Inhibitory RNA targeting huntingtin expression

Abstract

The present invention features RNA polynucleotide constructs comprising a sequence that targets Huntingtin mRNA, polynucleotide constructs comprising a sequence encoding such RNA constructs, and primary microRNA scaffolds. The sequences that comprise and / or encode targeted Huntingtin mRNA may be used, for example, to inhibit the expression of mutant HTT and / or to treat Huntington's disease.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480054282.5 (22) Application Date 2024.06.28 (30) Priority Data 63 / 511,187 2023.06.30 US 63 / 591,868 2023.10.20 US 63 / 557,370 2024.02.23 US 63 / 654,508 2024.05.31 US (85) PCT International Application Entering National Phase Date 2026.02.24 (86) PCT International Application Application Data PCT / US2024 / 036090 2024.06.28 (87) PCT International Application Publication Data WO2025 / 006937 EN 2025.01.02 (71) Applicant Spark Medical Inc. Address: Pennsylvania, USA Applicant: Genentech Inc. (72) Inventors: C. Cali, F. Kagnine, B. J. Harry, J. Savola, Q. Lee, M. M. Lee, P. L. Price, E. Ramsburg (74) Patent Agency: Beijing Kunrui Law Firm, 11494 Patent Attorney: Feng Xinqin (51) Int.Cl. C12N 15 / 113 (2006.01) A61K 31 / 7105 (2006.01) A61P 25 / 14 (2006.01) (54) Invention Title: Repressive RNA Targeting Huntington Protein Expression (57) Abstract: The present invention is characterized by an RNA polynucleotide construct comprising a sequence of a target huntingtin protein mRNA, a polynucleotide construct comprising a sequence encoding such an RNA construct, and a primary microRNA scaffold. Constructs containing a sequence targeting huntingtin protein mRNA and / or encoding a sequence targeting huntingtin protein mRNA can be used, for example, to inhibit mutant HTT expression and / or to treat Huntington's disease. Claims 8 pages, Description 104 pages, Sequence Listing (electronic publication), Figures 27 pages. CN 122095088 A 2026.05.26 CN 1 22 09 50 88 A 1. An RNA polynucleotide comprising a targeting RNA sequence that is at least 80% identical to the sequence of any one of SEQ ID NO: 1-19; provided that if the targeting sequence is at least 80% identical to SEQ ID NO: 18, then the RNA polynucleotide is (a) a primary amiRNA comprising the targeting sequence embedded in a scaffold selected from the S155e scaffold, S-26a scaffold, and S-33 scaffold; or (b) the RNA polynucleotide comprising the sequence of SEQ ID NO: 117 or 118.2. The RNA polynucleotide of claim 1, wherein the targeting RNA sequence is at least 90% identical to any one of SEQ ID NO: 1-3, 5, 6, and 16-19. 3. The RNA polynucleotide of claim 2, wherein the targeting RNA sequence comprises the sequence of any one of SEQ ID NO: 1-3, 5, 6, and 16-19. 4. The RNA polynucleotide of claim 2 or 3, wherein the RNA polynucleotide further comprises a second RNA sequence, wherein the second RNA sequence is substantially complementary to the targeting RNA sequence. 5. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a primary miRNA comprising a primary miRNA scaffold, a guide sequence, and a guest sequence, wherein the guide sequence comprises the targeting sequence and the guest sequence comprises the second RNA sequence. 6. The RNA polynucleotide of claim 5, wherein the scaffold is an S155e scaffold, an S26a scaffold, an S33 scaffold, or an S155 scaffold. 7. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 51-62, 64-68, and 78-87. 8. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises a sequence of any one of SEQ ID NO: 51-62, 64-68, and 78-87. 9. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide is composed of a sequence of any one of SEQ ID NO: 51-62, 64-68, and 78-87. 10. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a premiRNA comprising a guide sequence and a guest sequence, wherein the guide sequence comprises the target sequence and the guest sequence comprises the second RNA sequence. 11. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a shRNA comprising a guide sequence and a guest sequence, wherein the guide sequence comprises the target sequence and the guest sequence comprises the second RNA sequence. 12. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 88-96, 98-101, and 111-118. 13. The RNA polynucleotide of claim 12, wherein the RNA polynucleotide comprises a sequence of any one of SEQ ID NO: 88-96, 98-101, and 111-118. 14. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide comprises a guide sequence and a passthrough sequence.A repressive RNA double strand of a sequence, wherein the guiding sequence comprises the targeting sequence and the transit sequence comprises the second RNA sequence. 15. The RNA polynucleotide of claim 14, wherein the repressive RNA duplex has a combination of a guide strand and a guest strand selected from the following: a) a guide strand comprising the sequence of SEQ ID NO: 1 and a guest strand comprising a sequence comprising at least 80% identical to the sequence of any one of SEQ ID NO: 20, 21 and 22; b) a guide strand comprising the sequence of SEQ ID NO: 2 and a guest strand comprising a sequence comprising at least 80% identical to the sequence of any one of SEQ ID NO: 23, 24 and 25; c) a guide strand comprising the sequence of SEQ ID NO: 3 and a guest strand comprising a sequence comprising at least 80% identical to the sequence of any one of SEQ ID NO: 26, 27 and 28; d) a guide strand comprising the sequence of SEQ ID NO: 4 and a guest strand comprising a sequence comprising at least 80% identical to the sequence of SEQ ID NO: 29; e) a guide strand comprising the sequence of SEQ ID NO: 1 and a guest strand comprising a sequence comprising at least 80% identical to the sequence of SEQ ID NO: 29; f) A guide chain containing the sequence of SEQ ID NO: 5 and a pass chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 30; g) A guide chain containing the sequence of SEQ ID NO: 6 and a pass chain containing a sequence that is at least 80% identical to the sequence of any one of SEQ ID NO: 31, 32 and 33; h) A guide chain containing the sequence of SEQ ID NO: 7 and a pass chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 34; i) A guide chain containing the sequence of SEQ ID NO: 8 and a pass chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 35; j) A guide chain containing the sequence of SEQ ID NO: 9 and a pass chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 36; k) A guide chain containing the sequence of SEQ ID NO: 10 and a pass chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 37; The following are possible sequences: l) a guide chain containing the sequence of SEQ ID NO: 11 and a pass chain containing a sequence at least 80% identical to the sequence of SEQ ID NO: 39; m) a guide chain containing the sequence of SEQ ID NO: 13 and a pass chain containing a sequence at least 80% identical to the sequence of SEQ ID NO: 40; n) a guide chain containing the sequence of SEQ ID NO: 11 and a pass chain containing a sequence at least 80% identical to the sequence of SEQ ID NO: 40.The following sequences are included: o) a guide chain containing the sequence of SEQ ID NO: 15 and a pass chain containing the sequence of SEQ ID NO: 42; p) a guide chain containing the sequence of SEQ ID NO: 16 and a pass chain containing the sequence of any one of SEQ ID NO: 43, 44 and 45; q) a guide chain containing the sequence of SEQ ID NO: 17 and a pass chain containing the sequence of SEQ ID NO: 46; r) a guide chain containing the sequence of SEQ ID NO: 19 and a pass chain containing the sequence of SEQ ID NO: 47; and s) a guide chain containing the sequence of SEQ ID NO: 18 and a pass chain containing the sequence of any one of SEQ ID NO: 48, 49 and 50. 16. The RNA polynucleotide of claim 1, wherein the target sequence is a guide sequence that is at least 90% identical to SEQ ID NO: 18, and the guide sequence is embedded in an S26a scaffold or an S33 scaffold. 17. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 117 or CN 122095088 A 118. 18. The RNA polynucleotide of claim 17, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 86 or 87. 19. An optionally modified repressive RNA comprising (a) a guide strand capable of hybridizing to a target sequence of any one of SEQ ID NO: 119-137; and (b) a substantially complementary guest sequence; wherein one or more nucleotides of the guide strand and the guest strand are optionally modified RNA. 20. A polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1-19. 21. An expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1-19, and one or more expression control elements operatively linked to the nucleic acid sequence encoding the RNA polynucleotide. 22. The expression cassette of claim 21, wherein the expression cassette comprises an upstream promoter and a downstream polyadenylation signal operatively linked to the nucleic acid sequence encoding the RNA polynucleotide. 23. The expression cassette of claim 22, wherein the expression cassette from 5' to 3' comprises an expression control element operatively linked to the nucleic acid sequence encoding the RNA polynucleotide.A promoter or promoter / enhancer operatively linked to a nucleotide nucleic acid sequence, a nucleic acid sequence encoding the RNA polynucleotide, and a polyadenylation signal. 24. The expression cassette of claim 22 or 23, wherein the promoter provides high CNS expression. 25. The expression cassette of claim 22 or 23, wherein the promoter is an EF-1α promoter. 26. The expression cassette of any one of claims 21-25, wherein the expression cassette is DNA. 27. The expression cassette of claim 26, wherein the expression cassette comprises a sequence at least 90% identical to any one of SEQ ID NO: 188-199, 201-205, and 215-224. 28. The expression cassette of claim 27, wherein the expression cassette comprises the sequence of SEQ ID NO: 223 or 224. 29. A recombinant viral vector nucleic acid comprising an expression cassette of any one of claims 1-28 and 5' and / or 3' viral elements providing viral packaging and / or replication. 30. The recombinant viral vector nucleic acid of claim 29, 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. 31. The recombinant viral vector nucleic acid of claim 30, wherein the 5' ITR and the 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, and AAV3B. 32. The recombinant viral vector nucleic acid of claim 30, wherein the recombinant viral vector nucleic acid is self-complementary. 33. The recombinant viral vector nucleic acid of claim 30 or 32, wherein the 5' ITR comprises at least 95% identical sequence to SEQ ID NO: 254, and the 3' ITR comprises at least 95% identical sequence to SEQ ID NO: 253. 34. The recombinant viral vector nucleic acid of claim 30, wherein the recombinant viral vector nucleic acid comprises at least 90% identical sequence to any one of SEQ ID NO: 233-235 and 237-242. 35. A recombinant viral nucleotide comprising the sequence of any one of SEQ ID NO: 237-242. 36. A delivery medium comprising a viral or non-viral vector and the sequence of claim 19. (Claims 3 / 8, page 4, CN 122095088 A)The delivery medium of claim 36, wherein the delivery medium is the viral vector. 37. The delivery medium of claim 37, wherein the viral vector is a recombinant AAV, a recombinant lentiviral vector, or a recombinant adenovirus vector. 38. The delivery medium of claim 38, wherein the viral vector is a recombinant AAV, a recombinant lentiviral vector, or a recombinant adenovirus vector. 40. The delivery medium of claim 39, wherein the recombinant AAV vector comprises a capsid having at least 90% identical sequences to 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: 257 or SEQ ID NO: 260. 41. The delivery medium of claim 40, wherein the capsid 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 capsid; or the capsid comprises VP1 of SEQ ID NO: 257 or SEQ ID NO: 260. 42. The delivery medium of claim 36, wherein the delivery medium is nanoparticles selected from: 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. 43. The delivery medium of claim 42, wherein the delivery medium is an LNP or LPNP. 44. A pharmaceutical composition comprising the inhibitory RNA of claim 19, the polynucleotide of claim 20, the expression cassette of any one of claims 21-28, the recombinant viral vector nucleic acid of any one of claims 29-35, or the delivery medium of any one of claims 36-43; and a pharmaceutically acceptable carrier.45. A method for reducing the expression of huntingtin protein in cells or a subject, the method comprising administering to the cells or subject an inhibitory RNA according to claim 19, a polynucleotide according to claim 20, an expression cassette according to any one of claims 21-28, a recombinant viral vector nucleic acid according to any one of claims 29-35, a delivery medium according to any one of claims 36-43, or a pharmaceutical composition according to claim 44. 46. A method for treating a subject with Huntington's disease, the method comprising administering to the subject an inhibitory RNA according to claim 19, a polynucleotide according to claim 20, an expression cassette according to any one of claims 21-28, a recombinant viral vector nucleic acid according to any one of claims 29-35, a delivery medium according to any one of claims 36-43, or a pharmaceutical composition according to claim 44. 47. The method according to claim 45 or 46, wherein administration comprises direct intracranial, intracisional, or intraventricular administration. 48. The method according to claim 45 or 46, wherein the initial administration is external to the CNS. 49. The method according to any one of claims 45-48, wherein the subject is a human. 50. An AAV vector genomic plasmid, said AAV vector genomic plasmid comprising recombinant viral nucleic acid according to any one of claims 30-35. 51. The AAV genomic plasmid according to claim 50, wherein said plasmid lacks rep and cap genes. Claims 4 / 8 pages 5 CN 122095088 A 52. A method of producing an rAAV vector, said method comprising the step of culturing an rAAV packaging cell line containing rAAV helper viral activity, wherein the genome of said producing cell comprises nucleic acid, rep gene, and cap gene according to any one of claims 30-35, wherein said rAAV vector is produced. 53. A method of producing an rAAV vector, said method comprising the step of culturing rAAV-permitting cells containing an rAAV genomic plasmid according to claim 51, wherein said 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 said cell genome and / or provided by one or more separate plasmids. 54. The method of claim 53, wherein the rAAV-allowed cell is a packaging cell, wherein the genome of the packaging cell comprises a cap gene and a rep gene. 55. The method of claim 53, 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 the rep / cap plasmid and the helper activity is provided by the helper plasmid. 56. A method for obtaining an rAAV vector, the method comprising the steps of: (a) generating the rAAV vector using the method according to any one of claims 52-55 and (b) purifying the rAAV vector. 57. A polynucleotide comprising the RNA sequence of SEQ ID NO: 255, wherein N01 to N42 are ribonucleotides, N01 is complementary to N42, N02 is 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; or corresponding DNA. 58. The polynucleotide according to claim 57, the polynucleotide further comprising a 5' flanking region and a 3' flanking region, wherein the polynucleotide comprises the RNA sequence of SEQ ID NO: 256; or corresponding DNA sequence. 59. A DNA polynucleotide comprising, in the 5' to 3' direction: (a) a 5' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO: 262; (b) a CAG promoter; (c) a preamiRNA coding sequence containing the sequence of SEQ ID NO: 261, wherein the CAG promoter is operatively linked to the preamiRNA coding sequence and a polyadenylation signal; and (d) a 3' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO: 263. 60. The polynucleotide of claim 59, wherein the polyadenylation signal comprises the sequence of SEQ ID NO: 264 or 252, and the CAG promoter comprises the sequence of SEQ ID NO: 250 or 265. 61. The polynucleotide of claim 59 or 60, wherein the end of the 5' ITR to the end of the 3' ITR is at most about 2.5 kb. 62. The polynucleotide of claim 59, wherein the polynucleotide comprises the sequence of SEQ ID NO: 266. 63. The polynucleotide of any one of claims 59-62, wherein the polynucleotide is a plasmid further comprising an origin of replication and a selectivity marker. 64. The polynucleotide of any one of claims 59-62, wherein the polynucleotide is a recombinant adeno-associated virus (rAAV) nucleic acid comprising a 5' ITR at the 5' end and a 3' ITR at the 3' end. 65. A recombinant adeno-associated virus (rAAV) vector comprising: (a) the rAAV nucleic acid of claim 64; and (b) an rAAV capsid comprising: a VP1 containing the amino acid sequence of SEQ ID NO: 257, a SEQ ID NO: 266, and a SEQ ID NO: 266.66. A recombinant adeno-associated virus (rAAV) vector comprising: (a) rAAV nucleic acid containing a preamiRNA coding sequence, the preamiRNA coding sequence containing a sequence of SEQ ID NO: 261 operatively linked to an upstream promoter and a downstream multi-A, and (b) an rAAV capsid containing: VP1 containing an amino acid sequence of SEQ ID NO: 257, VP2 containing an amino acid sequence of SEQ ID NO: 258, and VP3 containing an amino acid sequence of SEQ ID NO: 259. 67. A pharmaceutical composition comprising about 1.0 x 10¹⁰ vg to about 1.0 x 10¹³ vg of the rAAV vector and pharmaceutically acceptable carrier according to claim 65 or 66. 68. The pharmaceutical composition of claim 67, wherein the pharmaceutical composition comprises about 1.0 x 10¹¹ vg to about 1.0 x 10¹² vg. 69. The composition of claim 67 or 68, wherein the composition further comprises an MRI imaging agent. 70. The composition of claim 69, wherein the MRI imaging agent is gadolinium. 71. A pharmaceutical composition comprising a sufficient amount of the rAAV carrier according to any one of claims 39-41, 65, and 66 to provide a 20% to 90% reduction in total huntingtin protein; and a pharmaceutically acceptable carrier. 72. The pharmaceutical composition of claim 71, wherein the composition comprises a sufficient amount of the rAAV carrier to provide a 20% to 65% reduction in total huntingtin protein. 73. The pharmaceutical composition of claim 72, wherein the composition comprises a sufficient amount of the rAAV carrier to provide a 25% to 40% reduction in total huntingtin protein. 74. A method of treating a subject with Huntington's disease, the method comprising administering an rAAV vector according to any one of claims 39-41, 65, and 66 or a pharmaceutical composition according to any one of claims 67-73 into the brain parenchyma of the subject. 75. A method of treating a subject with Huntington's disease, the method comprising: (a) determining the volume of the putamen and / or caudate nucleus in the right and / or left hemisphere of the subject; and (b) administering a recombinant adeno-associated virus (rAAV) vector into the brain parenchyma of the subject at a dose of about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³ to the right hemisphere and / or at a dose of about 2.0 x 10⁷ vg / mm³.A dose of approximately 2.0 x 10⁸ vg / mm³ is applied to the left hemisphere; wherein the dose is based on the volume determined in step (a). 76. The method of claim 75, wherein application to the right hemisphere comprises direct application to the putamen and caudate nucleus of the right hemisphere; and application to the left hemisphere comprises direct application to the putamen and caudate nucleus of the left hemisphere. 77. The method of claim 76, wherein the volumes of the putamen and caudate nucleus of the right hemisphere are measured, and the volumes of the putamen and caudate nucleus of the left hemisphere are measured, and each hemisphere independently receives a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ based on the measured volumes. 78. The method of any one of claims 75-77, wherein a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is applied to the right hemisphere's putamen and caudate nucleus in a proportion approximately equal to the volume of the putamen and caudate nucleus in the right hemisphere. 79. The method of any one of claims 75-78, wherein a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is applied to the right hemisphere's putamen and caudate nucleus in a ratio of approximately 67% putamen to approximately 33% caudate nucleus. 80. The method of any one of claims 75-79, wherein for the left hemisphere, a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is applied to the left hemisphere's putamen and caudate nucleus in a proportion approximately equal to the volume of the putamen and caudate nucleus in the left hemisphere. 81. The method of any one of claims 75-80, wherein a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is applied to the left hemisphere putamen and caudate nucleus at a ratio of about 67% putamen to about 33% caudate nucleus. Claims 6 / 8, page 7, CN 122095088 A 82. The method of any one of claims 75-81, wherein the same dose is applied to the right hemisphere and the left hemisphere. 83. The method of any one of claims 75-82, wherein application to the caudate nucleus includes a parietal lobe approach. 84. The method of any one of claims 75-83, wherein application to the putamen includes an occipital lobe approach. 85. The method of any one of claims 75-84, wherein 1.0 x 10¹⁰ vg to 1.0 x 10¹³ vg of rAAV carrier is administered to each hemisphere. 86. The method of claim 85, wherein 1.0 x 10¹¹ vg to 1.0 x 10¹² vg of rAAV carrier is applied to each hemisphere.87. The method of any one of claims 75-86, wherein the rAAV carrier is the rAAV carrier of any one of claims 39-41, 65, and 66. 88. The method of any one of claims 75-87, wherein administration comprises convection-enhanced delivery. 89. The method of any one of claims 75-88, the method comprising: (e) determining the volume of the putamen and / or the caudate nucleus in mm³ in the right hemisphere and / or the left hemisphere; and (f) multiplying the volume obtained in step (a) by a desired dose in vg / mm³ to further obtain the desired dose in vg / putamen and caudate nucleus; and (g) manipulating the dose obtained in step (b) with a given drug concentration in vg / ml to obtain a desired drug dose volume; and (h) administering a dose of the rAAV carrier into the brain parenchyma of the subject, wherein the dose in ml is obtained according to steps (b) and (c). 90. The method of claim 89, wherein the desired drug concentration is about 6.2 x 10¹¹ vg / ml. 91. A method of treating a subject with Huntington's disease, the method comprising: (e) determining the volume of the putamen and / or caudate nucleus in the right and / or left hemisphere, in mm³; and (f) multiplying the volume obtained in step (a) by a desired dose in vg / mm³ to further obtain the desired dose in vg / putamen and / or caudate nucleus; and (g) manipulating the dose obtained in step (b) with a given drug concentration in vg / ml to obtain a desired drug dose volume; and (h) administering a dose of recombinant adeno-associated virus (rAAV) vector into the brain parenchyma of the subject, wherein the dose in ml is obtained according to steps (b) and (c). 92. The method of claim 91, wherein the desired dose is about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³. 93. The method of claim 92, wherein the desired drug concentration is about 6.2 x 10¹¹ vg / ml. 94. The method of any one of claims 89-93, wherein step (a) measures the putamen and caudate nucleus in one or both hemispheres, and step (b) obtains a dose for both the putamen and the caudate nucleus. 95. The method of any one of claims 74-94, wherein the rAAV nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 261, and the expressed repressive RNA is measured in cerebrospinal fluid by detecting the miR155 scaffold. 96. The method of claim 95, wherein the detection of the expressed repressive nucleic acid comprises the step of: (a) from the claims...Page 7 / 8 CN 122095088 A Purification of RNA from cerebrospinal fluid; (b) reverse transcriptase; and (c) quantitative polymerase chain reaction (qPCR). 97. The method of claim 96, wherein step (a) comprises incubating cerebrospinal fluid in lysis buffer and ethanol for about 5 minutes. 98. The method of claim 96 or 97, wherein step (b) comprises about 20 µL of RNA and about 40 µL of RT. 99. The method of any one of claims 74-98, wherein the subject is a human. Claims 8 / 8 Page 9 CN 122095088 A Repressive RNA Targeting Huntington Protein Expression Cross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 511,187, filed June 30, 2023; U.S. Provisional Application No. 63 / 591,868, filed October 20, 2023; U.S. Provisional Application No. 63 / 557,370, filed February 23, 2024; and U.S. Provisional Application No. 63 / 654,508, filed May 31, 2024, the disclosure of each of which is incorporated herein by reference in its entirety. Reference to Electronically Filed Sequence Listing

[0002] The contents of the electronic sequence listing (065830.19WO.xml; size: 294,358 bytes; creation date: June 20, 2024) are incorporated herein by reference in their entirety. Background Art

[0003] Huntington's disease is a neurodegenerative disease caused by abnormal CAG amplification in exon 1 of the huntingtin protein gene. CAG amplification produces a mutant huntingtin protein with increased polyglutamine bundles. Symptoms of Huntington's disease include motor impairment, cognitive impairment, and mental disorders. A large number of different repressive RNAs have been proposed to treat Huntington's disease. (See, for example, Prister et al., Mol. Ther. Nucleic Acids. (2017) June 16; 7:324–334; Aguiar et al., Transl. Neurodegener. (2017) November 27; 6:30; Miniarkova et al., Mol. Ther. Nucleic Acids (2016) March 22; 5(3):e297; Paul et al., Cells (2020) July 15; 9(7): 1698; Wang et al., Hum. Gene. Ther. January 2022; 33(1–2):37–60; U.S. Patent No. 7,947,658; U.S. Patent No. 10,457,940; U.S. Patent No. 10,767,180; U.S. Patent No. 10,774),327; US Patent No. 10,174,321; US ​​Patent Application Publication No. 2020 / 0155624; US Patent Application Publication No. 2020 / 0377887; International Publication No. WO 2021 / 127455; and International Publication No. WO 2021 / 016505. ) Summary of the Invention

[0004] The present invention is characterized by an RNA polynucleotide construct comprising a sequence targeting HTT mRNA and a nucleotide sequence that can be used as, for example, a scaffold for the targeting sequence; and encoding a nucleic acid. The RNA polynucleotide construct comprises a repressive RNA polynucleotide comprising a sequence targeting huntingtin protein mRNA, such as artificial primary microRNA (primary amiRNA), pre-microRNA (pre-amiRNA), short hairpin RNA (shRNA), artificial microRNA (amiRNA), and optionally modified amiRNA. Constructs comprising a sequence targeting HTT mRNA and / or a coding sequence can be used, for example, in methods for inhibiting mutant HTT expression and / or treating Huntington's disease.

[0005] The HTT mRNA “target” sequence is substantially complementary to the HTT mRNA target sequence. The HTT mRNA target sequences described herein include sequences of any one of SEQ ID NO: 119-137. Preferred target sequences can be used as guide sequences for repressive RNAs. Examples of target sequences described herein include sequences that are at least 80% identical to any one of SEQ ID NO: 1-19.

[0006] The reference to “repressive” RNA polynucleotide indicates that the polynucleotide contains a sequence of a target RNA that can inhibit RNA activity. Inhibition of RNA activity leads to a reduction in protein expression from the targeted mRNA. Repressive RNAs include, for example, amiRNAs and amiRNA precursors, such as preamiRNA, shRNA, and primary amiRNA.

[0007] Therefore, a first aspect of the invention describes an RNA polynucleotide containing an RNA sequence that targets HTT mRNA. The RNA polynucleotide comprises a target RNA sequence that is at least 80% identical to any one of SEQ ID NO: 1-19; provided that if the target sequence is at least 80% identical to SEQ ID NO: 18, then the RNA polynucleotide is (a) a primary amiRNA comprising the target sequence embedded in a scaffold, the scaffold being selected from the S155e scaffold, the S26a scaffold, or the S33 scaffold; or (b) the RNA polynucleotide comprises the sequence of SEQ ID NO: 117 or 118.

[0008] A second aspect of the invention relates to an optionally modified repressive RNA duplex, the repressive RNA duplex being...The body comprises (a) a guide strand capable of hybridizing with a target sequence of any one of SEQ ID NO: 119-137; and (b) a substantially complementary guest sequence; wherein one or more nucleotides of the guide strand and the guest strand are optionally modified RNA.

[0009] A third aspect of the invention relates to a polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide, the RNA polynucleotide comprising an HTT mRNA targeting sequence.

[0010] A fourth aspect of the invention relates to an expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide and one or more expression control elements operatively coupled to the encoding nucleic acid sequence, the RNA polynucleotide comprising an HTT mRNA targeting sequence.

[0011] 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 and one or more expression control elements operatively coupled to the encoding nucleic acid sequence, the RNA polynucleotide comprising an HTT mRNA targeting sequence; and (b) 5' and / or 3' viral elements providing viral packaging and / or replication.

[0012] 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 HTT mRNA; (b) an optionally modified repressive RNA duplex targeting HTT mRNA; or (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding an RNA polynucleotide, the RNA polynucleotide comprising an HTT mRNA targeting sequence.

[0013] A seventh aspect of the invention relates to a pharmaceutical composition comprising (a) an RNA polynucleotide comprising a sequence targeting HTT mRNA, (b) an optionally modified repressive RNA duplex targeting HTT mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding a HTT mRNA targeting sequence, or (d) a delivery medium comprising (a), (b), or (c); and a pharmaceutically acceptable carrier.

[0014] An eighth aspect of the invention relates to an RNA sequence comprising SEQ ID NO: 255, wherein N01 to N42 are ribonucleotides, N01 is complementary to N41, N02 is 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; or corresponding DNA.

[0015] The DNA corresponding to the ribonucleotide provides deoxyribonucleotides, which include nitrogenous bases of thymine (T), cytidine (C), adenosine (A) and guanosine (G), wherein the complementary deoxyribonucleotides are A:T or C:G.

[0016] A ninth aspect of the present invention relates to a method for reducing mutant HTT expression and / or treating Huntington's disease in a subject, the method comprising administering (a) an RNA polynucleotide comprising a sequence targeting HTT mRNA; (b) an optionally modified repressive RNA duplex targeting HTT mRNA; (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding a target sequence of HTT mRNA; (d) a pharmaceutical composition comprising (a), (b), or (c) and a pharmaceutically acceptable carrier; or (e) a delivery medium comprising (a), (b), (c), or (d).

[0017] The tenth aspect relates to a DNA polynucleotide comprising, in the 5' to 3' direction: (a) a 5' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO: 262; (b) a CAG promoter; (c) a preamiRNA coding sequence containing the sequence of SEQ ID NO: 261, wherein the CAG promoter is operatively linked to the amiRNA coding sequence and a polyadenylation signal of the previous specification 2 / 104 pages 11 CN 122095088 A; and (d) a 3' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO: 263.

[0018] The eleventh aspect relates to a recombinant adeno-associated virus (rAAV) vector comprising: (a) rAAV nucleic acid, the rAAV nucleic acid comprising a preamiRNA coding sequence operatively linked to a promoter and a polyadenylation signal, the preamiRNA coding sequence comprising the sequence of SEQ ID NO: 261, and (b) an rAAV capsid comprising: VP1 containing the amino acid sequence of SEQ ID NO: 257, VP2 containing the amino acid sequence of SEQ ID NO: 258, and VP3 containing the amino acid sequence of SEQ ID NO: 259.

[0019] The twelfth aspect relates to a method of treating a subject with Huntington's disease, the method comprising (a) determining the volume of the putamen and / or caudate nucleus in the right and / or left hemisphere of the subject; and (b) administering a recombinant adeno-associated virus (rAAV) vector into the brain parenchyma of the subject at a dose of about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³ to the right hemisphere and / or at a dose of about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³ to the left hemisphere; wherein the dose is based on the volume determined in step (a).

