Modified nucleic acids and vectors encoding aspartoacylase (ASPA) for gene therapy

Abstract

The present disclosure relates to recombinant nucleic acids and gene therapy vectors comprising modified nucleic acids encoding aspartoacylase (ASPA) and variants thereof for use in the treatment of diseases and disorders associated with defects or dysfunction of ASPA, in particular Canavan disease.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 016,507, filed April 28, 2020, and U.S. Provisional Application No. 63 / 077,144, filed September 11, 2020. The contents of these applications are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to modified nucleic acids encoding aspartate acyltransferase (ASPA), methods using modified nucleic acids encoding ASPA, vectors comprising modified nucleic acids encoding ASPA, and the use of the vectors in treating diseases, symptoms, and conditions associated with reduced functional ASPA levels, including diseases, symptoms, and conditions associated with reduced cellular catabolism of N-acetyl-L-aspartate, such as Canavan disease. Background Technology

[0004] Canavan disease (CD) is associated with reduced and / or mutated expression of the ASPA gene, which encodes aspartate acylase (ASPA) (also known as aminoacylase 2). Reduced aspartate acylase activity leads to the accumulation of N-acetylaspartate (NAA) (also known as N-acetyl-L-aspartate) due to decreased conversion of NAA to aspartate and acetate. ASPA enzymes are involved in maintaining the metabolic integrity of myelin-forming cells. In the brain, ASPA gene expression is primarily limited to oligodendrocytes, which produce white matter. NAA accumulation in the brain is associated with oligodendrocyte dysfunction, disruption of myelin development, and destruction of existing myelin associated with neurons.

[0005] CD is an autosomal recessive genetic disorder that primarily presents in the neonatal / infant form. Children affected by this form exhibit symptoms associated with myelin degeneration in the brain and spinal cord during infancy. Symptoms include intellectual disability, loss of previously acquired motor skills, feeding difficulties, abnormal muscle tone, macrosomia, paralysis, and seizures. Life expectancy for children with neonatal / infant CD is typically limited to the first ten years. Individuals with a mild / adolescent form of CD may exhibit developmental delays in language and motor skills and have a shorter average lifespan.

[0006] To date, there are no treatments available to stop or slow the neurodegenerative effects of CD. Current treatments in clinical use or under evaluation aim to alleviate symptoms and maximize quality of life. Physical therapy, feeding tubes, and antiepileptic drugs are available to treat some symptoms and improve quality of life. Therefore, there is a great need for novel treatments for CD. Invention Overview

[0008] This article discloses and illustrates, by way of example, a modified nucleic acid encoding aspartate acylase (ASPA), a vector containing the modified nucleic acid (e.g., an rAAV vector), and a method of treating diseases, symptoms, or conditions mediated by a reduction in the level of the ASPA protein by administering the modified nucleic acid or a vector containing the modified nucleic acid to a patient in need.

[0009] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experiments. Such equivalents are intended to be covered by the following embodiments (E).

[0010] E1. An isolated nucleic acid encoding an aspartate acyltransferase (ASPA) comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:2.

[0011] E2. An isolated nucleic acid encoding an aspartate acyltransferase (ASPA), comprising a nucleic acid sequence containing or constituting the sequence of SEQ ID NO:2.

[0012] E3. An isolated nucleic acid encoding an aspartate acyltransferase (ASPA) comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:1.

[0013] E4. An isolated nucleic acid encoding an aspartate acyltransferase (ASPA), comprising a nucleic acid sequence containing or constituting the sequence of SEQ ID NO:1.

[0014] E5. An isolated nucleic acid encoding an aspartate acyltransferase (ASPA) comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:3.

[0015] E6. An isolated nucleic acid encoding an aspartate acyltransferase (ASPA), comprising a nucleic acid sequence containing or constituting the sequence of SEQ ID NO:3.

[0016] E7. A modified nucleic acid encoding an aspartate acyltransferase (ASPA) comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:2.

[0017] E8. A modified nucleic acid encoding an aspartate acyltransferase (ASPA), comprising a nucleic acid sequence containing or constituting the sequence of SEQ ID NO:2.

[0018] E9. A modified nucleic acid encoding an aspartate acyltransferase (ASPA) comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:1.

[0019] E10. A modified nucleic acid encoding an aspartate acyltransferase (ASPA), comprising a nucleic acid sequence containing or constituting the sequence of SEQ ID NO:1.

[0020] E11. A modified nucleic acid encoding an aspartate acyltransferase (ASPA) comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:3.

[0021] E12. A modified nucleic acid encoding an aspartate acyltransferase (ASPA), comprising a nucleic acid sequence containing or constituting the sequence of SEQ ID NO:3.

[0022] E13. A recombinant nucleic acid comprising a modified nucleic acid encoding an aspartate acyltransferase (ASPA), the modified nucleic acid comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical nucleic acid sequence to the nucleic acid sequence of SEQ ID NO:2.

[0023] E14. A recombinant nucleic acid comprising a modified nucleic acid encoding an aspartate acyltransferase (ASPA), the modified nucleic acid comprising or constituting the nucleic acid sequence of SEQ ID NO:2.

[0024] E15. A recombinant nucleic acid comprising a modified nucleic acid encoding an aspartate acyltransferase (ASPA), the modified nucleic acid comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical nucleic acid sequence to the nucleic acid sequence of SEQ ID NO:1.

[0025] E16. A recombinant nucleic acid comprising a modified nucleic acid encoding an aspartate acyltransferase (ASPA), the modified nucleic acid comprising or constituting the nucleic acid sequence of SEQ ID NO:1.

[0026] E17. A recombinant nucleic acid comprising a modified nucleic acid encoding an aspartate acyltransferase (ASPA), the modified nucleic acid comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical nucleic acid sequence to the nucleic acid sequence of SEQ ID NO:3.

[0027] E18. A recombinant nucleic acid comprising a modified nucleic acid encoding an aspartate acyltransferase (ASPA), the modified nucleic acid comprising or constituting the nucleic acid sequence of SEQ ID NO:3.

[0028] E19. The recombinant nucleic acid as described in any one of E13-E18, further comprising at least one element selected from the group consisting of: enhancers, promoters, exons, introns and polyadenylation (polyA) signal sequences.

[0029] E20. The recombinant nucleic acid as described in E19, wherein the enhancer comprises at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:6, SEQ ID NO:17, or both.

[0030] E21. The recombinant nucleic acid as described in any one of E19-E20, wherein the enhancer comprises or constitutes a nucleic acid sequence of SEQ ID NO:6, SEQ ID NO:17 or both.

[0031] E22. A recombinant nucleic acid as described in any one of E19-E21, wherein the promoter is constitutive or regulated.

[0032] E23. A recombinant nucleic acid as described in any one of E19-E22, wherein the promoter is inducible or repressible.

[0033] E24. The recombinant nucleic acid as described in any one of E19-E23, wherein the promoter comprises a nucleic acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:7.

[0034] E25. The recombinant nucleic acid as described in any one of E19-E24, wherein the promoter comprises or constitutes the nucleic acid sequence of SEQ ID NO:7.

[0035] E26. The recombinant nucleic acid as described in any one of E19-E25, wherein the exon contains at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:8, SEQ ID NO:18, or both.

[0036] E27. The recombinant nucleic acid as described in any one of E19-E26, wherein the exon comprises or constitutes a nucleic acid sequence of SEQ ID NO:8, SEQ ID NO:18, or both.

[0037] E28. The recombinant nucleic acid as described in any one of E19-E27, wherein the intron comprises at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:9, SEQ ID NO:10, or both.

[0038] E29. The recombinant nucleic acid as described in any one of E19-E28, wherein the intron comprises or constitutes a nucleic acid sequence of SEQ ID NO:9, SEQ ID NO:10, or both.

[0039] E30. The recombinant nucleic acid as described in any one of E19-E29, wherein the polyA sequence comprises at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:11.

[0040] E31. The recombinant nucleic acid as described in any one of E19-E30, wherein the polyA sequence comprises or constitutes the nucleic acid sequence of SEQ ID NO:11.

[0041] E32. The recombinant nucleic acid as described in any one of E19-E31, wherein the enhancer is operatively linked to the modified nucleic acid.

[0042] E33. The recombinant nucleic acid as described in any one of E19-E32, wherein the promoter is operatively linked to the modified nucleic acid.

[0043] E34. The recombinant nucleic acid as described in any one of E13-E18, further comprising at least one element selected from: cytomegalovirus (CMV) enhancer, heterozygous form of CBA promoter (CBh promoter), chicken β-actin (CBA) exon, CBA intron, mouse parvovirus (MVM) intron, and bovine growth hormone (BGH) polyA.

[0044] E35. The recombinant nucleic acid as described in any one of E13-E18, further comprising at least one element selected from: a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17; a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:7; a CBA exon comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18; a CBA intron comprising the nucleic acid sequence of SEQ ID NO:9; an MMV intron comprising the nucleic acid sequence of SEQ ID NO:10; and a BGH polyA comprising the nucleic acid sequence of SEQ ID NO:11.

[0045] E36. A vector genome comprising a modified nucleic acid as described in any one of E7-E12 or a recombinant nucleic acid as described in any one of E13-E35, wherein the vector genome further comprises at least one AAV ITR repeat sequence comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical nucleic acid sequences to the nucleic acid sequences of SEQ ID NO:5, SEQ ID NO:12, or both.

[0046] E37. The vector genome as described in E36, wherein the at least one AAV ITR repeat sequence comprises or constitutes a nucleic acid sequence or a combination thereof of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19.

[0047] E38. A vector genome as described in E36 or E37, comprising two AAV2ITR sequences flanking a nucleic acid sequence encoding ASPA and a CBh promoter upstream of the ASPA-coding sequence.

[0048] E39. The vector genome as described in any one of E36-E38, wherein the ASPA sequence comprises the nucleic acid sequence of SEQ ID NO:2.

[0049] E40. The vector genome as described in any one of E36-E39, wherein the at least one AAV2ITR sequence comprises the nucleic acid sequences of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof.

[0050] E41. The vector genome as described in any one of E36-E40, wherein the CBh promoter comprises the nucleic acid sequence of SEQ ID NO:7.

[0051] E42. A vector genome comprising nucleic acid, wherein the nucleic acid comprises from 5' to 3':

[0052] a) AAV2 ITR containing the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12 or SEQ ID NO:19;

[0053] b) A CMV enhancer containing a nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17, preferably SEQ ID NO:6;

[0054] c) A CBh promoter containing the nucleic acid sequence of SEQ ID NO:7;

[0055] d) CBA exons containing the nucleic acid sequence of SEQ ID NO:8 and SEQ ID NO:18, preferably SEQ ID NO:18;

[0056] e) CBA introns containing the nucleic acid sequence of SEQ ID NO:9;

[0057] f) MMV introns containing the nucleic acid sequence of SEQ ID NO:10;

[0058] g) A modified nucleic acid containing the nucleic acid sequence of any one of SEQ ID NO:1-3 encoding an aspartate acyltransferase (ASPA);

[0059] h) BGH polyA containing the nucleic acid sequence of SEQ ID NO:11; and

[0060] i) AAV2 ITR containing nucleic acid sequences of SEQ ID NO:5, SEQ ID NO:12, and SEQ ID NO:19.

[0061] E43. A vector genome containing nucleic acid, wherein the nucleic acid comprises from 5' to 3':

[0062] a) AAV ITR containing the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12 or SEQ ID NO:19;

[0063] b) An enhancer containing a nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17, preferably SEQ ID NO:6;

[0064] c) A promoter containing the nucleic acid sequence of SEQ ID NO:7;

[0065] d) Exons containing the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18, preferably SEQ ID NO:18;

[0066] e) Introns containing the nucleic acid sequence of SEQ ID NO:9;

[0067] f) Introns containing the nucleic acid sequence of SEQ ID NO:10;

[0068] g) A modified nucleic acid containing the nucleic acid sequence of any one of SEQ ID NO:1-3 encoding an aspartate acyltransferase (ASPA);

[0069] h) PolyA containing the nucleic acid sequence of SEQ ID NO:11; and

[0070] i) An AAV terminal repeat sequence containing a nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12 or SEQ ID NO:19.

[0071] E44. A vector genome as described in any one of E36-43, wherein the vector genome is self-complementary.

[0072] E45. A recombinant adeno-associated virus (rAAV) vector comprising a vector genome and capsid as described in any one of E36-E44.

[0073] E46. An rAAV vector containing a vector genome, the vector genome containing approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% identical nucleic acid sequences to the nucleic acid sequence of SEQ ID NO:2.

[0074] E47. The rAAV carrier as described in E46, comprising a capsid selected from the following: Oligo001, Oligo002, Oligo003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15- 1. Capsule of RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAVhu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N and AAV-LK03.

[0075] E48. An rAAV vector containing a vector genome, the vector genome containing approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% identical nucleic acid sequences to the nucleic acid sequence of SEQ ID NO:1.

[0076] E49. The rAAV carrier as described in E48, comprising a capsid selected from the following: Oligo001, Oligo002, Oligo003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15- 1. Capsule of RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAVhu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N and AAV-LK03.

[0077] E50. An rAAV vector containing a vector genome, the vector genome containing approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% identical nucleic acid sequences to the nucleic acid sequence of SEQ ID NO:3.

[0078] E51. The rAAV carrier as described in E50, comprising a capsid selected from the following: Oligo001, Oligo002, Oligo003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15- 1. Capsule of RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAVhu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N and AAV-LK03.

[0079] E52. The rAAV carrier as described in any one of E45-E51, wherein the capsid is selected from Olig001, Olig002 and Olig003 capsids.

[0080] E53. The rAAV vector as described in any one of E45-E52, wherein the capsid is an Olig001 capsid containing viral protein 1 (VP1) and wherein the VP1 contains at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:14.

[0081] E54. The rAAV vector as described in any one of E45-E53, wherein the capsid is an Oligo001 capsid containing viral protein 1 (VP1) and wherein the VP1 contains the amino acid sequence of SEQ ID NO:14.

[0082] E55. The rAAV vector as described in any one of E45-E52, wherein the capsid is an Olig002 capsid containing viral protein 1 (VP1) and wherein the VP1 contains at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:15.

[0083] E56. The rAAV vector as described in any one of E45-E52 and E55, wherein the capsid is an Oligo002 capsid containing viral protein 1 (VP1) and wherein the VP1 contains the amino acid sequence of SEQ ID NO:15.

[0084] E57. The rAAV vector as described in any one of E45-E52, wherein the capsid is an Olig003 capsid containing viral protein 1 (VP1) and wherein the VP1 contains at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:16.

[0085] E58. The rAAV vector as described in any one of E46-E52 and E57, wherein the capsid is an Oligo003 capsid containing viral protein 1 (VP1) and wherein the VP1 contains the amino acid sequence of SEQ ID NO:16.

[0086] E59. The rAAV vector as described in any one of E45-E58, wherein the vector genome is self-complementary.

[0087] E60. The rAAV vector as described in any one of E46-E59, wherein the vector genome contains at least one element selected from: at least one AAV inverted terminal repeat (ITR) sequence, enhancer, promoter, exon, intron and polyadenylation (polyA) signal sequence.

[0088] E61. The rAAV vector as described in E60, wherein the enhancer comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical nucleic acid sequences to the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17.

[0089] E62. The rAAV vector as described in E60 or E61, wherein the enhancer comprises or constitutes a nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17.

[0090] E63. The rAAV vector as described in any one of E60-E62, wherein the promoter is constitutive or regulated.

[0091] E64. The rAAV vector as described in any one of E60-E63, wherein the promoter is inducible or repressible.

[0092] E65. The rAAV vector as described in any one of E60-E64, wherein the promoter comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:7.

[0093] E66. The rAAV vector as described in any one of E60-E65, wherein the promoter comprises or constitutes the nucleic acid sequence of SEQ ID NO:7.

[0094] E67. The rAAV vector as described in any one of E60-E66, wherein the exon contains at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical nucleic acid sequence to the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18.

[0095] E68. The rAAV vector as described in any one of E60-E67, wherein the exon comprises or constitutes a nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18.

[0096] E69. The rAAV vector as described in any one of E60-E68, wherein the intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical nucleic acid sequences to those of SEQ ID NO:9, SEQ ID NO:10, or both.

[0097] E70. The rAAV vector as described in any one of E60-E69, wherein the intron comprises or constitutes a nucleic acid sequence of SEQ ID NO:9, SEQ ID NO:10, or both.

[0098] E71. The rAAV vector as described in any one of E60-E70, wherein the polyA sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:11.

[0099] E72. The rAAV vector as described in any one of E60-E71, wherein the polyA sequence comprises or constitutes the nucleic acid sequence of SEQ ID NO:11.

[0100] E73. The rAAV vector as described in any one of E60-E72, wherein the at least one AAV ITR repeat sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical nucleic acid sequences to or in combination with the nucleic acid sequences of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or combinations thereof.

[0101] E74. The rAAV vector as described in any one of E60-E73, wherein the at least one AAV ITR repeat sequence comprises or constitutes a nucleic acid sequence or a combination thereof of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19.

[0102] E75. The rAAV vector as described in any one of E46-E59, wherein the vector genome further comprises at least one element selected from: at least one AAV2 ITR sequence, CMV enhancer, CBh promoter, CBA exon 1, CBA intron 1, MVM intron, and BGH polyA.

[0103] E76. The rAAV vector as described in any one of E46-E59, wherein the vector genome further comprises at least one element selected from: at least one AAV2 ITR sequence comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19 or a combination thereof; a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17; a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:7; a CBA exon 1 comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18; a CBA intron 1 comprising the nucleic acid sequence of SEQ ID NO:9; an MMV intron comprising the nucleic acid sequence of SEQ ID NO:10; and a BGH polyA comprising the nucleic acid sequence of SEQ ID NO:11.

[0104] E77. The rAAV vector as described in any one of E46-E59, wherein the vector genome comprises two AAV2 ITR sequences flanked by sequences encoding ASPA and a CBh promoter upstream of the sequences encoding ASPA.

[0105] E78. The rAAV vector as described in E77, wherein the ASPA sequence comprises the nucleic acid sequence of SEQ ID NO:2.

[0106] E79. rAAV vectors such as E77 or E78, wherein the AAV ITR sequence comprises nucleic acid sequences of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19 or combinations thereof.

[0107] E80. The rAAV vector as described in any one of E77-E79, wherein the CBh promoter comprises the nuclear sequence of SEQ ID NO:7.

[0108] E81. An rAAV vector containing a vector genome, the vector genome comprising, from 5' to 3':

[0109] a) AAV2ITR containing nucleic acid sequences or combinations thereof, including SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19;

[0110] b) A CMV enhancer containing a nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:16;

[0111] c) A CBh promoter containing the nucleic acid sequence of SEQ ID NO:7;

[0112] d) CBA exon 1 containing the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18;

[0113] e) CBA intron 1 containing the nucleic acid sequence of SEQ ID NO:9;

[0114] f) MMV introns containing the nucleic acid sequence of SEQ ID NO:10;

[0115] g) A modified nucleic acid containing the nucleic acid sequence of any one of SEQ ID NO:1-3 encoding an aspartate acyltransferase (ASPA);

[0116] h) BGH polyA containing the nucleic acid sequence of SEQ ID NO:11; and

[0117] i) AAV2ITR containing nucleic acid sequences or combinations thereof of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19.

[0118] E82. An rAAV vector containing a vector genome, the vector genome comprising, from 5' to 3':

[0119] a) AAVITR containing nucleic acid sequences or combinations thereof, including SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19;

[0120] b) Enhancers containing nucleic acid sequences of SEQ ID NO:6 or SEQ ID NO:17;

[0121] c) A promoter containing the nucleic acid sequence of SEQ ID NO:7;

[0122] d) Exons containing the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18;

[0123] e) Introns containing the nucleic acid sequence of SEQ ID NO:9;

[0124] f) Introns containing the nucleic acid sequence of SEQ ID NO:10;

[0125] g) A modified nucleic acid containing the nucleic acid sequence of any one of SEQ ID NO:1-3 encoding an aspartate acyltransferase (ASPA);

[0126] h) PolyA containing the nucleic acid sequence of SEQ ID NO:11; and

[0127] i) An AAV terminal repeat sequence comprising nucleic acid sequences or combinations thereof, including SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19.

[0128] E83. The rAAV vector as described in E81 or E82, wherein the vector genome is self-complementary.

[0129] E84. The rAAV vector as described in any one of E81-E83, wherein the vector comprises an Olig001 capsid containing a VP1 protein, wherein the VP1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14.

[0130] E85. The rAAV vector as described in any one of E81-E83, wherein the vector comprises an Olig002 capsid containing a VP1 protein, wherein the VP1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:15.

[0131] E86. The rAAV vector as described in any one of E81-E83, wherein the vector comprises an Olig003 capsid containing a VP1 protein, wherein the VP1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:16.

[0132] E87. An rAAV vector comprising: i) an Olig001 capsid containing a VP1 protein, wherein the VP1 contains the amino acid sequence of SEQ ID NO:14; and ii) a self-complementary vector genome comprising, from 5' to 3':

[0133] a) AAV2ITR containing nucleic acid sequences or combinations thereof, including SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19;

[0134] b) A CMV enhancer containing a nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17;

[0135] c) A CBh promoter containing the nucleic acid sequence of SEQ ID NO:7;

[0136] d) CBA exon 1 containing the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18;

[0137] e) CBA intron 1 containing the nucleic acid sequence of SEQ ID NO:9;

[0138] f) MMV introns containing the nucleic acid sequence of SEQ ID NO:10;

[0139] g) A modified nucleic acid containing the nucleic acid sequence of any one of SEQ ID NO:1-3 encoding an aspartate acyltransferase (ASPA);

[0140] h) The nucleic acid sequence BGH polyA containing SEQ ID NO:11; and

[0141] i) AAV2 ITR containing the nucleic acid sequence of SEQ ID NO:5 or SEQ ID NO:12.

[0142] E88. An rAAV vector comprising: i) an Olig001 capsid containing a VP1 protein, wherein the VP1 contains the amino acid sequence of SEQ ID NO:14; and ii) a self-complementary vector genome comprising, from 5' to 3':

[0143] a) AAVITR containing nucleic acid sequences or combinations thereof, including SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19;

[0144] b) Enhancers containing nucleic acid sequences of SEQ ID NO:6 or SEQ ID NO:17;

[0145] c) A promoter containing the nucleic acid sequence of SEQ ID NO:7;

[0146] d) Exons containing the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18;

[0147] e) Introns containing the nucleic acid sequence of SEQ ID NO:9;

[0148] f) Introns containing the nucleic acid sequence of SEQ ID NO:10;

[0149] g) A modified nucleic acid containing the nucleic acid sequence of any one of SEQ ID NO:1-3 encoding an aspartate acyltransferase (ASPA);

[0150] h) PolyA containing the nucleic acid sequence of SEQ ID NO:11; and

[0151] i) AAVITR containing nucleic acid sequences of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19 or combinations thereof.

[0152] E89. The rAAV vector as described in any one of E45-E88, wherein the vector reduces the level of NAA in the cell upon introduction into the cell.

[0153] E90. The rAAV vector as described in E89, wherein the cell is a brain cell.

[0154] E91. The rAAV vector as described in E89 or E90, wherein the cell is an oligodendrocyte.

[0155] E92. The rAAV vector as described in any one of E45-E91, wherein administration of the vector to the subject increases the subject's balance, grip strength, and / or motor coordination compared to the balance, grip strength, and / or motor coordination of a subject with an ASPA gene mutation prior to administration of the vector.

[0156] E93. The rAAV vector as described in any one of E45-E92, wherein administration of the vector to the subject increases the subject's generalized motor function compared to the subject's generalized motor function prior to administration of the vector.

[0157] E94. The rAAV vector as described in any one of E45-E93, wherein administration of the vector to the subject reduces the NAA level in the subject compared to the NAA level in the subject with an ASPA gene mutation prior to administration of the vector.

[0158] E95. The rAAV vector as described in any one of E45-E94, wherein administration of the vector to the subject reduces the vacuolar volume fraction of the thalamus of the subject compared to the vacuolar volume fraction of the thalamus of a subject with an ASPA gene mutation prior to administration of the vector.

[0159] E96. The rAAV vector as described in any one of E45-E95, wherein administration of the vector to the subject reduces the vacuolar volume fraction of the cerebellar white matter / pons of the subject compared to the vacuolar volume fraction of the cerebellar white matter / pons of a subject with an ASPA gene mutation prior to administration of the vector.

[0160] E97. The rAAV vector as described in any one of E45-E96, wherein administration of the vector to the subject increases the number of oligodendrocytes in the thalamus of the subject compared to the number of oligodendrocytes in the thalamus of a subject with an ASPA gene mutation prior to administration of the vector.

[0161] E98. The rAAV vector as described in any one of E45-E97, wherein administration of the vector to the subject increases the number of oligodendrocytes in the subject's cerebral cortex compared to the number of oligodendrocytes in the subject's cerebral cortex prior to administration of the vector.

[0162] E99. The rAAV vector as described in any one of E45-E98, wherein administration of the vector to the subject increases the number of neurons in the subject's thalamus compared to the number of neurons in the subject's thalamus prior to administration of the vector.

[0163] E100. The rAAV vector as described in any one of E45-E99, wherein administration of the vector to the subject increases the number of neurons in the subject's cerebral cortex compared to the number of neurons in the subject's cerebral cortex prior to administration of the vector.

[0164] E101. The rAAV vector as described in any one of E45-E100, wherein administration of the vector to the subject increases the subject's cortical myelin formation compared to that of a subject with an ASPA gene mutation prior to administration of the vector.

[0165] E102. The rAAV vector as described in any one of E92-E101, wherein the subject is a human patient.

[0166] E103. The rAAV vector as described in any one of E92-E102, wherein the subject is a human patient with canavan disease or at risk of developing canavan disease.

[0167] E104. The rAAV vector as described in any one of E92-E103, wherein the subject has at least one ASPA gene mutation.

[0168] E105. A pharmaceutical composition comprising a modified nucleic acid as described in any one of E7-E12, a recombinant nucleic acid as described in any one of E13-E35, a vector genome as described in any one of E36-E44, or an rAAV vector as described in any one of E45-E104.

[0169] E106. A pharmaceutical composition comprising a modified nucleic acid as described in any one of E7-E12, a recombinant nucleic acid as described in any one of E13-E35, a vector genome as described in any one of E36-E44, or an rAAV vector as described in any one of E45-E104, and a pharmaceutically acceptable carrier.

[0170] E107. A method for treating and / or preventing diseases, symptoms, or conditions associated with defects or dysfunction of ASPA, the method comprising administering to a subject requiring treatment a therapeutically effective amount of a modified nucleic acid as described in any one of E7-E12, a recombinant nucleic acid as described in any one of E13-E35, a vector genome as described in any one of E36-E44, an rAAV vector as described in any one of E45-E104, or a pharmaceutical composition as described in E105 or E106.

[0171] E108. The method as described in E107, wherein the disease, condition or ailment associated with a deficiency or dysfunction of ASPA is canavan disease.

[0172] E109. The method as described in E107 or E108, wherein the modified nucleic acid, recombinant nucleic acid, vector genome, rAAV vector, or pharmaceutical composition is administered directly to the brain of the subject requiring treatment.

[0173] E110. The method of any one of E107-E109, wherein the modified nucleic acid, recombinant nucleic acid, vector genome, rAAV vector or pharmaceutical composition is directly administered to the central nervous system of the subject requiring treatment.

[0174] E111. The method of any one of E107-E110, wherein the modified nucleic acid, recombinant nucleic acid, vector genome, rAAV vector or pharmaceutical composition is applied to at least one region of the central nervous system selected from the following: brain parenchyma, spinal canal, subarachnoid space, ventricles, cisterna magna and any combination thereof.

[0175] E112. The method of any one of E107-E111, wherein the modified nucleic acid, recombinant nucleic acid vector genome, rAAV vector or pharmaceutical composition is administered by at least one method selected from: intraparenchymal administration, intrathecal administration, intraventricular administration, intracerebellar administration, and any combination thereof.

[0176] E113. The method as described in any one of E107-E112, wherein the subject is a human patient.

[0177] E114. The method of any one of E107-E113, wherein the subject is a human patient with canavan disease or at risk of developing canavan disease.

[0178] E115. The method as described in any one of E107-E114, wherein the subject has at least one ASPA gene mutation.

