. Rep. 10, 864, which is hereby incorporated herein by reference in its entirety.)

[0376] In some embodiments, polynucleotide constructs, viral vectors and nonviral vectors may be used in combination with agents that block, inhibit or reduce the interaction of IgG with neonatal Fc receptors (FcRn), such as anti-FcRn antibodies, to reduce IgG recycling and enhance in vivo IgG clearance; and / or in combination with agents that reduce the binding of circulating antibodies to recombinant viral vectors, or to nucleic acids or polynucleotide-encoded polypeptides, proteins or peptides encapsulated by recombinant viral vectors, or to polynucleotides. In some embodiments, antibodies binding to viral vectors are reduced or inhibited by agents (proteases or glycosidases) that reduce the interaction of IgG with FcRn.

[0377] In some embodiments, the polynucleotide constructs, viral vectors and nonviral vectors described herein may be used in combination with a peptidase (e.g., IdeS from Streptococcus pyogenes) or a modified variant thereof, or a glycoside endonuclease (e.g., EndoS from S. pyogenes) or a modified variant thereof. For example, such treatments may be performed to reduce or eliminate neutralizing antibodies against gene delivery media (e.g., viral vector capsids) and make it possible to treat patients previously considered unsuitable for gene therapy or patients who have developed antibodies produced by gene therapy. Such strategies are described, for example, in Leborgne et al., (2020) Nat. Med., 26:1096–1101.

[0378] VII. Kits

[0379] The present invention includes kits having packaging materials and one or more components therein. Kits typically include a label or packaging insert that includes a description of the components or instructions for the in vitro, in vivo or ex vivo use of the components. The kit may contain a collection of such components, for example, polynucleotide constructs, viral or nonviral vectors, and optionally a second active ingredient, such as another compound, agent, drug, or composition.

[0380] A kit refers to a physical structure that contains one or more components. The packaging material can sterilely hold the components and may be made of materials commonly used for such purposes, such as paper, corrugated fiber, glass, plastic, foil, ampoules, vials, and tubes.

[0381] The label or insert may include identification information for one or more of the components, dosage, and clinical pharmacology (including mechanism of action, pharmacokinetics, and pharmacodynamics) of the active ingredient. The label or insert may include information identifying the manufacturer, batch number, place and date of manufacture, and expiration date. The label or insert may include information that can be used with the kit components.Information about the disease. Labels or inserts may include instructions for clinicians or subjects regarding the use of one or more kit components in a method, use, or treatment plan or regimen. Instructions may include dosage, frequency, or duration, and instructions for implementing any of the methods, uses, treatment plans, or prevention or treatment regimens described herein.

[0382] Labels or inserts may include information about one or more benefits that the components may provide, such as prevention or treatment benefits. Labels or inserts may include information about potential adverse side effects, complications, or reactions, such as warnings to subjects or clinicians about situations where the particular composition is unsuitable. Adverse side effects or complications may also occur when subjects are taking, will take, or are currently taking one or more other drugs that may be incompatible with the composition, or when subjects are experiencing, will take, or are currently experiencing another treatment plan or regimen that may be incompatible with the composition; therefore, instructions may include information about such incompatibilities.

[0383] Labels or inserts include “printed matter,” such as paper or cardboard, or separately or adhered to components, kits, or packaging materials (e.g., boxes), or attached to ampoules, tubes, or vials containing kit components. Labels or inserts may also include computer-readable media, such as barcode printed labels, disks, optical discs such as CD-ROM or DVD-ROM / RAM, DVDs, MP3s, magnetic tapes, or electrical storage media such as RAM and ROM, or mixtures of these media such as magnetic / optical storage media, flash memory media, or memory cards.

[0384] VIII. Additional Aspects and Examples

[0385] Additional aspects, examples, and combinations thereof include the following:

[0386] 1. An RNA polynucleotide comprising a sequence identity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with respect to the sequence of any one of SEQ ID NO: 36, 38, 32-35, 37, or 39-62, and / or a targeting RNA sequence that differs from any one of SEQ ID NO: 36, 38, 32-35, 37, or 39-62 by one or two nucleotides.

[0387] 2. The RNA polynucleotide according to claim 1, wherein the targeting RNA sequence comprises a sequence selected from the group consisting of:

[0388] a) a sequence differing from SEQ ID NO: 34 in that it consists of 0, 1, 2, or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 108;

[0389] b) a sequence differing from SEQ ID NO: 36 in that it consists of 0, 1, 2, or 3 nucleotides.

[0390] c) A sequence of 0, 1, 2, or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO: 109;

[0391] d) A sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 37, wherein the target sequence comprises the sequence of SEQ ID NO: 110;

[0391] d) A sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 38, wherein the target sequence comprises the sequence of SEQ ID NO: 111;

[0392] e) A sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 39, wherein the target sequence comprises the sequence of SEQ ID NO: 112;

[0393] f) A sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 41, wherein the target sequence comprises the sequence of SEQ ID NO: 113; and

[0394] g) A sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 42, wherein the target sequence comprises the sequence of SEQ ID NO: 113; and

[0394] g) A sequence of 0, 1, 2, or 3 nucleotides, differing from SEQ ID NO: 42. 3. The RNA polynucleotide according to claim 1, wherein the target RNA sequence comprises the sequence of SEQ ID NO: 114.

[0395] 4. The RNA polynucleotide according to claim 3, wherein the target RNA sequence comprises the sequence of SEQ ID NO: 36 or SEQ ID NO: 38.

[0396] 5. The RNA polynucleotide according to any one of claims 1 to 4, wherein the RNA polynucleotide further comprises a second RNA sequence, wherein the second RNA sequence is substantially complementary to the target RNA sequence.

[0398] 6. The RNA polynucleotide according to claim 5, wherein the RNA polynucleotide is a pre-miRNA, wherein the pre-miRNA comprises a pre-miRNA scaffold, an embedded guide sequence, and an embedded passenger sequence, wherein the guide sequence comprises the target sequence and the passenger sequence comprises the second sequence.

[0399] 7. The RNA polynucleotide according to claim 6, wherein the RNA polynucleotide comprises having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of any one of SEQ ID NO: 122-128 and 207-224.Sequence identity sequence.

[0400] 8. The RNA polynucleotide according to claim 1, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 123 or 125.

[0401] 9. The RNA polynucleotide according to claim 5, wherein the RNA polynucleotide is shRNA, wherein the shRNA comprises an shRNA scaffold, an embedded guide sequence and an embedded passenger sequence, wherein the guide sequence comprises the target sequence and the passenger sequence comprises the second sequence.

[0402] 10. The RNA polynucleotide according to claim 5, wherein the RNA polynucleotide is pri-miRNA, wherein the pri-miRNA comprises a pri-miRNA scaffold, an embedded guide sequence and an embedded passenger sequence, wherein the guide sequence comprises the target sequence and the passenger sequence comprises the second sequence.

[0403] 11. The RNA polynucleotide according to claim 10, wherein the scaffold is miR-1, miR-26, miR16-1, miR-30, miR-33, miR-R101, miR-64, miR-122, miR-125, miR-135, miR-155, enhanced miR-155 (eSIBR), or miR-451 scaffold.

[0404] 12. The RNA polynucleotide according to claim 10, wherein the scaffold is an S155e scaffold, S26, S126 scaffold, S33 scaffold, or S155 scaffold. In a further embodiment, the scaffold comprises SEQ ID NO: 186 or 187.

[0405] 13. The RNA polynucleotide of claim 10, wherein the RNA polynucleotide comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 115-121, 207-224 or RNA version SEQ ID NO: 243-260 (ribose backbone, and U replaces T). In a further embodiment, the RNA polynucleotide comprises the passenger / guide combination provided in Tables 2, 3, or 4.

[0406] 14. The RNA polynucleotide of claim 11, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 116 or SEQ ID NO: 118.

[0407] 15. An optionally modified repressive RNA comprising (a) a guide strand capable of hybridizing to a target sequence of any of SEQ ID NO: 129-135; and (b)Essentially complementary passenger sequences; wherein one or more nucleotides of the guide strand and passenger strand are optionally modified RNA.