[0020] The reference to "based on" with respect to the volume determined in step (a) takes into account the volume of the putamen in the right and / or left hemisphere.The dosage is provided by measuring the volume of the capsid and / or caudate nucleus. For example, 2.0 x 10⁷ vg / mm³ provides a ratio of (a) 10⁷ vg to (b) mm³ of the capsid and caudate nucleus volumes in the hemisphere. The actual unit of the ratio does not necessarily have to be vg / mm³, as long as the ratio can be converted to a reference vg:mm³.

[0021] Further aspects of the invention include (a) an RNA polynucleotide comprising a sequence targeting HTT mRNA; (b) an optionally modified repressive RNA duplex targeting HTT mRNA; (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding a target sequence of HTT mRNA; or (d) a delivery medium comprising (a), (b), or (c) and a pharmaceutically acceptable carrier for use in medicines for reducing HTT or treating Huntington's disease; and (a), (b), (c), or (d) use in the preparation of a medicament (e.g., for use in medicines for reducing HTT or treating Huntington's disease).

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

[0023] Figure 1 shows SEQ ID NO: 256 in the primary amiRNA configuration. The predicted Dicer and Drosha cleavages are shown.

[0024] Figures 2A, 2B, and 2C show the encoding constructs and results from miRNA screening. Figure 2A shows some regions present in the recombinant adeno-associated virus (rAAV) nucleic acid used in the experiments: 5' ITR, CAG promoter, primary amiRNA containing the miR155 scaffold, polyadenylation site, and 3'-ITR. Figure 2B shows the level of endogenous HTT protein in HEK293 cells transfected with a plasmid encoding different primary amiRNAs containing the miR155 scaffold. Figure 2C shows the total HTT mRNA levels from HEK293 cells transfected with different plasmids encoding different primary amiRNAs containing the S155 scaffold. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0025] Figures 3A, 3B, 3C, 3D, and 3E show the results of miRNA screening in the YAC128 HD mouse model. (See Figure 3 / 104 pages, CN 122095088 A)Figure 3A shows the biodistribution of rAAV nucleic acids from the striatum of mice injected with different rAAV vectors, each containing nucleic acids encoding different primary amiRNAs. Figure 3B shows the miRNA abundance from the striatum of mice injected with different rAAV vectors, each encoding different primary amiRNAs. Figure 3C shows the level of mutant HTT protein in the striatum of mice injected with rAAV vectors, as detected by WES immunoassay using the polyQ-specific antibody MW1. Figure 3D shows the analysis of total human HTT mRNA by RT-qPCR in mice injected with the ss.CAG.S155.miR18 vector at dilution or 1e10 vg / mouse or 3.4e10 vg / mouse. Figure 3E shows the level of mutant HTT protein in the striatum of mice injected with the ss.CAG.S155.miR18 vector at dilution or 1e10 vg / mouse or 3.4e10 vg / mouse.

[0026] Figures 4A, 4B, 4C, and 4D illustrate miR18 embedded in different primary miRNA scaffolds and the predicted Drosha and Dices cleavage sites. Figure 4A shows miR18 in the S155 scaffold. Figure 4B shows miR18 embedded in the S26a scaffold. Figure 4C shows miR18 embedded in the S155e scaffold. Figure 4D shows miR18 embedded in the S33 scaffold. Each figure shows complementary bases and, in some cases, G:U wobble.

[0027] Figures 5A, 5B, and 5C illustrate the screening results using miR18 embedded in different primary miRNA scaffolds. Figure 5A shows miR18 expression from cells transfected with the indicated construct, as measured by RT-qPCR. Figure 5B shows HTT mRNA levels from cells transfected with the indicated construct, as measured by RT-qPCR. Figure 5C shows the HTT protein levels measured by WES capillary electrophoresis using mab2166 and cells transfected with the indicated construct. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0028] Figures 6A, 6B, and 6C show the small RNA sequencing results of HEK293 cells transfected with different miR18 expression constructs. Figure 6A shows small RNA sequencing reads aligned to the guide strand (G) or guest strand (P) of the miRNA duplex from HEK293 cells transfected with the indicated miRNA expression construct. Figure 6B shows the ratio of guide sequence to guest sequence for different constructs. Figure 6C shows read pileup in cells transfected with the indicated construct.

[0029] Figures 7A and 7B show the results of in vitro tests from different miRNA and scaffold combinations. Figure 7A shows miRNA expression from HEK293 cells transfected with the indicated construct, and the expression was analyzed at 48 hours post-transfection, with results provided as copy number (CN) of miRNA per microgram of total RNA. Figure 7B shows the HTT protein level from cells transfected with the indicated construct at 72 hours post-transfection, measured by WES capillary electrophoresis using mab2166, with the results provided as HTT protein / focal adhesion protein. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0030] Figures 8A, 8B, 8C, and 8D show the results of in vitro tests from different miRNA and scaffold combinations expressed using the EF1α long promoter or the CAG promoter. Figure 8A shows miRNA expression from HEK293 cells transfected with the indicated construct. Figure 8B shows miRNA expression from HEK293 cells transfected with the indicated construct. Figure 8C shows HTT mRNA levels from cells transfected with the indicated construct, measured by RT-qPCR. Figure 8D shows HTT mRNA levels from cells transfected with different constructs, measured by RT-qPCR.

[0031] Figures 9A, 9B, 9C, 9D, and 9E show the results of guide and guest sequence processing and biodistribution of different miRNA sequences and primary miRNA scaffold combinations in YAC128 mice. Different constructs were expressed using the EF-1α promoter. Figure 9A shows the guide and guest read counts from YAC128 mice injected with vectorized miRNA / scaffold constructs, determined by small RNA sequencing. Figure 9B shows the ratio of guide to guest sequences, determined by small RNA sequencing. Figure 9C shows the reduction of mutant HTT protein in the striatum. Figure 9D shows the quantification of total mutant HTT protein from YAC128 mice injected with each miRNA / scaffold construct or diluent. Figure 9E shows the vector biodistribution and mutant HTT protein levels of the miR18-containing construct. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Specification 4 / 104 pages 13 CN 122095088 A

[0032] Figure 10 shows the effects of rAAV-miR21 administered to non-human primates (NHPs) at different doses via different routes of administration three months post-injection. NHPs (N = 6 mice / group) were injected at the indicated doses via the following routes: IPTo the caudate nucleus and putamen, ICV to the lateral ventricle, or IT to the intrathecal space of the lumbar vertebral sheath. HTT = Huntington's protein; ICV = intraventricular; IP = intraparenchymal; IT = intrathecal.

[0033] Figure 11 shows the effect and persistence of different doses of rAAV-miR21 on HTT reduction in nonhuman primates (NHP) after three and twelve months. HTT = Huntington's protein; vg = vector genome. The 25% dashed line and 50% dashed line are controls relative to 0 vg / brain.

[0034] Figures 12A-12D show the analysis of vector copy number in caudate nucleus and putamen tissue samples in NHPs administered different amounts of rAAV-miR21. Figures 12A and 12C provide the results in the caudate nucleus. Figures 12B and 12D provide the results in the putamen. rAAV-miR21 levels were reported as VGCN / µg total genomic DNA and plotted as individual values ​​(symbols) or group mean ± SD (columns and whiskers). M = 1 / μg; VGCN = vector genome copy number.

[0035] Figures 13A-13D show the analysis of HTT protein in caudate nucleus and putamen tissue samples in NHP with different amounts of rAAV-miR21. Figures 13A and 13C provide the results in the caudate nucleus. Figures 13B and 13D provide the results in the putamen. HTT = Huntington protein; M = 1 / μg; vg = vector genome.

[0036] Figures 14A and 14B show the analysis of vector copy number in caudate nucleus and putamen tissue samples in NHP with different amounts of rAAV-miR21. Figure 14A provides the results in the caudate nucleus. Figure 14B provides the results in the putamen. rAAV-miR21 levels were reported as VGCN / µg total genomic DNA and plotted as individual values ​​(symbols) or group mean ± SD (columns and whiskers). An "x" indicates the absence of a 1 x 10⁹ twelve-month cohort. M = month; VGCN = vector genome copy number; BLOQ = below limit of quantitation.

[0037] Figures 15A and 15B show the percentage of HTT protein knockdown in the caudate nucleus (Figure 15A) and putamen (Figure 15B) at different rAAV-miR21 doses at three and twelve months. HTT = huntingtin protein; SD = standard deviation; vg = vector genome; BLOQ = below limit of quantitation. HTT protein levels were reported as ng / mg total protein and plotted as individual values ​​(symbols) or group mean ± SD (columns and whiskers). An "x" indicates the absence of a 1 x 10⁹ twelve-month cohort.

[0038] Figures 16A-16E show the results from studies 005-017. Figure 16A shows the results after applying different amounts of rAAV-Quantitative analysis of mature miR21 in the CSF of NHPs with miR21. miR21 levels were reported as RCN / µg total RNA and plotted as individual values ​​(symbols) or group mean ± SD (columns and whiskers). M = month; RCN = RNA copy number. Figure 16B shows the quantitative analysis of NF-L in the CSF of NHPs administered with different amounts of rAAV-miR21, D = day; D0 = pre-injection, D92 = end point of cohort 1, D365 = end point of cohort 2, NF-L = neurofilament light chain protein, and vg = vector genome. Figure 16C shows the change in % of NF-L in the CSF from baseline (pre-injection) to the final sample collection (3 or 12 months post-injection) in NHPs administered with different amounts of rAAV-miR21, M = month, NF-L = neurofilament light chain protein, and Vg = vector genome. Figure 16D shows longitudinal measurements of caudate nucleus structure volume from D0 (pre-injection) to D365 (12 months post-injection) in NHPs administered different amounts of rAAV-miR21. Each line represents one animal, D = day, M = month, L = left, R = right, and vg = vector genome. Figure 16E shows longitudinal measurements of capsid nucleus structure volume from D0 (pre-injection) to D365 (12 months post-injection) in NHPs administered different amounts of rAAV-miR21. Each line represents one animal, D = day, M = month, L = left, R = right, and vg = vector genome. Figure 16F shows longitudinal measurements of lateral ventricle volume in NHP patients treated with different amounts of rAAV-miR21, from D0 (pre-injection) to D365 (12 months post-injection). Each line represents one animal, D = day, M = month, L = left, R = right, and vg = vector genome.

[0039] Figure 17 shows an estimate of the reduction in HTT in the putamen based on NHP studies 3 months post-rAAV-miR21 administration, expressed as vector copy number in the putamen / m³ (vg / mm3). CI = confidence interval; HTT = huntingtin protein; KD% (Instructions for Use 5 / 104 pages 14 CN 122095088) A = knockdown percentage.

[0040] Figures 18A, 18B, and 18C show the effect of rAAV.EF1α.S26.miR18 on improving motor impairment, assessed by mean time on a rotating bar. Figure 18A depicts the time, measured in seconds, of male mice remaining on the rotating bar at each time point; the time is the average of three trials at each time point. Figure 18B depicts the time males remained on the rotating bar at all time points.The average time in seconds. Figure 18C shows the average time in seconds for female mice on the rotating bar at all time points. ** indicates a P-value < 0.01, *** indicates a P-value < 0.001, and ns = not significant. Detailed Description

[0041] The present invention is characterized by an RNA polynucleotide construct comprising a sequence targeting HTT mRNA and encoding DNA, as well as a nucleotide sequence that can be used as a scaffold, for example, primary amiRNA and preamiRNA. The RNA polynucleotide constructs targeting HTT mRNA provided herein include repressive RNA polynucleotides, preamiRNA, shRNA, and optionally modified repressive RNA.

[0042] In some embodiments, the repressive RNA polynucleotide is a repressive RNA duplex. The reference to “repressive RNA duplex” refers to a duplex RNA comprising a guide strand and a guest strand targeting the RNA target region. The guest strand is sufficiently complementary to the guide strand to hybridize under physiological conditions. Association of the guide strand with an RNA-induced silencing complex (RISC) can inhibit the activity of the target RNA. In the case of mRNA targets, for example, protein expression from the targeted mRNA can be inhibited.

[0043] Repressive RNA duplexes such as amiRNA can be generated, for example, by gene expression of primary amiRNA, preamiRNA, or shRNA, followed by biogenesis; and chemical synthesis.

[0044] Different guide sequences and guest sequences can be embedded in the primary miRNA scaffold to generate primary amiRNA. The guide strand contains the target sequence and can be selected to target different RNA sequences or regions, such as 5' UTR, 3' UTR, or mRNA. For a given guide sequence, different guest sequences can be embedded in the primary miRNA scaffold. The guest strand can be optimized for a specific scaffold and provide sufficient complementarity with the guide strand to hybridize under physiological conditions.

[0045] Constructs containing sequences targeting HTT mRNA can be used, for example, in repressive RNA or for generating repressive RNA, wherein the repressive RNA can inhibit mutant HTT expression and / or treat Huntington's disease. Inhibition of mutant HTT expression can be carried out, for example, for therapeutic and research purposes. The study aims to examine the effect of suppressing mutant HTT expression in animal models.

[0046] 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 subjects.

[0047] The terms "identical," "sameness," "percentage of similarity," and similar terms refer to two animals with the largest comparison in a particular region.A sequence. The region provided is relative to the indicated reference sequence. For example, the sequence “identical” to the target sequence or the “identity” with the target sequence can be calculated by determining the number of identical nucleotides in the sequence that is aligned to provide the maximum identity, dividing that number by the total number of nucleotides in the target sequence, and multiplying by 100. Differences between aligned sequences may include deletions, substitutions, and additions.

[0048] References to the indicated percentage of identity or similarity with one or more reference sequences, and similar wording throughout the specification that indicates the indicated percentage or similarity with one or more reference sequences, provide the indicated percentage or percentage range independently of each of the reference sequences. Unless otherwise indicated (e.g., the molecule is referred to as DNA or RNA), RNA and the corresponding DNA are considered identical when determining the percentage of identity or similarity. The corresponding RNA for DNA contains uracil instead of thymine and a ribose backbone instead of a deoxyribose backbone. Specification 6 / 104 pages 15 CN 122095088 A

[0049] Unless otherwise indicated, the terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to all forms of nucleic acids and 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.

[0050] 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 have any length.

[0051] 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 microcircle, 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.

[0052] “Transgenic” refers to a nucleic acid encoding an RNA sequence, which is intended or has been introduced into a cell and is operatively linked to a promoter. Transgenics include, for example, nucleic acids encoding heterologous polynucleotide sequences and / or repressive RNA, as well as heterologous promoters.

[0053] In some embodiments, the polynucleotide construct is “CpG-reduced” or “CpG-depleted”. In some embodiments, the “CpG-reduced” or “CpG-depleted” region is those regions outside the primary amiRNA or pre-amiRNA sequence. “CpG-reduced” or “CpG-depleted” refers to (i) a nucleotide sequence in which CpG binucleates are removed from a reference nucleic acid sequence.One or more of the nucleotides (or motifs); and / or (ii) the percentage of CpG in the polynucleotides mentioned is 0% to 10%. In different 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%.

[0054] In some embodiments, the CpG motif is reduced in the 5' and / or 3' untranslated region (UTR), filler sequence, promoter, enhancer, polyadenylation signal, 5' and / or 3' ITR and / or intron.

[0055] Unless the context clearly indicates otherwise, the singular forms “an / a (a)”, “an / a (an)”, and “the” include plural indicators.

[0056] The connecting term “and / or” between multiple statement elements covers both individual options and combined options. For example, in the case of two elements combined 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 one of the options is understood to fall within the meaning of the term “and / or” and thus satisfies the requirements of the term “and / or”. The coexistence of more than one of the options is also understood to fall within the meaning of the term “and / or”.

[0057] Unless the context explicitly indicates otherwise, the terms “or” and “and” have the same meaning as “and / or”.

[0058] References to terms such as “including,” “for example,” “e.g.,” “such as,” and subsequent different members or examples are open-ended descriptions, wherein the listed members or examples are illustrative and other members or examples may be provided or used.

[0059] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably to refer to an amino acid sequence 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 cell modification.

[0060] References to the word "comprise" and variations such as "comprises" and "comprising" as used with respect to an element or group of elements are open-ended and do not exclude additional elements or method steps not listed. Terms such as "comprising," "containing," and "characterized in" are synonymous with "comprising." In the various aspects and embodiments described herein, references to open-ended terms such as "comprising" may be replaced with "consisting of" or "substantially consisting of."

[0061] The reference to “consisting of” excludes any element, step, or component not specified in the listed elements of the claim, wherein such element, step, or component relates to the claimed invention.

[0062] The reference to “consisting substantially of” limits the scope of the claim to the specified materials or steps and those materials or steps that do not materially affect one or more basic and novel features of the claimed invention.

[0063] 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, 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 value by 10%. In some cases, the term “about” refers to a value within 10% of the base parameter.

[0064] Unless the context clearly indicates otherwise, all numerical or numerical ranges include integers within such ranges as well as fractions of said values ​​or integers within such ranges. Therefore, for clarification, mentioning a reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc., and mentioning numerical ranges such as "1-4" includes 1, 2, 3, 4, as well as 1.1, 1.2, 1.3, 1.4, etc. As a further explanation, "1 to 4 weeks" includes 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

[0065] Furthermore, references to numerical ranges such as "0.01 to 10" include 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, 9.9 mg / kg, etc.

[0066] References to integers greater than or less than reference numbers include numbers greater than or less than reference numbers, respectively. Therefore, for example, mentioning more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more; and applying "two 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.

[0067] 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 acknowledged as prior art concerning any disclosed or claimed invention. In some cases, a particular reference is indicated to be incorporated herein by reference to emphasize the incorporation.

[0068] The definitions provided herein, including those in this section and other parts of this application, apply throughout this application.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0070] The specification has been divided into sections and paragraphs, and examples of various embodiments are provided. These divisions should not be considered to separate the substance of one paragraph or section or embodiment from the substance of another paragraph or section or embodiment. The description provided has a broad application and covers all combinations of various sections, paragraphs, and sentences that can be contemplated. The discussion of any embodiments is intended to be exemplary only and is not intended to imply that the scope of this disclosure (including the claims (unless otherwise stated in the claims)) is limited to these examples.

[0071] The present invention is disclosed herein using affirmative language in general to describe numerous embodiments thereof. The present invention also explicitly includes embodiments in which specific subjects, such as substances or materials, method steps and conditions, schemes or procedures, are excluded in whole or in part. For example, in some embodiments of the present invention, materials and / or method steps are excluded. Therefore, even if the present invention is not generally expressed herein as not being included in the present invention, embodiments not explicitly excluded in the present invention are still disclosed herein. Specification 8 / 104 pages 17 CN 122095088 A

[0072] I. RNA polynucleotides targeting HTT mRNA

[0073] Different HTT mRNA target regions are provided by SEQ ID NO: 119-137. RNA polynucleotides targeting specific target regions contain sequences substantially complementary to said target regions. In some embodiments, constructs containing HTT mRNA targeting sequences are repressive RNA duplexes, primary amiRNAs, preamiRNAs, or shRNAs. The HTT mRNA targeting sequences present in these constructs provide a guide strand sequence capable of hybridizing with HTT mRNA. Such constructs also contain a guest sequence that is substantially complementary to the guide sequence.

[0074] References to "substantially complementary" and similar terms indicate a region of at least 10 nucleotides that is at least 70% complementary, at least 80% complementary, at least 90% complementary, or 100% complementary. The degree of complementarity is determined based on the sequence aligned for maximum complementarity.Differences between complementary sequences include additions, deletions, and / or non-complementary bases. Complementary RNA bases are A:U and C:G. Preferably, substantially complementary sequences can hybridize with each other under physiological conditions.

[0075] In a further embodiment, the substantially 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, said region being fully complementary, at least 70% complementary, at least 80% complementary, at least 90% complementary, or 100% complementary when aligned for maximum complementarity.

[0076] In a further embodiment, the substantially 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, which is either completely complementary or differs from complete complementarity by 1, 2 or 3 nucleotides when aligned for maximum complementarity.

[0077] In some embodiments, an RNA polynucleotide is involved, said RNA polynucleotide: (1) comprising 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 sequence of any one of SEQ ID NO: 1-19; (2) comprising 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: 1-19; said consecutive nucleotides being identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 ...1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 differing from each other by 0, 1, 2 or 3 nucleotides; and / or (3) consisting of any one of SEQ ID NO: 1-19 or a sequence differing from any one of SEQ ID NO: 1-19 by 1, 2 or 3 nucleotides.

[0078] In some embodiments, regarding the target sequence and the substantially complementary targeted sequence, the nucleotide difference between the target sequence and the targeted sequence (if present) is preferably located outside the seed region (e.g., nucleotides 2-8).

[0079] In some embodiments, the guiding sequence comprises, consists of, or consists substantially of the target sequence, and the RNA polynucleotide further comprises a guest sequence substantially complementary to the guiding sequence.

[0080] In some embodiments, the guiding sequence comprising the target sequence and the guest sequence comprising a substantially complementary sequence are provided in the combination shown in Table 1, wherein the guiding sequence comprises, consists substantially of, or consists of the indicated sequence, and the guest sequence comprises, consists substantially of, or consists of the indicated sequence or a sequence differing from the indicated sequence by 1, 2, or 3 nucleotides. Table 1 Specification 9 / 104 pages 18 CN 122095088 A Nucleotides underlined in bold are not complementary to the target.

[0081] In some embodiments, the combinations of the encoding guest strand and guide strand provided in Table 1 are present in a primary amiRNA, preamiRNA, shRNA, or repressive RNA duplex.

[0082] Nucleotide differences between the guide sequence and the target sequence complement can be provided, for example, to embed the guide sequence into a specific scaffold to enhance guide strand processing of the specific scaffold (e.g., incorporation into a RISC for targeting and repressing the target RNA) and / or reduce the complementarity of the guide strand with the host RNA.

[0083] Nucleotide differences between the guide strand and the guest strand complement can be introduced into the guest sequence for different purposes (e.g., embedding into a specific scaffold) to enhance guest strand processing (e.g., guest strand degradation and non-incorporation into a RISC for targeting and repressing RNA) and / or reduce the complementarity of the guest strand with the host mRNA.

[0084] IA Repressive RNA Double-Stranded Monomer Specification 10 / 104 pages 19 CN 122095088 A

[0085] The repressive RNA double-stranded monomer can bind to an RNA-induced silencing complex (RISC) to inhibit mRNA activity. References to RISC include related proteins, such as the core argonaute (AGO) protein. Without being bound by any particular theory, the guide strand is loaded onto the RISC and hybridized with the target mRNA, wherein hybridization begins at the seed region (located at n-2 to approximately n-8 from the 5' end of the guide strand).

[0086] In some embodiments, the seed region is either perfectly complementary to its mRNA target or differs from perfect complement by 1 nucleotide.

[0087] Preferably, the guest strand is discarded by the RISC and degraded. In different embodiments, the guide strand to guest strand ratio is about 100:1, about 1000:1, or about 3000:1.

[0088] 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 incorporated herein by reference in its entirety.)

[0089] In some embodiments, the guide strand and the guest strand are independently 18 to 25 nucleotides in length and each contains a 3' overhang of 1 to 5 nucleotides. In a further embodiment, each overhang is 1, 2, 3, or 4 nucleotides. In a further embodiment, each guide strand and the guest strand are independently 20, 21, 22, or 23 nucleotides; and each overhang is 2 or 3 nucleotides. In a further embodiment, each guide strand and the guest strand are 21, 22, or 23 nucleotides, and each overhang is 2 nucleotides.

[0090] IB amiRNA

[0091] Precursor constructs such as primary amiRNA, preamiRNA, and shRNA facilitate the delivery of repressive RNA duplexes to the subject. These different constructs contain stem-loop structures, a guide strand sequence, and a guest strand sequence; and utilize intracellular miRNA biogenesis mechanisms to form repressive RNA duplexes. For example, such constructs can be provided using transgenes encoding repressive RNA.

[0092] Naturally occurring miRNA production and processing involves transcription of primary miRNA from miRNA genes and cleavage of the 3' and 5' regions mediated by drosha-DGCR8, thereby forming premiRNA. The resulting premiRNA contains a stem and a apical stem-loop of approximately 22 nucleotides, the stem having a 2-nucleotide overhang at the 3' end. (See, for example, Jin et al., (2020) Mol. Cell. May 7, 2020;78(3):423-433, which is incorporated herein by reference in its entirety.)

[0093] The primary 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 located at the 5' or 3' of the loop. When the guide strand sequence is located at the 5' of the loop, the passenger strand sequence is located at the 3' of the apical loop; and when the guide strand sequence is located at the 3' of the loop, the passenger strand sequence is located at the 5' of the loop.

[0094] The 5' and 3' flanking regions form part of the stem region and further comprise single-stranded regions. The length of the flanking regions may vary independently. In some embodiments, the 5' and 3' primary miRNA flanking regions are each at least 20 nucleotides, at least 50 nucleotides, or at least 100 nucleotides.

[0095] A primary miRNA scaffold refers to a primary miRNA region that does not include the guide strand and the guest strand sequence. The scaffold can be used as a delivery medium for the biogenesis of different repressive RNA duplexes, wherein the guide strand and the guest strand sequence are embedded in the scaffold.

[0096] Scaffolds capable of incorporating the guide strand and the guest strand sequence can be based on naturally occurring primary miRNAs, modifications of naturally occurring miRNAs, or artificially designed considering the characteristics of primary miRNAs. (See, for example, Xie et al., Mol. Ther. (2020) 28(2):422-430; Owler et al., Nucleic Acids Research (2016), 44(5):e48; Roden et al., (2017) Genome Res. 27(3):374-384; Jin et al., (2020) Mol. Specification 11 / 104 pages 20 CN 122095088 A Cell. 7;78(3): 423-433; and Fang and Bartel Genes. Mol Cell. (2015) 60(1):131-145; each of which is incorporated herein by reference in its entirety.)

[0097] Design considerations for primary miRNA scaffolds include stem length, loop size, and the presence of specific motifs that can enhance biogenesis and / or repressive RNA. The primary miRNA scaffold may contain different structures, such as G-U or U-G wobble, single base pair mismatch, protrusion, and multi-base pair mismatch.

[0098] In some embodiments, the primary miRNA scaffold includes 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.

[0099] In some embodiments, the primary miRNA scaffold is a miR-1, miR-26, miR16-1, miR-30, miR-33, miR-101, 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).

[0100] In some embodiments, the primary miRNA scaffold is S155, S155e, S26, or S33. The S155 scaffold corresponds to the scaffold present in miR155, while the S155e scaffold corresponds to the enhanced miR155 scaffold. (See Fowler et al., Nucleic Acids Research (2016), 44(5):e48). The S33 scaffold corresponds to the scaffold present in miR33. (See Xie et al., Molecular Therapy (2020) 28:2 422-430 2020.) The S26a scaffold is an artificial scaffold generated using the miR26a scaffold as a starting point. Examples of primary miRNA scaffolds containing embedded guide sequences and guest sequences are shown in Figures 4A (S155), 4B (S26), 4C (S155e), and 4D (S33).

[0101] In some embodiments, the primary amiRNA contains a mismatched GHG motif in the 3' arm of the stem.

[0102] In some embodiments, the primary amiRNA stem length is 33, 34, 35, 36, 37, 38, or 39 nucleotides.

[0103] In some embodiments, the primary amiRNA stem length is 34, 35, or 36 nucleotides.

[0104] In some embodiments, the primary amiRNA apical loop is 3 to 23 nucleotides. In a further embodiment, the loop is 10 to 23 nucleotides. In some embodiments, the guide strand and the guest strand do not extend into the loop. In some embodiments, the guide strand and / or the guest strand extend into the loop.

[0105] In some embodiments, the primary amiRNA contains a 5', 7-methylguanylic acid (m7G) cap.

[0106] In some embodiments, the primary amiRNA contains a sequence of any one of SEQ ID NO: 51-87; or a sequence that differs from any one of SEQ ID NO: 51-87 by 0, 1, 2, 3, or 4 nucleotides, and is composed of or substantially composed of therein. Nucleotide differences can be introduced at different positions, taking into account the guidance provided herein, such as different primary miRNA scaffold characteristics, complementarity of the guide strand to the target, complementarity of the guest strand to the guide strand, and adjustments for a specific scaffold.

[0107] The premiRNA produced by drosha cleavage of the primary miRNA is exported from the nucleus to the cytoplasm, where it is cleaved by dicer to produce a repressive RNA duplex. The reference to “premiRNA” indicates that RNA polynucleotides (such as those containing the guide strand and guest strand as described herein) can be cleaved by dicer to produce a repressive RNA duplex.

[0108] In some embodiments, the repressive RNA duplex produced by dicer comprises a guide strand and a guest 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.)

[0109] In some embodiments, the preamiRNA comprises a sequence of any one of SEQ ID NO: 88-118; or a sequence differing by 0, 1, 2, 3, or 4 nucleotides from any one of SEQ ID NO: 88-118 in the specification 12 / 104 pages 21 CN 122095088 A, consisting of or substantially consisting of such a sequence. Nucleotide differences may be introduced at different locations (e.g., guide sequences, loop sequences, and / or guest sequences).

[0110] In some embodiments, a transgene is used to introduce primary amiRNA into cells, the transgene comprising a sequence encoding a primary amiRNA sequence. Biogenesis of the primary amiRNA results in the production of a repressive RNA duplex. An expression cassette containing the transgene may be introduced into cells using a viral or non-viral delivery medium.