[0179] E116. A method for treating or preventing canavan disease, the method comprising the steps of: i) assessing whether a subject has at least one ASPA gene mutation, and ii) administering to the subject a therapeutically effective amount of a modified nucleic acid as described in any one of E7-E12, a recombinant nucleic acid as described in any one of E13-E35, a vector genome as described in any one of E36-E44, an rAAV vector as described in any one of E45-E104, or a pharmaceutical composition as described in E105 or E106, thereby treating or preventing canavan disease in the subject.

[0180] E117. The method as described in E116, wherein the subject is diagnosed with canavan disease or is diagnosed at risk of developing canavan disease.

[0181] E118. A method for treating or preventing an ASPA deficiency-related disease in a subject with such need, comprising administering to the subject a therapeutically effective amount of a modified nucleic acid encoding ASPA, wherein the modified nucleic acid encoding ASPA is codon-optimized.

[0182] E119. The method as described in E118, wherein the modified nucleic acid encoding ASPA comprises the nucleic acid sequence of SEQ ID NO:2.

[0183] E120. The method as described in E118 or E119, wherein the modified nucleic acid encoding ASPA encodes an ASPA protein having the amino acid sequence of SEQ ID NO:4.

[0184] E121. The method of any one of E118-E120, wherein the modified nucleic acid encoding ASPA is expressed in a target cell and wherein the target cell is an oligodendrocyte.

[0185] E122. The method of any one of E118-E121, wherein the modified nucleic acid encoding ASPA is delivered to the target cell in a vector.

[0186] E123. The method as described in E122, wherein the vector is a viral vector or a non-viral vector.

[0187] E124. The method of any one of E118-E123, wherein the carrier is administered to the subject by systemic injection, direct intracranial injection or direct spinal canal injection.

[0188] E125. A host cell comprising a modified nucleic acid as described in any one of E7-E12, a recombinant nucleic acid as described in any one of E13-E35, a vector genome as described in any one of E36-E44, or an rAAV vector as described in any one of E45-E104.

[0189] E126. The host cell as described in E125, wherein the cell is selected from the following: VERO, WI38, MRC5, A549, HEK293, B-50 or any other HeLa cell, HepG2, Saos-2, HuH7 and HT1080.

[0190] E127. The host cell as described in E125-E126, wherein the cell is a HEK293 cell adapted for growth in a suspension culture.

[0191] E128. The host cell as described in any one of E125-E127, wherein the cell is a HEK293 cell with American Type Culture Collection (ATCC) number PTA 13274.

[0192] E129. The host cell of any one of E125-E128, wherein the cell contains at least one nucleic acid encoding at least one protein selected from the following: AAV Rep protein, AAV capsid (Cap) protein, adenovirus early region 1A (E1a) protein, E1b protein, E2a protein, E4 protein, and virus-associated (VA) RNA.

[0193] E130. A kit for treating canavan disease (CD) comprising a therapeutically effective amount of i) an rAAV carrier as described in any one of E45-E104 or ii) a pharmaceutical composition as described in E105 or E106.

[0194] E131. The kit as described in E130, wherein the kit further includes a label or insert containing instructions for using one or more of the kit components.

[0195] E132. The modified nucleic acid as described in any one of E7-E12, the recombinant nucleic acid as described in any one of E13-E35, the vector genome as described in any one of E36-E44, the rAAV vector as described in any one of E45-E104, or the pharmaceutical composition as described in E105 or E106, for the treatment or prevention of diseases, symptoms, or conditions associated with defects or dysfunction of ASPA.

[0196] E133. Modified nucleic acids, recombinant nucleic acids, vector genomes, rAAV vectors, or pharmaceutical compositions used as described in E132, wherein the disease, condition, or illness is canavan disease.

[0197] E134. Use of any of the modified nucleic acids described in any of E7-E12, any of the recombinant nucleic acids described in any of E13-E35, any of the vector genomes described in any of E36-E44, any of the rAAV vectors described in any of E45-E104, or any of the pharmaceutical compositions described in E105 or E106 in the preparation of a medicament for the treatment and / or prevention of diseases, symptoms, or conditions associated with defects or dysfunctions of ASPA.

[0198] E135. Use as described in E134, wherein the disease, symptom, or condition is canavan disease.

[0199] E136. A method for determining the biodistribution of a transgene delivered to the brain of a subject via an rAAV vector containing an Olig001 capsid, wherein a protein encoded by the transgene is expressed, the method comprising:

[0200] a) Administer the rAAV vector to the subject;

[0201] b) Fix brain tissue;

[0202] c) Electrophoretic transparentization of the brain;

[0203] d) Perform 3D microscopic imaging on brain tissue slices;

[0204] e) Detect the protein;

[0205] f) Optionally, the amount of protein present in brain tissue is quantified.

[0206] E137. The method as described in E136, wherein the administration is performed via intraventricular (ICV) injection, intraparenchymal (IP) injection, intrathecal (IT) administration, intracisternal (ICM) injection, or a combination thereof.

[0207] E138. The method as described in E136 or 137, wherein the brain tissue is fixed using, for example, paraformaldehyde or formalin.

[0208] E139. The method as described in any one of E136-E138, wherein the quantification includes stereo rendering.

[0209] E140. The method of any one of E136-E139, wherein the transgene encodes green fluorescent protein (GFP).

[0210] E141. The method as described in any one of E136-E140, wherein the level of transgene expression detected in the tissue is correlated with the rAAV vector transduction efficiency.

[0211] E142. The method of any one of E136-E141, further comprising (g) the step of assessing cell-type vector tropism by cell morphology assessment and determination of the spatial location of GFP expression.

[0212] E143. A modified nucleic acid encoding an aspartate acyltransferase (ASPA) comprising at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical nucleic acid sequence and promoter to any one of SEQ ID NO: 1-3.

[0213] E144. A modified nucleic acid encoding an aspartate acyltransferase (ASPA) comprising a nucleic acid containing or constituting the sequence of SEQ ID NO:2 and a promoter.

[0214] E145. A nucleic acid comprising a nucleic acid sequence encoding a promoter, and further comprising a modified nucleic acid sequence encoding ASPA, wherein the modified nucleic acid sequence comprises or constitutes the sequence of SEQ ID NO:2.

[0215] E146. An isolated nucleic acid that comprises a nucleic acid sequence containing a specified promoter and further comprises a nucleic acid sequence containing or constituting the nucleic acid sequence of SEQ ID NO:2.

[0216] E147. The pharmaceutical composition as described in E105, further comprising 350 mM NaCl and 5% D-sorbitol in PBS.

[0217] E148. A pharmaceutical composition as described in E106, wherein the pharmaceutically acceptable carrier comprises 350 mM NaCl and 5% D-sorbitol in PBS.

[0218] Other features and advantages of the invention will become apparent from the following detailed description, drawings, exemplary embodiments and claims. Attached Figure Description

[0219] Figure 1 An exemplary dose-responsive reduction of NAA, as determined by HPLC in cells, is described, in which the cells are transfected with a plasmid expressing NAA synthase (Nat8L) at a dose of 1.0 μg and co-transfected with plasmids containing wild-type human ASPA sequence (SEQ ID NO:3) or modified ASPA sequence, such as codon-optimized original (form 1) (SEQ ID NO:1) or codon-optimized ASPA sequence, such as novel (form 2) (SEQ ID NO:2).

[0220] Figure 2 An exemplary sampling of GFP-positive cells transduced via the rAAV vector administered via the intraplasmic administration (IP) route (ROA) is described. GFP-positive somatic cells (arrows) are scored in each region of interest to generate an estimate of the number (N) of transduced cells.

[0221] Figure 3 This describes exemplary numbers of GFP-positive cells (N) in the cortex, subcortical white matter of the corpus callosum and external bursa, striatum and cerebellum of 6-week-old NUR7 mice after intraparenchymal (IP) administration of AAV / Olig001-GFP, and the number of GFP-positive cells (N) after administration of 1×10⁻⁶ cells via IP ROA. 11 Representative images of native GFP fluorescence in sagittal sections of mouse brains containing the AAV / Olig001-GFP vector genome, showing concentrated GFP expression near the injection site. Estimates of N were generated using a spectrometer (k=4) across 144 sections. Mean + / - sem values ​​for each group (n=5 animals) are presented. Significant differences in the number of GFP-positive cells between dose groups within each region of interest are indicated by an asterisk.

[0222] Figure 4 This describes the exemplary number (N) of GFP-positive cells in the cortex, subcortical white matter, striatum, and cerebellum of 6-week-old NUR7 mice following intrathecal (IT) administration of AAV / Olig001-GFP, and the effect of administering 1×10⁶ GFP via intrathecal (IT) ROA. 11 Representative images of native GFP fluorescence in sagittal sections of mouse brains containing the AAV / Olig001-GFP vector genome, showing diffuse cortical marker expression demonstrating transduction via the vector, as well as moderate white matter tract cell expression demonstrating transduction in this region. Mean + / - SEM values ​​for each group (n = 5 animals) are presented. Significant differences in the number of GFP-positive cells between dose groups within each region of interest are indicated by an asterisk.

[0223] Figure 5 This describes the exemplary number (N) of GFP-positive cells in the cortex, subcortical white matter, striatum, and cerebellum of 6-week-old NUR7 mice after administration of AAV / Olig001-GFP to the intraventricular ventricle (ICV), and the effect of administration of 1×10⁻⁶ cells via ICV ROA. 11 Representative images of native GFP fluorescence in sagittal sections of mouse brains containing the AAV / Olig001-GFP vector genome, demonstrating strong white matter bundle GFP expression confirming cellular vector transduction in this region. Mean + / - sem for each group (n = 5 animals) are presented. Significant differences in the number of GFP-positive cells between dose groups within each region of interest are indicated by an asterisk.

[0224] Figure 6 This describes the exemplary number (N) of GFP-positive cells in the cortex, subcortical white matter, striatum, and cerebellum of 6-week-old NUR7 mice after administration of AAV / Olig001-GFP in the cisterna magna (ICM), and the effect of administration of 1×10⁻⁶ cells via ICM ROA. 11 Representative images of native GFP fluorescence in sagittal sections of mouse brains containing the AAV / Olig001-GFP vector genome, demonstrating intermediate white matter bundle GFP marker expression in this region, indicating cellular transduction. Mean + / - sem for each group (n = 5 animals) are presented. Significant differences in the number of GFP-positive cells between dose groups within each region of interest are indicated by an asterisk.

[0225] Figure 7 The study described the effects of administering 1×10⁶ doses to animals via four different routes of administration (IP, IT, ICV, and ICM). 11 VG dose, direct comparison of exemplary AAV / Olig001-GFP transduction efficiency in the following four regions of interest: cortex, subcortical white matter, striatum, and cerebellum, and representative images of native GFP fluorescence in sections transverse to the injection site within the parenchyma and ventricles. In ICV brain transverse sections, the cortical and subcortical white matter tracts showed a greater number of transgenic positive cells. For each group, n = 5 animals, mean + / - SEM values ​​were used. Significant differences in the number of GFP-positive cells between individual regions of interest are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001).

[0226] Figure 8 Exemplary oligotropy of AAV / Olig001-GFP in the cortex of 6-week-old NUR7 mice following administration of the vector in the intraparenchymal (IP), intrathecal (IT), intraventricular (ICV), and cisterna magna (ICM) regions was described. Cortical sections were analyzed by IHC using Olig2 and NeuN antibodies. For each group, n = 5 animals, the mean percentage + / - SEMs co-labeled with each indicator antigen were used. Asterisks indicate significant differences between groups.

[0227] Figure 9 Exemplary oligotropy of AAV / Olig001-GFP in the subcortical white matter of 6-week-old NUR7 mice following administration of the vector in the intraparenchymal (IP), intrathecal (IT), intraventricular (ICV), and cisterna magna (ICM) regions was described. Subcortical white matter sections were analyzed by IHC using Olig2 and NeuN antibodies. For each group, n = 5 animals, the mean percentage + / - SEMs co-labeled with each indicator antigen were used.

[0228] Figure 10 Exemplary oligotropy of AAV / Olig001-GFP in the striatum of 6-week-old NUR7 mice following administration of the vector in the intraparenchymal (IP), intrathecal (IT), intraventricular (ICV), and intracranial (ICM) regions was described, with marker detection demonstrating cellular transduction via the vector. Striatal sections were analyzed by IHC using Olig2 and NeuN antibodies. For each group, n = 5 animals, the mean percentage + / - SEMs co-labeled with each indicator antigen were used.

[0229] Figure 11 Exemplary oligotropy of AAV / Olig001-GFP in the cerebellum of 6-week-old NUR7 mice following administration of the vector in the intraparenchymal (IP), intrathecal (IT), intraventricular (ICV), and cisterna magna (ICM) regions was described, with marker detection demonstrating transduction via the vector. Cerebellar sections were analyzed by IHC using Olig2 and NeuN antibodies. For each group, n = 5 animals, the mean percentage + / - SEMs co-labeled with each indicator antigen were used.

[0230] Figure 12 Describes ICV application of 1×10 11 Exemplary efficiency of AAV / Olig001-GFP transduction in the cortical and subcortical white matter of age-matched wild-type (WT) and NUR7 mice two weeks after vector genome administration, and representative images of native GFP fluorescence in wild-type brains after AAV / Olig001-GFP administration, showing relatively restricted expression, thus demonstrating transduction via the vector, particularly in the subcortical white matter. For each group, n = 5 animals, mean number of GFP-positive cells per group + / - sem, *p < 0.05, **p < 0.01.

[0231] Figure 13 The expression plasmids for ASPA coding sequences and regulatory elements with codon optimization are described.

[0232] Figure 14 Exemplary rotarod drop wait times during life-long studies were described in AAV / Olig001-ASPA treated mice (at three dose levels), wild-type mice, and nur7 sham-treated mice. Data are presented as mean + / - sem, with n = 12 animals per group.

[0233] Figure 15 Exemplary open field activity is described during the life-long study of wild-type (WT) mice, AAV / Olig001-ASPA treated mice (at three dose levels), and sham-treated NUR7 mice. Data are presented as mean + / - sem, with n = 12 animals per group.

[0234] Figure 16 Exemplary NAA content in the brains of wild-type (WT), nur7 sham-treated, and AAV / Olig001-ASPA-treated mice (at three dose levels) is described. Data are presented as mean + / - sem. NAA is expressed in millimoles per gram of wet tissue weight (n = 6 animals per group). The dose of AAV / Olig001-ASPA is indicated on the x-axis.

[0235] Figure 17 Exemplary mean vector genome copy number (vg / mg) per mg of brain tissue in NUR7 mice treated with AAV / Olig001-ASPA at three different dose levels is described at 22 weeks of age. Mean vg / mg values ​​are presented as + / - sem (n = 6 animals / dose group).

[0236] Figure 18 Representative H&E-stained brain sections from nur7 sham-treated, AAV / Olig001-ASPA-treated, and wild-type mice are described, showing vacuolated regions.

[0237] Figure 19 Exemplary vacuolar volume fractions are described as the percentages of regions of interest (ROIs) in the thalamus and white matter / pons of 22-week-old sham-treated and AAV / Olig001-ASPA-treated nur7 mice. An asterisk indicates significant differences between groups.

[0238] Figure 20 The sham treatment for Olig2 staining and AAV / Olig001-ASPA treatment (2.5 × 10⁻⁶) for demonstrating oligodendrocytes are described. 11 Representative images of the thalamus and cortex of NUR7 mice (vg dose).

[0239] Figure 21 Exemplary counts of Olig2-positive cells in the thalamus and cortex of 22-week-old wild-type, sham-treated, and AAV / Olig001-ASPA-treated nur7 mice are described. Data are presented as mean Olig2-positive cell + / - sem (n = 6 animals per group). An asterisk indicates significant differences between groups.

[0240] Figure 22 The sham treatment and AAV / Olig001-ASPA treatment (2.5 × 10⁻⁶) for NeuN staining are described. 11 Representative images of the thalamus and cortex of NUR7 mice (vg dose).

[0241] Figure 23Exemplary counts of NeuN-positive cells in the thalamus and cortex of 22-week-old wild-type, sham-treated, and AAV / Olig001-ASPA-treated nur7 mice are described. Data are presented as mean NeuN-positive cell + / - sem (n = 6 animals per group). An asterisk indicates significant differences between groups.

[0242] Figure 24 The sham treatment and AAV / Olig001-ASPA treatment (2.5 × 10⁻⁶) for myelin basic protein (MBP) staining are described. 11 Representative images of the cortex of NUR7 mice (vg dose).

[0243] Figure 25 Exemplary myelin basic protein positive fiber length density (MBP-LD) (μm / mm) in the cortex of wild-type, sham-treated, and AAV / 001-ASPA-treated NUR7 mice is described. 3 Data are presented as mean MBP-LD+ / - sem (n = 6 animals per group). An asterisk indicates a significant difference between groups.

[0244] Figure 26 Exemplary brain images (from left to right) are described, including initially fixed pre-cleared samples from ICV-injected mice, samples after tissue clearing, 3DGFP fluorescence images, hemisphere volume segmentation analysis, and intensity heatmaps.

[0245] Figure 27 Intensity heatmaps for all four ICV-injected hemispheres are depicted. The total hemisphere volume is calculated and represented as a gray area. Calculated "low" GFP intensity is indicated in the gray area; "high" GFP intensity is indicated in the white area.

[0246] Figure 28 3D light-panel GFP fluorescence microscopy images of the transparent brains of animals administered AAV / Oligo001-GFP via ICV and IP administration routes are described.

[0247] Figure 29A Representative high-magnification images showing scores of GFP-positive cells co-labeled with Olig2 or NeuN are described. Total GFP cells are scored in each field of view, and the percentage of Olig2 and NeuN co-labeled cells is scored in the same field of view.

[0248] Figure 29B Representative images of GFP co-labeled with Olig2 in SCWM bundle cells of animal brains treated with AAV / Olig001-GFP via ICV ROA are described, showing near 100% oligotropy and almost complete absence of neurotropism.

[0249] Figure 29CA representative image depicting cerebellar GFP transgene expression in Purkinje neurons, with sparse Olig2 co-labeling in the white matter (arrows).

[0250] Figure 29D Representative images of GFP co-labeled with Olig2 in the striatum of the ICV ROA brain are depicted, showing a contrast with cerebellar orientation.

[0251] Figure 29E Representative images of white matter tracts in 8-week-old nur7 and age-matched wild-type naïve brains are depicted after processing for BrdU labeling and Olig2.

[0252] Figure 29F Exemplary counts of BrdU cells in wild-type and nur7 white matter bundles at 2 and 8 weeks are described. Mean BrdU-positive cell + / - sem for each group is presented. For each group (genotype at each week of age), n = 6.

[0253] Figure 29G Representative images of BrdU / GFP co-labeled cells in the subcortical white matter of the nur7 brain treated with AAV / Olig001-GFP via ICV ROA are described.

[0254] Figures 30A, 30B and 30C Biological distribution volume analysis is described. (A) Tissue volume as imaged by ICV and IP. (B) Mean and median GFP fluorescence intensity in the two ROAs. (C) Volume fraction of GFP positivity representing low and high intensities in the ROA.

[0255] Figures 31A, 31B and 31C The CLARITY and SWITCH workflows used for pharmacodynamic efficacy assessment are described. (A) Tissue clearing and labeling methods. From left to right: intact mouse brain, a 2 mm central section of the right hemisphere before clearing, the same tissue 1 day and 3 days after passive clearing, and a 3D image showing the fluorescence signal of previously labeled proteins (green: cell nuclei, red: myelin basic protein (MBP)). (B) Representative 2 mm sections of tissues treated with Nur7, WT, and Olig1-ASPA. Red arrows in each image indicate thalamic regions. (C) Tissue clarity 1 day after passive clearing.

[0256] Figures 32A, 32B and 32C Cell counting of tissues is described based on 2D regions. (A) Extracted single 2D slices from 3D images of all three groups with similar anatomical orientations. Red boxes identify the regions in the thalamus and cortex where cell counting was performed. (B) Enlarged image data from the red boxes in (A), and the corresponding cell segmentation. (C) Mean nuclear density (counts normalized by segmentation regions).

[0257] Figures 33A, 33B, 33C, 33D, 33E, 33F, 33G, 33H, and 33I depict 3D volumetric analysis of pharmacodynamic therapeutic effects. (A) Determining the complete 3D volume of a 2 mm tissue section. (B) Mean fluorescence intensity calculated within the 3D volume. (C) MBP characterization via a more restrictive threshold set at fluorescence values ​​exceeding 2000 (left panel) or a more inclusive threshold at 1000 (left panel). (D) MBP deficiency observed in Nur7 in both cases. The effect in the Olig1-ASPA group is seen at the lower threshold, where the overall value is close to the WT level. (E) Region-based 3D analysis in a thalamic region, where manually segmented portions of the region are shown in yellow. (F) Mean fluorescence of nuclear (SYTO) and myelin (MBP) markers within this region. (G) Region-based analysis on a portion of the cortex, where manually segmented portions are shown in yellow. (H) Mean fluorescence of nuclear (SYTO) and myelin (MBP) markers in this cortical region. (I) 3D cell concentration (nuclei / 100 μm) 2 ). Invention Details

[0259] definition

[0260] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to also include the plural forms as used in the specification and appended claims of this invention. The following terms have the given meanings:

[0261] As used herein, the terms “about” or “approximately” refer to measurable values ​​such as the amount of biological activity, the length of a polynucleotide or polypeptide sequence, the content of G and C nucleotides, the codon fitness index, the number of CpG dinucleotides, dosage, time, temperature, etc., and are intended to cover variations of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or even 0.1% in either direction (greater or less than) the specified amount.

[0262] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items, as well as combinations that are missing when interpreted in an alternative manner (“or”).

[0263] As used herein, the terms “adeno-associated virus” and / or “AAV” refer to parvoviruses and their variants that have a linear single-stranded DNA genome. Unless otherwise required, the term encompasses all subtypes as well as naturally occurring and recombinant forms. The wild-type genome contains 4,681 bases (Berns and Bohenzky (1987) Advances in Virus Research 32:243-307) and includes terminal repeat sequences (e.g., inverted terminal repeats (ITRs)) at each end, which serve as the origin of DNA replication in cis and as the viral packaging signal. The genome includes two large open reading frames, referred to as AAV replication (“AAV rep” or “rep”) and capsid (“AAV cap” or “cap”) genes. AAV rep and cap may also be referred to herein as AAV “packaging genes.” These genes encode viral proteins involved in viral genome replication and packaging.

[0264] In wild-type AAV viruses, the overlap of the three capsid genes VP1, VP2, and VP3 within a single open reading frame, along with alternative splicing, results in the production of VP1, VP2, and VP3. (Grieger and Samulski (2005) J. Virol. 79(15): 9933-9944.) A single P40 promoter allows all three capsid proteins to be expressed for VP1, VP2, and VP3 in a ratio of approximately 1:1:10, thereby complementing the production of the AAV capsid. More specifically, VP1 is a full-length protein, while VP2 and VP3 are gradually shortened due to increased N-terminal truncation. A well-known example is the capsid of AAV9 as described in U.S. Patent No. 7,906,111, in which VP1 contains amino acid residues 1 to 736 of SEQ ID NO: 123, VP2 contains amino acid residues 138 to 736 of SEQ ID NO: 123, and VP3 contains amino acid residues 203 to 736 of SEQ ID NO: 123. As used herein, the term “AAV Cap” or “cap” refers to AAV capsid proteins VP1, VP2 and / or VP3, as well as their variants and analogs.

[0265] At least four viral proteins, Rep 78, Rep 68, Rep 52, and Rep 40, are synthesized from the AAV rep gene and are named according to their apparent molecular weight. As used herein, “AAV rep” or “rep” means the AAV replication proteins Rep 78, Rep 68, Rep 52, and / or Rep 40, as well as their variants and analogs. As used herein, rep and cap refer to wild-type and recombinant (e.g., modified chimeras, etc.) rep and cap genes and the polypeptides they encode. In some embodiments, the nucleic acid encoding rep contains nucleotides from more than one AAV serotype. For example, the nucleic acid encoding rep may contain nucleotides from the AAV2 serotype and nucleotides from the AA3 serotype (Rabinowitz et al. (2002) J. Virology 76(2):791-801).

[0266] As used herein, the terms “recombinant adeno-associated virus vector,” “rAAV,” and / or “rAAV vector” refer to an AAV containing a vector genome in which the polynucleotide sequence is not or not entirely of AAV origin (e.g., polynucleotides heterologous to AA), and in which the rep and / or cap genes of the wild-type AAV viral genome have been removed from the viral genome. When the rep and / or cap genes of a typical AAV have been removed or are absent (and in which the side-connected ITR is typically derived from the ITR of a different serotype, e.g., but not limited to the AAV2 ITR in which the capsid is not AAV2), the nucleic acids within the AAV, including any ITR and any nucleic acids between it, are referred to as the “vector genome.” Therefore, the term rAAV vector encompasses an rAAV viral particle containing a capsid and heterologous nucleic acids, i.e., nucleic acids not originally present in the capsid in nature, and is referred to below as the “vector genome.” Therefore, the "rAAV vector genome" (or "vector genome") involves a heteropolynucleotide sequence (including at least one ITR, which is usually but not necessarily an ITR unrelated to the original nucleic acid present in the original AAV), which may but must be contained within the AAV capsid. The rAAV vector genome can be double-stranded (dsAAV), single-stranded (ssAAV), and / or self-complementary (scAAV).

[0267] As used herein, the terms “rAAV vector,” “rAAV viral particle,” and / or “rAAV vector particle” refer to an AAV capsid containing at least one AAV capsid protein (although all AAV capsid proteins are typically present, such as VPI, VPS, and VP3 or variants thereof) and containing a vector genome containing heterologous nucleic acid sequences not originally present in the original AAV capsid. These terms are distinguished from non-recombinant “AAV viral particle” or “AAV virus,” where the capsid contains a viral genome encoding the rep and cap genes, and AAV viruses are capable of replication if present in cells that also contain helper viruses such as adenoviruses and / or herpes simplex viruses, and / or essential helper genes produced by them. Therefore, the generation of rAAV vector particles must involve the use of recombinant DNA technology to produce a recombinant vector genome, which is thus contained within the capsid to form an rAAV vector, rAAV viral particle, or rAAV vector particle.

[0268] Genomic sequences of various serotypes of AAV, which are found in nature and / or in its mutants and variants, as well as inverted terminal repeat (ITR) sequences, rep protein and capsid subunit sequences, are known in the art. Such sequences are available in the literature or in public databases such as GenBank. See, for example, GenBank accession numbers NC-002077 (AAV-1), AF063497 (AAV-1), NC-001401 (AAV-2), AF043303 (AAV-2), NC-001729 (AAV-3), NC_001863 (AAV-3B), NC-001829 (AAV-4), U89790 (AAV-4), NC-006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8), and NC-006261 (AAV-8), the disclosures of which are incorporated herein by reference. See also, for example, Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; International Patent Publication WO 00 / 28061, WO WO 99 / 61601, WO 98 / 11244; WO 2013 / 063379, WO 2014 / 194132, WO 2015 / 121501; and U.S. Patent Nos. 6,156,303 and 7,906,111.

[0269] As used herein, the term “improvement” means a detectable or measurable improvement in a subject’s disease, condition or illness, or its symptoms, or underlying cellular responses. Detectable or measurable improvement includes a subjective or objective reduction, decrease, inhibition, suppression, limitation, or control of the occurrence, frequency, severity, progression, or duration of the disease, condition or illness, complications arising from or related to it, or improvement or reversal of its symptoms.

[0270] As used herein, the term "associated with" refers to a relationship between two entities when the presence, level, and / or form of one entity is associated with the presence, level, and / or form of another. For example, if the presence, level, and / or form of a particular entity (e.g., peptide, genetic trait, metabolite, microorganism, etc.) is associated with the incidence and / or susceptibility to a disease, symptom, or condition (e.g., in the entire relevant population), then that particular entity is considered associated with that particular disease, symptom, or condition. In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly such that they are physically close to each other and / or remain physically close to each other. In some embodiments, two or more physically associated entities are covalently linked; in some embodiments, two or more physically associated entities are non-covalently associated, for example, through hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0271] As used herein, the terms “cis motif” or “cis element” include conserved sequences, such as those found at or near the ends of a genomic sequence and identified as being used to initiate replication; and cryptic promoters or sequences located at internal positions that may be used for transcription initiation, splicing, or termination. Cis motifs or cis elements and the sequences they interact with reside on the same nucleic acid molecule. This differs from “trans motif” sequences, which are other sequences located on different nucleic acid molecules and function in a “trans” manner.