[0408] 16. A polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of 1 to 14.

[0409] 17. An expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of 1 to 14, the nucleic acid sequence being operatively linked to one or more expression control elements.

[0410] 18. The expression cassette according to 17, wherein the expression cassette comprises an upstream promoter and a downstream polyadenylation signal, the upstream promoter and the downstream polyadenylation signal being operatively linked to the nucleic acid sequence encoding the RNA polynucleotide.

[0411] 19. The expression cassette according to claim 18, wherein the expression cassette comprises, along the 5' to 3' axis, a promoter or promoter / enhancer, the nucleic acid sequence encoding the RNA polynucleotide, and a polyadenylation signal, the promoter or promoter / enhancer and the polyadenylation signal being operatively linked to the nucleic acid sequence encoding the RNA polynucleotide.

[0412] 20. The expression cassette according to claim 18 or 19, wherein the promoter provides CNS expression. Specification 47 / 106 pages 53 CN 122374456 A

[0413] 21. The expression cassette according to claim 18 or 19, wherein the promoter is an EF-1α promoter.

[0414] 22. The expression cassette according to any one of claims 17 to 21, wherein the expression cassette is DNA.

[0415] 23. The expression cassette of claim 22, wherein the expression cassette comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 168-174.

[0416] 24. The expression cassette of claim 23, wherein the expression cassette comprises the sequence of SEQ ID NO: 169 or 171.

[0417] 25. The expression cassette of any one of claims 17 to 24, further comprising a nucleic acid sequence encoding glial cell-derived neurotrophic factor (GDFN), the nucleic acid sequence being operatively linked to the promoter or operatively linked to a second promoter.

[0418] 26. A recombinant viral vector nucleic acid comprising the expression cassette of any one of claims 17 to 25, and 5' and 3' viral elements providing viral packaging and / or replication.

[0419] 27. The recombinant viral vector nucleic acid according to 26, wherein the recombinant viral vector nucleic acid is recombinant DNA and contains a 5' portion located in the recombinant viral vector nucleic acid.Adeno-associated virus (AAV) inverted repeat (ITR) sequence flanking the terminal and an AAV ITR located on the 3' terminal flanking of the recombinant viral vector nucleic acid.

[0420] 28. The recombinant viral vector nucleic acid according to 27, wherein the 5' ITR and 3' ITR are selected from the 5' ITR and 3' ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74 or AAV3B.

[0421] 29. The recombinant viral vector nucleic acid according to 26, wherein the recombinant viral vector nucleic acid is self-complementary.

[0422] 30. The recombinant viral vector nucleic acid according to claim 27, wherein the 5' ITR comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 182, and the 3' ITR comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 183.

[0423] 31. The recombinant viral vector nucleic acid according to claim 27 or 30, comprising a sequence having at least 90%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the sequences in SEQ ID NO: 175-181.

[0424] 32. The recombinant viral vector nucleic acid according to claim 27, comprising a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 176 or 178.

[0425] 33. A delivery medium comprising a viral vector or a non-viral vector and an RNA polynucleotide according to any one of 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of 17 to 25, or a recombinant viral vector nucleic acid according to any one of 26 to 32.

[0426] 34. The delivery medium according to claim 33, wherein the delivery medium is the viral vector.

[0427] 35. The delivery medium according to claim 34, wherein the viral vector is a recombinant AAV, a recombinant lentiviral vector, or a recombinant adenovirus vector.

[0428] 36. The delivery medium according to claim 35, wherein the viral vector is recombinant AAV, the recombinant AAV comprising the components according to claims 26 to 32.The recombinant viral vector nucleic acid and capsid as described in any one of the following.

[0429] 37. The delivery medium according to 36, wherein the recombinant AAV vector comprises a capsid having at least 90% sequence identity with VP1, VP2 or VP3 of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS or AAV1 / rh.10; or VP1 of SEQ ID NO: 164 or SEQ ID NO: 167.

[0430] 38. The delivery medium according to 36, wherein the coating is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10 coating; or the coating comprises VP1 of SEQ ID NO: 164 or SEQ ID NO: 167.

[0431] 39. The delivery medium according to 36, wherein the coating comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166.

[0432] 40. The delivery medium according to any one of 36 to 39, wherein the capsid is an AAV2 capsid comprising a targeting sequence of SEQ ID NO: 279, 280, 281 or 282 inserted between positions N587 and R588 corresponding to VP1.

[0433] 41. The delivery medium according to 39 or 40, wherein the recombinant viral vector nucleic acid comprises nucleic acid sequences of SEQ ID NO: 137 and 151.

[0434] 42. The delivery medium according to 41, wherein the recombinant viral vector nucleic acid comprises nucleic acid sequence of SEQ ID NO: 158.

[0435] 43. The delivery medium according to 41, wherein the recombinant viral vector nucleic acid comprises nucleic acid sequence of SEQ ID NO: 169.

[0436] 44. According to 41The delivery medium, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 176.

[0437] 45. The delivery medium according to any one of 41 to 44, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166.

[0438] 46. The delivery medium according to any one of 41 to 44, wherein the capsid is an AAV2 capsid comprising a targeting sequence of SEQ ID NO: 279 or 282 inserted between the positions N587 and R588 corresponding to VP1.

[0439] 47. The delivery medium according to claim 33, wherein the delivery medium is a nanoparticle selected from the group consisting of: lipid nanoparticles (LNP), polymer nanoparticles, lipid polymer nanoparticles (LPNP), protein- or peptide-based nanoparticles, DNA dendritic polymers or DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles and exosomes.

[0440] 48. The delivery medium according to claim 47, wherein the delivery medium is an LNP or an LPNP.

[0441] 49. A recombinant gland-associated vector comprising rAAV nucleic acid encapsulated in a capsid, wherein the rAAV nucleic acid comprises (a) a 5' ITR of SEQ ID NO: 182; (b) an EF1a promoter of SEQ ID NO: 184; (c) a pri-miRNA coding sequence comprising the sequence of SEQ ID NO: 158; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 185; and (e) a 3' ITR of SEQ ID NO: 183, wherein (c) is operatively linked to (b) and (d).

[0442] 50. The recombinant AAV vector according to claim 49, comprising at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the sequence or comprising the sequence of SEQ ID NO: 169.

[0443] 51. The recombinant AAV vector according to claim 49 or 50, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166.

[0444] 52. A pharmaceutical composition comprising: an RNA polynucleotide according to any one of 1 to 14, and a [specification missing - likely a date or date].122374456 A The repressive RNA according to 15, the polynucleotide according to 16, the expression cassette according to any one of 17 to 25, the recombinant viral vector nucleic acid according to any one of 26 to 32, the delivery medium according to any one of 33 to 48, or the rAAV vector according to any one of 49 to 51, and a pharmaceutically acceptable carrier.

[0445] 53. A method for reducing or inhibiting α-synuclein aggregation in a cell or a subject, the method comprising administering to the cell or the subject an RNA polynucleotide according to any one of 1 to 14, a repressive RNA according to 15, a polynucleotide according to 16, an expression cassette according to any one of 17 to 25, a recombinant viral vector nucleic acid according to any one of 26 to 32, a delivery medium according to any one of 33 to 48, an rAAV vector according to any one of 49 to 51, or a pharmaceutical composition according to 52.

[0446] 54. A method of treating a subject with a synucleinosis disease or condition, the method comprising administering to the subject an RNA polynucleotide according to any one of 1 to 14, a repressive RNA according to 15, a polynucleotide according to 16, an expression cassette according to any one of 17 to 25, a recombinant viral vector nucleic acid according to any one of 26 to 32, a delivery medium according to any one of 33 to 48, an rAAV vector according to any one of 49 to 51, or a pharmaceutical composition according to 52.

[0447] 55. The method according to 54, wherein the synucleinosis disease or condition is Lewy body disease or multiple system atrophy.

[0448] 56. The method of claim 54, wherein the synucleinosis disease or condition is infantile axonal dystrophy, atypical axonal dystrophy, adult paroxysmal dystonia-Parkinson's disease, autosomal recessive early-onset Parkinson's disease, POLG-related neurodegeneration, Niemann-Pick disease C1, or Krabby's disease.