[0111] In some embodiments, a transgene is used to introduce shRNA into cells, the transgene comprising a sequence encoding a shRNA sequence. shRNA is structurally and functionally similar to premiRNA. shRNA comprises an RNA duplex containing substantially complementary arms and loops, wherein the shRNA can be exported from the nucleus to the cytoplasm and cleaved by a dicer to form a repressive RNA duplex. (Aguiardr, Transl. Neurodegener. (2017) Nov 27; 6:30.)

[0112] IC S126-based scaffolds

[0113] S126 (also referred to herein as S26a or S26) scaffolds can be used to incorporate different guide strands and transit strands. Some embodiments involve RNA containing the sequence of SEQ ID NO: 255: N01 N02 N03 N04 N05 N06 N07 N08 N09 N10 N11 N12 N13 N14 N15 N16 N17 N18 N19 N20 N21 UUGGCAGGUCCCAN22 N23 N24N25N26N27N28N29N30N31N32N33N34N35N36N37N38N39N40N41N42CG, where N01 to N42 are ribonucleotides, N01 and N42 are complementary, N02 and N41 are not complementary, N03-N10 are complementary to N33-N40, N11 and N32 are not complementary, and N12-N21 are complementary to N22-N31. In some embodiments, the guide strand after cellular processing (e.g., Dosha and Dicer cleavage) is N01-N21U + 1 nucleotide (in a further embodiment, N01-N21U), where a target sequence can be inserted into N01-N21. In some embodiments, the cell-processed transit chain is provided by N23-N42C + 1 nucleotide, and in further embodiments, the processed transit chain is N23-N42C or N23-N42CG.

[0114] In some embodiments, the RNA sequence containing SEQ ID NO: 255 has the following structure: .

[0115] In different embodiments, the RNA sequence containing SEQ ID NO: 255 can be cleaved by Dicer; or it can be cleaved by Drosha and Dicer. Figure 1 shows the predicted Dosha and Dicer cleavage sites of the RNA sequence containing SEQ ID NO: 255, and further includes 5' flanking regions and 3' flanking regions.

[0116] In some embodiments involving an RNA sequence comprising SEQ ID NO: 255, the RNA sequence further comprises a 5' flanking region and a 3' flanking region, wherein the polynucleotide comprises the RNA sequence SEQ ID NO: 256: GUGGCCGN01N02N03N04N05N06N07N08N09N10N11N12N13N14N15N16N17N18N19N20N21UGUGCAGGUCCC AN22N23N24N25N26N27N28N29N30N31N32N33N34N35N36N37N38N39N40N41N42CGGGGACGC, Wherein N01 to N42 are ribonucleotides, N01 is complementary to N42, N02 is 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 the RNA sequence.

[0117] ID Modified repressive RNA

[0118] An alternative to providing repressive RNA duplexes via transgenic expression and biogenesis is direct application of the repressive RNA duplexes. Direct application can be facilitated using modified RNA. Modified repressive RNA duplexes can be, for example, throughIt is produced 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.

[0119] Modifications to the repressive RNA can be made at different positions (e.g., 5' end, 3' end, sugar moiety, phosphate group, and nucleobase). A variety of different modifications can be made to allow or promote Watson-Crick hybridization with the target and repressive RNA activity. References describing different modifications and modification modes 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 which is hereby incorporated herein by reference in its entirety.

[0120] In some embodiments, the repressive RNA comprises one or more modifications selected from: 5' phosphate mimics (e.g., 5-(E)-vinyl phosphate, 5'-methylene phosphonate, 5'-(R)-methyl phosphate, 5'-(S)-methyl phosphate, 5'(R)-MeOCH3 phosphate, 5'-(S)-methyl-F phosphate, 5-deoxy-5'-morpholino-2'O-methyluridine, and thiophosphate); internal phosphate modifications (e.g., Rp thiophosphate, Sp thiophosphate, dithiophosphate, methoxyphosphonate, phenylethyl phosphate, 2'-5' phosphate bond, and amide bond); 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 ribonucleotide; 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'-arabinose-fluorine, 2'O-benzyl, 2'O-methyl-4-pyridine, locked nucleic acids, (s)-cET-BNA, tricyclic-DNA, PMO, unlocked nucleic acids, diol nucleosidesAcids, alpha-lipitols, and nucleic acids); and conjugated groups (e.g., lipophilic groups, hydrophilic groups, cholesterol, GalNAc, docosahexaenoic acid, or docosahexaenoic acid with a phosphocholine head group).

[0121] In some embodiments, at least 50%, at least 60%, at least 70%, at least 90%, or 100% of the nucleotides are modified.

[0122] 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 the following: 2'-OMe, 2'-F, thiophosphate, phosphodiester, and 5'-vinylphosphonate. (See, for example, Alterman et al., Nature Biotechnology (2019) 37:884-894, which is incorporated herein by reference in its entirety.)

[0123] 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 by using a coding nucleic acid.

[0124] II. Nucleic acid encoding RNA polynucleotide containing a target sequence

[0125] The RNA polynucleotide containing the target sequence can be generated from the polynucleotide using a nucleic acid sequence encoding the RNA polynucleotide. Regarding the RNA polynucleotide, the coding nucleic acid sequence provides the same sequence as the corresponding RNA polynucleotide, wherein if the coding nucleic acid is DNA, the DNA will have the nucleobase thymine instead of uracil and deoxyribose instead of ribose. The RNA polynucleotide is generated from a template strand complementary to the coding strand. The coding strand can be provided, for example, together with the template and / or used to generate the template strand.

[0126] The polynucleotide containing the nucleic acid sequence encoding the RNA polynucleotide can 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. Additional activity can be provided, for example, by encoding a protein and / or encoding a sequence that provides one or more additional repressive RNAs.

[0127] The sequences providing additional repressive RNAs can be the same or different, and can target the same or different targets. Examples of different conformations include two or more primary amiRNAs encoding the same sequence; two or more primary amiRNAs encoding the same guide strand and different scaffolds; and / or two or more primary amiRNAs encoding different guide strands. The different guide strands can target 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 primary amiRNAs, each of which can be [specification 14 / 104, page 23, CN].122095088 A Same or different.

[0128] The same type of encoding conformation for primary amiRNA can be used for a polynucleotide encoding shRNA. In some embodiments, the polynucleotide encodes 1, 2, 3, 4, or 5 shRNAs, wherein each shRNA can be the same or different.

[0129] 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 may contain, for example, nucleic acid sequences encoding different RNA polynucleotides (e.g., different primary amiRNAs or shRNAs), which are the same or different, wherein the different polynucleotides can be operatively linked to the same or different expression elements. For example, the same promoter can be coupled to nucleic acid sequences providing multiple primary amiRNAs, or two or more primary amiRNAs can be operatively linked to different promoters.

[0130] In some embodiments, the polynucleotide encoding the RNA polynucleotide comprises the following sequences, wherein the sequences (1) are 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 sequences of any one of SEQ ID NO: 138-156; (2) comprise 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: 138-156; and / or (3) comprise sequences identical to those of SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 156. Sequences differing from SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, or SEQ ID NO: 156 by 0, 1, 2, or 3 nucleotides.

[0131] In some embodiments, the target sequence is a guide strand sequence, and the sequence encoding the RNA polynucleotide further comprises a guest sequence substantially complementary to the target sequence.

[0132] In some embodiments, a guide sequence comprising a target sequence and a guest sequence comprising a substantially complementary sequence are provided in the combination shown in Table 2, wherein the guide strand comprises, substantially constitutes, or is composed of the indicated sequence, and the guest sequence comprises, substantially constitutes, or is composed of the indicated sequence or a sequence differing from the indicated sequence by 1, 2, or 3 nucleotides. Table 2 Specification 15 / 104 pages 24 CN 122095088 A

[0133] Bold underline indicates non-complementary to the target sequence.

[0134] In some embodiments, the combination of the guest strand and guide strand provided in Table 2 encodes a primary amiRNA, pre-amiRNA, or shRNA.

[0135] In some embodiments, the polynucleotide encoding RNA polynucleotide comprises a sequence that (1) 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 one of SEQ ID NO: 188-224 and 226-229; (2) comprises 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: 188-230; and / or (3) comprises the same as SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 188, SEQ ID NO: 190 ... 191. SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214. SEQ ID NO: 215. SEQ ID NO:216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ ID NO: 229 sequences differing by 0, 1, 2 or 3 nucleotides.

[0136] II.A. Expression cassette element

[0137] 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 encoding an RNA polynucleotide targeting HTT mRNA and one or more regulatory sequences, and may also comprise additional sequences. Examples of additional sequences include non-coding sequences (such as filler sequences) and coding sequences (such as sequences encoding additional RNA polynucleotides and / or sequences encoding one or more polypeptides).

[0138] An “expression control element” influences the expression of a sequence operatively linked to it. Protein expression control elements can influence, for example, transcription, translation, splicing, and information stability. Expression control elements that influence the production of RNA polynucleotides can influence, for example, transcriptional levels.

[0139] Expression control elements are typically located at the 5' (“upstream”) or 3' (“downstream”) of the transcribed nucleic acid. Expression control elements can also be located within the transcript (e.g., in an intron). Expression control elements can be located near 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, cozak sequences, posttranscriptional regulatory elements, and termination sequences.

[0140] A promoter is a DNA region where transcription begins. Typically, the transcribed nucleic acid is located at the 3' of the promoter sequence. In some embodiments, the promoter sequence is coupled to an enhancer. An enhancer is a DNA region that increases transcription of the promoter. An enhancer may be adjacent to the promoter or may be distal to it. Typically, enhancers are located upstream of the promoter, but they can also be located downstream of or within the promoter sequence.

[0141] 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 transcriptional activating proteins or other transcriptional regulators unique to that cell, tissue, or organ type. (See, for example, Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Volume II, Cold Spring)Harbor Laboratory Press, New York; and Ausubel et al., (2010) Current protocols in molecular biology, John Wiley & Sons, New York. )

[0142] Incorporation of tissue-specific regulatory elements into the expression construct provides at least partial tissue tropism for the expression of the encoded RNA polynucleotide. Refers to promoters or enhancers that are specific to a particular cell type of tissue, indicating 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), metabolotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or neurofilament heavy chain (NFH), β-globin small gene nβ2, proenkephalinogen (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters; astrocyte-specific promoters, such as glial cell proficiency promoters. (See page 26 of 17 / 104 for details. CN 122095088 A) Promoters for myelin-associated protein (GFAP) and EAAT2; oligodendrocyte-specific promoters, such as myelin basic protein (MBP) / myelin-associated glycoprotein and oligodendrocyte transcription factor 2 promoters; neuron / hypothalamus-specific promoters, such as pro-melanocortin (POMC) promoters; and neuron / spinal cord-specific promoters, such as superoxide dismutase 1 (SOD1). (See, for example, U.S. Patent Publication No. 2021 / 214749 and Adeno-Associated Virus Vectors (2019), edited Castle., 1st edition, Springer New York, New York, NY.; both of which are incorporated herein by reference in their entirety.)

[0143] Expression control elements also include ubiquitous promoters or promiscuous promoters and promoters / enhancers capable of driving polynucleotide expression in many different cell types. These components include cytomegalovirus (CMV) immediate early promoter / enhancer sequences, Rous sarcoma virus (RSV) promoter / enhancer sequences, phosphoglycerate kinase (PKG) promoters, CAG (CMV enhancers, chicken β-actin promoters)(CBA) and rabbit β-globin intron complex (see, for example, Boshart et al., (1985) Cell, 41:521-530), SV40 promoter, dihydrofolate reductase promoter and cytoplasmic b actin promoter.

[0144] Other promoters include U6 promoter, 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, heterozygous promoters and promoters with multiple tissue specificity.

[0145] In some embodiments, the promoter is the 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, is composed of, or is substantially composed of, a sequence that is at least 95%, 97%, 99%, or 100% identical to, SEQ ID NO: 251.

[0146] Expression control elements can also influence expression in a manner modulated by signals or stimuli that can increase or decrease expression. A modulating 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 repression system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline induction system (Gossen et al., Science 268: 1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); the RU486 induction system (Wang et al., Nat. Biotech. 15:239-243 (1997) and Wang et al., Gene Ther. 4:432-441). (1997); and the rapamycin induction 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.

[0147] 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 a 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 the pLIVE vector, Mirus Bio, Madison, Wisconsin); synthetic and optimized introns of human hemoglobin subunit β (HBB2); and chimeric introns, such as introns composed of the 5'- of the first human β-globin intron. Instructions 18 / 104 pages 27 CN 122095088 A Splice donor, and branches and 3'-receptor sites from an intron located between the leader sequence and the body of the immunoglobulin gene heavy chain variable region. (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.)

[0148] In some embodiments, the nucleic acid encoding an RNA polynucleotide, primary amiRNA, or shRNA containing the target sequence is located within the intron.

[0149] In some embodiments, the expression cassette contains a post-transcriptional regulatory element. Post-translational regulatory elements such as the marmot 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.)

[0150] Polyadenylation signaling sequences provide for the formation of multi-A tails, which promote nuclear export, translation, and / or mRNA stability, and may also participate in transcription termination. Examples of polyadenylation signaling sequences include the SV40 late polyadenylation signal, bovine growth hormone multi-A (bGHpA) signal sequence, synthetic multi-A, mouse β-globin pA, rabbit β-globin pA, and H4-based pA (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197.)21, 4197).

[0151] 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. The Kozak concordant sequence and variants are provided, for example, in McClements et al., (2021) Molecular Vision, 27, 233–242.

[0152] 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.

[0153] In some embodiments, the expression cassette contains, from 5' to 3', a promoter or promoter / enhancer, an intron, a nucleic acid sequence encoding an RNA polynucleotide, and a polyadenylation signal operatively linked to a nucleic acid sequence encoding an RNA polynucleotide.

[0154] In some embodiments, the expression cassette further contains a miRNA target sequence, in a further embodiment, said miRNA target sequence being incorporated into the 3' UTR of the expression cassette. The miRNA target sequence is recognized by miRNAs present in a particular cell or tissue, leading to degradation of the mRNA transcript. Based on the presence of a specific miRNA in a particular cell, the incorporation of a miRNA target sequence can be used to reduce its expression in certain cell or tissue types. Multiple tandem repeats of the miRNA target sequence can be used to increase degradation. (Geisle et al., (2016) World Journal of Experimental Medicine 6(2): 37-54.)

[0155] In some embodiments, the expression cassette encoding a nucleotide sequence but not including a sequence encoding a primary miRNA or shRNA contains any of the following: 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 contains 0% to 5%, 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0% or about 5.0% CpGs.

[0156] II.B. Recombinant Viral Vector Nucleic Acid

[0157] 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 concatermerization. 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 naturally occurring or modified forms of naturally occurring sequences. Examples of 5' and 3' elements include adenovirus ITRs, adeno-associated virus ITRs, and packaging sequences;And retroviral 5' and 3' long terminal repeat (LTR) sequences 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.) Specification 19 / 104 pages 28 CN 122095088 A

[0158] The term “recombinant” as a modifier of nucleic acid or vector indicates 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 that are not naturally linked to said 5' and / or 3' elements. Similarly, viral vectors (such as rAAV vectors) may contain a naturally occurring or modified capsid that capsids the recombinant viral vector nucleic acid.

[0159] The polynucleotides, expression cassettes, and viral vector nucleic acids are compatible with the specific viral vector. For example, rAAV containing ssDNA or dsDNA can be generated, adenoviral vectors containing dsDNA can be generated, and retroviral vectors containing ssRNA can be generated.

[0160] In some embodiments, the viral vector nucleic acid contains any of the following outside the primary amiRNA or shRNA coding region: 0-5, 0-10, 0-15, 0-50, 0-100, or 0 to 150 CpG; 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, 44, 46, 47, 48, 49 or 50 CpG; and / or 0% 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.

[0161] II.C. miR21

[0162] The examples provided below include data demonstrating the role of an rAAV vector containing miR21-encoded nucleic acid (SEQ ID NO: 261) in animal models of Huntington's disease and NHP. miR21 is employed using a miR155 scaffold.

[0163] Some embodiments relate to a DNA polynucleotide comprising, in the 5' to 3' direction: (a) a 5' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO: 262; (b)(c) a CAG promoter; (d) a preamiRNA coding sequence containing the sequence of SEQ ID NO: 261, wherein the CAG promoter is operatively linked to the preamiRNA coding sequence and the polyadenylation signal; and (e) a 3' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO: 263.

[0164] In some further embodiments, the polyadenylation signal comprises the sequence of SEQ ID NO: 252 or 264.

[0165] In a further embodiment, the CAG promoter comprises the sequence of SEQ ID NO: 250 or 265.

[0166] In some embodiments, the DNA polynucleotide is self-complementary rAAV and is at most about 2.5 kb.

[0167] In some further embodiments, the DNA polynucleotide comprises the sequence of SEQ ID NO: 266.

[0168] In some embodiments, the polynucleotide is a plasmid further comprising an origin of replication and a selectivity marker.

[0169] In some embodiments, the polynucleotide is a recombinant adeno-associated virus (rAAV) nucleic acid containing a 5' ITR at the 5' end and a 3' ITR at the 3' end. III. Viral Vector

[0170] In some embodiments, the gene delivery medium is a viral vector. A viral vector contains a protein capsid that coats the 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 vectors.

[0171] 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 both serologically different viruses and viruses that may not be serologically different within a subgroup or variant of a given serotype. Serological distinctiveness can be determined based on the lack of cross-reactivity between antibodies against one capsid compared to another. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). Specification 20 / 104 pages 29 CN 122095088 A

[0172] As more naturally occurring viral isolates are discovered or capsid mutants are generated, serological differences with any of the existing serotypes may or may not exist. Therefore, in the absence of serological differences in a new virus, the new virus will be a subgroup or variant of the corresponding serotype.

[0173] III.A. Adenovirus Vectors

[0174] Adenoviruses are non-enveloped double-stranded DNA viruses. Recombinant adenovirus vectors contain a protein lacking one of the proteins involved in viral replication.Recombinant adenovirus nucleic acid containing multiple proteins, and further comprising 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) at its ends, the length of which varies between 30 and 371 bp. The ITRs serve as self-priming structures that facilitate non-primase-dependent DNA replication. The 5' and 3' inverted repeat sequences are not required to be exact inverted repeat sequences. The packaging signal located on the left arm of the genome is required for viral genome packaging. (Liu and Seol (2020) BMB Reports; 53(11):565–575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)

[0175] In some embodiments, the recombinant adenovirus vector is a third-generation vector, also referred to as “gutless” or “helper-dependent”. Virus-free gene vectors can be generated from recombinant adenovirus nucleic acid, in which all or substantially all viral sequences are absent except for the ITR and packaging signals. Virus-free gene-free adenovirus vectors are high-capacity vectors capable of holding up to about 36 kb of DNA insert. 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 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 incorporated herein by reference in its entirety.)

[0176] In some embodiments, the recombinant adenovirus vector may be generated based on rare human or chimpanzee serotypes. The use of chimpanzee serotypes and rare human serotypes may be helpful in reducing 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.)

[0177] Adenoviral vectors can be produced using, for example, appropriate helper viruses or plasmids and cell lines via trans-supply vectors to produce the desired viral proteins. (Liu and Seol (2020) BMB Reports;53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53. )

[0178] III.B. AAV Vector

[0179] Recombinant adeno-associated virus (referred to herein as “rAAV”) vectors are 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 rAAV 5' and 3' ITRs can be different, and the 5' and 3' inverted repeat sequences do not need to be exact inverted repeat sequences.

[0180] The rAAV vector contains recombinant AAV nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks one or more AAV proteins involved in viral replication. In some embodiments, the rAAV vector contains AAV 5' and / or 3' ITRs as well as DNA inserts. In some embodiments, the rAAV nucleic acid comprises a 5' ITR and / or a 3' ITR independently selected from the following 5' and 3' ITRs provided: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3B ITR. In a further embodiment, both 5' and 3' ITRs are present, and the two ITRs originate from the same serotype genome.

[0181] In a further embodiment, the 5' ITR contains at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence identical to SEQ ID NO: 254, and the 3' ITR independently contains at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence identical to SEQ ID NO: 253.

[0182] Recombinant adeno-associated virus vectors typically accept DNA inserts ranging in size from about 4 kb to about 5.2 kb. If desired, filler sequences can be used to increase the rAAV nucleic acid size and packaging efficiency. In different implementations, the rAAV nucleic acid containing the filler sequence 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, about 4.4 kb, about 4.5 kb, about 4.6 kb, or about 4.7 kb. Preferred filler sequences avoid coding sequences, repetitive sequences, recombinant sequences, and immunogenic sequences.

[0183] In some implementations, 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 the AAV capsid. scAAV and shAAV contain an inverse dimer repeat sequence providing intramolecular double-stranded DNA. scAAV can be generated by mutating the ITR terminal cleavage site, preventing Rep from creating a nick at the terminal cleavage site. shAAV can be generated using a short hairpin to produce dsAAV. scAAV and shAAV offer the advantage of using double-stranded DNA to avoid the DNA synthesis steps required for single-stranded rAAV nucleic acid after entering 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 incorporated herein by reference in its entirety.)

[0184] 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.

[0185] Recombinant AAV capsids and nucleic acids can be based on the same serotype (or subgroup or variant), or they can be based on different serotypes. In some embodiments, the rAAV nucleic acid has the same serotype genome (e.g., ITR) as the capsid protein.

[0186] In different embodiments, the rAAV capsid comprises proteins 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 at least 99.4%, at least 99.5%, at least 99.9%, or 100% identical to the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10,VP1, VP2, or VP3 of any one of AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10; or VP1 of SEQ ID NO: 257 or SEQ ID NO: 260.

[0187] In some embodiments, the AAV capsid comprises VP1, VP2, and VP3, each independently having a sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to 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: 257 or SEQ ID NO: 260; 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, which disclose RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6; U.S. Patent Publication No. 2013 / 0059732 and U.S. Patent No. 9,169,299, which disclose LK01, LK02, and LK03; and U.S. Patent No. 11,110,153; the disclosures of these patents are incorporated herein by reference in their entirety.)

[0188] In some embodiments, the capsid comprises VP1 having the sequence of SEQ ID NO: 257; VP2 having the sequence of SEQ ID NO: 258; and VP3 having the sequence of SEQ ID NO: 259. Specification 22 / 104 pages 31 CN 122095088 A

[0189] In some embodiments, the rAAV vector comprises (1) an rAAV nucleic acid containing a sequence encoding miR21 operatively linked to a promoter and a polyadenylation signal, and (2) an rAAV capsid containing (a) a VP1 containing the amino acid sequence of SEQ ID NO: 257, (b) a VP2 containing the amino acid sequence of SEQ ID NO: 258, and (c) a VP3 containing the amino acid sequence of SEQ ID NO: 259.

[0190] 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 the following literature: Chen et al., (2021) J.Control. Release 333, 129–138 (e.g., AAV9, AAV-PHP⋅B, AAV-PHP.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), each incorporated herein by reference in its entirety.

[0191] The AAV genome contains two major genes: rep and cap. Transcription from the rep gene begins at two distinct promoters, resulting in the production of non-structural proteins named Rep78, Rep68, Rep52, and Rep40. The rep proteins play a role in genome replication and / or capsid formation. The cap gene encodes structural proteins (VP1, VP2, and Vp3) that make up the capsid; non-structural assembly activation proteins (APP) that perform functions related to capsid assembly; and membrane-associated accessory proteins that may be associated with the generation phase of the replication cycle. (Maurer and Weitzman (2020) Hum. Gene Ther. 31(9–10):499–511, which are incorporated herein by reference in their entirety.)

[0192] AAV requires helper viral functions to complete its replication cycle. Helper viral functions can be supplied by different viruses in permissive cell lines. Permissive cell lines are cell lines that are 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, permissive primate cells; and baculoviruses, which can be used in combination with, for example, permissive insect cells such as sf9. (Maurer and Weitzman (2020) 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.)

[0193] Recombinant AAV can be produced using, for example, a suitable helper virus or plasmid and cell line via a trans-supply vector to produce the desired viral proteins. In some embodiments, rAAV is produced using an rAAV vector genomic plasmid. The plasmid contains a portion of the rAAV nucleic acid that is ultimately packaged or capsidated to form a viral (e.g., rAAV) vector. The “plasmid backbone” contains elements important for replication and the production of recombinant viruses. Apart from possible 3' ITR and / or 5' ITR clonal remnants, the plasmid backbone itself is not packaged or capsidated into the viral particles.

[0194] The vector genomic plasmid may contain regions such as origin of replication and selectivity markers. Other sites that may be present include cloning sites.

[0195] Recombinant AAV can be generated from different types of cell lines, including HeLa, A549, BHK, Vero, and HEK293 or derivatives thereof. In some embodiments, HEK293 cells (American Type Culture Collection accession number ATCC CRL1573) are used. Other host cell lines suitable for rAAV vector generation are described, for example, in the following literature: Robert et al., Biotechnol. J. (2017) 12(3), 1600193; and International Application No. PCT / US2017 / 024951, the disclosures of which are incorporated herein by reference in their entirety.

[0196] Recombinant AAV 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), edited Castle., 1st edition, Springer New York, New York, NY.; each of which is incorporated herein by reference in its entirety. Specification 23 / 104 pages 32 CN 122095088 A

[0197] In some embodiments, AAV helper functionality is introduced into host cells by transfecting host cells with an AAV helper construct prior to or simultaneously with transfection of the AAV expression vector. Host cells with AAV helper functionality may be referred to as “helper cells” or “packaging helper cells”. Therefore, 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 cannot replicate or package themselves. These constructs can take the form of, for example, plasmids, phages, transposons, colloids, viruses, or viral particles. 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. Recombinant AAV can be generated, for example, as described in the following documents: U.S. Patent 9,408,904; and International Applications PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are incorporated herein in their entirety.

[0198] In some embodiments, the rAAV vector is generated from rAAV-producing cells containing rAAV helper viral activity. The genome of the rAAV-producing cell contains rAAV nucleic acid, the rep gene, and the cap gene.

[0199] In some embodiments, the rAAV vector is generated by culturing rAAV-allowing cells containing an AAV genome plasmid, wherein the rAAV-allowing cells further contain the rep and cap genes provided as part of the cell genome and / or provided by one or more separate plasmids; and helper viral activity provided as part of the cell genome and / or provided by one or more separate plasmids. In a further embodiment, (a) the rAAV cell line is a packaging cell, wherein the genome of the packaging cell contains a cap gene and a rep gene; (b) the rep gene, cap gene, and helper activity are provided by the same plasmid; or (c) the rep gene and cap gene are provided by a rep / cap plasmid, and the helper activity is provided by a helper plasmid.

[0200] 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.

[0201] 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 a helper plasmid, wherein additional helper functions are provided by a host line.

[0202] In some embodiments, the rAAV vector is obtained by generating rAAV using the methods described herein and purifying said rAAV. Purification of rAAV can be performed using techniques such as gradient-based purification, column-based methods, and combination methods. (See, for example, Ayuso et al., Curr Gene Ther. (2010) 10(6):423-36, which is incorporated herein by reference in its entirety.)

[0203] III.C. Retroviral Vectors

[0204] Retroviruses are enveloped single-stranded RNA viruses containing 5' and 3' LTRs and a signal packaging sequence located just outside the LTR. 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 viral RNA packaging, reverse transcription, and proviral DNA.All required cis-acting sequences are integrated, while all HIV protein-coding genes are removed. The lentiviral vector has a packaging capacity of up to about 9 kb. If desired, the rAAV nucleic acid size and packaging efficiency can be increased using filler sequences. The lentiviral vector can be produced using appropriate plasmids and cell lines via trans-supply vectors to generate the desired viral proteins. (Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.) IV. Non-viral vectors

[0205] In some embodiments, the gene delivery medium is a non-viral vector. Preferred non-viral vectors are nanoparticles. A variety of different nanoparticles can be used, including lipid nanoparticles (LNP), polymer nanoparticles, lipid polymer nanoparticles (LPNP), 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 the instruction manual, page 24 / 104, 33 CN 122095088 A 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., Biomedicines, 10, 50; and Qin et al., Signal Transduct Target Ther. (2022)) May 21; 7(1):166; each of these is incorporated herein by reference in its entirety.)

[0206] If desired, nanoparticles may be targeted to cell types using, for example, targeting ligands that recognize receptors on target cells. 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.)

[0207] Nanoparticles can be used to deliver repressive RNA or encoding polynucleotide constructs into cells. In different 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).

[0208] The generation of different nanoparticles and the incorporation of nucleic acids and other compounds are well known in the art. Examples of publications illustrating the incorporation of nucleic acids in 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 that may affect 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.) IV.A. Lipid-Based Delivery Systems

[0209] Lipid-based delivery systems include those that use lipids as a component. Examples of lipid-based delivery systems include liposomes, LNPs, micelles, and extracellular vesicles.

[0210] "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.

[0211] DNA is negatively charged. Therefore, it may be advantageous for LNPs to contain cationic lipids (e.g., aminolipids). Exemplary aminolipids are described in the following documents: 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.U.S. Patent Publications 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411, and 2010 / 0117125 are all incorporated herein in their entirety. In some embodiments, the LNP comprises the aminolipids described in U.S. Patent No. 9,512,073, which is hereby incorporated herein in its entirety.