[0272] As used herein, the term "coding sequence" or "coding nucleic acid" refers to a nucleic acid sequence that encodes a protein or polypeptide and indicates a sequence that, when transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo under the control of appropriate regulatory sequences (operably ligated). The boundaries of a coding sequence are typically determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0273] As used herein, the term "chimera" refers to a viral capsid whose capsid sequence is derived from different parvoviruses, preferably different AAV serotypes, as described in Rabinowitz et al., U.S. Patent No. 6,491,907, the disclosure of which is incorporated herein by reference in its entirety. See also Rabinowitz et al. (2004) J.Virol. 78(9):4421-4432. In some embodiments, the chimeric viral capsid is an AAV2.5 capsid having a sequence of an AAV2 capsid with the following mutations: 263Q to A; 265 insert T; 705 N to A; 708 V to A; and 716 T to N. The nucleotide sequence encoding this capsid is defined as SEQ ID NO:15, as described in WO2006 / 066066. Other preferred chimeric AAV capsids include, but are not limited to: AAV2i8 described in WO 2010 / 093784; AAV2G9 and AAV8G9 described in WO 2014 / 144229; and AAV9.45 (Pulicherla et al. (2011) Molecular Therapy 19(6):1070-1078); AAV-NP4, NP22, NP66, AAV-LK01 to AAV-LK019 described in WO 2103 / 029030; RHM4-1 and RHM15-1 to RHM5-6 described in WO 205 / 013313; and AAV-DJ, AAV-DJ / 8, and AAV-DJ / 9 described in WO 2007 / 120542.

[0274] As used herein, the term "conservative substitution" refers to the replacement of an amino acid with a biologically, chemically, or structurally similar residue. Biological similarity means that the substitution does not impair biological activity. Structural similarity means that the amino acid has side chains of similar length, such as alanine, glycine, and serine, or has similar sizes. Chemical similarity means that the residues have the same charge or both are hydrophilic or hydrophobic. Specific examples include: replacing a hydrophobic residue with another hydrophobic residue, such as isoleucine, valine, leucine, or methionine; or replacing one polar residue with another polar residue, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc. Specific examples of conserved substitution include: hydrophobic residues, such as isoleucine, valine, leucine, or methionine, replacing each other; and polar residues replacing another polar residue, such as arginine replacing lysine, glutamic acid replacing aspartic acid, or glutamine replacing asparagine, etc. Conservative amino acid substitutions typically include substitutions within the following group: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. "Conservative substitution" also includes using a substituted amino acid to replace an unsubstituted parental amino acid.

[0275] As used herein, the term "sidejoining" refers to a sequence being sidejoined by other elements and indicates the presence of one or more sidejoining elements relative to the upstream and / or downstream of the sequence, i.e., 5' and / or 3'. The term "sidejoining" is not intended to indicate that the sequence must be continuous. For example, an intervening sequence may exist between the nucleic acid encoding a transgene and a sidejoining element. A sequence (e.g., a transgene) being "sidejoined" by two other elements (e.g., an ITR) indicates that one element is located at the 5' of the sequence and the other at the 3' of the sequence; however, an intervening sequence may exist between them.

[0276] As used herein, the term "fragment" refers to a material or entity having a structure that comprises discrete parts of the whole but lacks one or more parts found in the whole. In some embodiments, a fragment consists of discrete parts. In some embodiments, a fragment comprises or is included as a characteristic structural element or part found in the whole. In some embodiments, the polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., amino acid residues, nucleotides) found throughout the polymer.

[0277] As used herein, the term "functional" refers to a biomolecule that exhibits the characterized properties and / or activities. Biomolecules may have two functions (i.e., bifunctional) or multiple functions (i.e., multifunctional).

[0278] As used herein, the term "gene" refers to a polynucleotide containing at least one open reading frame capable of encoding a specific polypeptide or protein after transcription and translation. "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non-integrating transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), and / or integration of transferred genetic material into the genomic DNA of host cells.

[0279] As used herein, the term “heterogeneous” or “exogenous” nucleic acid refers to a nucleic acid inserted into a vector (e.g., an rAAV vector) for the purpose of vector-mediated transfer / delivery of nucleic acid into cells. Heterogeneous nucleic acids are generally different from the vector (e.g., AAV) nucleic acid; that is, heterogeneous nucleic acids are non-natural relative to viral (e.g., AAV) nucleic acids found in naturally occurring AAVs. Once transferred (e.g., transduced) or delivered into cells, the heterogeneous nucleic acid contained within the vector can be expressed (e.g., transcribed and translated, if appropriate). Alternatively, the heterogeneous nucleic acid contained within the vector that has been transferred (transduced) or delivered into cells does not necessarily need to be expressed. While the term “heterogeneous” is not always used herein to refer to nucleic acids, references to nucleic acids are intended to include heterogeneous nucleic acids even in the absence of the modifier “heterogeneous.” For example, a heterogeneous nucleic acid is a nucleic acid encoding an ASPA polypeptide, such as a codon-optimized nucleic acid encoding ASPA used to treat canavanine disease.

[0280] As used herein, the term "homology" or "homology" refers to two or more reference entities (e.g., nucleic acid or polypeptide sequences) that share at least partial identity over a given region or portion. For example, peptides are homologous at that position when the amino acid positions in two peptides are occupied by the same amino acid. It is worth noting that homologous peptides will retain the activity or function associated with the unmodified or reference peptide, and modified peptides generally have an amino acid sequence that is "substantially homologous" to the amino acid sequence of the unmodified sequence. When referring to polypeptides, nucleic acids, or fragments thereof, "substantial homology" or "substantial similarity" means that there is sequence identity in at least about 95% to 99% of the sequence when optimally aligned with another polypeptide, nucleic acid (or its complementary strand), or fragment thereof with appropriate insertions or deletions. The degree of homology (identity) between two sequences can be determined using computer programs or mathematical algorithms. Such algorithms for calculating the percentage of sequence homology (or identity) typically take into account sequence gaps and mismatches over the comparison region or range. Exemplary programs and algorithms are provided below.

[0281] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny of these cells. Host cells include “transfectants,” “transformants,” “transformed cells,” and “transduced cells,” encompassing primary transfected, transformed, or transduced cells and their derived progeny, regardless of passage number. In some embodiments, the host cell is a packaging cell used to generate the rAAV vector.

[0282] As used herein, the term "identity" or "same as" refers to the overall correlation between polymer molecules, such as between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical.

[0283] The percentage of identity between two nucleic acid or polypeptide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, vacancies can be introduced into one or both of the first and second sequences, and dissimilar sequences can be ignored for comparison purposes). In some embodiments, the length of the sequence to be aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. Nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., a nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of shared positions between the sequences, taking into account the number and length of vacancies, which need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms.

[0284] To determine the percentage of identity or homology, sequences can be aligned using methods and computer programs, including BLAST, available at ncbi.nlm.nih.gov / BLAST / on the World Wide Web. Another alignment algorithm is FASTA, which is available in the Genetics Computing Group (GCG) package from Madison, Wis., USA. Other techniques for alignment are described in Methods in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Alignment procedures that allow gaps in sequences are of particular interest. The Smith-Waterman algorithm is one that allows gaps in sequence alignment. See Meth. Mol. Biol. 70:173-187 (1997). Similarly, the GAP procedure using the Needleman and Wunsch alignment methods can be used to align sequences. See J. Mol. Biol. 48:443-453 (1970).

[0285] Interest has also arisen in the BestFit procedure for determining sequence identity using the local homology algorithm of Smith and Waterman (1981, Advances in Applied Mathematics 2:482-489). The gap-generating penalty typically ranges from 1 to 5, usually 2 to 4, and in some embodiments is 3. The gap-extending penalty typically ranges from about 0.01 to 0.20, and in some cases is 0.10. The procedure has default parameters determined by the input sequences for comparison. Preferably, sequence identity is determined using the default parameters determined by the procedure. This procedure is also available in the Genetics Computing Group (GCG) package from Madison, WI, USA.

[0286] Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. The percentage of sequence identity is calculated by FastDB based on the following parameters: mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; and connection penalty: 30.0.

[0287] As used herein, the terms “increase,” “improve,” or “decrease” indicate a value relative to a baseline measurement, such as a measurement in the same individual prior to the commencement of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. In some embodiments, a “control individual” is an individual suffering from the same form of disease or lesion as the individual to be treated.

[0288] As used herein, the terms “inverted terminal repeat,” “ITR,” “terminal repeat,” and “TR” refer to palindromic terminal repeat sequences located at or near the ends of the AAV genome, primarily consisting of complementary, symmetrically arranged sequences. These ITRs can fold to form T-shaped hairpin structures, acting as primers during DNA replication initiation. They are also required for viral genome integration into the host genome; for rescue from the host genome; and for capsidating viral nucleic acids into mature viral particles. Cis-ITRs are required for vector genome replication and its packaging into viral particles. “5'ITR” refers to the 5' end of the AAV genome and / or the 5' ITR of recombinant transgenes. “3'ITR” refers to the 3' end of the AAV genome and / or the 3' ITR of recombinant transgenes. The wild-type ITR is approximately 145 bp in length. Modified or recombinant ITRs may contain fragments or portions of wild-type AAV ITR sequences. Those skilled in the art will understand that during successive rounds of DNA replication, ITR sequences can be exchanged, causing a 5' ITR to become a 3' ITR and vice versa. In some implementations, at least one ITR is present at the 5' and / or 3' end of the recombinant vector genome, such that the vector genome can be packaged into a capsid to produce an rAAV vector containing the vector genome (also referred to herein as "rAAV vector particle" or "rAAV virus particle").

[0289] As used herein, the term “isolated” means 1) a substance or composition that is designed, produced, prepared, and / or manufactured artificially and / or 2) separated from at least one of its components at the time of its initial production (whether in nature and / or in an experimental setting). Generally, isolated compositions are substantially free of one or more materials that normally associate with them in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, and / or cell membranes. The term “isolated” does not exclude artificial combinations, such as recombinant nucleic acids, recombinant vector genomes (e.g., rAAV vector genomes), rAAV vector particles packaging, for example, capsidated vector genomes (e.g., but not limited to rAAV vector particles containing an AAV / Olig001 capsid), and pharmaceutical preparations. The term “isolated” also does not exclude alternative physical forms of the composition, such as heterozygous / chimeras, multimers / oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation), variants or derivatives, or artificial forms expressed in host cells.

[0290] The isolated substance or composition may be separated from about 10%, about 20%, about 30%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components originally associated with it. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as those skilled in the art will understand, a substance may still be considered “separated” or even “pure” after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percentage of separation or purity of the substance is calculated without the presence of such carriers or excipients.

[0291] As used herein, the terms “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide” are interchangeable to refer to any molecule that is composed of or contains monomeric nucleotides linked by phosphodiester bonds. Nucleic acids can be oligonucleotides or polynucleotides. Nucleic acid sequences are presented herein in a 5’ to 3’ orientation. The nucleic acid sequences (i.e., polynucleotides) disclosed herein can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules, and refer to all forms of nucleic acids such as double-stranded molecules, single-stranded molecules, small or short hairpin RNA (shRNA), microRNA, small or short interfering RNA (siRNA), trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA, and ribosomal RNA. When the polynucleotide is a DNA molecule, the molecule can be a gene, cDNA, an antisense molecule, or a fragment of any of the aforementioned molecules. Nucleotides are indicated herein by single-letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U). Nucleotide sequences can be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as gamma-hydroxyl nucleic acids (GNA) and threonine nucleic acids (TNA). Each of these sequences can be distinguished from naturally occurring DNA or RNA by altering the molecular backbone. Phosphothiophosphate nucleotides can also be used. Other deoxynucleotide analogs include methylphosphonates, aminophosphates, dithiophosphates, N3'-P5'-aminophosphates, and oligonucleotide phosphothiophosphates and their 2'-O-allyl analogs, and 2'-O-methylribonucleotide methylphosphonates, which can be used in the nucleotide sequences disclosed herein.

[0292] As used herein, the term "nucleic acid construct" refers to a non-naturally occurring nucleic acid molecule (e.g., recombinant nucleic acid) produced by the use of recombinant DNA technology. Nucleic acid constructs are single-stranded or double-stranded nucleic acid molecules modified to contain nucleic acid sequence strands arranged and combined in a manner not found in nature. Nucleic acid constructs can be "vectors" (e.g., plasmids, rAAV vectors, genomic vectors, expression vectors, etc.), that is, nucleic acid molecules designed to deliver exogenously generated DNA into host cells.

[0293] As used herein, the term "operably linked" refers to nucleic acid sequence (or polypeptide) elements linked in a functional relationship. A nucleic acid is operably linked to another nucleic acid sequence when it is in a functional relationship with that sequence. For example, if a promoter or other transcriptional regulatory sequence (e.g., an enhancer) affects the transcription of a coding sequence, it is operably linked to the coding sequence. In some embodiments, operably linked means that the linked nucleic acid sequences are contiguous. In some embodiments, operably linked does not mean that the nucleic acid sequences are contiguously linked, but rather that there are inserted sequences between those linked nucleic acid sequences.

[0294] As used herein, the terms “pharmaceutical acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gas, liquid, or solid or mixture thereof suitable for one or more routes of administration, in vivo delivery, or contact.

[0295] As used herein, the terms “polypeptide,” “protein,” “peptide,” or “encoded by a nucleic acid sequence” (i.e., encoded by a polynucleotide sequence, encoded by a nucleotide sequence) refer to the full-length natural sequence identical to that of a naturally occurring protein, as well as functional sequences, modified forms, or sequence variants, provided that the sequence, modified form, or variant retains a degree of functionality of the natural full-length protein. In the methods and uses of this disclosure, such polypeptides, proteins, and peptides encoded by nucleic acid sequences may, but are not necessarily, identical to defective endogenous proteins, or proteins whose expression is insufficient or absent in subjects treated with gene therapy.

[0296] As used herein, the terms “prevention” or “avoidance” refer to a delay in the onset and / or a reduction in the frequency and / or severity of one or more signs or symptoms of a particular disease, condition, or illness (e.g., canavanine disease). In some implementations, prevention is assessed on a population basis, such that the agent is considered to “prevent” the particular disease, condition, or illness if a statistically significant reduction in the progression, frequency, and / or intensity of one or more signs or symptoms of the disease, condition, or illness is observed in a population susceptible to the disease, condition, or illness. Prevention is considered complete when the onset of the disease, condition, or illness is delayed for a predetermined period of time.

[0297] As used herein, the term "recombinant" refers to a vector, polynucleotide (e.g., recombinant nucleic acid), polypeptide, or cell that is the product of various combinations of procedures as part of cloning, restriction, or ligation steps (e.g., related to the polynucleotides or polypeptides contained therein) and / or the production of constructs different from those found in nature. Recombinant viruses or vectors (e.g., rAAV vectors) contain a vector genome containing recombinant nucleic acids (e.g., nucleic acids containing transgenes and one or more regulatory elements, such as codon-optimized nucleic acids encoding ASPA and CBh promoters). The term includes both copies of the original polynucleotide construct and progeny of the original viral construct.

[0298] As used herein, the term "subject" refers to an organism, such as a mammal (e.g., human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, gerbil, cat, dog). In some embodiments, the subject is a NUR7 mouse. In some embodiments, the human subject is an adult, adolescent, or child subject. In some embodiments, the subject has a disease, condition, or illness, such as a disease, condition, or illness that can be treated as provided herein. In some embodiments, the subject has a disease, condition, or illness associated with a deficiency or dysfunction of aspartate acylase activity, such as canavan disease. In some embodiments, the subject is susceptible to a disease, condition, or illness. In some embodiments, susceptible subjects are susceptible to a disease, condition, or illness and / or have an increased risk of developing a disease, condition, or illness (e.g., compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of a disease, condition, or illness. In some embodiments, the subject does not exhibit specific symptoms (e.g., clinical manifestations of a disease) or characteristics of a disease, condition, or illness. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, symptom, or condition. In some embodiments, the subject is a human patient. In some embodiments, the subject is an individual who has received and / or has received a diagnostic and / or therapeutic treatment (e.g., gene therapy for canavan disease). In some embodiments, the subject is a human patient with canavan disease.

[0299] As used herein, the term "substantially" refers to qualitative conditions that exhibit all or nearly all of the range or extent of the features or properties of interest. Those skilled in the art will understand that biological and chemical phenomena rarely (if any) reach completion and / or proceed to a complete or attainable or absolute result. Therefore, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0300] As used herein, the term “symptom reduction” or “symptom reduction” refers to a reduction in the magnitude (e.g., intensity, severity, etc.) and / or frequency of one or more symptoms of a particular disease, condition, or illness. For clarity, a delay in the onset of a particular symptom is considered a form of reduced frequency of that symptom.

[0301] As used herein, the term "therapeutic peptide" is a peptide, polypeptide, or protein (e.g., enzyme, structural protein, transmembrane protein, transporter protein) that can alleviate or reduce symptoms caused by the absence or deficiency of a protein in target cells (e.g., isolated cells) or an organism (e.g., a subject). A therapeutic peptide or protein encoded by a transgene is a peptide or protein that provides a benefit to a subject, such as correcting gene defects or correcting gene defects related to expression or function. Similarly, a "therapeutic transgene" is a transgene that encodes a therapeutic peptide. In some embodiments, the therapeutic peptide expressed in host cells is an enzyme expressed by a transgene (i.e., a foreign nucleic acid introduced into the host cell). In some embodiments, the therapeutic peptide is an ASPA protein expressed by a therapeutic transgene transduced into cerebral cortical cells (e.g., oligodendrocytes).

[0302] As used herein, the term "therapeuticly effective amount" refers to the amount that produces the desired therapeutic effect when administered. In some embodiments, the term refers to an amount sufficient to treat a disease, condition, or illness when administered to a population suffering from or susceptible to such a disease, condition, or illness according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity and / or delays the onset of one or more symptoms of a disease, condition, and / or illness. Those skilled in the art will understand that the term "therapeuticly effective amount" does not actually require successful treatment in a particular individual. Rather, a therapeutically effective amount can be an amount that provides a particularly desired pharmacological response in a large number of subjects when administered to patients requiring such treatment.

[0303] As used herein, the term "transgenic" is used to mean any heterologous polynucleotide delivered to and / or expressed in a host cell, target cell, or organism (e.g., a subject). This "transgenic" may be delivered to a host cell, target cell, or organism using a vector (e.g., an rAAV vector). The transgenic may be operatively linked to a control sequence such as a promoter. Those skilled in the art will understand that the expression of the control sequence may be selected based on its ability to promote the expression of the transgenic in a host cell, target cell, or organism. Typically, the transgenic may be operatively linked to an endogenous promoter that associates with the transgenic in its natural state; however, more typically, the transgenic may be operatively linked to a promoter to which the transgenic does not associate in its natural state. Examples of transgenics are nucleic acids encoding therapeutic polypeptides such as the ASPA polypeptide, and exemplary promoters are promoters that are not operatively linked to nucleotides encoding ASPA in their natural state. This non-endogenous promoter may include the CBh promoter, as well as many other promoters known in the art.

[0304] Nucleic acids of interest can be introduced into host cells using a variety of techniques well known in the art, including transfection and transduction.

[0305] “Transfection” is generally referred to as a technique for introducing exogenous nucleic acids into cells without the use of a viral vector. As used herein, the term “transfection” refers to the transfer of recombinant nucleic acids (e.g., expression plasmids) into cells (e.g., host cells) without the use of a viral vector. Cells into which recombinant nucleic acids are introduced are referred to as “transfected cells.” Transfected cells may be host cells (e.g., CHO cells, Pro10 cells, HEK293 cells) containing expression plasmids / vectors for generating recombinant AAV vectors. In some embodiments, transfected cells (e.g., packaging cells) may contain plasmids containing transgenes (e.g., ASPA transgenes), plasmids containing the AAV rep gene and the AAV cap gene, and plasmids containing accessory genes. Many transfection techniques are known in the art, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals.

[0306] As used herein, the term "transduction" refers to the transfer of nucleic acids (e.g., vector genomes) into cells (e.g., target cells, including but not limited to oligodendrocytes) via a viral vector (e.g., rAAV vector). In some embodiments, gene therapy for canavan disease includes transducing a vector genome containing a modified nucleic acid encoding ASPA into oligodendrocytes. Cells in which the transgene is introduced via a virus or viral vector are referred to as "transduced cells." In some embodiments, the transduced cells are isolated cells and the transduction occurs in vitro. In some embodiments, the transduced cells are cells within an organism (e.g., a subject) and the transduction occurs in vivo. The transduced cells may be target cells of an organism, transduced via a recombinant AAV vector, such that the target cells of the organism express polynucleotides (e.g., transgenes, such as modified nucleic acids encoding ASPA).

[0307] Transducible cells include cells of any tissue or organ type or of any origin (e.g., mesoderm, ectoderm, or endoderm). Non-limiting examples of cells include: liver cells (e.g., hepatocytes, sinusoidal endothelial cells), pancreatic cells (e.g., β-islet cells, exocrine cells), lung cells, central or peripheral nervous system cells (e.g., brain cells (e.g., nerve or ependymal cells, oligodendrocytes) or spinal cells), kidney cells, eye cells (e.g., retinal cells), spleen cells, skin cells, thymocytes, testicular cells, lung cells, diaphragmatic cells, heart (cardiac) cells, muscle or psoas muscle cells, or intestinal cells (e.g., endocrine cells), adipose tissue (white, brown, or beige) cells, muscle cells (e.g., fibroblasts, myocytes), synovial cells, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic (e.g., blood or lymphocytes). Other examples include stem cells, such as those that develop or differentiate into pluripotent or multipotent progenitor cells, such as: liver cells (e.g., hepatocytes, sinusoidal endothelial cells), pancreatic cells (e.g., β-islet cells, exocrine cells), lung cells, central or peripheral nervous system cells (e.g., brain cells (e.g., nerve or ependymal cells, oligodendrocytes) or spinal cells), kidney cells, eye cells (e.g., retinal cells), spleen cells, skin cells, thymocytes, testicular cells, lung cells, diaphragmatic cells, heart (cardiac) cells, muscle or psoas muscle cells, or intestinal cells (e.g., endocrine cells), adipose tissue (white, brown, or beige) cells, muscle cells (e.g., fibroblasts, myocytes), synovial cells, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic (e.g., blood or lymphocytes).

[0308] In some embodiments, cells located in specific regions of a tissue or organ (e.g., the brain) can be transduced by administering an rAAV vector (e.g., rAAV containing the ASPA transgene) to the tissue or organ. In some embodiments, brain cells are transduced using rAAV containing the ASPA transgene. In some embodiments, cells of the cerebral cortex are transduced using rAAV containing the ASPA transgene. In some embodiments, striatal cells are transduced using rAAV containing the ASPA transgene. In some embodiments, subcortical white matter cells of the brain are transduced using rAAV containing the ASPA transgene. In some embodiments, cerebellar cells of the brain are transduced using rAAV containing the ASPA transgene.

[0309] As used herein, the terms “treatment,” “management,” or “therapy” refer to the application of a therapy that partially or completely relieves, improves, reduces, or inhibits one or more symptoms, features, and / or causes of a particular disease, condition, or illness, delays its onset, reduces its severity, and / or reduces its incidence.

[0310] As used herein, the term "vector" refers to plasmids, viruses (e.g., rAAV), granules, or other media that can be manipulated by inserting or introducing nucleic acids (e.g., recombinant nucleic acids). Vectors can be used for a variety of purposes, including, for example, gene manipulation (e.g., cloning vectors), introducing / transferring nucleic acids into cells, and transcribing or translating the inserted nucleic acids in cells. In some embodiments, the vector nucleic acid sequence contains at least an origin of replication for proliferation in cells. In some embodiments, the vector nucleic acid comprises a heterologous nucleic acid sequence, expression control elements (e.g., promoters, enhancers), selection markers (e.g., antibiotic resistance), a polyadenosine (polyA) sequence, and / or an ITR. In some embodiments, the nucleic acid sequence is amplified upon delivery to a host cell. In some embodiments, the cell expresses a polypeptide encoded by the heterologous nucleic acid sequence when delivered to a host cell in vitro or in vivo. In some embodiments, the nucleic acid sequence or a portion thereof is packaged into a capsid upon delivery to a host cell. The host cell can be an isolated cell or a cell within a host organism. In addition to the nucleic acid sequence encoding a polypeptide or protein (e.g., a transgene), an additional sequence (e.g., a regulatory sequence) may be present in the same vector (i.e., cis-associated with the gene) and side-attached to the gene. In some embodiments, the regulatory sequence may be present in a separate (e.g., a second) vector that acts in a trans-associated manner to regulate gene expression. Plasmid vectors may be referred to herein as “expression vectors”.

[0311] As used herein, the term "vector genome" refers to the recombinant nucleic acid sequence packaged or enclosed within a capsid to form an rAAV vector. Typically, a vector genome includes heterologous polynucleotide sequences, such as transgenes, regulatory elements, or ITRs not initially present in the capsid. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors (e.g., rAAV vectors), the vector genome does not include the entire plasmid but only the sequences intended for delivery via a viral vector. This non-vector genome portion of the recombinant plasmid is often referred to as the "plasmid backbone," and it is essential for the cloning, selection, and amplification of the plasmid (processes required for the proliferation of recombinant viral vectors), but it is not itself packaged or enclosed within the capsid of the rAAV vector.

[0312] As used herein, the term "viral vector" generally refers to a viral particle that acts as a medium for nucleic acid delivery and contains a vector genome (e.g., containing transgenic nucleic acids instead of encoding AAV rep and cap) packaged within the viral particle (i.e., the capsid), and includes, for example, lentiviruses and parvoviruses, including AAV serotypes and variants (e.g., rAAV vectors). Recombinant viral vectors do not contain a vector genome containing rep and / or cap genes.

[0313] This disclosure provides modified nucleic acids comprising a modified ASPA coding sequence, and their use in gene therapy pharmaceutical compositions. As used herein, “modified” means that the nucleic acid sequence encoding a polypeptide present in its natural state is altered such that, in one embodiment, the modified nucleic acid sequence drives a higher level of protein expression in the cell compared to the protein expression level of an unmodified (i.e., naturally occurring (including mutant forms of the gene)) nucleic acid sequence from the same cells elsewhere. This disclosure also provides recombinant nucleic acids comprising a vector genome that includes a modified ASPA coding sequence as part of its sequence. Furthermore, this disclosure provides packaged gene delivery media, such as rAAV vectors, that include a modified ASPA coding sequence. This disclosure also includes methods for delivering and preferably expressing the modified ASPA coding sequence in cells. This disclosure also provides gene therapy methods that administer a modified ASPA coding sequence, for example, as a component of a vector and / or packaged as a component of a viral gene delivery media (e.g., rAAV vector), to a subject. For example, treatment can be performed to increase ASPA levels in a subject and to treat ASPA deficiency in a subject. Each of these aspects of this disclosure is further discussed in subsequent sections.

[0314] AAV and rAAV carriers

[0315] AAV

[0316] As previously stated, the terms “adeno-associated virus” and / or “AAV” refer to parvoviruses and their variants having a linear single-stranded DNA genome. Unless otherwise required, the term covers all subtypes as well as naturally occurring and recombinant forms. Parvoviruses, including AAV, can be used as gene therapy vectors because they can penetrate cells and introduce nucleic acids (e.g., transgenes) into the cell nucleus. In some embodiments, the introduced nucleic acid (e.g., the rAAV vector genome) forms a circular polymorph that persists as an episome in the nucleus of the transduced cell. In some embodiments, the transgene is inserted into a specific site in the host cell genome, such as a site on human chromosome 19. In contrast to random integration, site-specific integration is believed to produce a predictable long-term expression profile. The insertion site of AAV in the human genome is called AAVS1. Once introduced into the cell, polypeptides encoded by the nucleic acid can be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, nucleic acids delivered via AAV can be used to express therapeutic polypeptides for treating diseases, symptoms, and / or conditions in human subjects.