[0449] 57. The method of claim 54, wherein the synucleinosis disease or condition is Parkinson's disease.

[0450] 58. A method for treating a disease or condition associated with low dopamine, the method comprising administering to the subject an RNA polynucleotide according to any one of 1 to 14, an inhibitory RNA according to 15, a polynucleotide according to 16, an expression cassette according to any one of 17 to 25, a recombinant viral vector nucleic acid according to any one of 26 to 32, or according to 33 to 48The delivery medium of any one of the following, the rAAV carrier of any one of the following 49 to 51, or the pharmaceutical composition of any one of the following 52.

[0451] 59. The method of any one of the following 53 to 58, wherein administration comprises direct intraparenchymal, intracisional, or intraventricular administration.

[0452] 60. The method of any one of the following 53 to 59, wherein initial administration is outside the CNS.

[0453] 61. The method of any one of the following 53 to 60, wherein the subject is human.

[0454] 62. The method of any one of the following 53 to 61, wherein the method reduces α-synuclein levels in a transduced brain region such as the substantia nigra to about 25% to about 70%, 30% to 65%, or 35% to 60% relative to baseline levels.

[0455] 63. The method according to any one of 53 to 62, wherein the recombinant AAV according to any one of 36 to 46 and 59 to 51 is provided at doses of 3e8 vg / SN to 3E11 vg / SN, about 3e8 vg / SN, about 3e9 vg / SN, about 3e10 vg / SN or about 3E11 vg / SN.

[0456] 64. An AAV vector genomic plasmid comprising recombinant viral nucleic acid according to any one of 26 to 32.

[0457] 65. The AAV genomic plasmid according to 64, wherein the plasmid lacks the rep and cap genes.

[0458] 66. A method for producing an rAAV vector, the method comprising the step of culturing an rAAV packaging cell line containing rAAV helper viral activity, wherein the genome of the producing cell contains nucleic acid, a rep gene, and a cap gene according to any one of 26 to 32, wherein the rAAV vector is produced.

[0459] 67. A method for producing an rAAV vector, the method comprising the step of culturing an rAAV-permitting cell containing an rAAV genome plasmid according to 64, wherein the rAAV-permitting cell further contains (a) rep and cap genes provided as part of the cell genome and / or provided by one or more separate plasmids, and (b) helper viral activity provided by the cell genome and / or provided by one or more separate plasmids.

[0460] 68. The method according to 67, wherein the rAAV-permitting cell is a packaging cell, wherein the genome of the packaging cell contains a cap gene and a rep gene.

[0461] 69. According to 67The method wherein (a) the rep gene, the cap gene, and the helper activity are provided in a single plasmid, or (b) the rep gene and the cap gene are provided in a rep / cap plasmid, and the helper activity is provided by a helper plasmid.

[0462] 70. A method for obtaining an rAAV vector, the method comprising the steps of: (a) producing the rAAV vector using the method according to any one of 66 to 69, and (b) purifying the rAAV vector.

[0463] IX. Sequences

[0464] Table 9 provides different nucleic acid and amino acid sequences. In different embodiments; the polynucleotide comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the nucleic acid sequences provided in Table 9; and the polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the amino acid sequences provided in Table 9.

[0465] Table 9 Specification 51 / 106 pages 57 CN 122374456 A

[0466] Specification 52 / 106 pages 58 CN 122374456 A Specification 53 / 106 pages 59 CN 122374456 A Specification 54 / 106 pages 60 CN 122374456 A Specification 55 / 106 pages 61 CN 122374456 A Specification 56 / 106 pages 62 CN 122374456 A Specification 57 / 106 pages 63 CN 122374456 A Specification 58 / 106 pages 64 CN 122374456 A Specification 59 / 106 pages 65 CN 122374456 A Specification 60 / 106 pages 66 CN 122374456 A Instruction manual, pages 61 / 106, 67 CN 122374456 A; pages 62 / 106, 68 CN 122374456 A; pages 63 / 106, 69 CN 122374456 A; pages 64 / 106, 70 CN 122374456 A; pages 65 / 106, 71 CN 122374456 A; pages 66 / 106, 72 CN 122374456 A; pages 67 / 106, 73 CN 122374456 A.Pages 68 / 106, 74 CN 122374456 A, Instruction Manual; Pages 69 / 106, 75 CN 122374456 A, Instruction Manual; Pages 70 / 106, 76 CN 122374456 A, Instruction Manual; Pages 71 / 106, 77 CN 122374456 A, Instruction Manual; Pages 72 / 106, 78 CN 122374456 A, Instruction Manual; Pages 73 / 106, 79 CN 122374456 A, Instruction Manual; Pages 74 / 106, 80 CN 122374456 A, Instruction Manual; Pages 85 / 106, 81 CN 122374456 A, Instruction Manual; Pages 76 / 106, 82 CN 122374456 A, Instruction Manual; Pages 77 / 106, 83 CN 122374456 A, Instruction Manual; Pages 78 / 106, 84 CN 122374456 A Instruction Manual 79 / 106 pages 85 CN 122374456 A Instruction Manual 80 / 106 pages 86 CN 122374456 A Instruction Manual 81 / 106 pages 87 CN 122374456 A Instruction Manual 82 / 106 pages 88 CN 122374456 A Instruction Manual 83 / 106 pages 89 CN 122374456 A Instruction Manual 84 / 106 pages 90 CN 122374456 A Instruction Manual 85 / 106 pages 91 CN 122374456 A Instruction Manual 86 / 106 pages 92 CN 122374456 A Instruction Manual 87 / 106 pages 93 CN 122374456 A Instruction Manual 88 / 106 pages 94 CN 122374456 A Specification 89 / 106 pages 95 CN 122374456 A Specification 90 / 106 pages 96 CN 122374456 A Specification 91 / 106 pages 97 CN 122374456 A Specification 92 / 106 pages 98 CN 122374456 A Specification 93 / 106 pages 99 CN 122374456 A

[0467] Table 10 references different miR constructs and provides SEQ ID NO: for guide RNA, passenger, seed region, pri-miRNA and pre-miRNA; and provides encoding / corresponding DNA (except for pre-miRNA). In different embodiments, the polynucleotide comprises having at leastNucleic acid sequences with 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0468] Table 10 Specification 94 / 106 pages 100 CN 122374456 A

[0469]

[0470] Examples

[0471] Examples are provided below to further illustrate different features of the invention and methods of carrying out the invention. The examples provided do not limit the claimed invention.

[0472] Example 1: Reduction of SNCA expression siRNA in HEK293 cells

[0473] The effects of different siRNAs on SNCA expression and cell survival were evaluated in HEK293T cells. Lipofectamine™ RNAiMAX (0.3 µL) containing 1 nM, 10 nM, or 100 nM siRNA was transfected into cells with a cell density of approximately 1.5 x 10⁴. SNCA expression was determined by PCR measurement of α-synuclein mRNA 72 hours after transfection. Cell viability was measured using Presto blue staining. Different constructs are summarized in Table 11. The siRNA sequences contain two 5'-deoxythymidines.

[0474] Table 11 Specification 95 / 106 pages 101 CN 122374456 A

[0475]

[0476] The effect of siRNA on SNCA expression is shown in Figure 1 and Table 12. Figure 1 shows the ability of different constructs to reduce SNCA expression, denoted as RQ. For each siRNA, the central concentration is 10 nM, the left side concentration is 1 nM, and the right side concentration is 100 nM.

[0477] Table 12 shows the siRNA names and provides some constructs with inhibition data at 100 mM.

[0478] Table 12 Specification 96 / 106 pages 102 CN 122374456 A

[0479]

[0480] Figures 2A, 2B and 2C show the survival percentage at different siRNA concentrations. Figure 2A shows the survival at 1 nM siRNA, Figure 2B shows the survival at 10 nM siRNA, and Figure 2C shows the survival at 100 nM siRNA.