[0212] The terms “cationic lipid” and “aminolipid” are used interchangeably herein to include lipids and their salts having one, two, three, or more fatty acid or aliphatic alkyl chains and pH-titrile amino groups (e.g., alkylamino or dialkylamino). Cationic lipids are typically protonated (i.e., positively charged) at pH values ​​below the cationic lipid pKa and substantially neutral at pH values ​​above the pKa. Cationic lipids can also be titratable cationic lipids. In some embodiments, the cationic lipid comprises a protonable tertiary amine (e.g., pH-titratable) group; a C18 alkyl chain, wherein each alkyl chain may independently have one or more double bonds or one or more triple bonds; and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0213] Cationic lipids include 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenicoxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenicoxy-N,N-dimethylaminopropane (γ-DLenDMA), and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K- C2-DMA, also known as DLin-C2K-DMA, XTC2 and C2K), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31Z)-hexadecene-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-M-C3-DMA, also known as MC3), their salts and mixtures thereof. Other cationic lipids include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleoyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, γ-DLen-C2K-DMA and (DLin-MP-DMA) (also known as 1-B11).

[0214] Other cationic lipids include 2,2-dilinoleoyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleoyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 2-Dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazino)propane (DLin-MPZ) 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylenylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-Dioleylenyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3-dioleylenyloxy)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-diolenoyloxy-N-[2(spermine-formamido)ethyl]-N,N-dimethyl-1-propanetrifluoroacetate ammonium (DOSPA), bis(octadecylamidoglycyl)Spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecyldienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxaproloxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecyldienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-diolenoyloxybenzylamine (DMOBA), 1,2-N,N′-diolenoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N′-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS), and disubstituted spermine (D2S), or mixtures thereof.

[0215] Many commercial formulations of cationic lipids can be used, such as LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO / BRL).

[0216] 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. Specification 26 / 104 pages 35 CN 122095088 A

[0217] In a further embodiment, the cationic lipid may be present in amounts of about 10% to about 85% of the LNP by molar ratio, or about 50% to about 75% of the LNP by molar ratio.

[0218] The LNP may contain neutral lipids. Neutral lipids may be included in lipid classes that are present at physiological pH in an uncharged or neutral zwitterionic form. Such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalins, and cerebrosides. The selection of neutral lipids is generally guided by considerations 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).

[0219] Lipids with various acyl chain groups having different chain lengths and saturation levels are available, or can be isolated or synthesized. In some embodiments, lipids containing saturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. In some embodiments, lipids having monounsaturated or diunsaturated fatty acids with carbon chain lengths ranging from C14 to C22 are used.The lipids can also be composed of a mixture of saturated and unsaturated fatty acid chains. Exemplary neutral lipids include 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), or phosphatidylcholine. Neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups such as serine and inositol.

[0220] In a further embodiment providing neutral lipids, the neutral lipids may be present in amounts ranging from about 0.1% to about 99% by weight of LNP, or from about 5% to about 15% by weight of LNP, for example, 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%.

[0221] The LNP may contain additional components, such as sterols and polyethylene glycol. Sterols can impart fluidity to the LNP. As used herein, “sterol” refers to naturally occurring sterols from plant (phytosterols) or animal (animal sterols) sources, as well as synthetic sterols not naturally occurring, all characterized by the presence of a hydroxyl group at the 3-position of the steroid A ring. Suitable sterols include those conventionally used in the field of liposome, lipovesicle, or lipogranule formulations, most commonly cholesterol. Phytosterols include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication No. 2011 / 0177156. In various embodiments providing sterols, the sterols are present in amounts ranging from about 1% to about 80% by weight of LNP, or from about 10% to about 25% by weight of LNP.

[0222] Polyethylene glycol (PEG) is a water-soluble polymer of ethylene PEG repeating units having terminal hydroxyl groups. PEGs are classified according to 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-succinimide succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), and monomethoxy polyethylene glycol-methyl amine.Benzenesulfonate (MePEG-TRES) and monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0223] 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 a further embodiment, 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 from about 2,000 Daltons or from about 750 Daltons.

[0224] PEG-modified lipids include PEG-dialkoxypropyl conjugates (PEG-DAA) as described in U.S. Patent Nos. 8,936,942 and 7,803,397. PEG-modified lipids (or lipid-polyoxyethylene conjugates) can have various "anchoring" lipid moieties to immobilize the PEG moieties to the surface of lipid vesicles. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) as described in U.S. Patent No. 5,820,873, PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropyl-3-amine. In some embodiments, the PEG-modified lipids may be PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, the PEG may be in amounts from about 0.1% to about 50% by weight of LNP, or from about 5% to about 15% by weight of LNP.

[0225] In a further embodiment involving LNP size, the LNP size 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 prior to encapsulation of nucleic acids.

[0226] In some embodiments involving LNP, the LNP is described in the following literature: Billingsley et al., Nano Lett. 2020, 20, 1578 or Billingsley et al., International Patent Publication No. WO 2021 / 077066 (both of which are incorporated herein by reference in their entirety). Billingsley et al. and WO2021 / 077066 describe LNPs containing lipid-anchored PEG, cholesterol, phospholipids, and ionizable lipids. In some embodiments, the LNP contains a C14-4 polyamine core and / or has a particle size of about 70 nm. C14-4 has the following structure.

[0227] In some embodiments, the LNP is composed of cationic lipids or lipopeptides as described in the following patents: U.S. Patent No. 10,493,031, U.S. Patent No. 10,682,374, or WO 2021 / 077066 (each of which is 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):

[0228] In some embodiments, the LNP contains a modified form of cKK-E12, referred herein as “bCKK-E12”, having the following structure: Specification 28 / 104 pages 37 CN 122095088 A

[0229] In some embodiments, the LNP contains lipids 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 as described in the following literature: Sabnis et al., Molecular Therapy 2018, 26:6, 1509-1519 (which is incorporated herein by reference in its entirety). In some embodiments, the LNP contains lipids 5, 8, 9, 10 or 11 as described in Sabnis et al.

[0230] Lipid 5 of Sabnis et al. has the following structure:

[0231] Lipid 9 of Sabnis et al. has the following structure:

[0232] Other lipids that can be used include those described in the following literature: 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, e.g. 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 Tanaka et al., Adv. Funct. Mater., 2020, 30, 1910575; each of which is incorporated herein by reference in its entirety.

[0233] In a further embodiment, the nanoparticles are LNPs. In a further embodiment, the LPN comprises, substantially consists of, or consists of, the following components on an olean basis: (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; or (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 phospholipids are neutral lipids; and the phospholipids are DOPE or DSPC.

[0234] In a further embodiment, LNP comprises, by molar percentage, the following components, substantially consisting of the following components or, as per specification page 29 / 104, 38 CN 122095088 A, consisting of the following components: (1) cKK-E12, about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (2) bCKK-E12, about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (3) lipid 9, about 50%; C14-PEG2000, about 1.5%; cholesterol, about 38.5%; and DSPC, about 10%; (4) lipid 5, about 50%; C14-PEG2000, about 1.5%; cholesterol, about 38.5%; and DSPC, about 10%; (5) Ionizable lipids, approximately 50%; DSPC, approximately 10%; cholesterol, approximately 37.5%; and stabilizers (PEG-lipids), approximately 2.5%; or (6) GenVoy-ILM™ LNP (Precision NanoSystems). IV.B. Polymer-based nanoparticles

[0235] 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 onto the surface of the 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, pachymannan, dextran and hyaluronic acid, and poly-β-amino esters. (Thomas et al., (2019) Molecules 24, 3744.)

[0236] Polymer-based nanoparticles can have different sizes, ranging from about 1 nm to about 1000 nm.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. VC Lipid Polymer Nanoparticles

[0237] Lipid polymer nanoparticles are hybrid nanoparticles that provide both lipid and polymer components, and can therefore be considered as LNPs or LPNPs. LPNP configurations can provide an external polymer and an internal lipid or an external lipid and an internal polymer. The presence of two different types of materials helps in designing nanoparticles to provide delayed release of components. Different lipid and polymer components can be selected by considering the materials to be delivered. (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.) IV.D. Protein and Peptide-Based Nanoparticles

[0238] Protein and peptide-based systems can employ a variety of different proteins and peptides. Examples of proteins that can be employed include gelatin and elastin. Peptide-based systems can employ, for example, CPP.

[0239] CPPs are short peptides (6-30 amino acid residues) that are potentially capable of intracellular permeation to deliver therapeutic molecules. Most CPPs are composed primarily of arginine and lysine residues, making them cationic and hydrophilic, 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, penetratin, 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 transportan, VT5, bovine antimicrobial peptide-7 (Bac7), proline-rich peptide (PPR), SAP (VRLPPP)3, TP10, pep-1, and MPG; and membrane-loving CPPs (exhibiting both hydrophobic and amphiphilic properties, and containing both large aromatic residues and small residues), such as H625, SPION-PEG-CPP, and NP.And hydrophobic CPPs (containing only nonpolar motifs or residues), such as SG3, PFVYLI, pep-7, and fibroblast growth factor.

[0240] 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. IV.E. Peptide Cage Nanoparticle Specification 30 / 104 pages 39 CN 122095088 A

[0241] Peptide cage-based delivery systems can be generated from protein materials capable of assembling into cage-like structures to form a confined internal environment. Peptide cages can comprise protein shells that self-assemble to form protein cages (e.g., structures with internal cavities that are naturally accessible to the solvent, or can be made so by changing the solvent concentration, pH, or equilibrium ratio). The monomers of the protein cages can be in naturally occurring forms or variant forms, including amino acid substitutions, insertions, and deletions (e.g., fragments).

[0242] Different types of protein “shells” can be assembled and loaded using different types of materials. Protein cages can be generated using one or more 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. 20040028694, and U.S. Patent Publication No. 20090035389, each of which is incorporated herein by reference in its entirety).

[0243] 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 the class of 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.

[0244] Protein cages can have different core 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. IV.F. Exosomes

[0245] 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 taken up by cells to deliver their cargo (e.g., expression cassettes containing nucleic acids encoding RNA polynucleotides containing HTT targeting sequences). The cargo can associate with exosome surface structures or can be encapsulated within an exosome bilayer.

[0246] Exosomes can be modified in various ways to facilitate cargo delivery and cell targeting. Modifications for facilitating cargo delivery include structures for association with the cargo, such as protein scaffolds and polymers. Modifications for cell targeting include targeting ligands and modifying surface charges. Publications describing the generation, modification, and use of exosomes for delivering different 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 by reference). V. Pharmaceutical Compositions

[0247] Pharmaceutical compositions can 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 HTT mRNA, (b) an optionally modified repressive RNA duplex targeting HTT mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid containing a sequence encoding the HTT mRNA targeting sequence, or (d) a delivery medium comprising (a), (b), or (c); and a pharmaceutically acceptable carrier.

[0248] The reference to “pharmaceutically acceptable” indicates that the component will not cause serious undesirable biological effects at the amounts 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 Publication No. WO 2021 / 071835.

[0249] In some embodiments, the formulation comprises about 10 mM sodium phosphate, about 150 mM sodium chloride, and about 0.001% Kolliphor®; and pH or about pH 7.3. Specification 31 / 104 pages 40 CN 122095088 A

[0250] 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, which are generally sterile and isotonic with the blood of the intended recipient. Illustrative examples include water, buffered saline, Hanks' solution, Ringer's solution, glucose, fructose, ethanol, animal oils, 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.

[0251] 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., sodium dihydrogen phosphate or disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, and mixtures of such salts), buffered saline solutions, sugars (e.g., glucose), and water for injection. The pharmaceutical composition includes a dried (e.g., lyophilized) composition that, upon addition of sterile water or physiological saline, allows for the formation of a solution suitable for administration.

[0252] Alternatively, the suspension can be prepared as a suitable oily injectable suspension. Suitable lipophilic solvents or mediators include fatty oils (e.g., sesame oil), or synthetic fatty acid esters (e.g., 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 a concentrated solution.

[0253] "Effective amount" or "sufficient amount" refers to an amount that provides the indicated or desired effect. Effective doses can be administered alone or in combination with one or more other compositions (e.g., additional therapeutic agents or immunosuppressants), treatments, regimens, or treatment regimens; and provide long-term or short-term responses.

[0254] A pharmaceutical composition comprising a transgenic RNA polynucleotide encoding a target HTT mRNA can be delivered to a subject to allow for 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 recipient to produce a therapeutically effective dose in the subject.

[0255] A “therapeutically effective dose” refers to the amount that elicits a desired or indicated biological or medical response in a subject. The therapeutically effective dose can be determined based on observed symptoms and / or by using biomarkers associated with a specific disease or disorder. The selection of a specific effective dose can be optimized by considering various factors, including the disease to be treated or prevented, the symptoms involved, the disease or disorder targeted, safety and efficacy in animal models, the patient’s weight, and the patient’s immune status. The optimal dose to be used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and can be evaluated 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.

[0256] In some embodiments, the pharmaceutical composition comprising the rAAV carrier comprises an empty AAV capsid. In some embodiments, in the pharmaceutical composition comprising the rAAV carrier and the empty AAV capsid, the ratio of the empty AAV capsid to the rAAV carrier is within or between the following: about 100:1-50:1, about 50:1-25:1, about 25:1-10:1, about 10:1-1:1, about 1:1-1:10, about 1:10-1:25, about 1:25-1:50, or about 1:50-1: 100. In some implementations, the ratio of empty AAV capsid to rAAV carrier is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0257] Further guidance and examples of pharmaceutical compositions and delivery systems are provided, for example, in the following literature: Remington: The Science and Practice of Pharmacy (2020) 23rd edition, University of the Sciences 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. Specification 32 / 104 pages 41 CN 122095088 A VI. Administration and Treatment

[0258] RNA polynucleotides containing an inhibitory sequence targeting HTT can be used in methods for reducing mutant HTT expression and / or treating Huntington's disease in subjects. Such methods may include, for example, administration of (a) an RNA polynucleotide containing a sequence targeting HTT mRNA, (b) an optionally modified repressive RNA duplex targeting HTT mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid containing a sequence encoding the HTT mRNA targeting sequence, or (d) a delivery medium containing (a), (b), or (c); and pharmaceutically acceptable carriers.

[0259] Symptoms of Huntington's disease include motor impairment, cognitive impairment, and mental disorders. A diagnosis of Huntington's disease can be confirmed, for example, by analyzing HTT protein (also referred to herein as Huntington's protein) that measures a glutamine repeat sequence or by measuring the nucleic acid encoding a CAG repeat sequence. In some embodiments, subjects are selected based on biomarkers or genetic markers associated with Huntington's disease; and / or subjects are diagnosed with Huntington's disease.

[0260] Measuring glutamine or CAG repeat sequences may also be used to identify patients with an increased likelihood of infection with Huntington's disease.The length of the CAG repeat sequence in the HTT gene appears to be negatively correlated with the age of onset and the initial progression of severity. Partial penetrance and late onset are associated with subjects having 36–39 CAG repeat sequences. (Langbehn et al., Am. J. Med. Genet. (2009) Part B 153B:397–408; and Langbehn Am. J. Med. Genet. (2022) 109:172–179, both of which are incorporated herein by reference in their entirety.)

[0261] In some embodiments, the treated subject has an HTT gene encoding HTT containing 36 or more CAG repeat sequence segments. In further embodiments, the subject has at least 40, at least 45, at least 50, at least 55, at least 70, or at least 100 CAG repeat sequence segments.

[0262] In some embodiments, the treated subject has an HTT protein comprising 36 or more glutamine repeat sequence segments. In a further embodiment, the subject has at least 40, at least 45, at least 50, at least 55, at least 70, or at least 100 glutamine repeat sequences.

[0263] In some embodiments, prior to initial treatment, the subject is diagnosed with an HTT gene comprising 36 or more CAG repeat sequence segments. In a further embodiment, prior to initial treatment, the subject is diagnosed with at least 40, at least 45, at least 50, at least 55, at least 70, or at least 100 CAG repeat sequence segments.

[0264] In some embodiments, prior to initial treatment, the subject is diagnosed with an HTT protein comprising 36 or more glutamine repeat sequence segments. In a further embodiment, prior to initial treatment, the subject is diagnosed with at least 40, at least 45, at least 50, at least 55, at least 70, or at least 100 glutamine repeat sequences.

[0265] Administration can be performed via various routes, such as subcutaneous, epidermal, intradermal, intrathecal, intraorbital, intramucosal, intranasal, intraperitoneal, intravenous, intrapleural, intraarterial, intracavitary, oral, intrahepatic, via portal vein, intramuscular, intracranial, intracisional, or intraventricular administration. In some embodiments, administration to a patient is performed via infusion in a drug carrier.

[0266] In some embodiments, CNS delivery is achieved using techniques and / or agents that facilitate crossing the blood-brain barrier; bypass the blood-brain barrier; or directly apply the drug to the brain.

[0267] Techniques facilitating 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. Common techniques for facilitating blood-brain barrier crossing can be utilized on both viral and nonviral delivery media.In addition, certain delivery mediators, 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; and Camalleri et al., J. Clin. Neurophysiol. (2020) Mar;37(2):104–117); and Fischell and Fishman, Front. Neurosci., September 23, 2021, Vol. 15, Article Description 33 / 104 pp. 42 CN 122095088 A 747726, doi: 10.3389 / fnins.2021.747726; each of which is incorporated herein by reference in its entirety.)

[0268] 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 No. 9,585,971, and U.S. Patent Publication No. US202 / 1214749, each of which is incorporated herein by reference in its entirety.

[0269] In some embodiments, CNS delivery involves the use of focused ultrasound and microbubbles. The combination of focused ultrasound and microbubbles can transiently disrupt the blood-brain barrier, thereby facilitating the entry of therapeutic agents, including transgenic delivery mediators. Focused ultrasound can store energy in selected areas of human anatomy and its use can be facilitated by MRI guidance. Different types of microbubbles can be used in conjunction with focused ultrasound to transiently disrupt the blood-brain barrier. (Cammalleri et al., J Clin Neurophysiol. (2020) Mar;37(2):104-117; Noroozian et al., Methods Mol Biol. (2019) 1950, 177–197; U.S. Patent No. 10,322,178; and International Patent Publication No. WO 2019 / 113538; each of which is incorporated herein by reference in its entirety.)

[0270] Microbubbles are typically administered intravenously. In some implementations, the microbubbles are: Definity™ (Lantheus Medical Imaging, Billerica, Massachusetts, USA), containing perfluoropropane lipid microspheres (average diameter 1.1–3.3 µm, maximum diameter 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, pyrogen-free suspension of human serum albumin and perfluoropropane microspheres (average diameter 3.0–4.5 µm, maximum diameter 32 µm). (See Camalleri et al., J Clin Neurophysiol. (2020) Mar;37(2):104-117, which is incorporated herein by reference in its entirety.)

[0271] In some embodiments, microbubbles are generated and applied as described in U.S. Patent No. 10,322,178 (which is incorporated herein by reference in its entirety).

[0272] 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. WO 2023 / 0044161; each of which is incorporated herein by reference in its entirety.)

[0273] 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. WO 2023 / 0044161.

[0274] In some embodiments, intranasal administration is used to achieve CNS vector delivery. Intranasal delivery bypasses the blood-brain barrier and reduces systemic exposure. In a further embodiment, intranasal delivery includes the use of focused ultrasound and microbubbles. It has been shown that combining focused ultrasound and microbubbles with intranasal delivery can facilitate the penetration of therapeutic agents already located in the perivascular space across the blood-brain barrier. (Ye et al., The Lancet (2022) October: Vol. 84: 1–14, incorporated herein by reference in its entirety.)

[0275] In some embodiments, administration is directed to the CNS, for example, via intraparenchymal, intracisional, or intraventricular administration.

[0276] In some embodiments, CNS administration is performed, for example, by direct administration 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 incorporated herein by reference in its entirety.)

[0277] Another example of a technique for CNS administration is convection-enhanced delivery. Convection-enhanced delivery involves surgically exposing the brain, placing a catheter directly into the target region, and subsequently infusing the therapeutic agent. (US Patent Publication No. 2022 / 010001; and Specification 34 / 104, page 43, CN 122095088 A Debinski et al. (2009) Expert Rev Neurother. 9(10):1519-27; both documents are incorporated herein by reference in their entirety.)

[0278] In different embodiments, intraparenchymal administration is performed via IP infusion to the caudate nucleus and putamen using any of the following: via a transfrontal approach along the axis of both structures; using a parietal approach (delivery to the caudate nucleus) and / or using an occipital approach (delivery to the putamen). In another embodiment, the carrier is delivered at a steady rate of about 5 µl / min. Administration may be performed via bilateral infusion, for example, using convection-enhanced delivery. In a further embodiment, administration is performed via bilateral stereotactic infusion. In a further embodiment, administration is performed under MRI guidance, wherein rAAV is provided as a formulation containing an imaging agent. Different devices can be used for infusion, including, for example, the ClearPoint® SmartFlow cannula and ClearPoint® neuronavigation system for MRI-guided convection-enhanced delivery (CED).

[0279] In some embodiments, the presence of a transgenic-encoded repressive nucleic acid is measured in cerebrospinal fluid (CSF) after IP infusion (e.g., to the caudate nucleus and / or putamen). The presence of the repressive nucleic acid in CSF can be monitored at different time points (e.g., at baseline and at 3, 6, 12, 18, and 24 months). In a further embodiment, a vector encoding miR21 is administered and the presence of miR21 in the CSF is measured. Measuring the repressive nucleic acid in CSF can be used as, for example, a persistent marker.

[0280] CNS delivery devices, systems, and techniques also include those described, for example, in the following documents: 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, and U.S. Patent No.Patent Publication No. 2021 / 0343397, US Patent Publication No. 2021 / 0282866, US Patent No. 9572928, US Patent No. 8337458, US Patent No. 10722265 and US Patent Publication No. 2021 / 214749, each of which is incorporated herein by reference in its entirety.

[0281] 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.

[0282] The optimal dose can vary depending on various factors, such as the specific therapeutic agent and the desired endpoint. The amount, frequency or duration of the dose can be increased or decreased proportionally, taking into account adverse side effects, complications or other risk factors of the treatment or therapy and the condition of the subject.

[0283] A “unit dosage form” refers to a physically discrete unit containing a predetermined effective amount of the active ingredient combined with a pharmaceutically acceptable carrier. Unit dosage forms may be provided, for example, in ampoules and vials that may include pharmaceutically acceptable carriers, or in compositions in a lyophilized or freeze-dried state. In the case of a lyophilized or freeze-dried state, a sterile liquid carrier may be added prior to administration. Individual unit dosage forms may be included in multi-dose kits or containers.

[0284] An “effective amount” achieves the desired or indicated effect. For example, an effective amount for treatment reduces one or more adverse symptoms, reduces the likelihood of one or more symptoms associated with a disease or disorder, or reduces the progression of a disease or disorder. Preferred effective amounts for treatment can effectively reduce multiple or all adverse symptoms.

[0285] In some embodiments, a pharmaceutical composition comprising a viral or non-viral vector is administered to a subject at a dose suitable for reducing mutant HTT or mutant HTT expression. In various embodiments, mutant HTT or mutant HTT expression is reduced by at least 50%, at least 60%, at least 70%, or at least 90%.

[0286] In some embodiments, the expression of total HTT protein and / or total HTT mRNA is reduced by about 20% to about 90%, about 20% to about 65%, about 25% to about 60%, about 25% to about 50%, about 25% to about 45%, about 25% to 40%, about 25% to 35%, about 25% to 30%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, or about 30% to about 35%.

[0287] In different 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. (Specification 35 / 104 pages 44)CN 122095088 A

[0288] In some embodiments, the rAAV vector dose ranges from at least 1 x 108 vector genomes / kg (vg / kg) of subject weight or more, for example, 1 x 109, 1 x 1010, 1 x 1011, 1 x 1012, 1 x 1013 or 1 x 1014 or more vector genomes / kg (vg / kg) of subject weight to achieve a therapeutic effect. In different implementations, 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 3 x 10¹² rAAV vg / kg; approximately 4 x 10¹² rAAV vg / kg or greater than approximately 4 x 10¹² rAAV vg / kg; approximately 5 x 10¹² rAAV vg / kg or greater than approximately 5 x 10¹² rAAV vg / kg; approximately 1 x 10¹³ rAAV vg / kg or greater than approximately 1 x 10¹³ rAAV vg / kg; approximately 2 x 10¹³ rAAV vg / kg or greater than about 2 x 10¹³ rAAV vg / kg; about 3 x 10¹³ rAAV vg / kg or greater than about 3 x 10¹³ rAAV vg / kg; about 4 x 10¹³ rAAV vg / kg or greater than about 4 x 10¹³ rAAV vg / kg; about 5 x 10¹³ rAAV vg / kg or greater than about 5 x 10¹³ rAAV vg / kg; about 6 x 10¹³ rAAV vg / kg or greater than about 6 x 10¹³ rAAV vg / kg.

[0289] Examples of dose ranges for rAAV vg / kg include a dose range of about 5 x 10¹¹ to about 6 x 10¹³ rAAV vg / kg; a dose range of about 5 x 10¹¹ to about 5.5 x 10¹¹ rAAV vg / kg; a dose range of about 5.5 x 10¹¹ to about 6 x 10¹¹ rAAV vg / kg; a dose range of about 6 x 10¹¹ to about 6.5 x 10¹¹ rAAV vg / kg; a dose range of about 6.5 x 10¹¹ to about 7 x 10¹¹ rAAV vg / kg; a dose range of about 7 x 10¹¹ to about 7.5 x 10¹¹ rAAV vg / kg; and a dose range of about 7.5 x 10¹¹ to about 8 x 10¹¹ rAAV vg / kg.Dosage range of approximately 8 x 10¹¹ to approximately 8.5 x 10¹¹ rAAV vg / kg; dosage range of approximately 8.5 x 10¹¹ to approximately 9 x 10¹¹ rAAV vg / kg; dosage range of approximately 9 x 10¹¹ to approximately 9.5 x 10¹¹ rAAV vg / kg; dosage range of approximately 9.5 x 10¹¹ to approximately 1 x 10¹² rAAV vg / kg; dosage range of approximately 1 x 10¹² to approximately 1.5 x 10¹² rAAV vg / kg; dosage range of approximately 1.5 x 10¹² to approximately 2 x 10¹² rAAV vg / kg; dosage range of approximately 2 x 10¹² to approximately 2.5 x 10¹² rAAV vg / kg; dosage range of approximately 2.5 x 10¹² to approximately 3 x 10¹² rAAV vg / kg; dosage range of approximately 3 x Dosage range from approximately 10¹² to approximately 3.5 x 10¹² rAAV vg / kg; dosage range from approximately 3.5 x 10¹² to approximately 4 x 10¹² rAAV vg / kg; dosage range from approximately 4 x 10¹² to approximately 4.5 x 10¹² rAAV vg / kg; dosage range from approximately 4.5 x 10¹² to approximately 5 x 10¹² rAAV vg / kg; dosage range from approximately 5 x 10¹² to approximately 5.5 x 10¹² rAAV vg / kg; dosage range from approximately 5.5 x 10¹² to approximately 6 x 10¹² rAAV vg / kg; dosage range from approximately 6 x 10¹² to approximately 6.5 x 10¹² rAAV vg / kg; dosage range from approximately 6.5 x 10¹² to approximately 7 x 10¹² rAAV vg / kg; dosage range from approximately 7 x 10¹² to approximately 7.5 x 10¹² rAAV vg / kg. Dosage range of 10¹² rAAV vg / kg; dosage range of approximately 7.5 x 10¹² to approximately 8 x 10¹² rAAV vg / kg; dosage range of approximately 8 x 10¹² to approximately 8.5 x 10¹² rAAV vg / kg; dosage range of approximately 8.5 x 10¹² to approximately 9 x 10¹² rAAV vg / kg; dosage range of approximately 9 x 10¹² to approximately 9.5 x 10¹² rAAV vg / kg; dosage range of approximately 9.5 x 10¹² to approximately 1 x 10¹³ rAAV vg / kg; dosage range of approximately 1 x 10¹³ to approximately 1.5 x 10¹³ rAAV vg / kg; dosage range of approximately 1.5 x 10¹³ to approximately 2 x 10¹³ rAAV vg / kg; dosage range of approximately 2 x 10¹³ to approximately 2.5 x 10¹³ rAAV vg / kg. Dosage range in vg / kg; approximately 2.5 x 10¹³ to approximately 3 x 10¹³ rAAVDosage ranges: approximately 3 x 10¹³ to approximately 3.5 x 10¹³ rAAV vg / kg; approximately 3.5 x 10¹³ to approximately 4 x 10¹³ rAAV vg / kg; approximately 4 x 10¹³ to approximately 4.5 x 10¹³ rAAV vg / kg; approximately 4.5 x 10¹³ to approximately 5 x 10¹³ rAAV vg / kg; approximately 5 x 10¹³ to approximately 5.5 x 10¹³ rAAV vg / kg; approximately 5.5 x 10¹³ to approximately 6 x 10¹³ rAAV vg / kg; approximately 6 x 10¹³ to approximately 1 x 10¹⁴ rAAV vg / kg.

[0290] In some embodiments, rAAV vg / kg is administered at the following doses: about 5 x 10¹¹ vg / kg, about 6 x 10¹¹ vg / kg, about 7 x 10¹¹ vg / kg, about 8 x 10¹¹ vg / kg, about 9 x 10¹¹ vg / kg, about 1 x 10¹² vg / kg, about 2 x 10¹² vg / kg, about 3 x 10¹² vg / kg, about 4 x 10¹² vg / kg, about 5 x 10¹² vg / kg, about 6 x 10¹² vg / kg, about 7 x 10¹² vg / kg, about 8 x 10¹² vg / kg, about 9 x 10¹² vg / kg, about 1 x 10¹³ vg / kg, about 2 x 10¹³ vg / kg. (Instructions for Use, pages 36 / 104, 45 CN) 122095088 A vg / kg, about 3 x 10¹³ vg / kg, about 4 x 10¹³ vg / kg, about 5 x 10¹³ vg / kg, or about 6 x 10¹³ vg / kg.