[0317] Multiple AAV serotypes exist in nature, and to date, at least fifteen wild-type serotypes from humans (i.e., AAV1-AAV15) have been identified. The difference between naturally occurring serotypes and variant serotypes lies in the presence of capsid proteins that are serologically different from other AAV serotypes. AAV types 1 (AAV1), 2 (AAV2), and 3 (AAV3), including AAV 3A (AAV3A) and AAV 3B (AAV3B), AAV types 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 12 (AAV12), AAVrh10, AAVrh74 (see WO 2016 / 210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV, as well as recombinant variants (e.g., capsid variants with insertions, deletions, and substitutions), such as those referred to as AAV. Variants of type 2i8 (AAV2i8), NP4, NP22, NP66, DJ, DJ / 8, DJ / 9, LK3, RHM4-1, and many others. "Primate AAV" refers to AAVs infecting primates, "Non-primate AAV" refers to AAVs infecting non-primate mammals, "Bovine AAV" refers to AAVs infecting bovine mammals, and so on. Serotype uniqueness is determined based on the lack of cross-reactivity between antibodies against one AAV and antibodies against another. This difference in cross-reactivity is typically due to differences in capsid protein sequences and antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). However, some naturally occurring AAVs or artificial AAV mutants (e.g., recombinant AAVs) may not exhibit serological differences from any currently known serotypes. Such viruses can be considered a subgroup of the corresponding type, or simply variant AAVs. Therefore, as used herein, the term "serotype" refers to serologically distinct viruses, such as AAV, as well as viruses that are not serologically distinct but can be found in a subgroup or variant of a given serotype, such as AAV.

[0318] A comprehensive list and alignment of the amino acid sequences of the capsids of known AAV serotypes is provided by Marsic et al. (2014) Molecular Therapy 22(11):1900-1909, especially in supplementation Figure 1 Provided by China.

[0319] Genomic sequences of various serotypes of AAV, as well as sequences of natural terminal repeats (ITRs), rep proteins, and capsid subunits, are known in the art. Such sequences are available in literature or public databases such as GenBank. See, for example, GenBank accessions NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001863 (AAV3B), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), NC_001862 (AAV6), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8), the publications of which are incorporated herein by reference. See also, for example, Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; International Patent Publication WO 00 / 28061, WO WO 99 / 61601, WO 98 / 11244; WO 2013 / 063379; WO 2014 / 194132; WO 2015 / 121501; and U.S. Patent Nos. 6,156,303 and 7,906,111. For illustrative purposes only, wild-type AAV2 comprises a small (20-25 nm) AAV icosahedral viral capsid composed of three proteins (VP1, VP2, and VP3; a total of 60 capsid proteins constituting the AAV capsid) with overlapping sequences. Proteins VP1 (735 aa; Genbank accession number AAC03780), VP2 (598 aa; Genbank accession number AAC03778), and VP3 (533 aa; Genbank accession number AAC03779) are present in the capsid in a ratio of 1:1:10. In other words, for AAV, VP1 is a full-length protein and VP2 and VP3 are stepwise shortened forms of VP1, with the N-terminus being truncated more than that of VP1.

[0320] Recombinant AAV

[0321] As previously mentioned, “recombinant adeno-associated virus” or “rAAV” is distinguished from wild-type AAV by replacing all or part of the endogenous viral genome with a non-natural sequence. Introducing a non-natural sequence into a virus defines a viral vector as a “recombinant” vector, and therefore as an “rAAV vector.” An rAAV vector may comprise a heteropolynucleotide encoding a desired protein or polypeptide (e.g., an ASPA polypeptide). The recombinant vector sequence may be encapsulated or packaged into an AAV capsid and referred to as an “rAAV vector,” “rAAV vector particle,” “rAAV viral particle,” or simply “rAAV.”

[0322] For the production of the rAAV carrier, the desired ratio of VP1:VP2:VP3 is in the range of about 1:1:1 to about 1:1:100, preferably in the range of about 1:1:2 to about 1:1:50, and more preferably in the range of about 1:1:5 to about 1:1:20. Although the desired ratio of VP1:VP2 is 1:1, the ratio of VP1:VP2 can vary from 1:50 to 50:1.

[0323] This disclosure provides rAAV vectors comprising polynucleotide sequences not derived from AAV (e.g., polynucleotides heterologous to AAV). The heteronucleotide may be side-joined with at least one, and sometimes two, AAV terminal repeat sequences (e.g., inverted terminal repeats (ITRs)). The heteronucleotide side-joined via the ITR, also referred to herein as a “vector genome,” typically encodes a polypeptide of interest or a gene of interest (“GOI”), such as a target for therapeutic treatment (e.g., a nucleic acid encoding ASPA for the treatment of canavanine disease). Delivery or administration of the rAAV vector to a subject (e.g., a patient) provides the subject with the encoded protein and peptide. Therefore, rAAV vectors can be used to transfer / deliver heteronucleotides for expression, for example, to treat various diseases, symptoms, and conditions.

[0324] The rAAV vector genome typically maintains a 145-base ITR in cis configuration with the heterologous nucleic acid sequences replacing the viral rep and cap genes. Such an ITR is essential for the generation of recombinant AAV vectors; however, modified AAV ITRs and non-AAV terminal repeat sequences, including partially or fully synthesized sequences, can also be used for this purpose. The ITR forms a hairpin structure and functions, for example, as a primer for host cell-mediated synthesis of complementary DNA strands after infection. The ITR also plays a role in viral packaging, integration, etc. The ITR is the only cis-configured AAV viral element required for AAV genome replication and packaging into the rAAV vector. The rAAV vector genome optionally contains two ITRs, typically located at the 5' and 3' ends of the vector genome containing heterologous sequences (e.g., transgenes encoding genes of interest or nucleic acid sequences of interest, including but not limited to antisense, siRNA, CRISPR molecules, and many other molecules). The 5' and 3' ITRs may both contain the same sequence or may each contain different sequences. AAV ITRs can come from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or any other AAV.

[0325] The rAAV vectors disclosed herein may contain an ITR from an AAV serotype (e.g., wild-type AAV2, fragments thereof, or variants) that is different from the capsid serotype (e.g., AAV8, Oligo001). Such rAAV vectors containing at least one ITR from a single serotype, but containing a capsid from a different serotype, may be referred to as hybrid viral vectors (see U.S. Patent No. 7,172,893). The AAV ITR may comprise the entire wild-type ITR sequence, or its variants, fragments, or modifications, while retaining functionality.

[0326] In some embodiments, the heterologous polypeptide comprises an ITR located at the left and right ends (i.e., the 5' and 3' ends, respectively) of the vector genome (e.g., an ITR from AAV2, but may include an ITR from any wild-type AAV serotype or its variant). In some embodiments, the left (e.g., 5') ITR comprises or constitutes a nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19. In some embodiments, the left (e.g., 5') ITR comprises about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19. In some embodiments, the right (e.g., 3') ITR comprises or constitutes a nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19. In some embodiments, the right (e.g., 3') ITR contains about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical nucleic acid sequences to those of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19. Each ITR is cis-separated from or with other elements in the vector genome, but can be separated by variable-length nucleic acid sequences such as recombinant nucleic acids containing modified nucleic acids encoding ASPA and regulatory elements. In some embodiments, the ITR is an AAV2 ITR or a variant thereof, and is side-gated with an ASPA transgene. In some embodiments, rAAV contains an ASPA transgene (e.g., containing the nucleic acid sequence of SEQ ID NO:2) side-gated with an AAV2 ITR (e.g., an ITR having the sequence shown in SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19).

[0327] In some implementations, the rAAV vector genome is linear, single-stranded, and side-linked with an AAV ITR. Before transcription and translation of the heterologous gene, the approximately 4700 nucleotide single-stranded DNA genome must be converted into a double-stranded form by initiating second-strand synthesis using a free 3'-OH from one of the self-initiating ITRs via a DNA polymerase (e.g., a transducing intracellular DNA polymerase). In some implementations, the full-length single-stranded vector genome (i.e., sense and antisense) is annealed to produce a full-length double-stranded vector genome. This can occur when multiple rAAV vectors with genomes of opposite polarity (i.e., sense or antisense) are simultaneously transduced into the same cell. Regardless of how it is produced, once the double-stranded vector genome is formed, the cell can transcribe and translate the double-stranded DNA and express the heterologous gene.

[0328] The efficiency of transgene expression from rAAV vectors can be hampered by the need to convert single-stranded rAAV genomes (ssAAV) into double-stranded DNA prior to expression. This step is circumvented by using self-complementary AAV genomes (scAAV) that can package inverted repeat genomes, which can fold into double-stranded DNA without the need for DNA synthesis or base pairing between multiple vector genomes (McCarty, (2008) Molec. Therapy 16(10):1648-1656; McCarty et al., (2001) Gene Therapy 8:1248-1254; McCarty et al., (2003) Gene Therapy 10:2112-2118). The limitation of scAAV vectors is that the size of the unique transgenes, regulatory elements, and IRTs to be packaged in the capsid is about half the size of the ssAAV vector genome (i.e., about 4,900 nucleotides, including two ITRs) (i.e., about 2,500 nucleotides, of which 2,200 nucleotides can be transgenes and regulatory elements, plus two copies of the about 145 nucleotide ITR).

[0329] The scAAV vector genome is prepared by using a nucleic acid that does not contain a terminal resolution site (TRS), or by altering the TRS of an rAAV ITR from a vector (e.g., a plasmid) containing the vector genome, thereby preventing replication from initiating at that end (see U.S. Patent No. 8,784,799). AAV replication within the host cell initiates at the wild-type ITR of the scAAV vector genome and continues to an ITR lacking or containing an altered terminal resolution site, then returns across the genome to produce a complementary strand. Thus, the resulting complementary single nucleic acid molecule is a self-complementary nucleic acid molecule that results in the vector genome having a mutated (unresolved) ITR in the middle and wild-type ITRs at each end. In some embodiments, the mutant ITR lacking a TRS or containing an altered TRS is located at the 5' end of the vector genome. In some embodiments, the mutant ITR lacking a TRS or containing an unresolved (cleaved) altered TRS is located at the 3' end of the vector genome. In some embodiments, the mutant ITR contains the nucleic acid of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19.

[0330] To avoid being bound by theory, although the two halves of the scAAV genome are complementary, it is impossible for a large number of base pairs to exist within the capsid because many bases are in contact with the amino acid residues of the inner capsid and the phosphate backbone is isolated towards the center (McCarty, Molec. Therapy (2008) 16(10): 1648-1656). The two halves of the scAAV genome may anneal during uncoating to form dsDNA hairpin molecules, with a covalently closed ITR at one end and two open ITRs at the other end. The ITRs are flanked by double-stranded regions that encode transgenes and cis-regulatory elements thereto.

[0331] The viral capsid of the rAAV vector can be derived from wild-type AAV or variant AAV, such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74 (see WO2016 / 210170), AAV12, AAV2i8, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO:5 of WO 2015 / 013313), RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAV... hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV (avian AAV), (cattle AAV), (dog AAV), (horse AAV), (primate AAV), (non-primate AAV), (snake AAV), (goat AAV), (shrimp AAV), (sheep AAV) and their variants (see, for example, Fields et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers). The capsid may be derived from U.S. Patent No. 7,906,111; Gao et al. (2004) J.Virol. 78:6381; Morris et al. (2004) Virol. 33:375; WO Numerous AAV serotypes are disclosed in WO 2013 / 063379 and WO 2014 / 194132; and include the truetype AAV (AAV-TT) variant disclosed in WO 2015 / 121501 and RHM4-1, RHM15-1 to RHM15-6 and their variants disclosed in WO 2015 / 013313. Those skilled in the art will recognize that other AAV variants performing the same or similar functions may exist but have not yet been identified. The complete complement of the AAV cap protein comprises VP1, VP2, and VP3. An ORF containing the nucleotide sequence encoding the AAV VP cap protein may contain less than the complete complement of the AAV cap protein or may provide the complete complement of the AAV cap protein.

[0332] In another embodiment, this disclosure provides the use of ancestral AAV vectors for therapeutic in vivo gene therapy. Specifically, the computer-derived sequences can be synthesized de novo and characterized for biological activity. In addition to assembly into rAAV vectors, the prediction and synthesis of ancestral sequences can be accomplished using the methods described in WO 2015 / 054653, the contents of which are incorporated herein by reference. Notably, rAAV vectors assembled from ancestral viral sequences can exhibit reduced sensitivity to pre-existing immunity in human populations compared to modern viruses or portions thereof.

[0333] In some embodiments, an rAAV vector comprising a capsid protein encoded by a nucleotide sequence derived from more than one AAV serotype (e.g., wild-type AAV serotype, variant AAV serotype) is referred to as a "chimeric vector" or "chimeric capsid" (see U.S. Patent No. 6,491,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, the chimeric capsid protein is encoded by a nucleic acid sequence derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes. In some embodiments, the recombinant AAV vector comprises a capsid sequence derived from, for example, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10, AAV2i8, or variants thereof, thereby producing a chimeric capsid protein comprising a combination of amino acids of any of the aforementioned AAV serotypes (see, Rabinowitz et al. (2002) J. Virology 76(2):791-801). Alternatively, the chimeric capsid may comprise a mixture of VP1 from one serotype, VP2 from a different serotype, VP3 from yet another different serotype, and combinations thereof. For example, a chimeric viral capsid may comprise an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit. Chimeric capsids may, for example, comprise AAV capsids having one or more B19 cap subunits, whereby the AAV cap protein or subunit may be replaced by the B19 cap protein or subunit. For example, in one embodiment, the VP3 subunit of the AAV capsid may be replaced by the VP2 subunit of B19. In some embodiments, the chimeric capsid is the Olig001 capsid as described in WO2014052789, which is incorporated herein by reference.

[0334] In some embodiments, the chimeric vector is engineered to exhibit altered tropism or tropism for a specific tissue or cell type. The term "tropism" refers to the preferential entry of a virus into certain cell or tissue types and / or the preferential interaction with the cell surface that promotes entry into certain cell or tissue types. AAV tropism is typically determined by the specific interaction between different viral capsid proteins and their homologous cell receptors (Lykken et al. (2018) J. Neurodev. Disord. 10:16). Preferably, once the virus or viral vector enters the cell, a sequence carried by the vector genome (e.g., rAAV vector genome) (e.g., a heterologous sequence such as a transgene) is expressed.

[0335] “Phosphoproteometry” refers to the transduction pattern of one or more target cells in various tissues and / or organs. For example, a chimeric AAV capsid may have a tropism characterized by effective transduction to oligodendrocytes and only low transduction to neurons, astrocytes, and other CNS cells. See WO2014 / 052789, which is incorporated herein by reference. If, when applied directly to the CNS, oligodendrocytes are preferentially transduced rather than neurons, astrocytes, and other CNS cell types, this chimeric capsid may be considered “oligodendrocyte-specific,” exhibiting an oligodendrocyte-specific tropism, and is referred to herein as “oligotropism.” In some embodiments, at least about 80% of the cells transduced by an oligodendrocyte-specific capsid are oligodendrocytes, for example, at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the transduced cells are oligodendrocytes.

[0336] In some embodiments, the rAAV vector can be used to treat or prevent “conditions associated with oligodendrocyte dysfunction.” As used herein, the term “associated with oligodendrocyte dysfunction” refers to a disease, condition, or symptom in which oligodendrocytes are damaged, lost, or misfunction compared to otherwise identical normal oligodendrocytes. This term includes diseases, conditions, and symptoms directly affecting oligodendrocytes, as well as diseases, conditions, or symptoms in which oligodendrocytes become dysfunctional secondary to the destruction of other cells. In some embodiments, the condition associated with oligodendrocyte dysfunction is Canavan disease (CD).

[0337] In some embodiments, the chimeric AAV capsid with oligodendrocyte tropism is Oligo001 (also known as BNP61) and comprises sequences from AAV1, AAV2, AAV6, AAV8, and AAV9 (see WO 2014 / 052789). In some embodiments, the Oligo001 capsid VP1 is encoded by a nucleic acid sequence comprising or constituting the nucleic acid sequence of SEQ ID NO:13. In some embodiments, the Oligo001 capsid VP1 is encoded by a nucleic acid sequence that is 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 nucleic acid sequence of SEQ ID NO:13.

[0338] The nucleic acid sequences encode overlapping AAV capsid proteins VP1, VP2, and VP3. The amino acid sequences of the Olig001 capsid proteins are shown in SEQ ID NO:14, wherein VP1 begins at amino acid residue 1 (methionine) of SEQ ID NO:14, VP2 begins at amino acid residue 148 (threonine) and VP3 begins at amino acid residue 203 (methionine).

[0339] In some embodiments, the chimeric AAV capsid with oligodendrocyte tropism is Olig002 (also known as BNP62) or Olig003 (also known as BNP63) (see WO 2014 / 052789). In some embodiments, the Oligo002 capsid VP1 comprises or is composed of the amino acid sequence of SEQ ID NO:15. In some embodiments, the amino acid sequence of the Oligo002 capsid VP1 is 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 SEQ ID NO:15. In some embodiments, the nucleic acid comprises a sequence encoding the amino acid sequence of SEQ ID NO:15. In some embodiments, the Oligo003 capsid comprises or is composed of the amino acid sequence of SEQ ID NO:16. In some embodiments, the Olig003 capsid VP1 amino acid sequence is 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 SEQ ID NO:16. In some embodiments, the nucleic acid comprises a sequence encoding the amino acid sequence of SEQ ID NO:16.

[0340] In some embodiments, rAAV vectors comprising a chimeric AAV capsid (e.g., Olign001) and a therapeutic transgene can be used to treat diseases, conditions, or illnesses associated with oligodendrocyte dysfunction. In these diseases, conditions, or illnesses, oligodendrocytes are damaged, lost, or misfunction. This can be a result of a direct effect on oligodendrocytes or when oligodendrocytes become dysfunctional secondary to the destruction of other cells. In some embodiments, rAAV vectors comprising an AAV / Olig001 capsid and a modified ASPA nucleic acid are used to treat canavan disease.

[0341] Recombinant nucleic acid

[0342] The recombinant nucleic acids disclosed herein include modified nucleic acids and plasmids and vector genomes containing the modified nucleic acids. The recombinant nucleic acid, plasmid, or vector genome may contain regulatory sequences to regulate (e.g., plasmid) amplification and / or control the expression of the modified nucleic acid (e.g., transgenes). Recombinant nucleic acids may also be provided as components of viral vectors (e.g., rAAV vectors). Typically, viral vectors comprise a vector genome containing recombinant nucleic acids packaged in a capsid.

[0343] Modified nucleic acids

[0344] Modified genes, nucleic acids, or polynucleotides, or variant forms thereof (e.g., transgenic), refer to nucleic acids that deviate from a reference sequence. The reference sequence can be a naturally occurring wild-type sequence (e.g., a gene) and can include naturally occurring variants (e.g., splice variants, alternative reading frames). Those skilled in the art will recognize that reference sequences can be found in publicly available databases such as GenBank (ncbi.nlm.nih.gov / genbank). Modified / variant nucleic acids can have substantially the same, greater, or less activity, function, or expression compared to the reference sequence. Preferably, modified or variant nucleic acids, as used interchangeably herein, exhibit improved protein expression, for example, the protein encoded thereby is expressed at a detectably higher level in cells compared to the expression level of a protein provided by an endogenous gene (e.g., a wild-type gene, a mutant gene) in otherwise otherwise identical cells. In some implementations, modified or variant nucleic acids, such as those interchangeable herein (e.g., modified nucleic acids encoding ASPA), exhibit improved protein expression, for example, the protein thereby encoded is expressed at a detectable higher level in cells compared to the expression level of a protein provided by an endogenous gene containing a mutation in otherwise identical cells.

[0345] Modifications to nucleic acids include substitutions of one or more nucleotides in the reference sequence (e.g., substitutions of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides), additions (e.g., insertions of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides), and deletions (e.g., deletions of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides, deletions of motifs, domains, fragments, etc.). The modified nucleic acid may be approximately 50%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence.

[0346] The modified nucleic acid may encode a polypeptide that has approximately 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with a reference polypeptide. In some embodiments, the modified nucleic acid encoding ASPA (e.g., SEQ ID NO:2) encodes a polypeptide that has 100% identity with a reference polypeptide (e.g., SEQ ID NO:4).

[0347] In some embodiments, the modified nucleic acid (e.g., a transgene) encodes a wild-type protein. This modified nucleic acid may be codon-optimized. As used interchangeably herein, "optimized" or "codon-optimized" means that the coding sequence has been optimized relative to a wild-type coding sequence or a reference sequence (e.g., the coding sequence for the ASPA peptide) to increase peptide expression, for example by minimizing the use of rare codons, reducing the number of CpG dinucleotides, removing cryptic splice donor or acceptor sites, removing the Kozak sequence, removing ribosome entry sites, etc. In some embodiments, the expression level of the protein from the codon-optimized sequence (e.g., the modified nucleic acid encoding ASPA) is increased compared to the expression level of the protein from the wild-type gene in otherwise identical cells. In some embodiments, the expression level of the protein from the codon-optimized sequence (e.g., the modified nucleic acid encoding ASPA) is not increased compared to the expression level of the protein from the wild-type gene in otherwise identical cells (e.g., expression is substantially similar). In some implementations, the expression levels of proteins derived from codon-optimized sequences (e.g., modified nucleic acids encoding ASPA) are increased compared to the expression levels of proteins from mutated genes in otherwise identical cells.

[0348] Examples of modifications include the removal of one or more cis-acting motifs and the introduction of one or more Kozak sequences. In some embodiments, one or more cis-acting motifs are removed and one or more Kozak sequences are introduced.

[0349] Examples of removable cis-acting motifs include internal TATA boxes; chi sites; ribosome entry sites; ARE, INS, and / or CRS sequence elements; repetitive sequences and / or RNA secondary structures; (covert) splicing donor and / or acceptor sites, branching points; and restriction sites.

[0350] In some embodiments, the modified nucleic acid encodes a modified or variant polypeptide. The modified polypeptide encoded by the modified nucleic acid may retain all or part of the function or activity of the polypeptide encoded by the wild-type or reference sequence. In some embodiments, the modified polypeptide has one or more non-conserved or conserved amino acid variations. In some embodiments, certain domains that have been shown to play a limited or no role in the function of the polypeptide are not present in the modified polypeptide (e.g., certain binding domains) (e.g., WO 2016 / 097219). Modified nucleic acids present in rAAV vectors may contain fewer nucleotides than those encoded by the wild-type or reference sequence due to the packaging capacity of the rAAV capsid (e.g., shortened miniature dystrophin transgenes, see WO 2001 / 83695; B-domain deletion human factor VIII transgenes, see WO 2017 / 074526), ​​and also include truncated and codon-optimized shortened transgenes (e.g., the codon-optimized miniature dystrophin transgene described in WO 2017 / 221145). In some embodiments, a polypeptide encoded by a modified nucleic acid has less, the same, or greater function or activity than a polypeptide encoded by a reference sequence, but at least a portion of the function or activity.

[0351] Compared to a reference and / or wild-type sequence (e.g., a sequence encoded by wild-type ASPA), a modified nucleic acid may have altered GC content (e.g., the number of G and C nucleotides present in the nucleic acid sequence), altered (e.g., increased or decreased) CpG dinucleotide content, and / or altered (e.g., increased or decreased) codon fitness index (CAI). See, for example, WO 2017 / 077451 (discussing various considerations known in the art for codon optimization of nucleic acid sequences of interest, including publicly available software for analyzing nucleic acid sequences for optimization). As used herein, alteration refers to a reduction or increase in a specific value, amount, or effect.

[0352] In some embodiments, the GC content of the modified nucleic acid sequence of this disclosure is increased relative to a reference and / or wild-type gene or coding sequence. The GC content of the modified nucleic acid is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 15%, at least 17%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% higher than that of the wild-type coding sequence (e.g., SEQ ID NO:3). In some embodiments, the GC content is expressed as a percentage of G (guanine) and C (cytosine) nucleotides in the sequence.

[0353] In some embodiments, the codon fitness index of the modified nucleic acid sequence of this disclosure is at least 0.74, at least 0.76, at least 0.77, at least 0.80, at least 0.85, at least 0.86, at least 0.87, at least 0.90, at least 0.95, or at least 0.98.

[0354] In some embodiments, the modified nucleic acid sequence of this disclosure has a reduced level of CpG dinucleotides compared to a wild-type or reference nucleic acid sequence, by about 10%, 20%, 30%, 50%, or more. In some embodiments, the modified nucleic acid has 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-45, or 45-50 or more dinucleotides less than the reference sequence (e.g., wild-type sequence).

[0355] CpG dinucleotide methylation is known to play a crucial role in the regulation of gene expression in eukaryotes. Specifically, CpG dinucleotide methylation in eukaryotes primarily silences gene expression by interfering with transcriptional mechanisms. Therefore, nucleic acids and vectors with reduced CpG dinucleotide numbers provide high and more durable transgene expression levels due to gene silencing induced by CpG motif methylation.

[0356] Modified nucleic acid sequences may include side-linked restriction sites to facilitate subcloning into expression vectors. Many such restriction sites are well known in the art, and include, but are not limited to, those mentioned above. Figure 13 The restricted sites shown are AvaI, XmaI, and XmaI.

[0357] This disclosure includes fragments of any one of the sequences shown in SEQ ID NO:1-3 and which encode functionally active fragments of the ASPA polypeptide. "Functionally active" or "functional ASPA polypeptide" indicates that the fragment provides the same or similar biological function and / or activity as the full-length ASPA polypeptide. That is, the fragment provides the same activity, including but not limited to the ability to convert NAA to acetate and aspartate. The biological activity of ASPA or its functional fragments also encompasses the reversal or prevention of neurodegenerative phenotypes associated with canavanine disease, as demonstrated elsewhere herein and in NUR7 mice.

[0358] This disclosure provides a modified ASPA nucleic acid sequence that encodes an ASPA polypeptide and contains at least one modification compared to a wild-type nucleic acid sequence (e.g., SEQ ID NO:3; GenBank accession number NM_000049.4 or NM_001128085.1, which has an alternative 5'UTR but encodes the same ASPA protein (SEQ ID NO:4)).

[0359] In some embodiments, the modified nucleic acid encoding ASPA is a codon-optimized nucleic acid encoding a wild-type ASPA polypeptide (e.g., SEQ ID NO:4) and comprises the sequence SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the modified nucleic acid encoding ASPA is a codon-optimized nucleic acid and consists of the sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the modified nucleic acid encoding ASPA is a codon-optimized nucleic acid and comprises at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0360] In some embodiments, cells containing a modified nucleic acid encoding ASPA exhibit increased protein expression, for example, the protein thereby encoded is expressed at a detectably higher level in the cell compared to the protein expression levels in otherwise otherwise identical cells containing wild-type ASPA nucleic acid or in otherwise identical cells containing mutant nucleic acids encoding ASPA. In some embodiments, the ASPA protein expression level in cells containing a modified nucleic acid encoding ASPA (e.g., a nucleic acid sequence containing SEQ ID NO:2) is increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 150%, about 200%, about 300%, about 400%, or more, compared to the ASPA protein expression level in otherwise otherwise identical cells containing wild-type ASPA nucleic acid. In some embodiments, the expression level of ASPA protein in cells containing a modified nucleic acid encoding ASPA (e.g., a nucleic acid sequence containing SEQ ID NO:2) is increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 150%, about 200%, about 300%, about 400%, or more, compared to the expression level of ASPA protein in otherwise identical cells containing a mutant nucleic acid encoding ASPA.

[0361] In some implementations, this may be referred to as “expression-optimized” or “enhanced expression” nucleic acid, or simply “modified nucleic acid”.

[0362] Those skilled in the art will understand that polypeptides encoded by the modified nucleic acids and their variants (e.g., SEQ ID NO:1, SEQ ID NO:2) of this disclosure are “functional ASPA polypeptides” that provide the same or similar biological functions and / or activities as ASPA polypeptides encoded by wild-type nucleic acids encoding ASPA (e.g., SEQ ID NO:3). That is, ASPA polypeptides encoded by modified nucleic acids encoding ASPA provide the same activities, including but not limited to the ability to convert NAA to acetate and aspartate. The biological activities of ASPA encompass the reversal or prevention of neurodegenerative phenotypes associated with canavanine disease, as demonstrated elsewhere herein and in NUR7 mice, including but not limited to improved rotarod latency tofall performance, improved open field passing distance, reduced NAA in brain tissue, reduced vacuolar volume in the brain (e.g., thalamus, cerebellar white matter / pons), increased Olig2-positive cells in the brain (e.g., thalamus, cortex), and / or increased cortical myelination.