[0481] Example 2: Reduced siRNA expression of SNCA in HEK293 cells

[0482] The effects of different siRNAs on SNCA expression and cell survival were evaluated in cell lines overexpressing SNCA fused to green fluorescent protein (GFP). The decrease in GFP indicates that SNCAReduced expression.

[0483] Cells were transfected with 100 nM siRNA 1-25 (see Table 11). Fluorescence from GFP was measured 72 hours after transduction (n=2). Scrambling control, commercially available siGLO (non-targeted control with red fluorescence), mismatch 11 (non-targeted control), and initial conditions were used as controls. The ability of different siRNAs to reduce SNCA expression is shown in Figure 3.

[0484] Example 3: siRNA inhibition of SNCB and SNCG expression

[0485] The effects of different constructs targeting SNCA expression on SNCB and SNCG expression were assessed using methods similar to those described in Example 1 (except for detecting SNCB and SNCG transcripts). SNCB encodes β-synuclein, and SNCG encodes γ-synuclein. β-synuclein and γ-synuclein have structures similar to α-synuclein.

[0486] Figures 4A and 4B show the effects of siRNA on off-target genes SNCB and SNCG 72 hours post-transfection using siRNA at 1 nM (left value), 10 nM (center value), and 100 nM (right value). siRNA constructs are summarized in Table 11. Figure 4A shows SNCB expression at 1 nM, 10 nM, or 100 nM siRNA. Figure 4B shows SNCG expression at 1 nM, 10 nM, or 100 nM siRNA.

[0487] Example 4: siRNA inhibition of SNCA expression in SH-SY5Y

[0488] The effects of different siRNAs on SNCA expression and cell survival were evaluated in SH-SY5Y cells (neuroblastoma cells). siRNA constructs are summarized in Table 11. As described in Example 1, cells were transduced with 10 nM or 100 nM siRNA, and expression was assessed by measuring SNCA mRNA transcripts using qPCR. Expression was measured 72 hours after transduction.

[0489] The ability of different siRNAs to inhibit expression in SH-SY5Y cells is shown in Figure 5. Cells treated with siRNA were compared with siGLO controls. The data confirmed the ability of the siRNAs tested in SH-SY5Y cells to inhibit SNCA expression.

[0490] Example 5: Differential Gene Expression Analysis

[0491] Figure 6 shows the siRNAs that targeted SNCA expression.Results of differential gene expression analysis on the ability to reduce or increase the expression of non-target genes. Generally, preferred repressive nucleic acids have a smaller effect on the expression of non-target genes.

[0492] Example 6: Pri-miRNA inhibition of SNCA

[0493] Pri-miRNA sequences were designed by modifying selected siRNA sequences based on repressive activity, effects on cell survival, and off-target effects. The siRNA guide strand was modified by replacing the 3' dT with a nucleotide complementary to the target and embedded in an S155 scaffold. The siRNA passenger strand was modified by replacing the 3' dT with a nucleotide complementary to the guide strand and removing two internal nucleotides used for embedding in the S155 scaffold.

[0494] Nucleic acids encoding different pri-miRNAs were inserted into plasmids containing reporter genes (mCherry driven by the CMV promoter) and the pri-miRNA coding sequences were placed under the control of the CBH promoter. The resulting plasmid contained CBH-miRNA-CMV-mCherry.

[0495] A plasmid encoding the human α-synuclein-GFP fusion protein was inserted into HEK293 cells. The GFP signal provided a measure of SCNA expression, while mCherry (red) positive cells indicated cells transfected with nucleic acids expressing pri-miRNA. Flow cytometry assays (CytoFLEX) were performed. The controls provided were initial (untransfected) and simulated (reagent-only transfected, without plasmid).

[0496] Table 13 provides the names of pri-miRNAs and summarizes the sequences encoding the guide DNA, passenger DNA, and pri-miRNA DNA, and provides references to the parental siRNA. Parental siRNA refers to siRNAs whose guide and passenger sequences are modified and embedded in the S155 scaffold.

[0497] Table 13

[0498]

[0499] The ability of different miRs to inhibit SCNA expression is shown in Figure 7. miR treatment significantly reduced normalized GFP signal (as a measure of SCNA expression). Specification 98 / 106 pages 104 CN 122374456 A

[0500] Example 7: In vitro inhibition of α-synuclein aggregation and expression

[0501] The ability of virally delivered nucleic acids containing nucleic acids encoding different miRNAs to inhibit α-synuclein aggregation and expression was tested in primary rat neurons. The miRNAs are summarized in Table 14. This was achieved by incorporating rAAV nucleic acids into VP1 containing SEQ ID NO: 164, VP2 containing SEQ ID NO: 165, and VP2 containing SEQ ID NO: 165.Recombinant AAV vectors were produced from the capsid of VP3 at 166. rAAV production was performed using triple transfection.

[0502] Primary rat cortical neurons were seeded in 96-well plates at 5e4 cells / well. On day 6, cells were (1) transduced with AAV1-CMV-A53Tα-Syn-WPRE (containing a recombinant viral vector encoding an α-synuclein with A53T substitution) at a multiplicity of infection (MOI) of 10,000 and with rAAV encoding miR at MOIs of 10,000 and 100,000; (2) on day 10, 1 µg / mL of human α-synuclein preformed fibrils (PFFs) were provided; and (3) on day 20, cells were fixed and permeated and stained with antibodies against α-synuclein and pS129.

[0503] The test constructs are summarized in Table 14. The pri-miRNA is operatively linked to the EF1 promoter (SEQ ID NO: 184). The control constructs are rAAV-CAG-EGFP containing a CAG promoter linked to EGFP; rAAV-EF1-mis11 containing a missense miRNA sequence; and rAAV1 / 2-3XmiR-GFP (AAV1 / 2-CAG-human-SNCA-3xmiR / GFP-WPRE-BGH-polyA) encoding a miRNA that targets α-synuclein-coding mRNA in 3 copies. (AAV1 / 2-CAG-human-SNCA-3xmiR / GFP-WPRE-BGH-polyA was obtained from Charles River).

[0504] Table 14

[0505]

[0506] Figure 8A shows the percentage of α-synuclein intensity normalized to the α-synuclein control. Different miRs significantly inhibited α-synuclein expression.

[0507] Figure 8B shows the percentage of pS129 intensity as a measure of aggregation. Aggregate formation was determined by measuring the fluorescence intensity of pS129 staining and taking images using an Opera Phenix 20X air objective. Different miRs significantly inhibited α-synuclein aggregation.

[0508] Example 8: In vivo inhibition in a cotransduction model

[0509] The ability of rAAV vectors containing viral nucleic acids encoding miR5 or miR7 to express and inhibit α-synuclein aggregation was evaluated in C57BL6 mice. Encapsulation of rAAV nucleic acids encoding miR5 or miR7 was performed as described in Example 7. The experimental design is shown in Table 15.

[0510] Table 15 ExplanationPage 99 / 106, 105 CN 122374456 A

[0511]

[0512] IPa refers to intracranial administration to both hemispheres (two values) or one hemisphere.

[0513] Inhibition of SCNA expression was assessed using qPCR to measure α-synuclein encoding mRNA transcripts. Figure 9A shows the fold change of α-synuclein relative to the rAAV1 / 2-A53T αSyn + rAAV-mis11 (mis11) control. Seven weeks after striatal injection in mice, recombinant AAV encoding miR5 and miR7 significantly reduced SCNA expression at doses of 5e9 and 1e10 vg / hemispheric. AAV1 / 2-3X miRNA was included as a positive control for knockdown.

[0514] The inhibition of α-synuclein aggregation in the frontal cortex was assessed using the α-synuclein-specific antibody syn204 at a dose of 1e10 vg / hemispheric. Protein analysis was performed using an antibody targeting human αSyn via JESS. The results are shown in Figure 9B. Both miR5 and miR7 reduced α-synuclein.

[0515] The average count of miRNA reads from small RNA sequencing showed that miR5 and miR7 were successfully expressed in the striatum of WT mice by the rAAV vector when co-transduced with AAV-A53T αSyn (Figure 9C).