[0291] 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 described above with respect to rAAV.

[0292] In some embodiments, the rAAV carrier dose is about 1.0 x 10¹⁰ vg to about 1.0 x 10¹³ vg, about 1.0 x 10¹¹ vg to about 1.0 x 10¹² vg, about 1.0 x 10¹¹ vg to about 1.8 x 10¹¹ vg, about 1.0 x 10¹¹ vg to about 1.5 x 10¹¹ vg, about 1.0 x 10¹¹ vg, about 1.1 x 10¹¹ vg, about 1.3 x 10¹¹ vg, about 1.4 x 10¹¹ vg, about 1.5 ...x 10¹¹ vg, about 1.6 x 10¹¹ vg, about 1.7 x 10¹¹ vg, about 1.8 x 10¹¹ vg, about 1.9 x 10¹¹ vg, about 1.0 x 10¹² vg, about 1.1 x 10¹² vg, about 1.3 x 10¹² vg, about 1.4 x 10¹² vg, about 1.5 x 10¹² vg, about 1.6 x 10¹² vg, about 1.7 x 10¹² vg, about 1.8 x 10¹² vg, about 1.9 x 10¹² vg, or about 1.0 x 10¹³ vg.

[0293] Some embodiments involve treating a patient, which considers (i) the right hemisphere; (2) the left hemisphere; or (3) the putamen and / or caudate nucleus volumes of both hemispheres. Preferably, the volumes of the putamen and caudate nucleus are measured.

[0294] The putamen and caudate nucleus may differ significantly among patients with Huntington's disease. Typically, the volumes of the putamen and caudate nucleus do not vary significantly in a particular patient.

[0295] In some embodiments, a viral vector (such as rAAV) is administered intraparenchymally to the subject to provide a dose of about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³ to the right or left hemisphere; or a dose of about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³ is administered to each of the right and left hemispheres; wherein mm³ is the volume of the putamen and caudate nucleus in the hemisphere.

[0296] In some embodiments, a subject with Huntington's disease is treated by methods including: (a) determining the volume of the putamen and / or caudate nucleus in the right and / or left hemisphere of the subject; and (b) administering a recombinant adeno-associated virus (rAAV) vector into the brain parenchyma of the subject at a dose of about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³ for the right hemisphere and / or at a dose of about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³ for the left hemisphere; wherein the dose is based on the volume determined in step (a).

[0297] The reference to “and / or” provides for each possible combination of variables, which can be combined with other combinations. For example, referring to determining the volume of the putamen and / or caudate nucleus in the right and / or left hemisphere includes: (1) determining the volume of the putamen in the right hemisphere; (2) determining the volume of the caudate nucleus in the right hemisphere; (3) determining the volume of the putamen in the left hemisphere; (4) determining the volume of the caudate nucleus in the left hemisphere; (5) determining the volumes of the caudate nucleus and putamen in the right hemisphere; (6) determining the volumes of the caudate nucleus and putamen in the left hemisphere; and (7) determining the volumes of the putamen and caudate nucleus in the right and left hemispheres.

[0298] In some embodiments, approximately 2.0 x 10⁷A dose of approximately 2.0 x 10⁸ vg / mm³ is administered independently to both hemispheres. The phrase “independently” indicates that the specific dose administered to the right and left hemispheres may vary within the indicated range.

[0299] In some embodiments, the dose is about 2.0 x 10⁷ vg / mm³, about 2.5 x 10⁷ vg / mm³, about 3.0 x 10⁷ vg / mm³, about 3.5 x 10⁷ vg / mm³, about 4.0 x 10⁷ vg / mm³, about 4.5 x 10⁷ vg / mm³, about 5.0 x 10⁷ vg / mm³, about 5.5 x 10⁷ vg / mm³, about 6.0 x 10⁷ vg / mm³, about 6.5 x 10⁷ vg / mm³, about 7.0 x 10⁷ vg / mm³, about 7.5 x 10⁷ vg / mm³, about 8.0 x 10⁷ vg / mm³, about 8.5 x 10⁷ vg / mm³, about 9.0 x 10⁷ vg / mm³, about 9.5 ... 107 vg / mm3 or about 1.0 x 108 vg / mm3.

[0300] In some embodiments, the putamen and caudate nucleus volumes of the first hemisphere (right or left) are obtained, and the dose for the second hemisphere is substantially the same as the dose for the first hemisphere.

[0301] In some embodiments, administration to the right hemisphere includes direct administration to the putamen and caudate nucleus of the right hemisphere; and administration to the left hemisphere includes direct administration to the putamen and caudate nucleus of the left hemisphere.

[0302] In some embodiments, the putamen and caudate nucleus volumes of the right hemisphere are measured, and the putamen and caudate nucleus volumes of the left hemisphere are measured, and based on the measured volumes, each hemisphere independently receives a dose of 2.0 x 107 vg / mm3 to 2.0 x 108 vg / mm3.

[0303] In some embodiments, a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is applied to the right hemisphere putamen and caudate nucleus in a proportion approximately equal to the volume of the putamen and caudate nucleus in the right hemisphere, and / or a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is applied to the left hemisphere putamen and caudate nucleus in a proportion approximately equal to the volume of the left hemisphere putamen and caudate nucleus.

[0304] The reference to approximate proportion indicates within 10% of each value to obtain 100%. For example, if the measured putamen to caudate nucleus volume is 60% : 40%, then a 10% approximate proportion provides a range of 70% : 30% to 50% : 50%. In further embodiments, the approximate proportion is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0305] In some embodiments, a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is administered to the right hemisphere putamen and caudate nucleus at a ratio of approximately 67% to approximately 33% of the putamen; and / or a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is administered to the left hemisphere putamen and caudate nucleus at a ratio of approximately 67% to approximately 33% of the caudate nucleus.

[0306] In some embodiments, the same dose is administered to both the right and left hemispheres.

[0307] In some embodiments, administration to the caudate nucleus includes a parietal approach, and / or administration to the putamen includes an occipital approach.

[0308] In some embodiments, 1.0 x 10¹⁰ vg to 1.0 x 10¹³ vg of rAAV carrier is administered to each hemisphere.

[0309] In some embodiments, a method of treating a subject for Huntington's disease includes (a) determining the volume of the putamen and / or caudate nucleus in the right and / or left hemisphere; and (b) administering a recombinant adeno-associated virus (rAAV) vector into the brain parenchyma of the subject, wherein a dose of about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³ is administered to the right and / or left hemisphere; wherein mm³ is the volume of the putamen and caudate nucleus in the hemisphere.

[0310] The dose administered to each hemisphere may be the same or different. In some embodiments, different amounts are provided to each hemisphere.

[0311] In some embodiments, the polynucleotide constructs, viral vectors, and nonviral vectors described herein are administered in combination with additional compounds or treatments for a particular disease or disorder; and / or in combination with compounds that reduce the immune response against the polynucleotide, delivery medium, and / or the resulting protein. Other compounds or treatments may be provided in different ways, such as by single administration; and may be administered or performed before, substantially simultaneously with, or after the administration of the polynucleotide constructs, viral vectors, and nonviral vectors described herein.

[0312] In some embodiments, the administration of the polynucleotide constructs, viral vectors, and nonviral vectors described herein is performed in combination with immunosuppressive agents or regimens. Such agents and regimens may be used as needed to achieve immune tolerance to the provided polynucleotides or delivery media, or to reduce the immune response to the provided polynucleotides or delivery media. Examples of immunosuppressive agents 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 removal.

[0313] In some embodiments, the polynucleotide construct, viral vector, or non-viral vector is administered in combination with one or more immunosuppressants, wherein one or more immunosuppressants are administered before, 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 simultaneously with the polynucleotide construct, viral vector, or non-viral vector. In some embodiments, one or more immunosuppressants are administered 1-12 hours, 12-24 hours, or 24-48 hours before the administration of the polynucleotide construct, viral vector, or non-viral vector; or 2-4 days, 4-6 days, 6-8 days, 8-10 days, 10-14 days, 14-20 days, 20-25 days, 25-30 days, 30-50 days, or more than 50 days after the administration of the polynucleotide construct, viral vector, or non-viral vector. In some embodiments, one or more immunosuppressants are administered 1–12 hours, 12–24 hours, or 24–48 hours after administration of the polynucleotide construct, viral vector, or non-viral vector; or 2–4 days, 4–6 days, 6–8 days, 8–10 days, 10–14 days, 14–20 days, 20–25 days, 25–30 days, 30–50 days, or more than 50 days after administration.

[0314] In some embodiments, the immunosuppressant is an anti-inflammatory agent. In some embodiments, the immunosuppressant is a steroid, such as a corticosteroid. In some implementations, the immunosuppressants are prednisone, prednisolone, calcineurin inhibitors (e.g., cyclosporine, tacrolimus), MMF (mycophenolate mofetil, e.g., CellCept®, Myfortic®), CD52 inhibitors (e.g., alemtuzumab), CTLA4-Ig (e.g., abatacept, beraccept), anti-CD3 mAb, anti-LFA-1 mAb (e.g., efazolin), anti-CD40 mAb (e.g., ASKP1240), anti-CD22 mAb (e.g., epazolizumab), anti-CD20 mAb (e.g., rituximab, olizumab, olfamumab, vetozumab), proteasome inhibitors (e.g., bortezomib), TACI-Ig (e.g., acecicept), anti-C5 mAb (e.g., eculizumab), mycophenolate mofetil, azathioprine, sirolimus everolimus, TNFR-Ig, and anti-TNF. mAb, tofacitinib, anti-IL-2R (e.g., baliximab), anti-IL-17 mAb (e.g., secukinumab), anti-IL-6 mAb (e.g., the anti-IL-6 antibody sirukumab), the anti-IL-6 receptor antibody tocilizumab (Actemra®), IL-10 inhibitorsTGF-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 mAb, e.g., belimumab), proliferation-inducing ligand (APRIL) inhibitors, anti-IL-1b mAb (e.g., canakinumab (Haris®)), C3a inhibitors, tregitope (see, for example, U.S. Patent No. 10,213,496), or combinations and / or derivatives thereof. In some embodiments, the immunosuppressant is a granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibitor, such as gimsilumab, lenzilumab, namilumab, otilimab, or mavrilimumab.

[0315] Strategies for reducing (overcoming), avoiding, or mitigating the effect 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, inhibit, prevent, or eradicate humoral immune responses against rAAV; altering the rAAV capsid serotype or engineering the rAAV capsid to make it less sensitive to neutralizing antibodies; using plasma exchange circulation to adsorb anti-AAV immunoglobulins, thereby reducing anti-AAV antibody titers; and using delivery techniques, such as balloon catheters followed by saline flushing. Such strategies are described, for example, in Mingozzi et al., (2013) Blood, 122:23-36. Similar techniques and strategies can be used for other types of viral vectors.

[0316] Empty capsids used as decoy probes, provided in formulations that are the same as or separate from 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 produced in a particular subject. In some implementations, the ratio of empty AAV capsid to rAAV carrier is within or between the following ranges: approximately 100:1 to 50:1, approximately 50:1 to 25:1, approximately 25:1 to 10:1, approximately 10:1 to 1:1, approximately 1:1 to 1:10, approximately 1:10 to 1:25, approximately 1:25 to 1:50, or approximately 1:50 to 1:10.100. In certain aspects, the ratio of the applied empty AAV capsid to the 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 type A in the rAAV serum specification, page 39 / 104, CN 122095088.

[0317] Other strategies for reducing humoral immunity against rAAV (which can be applied to other viral vectors) include methods for removing, depleting, capturing, and / or inactivating AAV antibodies, commonly referred to as apheresis, and more specifically as plasma removal (in the case of blood products). Apheresis or plasma removal is a process in which plasma of a human subject is circulated outside the body (in vitro) via a device and then returned to the patient, the device altering the plasma by adding, removing, and / or replacing components. Plasma removal 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-binding immunoglobulins (antibodies), thereby lowering the titer of AAV antibodies that may promote rAAV neutralization in the treated subject. An example is the use of a device comprising an AAV capsid affinity matrix column, and the passage of blood products (e.g., plasma) through the AAV capsid affinity matrix, resulting in the binding of different isotypes of AAV antibodies. (See, for example, Bertin et al., 2020, Sci. Rep. 10, 864, which is incorporated herein by reference in its entirety.)

[0318] In some embodiments, the polynucleotide construct, viral vector, and nonviral vector 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 IgG clearance in vivo; and / or in combination with agents that reduce circulating antibodies that bind to the recombinant viral vector, or to nucleic acids or polypeptides, proteins, or peptides encoded by polynucleotides capped by the recombinant viral vector, or to polynucleotides. In some embodiments, agents that reduce the interaction of IgG with FcRn (i.e., proteases or glycosidases) reduce or inhibit antibodies that bind to the viral vector.

[0319] In some embodiments, the polynucleotide constructs, viral vectors, and nonviral vectors described herein can be used in combination with endopeptidases (e.g., IdeS from Streptococcus pyogenes) or modified variants thereof, or endoglucosidases (e.g., EndoS from Streptococcus pyogenes) or modified variants thereof. For example, it can beSuch treatment is performed to reduce or eliminate neutralizing antibodies against gene delivery vectors (e.g., viral vector capsids) and to enable treatment of patients previously considered unsuitable for gene therapy or who have developed antibodies derived from gene therapy. Such strategies are described, for example, in Leborgne et al., (2020) Nat. Med., 26:1096-1101.

[0320] VII. Kits

[0321] The present invention includes kits having packaging materials and one or more components therein. Kits typically include labels or packaging inserts that include a description of the components or instructions for use of the components in vitro, in vivo, or ex vivo. Kits may contain a collection of such components, such as polynucleotide constructs, viral or nonviral vectors, and optionally a second active substance, such as another compound, agent, drug, or composition.

[0322] A kit refers to a physical structure that contains one or more components. Packaging materials can sterilely retain the components and can be made of materials commonly used for such purposes, such as paper, corrugated fiber, glass, plastic, foil, ampoules, vials, and tubes.

[0323] Labels or inserts may include identification information for one or more of the components, dosage amounts, and clinical pharmacology (including mechanism of action), pharmacokinetics, and pharmacodynamics of one or more active ingredients. Labels or inserts may include information identifying the manufacturer, batch number, place and date of manufacture, and expiration date. Labels or inserts may include information about the diseases that the kit components may target. Labels or inserts may include instructions for clinicians or subjects to use one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions may include dosage amounts, frequency, or duration, and instructions for carrying out any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0324] Labels or inserts may include information about one or more benefits that the components may provide, such as prophylactic or therapeutic benefits. Labels or inserts may include information about potential adverse side effects, complications, or reactions, such as warnings to subjects or clinicians regarding situations where the specific composition is unsuitable for use, as per CN 122095088 A, page 40 / 104. 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 undergoing another treatment protocol or therapeutic regimen that will be incompatible with the composition; therefore, the package insert may include information about such incompatibilities.

[0325] Labels or inserts include “printed matter,” such as paper or cardboard, either alone or attached to components, reagent kits, or packaging materials (e.g., boxes), or affixed to ampoules, tubes, or vials containing reagent kit components. Labels or inserts may also include computer-readable media such as barcode-printed labels, disks, optical discs (e.g., CD-ROM / RAM or DVD-ROM / RAM, DVD), MP3 players, magnetic tapes, or electrical storage media (e.g., RAM and ROM), or hybrids of these media (e.g., magnetic / optical storage media, flash memory media, or memory cards). VIII. Other Aspects and Embodiments

[0326] Other aspects, embodiments, and combinations thereof include the following: 1. An RNA polynucleotide comprising a target RNA sequence that is at least 80% identical to the sequence of any one of SEQ ID NO: 1-19; provided that if the target sequence is at least 80% identical to SEQ ID NO: 18, then the RNA polynucleotide is (a) a primary amiRNA comprising the target sequence embedded in a scaffold selected from the S155e scaffold, the S-26a scaffold, and the S-33 scaffold; or (b) the RNA polynucleotide comprising the sequence of SEQ ID NO: 117 or 118. In various embodiments, the sequence 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 one of SEQ ID NOs: 1-3, 5, 6, and 16-19. 3. The RNA polynucleotide according to claim 2, wherein the target RNA sequence comprises the sequence of any one of SEQ ID NOs: 1-3, 5, 6, and 16-19. 4. The RNA polynucleotide according to claim 2 or 3, wherein the RNA polynucleotide further comprises a second RNA sequence, wherein the second RNA sequence is substantially complementary to the target RNA sequence. 5. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a primary miRNA comprising a primary miRNA scaffold, a guide sequence, and a guest sequence, wherein the guide sequence comprises the target sequence and the guest sequence comprises the second RNA sequence. 6. The RNA polynucleotide of claim 5, wherein the scaffold is an S155e scaffold, an S26a scaffold, an S33 scaffold, or an S155 scaffold. 7. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide comprises the sequence described in SEQ ID NO:8. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises a sequence of any one of SEQ ID NO: 51-62, 64-68, and 78-87. 9. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises a sequence of any one of SEQ ID NO: 51-62, 64-68, and 78-87. 10. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a premiRNA comprising a guide sequence and a guest sequence, wherein the guide sequence comprises the target sequence and the guest sequence comprises the second RNA sequence. (Specification 41 / 104 pages 50 CN 122095088 A) 11. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a shRNA comprising a guide sequence and a guest sequence, wherein the guide sequence comprises the target sequence and the guest sequence comprises the second RNA sequence. 12. The RNA polynucleotide of claim 10, wherein the RNA polynucleotide comprises a sequence that is at least 90% or at least 95% identical to any one of SEQ ID NO: 88-96, 98-101, and 111-118. 13. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises a sequence of any one of SEQ ID NO: 88-96, 98-101, and 111-118. 14. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a repressive RNA duplex comprising a guide sequence and a guest sequence, wherein the guide sequence comprises the target sequence and the guest sequence comprises the second RNA sequence. 15. The RNA polynucleotide according to claim 14, wherein the repressive RNA duplex has a combination of a guide strand and a guest strand selected from the following: a) a guide strand comprising the sequence of SEQ ID NO: 1 and a guest strand comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to any one of SEQ ID NO: 20, 21, and 22; b) a guide strand comprising the sequence of SEQ ID NO: 2 and a guest strand comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to any one of SEQ ID NO: 23, 24, and 25; c) a guide strand comprising the sequence of SEQ ID NO: 3 and a guest strand comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to any one of SEQ ID NO: 26, 27, and 28; d) a guide strand comprising the sequence of SEQ ID NO: 1 and a guest strand comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to any one of SEQ ID NO: 26, 27, and 28;e) A guide chain containing the sequence of SEQ ID NO: 4 and a pass chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 29; f) A guide chain containing the sequence of SEQ ID NO: 5 and a pass chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 30; g) A guide chain containing the sequence of SEQ ID NO: 6 and a pass chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of any one of SEQ ID NO: 31, 32, and 33; h) A guide chain containing the sequence of SEQ ID NO: 7 and a pass chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 34; The following are examples of sequences: i) a guide chain containing the sequence of SEQ ID NO: 35 and a guest chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 35; j) a guide chain containing the sequence of SEQ ID NO: 10 and a guest chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 37; k) a guide chain containing the sequence of SEQ ID NO: 11 and a guest chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 38; l) a guide chain containing the sequence of SEQ ID NO: 12 and a guest chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 35. 39. A transit chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical sequences to SEQ ID NO: 40; m) A guide chain containing the sequence of SEQ ID NO: 13 and a transit chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical sequences to SEQ ID NO: 40; n) A guide chain containing the sequence of SEQ ID NO: 14 and a transit chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical sequences to SEQ ID NO: 41; Description 42 / 104 pages 51 CN 122095088 A o) Containing SEQp) A guiding chain containing the sequence of SEQ ID NO: 15 and a transit chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% of the sequence identical to SEQ ID NO: 42; p) A guiding chain containing the sequence of SEQ ID NO: 16 and a transit chain containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% of the sequence identical to any one of SEQ ID NO: 43, 44, and 45. q) A guide strand containing the sequence of SEQ ID NO: 17 and a guest strand containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 46; r) A guide strand containing the sequence of SEQ ID NO: 19 and a guest strand containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 47; and s) A guide strand containing the sequence of SEQ ID NO: 18 and a guest strand containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of any one of SEQ ID NO: 48, 49, and 50. 16. The RNA polynucleotide according to claim 1, wherein the target sequence is a guide sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 18, and the guide sequence is embedded in an S26a scaffold or an S33 scaffold. 17. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 117 or 118. 18. The RNA polynucleotide of claim 17, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 86 or 87. 19. An optionally modified repressive RNA comprising (a) a guide strand capable of hybridizing to a target sequence of any one of SEQ ID NO: 119-137; and (b) a substantially complementary guest sequence; wherein one or more nucleotides of the guide strand and the guest strand are optionally modified RNA. 20. A polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of 1-19. 21. An expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of 1-19, and one or more expression control elements operatively linked to the nucleic acid sequence encoding the RNA polynucleotide. 22. The expression cassette of claim 21, wherein the expression cassette comprises an upstream promoter and a downstream polyadenylation signal operatively linked to a nucleic acid sequence encoding the RNA polynucleotide.23. The expression cassette of claim 22, wherein the expression cassette comprises, from 5' to 3', a promoter or promoter / enhancer operatively linked to a nucleic acid sequence encoding the RNA polynucleotide, the nucleic acid sequence encoding the RNA polynucleotide, and a polyadenylation signal. 24. The expression cassette of claim 22 or 23, wherein the promoter provides high CNS expression. 25. The expression cassette of claim 22 or 23, wherein the promoter is an EF-1α promoter. 26. The expression cassette of any one of claims 21-25, wherein the expression cassette is DNA. 27. The expression cassette of claim 26, wherein the expression cassette comprises at least 90%, at least 95%, at least 97%, or 100% identical sequences to any one of SEQ ID NO: 188-199, 201-205, and 215-224. 28. The expression cassette of claim 26, wherein the expression cassette comprises the sequence of SEQ ID NO: 223 or 224. 29. A recombinant viral vector nucleic acid comprising an expression cassette according to any one of 21-28 and 5' and / or 3' viral elements for viral packaging and / or replication. 30. The recombinant viral vector nucleic acid according to 29, wherein the recombinant viral vector nucleic acid is DNA and comprises an adeno-associated virus (AAV) inverted repeat sequence (ITR) located on the 5' end flanking the recombinant viral vector nucleic acid and an AAV ITR located on the 3' end flanking the recombinant viral vector nucleic acid. 31. The recombinant viral vector nucleic acid of claim 30, wherein the 5' ITR and the 3' ITR are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3B. 32. The recombinant viral vector nucleic acid of claim 30, wherein the recombinant viral vector nucleic acid is self-complementary. 33. The recombinant viral vector nucleic acid of claim 30 or 32, wherein the 5' ITR contains at least 90%, at least 95%, at least 97%, or 100% of the sequence identical to SEQ ID NO: 254, and the 3' ITR contains at least 95%, at least 95%, at least 97%, or 100% of the sequence identical to SEQ ID NO: 253. 34. The recombinant viral vector nucleic acid according to claim 30, wherein the recombinant viral vector nucleic acid comprises at least 90%, at least 95%, at least 97%, or 100% identical sequences to any one of SEQ ID NO: 233-235 and 237-242. 35. A recombinant viral nucleotide comprising SEQ ID NO:36. A delivery medium comprising a viral or non-viral vector and an inhibitory RNA according to claim 19, a polynucleotide according to claim 20, an expression cassette according to any one of claims 21-28, or a recombinant viral vector nucleic acid according to any one of claims 29-35. 37. The delivery medium according to claim 36, wherein the delivery medium is the viral vector. 38. The delivery medium according to claim 37, wherein the viral vector is a recombinant AAV, a recombinant lentiviral vector, or a recombinant adenovirus vector. 39. The delivery medium according to claim 38, wherein the viral vector is a recombinant AAV. 40. The delivery medium according to claim 39, wherein the recombinant AAV vector comprises a capsid having at least 90% identical sequences to 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: 257 or SEQ ID NO: 260. 41. The delivery medium of claim 40, wherein the capsid 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 capsid; or the capsid comprises VP1 of SEQ ID NO: 257 or SEQ ID NO: 260. 42. The delivery medium of claim 36, wherein the delivery medium is a nanoparticle selected from: 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. 43. The delivery medium according to claim 42, wherein the delivery medium is an LNP or LPNP. 44. A pharmaceutical composition comprising an inhibitory RNA according to claim 19, a polynucleotide according to claim 20, an expression cassette according to any one of claims 21-28, a recombinant viral vector nucleic acid according to any one of claims 29-35, or a delivery medium according to any one of claims 36-43; and a pharmaceutically acceptable carrier.45. A method for reducing the expression of huntingtin protein in cells or a subject, the method comprising administering to the cells or subject an inhibitory RNA according to 19, a polynucleotide according to 20, an expression cassette according to any one of 21-28, a recombinant viral vector nucleic acid according to any one of 29-35, a delivery medium according to any one of 36-43, or a pharmaceutical composition according to 44. 46. A method for treating a subject with Huntington's disease, the method comprising administering to the subject an inhibitory RNA according to 19, a polynucleotide according to 20, an expression cassette according to any one of 21-28, a recombinant viral vector nucleic acid according to any one of 29-35, a delivery medium according to any one of 36-43, or a pharmaceutical composition according to 44. 47. The method according to 45 or 46, wherein administration comprises direct intracranial, intracisional, or intraventricular administration. 48. The method according to 45 or 46, wherein the initial administration is external to the CNS. 49. The method according to any one of 45-48, wherein the subject is a human. 50. An AAV vector genomic plasmid comprising recombinant viral nucleic acid according to any one of 30-35. 51. The AAV genomic plasmid according to 50, wherein the plasmid lacks the rep and cap genes. 52. A method of producing an rAAV vector, the method comprising the step of culturing an rAAV production cell line containing rAAV helper viral activity, wherein the genome of the production cell comprises nucleic acid, a rep gene, and a cap gene according to any one of 30-35, wherein the rAAV vector is produced. 53. A method of producing an rAAV vector, the method comprising the step of culturing an rAAV-permitting cell containing an rAAV genomic plasmid according to 51, wherein the rAAV-permitting cell further comprises (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. 54. The method of claim 53, wherein the rAAV-allowing cell is a packaging cell, wherein the genome of the packaging cell comprises a cap gene and a rep gene. 55. The method of claim 53, 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. 56. A method for obtaining an rAAV vector, the method comprising the steps of: (a) using any of the methods described in claims 52-55(a) The method produces the rAAV vector and (b) purifies the rAAV vector. 57. A polynucleotide comprising the RNA sequence of SEQ ID NO: 255, wherein N01 to N42 are ribonucleotides, N01 is complementary to N42, N02 is not complementary to N41, N03–N10 is complementary to N33–N40, N11 is not complementary to N32, and N12–N21 is complementary to N22–N31; or corresponding to DNA. 58. The polynucleotide according to claim 57, further comprising a 5' flanking region and a 3' flanking region, wherein the polynucleotide comprises the RNA sequence of SEQ ID NO: 256; or corresponding to DNA sequence. 59. A DNA polynucleotide comprising, in the 5' to 3' direction: (a) a 5' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO: 262; (b) a CAG promoter; (c) a preamiRNA coding sequence containing the sequence of SEQ ID NO: 261, wherein the CAG promoter is operatively linked to the preamiRNA coding sequence and a polyadenylation signal; and (d) a 3' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO: 263. 60) The polynucleotide according to claim 59, wherein the polyadenylation signal comprises the sequence of SEQ ID NO: 264 or 252. 61) The polynucleotide according to claim 59 or 60, wherein the CAG promoter comprises the sequence of SEQ ID NO: 250. 62) The polynucleotide according to claim 59 or 60, wherein the CAG promoter comprises the sequence of SEQ ID NO: 265. 63) The polynucleotide according to any one of 59-62, wherein the end of the 5' ITR to the end of the 3' ITR is at most about 2.5 kb. 64) The polynucleotide according to claim 59, wherein the polynucleotide comprises the sequence of SEQ ID NO: 266. 65) The polynucleotide according to any one of 59-64, wherein the polynucleotide is a plasmid further comprising an origin of replication and a selectivity marker. 66) The polynucleotide according to any one of 59-64, wherein the polynucleotide is a recombinant adeno-associated virus (rAAV) nucleic acid comprising a 5' ITR at the 5' end and a 3' ITR at the 3' end. 67) A recombinant adeno-associated virus (rAAV) vector, said recombinant adeno-associated virus vector comprising: (a) rAAV nucleic acid according to 66, and (b) an rAAV capsid comprising: VP1 containing the amino acid sequence of SEQ ID NO: 257, and SEQ ID NO:68) A recombinant adeno-associated virus (rAAV) vector comprising: (a) rAAV nucleic acid containing a preamiRNA coding sequence operatively linked to a promoter and a polyadenylation signal, the preamiRNA coding sequence containing the sequence of SEQ ID NO: 261; and (b) an rAAV capsid containing: VP1 containing the amino acid sequence of SEQ ID NO: 257, VP2 containing the amino acid sequence of SEQ ID NO: 258, and VP3 containing the amino acid sequence of SEQ ID NO: 259. 69) A pharmaceutical composition comprising about 1.0 x 10¹⁰ vg to about 1.0 x 10¹³ vg of the rAAV vector according to 67 or 68 and a pharmaceutically acceptable carrier. 70) The pharmaceutical composition according to 69 comprises about 1.0 x 10¹¹ vg to about 1.0 x 10¹² vg and a pharmaceutically acceptable carrier. 71) The pharmaceutical composition according to claim 69, wherein the pharmaceutical composition comprises about 1.0 x 10¹¹ vg, about 1.1 x 10¹¹ vg, about 1.2 x 10¹¹ vg, about 1.3 x 10¹¹ vg, about 1.4 x 10¹¹ vg, about 1.5 x 10¹¹ vg, about 1.6 x 10¹¹ vg, about 1.7 x 10¹¹ vg, about 1.8 x 10¹¹ vg, about 1.9 x 10¹¹ vg, about 1.0 x 10¹² vg, about 1.1 x 10¹² vg, about 1.2 x 10¹² vg, about 1.3 x 10¹² vg, about 1.4 x 10¹² vg, about 1.5 x 10¹² vg, about 1.6 x 10¹² vg. 72) A pharmaceutical composition according to any one of 44 or 69-71, wherein the pharmaceutical composition further comprises an MRI imaging agent. 73) A pharmaceutical composition according to 72, wherein the MRI imaging agent is gadolinium. 74) A pharmaceutical composition comprising a sufficient amount of the rAAV carrier according to any one of 38-41, 67 or 68 to provide a 20% to 90% reduction in total huntingtin protein; and a pharmaceutically acceptable carrier. 75) A pharmaceutical composition according to 74, wherein the pharmaceutical composition comprises a sufficient amount of the rAAV carrier to provide a 20% to 65% reduction in total huntingtin protein.76) A pharmaceutical composition according to claim 75, wherein the pharmaceutical composition comprises a sufficient amount of the rAAV carrier to provide a 25% to 40% reduction in total huntingtin protein; and a pharmaceutically acceptable carrier. 77) A pharmaceutical composition comprising a sufficient amount of the rAAV carrier according to any one of 39-41, 67, or 68 to reduce total HTT protein and / or total HTT mRNA to about 25% to about 60%, about 25% to about 50%, about 25% to about 45%, about 25% to 40%, about 25% to 35%, about 25% to 30%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, or about 30% to about 35%; and a pharmaceutically acceptable carrier. 78) A method of treating a subject with Huntington's disease, the method comprising administering, into the brain parenchyma of the subject, the rAAV vector according to claim 67 or 68 or the pharmaceutical composition according to any one of 69-77. 79) A method of treating a subject with Huntington's disease, the method comprising (a) determining the volume of the putamen and / or caudate nucleus in the right and / or left hemisphere of the subject; and (b) administering, into the brain parenchyma of the subject, a recombinant adeno-associated virus (rAAV) vector at a dose of about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³ to the right hemisphere and / or at a dose of about 2.0 x 10⁷ vg / mm³ to the left hemisphere; wherein the dose is based on the volume determined in step (a). In a further embodiment, the following volumes are determined: (1) the putamen in the right hemisphere; (2) the caudate nucleus in the right hemisphere; (3) the putamen in the left hemisphere; (4) the caudate nucleus volume in the left hemisphere; (5) the caudate nucleus and putamen volume in the right hemisphere; (6) the caudate nucleus and putamen volume in the left hemisphere; or (7) the caudate nucleus and putamen volume in both the right and left hemispheres. 80) The method according to 79, wherein application to the right hemisphere comprises direct application to the putamen and caudate nucleus of the right hemisphere; and application to the left hemisphere comprises direct application to the putamen and caudate nucleus of the left hemisphere. 81) The method according to 80, wherein the putamen and caudate nucleus volumes of the right hemisphere are measured, and the putamen and caudate nucleus volumes of the left hemisphere are measured, and based on the measured volumes, each hemisphere independently receives a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³. 82) The method according to any one of 79-81, wherein 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / 83) The method according to any one of 79-82, wherein a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is applied to the right hemisphere's putamen and caudate nucleus in a ratio of about 67% putamen to about 33% caudate nucleus. 84) The method according to any one of 79-83, wherein for the left hemisphere, a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is applied to the left hemisphere's putamen and caudate nucleus in a ratio of about 67% putamen to about 33% caudate nucleus. 85) The method according to any one of 79-84, wherein a dose of 2.0 x 10⁷ vg / mm³ to 2.0 x 10⁸ vg / mm³ is applied to the left hemisphere putamen and caudate nucleus at a ratio of approximately 67% putamen to approximately 33% caudate nucleus. 86) The method according to any one of 79-85, wherein the same dose is applied to the right hemisphere and the left hemisphere. 87) The method according to any one of 79-86, wherein application to the caudate nucleus includes a parietal approach. 88) The method according to any one of 79-87, wherein application to the putamen includes an occipital approach. 89) The method according to any one of 79-88, wherein 1.0 x 10¹⁰ vg to 1.0 x 10¹³ vg of rAAV carrier is administered to each hemisphere. 90) The method according to 89, wherein 1.0 x 10¹¹ vg to 1.0 x 10¹² vg of rAAV carrier is applied to each hemisphere. 91) The method according to any one of 79-88, wherein approximately 1.0 x 10¹¹ vg, approximately 1.1 x 10¹¹ vg, approximately 1.2 x 10¹¹ vg, approximately 1.3 x 10¹¹ vg, approximately 1.4 x 10¹¹ vg, approximately 1.5 x 10¹¹ vg, approximately 1.6 x 10¹¹ vg, approximately 1.7 x 10¹¹ vg, approximately 1.8 x 10¹¹ vg, approximately 1.9 x 10¹¹ vg, approximately 1.0 x 10¹² vg, approximately 1.1 x 10¹² vg, approximately 1.2 x 10¹² vg, approximately 1.3 x 10¹² vg, approximately 1.4 x 10¹² vg, approximately 1.5 x 10¹¹ vg, are applied to each hemisphere. 10¹² vg, approximately 1.6 x 10¹² vg, approximately 1.7 x 10¹² vg, approximately 1.8 x 10¹² vg, approximately 1.9 x 10¹² vg, or approximately 1.0 x 10¹³ vg.92) The method according to any one of 79-91, wherein the rAAV carrier is the rAAV carrier according to any one of claims 39-41, 67, and 68. 93) The method according to any one of 79-92, wherein administration includes convection-enhanced delivery. 94) The method according to any one of 79-93, the method comprising: (a) determining the volume of the putamen and / or the caudate nucleus in mm³ in the right hemisphere and / or the left hemisphere; and (b) multiplying the volume obtained in step (a) by a desired dose in vg / mm³ to further obtain the desired dose in vg / putamen and caudate nucleus; and (c) manipulating the dose obtained in step (b) with a given drug concentration in vg / ml to obtain a desired drug dose volume; and (d) administering a dose of the rAAV carrier into the brain parenchyma of the subject, wherein the dose in ml is obtained according to steps (b) and (c). 95) The method of claim 94, wherein the desired drug concentration is about 6.2 x 10¹¹ vg / ml. 96) A method of treating a subject with Huntington's disease, the method comprising: (a) determining the volume of the putamen and / or caudate nucleus in the right and / or left hemisphere, in mm³; and (b) multiplying the volume obtained in step (a) by a desired dose in vg / mm³ to further obtain the desired dose in vg / putamen and / or caudate nucleus; and (c) manipulating the dose obtained in step (b) with a given drug concentration in vg / ml to obtain a desired drug dose volume; and (d) administering a dose of recombinant adeno-associated virus (rAAV) vector into the brain parenchyma of the subject, wherein the dose in ml is obtained according to steps (b) and (c). 97) The method of claim 96, wherein the desired dose is about 2.0 x 10⁷ vg / mm³ to about 2.0 x 10⁸ vg / mm³. 98) The method of claim 97, wherein the desired drug concentration is about 6.2 x 10¹¹ vg / ml. 99) The method of any one of 96-98, wherein step (a) measures the putamen and caudate nucleus in one or both hemispheres, and step (b) obtains a dose for both the putamen and the caudate nucleus. 100) The method of any one of 78-99, wherein the presence of the inhibitory nucleic acid encoded by the rAAV vector is measured in cerebrospinal fluid. In a further embodiment, the presence of the inhibitory nucleic acid encoded by the rAAV vector is measured before or simultaneously with vector administration, and at least one month after vector administration; or before or simultaneously with vector administration,And the presence of the repressive nucleic acid encoded by the rAAV vector is measured at least 3 months, at least 6 months, at least 12 months, at least 18 months and / or at least 24 months after vector administration. 101) The method according to 100, wherein the repressive nucleic acid is encoded by an rAAV nucleic acid comprising a miR155 scaffold, and the expressed repressive RNA is measured in cerebrospinal fluid by detecting the miR155 scaffold. 102) The method according to 101, wherein the repressive nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 261. 103) The method according to 101 or 102, wherein the detection of the expressed repressive nucleic acid comprises the steps of: (a) purifying RNA from cerebrospinal fluid; (b) reverse transcriptase; and (c) polymerase chain reaction (qPCR). 104) The method according to 103, wherein step (a) comprises incubating cerebrospinal fluid in lysis buffer and ethanol for approximately 5 minutes. 105) The method according to any one of 102-104, wherein step (b) comprises about 20 µL of RNA and about 40 µL of RT. 106) The method according to any one of 102-105, wherein step (c) comprises cycling between about 95ºC and about 60ºC. In a further embodiment, the cycling at 95ºC lasts for about 15 seconds and the cycling at 60ºC lasts for about one minute; and / or about 40 cycles are performed. In another embodiment, prior to cycling, the qPCR mixture is placed at 50ºC for about two minutes, and then at 95ºC for about 10 minutes. 107) The method according to any one of 78-106, wherein the subject is a human. IX. Sequence