[0363] Control element

[0364] This disclosure includes recombinant nucleic acids comprising modified nucleic acids encoding ASPA and various regulatory or control elements. Typically, regulatory elements are nucleic acid sequences that affect the expression of operatively linked polynucleotides. The precise nature of regulatory elements available for gene expression varies between organisms and cell types, including, for example, promoters, enhancers, introns, etc., aimed at promoting proper heteropolynucleotide transcription and translation. Regulatory control can be affected at the levels of transcription, translation, splicing, signal stability, etc. Typically, regulatory control elements regulating transcription are located near the 5' end (i.e., upstream) of the transcribed polynucleotide. Regulatory control elements can also be located at the 3' end (i.e., downstream) of the transcribed sequence or within the transcript (e.g., in an intron). Regulatory control elements can be located remotely from the transcribed sequence (e.g., 1 to 100, 100 to 500, 500 to 1000, 1000 to 5000, 5000 to 10,000 or more nucleotides). However, due to the length of the AAV vector genome, regulatory control elements are typically in the range of 1 to 1000 nucleotides from the polynucleotide.

[0365] promoter

[0366] As used herein, the term "promoter," such as "eukaryotic promoter," refers to a nucleotide sequence that initiates transcription of a specific gene or one or more coding sequences (e.g., the ASPA coding sequence) in eukaryotic cells (e.g., oligodendrocytes). Promoters may work in conjunction with other regulatory elements or regions to direct the transcriptional level of a gene or coding sequence. Such regulatory elements include, for example, transcription binding sites, repressor and activator protein binding sites, and other nucleotide sequences known to function directly or indirectly to regulate the amount of transcription from the promoter, including, for example, attenuators, enhancers, and silencers. Promoters are typically located on the same strand and close to the transcription start site, i.e., the 5' of the gene or coding sequence to which they are operatively linked. Promoter length is typically 100–1000 nucleotides. Promoters generally increase gene expression relative to the expression of the same gene in the absence of a promoter.

[0367] As used herein, a “core promoter” or “minimum promoter” refers to the smallest portion of the promoter sequence required to properly initiate transcription. It may include any of the following: a transcription start site, an RNA polymerase binding site, and a general transcription factor binding site. A promoter may also contain a proximal promoter sequence (5' of the core promoter) and a distal promoter sequence (3' of the core promoter) containing other major regulatory elements (e.g., enhancers, silencers, boundary elements, insulators).

[0368] Examples of suitable promoters include: adenovirus promoters, such as the adenovirus major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; respiratory syncytial virus promoters; Rous sarcoma virus (RSV) promoters; albumin promoters; inducible promoters, such as the mouse mammary tumor virus (MMTV) promoter; metallothionein promoters; heat shock promoters; α-1-antitrypsin promoters; hepatitis B surface antigen promoters; transferrin promoters; apolipoprotein A-1 promoters; chicken β-actin (CBA) promoters, elongation factor 1a promoters (EF1a), hybrid forms of CBA promoters (CBh promoters) and CAG promoters (cytomegalovirus early enhancer and promoter, the first exon and first intron of the chicken β-actin gene and the splice acceptor of the rabbit β-globin gene) (Alexopoulou et al. (2008) BioMed. Central Cell Biol. 9:2); and the human ASPA gene promoter. In some implementations, the promoter is a fragment or variant of the CBh promoter and contains or constitutes a nucleic acid sequence of SEQ ID NO:7.

[0369] In some embodiments of this disclosure, a eukaryotic promoter sequence (e.g., the CBh promoter) is operatively linked to a modified nucleic acid encoding ASPA. In some embodiments, a promoter comprising the nucleic acid sequence of SEQ ID NO:7 (e.g., the CBh promoter) is operatively linked to a modified nucleic acid encoding ASPA. In some embodiments, a promoter comprising or constituting a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:7 is operatively linked to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:2. In some embodiments, a promoter comprising at least 95% identical to the nucleic acid sequence of SEQ ID NO:7 is operatively linked to a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:2 and induces expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 in oligodendrocytes. In some embodiments, the expression level of a polypeptide encoded by a nucleic acid containing a nucleic acid sequence containing a nucleic acid containing a promoter containing a nucleic acid containing SEQ ID NO:2 is detectably higher in cells than the expression level of a polypeptide encoded by a nucleic acid containing a promoter containing a nucleic acid containing SEQ ID NO:7, which is inoperably linked to the nucleic acid sequence containing SEQ ID NO:7. In some embodiments, a recombinant nucleic acid containing a promoter containing a nucleic acid sequence at least 95% identical to the nucleic acid sequence containing SEQ ID NO:7 is operatively linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence containing SEQ ID NO:2 and induces the expression of a polypeptide encoded by the nucleic acid sequence containing SEQ ID NO:2 in oligodendrocytes.

[0370] Promoters can be constitutive, tissue-specific, or regulated. A constitutive promoter is one that results in the expression of a gene that is operatively linked at virtually all times. In some implementations, constitutive promoters are active in most eukaryotic tissues under most physiological and developmental conditions.

[0371] Regulated promoters are promoters that can be activated or inactivated. Regulated promoters include inducible promoters, which are normally "off" but can be induced to "on," and repressive promoters, which are normally "on" but can be "off." Many different regulatory factors are known, including temperature, hormones, cytokines, heavy metals, and regulatory proteins. The distinction is not absolute; constitutive promoters can often be regulated to some extent. In some cases, endogenous pathways can be used to provide regulation of transgene expression, for example, by using promoters that are naturally downregulated when the pathological condition improves.

[0372] Tissue-specific promoters are promoters that are active only in a specific type of tissue, cell, or organ. Typically, tissue-specific promoters are recognized by transcriptional activation elements that are specific to a particular tissue, cell, and / or organ. For example, tissue-specific promoters may be more active in one or more specific tissues (e.g., two, three, or four) compared to other tissues. In some embodiments, the expression of genes regulated by tissue-specific promoters is much higher in the tissue to which the promoter is specific compared to other tissues. In some embodiments, the promoter may have little or no activity in any tissue other than the tissue to which the promoter is specific. Promoters can be tissue-specific promoters, such as the mouse albumin promoter or the transthyretin promoter (TTR), which are active in liver cells. Other examples of tissue-specific promoters include promoters from genes encoding skeletal α-actin, myosin light chain 2A, dystrophin, and muscle creatine kinase, which induce expression in skeletal muscle (Li et al. (1999) Nat. Biotech. 17:241-245). Liver-specific expression can be induced using promoters derived from the albumin gene (Miyatake et al. (1997) J.Virol. 71: 5124-5132), the hepatitis B virus core promoter (Sandig et al. (1996) Gene Ther. 3: 1002-1009), and α-fetoprotein (Arbuthnot et al. (1996) Hum. Gene. Ther. 7: 1503-1514).

[0373] Enhancer

[0374] On the other hand, the modified nucleic acid encoding a therapeutic peptide further includes an enhancer that increases the expression of the therapeutic peptide (e.g., ASPA protein). Typically, the enhancer element is located upstream of the promoter element, but it can also be located downstream or within another sequence (e.g., a transgene). The enhancer may be located 100, 200, 300, or more nucleotides upstream or downstream of the modified nucleic acid. The enhancer typically increases the expression of the modified nucleic acid (e.g., encoding a therapeutic peptide, such as ASPA) beyond the increase provided by the promoter element alone.

[0375] Many enhancers are known in the art, including but not limited to the cytomegalovirus (CMV) major immediate early enhancer. More specifically, the CMV MIE promoter comprises three regions: a regulator, a unique region, and an enhancer (Isomura and Stinski (2003) J. Virol. 77(6):3602-3614). The CMV enhancer region can be combined with another promoter or a portion thereof to form a heterozygous promoter, thereby further increasing the expression of the nucleic acid operatively linked to it. For example, the chicken β-actin (CBA) promoter or a portion thereof can be combined with the CMV promoter / enhancer or a portion thereof to prepare a CBA form of the chicken β-actin heterozygous promoter called the “CBh” promoter, as described by Gray et al. (2011, Human Gene Therapy 22:1143-1153). Like promoters, enhancers can be constitutive, tissue-specific, or regulated.

[0376] In some embodiments of this disclosure, an enhancer sequence (e.g., a CMV enhancer) is operatively linked to a modified nucleic acid encoding ASPA. In some embodiments, an enhancer (e.g., a CMV enhancer) comprising or constituting a nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17 is operatively linked to a modified nucleic acid encoding ASPA. In some embodiments, an enhancer comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17 is operatively linked to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:2, and optionally operatively linked to a promoter comprising the nucleic acid sequence of SEQ ID NO:7. In some embodiments, an enhancer comprising a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17 is operatively linked to a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:2 and induces expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 in oligodendrocytes. In some embodiments, an enhancer comprising a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17 is operatively linked to a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:7, and is operatively linked to a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:2, and the nucleic acid sequences of SEQ ID NO:6 (or SEQ ID NO:17) and SEQ ID NO:7 together induce expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 in oligodendrocytes. In some implementations, the expression level of a polypeptide encoded by a nucleic acid sequence SEQ ID NO:2, which is operatively linked to an enhancer containing a nucleic acid sequence of SEQ ID NO:6 (or SEQ ID NO:17), is detectably higher in cells than that of a polypeptide encoded by an enhancer operatively linked to a nucleic acid sequence of SEQ ID NO:2, which is otherwise identical.In some embodiments, the recombinant nucleic acid comprises an enhancer containing at least 95% identical nucleic acid sequences to the nucleic acid sequences of SEQ ID NO:6 or SEQ ID NO:17, operably linked to at least 95% identical nucleic acid sequences to the nucleic acid sequences of SEQ ID NO:7, and operably linked to at least 95% identical nucleic acid sequences to the nucleic acid sequences of SEQ ID NO:2, and the nucleic acid sequences of SEQ ID NO:6 (or SEQ ID NO:17) and SEQ ID NO:7 together induce expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 in oligodendrocytes.

[0377] Filler, spacer, and stuffer

[0378] As disclosed herein, recombinant nucleic acids intended for use in rAAV vectors may include additional nucleic acid elements to adjust the length of the nucleic acid to near or to the normal size (e.g., approximately 4.7 to 4.9 kilobases) of an acceptable viral genome sequence for packaging AAV into an rAAV vector (Grieger and Samulski (2005) J. Virol. 79(15): 9933-9944). This sequence may be interchangeably referred to as a filler region, spacer region, or filling region. In some embodiments, the filler DNA is an untranslated (non-protein-coding) segment of the nucleic acid. In some implementations, the filling or filling region polynucleotide sequence is a sequence with a length between about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90-90-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1000, 1000-1500, 1500-2000, 2000-3000 or longer.

[0379] AAV vectors typically accept DNA inserts ranging in size from about 4 kb to about 5.2 kb or from about 4.1 to 4.9 kb to optimally package nucleic acids into the AAV capsid. In some embodiments, the rAAV vector comprises a vector genome of total length between about 3.0 kb to about 3.5 kb, about 3.5 kb to about 4.0 kb, about 4.0 kb to about 4.5 kb, about 4.5 kb to about 5.0 kb, or about 5.0 kb to about 5.2 kb. In some embodiments, the rAAV vector comprises a vector genome of total length of about 4.7 kb. In some embodiments, the rAAV vector comprises a self-complementary vector genome. Although the total length of the self-complementary (sc) vector genome in the rAAV vector is equal to that of the single-stranded (ss) vector genome (i.e., approximately 4 kb to approximately 5.2 kb), the nucleic acid sequence encoding the sc vector genome (i.e., containing transgenes, regulatory elements, and ITRs) must be only half the length of the nucleic acid sequence encoding the ss vector genome in order to encapsulate the sc vector genome.

[0380] Introns and exons

[0381] In some implementations, the recombinant nucleic acid includes, for example, introns, exons, and / or portions thereof. Introns may function as filler or padding polynucleotide sequences to achieve the appropriate length for packaging the vector genome into the rAAV vector. Intron and / or exon sequences may also enhance the expression of peptides (e.g., transgenes) compared to expression in the absence of intron and / or exon elements (Kurachi et al. (1995) J. Biol. Chem. 270(10):576-5281; WO 2017 / 074526). Furthermore, filler / padding polynucleotide sequences (also referred to as “insulators”) are well known in the art and include, but are not limited to, the polynucleotide sequences described in WO 2014 / 144486 and WO 2017 / 074526.

[0382] Intronic elements may be derived from a gene identical to a heterologous polynucleotide, or from a completely different gene or other DNA sequence (e.g., the chicken β-actin gene, mouse parvovirus (MVM)). In some embodiments, the recombinant nucleic acid includes at least one element selected from introns and exons derived from a non-homologous gene (i.e., not derived from a modified nucleic acid, e.g., a transgene). In some embodiments, the intron is derived from the chicken β-actin gene, for example, comprising or constituting the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the intron comprises about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the intron is derived from an MVM, for example, comprising or constituting the nucleic acid sequence of SEQ ID NO:10. In some embodiments, the intron comprises about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:10. In some embodiments, the exon is derived from the chicken β-actin gene, for example, comprising or constituting the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the exon comprises about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the recombinant nucleic acid comprises at least one of an enhancer sequence (e.g., SEQ ID NO:6 or SEQ ID NO:17), a promoter sequence (e.g., SEQ ID NO:7), an exon (e.g., SEQ ID NO:8 or SEQ ID NO:18), and an intron (e.g., SEQ ID NO:9, SEQ ID NO:10) and regulates the expression of a heterologous polypeptide optionally encoded by the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the expression level of a polypeptide encoded by a nucleic acid sequence of SEQ ID NO:2 that is operatively linked to such a regulatory element is detectably higher in cells than the expression level of a polypeptide encoded by a nucleic acid sequence of SEQ ID NO:2 that is operatively linked to such a regulatory element, which includes at least one of the following: enhancer sequences (e.g., SEQ ID NO:6 or SEQ ID NO:17), promoter sequences (e.g., SEQ ID NO:7), exons (e.g., SEQ ID NO:8 or SEQ ID NO:18), and introns (e.g., SEQ ID NO:9, SEQ ID NO:10).

[0383] In some embodiments, the recombinant nucleic acid comprises a modified nucleic acid of SEQ ID NO:2, which is operatively linked to a regulatory element comprising at least one of the following: an enhancer sequence (e.g., SEQ ID NO:6 or SEQ ID NO:17), a promoter sequence (e.g., SEQ ID NO:7), an exon (e.g., SEQ ID NO:8 or SEQ ID NO:18), and an intron (e.g., SEQ ID NO:9, SEQ ID NO:10).

[0384] Polyadenylation signal sequence (polyA)

[0385] Other regulatory elements may include stop codons, termination sequences, and polyadenylation (polyA) signaling sequences, such as, but not limited to, the bovine growth hormone polyA signaling sequence (BHG polyA). The polyA signaling sequence drives the efficient addition of a polyadenosine "tail" at the 3' end of eukaryotic mRNA to guide gene transcription termination (see, e.g., Goodwin and Rottman, J. Biol. Chem. (1992) 267(23):16330-16334). The polyA signal acts as a signal for the cleavage of the endonuclease at the 3' end of newly formed precursor mRNA and the addition of an RNA fragment consisting only of adenine bases to this 3' end. The polyA tail is important for nuclear export, translation, and stability of mRNA. In some implementations, polyA is an early polyadenylation signal of SV40, a late polyadenylation signal of SV40, a polyadenylation signal of HSV thymidine kinase, a polyadenylation signal of protamine gene, a polyadenylation signal of adenovirus 5E1b, a polyadenylation signal of growth hormone, a polyadenylation signal of PBGD, or a computer-designed polyadenylation signal.

[0386] In some embodiments, and optionally in combination with one or more other regulatory elements described herein, the polyA signal sequence of the recombinant nucleic acid is a polyA signal capable of directing and influencing endonuclease cleavage and polyadenylation of the precursor mRNA produced by transcription of a modified nucleic acid encoding ASPA (e.g., SEQ ID NO:2). In some embodiments, the polyA sequence comprises or constitutes the nucleic acid sequence of SEQ ID NO:11. In some embodiments, the polyA sequence comprises about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:11. In some embodiments, the recombinant nucleic acid comprises at least one of an enhancer sequence (e.g., SEQ ID NO:6 or SEQ ID NO:17), a promoter sequence (e.g., SEQ ID NO:7), an exon (e.g., SEQ ID NO:8 or SEQ ID NO:18), an intron (e.g., SEQ ID NO:9, SEQ ID NO:10), and polyA (SEQ ID NO:11) and regulates the expression of a heterologous polypeptide optionally encoded by the nucleic acid sequence of SEQ ID NO:2.

[0387] In some implementations, the oligodendrocyte-specific rAAV vector (e.g., AAV / Olig001-ASPA) contains a self-complementary vector genome containing an AAV ITR (e.g., AAV2 ITR) and a recombinant nucleic acid containing a modified (i.e., codon-optimized) nucleic acid encoding ASPA and at least one of the following regulatory elements: enhancer (e.g., CMV enhancer), promoter (e.g., CBh promoter), exon (e.g., CBA exon 1), intron (e.g., CBA intron, MVM intron), and polyA (e.g., BHG polyA).

[0388] In some embodiments, the oligodendrocyte-specific rAAV vector (e.g., AAV / Olig001-ASPA) contains a self-complementary genome comprising an AAV ITR (e.g., SEQ ID NO:5, SEQ ID NO:12 and / or SEQ ID NO:19) and a recombinant nucleic acid comprising a modified (i.e., codon-optimized) nucleic acid encoding ASPA (e.g., SEQ ID NO:2) and at least one of the following regulatory elements: an enhancer (e.g., SEQ ID NO:6 or SEQ ID NO:17), a promoter (e.g., SEQ ID NO:7), an exon (e.g., CBA exon SEQ ID NO:8 or SEQ ID NO:18), an intron (e.g., SEQ ID NO:9 and SEQ ID NO:10), and a polyA (e.g., SEQ ID NO:11).

[0389] In some implementations, the oligodendrocyte-specific rAAV vector (e.g., AAV / Olig001-ASPA) contains a self-complementary genome including SEQ ID NO:20.

[0390] Bioactivity of the rAAV vector disclosed herein

[0391] In some embodiments, the rAAV vector of this disclosure (e.g., containing an ASPA transgene) transduces target cells (e.g., oligodendrocytes) and mediates biological activity. In some embodiments, the rAAV vector (e.g., AAV / Olig001-ASPA) transduces target cells (e.g., oligodendrocytes) and mediates at least one detectable activity selected from:

[0392] (i) Reduce NAA levels in cells in vitro;

[0393] (ii) Improve, increase and / or enhance balance, grip strength and / or motor coordination;

[0394] (iii) Improve, increase and / or enhance drop wait time (seconds);

[0395] (iv) Improve, increase and / or enhance generalized motor function;

[0396] (v) Reduce, inhibit and / or neutralize the accumulation of NAA levels in the body;

[0397] (vi) Reduce, inhibit and / or neutralize the vacuolar volume fraction in the thalamus;

[0398] (vii) Reduce, inhibit and / or neutralize the volume fraction of vacuoles in the cerebellar white matter / pons;

[0399] (viii) Improve, increase and / or enhance the number of oligodendrocytes in the thalamus;

[0400] (ix) Improve, increase and / or enhance the number of oligodendrocytes in the dermis;

[0401] (x) Improve, increase and / or enhance the number of neurons in the thalamus;

[0402] (xi) Improve, increase, and / or enhance the number of neurons in the cortex; and

[0403] (xii) Improve, increase and / or cortical myelination.

[0404] In some embodiments, the rAAV vector that transduces target cells (e.g., oligodendrocytes) and mediates at least one detectable activity of (i) to (xii) is AAV / Oligo001-ASPA.

[0405] In some embodiments, the NAA levels in cells transduced with an rAAV vector (e.g., AAV / Olig001-ASPA) are lower than those in otherwise otherwise identical cells transduced with rAAV containing a wild-type nucleic acid sequence encoding ASPA (e.g., SEQ ID NO:3). In some embodiments, the NAA levels in cells transduced with an rAAV vector (e.g., AAV / Olig001-ASPA) are lower than those in otherwise otherwise identical cells transduced with rAAV containing a nucleic acid optimized with an alternative codon encoding ASPA (e.g., SEQ ID NO:1). In some embodiments, the NAA levels in cells transduced with an rAAV vector (e.g., AAV / Olig001-ASPA) are lower than those in otherwise otherwise identical cells containing a mutant nucleic acid encoding ASPA that is not transduced.

[0406] In some embodiments, the NAA levels in cells transduced in vivo with an rAAV vector (e.g., AAV / Olig001-ASPA) are lower than those in otherwise otherwise identical cells transduced in vivo with rAAV containing a wild-type nucleic acid sequence encoding ASPA (e.g., SEQ ID NO: 3). In some embodiments, the NAA levels in cells transduced in vivo with an rAAV vector (e.g., AAV / Olig001-ASPA) are lower than those in otherwise otherwise identical cells transduced in vivo with rAAV containing a nucleic acid optimized with an alternative codon encoding ASPA (e.g., SEQ ID NO: 1). In some embodiments, the NAA levels in cells transduced in vivo with an rAAV vector (e.g., AAV / Olig001-ASPA) are lower than those in otherwise otherwise identical cells containing a mutant nucleic acid that are not transduced.

[0407] In some implementations, such as by means of, for example, rotarod performance, balance, grip strength, and / or motor coordination are significantly improved in subjects with ASPA gene mutations who have not received rAAV vectors, compared to other subjects who are similar in other respects but have not received rAAV vectors, or compared to the same subjects before receiving rAAV vectors.

[0408] In some embodiments, as measured by, for example, rotarod performance, the balance, grip strength, and / or motor coordination of subjects with an ASPA gene mutation who have been given an rAAV vector (e.g., AAV / Olig001-ASPA) are not different from those of otherwise similar subjects without an ASPA gene mutation who have not been given an rAAV vector. In some embodiments, the rAAV vector (e.g., AAV / Olig001-ASPA) is administered via an intraventricular (ICV) administration route. In some embodiments, rotarod performance is measured as drop wait time in seconds.

[0409] In some implementations, generalized motor function in subjects with the ASPA gene mutation who have received an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly improved, as measured by, for example, open field activity, compared to generalized motor function in otherwise similar subjects without rAAV vector administration, or compared to function in subjects before rAAV vector administration. In some implementations, the rAAV vector (e.g., AAV / Olig001-ASPA) is administered via an intraventricular (ICV) administration route.

[0410] In some implementations, as measured by, for example, open field activity, generalized motor function in subjects with an ASPA gene mutation who have been administered an rAAV vector (e.g., AAV / Olig001-ASPA) is indistinguishable from generalized motor function in otherwise similar subjects without an ASPA gene mutation who have not been administered rAAV. In some implementations, the rAAV vector (e.g., AAV / Olig001-ASPA) is administered via an intraventricular (ICV) administration route.

[0411] In some embodiments, the NAA levels in the brains of subjects with ASPA gene mutations who have received an rAAV vector (e.g., AAV / Olig001-ASPA) are significantly lower than those in the brains of otherwise similar subjects with the ASPA gene mutation who have not received an rAAV vector, or compared to the NAA levels in subjects before rAAV vector administration. In some embodiments, the NAA levels in the brains of subjects with ASPA gene mutations who have received an rAAV vector (e.g., AAV / Olig001-ASPA) are lower or not different compared to those in the brains of otherwise similar subjects without the ASPA gene mutation who have not received an rAAV vector.

[0412] In some embodiments, the vacuolar fraction in the thalamus of subjects with the ASPA gene mutation who have received an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly reduced compared to the vacuolar fraction in the thalamus of otherwise similar subjects with the ASPA gene mutation who have not received an rAAV vector, or compared to subjects before rAAV vector administration, wherein the vacuolar fraction is measured, for example, by unbiased stereometry. In some embodiments, the vacuolar fraction in the cerebellar white matter / pons of subjects with the ASPA gene mutation who have received an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly reduced compared to the vacuolar fraction in the cerebellar white matter / pons of otherwise similar subjects with the ASPA gene mutation who have not received an rAAV vector, or compared to subjects before rAAV vector administration, wherein the vacuolar fraction is measured, for example, by unbiased stereometry.

[0413] In some embodiments, the number of oligodendrocytes in the thalamus of subjects with an ASPA gene mutation who have received an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly increased compared to the number of oligodendrocytes in the thalamus of otherwise similar subjects with an ASPA gene mutation who have not received the vector, or compared to subjects before vector administration. The number of oligodendrocytes in the thalamus is measured, for example, by IHC and unbiased stereometry using an Oligo2 antibody. In some embodiments, the number of oligodendrocytes in the cerebral cortex of subjects with an ASPA gene mutation who have received an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly increased compared to the number of oligodendrocytes in the cerebral cortex of otherwise similar subjects with an ASPA gene mutation who have not received an rAAV vector, or compared to the same subject before vector administration. The number of oligodendrocytes in the cerebral cortex is measured, for example, by IHC and unbiased stereometry using an Oligo2 antibody. In some implementations, the number of oligodendrocytes in the cortex of subjects with an ASPA gene mutation who have been given an rAAV vector (e.g., AAV / Olig001-ASPA) is not different from the number of oligodendrocytes in the cortex of otherwise similar subjects who have not been given an ASPA gene mutation and have not been given an rAAV vector, wherein the number of oligodendrocytes in the cortex is measured by, for example, IHC and unbiased stereometry using an Olig2 antibody.

[0414] In some embodiments, the number of neurons in the thalamus of subjects with the ASPA gene mutation who have received an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly increased compared to the number of neurons in the thalamus of otherwise otherwise similar subjects with the ASPA gene mutation who have not received an rAAV vector, or compared to subjects before vector administration. The number of neurons in the thalamus is measured, for example, by IHC and unbiased stereometry using a NeuN antibody. In some embodiments, the number of neurons in the cerebral cortex of subjects with the ASPA gene mutation who have received an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly increased compared to the number of neurons in the cerebral cortex of otherwise otherwise similar subjects with the ASPA gene mutation who have not received an rAAV vector, or compared to subjects before vector administration. The number of neurons in the cerebral cortex is measured, for example, by IHC and unbiased stereometry using a NeuN antibody. In some implementations, the number of neurons in the cortex of subjects with an ASPA gene mutation who have been given an rAAV vector (e.g., AAV / Olig001-ASPA) is not different from the number of neurons in the cortex of otherwise similar subjects who have not been given an ASPA gene mutation and have not been given an rAAV vector, wherein the number of neurons in the cortex is measured by, for example, IHC and unbiased stereometry using NeuN antibody.

[0415] In some implementations, cortical myelination in the brains of subjects with ASPA gene mutations who have been given an rAAV vector (e.g., AAV / Oligo001-ASPA) is significantly increased compared to cortical myelination in the brains of otherwise similar subjects who have not been given an rAAV vector, or compared to cortical myelination in the brains of subjects before administration of the vector, wherein cortical myelination is measured by, for example, cortical myelin basic protein positive fiber length density (MBP-LD).

[0416] Assembly of viral vectors

[0417] Viral vectors carrying transgenes (e.g., ASPA) (e.g., rAAV vectors) are assembled from polynucleotides encoding the transgene, suitable regulatory elements, and elements necessary for the production of viral proteins that mediate cell transduction. Examples of viral vectors include, but are not limited to, adenoviruses, retroviruses, lentiviruses, herpesviruses, and adeno-associated virus (AAV) vectors, particularly rAAV vectors (as previously described).

[0418] The vector genome component of the rAAV vector generated according to the method of this disclosure includes at least one transgene, such as a modified nucleic acid encoding ASPA and an associated expression control sequence that controls the expression of the modified nucleic acid encoding ASPA.

[0419] In a preferred embodiment, the vector genome comprises a portion of the parvovirus genome, such as the AAV genome in which rep and cap are missing and / or replaced by modified nucleic acids (e.g., transgenes, such as modified nucleic acids encoding ASPA) and their associated expression control sequences. The modified nucleic acid encoding ASPA is typically inserted adjacently to one or two (i.e., lateral to) AAV ITR or ITR elements sufficient for viral replication (Xiao et al. (1997) J.Virol. 71(2):941-948), replacing the nucleic acid encoding the viral rep and cap proteins. Additional regulatory sequences suitable for promoting tissue-specific expression of the modified nucleic acid encoding ASPA in target cells (e.g., oligodendrocytes) may also be included.