[0516] The effect of the viral vector on endogenous mouse SNCA (mSCNA) is shown in Figure 9D. Overexpression of human A53T α-synuclein did not affect the expression of endogenous α-synuclein. miR7 has a one-base mismatch with mouse α-synuclein mRNA (not complementary at position 6), while miR5 has two-base mismatches with mouse α-synuclein mRNA (not complementary at positions 7 and 10). miR7 reduces α-synuclein in the striatum, while miR5 has no significant effect. Specification 100 / 106 pages 106 CN 122374456 A

[0517] Example 9: Inhibition of endogenous mSNCA expression in the substantia nigra

[0518] The ability of the rAAV vector to inhibit endogenous mSCNA expression in the substantia nigra was evaluated in mice. The experimental design is provided in Table 16.

[0519] Table 16

[0520]

[0521] SN refers to substantia nigra injection.

[0522] Figures 10A to 10D show the biodistribution and the effect on mSCNA expression. Figure 10A shows the biological distribution in the substantia nigra (SN) and striatum (Str). In the striatum, at lower...Recombinant AAV-miR5 and rAAV-miR7 vectors injected into the substantia nigra were detected under VCGN, indicating anterograde movement of the viral vector.

[0523] Figure 10B shows mSCNA expression in the substantia nigra (SN) and striatum (Str). The level of mouse endogenous α-synuclein mRNA in the striatum was significantly higher than that in the substantia nigra.

[0524] Figure 10C shows mSCNA expression in the substantia nigra (SN). Mouse endogenous α-synuclein mRNA in the substantia nigra was significantly reduced by rAAV-miR7 (1 mismatch), but not by rAAV-miR5 (two mismatches).

[0525] Figure 10D shows mSCNA expression in the striatum (Str). Mouse endogenous α-synuclein mRNA in the striatum was significantly reduced by both rAAV-miR7 (1 mismatch) and rAAV-miR5 (two mismatches).

[0526] Example 11: miRNA Processing

[0527] Figure 11 illustrates the guide and passenger processing of miR5 and miR7. Next-generation sequencing (NGS) was performed on small RNAs to evaluate miRNA processing after intraparenchymal (IP) delivery of the miRNA rAAV vector into the mouse striatum. Briefly, for C57BL / 6 mice, rAAV1 / 2-A53T αSyn (expressing human SNCA with an A53T mutation) plus rAAV-mis11 (encoding a missense miRNA) or rAAV-miR5 or rAAV-miR7 were co-injected into the mouse striatum at two doses. Seven weeks later, tissue was collected and processed for NGS sequencing of the small RNAs. The amounts of guide RNA and passenger RNA generated from two different rAAV-miRNA vectors at two different doses indicate a favorable guide-to-passenger ratio (where the guide is the dominant species), thereby driving the specific degradation of the target mRNA (Fig. 11).

[0528] Example 12: Ten-week rAAV-miR5 study

[0529] The effects of rAAV vectors containing viral nucleic acids encoding miR5, bilaterally delivered to the substantia nigra of C57BL6 mice, were evaluated ten weeks after administration. Various parameters, including body weight, biodistribution, α-synuclein knockdown, dopamine levels, and dopamine turnover, were measured. In addition, miRNA processing and effects on endogenous miRNA mechanisms were evaluated.

[0530] Recombinant rAAV nucleic acids were produced by incorporating them into capsids containing VP1 (SEQ ID NO: 164), VP2 (SEQ ID NO: 165), and VP3 (SEQ ID NO: 166).AAV vector. rAAV was generated using triple transfection.

[0531] Mice were administered a fixed dose of human αSyn (AAV1 / 2-A53TαSyn) to induce dopamine loss, as observed in synucleopathies including Parkinson's disease, and treated animals received 5e7, 5e8, or 5e9 vector genome (vg) doses of rAAV-miR5. The control was administered a diluent or miss11 (missense construct) with 5e9 vg + αSyn. Specification 101 / 106 pages 107 CN 122374456 A

[0532] Figure 12 shows the statistical significance of the endpoint weight. The endpoint weight analysis showed that the human αSyn (AAV1 / 2-A53TαSyn) and control miRNA (rAAV-CAG-mis11) treatment groups had significantly reduced weight compared to the diluent-treated animals. Compared to the control, 5e7 vg of rAAV-miR5 showed weight loss. No statistically significant weight loss was observed in the high- and medium-dose (5e9 and 5e8 vg) groups treated with rAAV-miR5.

[0533] The distribution of rAV-miR5 to the substantia nigra is shown in Figures 13A and 13B. The presence of vectors was assessed using specific primers designed based on BgH and RbG polyA sequences. The AAV1 / 2-A53TαSyn vector has BgH polyA; and rAAV-miR5 and rAAV-CAG-mis11 have RbG polyA. Figure 13A shows the measurement results of BgH polyA. Figure 13B shows the measurement results of RbG polyA.

[0534] Figure 14 shows the ability of rAAV-miR5 to knock down α-Syn as measured by qRT-PCR. Substantia nigra brain tissue was analyzed by qRT-PCR using α-Syn primers. No signal was observed in the diluent injection group, demonstrating specificity for human α-Syn. Compared with rAAV-CAG-mis11 (control miRNA), statistically significant knockdown of human α-Syn was observed in the rAAV-miR5 treatment group at doses of 5e9 (highest dose) and 5e8 (medium dose), but no statistically significant knockdown of human α-Syn was observed at 5e7 (lowest dose). Statistical analysis was performed using ordinary one-factor Anova and Tukey multiple comparison tests.

[0535] Figure 15 shows the ability of rAAV-miR5 to knock down α-Syn in the cortex as measured by JESS assay. The first diluent lacked human α-Syn, while the second diluent contained human α-Syn.α-Syn. Compared with rAAV-CAG-mis11, statistically significant knockdown of human α-Syn was observed in the rAAV-miR5 treatment group at all doses of 5e9 vg (highest dose), 5e8 vg (medium dose), and 5e7 vg (lowest dose).

[0536] In Parkinson's disease, dopaminergic neurons in the substantia nigra project into the striatum, where they release the neurotransmitter dopamine. Dopamine levels and turnover were measured to assess whether rAAV-miR5 treatment inhibited dopamine loss.

[0537] The ability of rAAV-miR5 to inhibit dopamine loss induced by A53T α-Syn is shown in Figure 16. Compared to the diluent group, significantly greater dopamine loss was observed in the striatum following administration of rAAV encoding human A53TαSyn (AAV1 / 2-A53TαSyn) and rAAV encoding the negative control miRNA (rAAV-CAG-mis11). Statistically significant inhibition of dopamine loss in the striatum was observed in all miRNA treatment groups (rAAV-miR5, 5e9 vg, 5e8 vg, and 5e7 vg) compared to the AAV1 / 2-A53TαSyn and negative control miRNA (rAAV-CAG-mis11) treatment groups.

[0538] In Parkinson's disease, the ratio of downstream dopamine metabolites (DOPAC and HVA) to dopamine (DOPAC + HVA) indicates stable dopamine turnover and healthy dopamine neurons. Figure 17 illustrates the ability of rAAV-miR5 to prevent impaired dopamine turnover. Impaired dopamine turnover was observed after treatment with AAV1 / 2-A53TαSyn and the negative control miRNA (rAAV-CAG-mis11). Protection against impaired dopamine turnover was observed in the rAAV-miR5-treated group at medium and low doses, but not at higher doses. This suggests that despite good protection against α-Syn knockdown and dopamine loss, the dopamine mechanism is impaired due to the highest total viral load.

[0539] Figure 18 shows the evaluation of microRNA processing by small RNA sequencing. Small RNA sequencing after rAAV-miR5 showed favorable expression of miRNA guide relative to the passenger chain.

[0540] Figure 19 summarizes the results of small RNA sequencing. The endogenous miRNA mechanism was not affected by rAAV-miR5 overexpression. Even at the highest dose of 5e9 vg, expressed exogenous mature miRNAs accounted for 1% of the total endogenous miRNA library. Neutralized to the lowest doses (5e8 and 5e7 vg), they accounted for [a certain percentage] of the total endogenous miRNA library.<0.1%. The diluent treatment group was the negative control group, which had no miRNA.