[0327] Table 3 provides different nucleic acid and amino acid sequences. In some embodiments, the polynucleotide comprises at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical nucleic acid sequences to any nucleic acid sequence provided in Table 3. Table 3 Instruction Manual 49 / 104 pages 58 CN 122095088 A Instruction Manual 50 / 104 pages 59 CN 122095088 A Instruction Manual 51 / 104 pages 60 CN 122095088 A Instruction Manual 52 / 104 pages 61 CN 122095088 A Instruction Manual 53 / 104 pages 62 CN 122095088 A Instruction Manual 54 / 104 pages 63 CN 122095088 A Instruction Manual 55 / 104 pages 64 CN 122095088 A Instruction Manual 56 / 104 pages 65 CN 122095088 A Instruction Manual 57 / 104 pages66 CN 122095088 A Instruction Manual, pages 58 / 104; 67 CN 122095088 A Instruction Manual, pages 59 / 104; 68 CN 122095088 A Instruction Manual, pages 60 / 104; 69 CN 122095088 A Instruction Manual, pages 61 / 104; 70 CN 122095088 A Instruction Manual, pages 62 / 104; 71 CN 122095088 A Instruction Manual, pages 63 / 104; 72 CN 122095088 A Instruction Manual, pages 64 / 104; 73 CN 122095088 A Instruction Manual, pages 65 / 104; 74 CN 122095088 A Instruction Manual, pages 66 / 104; 75 CN 122095088 A Instruction Manual, pages 67 / 104; 76 CN 122095088 A Instruction manual 68 / 104 pages 77 CN 122095088 A Instruction manual 69 / 104 pages 78 CN 122095088 A Instruction manual 70 / 104 pages 79 CN 122095088 A Instruction manual 71 / 104 pages 80 CN 122095088 A Instruction manual 72 / 104 pages 81 CN 122095088 A Instruction manual 73 / 104 pages 82 CN 122095088 A

[0328] Table 4 provides a summary of the correspondences between the guide RNA, guest RNA, primary amiRNA, pre-amiRNA and target sequence and the corresponding / coding DNA of the guide sequence, guest sequence and primary amiRNA. Table 4 Instruction manual 74 / 104 pages 83 CN 122095088 A

[0329] The scaffold names in Table 4 are provided for primary amiRNA and are applicable to primary miRNA corresponding / coding DNA. Examples

[0330] Examples are provided below, which further illustrate different features of the invention and methods for practicing the invention. The examples provided do not limit the claimed invention. Specification 75 / 104 pages 84 CN 122095088 A

[0331] Example 1: miRNA screening for HTT protein knockdown

[0332] The ability of different miRNA guide sequences to inhibit HTT expression was evaluated using plasmids containing rAAV nucleic acids encoding different primary amiRNA constructs. Primary amiRNA constructs were designed by embedding different guide sequences and guest sequences into an S155 scaffold. The complement of the guide sequence was taken and two base pairs at positions 9 and 10 were removed to create a central bulge.The guest sequence was designed. The S155 scaffold provides a 27-base 5' flanking region and a 41-base 3' flanking region.

[0333] Figure 2A shows the positions of the different components of the rAAV nucleic acid encoding the primary amiRNA: 5' ITR, CAG promoter, S155 scaffold-based primary amiRNA, polyadenylation signal (pA), and 3' ITR.

[0334] Recombinant AAV nucleic acids encoding different primary amiRNAs were cloned into plasmids to obtain miRNA expression plasmids. rAAV includes AAV2 ITR, rabbit β-globin (RBG) polyadenylation signal (pA), and CAG promoter. The encoded primary amiRNA was inserted downstream of the promoter. Table 5 provides (a) the construct name, promoter, scaffold, and miRNA name indicating that the generated rAAV nucleic acid is single-stranded (“ss”); and (b) the target sequence; and (c) the DNA encoding the primary amiRNA of different constructs. Table 5 Instructions 76 / 104 pages 85 CN 122095088 A

[0335] The plasmid was transfected into HEK293 cells, and cell lysates were collected 72 hours post-transfection. Figure 2B shows the HTT protein content in the lysates quantified and normalized using the quinoline carboxylic acid assay (BCA). Protein quantification was performed using a custom WES capillary electrophoresis assay to identify the total human HTT protein using an antibody (Millipore mab2166-clone 1HU-4C8). Data were normalized relative to out-of-order controls. N = 3 experiments. One-way ANOVA and comparisons with controls (Dunnett test).

[0336] The constructs summarized in Table 5 were further evaluated by transfecting HEK293 cells with miRNA expression plasmids and measuring HTT mRNA levels. Figure 2C shows the total HTT mRNA levels from HEK293 cells transfected with different miRNA expression plasmids. RNA was extracted from cells transfected with 1 µg plasmid 72 hours post-transfection and analyzed using TaqMan RT-qPCR primers / probes. HTT mRNA levels were plotted against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and normalized relative to a random control. N = 3–6 transfections. ss.CAG.S155.miR18 showed the highest levels of reduced HTT mRNA.

[0337] Example 2: Screening in the YAC128 HD mouse model

[0338] Recombinant AAVs encoding different miRNAs were generated using triple transfection and tested in the YAC128 HD mouse model. S155.miR1, S155.miR2, S155.miR16, and S155.miR18 constructs were capsidated into AAV particles, andThe vector was bilaterally delivered to the striatum using a stereotactic injection device at a total dose of 1e10 vector genomes / mouse. Mice were 6 weeks old at injection and fed for two months post-injection. The AAV particles contained VP1 SEQ ID NO: 257, VP2 SEQ ID NO: 258, and VP3 SEQ ID NO: 259. The different constructs are summarized in Table 6. Table 6.

[0339] Two months post-injection, the copy number of the injected vector was assessed by extracting DNA from mouse striatal tissue and performing qPCR using primers targeting the vector DNA. Total DNA was extracted from freshly frozen mouse striatal tissue and measured using TaqMan qPCR primers / probes targeting the transgenic multi-A sequence. N = 6 mice / group. No significant differences were observed between any miRNA vector injection groups (Kruskal-Wallis nonparametric ANOVA + Dunnett test). The results are shown in Figure 3A.

[0340] The miRNA abundance from mouse striatum injected with rAAV vectors encoding different miRNA constructs is shown in Figure 3B. Total RNA was extracted from freshly frozen mouse striatum tissue, and miRNA levels were assessed using a custom-made TaqMan microRNA reverse transcription kit (T-qPCR). N = 5–6 mice / group. rAAV.ss.CAG.S155.miR18 had the highest miRNA abundance compared to other vectorized miRNA injection groups (Kruskal-Wallis nonparametric ANOVA + Dunnett test). miR16 had significantly lower miRNA levels than other groups, while miR18, miR1, and miR2 had similar miRNA levels in the striatum.

[0341] The mutant HTT protein from striatum lysates was evaluated using WES capillary electrophoresis with an antibody (Millipore, MABN2427) specific for the multiQ mutant. All tested vectors significantly reduced HTT mutant protein, with miR18 showing the largest reduction, approximately 40% of the diluent control (Figure 3C). Specification 77 / 104 pages 86 CN 122095088 A

[0342] Total HTT mRNA levels were measured in mice injected with the diluent or 1e10 vg / mouse or 3.4e10 vg / mouse r.AAV.ss.CAG.S155.miR18 vector. HTT mRNA levels were assessed by RT-qPCR (Figure 3D). N = 5–12 mice / group. HTT mRNA levels, as assessed by RT-qPCR, were reduced by approximately 30.1% at low doses and by approximately 61.4% at high doses.

[0343] Total HTT mRNA levels were measured in mice injected with the diluent or 1e10 vg / mouse or 3.4e10 vg / mouse r.AAV.ss.CAG.S155.miR18 vector.Mutant HTT protein was assessed in the striatum of mice injected with the ss.CAG.S155.miR18 vector from vg / mouse. Protein was detected by WES immunoassay using the multi-Q specific antibody MW1 and normalized relative to a dilution control. N = 5–12 / group. The level of mutant HTT protein, as assessed by WES capillary electrophoresis, was reduced by 40.3% and 65.5% in the low-dose and high-dose tests, respectively (Figure 3E).

[0344] Example 3: In vitro screening of miRNA scaffolds

[0345] Guide sequences and guest sequences from miR18 were embedded into 10 different miRNA scaffolds: Scr6 (randomized control), S451 (miR451 scaffold), S122 (miR122 scaffold), S155e (eSIBR scaffold), S26, S33 (miR33 scaffold), S186 (miR186 scaffold), S10, S16 (miR-16 scaffold), S155 (miR155 scaffold), and Slet7. S451, S122, S33, S186, SR16, and S155 scaffolds were generated from endogenous human or mouse miRNA sequences by replacing the endogenous guide / guest sequences with artificial guide sequences and guest sequences. The “S155e” backbone is the modified miR155 backbone described by Fang and Bartel Genes. Mol Cell. (2015) 60 (1):131-1452015. The S26, S10, and Slet7 scaffolds are artificial scaffolds generated by modifying miR26, miR10, and Slet7. Artificial guest sequences are manipulated by generating mismatches with the guide sequence to maintain the secondary structure of the endogenous miRNA.

[0346] The nucleic acid encoding the scaffold was used to generate a miRNA expression plasmid (CAG promoter) designed according to Example 1 and transfected into HEK293 cells as described in Example 1. The scaffold has embedded guide and guest sequences as well as flanking sequences at the 5' and 3' ends. The constructs are summarized in Table 7. Table 7

[0347] Figure 5A shows the miRNA sequence levels from cells transfected with the indicated constructs as measured by RT-qPCR. Data are normalized relative to miR18 expressed from the S155 scaffold. N = 3–6 transfections. The S26a and S33 scaffolds showed significantly higher miRNA levels (approximately 2.5-fold and 3.5-fold, respectively) compared to the S155 scaffold. The S155e scaffold also tended to have higher miRNA levels (approximately 2-fold) compared to the original S155 scaffold.

[0348] Figure 5B shows the HTT mRNA levels from cells transfected with the indicated constructs as measured by RT-qPCR.Data were normalized relative to the out-of-order control. N = 3–6 transfections. The S155e, S26a, and S33 scaffolds exhibited the most effective HTT mRNA knockdown, with a reduction of approximately 60%.

[0349] Figure 5C shows protein levels from cells transfected with the indicated constructs, measured by WES capillary electrophoresis using mab2166. Data were normalized relative to the out-of-order control. N = 3–6 transfections. The 155e, S26a, and S33 scaffolds tended to have the largest HTT protein knockdown, with a knockdown level of approximately 40% compared to the out-of-order control. The results suggest that miRNA scaffolds with embedded miRNA guide sequences and guest sequences may affect efficacy.

[0350] Example 4: Evaluation of miR18 processing

[0351] To further characterize the effect of miRNA scaffolds on miRNA processing, small RNA sequencing was performed on HEK293 cells transfected with miR18 guide sequences embedded in scaffolds S155, S155e, S451, S26a, or S33 as described in Table 7. Sequencing was performed using Illumina TruSeq libraries with reads having a single 150-base-pair terminal. Read counts were normalized relative to per million reads, with a total of approximately 2 million reads per sample. The Scr6 group expressed a scrambled control, while the other groups expressed miR18. N = 3 samples / group.

[0352] Figure 6A shows the read counts for the guide strand and the guest strand. Compared to the S155 scaffold, the miR18 guide strand level was approximately 2–3 times higher when expressed from the S26a or S33 scaffold. The S26a scaffold showed the lowest amount of guest strand expression.

[0353] Figure 6B shows the ratio of the guide sequence to the guest sequence in the miRNA construct. N = 3 samples / group. Compared with the S155 scaffold, the S26, S33 and S451 scaffolds all produced a higher ratio of guide sequence to guest sequence.

[0354] Figure 6C shows the read stacking along the miRNA transgene in cells transfected with the indicated constructs. Analysis of the read stacking showed that the S26a and S33 scaffolds had the most precise processing of the guide miRNA, and the level of the guest chain was almost undetectable. Since guest chain activity can lead to dysregulation of off-target genes, the low amount of guest chains produced by these scaffolds indicates the potential improvement in the safety and efficacy of these constructs.

[0355] Example 5: In vitro testing of different miRNA and scaffold combinations

[0356] Guide sequences and guest sequences of miR1, miR2, miR16 and miR18 were embedded in S155, S155e, S26a or S33 scaffolds. The nucleic acid encoding the miRNA was cloned into the plasmid construct described in Example 1. The construct contained the CAG promoter and is summarized in Table 8. The plasmid was transfected into HEK293 cells, and miRNA expression and HTT protein levels were measured. Table 8 Instructions for UsePage 79 / 104, 88 CN 122095088 A

[0357] Figures 7A and 7B illustrate the effects of different combinations of miRNA guide sequences, transit sequences, and scaffolds on the processing efficiency and potency of target knockdown. Figure 7A shows miRNA expression from HEK293 cells transfected with the indicated constructs, and the expression was analyzed 48 hours post-transfection. miRNA levels were detected by RT-qPCR. N = 2–3 transfections / group. The indicated groups were significantly different from each other (one-way ANOVA + Bonferroni test). miRNA expression levels varied depending on the specific miRNA sequence and scaffold. For example, the S33 scaffold produced higher levels of miR1, miR2, and miR18 compared to the S155 scaffold; and the S26a scaffold produced higher levels of miR1 and miR2 compared to the S155 scaffold.

[0358] Figure 7B shows the HTT protein levels from cells transfected with the indicated construct, measured by WES capillary electrophoresis using mab2166 at 72 hours post-transfection. The data were plotted against the loaded control protein, plaque protein (a cytoskeletal protein), and then normalized against the randomized control. N = 6 transfections, 2 experiments. The indicated groups were significantly different from each other (one-way ANOVA + Dunnett test). For example, the S33 scaffold induced greater knockdown in the miR2 and miR16 groups compared to the S155 scaffold. The knockdown of HTT protein levels depended on the specific miRNA sequence and scaffold.

[0359] Example 6: EF-1α Promoter

[0360] When the AAV vector for in vivo testing of the CAG promoter-miRNA construct was generated, heterogeneity of the AAV genome was observed by cesium chloride gradient and capillary electrophoresis of the extracted AAV genomic DNA. A novel construct was designed, incorporating approximately 2.5 kb of the human EEF1A1 gene (abbreviated as EF-1α, SEQ ID NO: 251) promoter into the CAG promoter construct (Example 1 plasmid). The use of the EF-1α promoter increased the rAAV nucleic acid insert from 2.2 kb to 3.1 kb and reduced the GC content of the promoter. The EF-1α construct is summarized in Table 9. Table 9

[0361] Figure 8A shows miRNA expression in HEK293 cells transfected with CAG and EF-1α promoter plasmids expressing miR18 from the S155 scaffold. miRNAs were evaluated by RT-qPCR. mm11 is mentioned to indicate miRNAs with out-of-order guide sequences that are not intended to target genes. Data were normalized relative to intracellular plasmid DNA content to account for differences in transfection efficiency. N = 6 timesTransfection / group. The EF-1α promoter construct produced miR18 at levels similar to the CAG promoter construct (one-way ANOVA + Tugi post-hoc test).

[0362] Figure 8B shows miRNA expression from HEK293 cells transfected with the following: different miRNA guide sequences embedded in S155, S26a, or S33 scaffolds and cloned into plasmids with a 2.5 kb EF-1α promoter; miR18 in an S155 scaffold with a CAG promoter; and mm11 in a CAG promoter. miRNAs were assessed by RT-qPCR. N = 2–3 transfections / group. The indicated groups were significantly different from each other (Welch nonparametric ANOVA + Dunnett test). The EF-1α construct produced miR18 at levels similar to the CAG promoter construct. miR18 embedded in the S26a or S33 scaffold showed approximately 2.7-fold higher miRNA abundance than that of the S155 scaffold (page 81 / 104, CN 122095088 A). Similarly, miR1 showed approximately 4.6-fold increased miRNA levels when embedded in the S26a or S33 scaffold. As shown in Figure 8B, miRNA expression levels were also influenced by the guide sequence selection.

[0363] Figure 8C shows HTT mRNA levels from cells transfected with the indicated constructs, measured by RT-qPCR. Data were normalized relative to mismatch controls. N = 6 transfections, 2 experiments. All miRNA plasmid transfection groups showed highly significant (p < 0.0001) HTT downregulation (one-way ANOVA + Dunnett test). miR18 embedded in the S33 scaffold showed the highest knockdown levels, with HTT mRNA levels reduced by approximately 65%. Given that the transfection efficiency of these experiments was approximately 70%, this is roughly the theoretical maximum knockdown. miR2 and miR3 also tended to be more potent when embedded in the S26a or S33 scaffolds, but the high knockdown levels in all samples indicated that the in vitro system was saturated, making comparisons difficult. Incorporation into the S26a and S33 scaffolds did not improve miRNA levels of miR16 (Figure 8C), and small RNA next-generation sequencing (NGS) confirmed that miR16 was not efficiently processed in any of the scaffolds tested (data not shown).

[0364] The miR16 construct was modified to optimize pyrimidine content. miR17 was provided in the S451 scaffold with the SEQ ID NO: 17 guide sequence (primary amiRNA of SEQ ID NO: 82), where the resulting modification increased cyclic pyrimidine content. miR19 was provided in the S155 scaffold with the SEQ ID NO: 19 guide sequence, and a corresponding modification to the guest sequence provided GG, thereby increasing GC content (SEQ ID NO: 82).83 primary amiRNA). Figure 8D shows the HTT mRNA levels from cells transfected with the construct as measured by RT-qPCR. N = 2 transfections / group.

[0365] Example 7: Testing of miR18 and miR2 constructs in a YAC mouse model

[0366] The recombinant AAV constructs (Table 10) containing an EF-1α promoter operatively linked to a different DNA encoding primary amiRNA were further tested in vivo using a YAC128 mouse model. The mice were bilaterally injected with rAAV virus particles (prepared by standard triple transfection) into the striatum at a dose of 5e9 vg / hemispheric (1e10 vg / mouse) and fed for 2 months post-injection. Small RNA sequencing was performed on the striatum of the injected mice to characterize miRNA processing. Sequencing was performed using an Illumina TruSeq library with single-terminal 150-base-pair reads, yielding approximately 3-5 million reads / sample. Table 10

[0367] Figure 9A shows the guide and guest read counts from YAC128 mice injected with vectored miRNA / scaffold constructs, determined by small RNA sequencing using the Illumina TruSeq platform. RNA was extracted from the striatum and sequenced in N = 3 mice / group. Constructs containing S26a or S33 scaffolds produced higher guide levels and lower guest levels compared to constructs with the S155 scaffold. miR18 generally produced higher levels of miRNA compared to miR2. The S26a scaffold moderately reduced the guest level of miR18, but reduced the guest level in miR2 constructs by 93-fold. Specification 82 / 104 pages 91 CN 122095088 A

[0368] Figure 9B shows the ratio of guide to guest sequences determined by small RNA sequencing. RNA was extracted from the striatum and sequenced in N = 3 mice / group. In this analysis, guide sequences matching at least 19 bp of the expected 21 bp were considered. The relative amounts of guide sequences to guest sequences can be used as an indicator of miRNA processing efficiency (Figure 9B). The S26a scaffold increased the guide sequence to guest sequence ratio of miR18 by 2.6-fold and the guide sequence to guest sequence ratio of miR2 by approximately 293-fold. The S33 scaffold increased the guide sequence to guest sequence ratio of miR18 by 3.3-fold and the guide sequence to guest sequence ratio of miR2 by 23.6-fold.

[0369] Table 11 provides the guide and guest chain read counts from YAC128 mice injected with vectored miRNA / scaffold constructs, determined by small RNA sequencing using the Illumina TruSeq platform. RNA was extracted from the striatum and compared with N =Sequencing was performed on 3 mice / group. miR18 in the S155 scaffold produced the largest heterogeneous mixture of guide sequences with an extra base at the 3' end.