[0420] Packaging cells

[0421] Those skilled in the art will understand that an rAAV vector containing transgenes but lacking viral proteins (e.g., cap and rep) necessary for viral replication cannot replicate, as such proteins are essential for viral replication and packaging. The cap and rep genes may be provided to the cell (e.g., host cell, such as packaging cell) as part of a plasmid separate from the plasmid that provides the transgenes to the vector genome.

[0422] "Packaging cells" or "production cells" refer to cells or cell lines that can be transfected with vectors, plasmids, or DNA constructs and trans-provide all the missing functions required for the complete replication and packaging of a viral vector. The required genes for rAAV vector assembly include the vector genome (e.g., modified nucleic acids encoding ASPA, regulatory elements, and ITRs), the AAV rep gene, the AAV cap gene, and certain accessory genes from other viruses such as adenoviruses. Those skilled in the art will understand that the genes required for AAV production can be introduced into packaging cells in various ways, including, for example, transfection with one or more plasmids. However, in some embodiments, some genes (e.g., rep gene, cap gene, accessory genes) may already be present in the packaging cells, integrated into the genome, or carried on episomes. In some embodiments, the packaging cells constitutively or inducibly express one or more missing viral functions.

[0423] Any suitable packaging cell known in the art can be used to generate packaging viral vectors. Mammalian or insect cells are preferred. Examples of cells that can be used to generate packaging cells in the practice of this disclosure include, for example, human cell lines such as PER.C6, WI38, MRC5, A549, HEK293 cells (which express functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cell line, HepG2, Saos-2, HuH7, and HT1080 cell lines. Suitable non-human mammalian cell lines include, for example, VERO, COS-1, COS-7, MDCK, BHK21-F, HKCC, or CHO cells.

[0424] In some embodiments, the packaging cells are capable of growth in suspension cultures. In some embodiments, the packaging cells are capable of growth in serum-free media. For example, HEK293 cells are grown in suspension in serum-free media. In another embodiment, the packaging cells are HEK293 cells as described in U.S. Patent No. 9,441,206 and deposited at the American Center for Type Culture Collection (ATCC) PTA 13274. Many rAAV packaging cell lines are known in the art, including but not limited to those disclosed in WO 2002 / 46359.

[0425] Cell lines used as packaging cells include insect cell lines. Any insect cell that allows AAV replication and can be maintained in culture can be used according to this disclosure. Examples include fall armyworm (Spodopterafrugiperda) cell lines (such as Sf9 or Sf21), Drosophila spp. cell lines, or mosquito cell lines (e.g., Aedes albopictus-derived cell lines). A preferred cell line is the fall armyworm Sf9 cell line. The following references are incorporated herein because their teachings relate to the use of insect cells for the expression of heterologous peptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in cultures: Methods in Molecular Biology, Richard ed., Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al. (1989) J. Virol. 63:3822-3828; Kajigaya et al. (1991) Proc. Nat'l. Acad. Sci. USA 88:4646-4650; Ruffing et al. (1992) J.Virol. 66:6922-6930; Kimbauer et al. (1996) Virol. 219:37-44; Zhao et al. (2000) Virol. 272:382-393; and U.S. Patent No. 6,204,059.

[0426] As a further alternative, the viral vector of this disclosure can be produced in insect cells using a baculovirus vector that delivers the rep / cap gene and the rAAV template, as described, for example, by Urabe et al. (2002) Human Gene Therapy 13:1935-1943. When AAV is produced using baculovirus, in some embodiments, the vector genome is self-complementary. In some embodiments, the host cell is a baculovirus-infected cell (e.g., an insect cell) that optionally contains additional nucleic acids encoding baculovirus auxiliary functions, thereby facilitating the production of the viral capsid.

[0427] Packaging cells typically include one or more viral vector functions, as well as auxiliary and packaging functions sufficient to cause replication and packaging of the viral vector. These different functions can be provided to the packaging cells together or separately using genetic constructs such as plasmids or amplicones, and they can be present extrachromosomally in the cell line or integrated into the chromosome of the host cell. In some embodiments, the packaging cells are transfected with at least the following: i) a plasmid containing a vector genome comprising codon-optimized human ASPA transgenes (e.g., SEQ ID NO:2) and AAV ITRs (e.g., SEQ ID NO:5 and SEQ ID NO:12) and further comprising at least one of the following regulatory elements: enhancer (e.g., SEQ ID NO:6), promoter (e.g., SEQ ID NO:7), exon (e.g., CBA exon SEQ ID NO:8), intron (e.g., SEQ ID NO:9 and SEQ ID NO:10), and polyA (e.g., SEQ ID NO:11); and ii) a plasmid containing a rep gene (e.g., AAV2rep) and a cap gene (e.g., Olig001 cap).

[0428] In some implementations, the host cell is provided with one or more packaging or auxiliary functions incorporated, for example, into a host cell line, which has one or more vector functions that are incorporated into or integrated into the chromosomal DNA of the cell from outside the chromosome.

[0429] Accessibility

[0430] AAV is a helper virus because it cannot replicate in cells without co-infection via helper viruses. Helper functions include helper viral elements required for the active infection of packaging cells, which are necessary for the packaging of the initiating viral vector. Helper viruses typically include adenoviruses or herpes simplex viruses. Adenoviral helper functions typically include adenoviral components such as adenoviral early region 1A (E1a), E1b, E2a, E4, and virus-associated (VA) RNA. Helper functions (e.g., E1a, E1b, E2a, E4, and VA RNA) can be provided to packaging cells by transfecting cells with one or more nucleic acids encoding different helper elements. Alternatively, the host cell (e.g., the packaging cell) may contain nucleic acids encoding helper proteins. For example, HEK293 cells are generated by transforming human cells with adenoviral 5 DNA and now express many adenoviral genes, including but not limited to E1 and E3 (see, for example, Graham et al. (1977) J. Gen. Virol. 36:59-72). Therefore, these auxiliary functions can be provided by HEK293 packaging cells without the need to supply them to the cells via, for example, plasmids that encode them. In some embodiments, the packaging cells are transfected with at least the following: i) a plasmid containing a vector genome containing codon-optimized human ASPA transgenes (e.g., SEQ ID NO:2) and AAV ITRs (e.g., SEQ ID NO:5 and SEQ ID NO:12) and further containing at least one of the following regulatory elements: enhancer (e.g., SEQ ID NO:6), promoter (e.g., SEQ ID NO:7), exon (e.g., CBA exon SEQ ID NO:8), intron (e.g., SEQ ID NO:9 and SEQ ID NO:10), and poly A (e.g., SEQ ID NO:11); ii) a plasmid containing a rep gene (e.g., AAV2 rep) and a cap gene (e.g., Olig001 cap); and iii) a plasmid containing auxiliary functions.

[0431] Any method can be used to introduce a nucleotide sequence with an auxiliary function into the cell host to achieve replication and packaging, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and combinations of liposomes with nuclear localization signals. In some embodiments, the auxiliary function is provided by transfection with a viral vector, or it can be provided using standard methods for generating viral infection by infection with an auxiliary virus.

[0432] The vector genome can be any suitable recombinant nucleic acid, such as DNA or RNA constructs, and can be single-stranded, double-stranded, or duplexed (i.e., self-complementary, as described in WO 2001 / 92551).

[0433] Production of packaging viral vectors

[0434] Viral vectors can be generated by a variety of methods known to those skilled in the art (see, for example, WO 2013 / 063379). A preferred method is described in Grieger et al. (2015) Molecular Therapy 24(2):287-297, the contents of which are incorporated herein by reference for all purposes. In short, efficient transfection of HEK293 cells was used as a starting point, with adherent HEK293 cell lines from qualified clinical seed cell banks used for growth in shake flasks and WAVE bioreactors under animal-free suspension conditions to allow for rapid and scalable rAAV production. Using a triple transfection method (e.g., WO 96 / 40240), HEK293 cell line suspensions yielded greater than 1 × 10⁻⁶ cells at harvest time 48 hours post-transfection. 5 One particle (vg) containing the vector genome per cell or greater than 1 × 10⁻⁶ 14 Cell cultures in vg / L. More specifically, triple transfection refers to a method in which packaging cells are transfected with three plasmids: one plasmid encodes the AAV rep and cap (e.g., Olig001 cap) genes, another plasmid encodes various auxiliary functions (e.g., adenovirus or HSV proteins such as E1a, E1b, E2a, E4, and VARNA), and a third plasmid encodes the transgene (e.g., ASPA) and various elements controlling transgene expression.

[0435] The single-stranded vector genome is packaged into the capsid in approximately equal proportions as either the positive or negative strand. In some embodiments of the rAAV vector, the vector genome is in the positive polarity (i.e., the sense or coding sequence of the DNA strand). In some embodiments of the rAAV vector, the vector is in the negative polarity (i.e., the antisense or template DNA strand). Given that the nucleotide sequence of the positive strand is 5' to 3' oriented, the nucleotide sequence of the negative strand with its 5' to 3' orientation can be identified as the inverse complement of the positive strand's nucleotide sequence.

[0436] To achieve the desired yield, various variables such as the selection of compatible serum-free suspension medium to support growth and transfection, the selection of transfection reagents, transfection conditions, and cell density were optimized.

[0437] rAAV vectors can be purified using standard methods in the art, such as column chromatography or cesium chloride gradient. Methods for purifying rAAV vectors are known in the art and include those described in Clark et al. (1999) Human Gene Therapy 10(6):1031-1039; Schenpp and Clark (2002) Methods Mol. Med. 69:427-443; U.S. Patent Nos. 6,566,118 and WO 98 / 09657.

[0438] High-purity carrier preparations of AAV serotypes 1-6, 8, 9 and various chimeric capsids (e.g., Oligo001) can be generated using a general purification strategy based on ion-exchange chromatography. In some embodiments, the process can be completed within one week, yielding a high filled-to-empty-capsule ratio (>90% filled capsid) and providing a purified yield (>1×10⁻⁶) suitable for clinical applications. 13 (vg / L) and purity. In some embodiments, this method is universal across all serotypes and chimeric capsids. Scalable preparation techniques can be used to prepare GMP clinical and commercial-grade rAAV vectors (e.g., for the treatment of canavanine disease).

[0439] After the rAAV carrier of this disclosure has been produced and purified, it can be titrated (e.g., to quantify the amount of rAAV carrier in a sample) to prepare a composition for administration to subjects such as those with canavan disease. The rAAV carrier titration can be performed using methods known in the art.

[0440] In some implementations, the number of viral particles, including those containing the vector genome and those with “empty” capsids, can be determined using electron microscopy, such as transmission electron microscopy (TEM). This TEM-based method can provide the number of vector particles (or viral particles in the case of wild-type AAV) in a sample.

[0441] In some implementations, the rAAV vector genome can be titrated using quantitative PCR (qPCR) with primers targeting sequences in the vector genome, such as ITR sequences (e.g., SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19) and / or sequences in transgenes (e.g., SEQ ID NO:2) or regulatory elements. A standard curve can be generated by performing qPCR in parallel with dilutions of standards of known concentration (e.g., plasmids containing sequences of the vector genome), allowing the concentration of the rAAV vector to be calculated as the amount of vector genome (vg) per unit volume, such as microliters or milliliters. The number of empty capsids can be determined by comparing the number of vector particles, as measured, for example, by electron microscopy, with the number of vector genomes in the sample. Because the vector genome contains therapeutic transgenes, the vg / kg or vg / ml of the vector sample may be a better indicator of the therapeutic dose of the vector received by the subject than the number of vector particles, since some of the vector particles may be empty and do not contain the vector genome. Once the concentration of the rAAV vector genome in the stock solution is determined, it can be diluted to a suitable buffer or dialyzed against the buffer to prepare a composition for administration to a subject (e.g., a subject with canavan disease).

[0442] Treatment

[0443] Modified nucleic acids (such as modified nucleic acids encoding ASPA) disclosed herein may be used for gene therapy to treat and / or prevent diseases, symptoms, or conditions associated with defects or dysfunction of the ASPA peptide (e.g., canavan disease), and upregulation of the ASPA gene may produce therapeutic benefits or improvements in any other condition and / or disease, such as diseases, symptoms, or conditions mediated or associated with a reduction in the level or function of the ASPA peptide compared to the level or function of the ASPA peptide in otherwise healthy individuals.

[0444] Vector genomes and / or rAAV vectors containing modified nucleic acids encoding ASPA, as disclosed herein, can be used for gene therapy to treat and / or prevent diseases, conditions, or illnesses associated with or caused by defects or dysfunction of the ASPA enzyme (e.g., canavan disease), as well as any other conditions and / or illnesses for which upregulation of the ASPA enzyme may produce a therapeutic benefit or improvement. In some embodiments, the methods of this disclosure include the use of an rAAV vector or a pharmaceutical composition thereof in treating a subject with canavan disease. In some embodiments, the methods of this disclosure include using an rAAV vector (e.g., AAV / Oligo001-ASPA) or a pharmaceutical composition thereof to increase the level of ASPA in a subject with this need.

[0445] The present disclosure discloses modified nucleic acids encoding ASPA, vector genomes containing modified nucleic acids encoding ASPA, and / or rAAV vectors containing modified nucleic acids encoding ASPA (e.g., AAV / Oligo001-ASPA), which can be used to prepare medicaments for the treatment and / or prevention of diseases, symptoms, or conditions associated with or caused by defects or dysfunctions of ASPA (e.g., reduced levels of functional ASPA enzymes, such as in canavan disease) and any other conditions or diseases for which upregulation of ASPA may produce therapeutic benefits or improvement.

[0446] In some embodiments, gene therapy treatments and / or preventions of diseases, conditions, or illnesses associated with defects or dysfunction of the ASPA enzyme (e.g., canavan disease) and upregulation of ASPA gene expression and / or increased expression of functional ASPA enzymes may produce therapeutic benefits or improvements, including administration to a subject in need of treatment (e.g., a patient) of a therapeutically effective amount of the modified nucleic acid encoding ASPA of this disclosure, a vector genome containing the modified nucleic acid encoding ASPA, and / or an rAAV vector containing the modified nucleic acid encoding ASPA (e.g., AAV / Oligo001-ASPA).

[0447] Compared to baseline measurements (such as measurements of the same individual prior to treatment initiation as described herein, or measurements of a control individual (or multiple control individuals to establish a comparative level) in the absence of treatment as described herein), treating a subject (e.g., a patient) with a therapeutically effective amount of the modified nucleic acid encoding ASPA of this disclosure, a vector genome containing the modified nucleic acid encoding ASPA, and / or an rAAV vector containing the modified nucleic acid ASPA (e.g., AAV / Oligo001-ASPA) can alleviate, improve, treat, prevent, or reduce one or more symptoms or severity of canavanine disease. In some embodiments, a “control individual” is an individual who suffers from the same form of disease or lesion as the treated individual but is currently untreated but may receive treatment in the future.

[0448] For example, compared with NAA accumulation in control individuals, or with NAA accumulation in the same individual before treatment, treating subjects with a therapeutically effective amount of a modified nucleic acid encoding ASPA, a vector genome containing a modified nucleic acid encoding ASPA, and / or an rAAV vector (e.g., AAV / Oligo001-ASPA) can reduce NAA accumulation. In some embodiments, NAA accumulation is reduced by approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 100% in treated subjects compared with control individuals, or with NAA accumulation in the same individual before treatment.

[0449] In some embodiments, treating subjects with a therapeutically effective amount of a modified nucleic acid encoding ASPA, a vector genome containing a modified nucleic acid encoding ASPA, and / or an rAAV vector (e.g., AAV / Oligo001-ASPA) can increase aspartate and / or acetate levels compared to control individuals or to the same individual before treatment. In some embodiments, the increase in aspartate and / or acetate levels in treated subjects is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to control individuals or to the same individual before treatment.

[0450] In some embodiments, treatment may also alleviate, improve, treat, prevent, or reduce myelin degeneration in the brain and spinal cord, intellectual disability, loss of previously acquired motor skills, difficulty eating, abnormal muscle tone, macrosomia, paralysis, and the severity of seizures and / or delays in the development of language and motor skills, compared to the condition in control individuals or pre-treatment subjects. In some embodiments, treatment of subjects (e.g., patients) with a therapeutically effective amount of the modified nucleic acid encoding ASPA of this disclosure, a vector genome containing the modified nucleic acid encoding ASPA, and / or an rAAV vector containing the modified nucleic acid encoding ASPA may also increase, improve, enhance balance, grip strength, and / or motor coordination and generalized motor function, or prevent further loss thereof, compared to the condition in control individuals or the same pre-treatment subjects. In some implementations, treating a subject (e.g., a patient) with a therapeutically effective amount of the modified nucleic acid encoding ASPA of this disclosure, a vector genome containing the modified nucleic acid encoding ASPA, and / or an rAAV vector containing the modified nucleic acid encoding ASPA can reduce the vacuolar volume fraction in the brain (e.g., thalamus, cerebellar white matter / pons), increase the number of oligodendrocytes in the brain (e.g., thalamus, cortex), increase the number of neurons in the brain (e.g., thalamus, cortex), and / or increase cortical myelination compared to the situation in a control individual or to the same subject before treatment.

[0451] Suitable subjects for treatment include any subject who has an insufficient amount of functional gene product (protein), is at risk of producing an insufficient amount of functional gene product (protein), has a defect in a functional gene product (protein), or produces an abnormal, partially functional, or non-functional gene product (protein, e.g., enzyme), which could lead to disease. In some embodiments, patients are treated with the vector or pharmaceutical composition of this disclosure before exhibiting any symptoms of the disease, condition, or illness (e.g., canavan disease). In some embodiments, patients diagnosed by genetic analysis as being at risk of a disease, condition, or illness (e.g., canavan disease) are treated with the rAAV vector or composition of this disclosure before exhibiting symptoms.

[0452] In some embodiments, the subject to be treated may be a mammal, and in particular, the subject is a human patient, for example, a patient with canavan disease. Because the ASPA protein has an incorrect amino acid sequence due to one or more mutations in the coding sequence of the ASPA gene, and thus its function is reduced or absent, expressed in the wrong tissue or at the wrong time, underexpressed or not expressed at all, the subject may require treatment. The modified nucleic acid encoding ASPA of the present invention can be applied to enhance, improve, or provide the production of a functional ASPA enzyme, which in turn catalyzes the breakdown of NAA into aspartate and acetate, as well as other biological functions as discussed elsewhere herein.

[0453] The target cells of the rAAV vector of the present invention are cells, especially oligodendrocytes, which are typically capable of endogenously expressing ASPA enzymes such as those found in the mammalian brain.

[0454] In embodiments that refer to the treatment methods described herein, such embodiments are also further embodiments used for the treatment, or alternatively for the preparation of a medicament for the treatment.

[0455] Pharmaceutical Composition

[0456] In certain embodiments, this disclosure provides pharmaceutical compositions or medicines for the prevention or treatment of diseases, symptoms, or conditions mediated or associated with reduced expression and / or activity of ASPA, such as canavan disease. In some embodiments, the pharmaceutical composition comprises a modified nucleic acid, a recombinant nucleic acid, a viral vector genome, an expression vector, a host cell or rAAV vector, and a pharmaceutically acceptable carrier.

[0457] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a vector (e.g., a viral vector genome, an expression vector, an rAAV vector) or host cell containing a modified nucleic acid encoding ASPA that can increase the expression level and / or activity level of ASPA in the cell.

[0458] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a vector (e.g., a viral vector genome, expression vector, rAAV vector) or host cell (e.g., for ex vivo gene therapy) containing a modified nucleic acid encoding ASPA, wherein the composition further comprises a pharmaceutically acceptable carrier, adjuvant, diluent, excipient, and / or other agent. The pharmaceutically acceptable carrier, adjuvant, diluent, excipient, or other agent is not a biologically or otherwise undesirable agent, for example, that would allow the material to be administered to a subject without causing an undesirable biological effect beyond the material's beneficial biological effect.

[0459] Any suitable pharmaceutically acceptable carrier or excipient may be used to prepare the pharmaceutical compositions according to the invention (see, for example, Remington, The Science and Practice of Pharmacy, Alfonso R. Gennaro (ed.), Mack Publishing Company, April 1997).

[0460] Pharmaceutical compositions are typically sterile, pyrogen-free, and stable under preparation and storage conditions. Pharmaceutical compositions may be formulated as solutions (e.g., water, saline, dextran solution, buffer solutions, or other pharmaceutically sterile fluids), microemulsions, liposomes, or other ordered structures suitable for accommodating high concentrations of products (e.g., viral vector particles, microparticles, or nanoparticles). In some embodiments, pharmaceutical compositions comprising modified nucleic acids of this disclosure, vector genomes comprising modified nucleic acids, host cells, or rAAV vectors are formulated in water or buffered saline solutions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability may be maintained, for example, in the case of dispersions, by using coatings (e.g., lecithin), by maintaining a desired particle size, and by using surfactants. In some embodiments, isotonic agents such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride are preferably included in the composition. Prolonged absorption of injectable compositions can be achieved by including delayed-absorption agents such as monostearate and gelatin in the composition. In some embodiments, the nucleic acids, carriers, and / or host cells of this disclosure can be administered in controlled-release formulations, for example, in compositions comprising sustained-release polymers or other carriers that protect the product from rapid release, including implantation and microencapsulation delivery systems.

[0461] In some embodiments, the pharmaceutical compositions disclosed herein are parenteral pharmaceutical compositions, including compositions suitable for intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, intraparenchymal (IP), intrathecal (IT), intraventricular (ICV), and / or intracranial (ICM) administration. In some embodiments, pharmaceutical compositions comprising an rAAV vector containing a modified nucleic acid encoding ASPA are formulated for administration via ICV injection.

[0462] In some implementations, the rAAV vector (e.g., AAV / Olig001 ASPA) is formulated in PBS with 350 mM NaCl and 5% D-sorbitol.

[0463] Application method

[0464] The modified nucleic acid encoding a transgene (e.g., ASPA) or a vector containing the modified nucleic acid (e.g., a vector genome, rAAV vector) disclosed herein can be administered to a subject (e.g., a patient) to treat the subject. Administration of the vector to a human subject or animal in need can be performed by any method known in the art for administering a vector. Target cells for the vectors of this disclosure include cells of the CNS, preferably oligodendrocytes.

[0465] The carrier can be administered as a supplement to and adjunct to standard care treatment. That is, the carrier can be administered simultaneously, concurrently, or at predetermined dosing intervals that can be determined by a person skilled in the art using conventional methods, with another agent, compound, drug, treatment, or treatment regimen. Uses disclosed herein include administering the rAAV carrier of this disclosure concurrently, in addition to, and / or at synchronized dosing schedules with standard care known in the art for canavan disease.

[0466] In some embodiments, the combination composition includes one or more immunosuppressants. In some embodiments, the combination composition includes an rAAV vector containing a transgene (e.g., a modified nucleic acid encoding ASPA) and one or more immunosuppressants. In some embodiments, the method includes administering or delivering the rAAV vector containing the transgene (e.g., a modified nucleic acid encoding ASPA) to a subject and administering the immunosuppressant to the subject prophylactically before or after administration of the vector (i.e., before or after symptoms of a response to the vector and / or the protein provided therein become apparent).

[0467] In some embodiments, the rAAV of the present invention may be co-administered with an empty capsid (i.e., a viral capsid that does not contain a nucleic acid molecule or vector genome) containing a capsid protein that is the same as or different from that of an rAAV vector containing a modified nucleic acid (e.g., encoding ASPA). Those skilled in the art will understand that co-administration of the empty capsid may reduce the immune response, e.g., neutralizing response, to the rAAV of this disclosure. Not wishing to be bound by any particular theory, the empty capsid may be used as an immune decoy to prevent an rAAV vector containing a modified nucleic acid (e.g., encoding ASPA) from a neutralizing antibody (Nab) immune response, as discussed, for example, in WO 2015 / 013313.

[0468] In one embodiment, the vector of this disclosure (e.g., an rAAV vector comprising a modified nucleic acid encoding ASPA) is administered systemically. Exemplary systemic administration methods include, but are not limited to, intravenous (e.g., portal vein), intraarterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic, and intramuscular administration, as well as direct tissue or organ injection. Those skilled in the art will understand that systemic administration can deliver the modified nucleic acid (e.g., a modified nucleic acid encoding ASPA) to all tissues. In some embodiments, direct tissue or organ administration includes administration to the liver. In some embodiments, direct tissue or organ administration includes administration to areas directly affected by ASPA deficiency (e.g., the brain and / or the central nervous system). In some embodiments, the vector of this disclosure and its pharmaceutical composition are administered to the brain parenchyma (i.e., via intraparenchymal administration), the spinal canal, or the subarachnoid space to reach cerebrospinal fluid (CSF) (i.e., via intrathecal administration), the ventricles (i.e., via intraventricular administration), and / or the cisterna magna (i.e., via intracranial administration).

[0469] Therefore, in some embodiments, the vector of this disclosure comprising a modified nucleic acid encoding ASPA is administered by direct injection into the brain (e.g., into the parenchyma, ventricles, cisterna magna, etc.) and / or into the CSF (e.g., into the spinal canal or subarachnoid space) to treat the neurodegenerative aspects of canavan disease. The target cells of the vector of this disclosure include cells located in the cortex, subcortical white matter of the corpus callosum, striatum, and / or cerebellum. In some embodiments, the target cells of the vector of this disclosure are oligodendrocytes. Additional administration routes may also include local application of the vector under direct visualization, e.g., cortical application or other non-stereotactic applications.

[0470] In some embodiments, the vector of this disclosure is administered via at least two routes. For example, the vector is administered systemically and also directly to the brain. If administered via at least two routes, the administration of the vector may, but does not have to, be simultaneous or concurrent. Alternatively, administration via different routes may be performed separately, with time intervals between each administration.

[0471] The modified nucleic acid encoding ASPA, the vector genome containing the modified nucleic acid encoding ASPA, and / or the rAAV vector containing the modified nucleic acid encoding ASPA disclosed herein can be used for in vitro transduction of cells or direct administration to a subject (e.g., direct administration to the CNS of a patient with canavan disease). In some embodiments, transduced cells (e.g., host cells) are administered to a subject to treat or prevent a disease, symptom, or condition (e.g., cell therapy for canavan disease). The rAAV vector containing the modified therapeutic nucleic acid (e.g., encoding ASPA) is preferably administered to cells in a biologically effective amount. In some embodiments, the biologically effective amount of the vector is sufficient to result in transduction and expression of the modified nucleic acid encoding ASPA (i.e., the transgene) in the target cells.

[0472] In some embodiments, this disclosure includes methods for increasing the level and / or activity of ASPA in cells by administering a modified nucleic acid encoding ASPA, alone or in a vector (including plasmids, viral vectors, nanoparticles, liposomes, or any known method of delivering nucleic acids to cells) to cells (in vivo, in vitro, or ex vivo).

[0473] The dosage of the rAAV vector depends on factors such as the administration method, the disease or condition to be treated, the stage and / or aggressiveness of the disease, the individual subject's condition (age, sex, weight, etc.), the specific viral vector, the stability of the protein to be expressed, the host immune response to the vector, and / or the gene to be delivered. Typically, the dosage range is at least 1 × 10⁻⁶. 8 or more, for example, 1×10 9 1×10 10 1×10 11 1×10 12 1×10 13 1×10 14 1×10 15 More vector genomes (vg) / kg subject weight to achieve therapeutic effects.

[0474] In some implementations, the modified nucleic acid encoding ASPA can be administered as a component of a DNA molecule (e.g., a recombinant nucleic acid) having regulatory elements (e.g., promoters) suitable for expression in target cells (e.g., oligodendrocytes). The modified nucleic acid encoding ASPA can also be administered as a component of a plasmid or viral vector such as an rAAV vector. The rAAV vector can be administered in vivo by direct delivery (e.g., direct delivery to the CNS) to a patient requiring treatment (e.g., a cannabinoid patient). The rAAV vector can also be administered ex vivo to a patient (e.g., cell therapy) by in vitro administration of the vector to cells of a donor patient requiring treatment, followed by returning the transduced cells to the donor.