[0541] Example 13: SNCA-OVX Mouse Study Manual 102 / 106 pages 108 CN 122374456 A

[0542] SNCA-OVX mice were given rAAV-miR5 at 5e8 vg / SN and 5e9 vg / SN. SNCA-OVX mice were genetically altered in that two copies of the mouse SNCA gene had been genetically deleted; and contained a BAC construct that provided one copy of the human SNCA gene under the control of its endogenous promoter, which provided approximately 1.6–1.9 times the normal level of α-Syn expression. (Janezic et al., Proc Natl Acad Sci USA 2013;110(42):E4016-25). Human SNCA mRNA levels were measured by RT-qPCR.

[0543] High variability in SNCA mRNA abundance in the substantia nigra was observed between untreated males and females, and even among members of the same sex. Furthermore, male SNCA-OVX mice had significantly higher body weight than female mice, leading to inaccurate targeting of the substantia nigra, and lower distribution of rAAV-miR5 in male mice compared to female mice in the same group. While this variability makes it difficult to interpret rAAV-miR5 dosing studies, in females, at the 6-week time point, a significant 45% reduction in SNCA mRNA was observed at 5e9 vg / SN per hemisphere (Figure 20).

[0544] Example 14: Dosage Range Non-Human Primate (NHP) Study

[0545] A two-month NHP dose range study was conducted to evaluate the bilateral targeting, pharmacology, and preliminary safety of rAAV-miR5 in the substantia nigra of green monkeys (Chlorocebus sabaeus, AGM, n=2 / group, 3 doses). Recombinant AAV vectors were produced by incorporating rAAV nucleic acid into a capsid containing VP1 (SEQ ID NO: 164), VP2 (SEQ ID NO: 165), and VP3 (SEQ ID NO: 166). rAAV production was performed using triple transfection. Classical hematoxylin and eosin (H&E) staining was performed to assess potential histopathology.

[0546] The primary objective of this study was to evaluate the delivery of the gene therapy AAV vector into the substantia nigra pars compacta of male green monkeys via MRI-guided, convection-enhanced bilateral intraparenchymal (IP) infusion, followed by an 8-week observation period.Brain biodistribution. The secondary readouts of this study focused on assessing the expression levels of target genes in target brain regions, including the substantia nigra and surrounding tissues and their processes.

[0547] Each NHP received bilateral doses of the mediator, control miRNA vector (missense 11 expressed under the EF-1α promoter), or rAAV-miR5. The study design is shown in Table 17 below.

[0548] Table 17

[0549]

[0550] * 15 μL of the left hemisphere of animal C1303 was administered. Instructions for use 103 / 106 pages 109 CN 122374456 A

[0551] On day 60±3, all NHPs were euthanized and the brains were removed, placed in pre-cooled brain matrix, cut into 4 mm sections, and divided into left and right hemispheres for processing. The left hemisphere was fixed for histology, and the right hemisphere tissue was dissected or perforated and processed for biodistribution analysis.

[0552] MRI images with gadolinium contrast images show successful targeting of the substantia nigra in the test animals. Table 18 shows the estimated substantia nigra compacta coverage for each test animal. These values ​​were calculated from post-infusion images and are estimates of the anatomical target and infusion coverage. Due to the limited resolution of the 1.5T MRI scanner, the substantia nigra compacta and reticular formation could not be distinguished. The values ​​provided in Table 18 correspond to coverage of the entire anatomical structure. Given that the compacta is located on the back side of the reticular formation, the overall coverage of the compacta is expected to be higher than the estimates.

[0553] Table 18: Substantia nigra compacta coverage %

[0554]

[0555] A dose-dependent distribution and expression of rAAV-miR5 was observed, corresponding to a decrease in SNCA mRNA lasting for 2 months (the longest time point tested) after bilateral infusion of rAAV-miR5 into the substantia nigra of NHP (Figures 21A, 21B, and 21C). Overall SNCA mRNA data also indicated that rAAV-miR5-mediated SNCA mRNA reductions were primarily observed in the substantia nigra (>40%). Quantitative immunohistochemical staining of cytoplasmic α-syn protein in TH-positive DA neurons of the SN indicated a dose-dependent rAAV-miR5-mediated reduction in cytoplasmic α-Syn protein (Fig. 21D).

[0556] The distribution of rAAV-miR5 administered at doses of 3e10 vg / SN (Fig. 22A), 3e9 vg / SN (Fig. 22B), and 3e8 vg / SN (Fig. 22C) was primarily contained in the substantia nigra, with limited diffusion to the basal ganglia structures connecting to the hypothalamus and globus pallidus. Correspondingly, SNCA mRNA levels were primarily observed in the SN and some adjacent basal ganglia regions.Dose-dependent reduction (Fig. 22D). RT-qPCR analysis of SNCA mRNA was performed using a validated primer-probe combination and showed a dose-dependent reduction in SNCA mRNA in the substantia nigra (>50% reduction at a dose of 3e10 vg / SN) compared to the control group (including diluent-treated animals and the mis11 control group to account for basal variability in endogenous SNCA levels) 2 months after autopsy. Very little diffusion into the spinal cord region was observed, and no vector diffusion into any peripheral tissues outside the CNS was observed. The expression of the transgenic product miR5 followed the distribution pattern of rAAV-miR5, showing significant presence only in the substantia nigra and limited expression only in adjacent basal ganglia structures.

[0557] Overall, the data show that the biodistribution of the vector genome and the transgenic product miR5 is dose-dependent and concentrated in the substantia nigra. rAAV-miR5 did not significantly diffuse outside the injected and anatomically connected brain regions. Instructions for Use, pages 104 / 106, 110 CN 122374456 A

[0558] Neurosurgical treatment was well tolerated, with no adverse events reported during the study lifespan, and minimal histopathological changes were observed only along the needle tract in all NHPs. No rAAV-miR5-related microscopic findings were observed. All microscopic findings in the study were incidental or related to the infusion procedure. Cannulation entry points and insertion tracts consistent with the infusion procedure were visible in the brains of all NHPs and primarily included brain parenchymal loss and vacuolation, pigmented macrophages, glial degeneration, and axonal degeneration. The incidence and severity of these findings were independent of rAAV-miR5 treatment.

[0559] The NHP study demonstrated that when delivered to the brains of NHPs via neurosurgical delivery, rAAV-miR5 induces a dose-dependent decrease in the stability of both SNCA mRNA and α-Syn protein. In NHP, the surgical administration procedure, similar to that used in human patients, was well tolerated and produced good coverage of the target brain regions with minimal histopathological changes.

[0560] The expression of the transgenic product (miR5) followed the distribution pattern of rAAV-miR5, showing significant presence only in the SN and limited expression only in adjacent basal ganglia structures. rAAV-miR5-mediated SNCA mRNA reduction was observed primarily in the SN (>40%), providing reductions across the target range of 25%–70%. Overall, the data suggest that the biodistribution of the vector genome and the transgenic product was dose-dependent and concentrated in the SN. rAAV-miR5 did not significantly diffuse beyond the injection site and anatomically connected brain regions.

[0561] Example 15: Four-Month NHP Dosage Study

[0562] Considering the target α-synuclein reduction of 25%–70% and prior preclinical data, a four-month dose range study was planned using cynomolgus monkeys at doses of 3e10 vg to 3e11 vg per hemisphere to provide additional biodistribution, pharmacology, and safety data. The study design is shown in Table 19.

[0563] Table 19

[0564]

[0565] A single dose was planned to be administered via direct convection-enhanced IP infusion into the substantia nigra using a ClearPoint Orchestra™ neurosurgical frame paired with a ClearPoint SmartFlow Neuro ventriculoscope and targeting with ClearPoint Neuro navigation software. Imaging was performed using MRI (3T Siemens Skyra).