[0370] Results are provided in Table 11. For miR2, in the S155 scaffold construct, the expected guide sequence (21 bases) from the algorithm was produced in <1% of the time, while the S26a and S33 scaffolds produced >94% of the guide sequences that matched the expected sequence. Results are provided for the sequenced DNA produced from the RNA guide sequences (labeled as guide sequences in Table 11). Table 11 Specification 83 / 104 pages 92 CN 122095088 A

[0371] Figure 9C shows the quantification of total mutant HTT protein from YAC128 mice injected with each miRNA / scaffold construct or diluent. N = 2–6 mice / group. Due to the variability of striatal injection, only mice with a vector biodistribution >1e7 CN / ug DNA were included in this analysis. One-way ANOVA (p < 0.0001) and subsequent Tuki post-hoc tests showed that mutant HTT protein was significantly reduced in all test groups compared to the diluent (p < 0.0001). ss.EF1α.S26.miR18 showed a significant difference compared to ss.EF1α.S33.miR18 (p < 0.05), but no significant difference compared to ss.EF1α.S155.miR18. Constructs containing miR2 showed no significant differences among themselves.

[0372] To further investigate the variability of carrier biodistribution generated by intrastriatal injection, the correlation between carrier biodistribution in the striatum and mutant HTT knockdown was examined. Figure 9D shows the relationship between carrier biodistribution from the right hemisphere and mutant HTT protein levels from the same hemisphere for constructs containing miR18. The analysis, on pages 84 / 104 of the specification (CN 122095088 A), included all mice (n = 6-8 mice / group). Linear regression analysis showed high linear correlation (EF1α.S155.miR18; R2 = 0.6455; EF1α.S26.miR18; R2 = 0.6831; EF1α.S33.miR18; R2 = 0.8683). The slopes were significantly different from each other (p = 0.0411).

[0373] Figure 9E shows the relationship between vector biodistribution and mutant HTT protein levels for constructs containing miR18. The analysis included all mice (n = 6 mice / group). Linear regression analysis showed a high linear correlation (EF1α.S155.miR2; R2 = 0.8644; EF1α.26.miR2; R2 = 0.9379; EF1α.S33.miR2; R2 = 0.8644).0.8944). The slopes did not differ significantly from each other (p = 0.0674). EF1α.S155.miR18 and EF1α.26.miR18 had similar overall potency, but EF1α.26.miR18 had a steeper slope, indicating that it was more effective at reducing HTT protein at lower biodistribution compared to other constructs (Fig. 9E). The EF1α.26.miR construct also tended to have increased potency when compared to other miR2 constructs, indicating that scaffold-improved miRNA processing can also improve efficacy.

[0374] The results indicate that miRNA processing efficiency is influenced by specific guide strand and scaffold selection. MiRNA processing efficiency that reduces unwanted transit strand levels and increases the level of active guide miRNAs can affect the safety and efficacy profile of therapeutic miRNA vectors targeting HTT.

[0375] Example 8: HTT Inhibition in YAC128 HD Mice

[0376] The ability of rAAV vectors containing nucleic acids encoding miR21 was evaluated in YAC128 HD mice. The YAC128 HD strain encodes the human HTT gene with amplified exon 1. The rAAV vectors administered were: (1) rAAV-miR21 containing a capsid of VP1 (SEQ ID NO: 257), VP2 (SEQ ID NO: 258), and VP3 (SEQ ID NO: 259); and rAAV nucleic acid (SEQ ID NO: 266) containing miR21 nucleic acid (SEQ ID NO: 261); or (2) an AAV9 capsid and rAAV nucleic acid, wherein the rAAV nucleic acid further contains nucleic acid encoding miR21 (AAV9). Recombinant AAV vectors were generated by triple transfection.

[0377] Different doses of rAAV-miR21 or AAV9 were administered to YAC128 HD mice. The effects of the rAAV vector on HTT protein and HTT mRNA are shown in Table 12. The results provided in Table 12 show the percentage of knockdown compared to untreated mice.

[0378] Table 12 shows statistically paired comparisons between vector AAV9 and rAAV-miR21 using the Tuki HSD test and are reported as follows: *p < 0.05, and ***p < 0.001.

[0379] Medium doses (6 × 10⁹) and low doses (6 × 10¹⁰) of rAAV-miR21 and AAV9 were well tolerated, while high doses (6 × 10¹⁰) showed adverse neurodegeneration. Another study using a dose of 1.9 × 10¹⁰ vg / brain was well tolerated and reduced HTT mRNA and protein. Specification 85 / 104 pages 94 CN 122095088 A

[0380] ≥1.9 × 10¹⁰A dose of vg / brain resulted in a dose-dependent increase in striatal vacuolation, microglial proliferation, and astrocyte proliferation, with additional signs of neurodegeneration at 6 × 10¹⁰ vg / brain, which was considered unfavorable. The neurodegeneration observed in mice at higher doses may be a result of higher carrier loads at escalating doses, which may be exacerbated by tissue damage due to surgical trauma. Histopathological findings at 6.0 × 10¹⁰ vg / brain showed effects on motor impairment, such as those observed through reduced rotator bar activity and increased mortality.

[0381] Example 9: HTT Inhibition in Non-Human Primates

[0382] rAAV-miR21 was further evaluated in a series of non-human primate (NHP) studies. These studies included examinations of different doses, routes of administration, and surgical procedures. Both efficacy and adverse effects were examined. Adverse effects could potentially arise from various sources, including rAAV, carrier dose, route of administration, surgical technique, and study implementation.

[0383] Studies 005-001 compared the efficiency of HTT protein reduction at 3 and 6 months after different doses administered intrathecally (IT), intraparenchymally (IP), and intraventricularly (ICV). IP administration was the most effective route, effectively reducing HTT with minimal rAAV diffusion to non-target brain regions or peripheral areas. Histopathological evaluation revealed adverse changes along the needle mark and cannula tip, including inflammation and tissue damage.

[0384] Studies 005-004 tested the safety and efficacy of rAAV-miR21 administered to rhesus monkeys at doses ranging from 3.17 x 10⁹ to 3.17 × 10¹² vg / brain using a ClearPoint-like system at 3 and 12 months, administered as a single IP injection via the frontal approach (caudate nucleus: 1 deposition point, 0.050 mL; putamen: 2 deposition points, 0.125 mL / deposition point; at a rate of 5 µL / min). Male and female rhesus monkeys were administered a single IP dose (qPCR titer) of rAAV-miR21 or a vector control.

[0385] Compared to single-route IT or ICV administration, IP administration of rAAV-miR21 in studies 005-001 resulted in higher levels of vector-derived transgenes and higher reductions in HTT mRNA and HTT protein (>50% and 65% reductions in the caudate nucleus and putamen, respectively) for up to 6 months. Figure 10 shows the effects of rAAV-miR21 administered at different doses and routes of administration 3 months post-injection.

[0386] In studies 005-004, rAAV-miR21 biodistribution analysis showed consistently high vector levels in regions near the injection site (e.g., putamen, lateral globus pallidus, caudate nucleus, and medial globus pallidus) in all administration groups.Further biodistribution analyses were performed to assess the high variability in biodistribution readouts within individual groups and between the 3-month and 12-month postoperative groups. Additional results confirmed that the rAAV vector DNA was detectable in a dose-dependent manner and that its concentration remained stable in the injected brain structures up to 12 months postoperatively. No statistically significant differences were identified between the vector copy numbers at 3 and 12 months. Peripheral tissues contained lower vector levels.

[0387] Results from studies 005-004 are shown in Figures 12A, 12B, 12C, 12D, 13A, 13B, 13C, and 13D. Biodistribution in the caudate nucleus and putamen after IP administration is shown in Figures 12A, 12B, 12C, and 12D. Additional analyses (Figures 12C and 12D) addressed caudate nucleus and putamen levels not included in the first round of analyses (Figures 12A and 12B). The miR21 levels in a given brain region within the same dose group were as variable as the biodistribution values ​​from the first round of analysis. Overall, the mean miR21 level was dose-dependent at each time point in animals receiving the same dose, and the level was variable from day 92 to day 365 ± 5. Regions with the highest miR21 levels corresponded to regions with the largest reduction in HTT mRNA and the largest reduction in HTT protein. The highest rAAV administration of 3.17 × 10¹² vg / brain was associated with a dose-dependent reduction in HTT mRNA in the studied brain tissue at the post-mortem intervals of day 92 and day 365 ± 5.

[0388] Analysis of HTT protein from tissue samples from studies 005–004 (Figures 13A and 13B) revealed reduced HTT protein levels at the application sites, primarily located in the caudate nucleus and putamen, as well as in surrounding structures. The reduction tended to be dose-dependent, and a significant reduction was observed in the putamen at all dose levels. At 90 days, other structures with reduced white matter levels were found in the medial prefrontal cortex, motor cortex, caudate nucleus, and medial part of the globus pallidus. At 12 months, a similar pattern of reduction in HTT protein levels was observed. Maximum reductions in protein levels were observed in the putamen, lateral part of the globus pallidus, caudate nucleus, medial part of the globus pallidus, dorsal and medial prefrontal cortex, motor cortex, and somatosensory cortex. Due to the high variability in HTT protein readout within individual groups and between the 3-month and 12-month postoperative groups, additional samples were collected and analyzed. These additional results, shown in Figures 13C and 13D, confirm that HTT protein decreased in a dose-dependent manner and that its concentration remained stable in the injected brain structures until 12 months postoperatively. No statistically significant differences in HTT protein concentration were identified between 3 and 12 months at each dose.

[0389] In both studies 005-001 and 005-004, which used the transfrontal approach / unoptimized surgical procedure, a dose-dependent increase in the extent and severity of local tissue damage was observed. The highest-dose cohort in study 005-004 (3.17 × 10¹² vg / brain) showed severe neurological findings, including limb dislocation / weakness, grasping weakness, ataxia, proprioceptive deficits, dehydration, and progressive limb paralysis / paralysis (Class 1). Neurological findings at lower doses included transient twitching and tremors, as well as more severe but still transient seizures (Class 2), from which the animals recovered. Class 2 effects were attributed to the surgery and study implementation. Tissue damage was observed at doses ≥ 1 × 10¹¹ vg / brain.

[0390] Adverse results are summarized in Table 13A: N / A = no administration to animals; vg = vector genome. Class 1 findings were not observed in animals receiving doses of 1 × 10¹² vg / brain or less.

[0391] Table 13A

[0392] Studies 005-010, 005-016, and 005-017 employed improved surgical procedures that significantly reduced histopathological findings. These improvements included real-time magnetic resonance imaging (MRI) of the infusion and refinement of the infusion pathway / parameters. Placing the cannula along the parietal and occipital lobe approaches (instead of the frontal approach) into the caudate and putamen (two approaches per hemisphere instead of three) achieved equivalent or higher coverage of the target structures compared to fewer injections and fewer safety findings (Table 13B). The modified approach allowed the infusion solution to be released in multiple smaller steps rather than in a single bolus. Additionally, the reduced infusion rate better preserved the integrity of the brain parenchyma. Table 13B Instructions for Use 87 / 104 pages 96 CN 122095088 A

[0393] Improvements to the study implementation included the use of additional stereotactic equipment, which shortened the total surgical time and thus limited the duration of animal exposure to anesthesia. In the modified study protocol, animals recovered in a dark and quiet room on the first night after surgery and were monitored more frequently.

[0394] Studies 005-010 included injections along the transfrontal approach and evaluated the effects of surgical parameters (such as injection rate) on the safety and efficacy of rAAV-miR21 IP administration to the caudate / putamen. Two groups received rAAV-miR21 at an incline rate (1–3–5 μL / min) at 1 × 10¹¹ or 1 × 10¹² vg / brain, and a third group received rAAV-miR21 at a steady rate of 5 μL / min at 1 × 10¹² vg / brain. Previous studies 005-001 and 005-004 employed 5μL / min injection rate. Reducing the IP infusion rate resulted in increased safety for the brain parenchyma. In studies 005-010, 1 × 10¹¹ vg / brain was the highest rAAV-miR21 dose, which was not related to the findings of the test article.

[0395] Studies 005-016 involved injections into the caudate nucleus and putamen via the parietal and occipital lobe approaches, respectively, at an incline rate (1–3–5 μL / min) instead of the frontal approach. Cannula placement and diffusion of the rAAV formulation mixed with gadolinium were also monitored in real time using MRI. Compared to studies 005-010, the shift from targeting the putamen via the frontal approach to the occipital lobe approach, coupled with a reduction in infusion volume, resulted in similar structural coverage (approximately 63%). Despite the same infusion volume, targeting the caudate nucleus via the parietal lobe approach instead of the frontal approach resulted in almost double the structural coverage (approximately 41%, compared to approximately 22% in studies 005-010). Animals were injected with escalating doses of rAAV-miR21: 1 × 10¹⁰, 1 × 10¹¹, and 1 × 10¹² vg / brain. Target structure coverage was closely associated with the biodistribution of rAAV-miR21 and the dose-dependent reduction of HTT mRNA and HTT protein in the caudate nucleus and putamen. Studies have shown that, with a maximum dose of 1 × 10¹² vg / brain, rAAV-miR21 safely induced a 60% to 70% reduction in HTT protein in both target structures with minimal local damage at the administration site.

[0396] Studies 005-017 confirmed the safety and efficacy of rAAV-miR21 at different doses. Figure 11 shows the effect of different doses on HTT protein. At three and twelve months post-rAAV-miR21 administration, doses between 1 × 10¹⁰ and 1 × 10¹² vg / brain reduced total HTT protein in NHP in a dose-dependent manner, with no significant difference between the different doses at the same time points, indicating durability of treatment. The 25% and 50% dashed lines represent the control relative to 0 vg / brain. For the 1 × 10¹² vg / brain dose, volumetric data from (3M) T1-weighted real-time MRI at the time of administration in study cohort 1 (67% of the total dose was allocated to the putamen and 33% to the caudate nucleus) were used to convert vg / mm³ of each target brain structure (putamen or caudate nucleus), providing an average of 6.8 × 10⁸ vg / mm³ to the putamen and 4.8 × 10⁸ vg / mm³ to the caudate nucleus.

[0397] Analysis of tissue samples from 3 and 12 months post-injection in studies 005-017 confirmed the persistence of rAAV-miR21 vector genome distribution at different doses in both the caudate nucleus (Fig. 14A) and putamen (Fig. 14B), as well as minimal diffusion of the viral genome into surrounding areas. The highest vector levels were consistently associated with the injection site (caudate nucleus and putamen) and the surrounding region.(e.g., lateral and medial parts of the globus pallidus and the VL thalamus) are associated. Compared with the carrier levels in or near the injection site, other brain tissues, spinal cord (sacrum, cervical vertebrae, thoracic vertebrae, lumbar vertebrae), dorsal root ganglia (sacrum, cervical vertebrae, thoracic vertebrae, lumbar vertebrae), spleen, and liver contain significantly lower carrier levels. Except for the spleen and liver, peripheral tissues have limited exposure to rAAV-miR21. All sciatic nerve, ovarian, and testicular tissues collected from animals given the carrier were negative. Specification 88 / 104 pages 97 CN 122095088 A

[0398] Figures 15A and 15B show the results from study 005-017 measuring HTT knockdown. Figure 15A shows HTT knockdown in the caudate nucleus. The mean HTT protein level quantified in animals injected with rAAV-miR21 at 1 × 10¹¹ or 1 × 10¹² vg / brain was significantly lower than the mean HTT protein level quantified in animals injected with the diluent. The mean HTT protein level quantified in animals administered 1 × 10¹² vg / brain was also significantly lower than the HTT protein level quantified in animals injected with lower doses.

[0399] Figure 15B illustrates HTT knockdown in the caudate nucleus. The mean HTT protein level quantified in animals injected with rAAV-miR21 at 1 × 10¹⁰, 1 × 10¹¹, or 1 × 10¹² vg / brain was significantly lower than the mean HTT protein level quantified in animals injected with the diluent. The mean HTT protein level quantified in animals administered 1 × 10¹¹ vg / brain was also significantly lower than the HTT protein level quantified in animals injected with 1 × 10⁹ or 1 × 10¹⁰ vg / brain.

[0400] The persistence of transgenic expression derived from rAAV-miR21 transduction was confirmed in brain tissue and further confirmed by measuring mature miR21 in CSF collected from animals at 3 and 12 months post-injection. Figures 16A and 16B illustrate the results of measuring brain miR21 and NF-L.

[0401] Neurofilament light chain (NF-L) is a neuronal cytoplasmic protein highly expressed in myelinated axons and an established biomarker for monitoring neurological disorders and traumatic events (such as IP surgery). The data reported in Figures 16C and 16D show elevated NF-L levels in CSF of all animals in Study 005–017 (cohort 1) at 3 months post-IP injection compared to baseline levels (pre-injection), confirming that the elevation of NF-L levels in CSF was solely due to the surgical procedure and not rAAV-miR21. Circulating NF-L levels 12 months post-surgery (cohort 2) were generally comparable to baseline levels, indicating that IP surgery induces acute rather than chronic changes in brain parenchymal homeostasis.

[0402] The safety of rAAV-miR21 IP delivery was confirmed by collecting and analyzing longitudinal brain MRI scans of all animals treated in Studies 005-017. No significant changes were observed in the volume measurements of the caudate nucleus (Fig. 16D), putamen (Fig. 16E), and lateral ventricles (Fig. 16E) of both hemispheres between pre-treatment and at the end of the 3- or 12-month post-operative period.

[0403] In cynomolgus monkeys treated in Studies 005-017, no significant treatment-related histopathological changes were revealed in the nervous system (hemispheric, meninges / dura mater [injection site], spinal cord, ganglia [dorsal root ganglia and trigeminal ganglia], spinal nerve roots, and nerves) and systemic tissues targeted by the selected protocol after infusion of up to 1 x 10¹² vg / brain of rAAV-miR21 into the bilateral brain parenchyma (caudate nucleus and putamen) at day 92 ± 4 post-administration. Neural tissue loss was limited to the target injection site (caudate nucleus and putamen) and the cannula trajectory (adjacent white matter tracts and / or adjacent cerebral cortex), which is typically documented as a surgical effect observed when using the direct intraparenchymal administration route. Test article-related changes at 3 and 12 months post-injection were limited to a slight increase in cuffing at the target site due to mononuclear cell infiltration (lymphocytes and / or macrophages) recorded in animals treated with the highest dose of rAAV-miR21.

[0404] Protocols and results from different studies are summarized in Tables 14–18.

[0405] Table 14 Specification 89 / 104 pages 98 CN 122095088 A

[0406] Table 15

[0407] Table 16 Specification 90 / 104 pages 99 CN 122095088 A

[0408] Table 17 Specification 91 / 104 pages 100 CN 122095088 A

[0409] Table 18 Specification 92 / 104 pages 101 CN 122095088 A

[0410] The percentage of dose-dependent reduction of HTT protein in NHP in different studies is summarized in Table 19.

[0411] Table 19 Specification 93 / 104 pages 102 CN 122095088 A

[0412] Observations from different studies include that rAAV-miR21-driven HTT protein reduction is generally dose-dependent and durable, with 1 × 10¹² vg / brain achieving maximum HTT protein knockdown of up to 62%–64% and 69%–73% in the caudate nucleus and putamen, respectively, at 3 and 12 months post-injection. In brain structures adjacent to the injection site, the HTT protein knockdown was up to 77%–59% and 50%–45% in the lateral and medial parts of the globus pallidus, respectively. At 3 months post-injection, the cerebellar cortex wasThe only test area without HTT protein reduction. At 12 months post-injection, a mean reduction of approximately 10% in HTT was observed in the cerebellum.

[0413] A single bilateral IP (caudate and putamen) infusion of up to 1 × 10¹² vg / brain of rAAV-miR21, evaluated at 3 and 12 months post-administration, did not reveal significant treatment-related histopathological microstructure changes in the nervous system (hemispheric, spinal cord, ganglia [sensory and autonomic ganglia], nerve roots and nerves) and selected systemic tissues (heart, kidney, liver, lung, lymph nodes).

[0414] In summary, the results indicate that administration of rAAV-miR21 at a dose up to 1 × 10¹² vg / brain using optimized surgical procedures was not associated with adverse clinical or neurological changes related to the test article.

[0415] Using 3-month NHP data from Studies 005-017 regarding the efficacy of various doses of rAAV-miR21 expressed in vg / mm3 / putamen, Figure 17 shows estimated dose-response curves with 95% confidence intervals across the entire dose range. For clinical low-dose and high-dose extrapolation, the lower limit of the 95% confidence interval was associated with 25% and 50% reductions in HTT in the putamen, respectively.

[0416] At 3 and 12 months following rAAV IP injections in the caudate nucleus and putamen via the parietal and occipital lobe pathways, miR21 was detectable in the CSF of cynomolgus monkeys, demonstrating that miR21 detection in the CSF provides a valuable biomarker for assessing the durability of the therapy. Absolute quantification of miR21 in CSF samples was performed using the enhanced assay procedure described in Example 12, employing reverse transcription and quantitative polymerase chain reaction (RT-qPCR) and synthetic miRNA oligonucleotide standards.

[0417] Longitudinal analysis of whole-brain MR scans revealed that the surgically driven injury had subsided two months after injection, and the volumes of the lateral ventricles and target structures remained unchanged up to 12 months after injection. Surgical injury regression and the lack of AAV-driven toxicity were also monitored by readouts of neurofilament light chain (NFL) protein in CSF (page 94 / 104, CN 122095088 A) and histopathological analysis of the brain, confirming that there was virtually no neuronal damage and anatomical changes at 1 x 10¹² vg / brain.

[0418] Example 10: Clinical Trials

[0419] Information from the NHP study can be used to guide human treatment and clinical trial administration. In NHP, bilateral IP infusion of rAAV-miR21 at a dose up to 1.00 × 10¹² vg / brain was independent of any effects on: body weight, heart rate, body temperature, or respiration, ophthalmic endpoints, hematological changes, coagulation, serum chemical parameters, or total CSF cells.

[0420] The putamen and caudate nucleus volumes of different Huntington's disease patients can vary within specific disease stages and at different disease stages.High variability is observed between them. Patients can receive personalized treatment based on the volume of the putamen and caudate nucleus, which is determined, for example, by vMRI (volumetric magnetic resonance imaging). Dosing can be administered to patients in an exposure-based manner, where the vector genome (vg) / mm3 of the target tissue remains constant, but the total IP infusion volume (and total vg / brain) is adjusted to accommodate different putamen and caudate nucleus volumes. Although inter-individual volume variability can be large, intra-subject volume variability between the left and right hemispheres is negligible (typically within 5%).

[0421] Based on the putamen to caudate nucleus ratio of approximately 67% and 33% at administration, a dose of 1 × 10¹² vg / brain represents 6.8 × 10⁸ vg / mm³ in the putamen (unilateral) and 4.8 × 10⁸ vg / mm³ in the caudate nucleus (unilateral). The lower bounds of the 95% confidence intervals for the estimated doses reflecting a 25% and 50% reduction in HTT levels in the putamen are 2.0 × 10⁷ vg / mm³ and 2.0 × 10⁸ vg / mm³, respectively (Figure 17).

[0422] The infusion volume can be adjusted to correspond to specific putamen and caudate nucleus volumes (e.g., based on baseline vMRI) to provide preferably at least 50% coverage of the target region. Coverage can be measured using an imaging agent such as gadolinium. For the putamen, the target region is the entire putamen, and for the caudate nucleus, the target region is the pre-commissural caudate nucleus.

[0423] Clinical trials are currently planned to administer two escalating doses of rAAV-mi21 bilaterally to the putamen and caudate nucleus. The low dose of 2.0 x 10⁷ vg / mm³ represents the estimated dose providing a 25% to 35% reduction (95% confidence interval) in putamen HTT levels in NHP. A high dose of 2.0 x 10⁸ vg / mm³ represents an estimated dose that provides a 50% to 60% reduction (95% confidence interval) in putamen HTT levels in NHP.

[0424] Table 20 provides examples of high-dose calculations for different sizes of putamen and caudate nucleus volumes using a dosage formulation of 6.2 x 10¹¹ vg / ml and the MRI contrast agent gadolinium. Parameters can vary, for example, different doses, formulation concentrations, and infusion volumes can be used; the expected putamen and caudate nucleus volumes will vary between patients; and other imaging agents can be used.

[0425] Table 20 Specification 95 / 104 pages 104 CN 122095088 A IP = Intraparenchymal; Max = Maximum value; Min = Minimum value; vg = Vector genome.

[0426] The formulation used for carrier application may contain, for example, 10 mM sodium phosphate, 150 mM sodium chloride, 0.001% Kolliphor, and pH 7.3.

[0427] The study will measure standard biomarkers for Huntington's disease, and the current plan is to also include the following three potential biomarkers as potential surrogate endpoints: (1) changes in the volume of the caudate nucleus or putamen as measured by vMRI; (2) the Huntington's Disease Motor Assessment (HDDMS), which uses a smartphone-based motor task to provide a comprehensive score to sensitively assess the progression of motor symptoms, the motor task assessing fine motor control (rapid tapping task), upper limb chorea (chorea task), trunk chorea (balance task), and gait (2-minute walk); and (3) changes in the neuropathology of Huntington's disease as measured by neurofilament light chain protein (NfL).

[0428] The clinical study plan evaluates the initial efficacy of a single IP infusion of rAAV-miR-21 into the caudate nucleus and putamen of adults with Huntington's disease. Initial efficacy and pharmacodynamics can be assessed in an exploratory manner by evaluating changes in selected biomarkers (MRI, HDDMS, and NfL, as potential surrogate endpoints) and clinical or participant-reported outcome measures (assessments of physical activity and sleep, clinical assessments, participant-reported outcomes, neuroimaging parameters, and biofluid biomarkers). Well-established clinical measures will also be evaluated.

[0429] rAAV-miR21 can be administered, for example, via convection-enhanced delivery to both structures via long-axis IP along the caudate nucleus and putamen. Administration can be performed under MRI guidance during surgery (to visualize and optimize coverage of the target area) and using the SmartFlow® neuroventricular cannula and ClearPoint® neuronavigation system.

[0430] Example 11: Long-term efficacy of rAAV.EF1α.S26.miR18

[0431] The efficacy of rAAV.EF1α.S26.miR18 was evaluated in a 12-month study in YAC128 HD mice. Three doses of the carrier (6 x 10⁸, 6 x 10⁹, and 6 x 10¹⁰ vg / brain) were delivered directly to the striatum via stereotactic injection for each animal. Motor function was assessed at baseline and every 2 months after treatment using the accelerated rotation bar test.

[0432] A constrained maximum likelihood model was fitted to the dataset, and significant effects of sex, time, group, and multifactorial interactions were shown (Table 21). Female YAC128 mice did not show any significant differences between mice treated with the diluent and those treated with rAAV.EF1α.S26.miR18, but males showed significantly improved motor performance in YAC128 mice at doses of 6 x 10⁸ and 6 x 10⁹ vg / brain (Table 22).

[0433] Table 21

[0434] Table 22

[0435] Figures 18A, 18B, and 18C show the average time on the rotating bar. Figure 18A depicts the time, in seconds, of male mice remaining on the accelerating rotating bar, which is the average of three trials at each time point. The motor phenotype of male mice showed significant rescue, which began at 4 months post-injection (6-month-old mice) and persisted throughout the study duration. Figure 18B depicts the average time, in seconds, of male mice on the rotating bar at all time points. Figure 18C shows the average time, in seconds, of female mice on the rotating bar at all time points. Female mice in the high-dose group tended to have improved motor function, but there was no significant difference between the diluent group and the treatment group. These results indicate that rAAV.EF1α.S26.miR18 is well tolerated and has a durable therapeutic effect in the YAC128 HD mouse model.

[0436] Example 12: Detection and quantification of miR21 in cerebrospinal fluid

[0437] Enhancements were made to the procedure for the detection and quantification of miR21 in cynomolgus monkey CSF. The general procedure involved purifying miRNA from CSF and then performing reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) analysis. Purification was performed using the Norgen Urine MicroRNA Purification Kit and under regulated conditions. RT-qPCR was performed using primers targeting the miRNA scaffold. Assay evaluation involved generating calibration curves obtained by incorporating known amounts of miR39 (a synthetic miRNA derived from cel-miR-39 found in Caenorhabditis elegans) and miR21 into monkey CSF, purifying the mixture, and analyzing it by RT-qPCR.

[0438] Enhancement Assay

[0439] Enhancement assays were identified by varying the reaction conditions (e.g., RNA volume) and incubation conditions. Enhancement assays involved the following reagents, purification procedures, and RT-qPCR.

[0440] Reagents: The sources of the reagents are summarized in Table 23. Table 23 Instruction Manual 97 / 104 pages 106 CN 122095088 A

[0441] The doping mixtures and QC controls (HQC, MQC, and LQC) are shown in Table 24. miRNA standards were prepared in 20 ng / µL carrier RNA solution, and doped miRNA controls were prepared in 400 ng / µL carrier RNA solution. The miRNA standard mixtures and QC controls of HQC, MQC, and LQC contained the same copy number of both miR21 and miR39. The copy number of the assay reagent is shown in Table 24: Table 24 Instruction Manual 98 / 104 pages 107 CN 122095088 A

[0442] CSF Sample Processing and miRNA Purification i. Dilute the miR21-doped solution with standard carrier RNA solution at a ratio of 1:100 to a low-doped miR21 solution (5E3 CN / 5 µL). ii.Add 42 mL of 96%–100% ethanol to concentrated wash solution A and mix by inverting. iii. Prepare complete lysis buffer A by adding 25 µL of β-mercaptoethanol to every 2.5 mL of lysis buffer A. iv. Transfer 200 µL of CSF to a new 1.5 mL tube. v. Add 300 µL of complete lysis buffer A to CSF ​​and vortex for 15 seconds. vi. Add 5 µL of miR39 stock solution and miR21 stock solution or diluted miR21, as shown in Table 25. Table 25 vii. Add 300 µL of ethanol and immediately mix by 5 up-and-down pipetting motions. viii. Incubate the sample tube at room temperature for 5 minutes. ix. Apply 500 µL of CSF lysis product to the column provided in a 2 mL collection tube and centrifuge at 8000 rpm for 1 minute. Discard the flow-through. x. Place the column on a new 2 mL collection tube, apply the remaining lysate to the column, centrifuge at 8000 rpm for 1 min, and discard the flow-through. xi. Place the column on a new 2 mL collection tube, add 400 µL of wash solution A to the column, centrifuge at 14,000 rpm for 1 min, and discard the flow-through. xii. Repeat the washing step twice. Each time, discard the flow-through and place the column on a new 2 mL collection tube (Instruction manual page 99 / 104, 108 CN 122095088 A). xiii. Rotate the column at 14,000 rpm for 2 min. xiv. Place the column on a new 1.7 mL elution tube. Add 50 µL of elution solution A to the center of the film at the bottom of the column. xv. Incubate the column at room temperature for at least 5 min. xvi. Centrifuge at 2,000 rpm for 2 min, then centrifuge again at 14,000 rpm for 2 min. xvii. Store the miRNA sample at -80ºC, or on ice if RT is to be performed immediately.