[0475] This disclosure includes an administration method that results in detectably higher levels of ASPA-encoding mRNA, ASPA protein expression, and / or ASPA activity in otherwise identical cells compared to those in which no modified nucleic acid (e.g., a modified nucleic acid encoding ASPA) has been administered.

[0476] In another embodiment, this disclosure includes an administration method that results in a detectably higher level of mRNA encoding functional ASPA and / or expression of functional (e.g., biologically active) ASPA protein compared to the levels of functional ASPA (mRNA and / or protein) present in otherwise identical cells to which the modified nucleic acid (e.g., the modified nucleic acid encoding ASPA) has not been administered. That is, the invention includes a method for increasing the level of functional ASPA in cells, wherein the cells produce normal levels of ASPA but the ASPA protein lacks activity or exhibits reduced activity compared to normal wild-type ASPA.

[0477] Those skilled in the art will understand that the cells may be cultured or grown in vitro or may exist in an organism (i.e., in vivo). Furthermore, the cells may express endogenous ASPA to increase the level of ASPA in the cells, and / or the cells may express endogenous ASPA that is a mutant or variant of wild-type ASPA, for example, ASPA having the sequence SEQ ID NO:3, especially when more than one wild-type allele of human ASPA may be present. Therefore, the level of ASPA is increased compared to the level of ASPA expressed in otherwise identical but untreated cells.

[0478] Reagent test kit

[0479] This disclosure provides kits containing packaging materials and one or more components. The kits typically include a label or packaging insert containing a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components. The kits may contain a collection of such components, such as modified nucleic acids, recombinant nucleic acids, vector genomes, rAAV vectors, and optionally a second active agent such as a compound, therapeutic agent, drug, or composition.

[0480] A kit refers to the physical structure containing one or more components of a kit. Packaging materials can maintain the components in a sterile manner and can be made of materials commonly used for this purpose (e.g., paper, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0481] The label or insert may include identification information, dosage, and clinical pharmacology (including mechanism of action, pharmacokinetics, and pharmacodynamics) of one or more of the components. The label or insert may include information identifying the manufacturer, batch number, place and date of manufacture, and expiration date. The label or insert may include information regarding the diseases for which the kit components can be specifically used (e.g., canavan disease). The label or insert may include instructions for clinicians or subjects to use one or more of the kit components in a method, use, or treatment protocol or regimen. The instructions may include dosage, duration, frequency, and instructions for practicing any of the methods, uses, treatment protocols, or prevention or treatment protocols described herein.

[0482] Labels or inserts may include information about potential adverse side effects, complications, or reactions, such as warnings to subjects or clinicians about situations where the particular composition is not suitable for use.

[0483] equivalent

[0484] The foregoing written description is considered sufficient to enable those skilled in the art to practice this disclosure. The foregoing description and examples detail certain exemplary embodiments of this disclosure. However, it should be understood that, however detailed the foregoing may be in the text, this disclosure can be practiced in many ways, and should be interpreted in accordance with the appended claims and any equivalents thereof.

[0485] All references cited in this article, including patents, patent applications, papers, textbooks, and the references cited therein, are incorporated herein by reference in their entirety, to the extent that they have not yet been cited.

[0486] Exemplary Implementation

[0487] The invention is described in further detail with reference to the following experimental embodiments. These embodiments are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the invention should in no way be construed as limited to the following embodiments, but should be understood to cover any and all variations that become apparent from the teachings provided herein. Example

[0488] Example 1: NAA dose response induced by using an rAAV vector containing codon-optimized nucleic acid encoding ASPA. Reduced adaptability

[0489] Human embryonic kidney (HEK) cells were transfected with a plasmid expressing NAA synthase (Nat8L) at 1.0 μg and co-transfected with plasmids containing 0.1, 0.2, 0.5, or 1.0 μg of the following: wild-type human ASPA nucleic acid sequence (SEQ ID NO:3), codon-optimized nucleic acid encoding ASPA (containing the nucleic acid sequence of SEQ ID NO:1, see Francis et al. (2016) Neurobiol. Dis. 96:323-334), or codon-optimized nucleic acid encoding ASPA containing the nucleic acid sequence of SEQ ID NO:2. NAA concentrations were measured by HPLC (n = 4 / group). A dose-responsive decrease in NAA was observed in cultures transfected with the codon-optimized nucleic acid encoding ASPA of SEQ ID NO:2, compared to cultures transfected with wild-type nucleic acid encoding ASPA or the codon-optimized nucleic acid encoding ASPA of SEQ ID NO:1. Figure 1 ).

[0490] Example 2: Biodistribution of oligotropic AAV / Olig001

[0491] This study aimed to determine the most effective dose and route of administration (ROA) for promoting extensive CNS oligodendrocyte transduction in a mouse model of inherited human leukodystrophy, Canavan disease, using an oligotropic AAV (AAV / Olig001; (WO2014 / 052789; Powell et al. (2016) Gen. Ther. 23:807-814)) capsid variants. Three doses of AAV / Olig001 delivered via four different ROAs were tested in adult, symptomatic Canavan mice (nur7), and vector propagation and transduction were quantified two weeks post-transduction by stereographic estimation of reporter gene green fluorescent protein (GFP)-positive cells generated in four anatomical regions of interest. Oligotropism was validated by scoring the incidence of lineage-specific antigens co-labeled with GFP in these same regions. The ROAs used were intraparenchymal (IP), intrathecal (IT), intraventricular (ICV), and intracerebral (ICM) routes. Three doses of the carrier were administered via each route: 1 × 10⁻⁶. 10 1×10 11 and 5×10 11 Total vector genome (VG), where the volume of delivered material was constant across all treatment cohorts, and direct pairwise comparisons were performed for each group to determine the optimal combination of dose and ROA for the application of AAV / Olig001 in canavan disease. Six-week-old aspartate acylase-deficient NUR7 mice (Traka et al. (2008) J. Neurosci 28:11537-11549) were used, representing the acute symptomatic phase of canavan disease. The results from this study form the basis for supporting further preclinical efficacy studies of AAV / Olig001 for clinical application in currently refractory white matter diseases such as canavan disease.

[0492] Material

[0493] AAV / Olig001 vector

[0494] Two batches of AAV / Olig001 vectors containing constitutive expression cassettes of the GFP reporter gene were generated (batch number 7660 and batch number LAV38A). All generated vectors contained the GFP reporter gene, driven by a heterozygous CMV / chicken β-actin promoter (CBh) with a side-complementary AAV ITR. Vectors were generated by transient transfection of HEK293 cells followed by iodixanol gradient centrifugation and ion-exchange chromatography (Gray et al., (2013) Gene Ther. 20:450-9). The concentration of the vector was defined as the total number of viral vector genomes (vg), which was determined by qPCR quantification of the DNAse-resistant AAV inverted terminal repeat (ITR) sequence in the stock formulation.

[0495] animal

[0496] All animals used in this study were generated from a population maintained under an approved institutional protocol at the Rowan School of Osteopathic Medicine animal facility. Initial animals were sourced from commercial sources (Jackson Laboratories). The nur7 mouse is a well-characterized model of canavan disease, containing an inactivating point mutation in the gene encoding the glial hydrolase aspartate acylase (aspa), rendering the protein nonfunctional (Traka et al. J. Neuroscience (2008) 28(45) 11537-11549). Homozygous nur7 mutant animals were generated by pairing heterozygous vector animals and genotyped using in-house customized SNP assays and real-time PCR.

[0497] AAV / Olig001-GFP diluted to an appropriate concentration in 0.9% saline was delivered via stereotactic injection to 6-week-old NUR7 mutant mice under inhalation anesthesia (4% induction and maintenance titration for efficacy). Four treatment cohorts were generated, each distinguished by a different route of administration (ROA): intrathecal (IT), intraparenchymal (IP), intraventricular (ICV), and intracisternal (ICM). Within each ROA cohort, animal subgroups (1 × 10⁻⁶) were established, defined by the dose of the carrier administered at each ROA. 10 1×10 11 and 5×10 11 Total vector genome).

[0498] Therefore, for each ROA, three dose-defined subgroups were generated, with n = 5 animals per dose under each ROA, providing a total of 60 NUR7 mice for the study. AAV / Olig001-GFP was administered to anesthetized mice, and for all procedures, regardless of dose or ROA, the total delivery volume of 5 μL was constant. IP ROA required five 1 μL injections of the vector at five stereotactic coordinates, using a digital pump at a rate of 0.1 μL / min, twice into the anterior and posterior subcortical white matter of each hemisphere (i.e., a total of four injections in the cingulate cortex) and one additional injection into the cerebellar white matter (to provide a total of five injections). IT ROA animals received a single 5 μL vector infusion into the subarachnoid space via lumbar puncture between L5 and L6. ICV ROA animals received two 2.5 μL vector injections, each at a rate of 0.1 μL / min into each lateral ventricle. ICM ROA animals received 5 μL of the vector delivered directly to the CSF via the cisterna magna at a rate of 0.1 μL / min. Twenty minutes prior to surgery, all animals received 0.5 mL of 20% mannitol (ip). Following AAV / Olig001-GFP delivery, all animals were grouped and fed for two weeks, then sacrificed for postmortem analysis.

[0499] Systemic BrdU (50 mg / kg, ip) was administered twice daily for two consecutive days to juvenile 2-week and 8-week-old wild-type and NUR7 mice, followed by sacrifice on day 3. BrdU was then administered to the animals at a concentration of 50 mg / kg. After DNA hydrolysis in 1 M HCl, brain tissue sections were stained with BrdU using a commercially available antibody (Millipore-Sigma).

[0500] method

[0501] Quantitative analysis of carrier biological distribution using unbiased stereometry

[0502] Two weeks after the vector surgery, animals were deeply anesthetized and perfused with 0.9% saline followed by freshly prepared buffered 4% paraformaldehyde to prepare the brain. The perfused brain was excised and post-fixed overnight in 4% PFA at 4°C. The fixed brain was cryopreserved and rapidly frozen in a dry ice / isopentane bath and stored at -80°C, followed by immunohistochemical treatment. Serial 40 μm sagittal sections (144 sections in total) were produced for each brain, and every fourth section was stained with GFP using a commercially available antibody (Sigma / Millipore). GFP-positive somatic cells in the cortex, subcortical white matter, striatum, and cerebellum were scored using an unbiased stereographic optical fractionator method. Figure 2(West et al., Anat. Rec. (1991) 231:482-97). GFP-positive somatic cell counts were generated in four distinct regions of interest (i.e., the cerebral cortex, subcortical white matter of the corpus callosum and external capsule, striatum, and cerebellum) using Stereology software (Stereologer, Stereology Resource Center) coupled to an upright bright-field microscope equipped with a motorized stage. Within each region of interest, GFP-positive cells in the sampling fraction were reconstituted into an absolute estimate using the formula ΣQ*(t / h)*(1 / asf)*(1 / ssf), where ΣQ = particle count, t = slice thickness, h = counting frame height, asf = area sampling fraction, and ssf = slice sampling fraction. For all datasets thus generated, intrasample variation was monitored by calculating the coefficient of error (CE) contributing less than 15% of the total variance (CV) threshold to reduce technical noise masking the true biological variance between samples. Significant differences in the estimated average group mean of N were determined by an unpaired two-tailed Student's t-test, with a threshold significance of p < 0.05.

[0503] Quantitative analysis of carrier orientation

[0504] Vector tropism in AAV / Olig001-GFP transduced brains was quantified by scoring lineage-specific antigens co-labeled with GFP fluorescence. Alternative sections were processed using commercially available antibodies (Sigma / Millipore) for immunohistochemistry of NeuN (present in most CNS and PNS neuronal cell types in vertebrates), GFAP (glial fibrillary acidic protein), or Olig2 (oligodendrocyte lineage transcription factor 2) to label neurons, astrocytes, and oligodendrocytes, respectively. Scanning confocal microscopy was used to generate multipoint image stacks for each region of interest. Total GFP-positive cells and the number of GFP-positive cells co-labeled with each lineage-specific antigen (Olig2 and NeuN) in each stack were counted using NIS-Elements Advanced Research software (Nikon). The number of individual brains was tallied (a total of 8 consecutive sections were sampled from each brain, with a sampling interval of 4). ROIs in individual sections were delineated using software, and individual spots were placed every 200 μm² for high-magnification sampling to score GFP immunofluorescence somatic cells and GFP / Olig2 or NeuN positive cell bodies. The total number of GFP-positive somatic cells co-labeled with Olig2 or NeuN was calculated by dividing the number of GFP-positive somatic cells by the lineage-specific co-labeling in each series of sections. The mean of each ROA was calculated (n = 5 animals).

[0505] result

[0506] Intra-infrared (IP) ROA dose response

[0507] Five separate injections targeting the subcortical white matter of the cerebral hemispheres and cerebellum were administered to IP ROA animals. Two weeks (8 weeks old) after vector administration, the treated animals were sacrificed and the brains were processed for GFP immunohistochemistry, and GFP-positive somatic cells in the cortex, subcortical white matter, striatum, and cerebellum were scored using unbiased stereochemistry with an optical fractionator to provide an absolute estimate of transduced cells in each region of interest. All three doses of AAV / Olig001-GFP administered resulted in significant transduction levels of cells throughout the brain. Figure 3 In the cortex, at 1×10 10 and 1×10 11 Between doses of VG, the number of transduced cells increased significantly (+1.6-fold, p = 0.0096), but at a maximum of 5 × 10⁶ cells / day. 11 There was no significant further increase in dosage (p = 0.659), indicating that saturation had been reached. Figure 3 High levels of transduction were observed in the subcortical white matter of the corpus callosum and external bursa, with positive cells most concentrated at the four injection sites. The number of GFP-positive cells in the subcortical white matter also increased in a dose-dependent manner, from 1 × 10⁻⁶ cells / year. 10 Up to 1×10 11 A 2.2-fold increase in dose was statistically significant (p = 0.0144), but from 1 × 10 11 Up to 5×10 11 The 1.3-fold increase did not reach statistical significance (p = 0.283). Moderate striatal transduction was present. From 1 × 10 10 Up to 1×10 11 The 3.4-fold increase was highly significant (p = 4.38 × 10⁻⁶). -5 However, in 5×10 11 At the specified dose, transduction did not show a significant further increase (p = 0.706). Transgenic expression in the cerebellum was limited to the area immediately surrounding the single injection site, with 1 × 10⁻⁶ transgenes expressed in this area. 11 and 5×10 11 All doses resulted in a significant increase beyond the previous dose (1×10). 11 : 1.5 times increase [p = 0.0016]; 5 × 10 11 The number of transducing cells was 1.4-fold increased [p = 0.0019]. The cortex showed the highest number of transducing cells (513, 477), followed by the subcortical white matter (178, 362), the cerebellum (86, 820), and finally the striatum (62, 706). GFAP co-labeling was less than 2%.

[0508] Intrathecal (IT) ROA dose response

[0509] IT administration of AAV / Olig001-GFP resulted in excellent transgene expression distribution throughout the brain, except for the subcortical white matter of the corpus callosum and external capsule. Figure 4 From 1×10 10 Up to 1×10 11 There was a significant increase in cortical transduction of vg (6.1-fold increase, p = 0.000026), especially at 5 × 10⁻⁶. 11 No significant increase was observed at the vg dose (p = 0.273). Although the distribution of GFP expression in the IT ROA cortex was excellent, the expression intensity was slightly lower compared to the IP brain. Unsurprisingly, considerable GFP expression was found in the lumbar region of the spinal cord, indicating some dilution of the vector in the spinal cord tissue along the pathway to the brain. The most compelling observation in the IT ROA brain was the lack of transgene expression in the corpus callosum and external bursa. Although when the dose increased from 1 × 10⁻⁶, the expression intensity was significantly lower. 10 Increased to 1×10 11 At 1×10⁻⁶, a highly significant increase (6.3-fold increase, p = 0.00021) was observed in white matter tract cells expressing GFP, but the absolute number of transducing white matter cells in the ITROA brain was relatively moderate. 11 At the specified dose, the average number of positive cells in the white matter tract regions of the brain was 64,970, compared to 178,362 in the IP brain at the same dose. Similar to cortical ROA, in the IT ROA brain, increasing the dose from 1×10⁻⁶... 11 Further increase to 5×10 11 The number of transduced white matter tract cells was not significantly increased (p = 0.203).

[0510] At each consecutive dose, the striatum showed a dose-responsive increase in transducing cells. The dose was increased from 1×10⁻⁶. 10 Increased to 1×10 11 This resulted in 2.7 times more GFP-positive cells in the striatum (p = 0.001). The number subsequently increased to 5 × 10⁶. 11 A 3.2-fold increase in positive cells was observed at the dose (p = 0.000037), which resulted in a number comparable to that of IP ROA striatal transduction (IT at 5 × 10⁻⁶). 11 The average value was 79,444, and the IP was 5×10. 11 The average value is 65,203.

[0511] IT ROA leads to strong expression of cerebellar transgenes, particularly when the expression rate increases from 1×10⁻⁶. 10 Move to 1×10 11At the initial dose, a significant 1.5-fold increase was observed (p = 0.0064), but at 5 × 10⁻⁶ doses... 11 No further increase was observed at the specified dose. Cerebellar transduction was comparable to that of IPROA brain, and at 1×10 11 and 5×10 11 Slightly higher at certain doses, but not significantly higher.

[0512] Intraventricular (ICV) ROA Dose Response

[0513] ICV administration of AAV / Olig001-GFP resulted in significant transgene expression in all regions of interest, particularly strong transduction in the subcortical white matter. Figure 5 When the dose is from 1×10 10 Increased to 1×10 11 At that time, all regions of interest showed a dose-responsive increase in the number of transduced cells, but compared with 1×10 11 Compared to the dosage, at 5×10 11 At the specified dose, a slight, insignificant increase was observed in most regions, except for the cerebellum. Cortical transgene expression was comparable to that in the IP ROA brain, where the expression increased at doses from 1 × 10⁻⁶ to 10⁻⁶. 10 Increased to 1×10 11 At that time, the number of transgenic positive cells increased twofold (p = 0.00029), and 5 × 10⁻⁶ cells were applied. 11 A further 1.2-fold increase after the initial dose did not reach statistical significance (p = 0.123).

[0514] Subcortical white matter transduction in the brain during ICV is significant, particularly when the dose increases from 1 × 10⁻⁶. 10 Increased to 1×10 11 At that time, a two-fold increase in GFP-positive white matter tract cells was observed (p = 0.00052). At a maximum of 5 × 10⁻⁶ 11 A moderate, non-significant increase was observed at the dose (p = 0.334). At 1 × 10 11 At the dose, subcortical white matter transduction in the ICV brain was significantly increased by 1.5-fold compared to the IP brain (p = 0.041) and by 4.2-fold compared to the IT ROA brain (p = 0.0001).

[0515] Very similar transgene expression patterns were observed in the striatum of the ICV dose groups, where the expression pattern varied from 1 × 10⁻⁶ to 1 × 10⁻⁶. 10 Increased to 1×10 11 At 1000 Hz, a significant 2-fold increase in GFP-positive cells was observed (p = 0.000043), but at 5 × 10⁻⁶ Hz... 11No significant further increase was observed at the dose level (p = 0.537). Strong striatal transgene expression was evident in the ICV brain, with a 2.5-fold increase in GFP-positive cells in this region compared to the IP brain (p = 0.00004).

[0516] Strong cerebellar ICV transduction was observed, with a dose-dependent increase in GFP-positive cells observed at two consecutive higher doses (at 1×10⁻⁶). 11 +2 times, p = 9.56 × 10 -6 ; in 5×10 11 (+1.5 times, p = 0.00073). In 1×10 11 At the specified dose, the number of GFP-positive cells increased 1.7-fold (p = 0.0001) relative to the IP brain and 1.4-fold (p = 0.0013) relative to the IT brain. In ICV ROA brains, the cerebellum was the only region showing a further significant increase in GFP-positive cells.

[0517] Throughout the sampling process, the most significant difference between the IP and ICV ROA brains was the greater distribution of the vector in the ICV group. In the IP brain, transgene expression was more intense at the injection site, but rapidly diluted from that site. In contrast, ICV transgene expression was relatively evenly distributed over a larger area of ​​the brain.

[0518] Intracaver magnum (ICM) ROA dose response

[0519] Application of AAV / Olig001-GFP to the ICM resulted in relatively widespread but moderate transgene expression in the cortex, striatum, and cerebellum. However, similar to the ITC ROA brain, significant transgene expression was lacking in the subcortical white matter of the ICM brain. Figure 6 Cortical transgene expression was dose-responsive, with each successive higher dose leading to a significant increase in GFP-positive cells (1×10⁻⁶). 11 A 2.2-fold increase, p = 0.018; 5 × 10 11 (1.3 times increase, p = 0.043). In 1×10 11 At the specified dose, a significant increase in GFP-positive cells was observed in the striatum and cerebellum (p = 2.49 × 10⁻⁶ for both the striatum and cerebellum). -6 And p = 0.0062), of which the highest is 5 × 10 11 The dose resulted only in a further increase in positive cells in the cerebellum (p = 0.061). Transduction of the subcortical white matter tracts via the ICM ROA was noticeably moderate. Although the administered dose was increased from 1 × 10 10 Increased to 1×10 11This resulted in a significant increase in GFP-positive cells (p = 0.00086), but the actual number of transgenic positive cells present was relatively negligible.

[0520] Compared to the ICV brain, the ICM subcortical white matter showed a 14.2-fold reduction in GFP-positive cells (ICV mean 271,274; ICM mean 18,996, p = 0.00002) and a 3.4-fold reduction in IT compared to the second-lowest subcortical white matter transducing ROA group (IT mean 64,970), making the ICM the least effective ROA for white matter transduction. Distribution in other regions of interest was comparable to other ROA treatments, with no significant difference in cortical transduction compared to all three other ROAs. Striatal transduction via the ICM was slightly reduced compared to the ICV ROA (p = 0.043). At 1 × 10⁻⁶ 11 At the specified dose, striatal ICM GFP expression was significantly higher than that of IP (+2.0-fold, p = 0.00005) and IT (+5.1-fold, p = 0.0000005). Among any of the four ROAs examined, the ICM brain showed the highest number of transduced cerebellar cells. At 1 × 10⁻⁶ 11 At the dose, cerebellar ICM transduction increased 1.5-fold relative to ICV (mean ICM 228,282; mean ICV 157,203), 2.6-fold relative to IP, and 2.2-fold relative to IT.

[0521] Comparison of routes of application (ROA)

[0522] For all ROAs studied in this paper, in all regions of interest, the carrier dose was increased from 1 × 10⁻⁶. 10 Increased to 1×10 11 It caused a 2-3 fold increase in the number of transduced cells, and further dose increases to 5 × 10⁻⁶. 11 This results in a negligible increase in the total number of transduced cells. Within each region of interest, 1×10-1 11 Direct comparison of all four ROAs at different doses revealed significant differences in the absolute number of transduced cells in all four ROIs. Figure 7 For each ROA, use 1×10. 11 The number of transduced cells in the cortex of the brain transduced by each vector genome did not differ significantly, with all resulting in an average of 44,000–50,000 positive somatic cells. In contrast, ICV ROA showed a clear advantage in the subcortical white matter, with a significantly higher number of transduced cells in the ICV brain compared to any other group. ICV and IP transduced brains produced the highest and second-highest numbers of transduced white matter tract cells, respectively. Transduced cells via ICV ROA were 1 × 10⁻⁶. 11The average number of white matter tracts transduced by the AAV / Olig001-GFP vector was 2.7 × 10⁻⁶. 5 The number of positive cells was significantly higher than the average of 1.8 × 10⁶ in the brain that had received the same dose of IP. 5 0.041 positive cells (p = 0.041).

[0523] Both IT and ICM ROA were inefficient in transducing subcortical white matter cells, with an average efficiency of 2.7 × 10⁻⁶ in the ICV group. 5 Compared to the ICM group, the average number of positive cells was 1.9 × 10⁶. 4 The number of cells was significantly reduced by 14-fold (p = 0.000083) in the individual group and by 4-fold (p = 0.0001) in the IT group. This may be noteworthy in disease model systems with myelin deficiency.

[0524] The ICV pathway also leads to efficient transduction of cells in the striatum, with a significantly higher number of GFP-positive cells in the ICV brain compared to all other ROAs (ICV vs. IP, p = 3.68 × 10⁻⁶). -5 Compared to IT, ICV has p = 1.61 × 10⁻⁶ -5 (ICV vs. ICM, p = 0.043). Among all IP, IT, and ICV ROAs, cerebellar transduction efficiency was comparable, but the ICM brain exhibited the highest number of transduced cerebellar cells (ICM vs. ICV, p = 0.045).

[0525] Although the absolute number of cells transduced via AAV / Olig001-GFP in specific regions was comparable in both IP and ICV ROA brains, the majority of positive cell counts in IP brains were due to slices immediately adjacent to the injection site, while the distribution of positive cells in ICV brains was relatively uniform. Systematic, non-random stereosampling allowed for the identification of variance (intra-sample variance) between slices sampled from individual brains, expressed as the coefficient of error (CE) in the dataset, calculated by dividing the standard error of the mean of repeated estimates by the mean. CE is half the total variance in the sampled population, with the other half being the true biological variance (CV), or the difference in mean between individual brains. The average CE for a single IP brain was calculated to be approximately 12% of the total variance, while the average CE for an ICV brain was approximately 3%, meaning that GFP-positive cells were more evenly distributed across all slices sampled in ICV brains. In IP brains, the actual number of positive cells in a single sampled slice decreased the lateral distance from the injection site, while the number of positive cells in ICV brains remained close to the intra-sample mean across all sampled slices. The end result of this difference is that the carrier spreads more extensively in the ICV ROA brain compared to the IP brain, especially in the cortex and subcortical white matter. Figure 7 ).

[0526] in conclusion

[0527] Using four different ROAs, dose-ROA combinations beneficial for overall CNS oligodendrocyte transduction were determined in acute symptomatic animals closely mimicking Canavan's brain at diagnostic time. Administration of the AAV / Olig001-GFP vector to all regions of interest except the cerebellum produced greater than 70% oligotropy without requiring lineage-specific expression elements. A dose-dependent increase in transgenic positive oligodendrocytes was evident across all ROAs, with intraventricular ROAs promoting a higher number of transduced white matter tract cells while maintaining greater than 90% oligotropy in this key region of interest. These data highlight that capsid-cell surface interactions are a major determinant of oligotropy, and this determinant is most relevant for clinical application in oligodendrocyte-specific abnormalities such as Canavan's disease. These data also demonstrate that the Olig001 capsid has potential as a therapeutic capsid for treating oligodendrocyte-related diseases, symptoms, and / or conditions, including Canavan's disease.

[0528] Example 3: Carrier tropism via route of administration (ROA)

[0529] A notable characteristic of AAV / Olig001 is its pronounced oligotropy compared to other AAV capsid variants (Powell et al. (2016) Gen. Ther. 23:807-814; Francis et al. (2016) Neurobiol. Dis. 96:323-334). For application in canavan disease (which is defined as a white matter disorder), the AAV / Olig001 vector must exhibit this tropism when applied via different ROAs. Oligopterygism can vary due to variables such as intervention age (Gholizadeh et al. Hum. Gene Ther. Methods (2013) 24:205-13; Foust et al. Nature Biotech. (2009) 27:59-65), and although previous work has demonstrated the oligoparticle potential of AAV / Olig001 in neonatal NUR7 mice (Francis et al. Neurobiol. Dis. (2016) 96:323-334), the transfer of this tropism potential to older symptomatic animals remains untested. For this purpose, the 1×10⁻⁶ nucleotides used in Example 2 were evaluated in 6-week-old animals. 11 The potential effects of all four ROAs at different doses on vector tropism were investigated. Co-labeling of GFP transgenes with lineage-specific antigens Olig2 (i.e., a target-specific marker for oligodendrocytes) and NeuN (i.e., a target-specific marker for neurons) was analyzed in the cortex, subcortical white matter, striatum, and cerebellum to determine the absolute number of GFP-positive cells generated.

[0530] result

[0531] All four ROAs produced comparable results, with oligotropy remaining intact. Non-oligodendrocyte transgene expression was attributable to neurons, with very few astrocytes expressing GFP (<5%) observed in all four ROA cohorts.