[0566] Various parameters and endpoints can be examined, such as one or more or all of the following: observations, body weight, physical examination, neurological FOB, ophthalmological examination, blood pressure, electrocardiogram, clinicopathological assessment, organ weight, and macroscopic and microscopic examinations; tissue samples collected at autopsy (multiple brain regions, DRGs [cervical, thoracic, and lumbar], adrenal glands, liver, lungs, heart, kidneys, spleen, testes, and ovaries), and blood and CSF at selected time points for biodistribution and / or biomarker analysis. Tissues for microscopic examination may include brain regions, spinal cord including nerve roots (cervical, thoracic, and lumbar), dorsal root ganglia (“DRGs”) (cervical, thoracic, and lumbar), sciatic nerve, bone, eyes, liver, spleen, heart, lungs, kidneys, skeletal muscle, stomach, testes, epididymis, ovaries, uterus, and lymph nodes (cervical and submandibular).

[0567] The levels of rAAV-miR5 genomic DNA and miRNA transgenes and cynomolgus monkey SNCA mRNA can be detected in the CNS to determine the biodistribution and magnitude (pharmacodynamics) of SNCA mRNA reduction at each dose level. The level of α-Syn protein can be measured using a variety of methods, including enhanced chemiluminescence and immunohistochemical detection methods. Specification 105 / 106 pages 111 CN 122374456 A

[0568] Example 16: Human Dosage

[0569] Dosing data from non-human primates can be used as a starting point for extrapolating to human brain targets, for example, targeting the substantia nigra to achieve a dose that reduces α-syn by 25% to 75%. For example, based on the volume difference between the substantia nigra volume of adult cynomolgus monkeys and Parkinson's disease patients at the same disease stage (modified Hoehn & Yahr 1-2.5) as the target patient population, the NHP dose can be extended to humans. Substantia nigra MRIVolume data are available from the Parkinson's Disease Progression Markers Initiative database and related published reports (Langley et al., medRxiv [Preprint]. Aug 19, 2023. 23294281).

[0570] While the invention has been described and illustrated with reference to certain specific embodiments thereof, those skilled in the art will understand that various adaptations, changes, modifications, substitutions, deletions or additions of procedures and schemes may be made without departing from the spirit and scope of the invention. Instruction Manual 106 / 106 Page 112 CN 122374456 A Figure 1 Figure 2A Instruction Manual Appendix 1 / 29 Page 113 CN 122374456 A Figure 2B Figure 2C Instruction Manual Appendix 2 / 29 Page 114 CN 122374456 A Figure 3 Figure 4A Figure 4B Instruction Manual Appendix 3 / 29 Page 115 CN 122374456 A Figure 5 Figure 6 Instruction Manual Appendix 4 / 29 Page 116 CN 122374456 A Figure 7 Figure 8A Instruction Manual Appendix 5 / 29 Page 117 CN 122374456 A Figure 8B Instruction Manual Appendix 6 / 29 Page 118 CN 122374456 A Figure 9A Figure 9B Instruction Manual Appendix 7 / 29 Page 119 CN 122374456 A Figure 9C Instruction Manual Appendix 8 / 29 Page 120 CN 122374456 A Figure 9D Instruction Manual Appendix 9 / 29 Page 121 CN 122374456 A Figure 10A Instruction Manual Appendix 10 / 29 Page 122 CN 122374456 A Figure 10B Instruction Manual Appendix 11 / 29 Page 123 CN 122374456 A Figure 10C Instruction Manual Appendix 12 / 29 Page 124 CN 122374456 A Figure 10B Instruction Manual Appendix 13 / 29 Page 125 CN 122374456 A Figure 11 Instruction Manual Appendix 14 / 29 Page 126 CN 122374456 A Figure 12 Instruction Manual Appendix 15 / 29 Page 127 CN 122374456 A Figure 13A Instruction Manual Appendix 16 / 29 Page 128 CN 122374456 A Figure 13B Instruction Manual Appendix 17 / 29 Page 129 CN 122374456 A Figure 14 Instruction Manual Drawings 18 / 29 Page 130 CN 122374456 A Figure 15 Instruction Manual Drawings 19 / 29Page 131 CN 122374456 A Figure 16 Instruction Manual Drawing 20 / 29 Page 132 CN 122374456 A Figure 17 Instruction Manual Drawing 21 / 29 Page 133 CN 122374456 A Figure 18 Figure 19 Instruction Manual Drawing 22 / 29 Page 134 CN 122374456 A Figure 20 Instruction Manual Drawing 23 / 29 Page 135 CN 122374456 A Figure 21A Instruction Manual Drawing 24 / 29 Page 136 CN 122374456 A Figure 21B Instruction Manual Drawing 25 / 29 Page 137 CN 122374456 A Figure 21C Instruction Manual Drawing 26 / 29 Page 138 CN 122374456 A Figure 21D Figure 22A Instruction Manual Drawing 27 / 29 Page 139 CN 122374456 A Figure 22B Figure 22C Appendix to the Instruction Manual, Page 28 / 29, 140 CN 122374456 A Figure 22D Appendix to the Instruction Manual, Page 29 / 29, 141 CN 122374456 A

Claims

1. An RNA polynucleotide comprising a target RNA sequence having at least 90% sequence identity with a sequence of any one of SEQ ID NO: 36, 38, 32-35, 37 or 39-62.

2. The RNA polynucleotide of claim 1, wherein the targeting RNA sequence comprises a sequence selected from the group consisting of: a) A sequence that differs from SEQ ID NO: 34 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 108; b) A sequence that differs from SEQ ID NO: 36 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 109; c) A sequence that differs from SEQ ID NO: 37 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 110; d) A sequence that differs from SEQ ID NO: 38 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 111; e) A sequence that differs from SEQ ID NO: 39 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 112; f) A sequence that differs from SEQ ID NO: 41 by 0, 1, 2, or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 113; and g) A sequence that differs from SEQ ID NO: 42 by 0, 1, 2 or 3 nucleotides, wherein the target sequence comprises the sequence of SEQ ID NO:

114.

3. The RNA polynucleotide of claim 1, wherein the targeting RNA sequence comprises the sequence of SEQ ID NO:36 or SEQ ID NO:

38.

4. The RNA polynucleotide of claim 3, wherein the targeting RNA sequence comprises the sequence of SEQ ID NO:95 or SEQ ID NO:

97.

5. The RNA polynucleotide according to any one of claims 1 to 4, wherein the RNA polynucleotide further comprises a second RNA sequence, wherein the second RNA sequence is substantially complementary to the target RNA sequence.

6. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide is a pre-miRNA, wherein the pre-miRNA comprises a pre-miRNA scaffold, an embedded guide sequence, and an embedded passenger sequence, wherein the guide sequence comprises the target sequence and the passenger sequence comprises the second sequence.

7. The RNA polynucleotide of claim 6, wherein the RNA polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 122-128 and 207-224.

8. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 123 or 125.

9. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide is shRNA, wherein the shRNA comprises an shRNA scaffold, an embedded guide sequence, and an embedded passenger sequence, wherein the guide sequence comprises the target sequence and the passenger sequence comprises the second sequence.

10. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide is pri-miRNA, wherein the pri-miRNA comprises a pri-miRNA scaffold, an embedded guide sequence, and an embedded passenger sequence, wherein the guide sequence comprises the target sequence and the passenger sequence comprises the second sequence.

11. The RNA polynucleotide of claim 10, wherein the scaffold is a miR-1, miR-26, miR16-1, miR-30, miR-33mi-R101, miR-64, miR-122, miR-125, miR-135, miR-155, enhanced miR-155 (eSIBR), or miR-451 scaffold.

12. The RNA polynucleotide of claim 10, wherein the scaffold is an S155e scaffold, an S26 scaffold, an S126 scaffold, an S33 scaffold, or an S155 scaffold.

13. The RNA polynucleotide of claim 10, wherein the RNA polynucleotide comprises a sequence having at least 90% sequence identity with any of SEQ ID NO: 115-121, 207-224, or the RNA version of SEQ ID NO: 243-260.

14. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 116 or SEQ ID NO:

118.

15. An optionally modified repressive RNA comprising (a) a guide strand capable of hybridizing to a target sequence of any of SEQ ID NO: 129-135; and (b) a substantially complementary passenger sequence; wherein one or more nucleotides of the guide strand and the passenger strand are optionally modified RNA.