[0443] Reverse transcription protocol i. Prepare the RT premix according to Table 26. Table 26 ii. Pipe 20 µL of the RT premix into the wells of the PCR strip. iii. Standard RNA dilution: 1. Transfer 50 µL of the miRNA standard mixture (2.5E6 CN / µL for each miRNA) into tube 1 of a standard 8-well strip. 2. Serially dilute the standards (Table 27) to construct a 7-point standard curve. Adjust the amounts in Table 27 as needed. Table 27 iv. RT reaction setup: 1. Pipe 20 µL of the RNA sample miRNA, QC sample, standards, and reagent controls into the PCR strip containing the RT premix solution according to the RT strip layout in Table 28: Instruction manual 100 / 104 pages 109 CN 122095088 A Table 28 v.Use a microcentrifuge to briefly centrifuge to collect samples and reagents at the bottom of the wells. vi. Load the tubes into the PCR thermal cycler and run according to the parameters provided in Table 29. Table 29 vii. After RT thermal cycling, add 10 μL of TE buffer to each well. Mix by at least 8 pipette transfers. viii. Store the remaining RNA in a -80ºC freezer.

[0444] qPCR Protocol

[0445] Prepare premix A (MMA) for miR21 qPCR as provided in Table 29 and premix A (MMB) for miR39 qPCR as provided in Table 30. Table 29 Table 30

[0446] Load 15 µL of MMA into columns 1 to 6, 15 µL of MMB into columns 7 to 12, and 10 µL of standard cDNA as provided in Table 31. Centrifuge at 2500 rpm for 5 minutes. Instructions 101 / 104 pages 110 CN 122095088 A Table 31

[0447] Thermal cycling and analysis were performed as provided in Tables 32 and 33. Table 32 Table 33

[0448] Results using different conditions

[0449] The experimental results for quantifying miR21 and miR39 from NHP CSF fluid samples doped with miRNA are described in runs 3, 4, and 5.

[0450] Run 3:

[0451] Table 34 summarizes the experimental protocol for run 3. Run 3 consisted of 200 µL CSF + 300 µL lysis buffer, doped with (a) miR39 at 5e6 CN in each sample; (b) miR21 at 5e6 CN in samples 1, 2, 5, and 6; and (c) miR21 at 5e4 CN in samples 3, 4, 7, and 8. Table 34 Instructions for Use, Pages 102 / 104, 111 CN 122095088 A

[0452] The following reverse transcriptase reactions were performed: (a) 15 µL RT: 5 µL RNA; (b) 25 µL RT: 10 µL RNA; or (c) 40 µL RT: 20 µL RNA. RNA standards for 15 µL RT: qPCR reads: 1e7, 1e6, 1e5, 1e4, 1e3, 1e2, 1e1, O. RNA standards for 25 µL RT: qPCR reads were twice the amount of 15 µL RNA. RNA standards for 40 µL RT: qPCR reads were four times the amount of 15 µL RNA. TE buffer was added to obtain a final reaction volume of 50 µL.

[0453] The miR21 and miR39 recoveries and detections using different volumes of RT products are summarized in Table 35. Table 35

[0454] The standard curves derived from different amounts of RNA and RT were close, so higher amounts of RT could be used to improve detection sensitivity. The slopes of miR21 and miR39 were comparable.

[0455] In all groups, the recoveries of both miR21 and miR39 ranged from >50% to <110%. The highest recoveries were observed with higher concentrations of RNA (20 µL) and RT (40 µL), and are shown in Tables 36 and 37. Table 36 Specification 103 / 104 pages 112 CN 122095088 A Table 37

[0456] For run 3, for samples analyzed with three RT reaction volumes, the average recovery of miR39 from samples purified without two incubation steps (ethanol incubation and incubation for elution) was 60.71%, and the average recovery was 70.07% with the additional incubation step. The average recovery rate of miR21 from the sample without two incubation steps was 77.75%, and the average recovery rate with two incubation steps was 88.23%.

[0457] Run 4:

[0458] In Run 4, RNA standard curves from 1e7 to 10 CN / reaction and various RT reaction volumes (including 15, 25, and 40 µL RT products) were used to investigate the sensitivity of RT-qPCR detection.

[0459] In summary, for Run 4, 10 copies of miRNA standards for both miR21 and miR39 were detected as 2 copies / µL, 1 copy / µL, and 0.5 copies / µL respectively in various RT products. Therefore, larger RT volumes can be used to detect low concentrations of miRNA. (Data not shown.)

[0460] Run 5:

[0461] The enhanced assays described above were used to analyze miRNA standards, QC controls (including HQC, MQC, and LQC), and protocols. The following results were obtained for miR21 and the QC control (basic data not shown): - Standard curve: Y = -3.4243X + 41.238, R² = 0.9997, E = 0.9590. The average Ct value for 10 copies / PCR was 38. - QC recovery was 95.79% for HQC, 92.07% for MQC, and 82.97% for LQC. The following results were obtained for miR39 and the QC control (basic data not shown): - Standard curve: Y = -3.3401X + 41.245, R² = 0.9992, E = 0.9925. The average Ct value for 10 copies / PCR was 37.54. - The QC recovery rate was 105.50% for HQC, 93.807% for MQC, and 80.99% for LQC.

[0462] Although the present invention has been described and illustrated with reference to certain specific embodiments thereof, those skilled in the art will understand that various adjustments, changes, modifications, substitutions, deletions or additions can be made to the procedures and schemes without departing from the spirit and scope of the present invention. Instruction Manual 104 / 104 Page 113 CN 122095088 A Figure 1 Figure 2A Instruction Manual Drawings 1 / 27 Page 114 CN 122095088 A Figure 2B Figure 2C Instruction Manual Drawings 2 / 27 Page 115 CN 122095088 A Figure 3A Figure 3B Instruction Manual Drawings 3 / 27 Page 116 CN 122095088 A Figure 3C Figure 3D Instruction Manual Drawings 4 / 27 Page 117 CN 122095088 A Figure 3E Instruction Manual Drawings 5 / 27 Page 118 CN 122095088 A Figure 4A Figure 4B Figure 4C Figure 4D Instruction Manual Drawings 6 / 27 Page 119 CN 122095088 A Figure 5A Figure 5B Instruction Manual Drawings 7 / 27 Page 120 CN 122095088 A Figure 5C Figure 6A Instruction Manual Drawings 8 / 27 Page 121 CN 122095088 A Figure 6B Figure 6C Instruction Manual Drawings 9 / 27 Page 122 CN 122095088 A Figure 7A Figure 7B Instruction Manual Drawings 10 / 27 Page 123 CN 122095088 A Figure 8A Figure 8B Instruction Manual Drawings 11 / 27 Page 124 CN 122095088 A Figure 8C Figure 8D Instruction Manual Drawings 12 / 27 Page 125 CN 122095088 A Figure 9A Figure 9B Instruction Manual Drawings 13 / 27 Page 126 CN 122095088 A Figure 9C Figure 9D Instruction Manual Drawings 14 / 27 Page 127 CN 122095088 A Figure 9E Figure 10 Instruction Manual Drawings 15 / 27 Page 128 CN 122095088 A Figure 11 Figure 12A Instruction Manual Drawings 16 / 27 Page 129 CN 122095088 A Figure 12B Figure 12C Instruction Manual Drawings 17 / 27 Page 130 CN 122095088 A Figure 12D Figure 13A Instruction Manual Drawings 18 / 27 Page 131 CN 122095088 A Figure 13B Figure 13C Instruction Manual Drawings 19 / 27 Page 132 CN 122095088 A Figure 13D Figure 14A Instruction Manual DrawingsFigure 20 / 27, page 133, CN 122095088 A; Figure 14B, Figure 15A; Instruction Manual Drawings; Figure 21 / 27, page 134, CN 122095088 A; Figure 15B, Figure 16A; Instruction Manual Drawings; Figure 22 / 27, page 135, CN 122095088 A; Figure 16B, Figure 16C; Instruction Manual Drawings; Figure 23 / 27, page 136, CN 122095088 A; Figure 16D, Figure 16E; Instruction Manual Drawings; Figure 24 / 27, page 137, CN 122095088 A; Figure 16F, Figure 17; Instruction Manual Drawings; Figure 25 / 27, page 138, CN 122095088 A; Figure 18A, Figure 18B; Instruction Manual Drawings; Figure 26 / 27, page 139, CN 122095088 A; Figure 18C; Instruction Manual Drawings; Figure 27 / 27, page 140, CN 122095088 A

Claims

1. An RNA polynucleotide comprising a target RNA sequence that is at least 80% identical to the sequence of any one of SEQ ID NO: 1-19; wherein if the target sequence is at least 80% identical to SEQ ID NO: 18, then the RNA polynucleotide is (a) a primary amiRNA comprising the target sequence embedded in a scaffold selected from the S155e scaffold, the S-26a scaffold, and the S-33 scaffold; or (b) the RNA polynucleotide comprising the sequence of SEQ ID NO: 117 or 118.

2. The RNA polynucleotide of claim 1, wherein the target RNA sequence is at least 90% identical to any one of SEQ ID NO: 1-3, 5, 6 and 16-19.

3. The RNA polynucleotide of claim 2, wherein the target RNA sequence comprises any one of SEQ ID NO: 1-3, 5, 6 and 16-19.

4. The RNA polynucleotide of claim 2 or 3, wherein the RNA polynucleotide further comprises a second RNA sequence, wherein the second RNA sequence is substantially complementary to the target RNA sequence.

5. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a primary miRNA comprising a primary miRNA scaffold, a guide sequence, and a guest sequence, wherein the guide sequence comprises the target sequence and the guest sequence comprises the second RNA sequence.

6. The RNA polynucleotide according to claim 5, wherein the scaffold is an S155e scaffold, an S26a scaffold, an S33 scaffold, or an S155 scaffold.

7. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 51-62, 64-68 and 78-87.

8. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises the sequence of any one of SEQ ID NO: 51-62, 64-68 and 78-87.

9. The RNA polynucleotide according to claim 1, wherein the RNA polynucleotide comprises the sequence of any one of SEQ ID NO: 51-62, 64-68 and 78-87.

10. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a premiRNA comprising a guide sequence and a visitor sequence, wherein the guide sequence comprises the target sequence and the visitor sequence comprises the second RNA sequence.

11. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is an shRNA comprising a guide sequence and a visitor sequence, wherein the guide sequence comprises the target sequence and the visitor sequence comprises the second RNA sequence.

12. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 88-96, 98-101 and 111-118.

13. The RNA polynucleotide of claim 12, wherein the RNA polynucleotide comprises the sequence of any one of SEQ ID NO: 88-96, 98-101 and 111-118.

14. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a repressive RNA duplex comprising a guide sequence and a visitor sequence, wherein the guide sequence comprises the target sequence and the visitor sequence comprises the second RNA sequence.

15. The RNA polynucleotide of claim 14, wherein the repressive RNA duplex has a combination of a guide strand and a guest strand selected from: a) A guide chain containing the sequence of SEQ ID NO: 1 and a transit chain containing a sequence that is at least 80% identical to any one of SEQ ID NO: 20, 21 and 22; b) A guide chain containing the sequence of SEQ ID NO: 2 and a transit chain containing a sequence that is at least 80% identical to any one of SEQ ID NO: 23, 24 and 25; c) A guide chain containing the sequence of SEQ ID NO: 3 and a transit chain containing a sequence that is at least 80% identical to any one of SEQ ID NO: 26, 27 and 28; d) A guide chain containing the sequence of SEQ ID NO: 4 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 29; e) A guide chain containing the sequence of SEQ ID NO: 5 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 30; f) A guide chain containing the sequence of SEQ ID NO: 6 and a transit chain containing a sequence that is at least 80% identical to any one of SEQ ID NO: 31, 32 and 33; g) A guide chain containing the sequence of SEQ ID NO: 7 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 34; h) A guide chain containing the sequence of SEQ ID NO: 8 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 35; i) A guide chain containing the sequence of SEQ ID NO: 9 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 36; j) A guide chain containing the sequence of SEQ ID NO: 10 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 37; k) A guide chain containing the sequence of SEQ ID NO: 11 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 38; l) A guide chain containing the sequence of SEQ ID NO: 12 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 39; m) A guide chain containing the sequence of SEQ ID NO: 13 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 40; n) A guide chain containing the sequence of SEQ ID NO: 14 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 41; o) A guide chain containing the sequence of SEQ ID NO: 15 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 42; p) A guide chain containing the sequence of SEQ ID NO: 16 and a transit chain containing a sequence that is at least 80% identical to any one of SEQ ID NO: 43, 44 and 45; q) A guide chain containing the sequence of SEQ ID NO: 17 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 46; r) A guide chain containing the sequence of SEQ ID NO: 19 and a transit chain containing a sequence that is at least 80% identical to the sequence of SEQ ID NO: 47; and s) A guide chain containing the sequence of SEQ ID NO: 18 and a passer chain containing a sequence that is at least 80% identical to any one of SEQ ID NO: 48, 49 and 50.

16. The RNA polynucleotide of claim 1, wherein the targeting sequence is a guide sequence that is at least 90% identical to SEQ ID NO: 18, and the guide sequence is embedded in an S26a scaffold or an S33 scaffold.

17. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 117 or 118.

18. The RNA polynucleotide of claim 17, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 86 or 87.

19. An optionally modified repressive RNA comprising (a) a guide strand capable of hybridizing with a target sequence of any one of SEQ ID NO: 119-137; and (b) a substantially complementary guest sequence; wherein one or more nucleotides of the guide strand and the guest strand are optionally modified RNA.

20. A polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1-19.

21. An expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1-19, and one or more expression control elements operatively linked to the nucleic acid sequence encoding the RNA polynucleotide.

22. The expression cassette of claim 21, wherein the expression cassette comprises an upstream promoter and a downstream polyadenylation signal operatively linked to a nucleic acid sequence encoding the RNA polynucleotide.

23. The expression cassette of claim 22, wherein the expression cassette from 5' to 3' comprises a promoter or promoter / enhancer operatively linked to a nucleic acid sequence encoding the RNA polynucleotide, a nucleic acid sequence encoding the RNA polynucleotide, and a polyadenylation signal.

24. The expression cassette of claim 22 or 23, wherein the promoter provides high CNS expression.

25. The expression cassette according to claim 22 or 23, wherein the promoter is the EF-1α promoter.

26. The expression cassette according to any one of claims 21-25, wherein the expression cassette is DNA.

27. The expression cassette of claim 26, wherein the expression cassette comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 188-199, 201-205 and 215-224.

28. The expression cassette of claim 27, wherein the expression cassette comprises the sequence of SEQ ID NO: 223 or 224.

29. A recombinant viral vector nucleic acid comprising an expression cassette according to any one of claims 1-28 and 5' and / or 3' viral elements for providing viral packaging and / or replication.

30. The recombinant viral vector nucleic acid of claim 29, 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.

31. The recombinant viral vector nucleic acid according to claim 30, wherein the 5' ITR and the 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 and AAV3B.

32. The recombinant viral vector nucleic acid according to claim 30, wherein the recombinant viral vector nucleic acid is self-complementary.

33. The recombinant viral vector nucleic acid according to claim 30 or 32, wherein the 5' ITR contains at least 95% identical sequence to SEQ ID NO: 254, and the 3' ITR contains at least 95% identical sequence to SEQ ID NO:

253.

34. The recombinant viral vector nucleic acid according to claim 30, wherein the recombinant viral vector nucleic acid comprises a sequence that is at least 90% identical to the sequence of any one of SEQ ID NO: 233-235 and 237-242.

35. A recombinant viral nucleotide comprising the sequence of any one of SEQ ID NO: 237-242.

36. A delivery medium comprising a viral or non-viral vector and an inhibitory RNA according to claim 19, a polynucleotide according to claim 20, an expression cassette according to any one of claims 21-28, or a recombinant viral vector nucleic acid according to any one of claims 29-35.

37. The delivery medium of claim 36, wherein the delivery medium is the viral vector.

38. The delivery medium of claim 37, wherein the viral vector is a recombinant AAV, a recombinant lentiviral vector, or a recombinant adenovirus vector.

39. The delivery medium of claim 38, wherein the viral vector is recombinant AAV.

40. The delivery medium of claim 39, wherein the recombinant AAV vector comprises a capsid having at least 90% identical sequences to 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: 257 or SEQ ID NO:

260.

41. The delivery medium of claim 40, 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: 257 or SEQ ID NO:

260.

42. The delivery medium of claim 36, wherein the delivery medium is a nanoparticle selected from: 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.

43. The delivery medium according to claim 42, wherein the delivery medium is an LNP or an LPNP.

44. A pharmaceutical composition comprising the inhibitory RNA according to claim 19, the polynucleotide according to claim 20, the expression cassette according to any one of claims 21-28, the recombinant viral vector nucleic acid according to any one of claims 29-35, or the delivery medium according to any one of claims 36-43; and a pharmaceutically acceptable carrier.

45. A method for reducing the expression of huntingtin protein in cells or a subject, the method comprising administering to the cells or subject an inhibitory RNA according to claim 19, a polynucleotide according to claim 20, an expression cassette according to any one of claims 21-28, a recombinant viral vector nucleic acid according to any one of claims 29-35, a delivery medium according to any one of claims 36-43, or a pharmaceutical composition according to claim 44.

46. ​​A method of treating a subject with Huntington's disease, the method comprising administering to the subject the inhibitory RNA of claim 19, the polynucleotide of claim 20, the expression cassette of any one of claims 21-28, the recombinant viral vector nucleic acid of any one of claims 29-35, the delivery medium of any one of claims 36-43, or the pharmaceutical composition of claim 44.

47. The method of claim 45 or 46, wherein administration comprises direct intraparenchymal, intracisional, or intraventricular administration.

48. The method of claim 45 or 46, wherein the initial application is performed outside the CNS.

49. The method according to any one of claims 45-48, wherein the subject is a human.

50. An AAV vector genome plasmid comprising recombinant viral nucleic acid according to any one of claims 30-35.

51. The AAV genomic plasmid of claim 50, wherein the plasmid lacks the rep and cap genes.

52. 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 cell contains a nucleic acid, a rep gene, and a cap gene according to any one of claims 30-35, wherein the rAAV vector is produced.

53. 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 51, wherein the rAAV-permitting cells further contain (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.

54. The method of claim 53, wherein the rAAV-allowed cell is a packaging cell, wherein the genome of the packaging cell comprises the cap gene and the rep gene.

55. The method of claim 53, 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.

56. A method for obtaining an rAAV vector, the method comprising the following steps: (a) The rAAV vector is generated using the method according to any one of claims 52-55 and (b) The rAAV vector is purified.

57. A polynucleotide comprising the RNA sequence of SEQ ID NO: 255, wherein N 01 To N 42 It is a ribonucleotide, N 01 With N 42 Complementary, N 02 With N 41 Not complementary, N 03 -N 10 With N 33 -N 40 Complementary, N 11 With N 32 Not complementary, and N 12 -N 21 With N 22 -N 31 Complementary; or corresponding DNA.

58. The polynucleotide of claim 57, wherein the polynucleotide further comprises a 5' flanking region and a 3' flanking region, wherein the polynucleotide comprises the RNA sequence of SEQ ID NO: 256; or the corresponding DNA sequence.

59. A DNA polynucleotide, said DNA polynucleotide comprising, in the 5' to 3' direction: (a) A 5' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO: 262; (b) CAG promoter; (c) A preamiRNA coding sequence containing the sequence of SEQ ID NO: 261, wherein the CAG promoter is operatively linked to the preamiRNA coding sequence and a polyadenylation signal; and (d) A 3' inverted terminal repeat (ITR) sequence containing the sequence of SEQ ID NO:

263.

60. The polynucleotide of claim 59, wherein the polyadenylation signal comprises the sequence of SEQ ID NO: 264 or 252, and the CAG promoter comprises the sequence of SEQ ID NO: 250 or 265.

61. The polynucleotide of claim 59 or 60, wherein the end of the 5' ITR to the end of the 3' ITR is at most about 2.5 kb.

62. The polynucleotide of claim 59, wherein the polynucleotide comprises the sequence of SEQ ID NO:

266.

63. The polynucleotide according to any one of claims 59-62, wherein the polynucleotide is a plasmid further comprising an origin of replication and a selectivity marker.

64. The polynucleotide according to any one of claims 59-62, wherein the polynucleotide is a recombinant adeno-associated virus (rAAV) nucleic acid comprising a 5' ITR at the 5' end and a 3' ITR at the 3' end.

65. A recombinant adeno-associated virus (rAAV) vector, said recombinant adeno-associated virus vector comprising: (a) the rAAV nucleic acid according to claim 64; and (b) an rAAV capsid, the rAAV capsid comprising: VP1 containing the amino acid sequence of SEQ ID NO: 257, VP2 containing the amino acid sequence of SEQ ID NO: 258, and VP3 containing the amino acid sequence of SEQ ID NO:

259.

66. A recombinant adeno-associated virus (rAAV) vector, said recombinant adeno-associated virus vector comprising: (a) rAAV nucleic acid, said rAAV nucleic acid comprising a preamiRNA coding sequence, said preamiRNA coding sequence comprising the sequence of SEQ ID NO: 261 operatively linked to an upstream promoter and a downstream multi-A, and (b) an rAAV capsid comprising: VP1 containing the amino acid sequence of SEQ ID NO: 257, VP2 containing the amino acid sequence of SEQ ID NO: 258, and VP3 containing the amino acid sequence of SEQ ID NO:

259.

67. A pharmaceutical composition comprising about 1.0 x 10 10 vg to approximately 1.0 x 10 13 vg’s rAAV carrier and pharmaceutically acceptable carrier as described in claim 65 or 66.

68. The pharmaceutical composition of claim 67, wherein the pharmaceutical composition comprises about 1.0 x 10 11 vg to approximately 1.0x 10 12 vg.

69. The composition according to claim 67 or 68, wherein the composition further comprises an MRI imaging agent.

70. The composition of claim 69, wherein the MRI imaging agent is gadolinium alcohol.

71. A pharmaceutical composition comprising a sufficient amount of the rAAV carrier according to any one of claims 39-41, 65 and 66 to provide a 20% to 90% reduction in total huntingtin protein; and a pharmaceutically acceptable carrier.

72. The pharmaceutical composition of claim 71, wherein the composition comprises a sufficient amount of the rAAV carrier to provide a 20% to 65% reduction in total huntingtin protein.

73. The pharmaceutical composition of claim 72, wherein the composition comprises a sufficient amount of the rAAV carrier to provide a 25% to 40% reduction in total huntingtin protein.

74. A method of treating a subject with Huntington's disease, the method comprising administering, into the brain parenchyma of the subject, an rAAV carrier according to any one of claims 39-41, 65 and 66 or a pharmaceutical composition according to any one of claims 67-73.

75. A method for treating a subject with Huntington's disease, the method comprising: (a) Determine the volume of the putamen and / or caudate nucleus in the right and / or left hemisphere of the subject; as well as (b) Injecting the recombinant adeno-associated virus (rAAV) vector into the brain parenchyma of the subject at a concentration of approximately 2.0 x 10⁻⁶. 7 vg / mm 3 From approximately 2.0 x 10 8 vg / mm 3 The dose was applied to the right hemisphere and / or at approximately 2.0 x 10⁻⁶. 7 vg / mm 3 From approximately 2.0 x 10 8 vg / mm 3 The dose is applied to the left hemisphere; wherein the dose is based on the volume determined in step (a).

76. The method of claim 75, wherein applying to the right hemisphere comprises applying directly to the right hemisphere putamen and caudate nucleus; and applying to the left hemisphere comprises applying directly to the left hemisphere putamen and caudate nucleus.

77. The method of claim 76, wherein the capsid and caudate nucleus volumes of the right hemisphere are measured, and the capsid and caudate nucleus volumes of the left hemisphere are measured, and each hemisphere independently receives 2.0 x 10⁻⁶ units based on the measured volumes. 7 vg / mm 3 Up to 2.0 x 10 8 vg / mm 3 The dosage.

78. The method according to any one of claims 75-77, wherein 2.0 x 10 7 vg / mm 3 Up to 2.0 x 10 8 vg / mm 3 The dose is applied to the right hemisphere putamen and caudate nucleus in a proportion approximately equal to the volume of the putamen and caudate nucleus in the right hemisphere.

79. The method according to any one of claims 75-78, wherein 2.0 x 10 7 vg / mm 3 Up to 2.0 x 10 8 vg / mm 3 The dose was applied to the right hemisphere putamen and caudate nucleus at a ratio of approximately 67% to approximately 33% of the putamen.

80. The method according to any one of claims 75-79, wherein for the left hemisphere, 2.0 x 10 7 vg / mm 3 Up to 2.0 x 10 8 vg / mm 3 The dose is applied to the putamen and caudate nucleus of the left hemisphere in a proportion approximately equal to the volume of the putamen and caudate nucleus of the left hemisphere.

81. The method according to any one of claims 75-80, wherein 2.0 x 10 7 vg / mm 3 Up to 2.0 x 10 8 vg / mm 3 The dose was applied to the left hemisphere putamen and caudate nucleus at a ratio of approximately 67% to approximately 33% of the putamen.

82. The method according to any one of claims 75-81, wherein the same dose is applied to the right hemisphere and the left hemisphere.

83. The method according to any one of claims 75-82, wherein the application to the caudate nucleus includes the parietal lobe approach.

84. The method according to any one of claims 75-83, wherein the application to the shell and core includes the occipital lobe access pathway.

85. The method according to any one of claims 75-84, wherein 1.0 x 10⁻⁶ is applied to each hemisphere. 10 vg to 1.0 x 10 13 vg's rAAV vector.

86. The method of claim 85, wherein 1.0 x 10⁻⁶ is applied to each hemisphere. 11 vg to 1.0 x 10 12 vg's rAAV vector.

87. The method according to any one of claims 75-86, wherein the rAAV vector is the rAAV vector according to any one of claims 39-41, 65 and 66.

88. The method according to any one of claims 75-87, wherein the application comprises convection-enhanced delivery.

89. The method according to any one of claims 75-88, the method comprising: (e) Determine the right hemisphere and / or the left hemisphere by mm 3 The volume of the shell and / or the tail nucleus; and (f) Multiply the volume obtained in step (a) by vg / mm 3 The expected dose is calculated, and the expected dose is further obtained in vg / shell and caudate nucleus. as well as (g) Manipulate the dose obtained in step (b) using a given drug concentration in vg / ml to obtain the desired drug dose volume; and (h) Administering a dose of the rAAV carrier into the brain parenchyma of the subject, wherein the dose in ml is obtained according to steps (b) and (c).

90. The method of claim 89, wherein the desired drug concentration is about 6.2 x 10⁻⁶. 11 vg / ml.

91. A method for treating a subject with Huntington's disease, the method comprising: (e) Determine the right and / or left hemisphere by mm 3 The volume of the capsid and / or caudate nucleus; and (f) Multiply the volume obtained in step (a) by vg / mm 3 The expected dose is calculated, and the expected dose is further obtained in vg / shell and / or tail nucleus; as well as (g) Manipulate the dose obtained in step (b) using a given drug concentration in vg / ml to obtain the desired drug dose volume; and (h) Administer a dose of recombinant adeno-associated virus (rAAV) vector into the brain parenchyma of the subject, wherein the dose in ml is obtained according to steps (b) and (c).

92. The method of claim 91, wherein the desired dose is about 2.0 x 10⁻⁶. 7 vg / mm 3 From approximately 2.0 x 10 8 vg / mm 3 .

93. The method of claim 92, wherein the desired drug concentration is about 6.2 x 10⁻⁶. 11 vg / ml.

94. The method according to any one of claims 89-93, wherein step (a) measures the putamen and caudate nucleus in one or both hemispheres, and step (b) obtains a dose for both the putamen and the caudate nucleus.

95. The method according to any one of claims 74-94, wherein the rAAV nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 261, and the repressive RNA expressed is measured in cerebrospinal fluid by detecting the miR155 scaffold.

96. The method of claim 95, wherein the detection of the expressed inhibitory nucleic acid comprises the following steps: (a) purification of RNA from cerebrospinal fluid; (b) reverse transcriptase; and (c) quantitative polymerase chain reaction (qPCR).

97. The method of claim 96, wherein step (a) comprises incubating the cerebrospinal fluid in lysis buffer and ethanol for about 5 minutes.

98. The method according to claim 96 or 97, wherein step (b) comprises about 20 µL of RNA and about 40 µL of RT.

99. The method according to any one of claims 74-98, wherein the subject is a human.

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