16. A polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1 to 14.

17. An expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1 to 14, said nucleic acid sequence being operatively linked to one or more expression control elements.

18. The expression cassette of claim 17, wherein the expression cassette comprises an upstream promoter and a downstream polyadenylation signal operatively linked to the nucleic acid sequence encoding the RNA polynucleotide.

19. The expression cassette of claim 18, wherein the expression cassette comprises, along the 5' to 3' direction, a promoter or promoter / enhancer, the nucleic acid sequence encoding the RNA polynucleotide, and a polyadenylation signal, the promoter or promoter / enhancer and the polyadenylation signal being operatively linked to the nucleic acid sequence encoding the RNA polynucleotide.

20. The expression box of claim 18 or 19, wherein the promoter provides CNS expression.

21. The expression cassette according to claim 18 or 19, wherein the promoter is the EF-1α promoter.

22. The expression cassette according to any one of claims 17 to 21, wherein the expression cassette is DNA.

23. The expression cassette of claim 22, wherein the expression cassette comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 168-174.

24. The expression box of claim 23, wherein the expression box comprises the sequence of SEQ ID NO: 169 or 171.

25. The expression cassette according to any one of claims 17 to 24, further comprising a nucleic acid sequence encoding glial cell-derived neurotrophic factor (GDFN), said nucleic acid sequence being operatively linked to said promoter or operatively linked to a second promoter.

26. A recombinant viral vector nucleic acid comprising an expression cassette according to any one of claims 17 to 25, and 5' and 3' viral elements for viral packaging and / or replication.

27. The recombinant viral vector nucleic acid of claim 26, wherein the recombinant viral vector nucleic acid is recombinant DNA and comprises an adeno-associated virus (AAV) inverted repeat sequence (ITR) located on the 5' end flanking of the recombinant viral vector nucleic acid and an AAV ITR located on the 3' end flanking of the recombinant viral vector nucleic acid.

28. The recombinant viral vector nucleic acid according to claim 27, wherein the 5' ITR and 3' ITR are selected from the 5' ITR and 3' ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74 or AAV3B.

29. The recombinant viral vector nucleic acid according to claim 27, wherein the recombinant viral vector nucleic acid is self-complementary.

30. The recombinant viral vector nucleic acid of claim 27, wherein the 5' ITR comprises a sequence having at least 95% sequence identity with SEQ ID NO: 182, and the 3' ITR comprises a sequence having at least 95% sequence identity with SEQ ID NO:

183.

31. The recombinant viral vector nucleic acid according to claim 27 or 30, comprising a sequence having at least 90% sequence identity with the sequence of any one of SEQ ID NO: 175-181.

32. The recombinant viral vector nucleic acid according to claim 27, comprising a sequence having at least 95% sequence identity with SEQ ID NO: 176 or 178.

33. A delivery medium comprising a viral vector or a non-viral vector and an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, or a recombinant viral vector nucleic acid according to any one of claims 26 to 32.

34. The delivery medium according to claim 33, wherein the delivery medium is the viral vector.

35. The delivery medium according to claim 34, wherein the viral vector is a recombinant AAV, a recombinant lentiviral vector, or a recombinant adenovirus vector.

36. The delivery medium of claim 35, wherein the viral vector is a recombinant AAV, the recombinant AAV comprising the recombinant viral vector nucleic acid of any one of claims 26 to 32 and a capsid.

37. The delivery medium of claim 36, wherein the recombinant AAV vector comprises a capsid having at least 90% sequence identity with any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10; or VP1 of SEQ ID NO: 164 or SEQ ID NO:

167.

38. The delivery medium of claim 36, wherein the capping is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10 capping; or the capping comprises VP1 of SEQ ID NO:164 or SEQ ID NO:

167.

39. The delivery medium of claim 36, wherein the outer casing comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO:

166.

40. The delivery medium of claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequences of SEQ ID NO: 137 and 151.

41. The delivery medium according to claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO:

158.

42. The delivery medium of claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO:

169.

43. The delivery medium according to claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO:

176.

44. The delivery medium of claim 33, wherein the delivery medium is a nanoparticle selected from the group consisting of: lipid nanoparticles (LNP), polymer nanoparticles, lipid polymer nanoparticles (LPNP), protein- or peptide-based nanoparticles, DNA dendritic polymers or DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles and exosomes.

45. The delivery medium according to claim 44, wherein the delivery medium is an LNP or an LPNP.

46. ​​A recombinant gland-associated vector comprising rAAV nucleic acid encapsulated in a capsid, wherein the rAAV nucleic acid comprises (a) a 5' ITR of SEQ ID NO: 182; (b) an EF1a promoter of SEQ ID NO: 184; (c) a pri-miRNA coding sequence comprising the sequence of SEQ ID NO: 158; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 185; and (e) a 3' ITR of SEQ ID NO: 183, wherein (c) is operatively linked to (b) and (d).

47. The recombinant AAV vector of claim 46, comprising at least 95% identical sequence to SEQ ID NO:

169.

48. The recombinant AAV vector according to claim 46 or 47, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO:

166.

49. A pharmaceutical composition comprising: an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery medium according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48; and a pharmaceutically acceptable carrier.

50. A method for reducing or inhibiting α-synuclein aggregation in cells or a subject, the method comprising administering to the cells or the subject an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery medium according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48, or a pharmaceutical composition according to claim 49.

51. A method of treating a subject with synucleinosis disease or condition, the method comprising administering to the subject an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery medium according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48, or a pharmaceutical composition according to claim 49.

52. The method of claim 51, wherein the synucleinosis disease or condition is Lewy body disease or multiple system atrophy.

53. The method of claim 51, wherein the synucleinosis disease or condition is infantile axonal dystrophy, atypical axonal dystrophy, adult paroxysmal dystonia-Parkinson's disease, autosomal recessive early-onset Parkinson's disease, POLG-related neurodegeneration, Niemann-Pick disease C1, or Krabby's disease.

54. The method of claim 51, wherein the synucleinosis disease or condition is Parkinson's disease.

55. A method of treating a disease or condition associated with low dopamine, the method comprising administering to the subject an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery medium according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48, or a pharmaceutical composition according to claim 46.

56. The method according to any one of claims 50 to 55, wherein the administration comprises direct intraparenchymal, intracisional, or intraventricular administration.

57. The method according to any one of claims 50 to 56, wherein the initial application is performed outside the CNS.

58. The method according to any one of claims 50 to 57, wherein the subject is a person.

59. The method according to any one of claims 50 to 58, wherein the method reduces α-synuclein levels in the transduced brain region by about 25% to about 70% relative to baseline.

60. The method of claim 59, wherein the transduced brain region is the substantia nigra.

61. An AAV vector genomic plasmid comprising recombinant viral nucleic acid according to any one of claims 26 to 32.

62. The AAV genome plasmid according to claim 61, wherein the plasmid lacks the rep and cap genes.

63. A method for producing an rAAV vector, the method comprising the step of culturing an rAAV packaging cell line containing rAAV helper virus activity, wherein the genome of the producing cells comprises the nucleic acid, rep gene, and cap gene according to any one of claims 26 to 32, wherein the rAAV vector is produced.

64. A method for producing an rAAV vector, the method comprising the step of culturing rAAV-permitting cells containing an rAAV genomic plasmid according to claim 62, wherein the rAAV-permitting cells further contain (a) rep and cap genes as part of a cell genome and / or provided by one or more separate plasmids, and (b) helper viral activity provided by the cell genome and / or provided by one or more separate plasmids.

65. The method of claim 64, wherein the rAAV-allowed cell is a packaging cell, wherein the genome of the packaging cell comprises a cap gene and a rep gene.

66. The method of claim 64, wherein (a) the rep gene, the cap gene, and the helper activity are provided in a single plasmid, or (b) the rep gene and the cap gene are provided in a rep / cap plasmid, and the helper activity is provided by a helper plasmid.

67. A method for obtaining an rAAV vector, the method comprising the following steps: (a) producing the rAAV vector using the method according to any one of claims 63 to 66, and (b) purifying the rAAV vector.

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