[0532] In IT, ICV, and ICM ROA, cortical co-labeling with Olig2 was comparable, with the percentage of total GFP-positive cells consistently around 75%. In IP-transduced brain, the percentage of GFP-positive cells co-labeled with Olig2 was approximately 62.3%, a smaller but significant reduction. Figure 8 In these identical brains, GFP-positive cells co-labeled with NeuN constituted the majority of the remaining transducing cortical population (35.1%). All three IT, ICV, and ICM ROA brains showed approximately 20% NeuN co-labeling. In the IT ROA brain, 75.5% of cortical GFP-positive cells were co-labeled with Olig2, and 20.2% were co-labeled with NeuN. The ICV ROA brain showed 70.8% oligotropic and 23.6% neurotropic in the cortex, while the ICM brain showed 76% GFP co-labeled with Olig2 and 17.4% GFP co-labeled with NeuN in the cortex. Oligotropy was relatively small across the four different ROAs, but IP ROA showed a significant increase in NeuN co-labeling (p = 0.0043 compared to IT; p = 0.0119 compared to ICV; p = 0.00059 compared to ICM). This increase corresponded to a slight but significant decrease in Olig2 co-labeling relative to the other three ROAs (p = 0.026 compared to IT; p = 0.048 compared to ICV; p = 0.0085 compared to ICM), suggesting that IP ROA contributes to a smaller increase in neurotropism at the expense of oligotropy. Furthermore, the increase in NeuN co-labeling in IP ROA is noteworthy (+1.5-fold, p = 0.012), indicating that the decrease in Olig2 co-labeling in this ROA is attributable to increased neuronal transduction. Most of the GFP-NeuN co-labeled markers in the IP ROA brain were clustered near the injection site, indicating that the number of AAV / Olig001-GFP markers immediately adjacent to the injection site had reached saturation.

[0533] In all four ROAs, Olig2 and GFP co-labeled >90% of the subcortical white matter. Figure 9In all four ROAs, NeuN co-labeling with GFP was <6%. No significant differences in the percentage of co-labeling with either antigen were observed between ROAs, indicating a strong preference for oligodendrocytes in white matter-rich regions, independent of ROA. Nearly ubiquitous Olig2 co-labeling and no NeuN co-labeling were observed in the corpus callosum via ICV transduction.

[0534] In all ROAs, Olig2 co-labeling with GFP in the striatum was comparable, with the percentage of total GFP-positive cells co-labeled with Olig2 >80%. Figure 10 The remaining GFP-positive cells in the striatum (<20%) were co-labeled with NeuN.

[0535] In all four ROAs, compared with other brain regions studied, cerebellar co-labeling showed a significantly different ratio of Olig2 and NeuN. In all four ROAs, the percentage of Olig2 co-labeled cells was 10% of total GFP-positive cells. Figure 11 Transgenic expression was dominated by neurons in the cerebellum, accounting for over 80% of GFP-expressing cells. Within the cerebellum, no significant differences were observed in the percentage of co-labeled antigens between ROA cohorts. Larger Purkinje neurons in the granule cell layer were strongly GFP-positive. Figure 29C Only sporadic Olig2 / GFP co-labeling was observed in the cerebellar white matter tracts. This is consistent with the near 100% oligotropy observed in the subcortical white matter. Figure 29B ), and the contrast between 70% to 80% oligotropy formation observed in relatively neuronal-dense regions such as the cortex and striatum ( Figure 29D ). In 1×10 11 At each dose, total GFP-positive cells for each ROA score were sorted in order of the highest to lowest average (+ / - sd) of total GFP-positive cells, where n=5: ICV 1104256.4 (106816.96); IP 841365.6 (121722.7); ICM 815486.9 (106979.7); IT 742143.1 (79496.5).

[0536] ICV ROA produced the highest number of total GFP-positive cells (the sum of all ROA counts in a single brain), which was 1.3 times higher than the next ranked ROA, IP (p = 0.0067). Among all ROAs, including ICM (p = 0.0027) and IT (p = 0.0003), the total number in ICV brains was significantly increased. The number of cells in IP ROA brains did not significantly increase compared to ICM (p = 0.730) or IT (p = 0.165), indicating that ICV ROAs were significantly better at transducing total cells. Approximately 75% (about 262,891) of the difference in total GFP-positive cell number between the ICV and IP cohorts was attributed to subcortical white matter (35%) and striatum (36%) ROAs, both exhibiting >80% oligotropy in both cohorts. This implies that ICV brains contained at least approximately 210,000 more transduced oligodendrocytes in certain regions than IP brains. If this analysis is limited to the subcortical white matter, i.e., ROIs exhibiting >90% oligotropy across all ROAs, then administration of AAV / Olig001 via ICV ROA is expected to result in at least an additional 83,000 oligodendrocytes / brain transduction. When evaluated against the ROA cohort exhibiting the worst level of GFP transgene expression, i.e., the ICM cohort, ICV administration resulted in an increase of over 200,000 oligodendrocytes / brain transduced with AAV / Olig001.

[0537] It is known that the adult mammalian CNS contains a significant number of oligodendrocyte precursor cells in the white matter (Dawson et al., Mol. Cell Neurosci. (2003) 24:476-488), and evidence of attempted myelin regeneration in the form of increased immature oligodendrocyte conversion has previously been shown in juvenile nur7 (Francis et al., J. Cerebral Blood Flow Metabolism (2012) 32:1725-36). Given the significant myelin regeneration capacity of the white matter even in the adult brain, the persistence of the resident population of immature oligodendrocytes in the adult nur7 white matter must be considered an ideal target for oligotropic gene delivery vectors.

[0538] To assess the relative number of proliferating oligodendrocyte progenitor cells / immature oligodendrocytes, NUR7 and wild-type mice were administered systemic BrdU twice daily for two days and sacrificed on day 3 for BrdU / Olig2 co-labeling. Figure 29E-G). BrdU administration was initiated in 2- and 8-week-old cohorts to quantify the potential persistence of proliferating oligodendrocytes in the juvenile and adult brains. Counts of BrdU-positive cells in the corpus callosum and external capsule of genotype cohorts at each age revealed a 1.8-fold increase in BrdU-positive cells in the 2-week-old NUR7 brain (p = 0.029) and a 1.6-fold increase in the 8-week-old NUR7 brain (p = 0.034) compared to wild-type. Figure 29F At both age levels, the vast majority of BrdU cells in the white matter of NUR7 mice were co-labeled with Olig2, indicating the persistence of proliferating progenitor cells / immature oligodendrocytes in the white matter of adult symptomatic NUR7 mice. A subset of three 6-week-old NUR7 mice were administered systemic BrdU for two consecutive days, followed by 1×10⁻⁶ doses. 11 Animals were transduced with AAV / Olig001-GFP using vg, and sacrificed 2 weeks post-transduction to obtain evidence of proliferating cell transduction in the white matter bundles. Numerous BrdU / GFP co-labeled cells were observed in the white matter bundles of these animals, indicating successful transduction of resident progenitor / immature cells.

[0539] via ICV ROA, a group of age-matched (i.e., 6 weeks old) healthy wild-type animals from the nur7 ROA cohort were fed 1×10 11 vg AAV / Olig001-GFP transduction, followed by sacrifice after 2 weeks to produce stereographic estimates of GFP-positive cells in the cortex and subcortical white matter tracts. Figure 8 Estimates of GFP-positive cells revealed a significant two-fold reduction in both cortical and subcortical white matter in the wild-type brain compared to the NUR7 brain (p = 0.00032 and p = 0.0116 for their respective ROIs). GFP transgene expression in the subcortical white matter of the wild-type brain was highly restricted to areas adjacent to the lateral ventricles, while cortical expression, although rather dispersed, was moderate in absolute numbers of transduced cells.

[0540] in conclusion

[0541] Examples 2 and 3 demonstrate that the intraventricular (ICV) administration route of the AAV / Olig001 GFP vector provides an optimal combination of vector diffusion and oligodendrocyte tropism. Crucially, this ROA appears perfectly suited for transducing subcortical white matter, tissue affected by the pathology of Canavan disease. Therefore, the ability to transduce hundreds of thousands of cells and maintain near 100% tropism for oligodendrocytes gives AAV / Olig001 a significant advantage over other AAV capsids. Four- to six-week-old nur7 corpus callosum / external bursa of callosity contain approximately 1,500,000 Olig2-positive cells; thus, administration of 1 × 10⁻⁶ cells via the ICV ROA is sufficient. 11The dose of AAV / Olig001 vector has the potential to transduce approximately 20% of the resident oligodendrocyte population. It should be noted that white matter tracts from NUR7 mice showed evidence of attempted myelin regeneration and contained a significant number of proliferating oligodendrocyte progenitor cells. Given that a single oligodendrocyte can myelinate multiple axons, the potential for myelin regeneration after transduction of white matter with the therapeutic AAV / Olig001 vector is significant.

[0542] Other CSF-targeted ROAs, namely IT and ICM, exhibit relatively poor white matter tract transduction and are not considered as the first choice for therapeutic ROAs. IP brain shows nearly comparable transduction levels in terms of the number of transduced cells, but most of these cells are concentrated at the injection site. Cells at these sites may have more vector genome copy number / cell than any other ROA, but the vector diffusion from these sites is significantly lower compared to ICV ROAs. Wider distribution of GFP transduction via ICV administration is advantageous because a suitable balance can be achieved between the number of transduced cells and the number of vector copies per transduced cell.

[0543] Indeed, the high concentrations of transgene expression in the IP brains of Examples 2 and 3 were associated with a smaller but significant reduction in oligodendrocyte tropism and a balanced increase in neurotropism within the cortex. This suggests that saturation of the region with AAV / Olig001 can lead to a specific reduction in oligodendrocytes. It should be noted that in the animals used in this study, the number of cortical oligodendrocytes was reduced relative to wild-type NUR7 mice, and there was evidence of stress and apoptosis (Francis et al. (2012) J. Cereb. Blood Fl. Metab. 32:1725-1736), which is expected to affect transduction efficiency.

[0544] Except for the cerebellum, carrier tropism was oligotropy in all regions of interest (ROAs) ranging from 75-90%. In all ROA groups, this region exhibited >80% neurotropism. Particularly strong transgene expression was observed in Purkinje neurons in the granular layer. The reason for this apparent reversal of tropism is not clear, but the cerebellum is clearly a unique anatomical entity relative to the resident cell type. Purkinje cells within the cerebellum express Olig2 at low but considerable levels, and the AAV / Olig001 capsid likely exhibits significantly different interactions with the surface of Purkinje neurons compared to the surfaces of other neurons in other brain regions.

[0545] The present embodiments demonstrate that AAV / Olig001 promotes strong expression of oligodendrocyte transgenes throughout the brain of NUR7 Canavan disease mice, with the cerebellum being a significant exception. In all other brain regions, >70% oligotropy was achieved without the need for lineage-specific promoters. The inherent affinity of the AAV / Olig001 capsid for oligodendrocyte surfaces is a significant advantage over selective promoter use in other non-oligotropy capsid serotypes, as it ensures that the vector is expressed in target cells at a dose as close as possible to the total delivered dose. These data identify the advantages of targeting different ROAs in the white matter of the brain, with ICV ROA demonstrating suitability for preclinical efficacy studies in symptomatic adult NUR7 mice as a model for treating Canavan disease.

[0546] Example 4: Difference in AAV / Olig001-GFP transduction efficiency between wild-type and NUR7 brains

[0547] The NUR7 mouse model of canavan disease exhibits symptoms of overall motor dysfunction at 2 weeks of age. By 6 weeks of age, the NUR7 brain suffers from significant cellular loss, white matter loss, and extensive vacuolation. Therefore, the 6-week-old NUR7 brain is a significantly different microenvironment compared to the healthy brain, potentially affecting the spread and transduction of AAV / Olig001-GFP. Indeed, compared to the transduction levels in the NUR7 mouse brain, in a cohort of 6-week-old wild-type mice, administration of 1×10⁻⁶ AAV / Olig001-GFP via ICV ROA significantly reduced the transduction levels. 11 The dose resulted in a significant reduction in transduction levels in both the cortex and subcortical white matter. Figure 12 (Each group has n = 5 animals, and the values ​​are shown as mean + / - sem, *p≤0.05, **p≤0.01).

[0548] Stereoscopic estimation of GFP-positive cells in the cortex and subcortical white matter demonstrated a significantly reduced incidence (at least 50%) of transgene expression in the wild-type brain. Strong GFP fluorescence was confined to regions immediately adjacent to the lateral ventricles, and moderate GFP fluorescence signals were observed in the cortex and subcortical white matter in the wild-type brain. Transgene expression was poor in the cerebellum. These data suggest a genotype-specific effect on the propagation and transduction efficiency of AAV / Olig001. Furthermore, because the nur7 brain, like the human canavan brain, exhibits severe vacuolation, excessively large ventricles, and significantly elevated NAA, these signs and symptoms could potentially influence the vector propagation and biodistribution of human AAV / Olig001 therapeutic agents.

[0549] Example 5: In vivo administration of AAV / Olig001-ASPA to NUR7 mice improved rotarod performance.

[0550] method

[0551] A single dose of AAV / Olig001-ASPA containing the codon-optimized ASPA sequence of SEQ ID NO:2 was administered to 6-week-old NUR7 mice. Expression plasmids encoding the codon-optimized ASPA and regulatory elements were then... Figure 13 As shown in the figure. 2.5 × 10 11 7.5×10 10 Or 2.5×10 10 The total dose of the vehicle was administered via the intraventricular (ICV) administration route (ROA). All dose groups were delivered in a total volume of 5 μl, with 2.5 μl injected into the lateral ventricle of each hemisphere of the brain. An age-matched control group of NUR7 animals was generated by injecting an equal volume of saline via the same ROA. Age-matched naive wild-type animals were used as calibration references for all motor function tests. Animals were tested monthly for four months, two weeks after vehicle administration, targeting waiting time from rotundus drop and overall activity in the open field activity room. All behavioral tests were performed by individuals unaware of the treatment.

[0552] result

[0553] Rotating bar performance

[0554] At the highest dose applied (2.5 × 10⁻⁶), 11 At a dose of 7.5 × 10⁻⁶, AAV / Olig001-ASPA rescued progressively deteriorating balance, grip strength, and / or motor coordination in NUR7 mice, as measured by rotarod performance, to levels indistinguishable from those in age-matched wild-type animals and significantly improved relative to sham-treated NUR7 controls. At this dose, the increased rotarod performance in AAV / Olig001-ASPA-treated animals was significant throughout the study period (p = 0.028), as determined by repeated measures ANOVA, and significantly higher at each individual time point, as determined by unpaired Student's t-test. 10 At the lowest dose (vg), AAV / Olig001-ASPA also promoted significantly improved rotator performance in NUR7 mice at each tested time point, but this improvement was not significant throughout the study period (repeated measures ANOVAp = 0.19). 10Under the vg) treatment, AAV / Olig001-ASPA effectively promoted improved rotator performance only at the last two time points tested (18 and 22 months). Table 1 provides the mean drop wait time in seconds (with standard deviation) for each treatment group. For each group, 12 mice (6 males and 6 females) were tested. Table 2 provides the p-values ​​for unpaired t-test comparisons between AAV / Olig001-ASPA treatment and sham treatment in NUR7 mice at each age. Except for administration of 2.5 × 10 at 10 and 14 weeks. 10 Statistically significant improvements were observed in all groups of mice except for those treated with the sham treatment.

[0555] Table 1. Waiting time for the rotor to fall.

[0556]

[0557] Table 2. P-values ​​for the difference in rotarod waiting time between AAV / Olig001-ASPA treated mice and sham-treated mice.

[0558]

[0559] Figure 14 The plotted mean rotundum drop wait times during the life study period are shown for each AAV / Olig001-ASPA nur7 dose group, the sham-treated nur7, and the naive wild-type control. Drop wait times increased in all three dose groups, with the highest dose being significant throughout the study period by repeated-measures ANOVA(*).

[0560] Open field activities

[0561] At each age during bar rotation, the animals' generalized locomotor function in the open activity room was also assessed. Figure 15 Animals were given a single 20-minute course each time, and the total distance traveled in each course was recorded. Compared to age-matched wild-type animals, the sham-treated NUR7 animals showed significant hyperactivity at all ages, especially at the last time point. At 22 weeks of age, the sham-treated NUR7 animals showed a significant 3-fold increase in activity (distance traveled; p = 0.0202) compared to the wild-type. Conversely, 2.5 × 10⁻⁶ NUR7 animals showed significantly less activity (distance traveled; p = 0.0202). 11 A dose of AAV / Olig001-ASPA resulted in normal activity levels in NUR7 mice, which were statistically significant relative to the sham-treated control (p = 0.0312) and indistinguishable from age-matched wild-type mice. (Lower 7.5 × 10⁻⁶) 10The dose of AAV / Olig001-ASPA resulted in an activity pattern more closely similar to the wild-type rather than the sham-treated nur7 pattern, but at 22 weeks of age, it was just below the threshold of statistical significance compared to the sham-treated pattern (p = 0.1181). The lowest (2.5 × 10⁻⁶) 10 The dose of AAV / Olig001-ASPA did not significantly normalize pathological hyperactivity and more closely resembled the sham-treated nur7 control than the wild-type reference.

[0562] Assessment of open field activity in these same animals demonstrated dose-dependent normalization of excessive activity in the AAV / Olig001-ASPA treated nur7 animals. Data are presented as mean + / - sem, with n = 6 animals per group.

[0563] NAA accumulation and vector genome (vg) copy number

[0564] After a 22-week rotator test, mice were euthanized and brain tissue was isolated. One hemisphere of each brain was processed for HPLC analysis of NAA, and the remaining hemispheres were processed for vector genome (vg) copy number analysis by quantitative PCR.

[0565] The brains of NUR7 mice treated with sham saline contained typically higher levels of NAA, as expected from the loss of ASPA function. Figure 16 A dose-responsive reduction in pathologically elevated NAA was observed in the AAV / Olig001-ASPA treatment group, with a maximum of 2.5 × 10⁻⁶. 11 The dose resulted in a very significant 2.6-fold reduction (p = 5.06 × 10⁻⁶). -6 ), medium 7.5×10 10 The dose resulted in a 1.6-fold reduction (p = 5.17 × 10⁻⁶). -5 ), and the lowest is 2.5×10 10 The dose resulted in a 1.4-fold reduction (p = 0.001). NAA in the nur7 brain treated with the highest dose of AAV / Olig001-ASPA was in fact significantly lower than NAA in the age-matched wild-type brain (p = 0.0012).

[0566] The vector genome (vg) copy number was quantified using quantitative PCR of the remaining hemispheres of the brain analyzed for NAA, employing a custom TaMan probe / primer set targeting the bovine growth hormone (BGH) polyadenylation sequence of the recombinant AAV / Olig001-ASPA expression cassette. Total DNA content in the hemispheres was isolated using commercially available DNA purification columns and kits (Qiagen), and the resulting DNA samples were run against a purified plasmid standard curve to produce vg / wet tissue weight for each sample. The resulting VG / mg tissue value reflects the dose of AAV / Olig001-ASPA administered (…). Figure 17 Its response to carrier dose is consistent with that of NAA.

[0567] Cavitation analysis

[0568] Unbiased stereoscopic analysis of the brains of NUR7 mice treated with AAV / Olig001-ASPA was used to quantify the vacuolar volume fraction in the thalamus and cerebellar white matter / pons as a function of carrier dose. Figure 18 The area occupied by cavitation within each region of interest is defined as a vacuole and presented as a percentage of the overall region of interest volume. At each dose, AAV / Olig001-ASPA treatment resulted in complete rescue of thalamic vacuolation, as shown by a significant reduction in the thalamic vacuolar volume fraction compared to sham-treated mice (2.5 × 10⁻⁶). 11 p = 4.6 × 10 -8 7.5×10 10 p = 6.4 × 10 -8 ; and 2.5×10 10 p = 6.2 × 10 -8 ()( Figure 19 Compared with sham-treated mice, vacuolation in the cerebellar white matter / pons was also significantly rescued at all doses (2.5 × 10⁻⁶). 11 p = 1.3 × 10 -5 7.5×10 10 p = 2.5 × 10 -5 ; and 2.5×10 10 (p = 0.0009), but the degree of rescue is proportional to the dose of the carrier applied. Minimum 2.5 × 10⁻⁶. 10 The vacuolar volume fraction in the dose group was significantly higher than that at the highest dose of 2.5 × 10⁻⁶. 11 The volume fraction of air bubbles in the dose (p = 5.74 × 10⁻⁶) -6 While still significantly less than the vacuolar volume fraction in the sham control (p = 0.0009) Figure 19 ).

[0569] Oligodendrocyte recovery

[0570] The same brain samples used for vacuolation analysis were processed for Olig2 immunohistochemistry to identify oligodendrocytes. Unbiased stereoscopy was used to sample the thalamus and cortex for Olig2-positive cells to identify significant differences in white matter-producing cells in regions affected and unaffected by vacuolation. Figure 20 Compared to age-matched wild-type brains, sham-treated NUR7 brains showed a significant 4.6-fold loss of Olig2-positive cells, representing only 21% of the normal wild-type content (p = 4.9 × 10⁻⁶). -7 Olig2 counts in the thalamus of AAV / Olig001-ASPA treated NUR7 mice and sham-treated NUR7 mice ( Figure 21 The results showed that, compared to the sham control, all three AAV / Olig001-ASPA-treated nur7 cohorts had a significant increase in oligodendrocytes (2.5 × 10⁻⁶). 11 vg, p = 6.75 × 10 -8 7.7×10 10 vg, p = 0.026; 2.3 × 10 10 vg, p = 3.18 × 10 -5 Olig2 loss in the cortical region was not drastic but significant (a 1.7-fold reduction in sham-treated Nur7 mice compared to wild-type mice; p = 0.0025). Compared to sham-treated Nur7 control mice, 2.5 × 10⁻⁶ Olig2 loss was observed. 11 Olig2 content in the cortex of NUR7 brain treated with VG ( Figure 21 The level was also significantly increased (p = 0.0002), but not significantly increased in the brains of the two lower dose groups.

[0571] Neuron recovery

[0572] In the same 22-week-old brains used for Olig2 staining, the thalamus and cortex were scored against NeuN-positive neurons. Figure 22 The number of thalamic neurons in the sham-treated NUR7 animals was approximately 35% of the value in age-matched wild-type animals (p = 2.8 × 10⁻⁶). -5 ()( Figure 23 ). Using 2.5×10 11 Nur7 mice treated with AAV / Olig001-ASPA contained a 2.3-fold increase in the number of thalamic neurons compared to sham-treated control mice (p = 0.0009) and approximately 84% of the thalamic neurons observed in wild-type mice. At two lower doses of 7.5 × 10⁻⁶, the number of neurons was significantly increased. 10 and 2.5×10 10Under these conditions, AAV / Olig001-ASPA promoted an increase in NeuN-positive cells in the thalamus, increasing by 1.8 and 1.6 times, respectively, compared to sham-treated control mice (p = 0.012; p = 0.042). In the cortex (motor and somatosensory), neuronal loss in the brains of sham-treated NUR7 mice was less severe but significant compared to age-matched wild-type mouse brains. The cortex of sham-treated mice contained approximately 80% of the NeuN-positive cells observed in wild-type mice, representing a 1.2-fold reduction (p = 0.005). Using 2.5 × 10⁻⁶ 11 The number of cortical neurons in AAV / Olig001-ASPA-treated Nur7 mice was approximately 98% of that observed in wild-type mice, and 1.2-fold higher than that observed in sham-treated Nur7 mice (p = 0.013). Continuous doses of AAV / Olig001-ASPA resulted in a stable 1.2-fold increase in cortical neurons relative to the sham treatment. For 7.5 × 10 10 The high variance of the sampling data made this increase insignificant (p = 0.113). At the lowest dose of 2.5 × 10⁻⁶... 10 At the specified dose, mice treated with AAV / Olig001-ASPA maintained a significant 1.2-fold increase in cortical neurons compared to the sham-treated control (p = 0.05).

[0573] Improved myelination

[0574] Cortical myelin-positive fiber length density (MBP-LD) in the entire cortex of 22-week-old NUR7 brains treated with unbiased stereoscopic quantitative sham treatment and AAV / Olig001-ASPA was used to provide an index of myelination recovery after AAV / Olig001-ASPA treatment. Computer-generated probes were used to sample MBP-positive fibers in the motor and somatosensory cortices to score isotropic probe fiber interactions in 3D tissue space. The final MBP length density (μm fibers / mm) was then obtained by dividing the total sum of MBP-positive fiber lengths within the cortex by the volume of the sampled tissue. 3 ()( Figure 24 When compared with age-matched wild-type brains, the sham-treated NUR7 brains showed a highly significant 2-fold reduction in cortical MBP-LD (p = 0.0001). Treatment with all three doses of AAV / Olig001-ASPA resulted in a significant increase in cortical MBP-LD relative to the sham-treated control, with the degree of improvement being dose-proportional (2.5 × 10⁻⁶). 11 p = 0.0014; 7.5 × 10 10 p = 0.003; 2.5 × 10 10p = 0.016). Brains of sham-treated and AAV / Olig001-ASPA-treated NUR7 mice were stained with antimyelin basic protein (MBP). Figure 25 ).

[0575] These data demonstrate that AAV / Olig001-ASPA treatment in a mouse model of canavan disease improves balance, grip strength and / or motor coordination, motor function, reduces the amount of NAA present in the brain, reduces brain vacuolation, increases the number of Olig2 and NeuN positive cells, and restores myelin formation.

[0576] Example 6: CLARITY-assisted biodistribution for Canavan gene therapy

[0577] The biodistribution of the oligodendrocyte-tropy rAAV vector (Olig001) with a green fluorescent protein (GFP) transgene in the brains of Nur7 mice exhibiting a canavan disease phenotype was assessed using three-dimensional (3D) tissue clearing and imaging methods. This allowed for a global representation and volumetric measurement of the vector biodistribution within the hemispheres of Nur7 mice administered via an alternative route of administration (ROA). The biodistribution efficacy of intraventricular (ICV) and intraparenchymal (IP) ROAs was compared, and this method was used to supplement conventional stereological data obtained from traditional two-dimensional (2D) histological assessments.

[0578] This embodiment demonstrates the applicability of the 3D method and its significance in assessing the biodistribution of AAV / Olig001-GFP in the adult mouse hemisphere of a canavan disease mouse model. The results are presented as 3D transparent brain images of light sheet microscopy data and a visual qualitative and quantitative representation of the list parameters for biodistribution estimation.

[0579] Sample preparation and imaging

[0580] Four adult mice of each ROA (eight mice in total) were given 5 × 10⁻⁶ doses at 6 weeks of age. 11 Vector genome (vg) / animals were administered and the animals were sacrificed two weeks later. PFA-fixed brains were received and prepared for 3D tissue clearing and volumetric light sheet microscopy imaging. Each brain was sagittally bisected, and the right hemisphere was cleared using CLARITY (Chung et al., Nature, 2013). Samples were similarly prepared by hydrogel embedding and polymerization, followed by electrophoretic clearing using a commercially available device (X-Clarity, Logos Biosystems) and commercially available reagents (Logos Biosystems). Macroscopic micrographs of the key steps during sample processing were obtained to document sample condition. Figure 26 ).

[0581] Full 3D microscopic imaging of each transparent hemisphere was performed using a Zeiss Z.1 light sheet microscope with 5× magnification objectives and tile-based acquisition covering the entire hemisphere. Imaging parameters were adjusted to detect GFP expression and kept constant across all samples to ensure consistency and enable relative comparisons. All samples were processed and imaged under identical conditions from tissue transparentization to image acquisition and analysis.

[0582] Image processing and analysis

[0583] The original dataset was preprocessed and reconstructed into complete, seamless 3D images using internally custom-designed algorithms for each hemisphere. The final images, each containing one hemisphere, were then input into commercial 3D image processing and analysis programs (Imaris, Bitplane) for holistic, quantitative biodistribution analysis. First, the overall mean and median (GFP) signal values ​​within the entire hemisphere volume were calculated. Furthermore, two GFP intensity thresholds were selected to specify “low” or “high” GFP expression. Figure 27 These thresholds were then kept constant across all samples to achieve consistency. The volumes of these classification intensity regions were then determined and compared with the whole hemisphere volume to generate “vol% high / low expression” (Table 3).

[0584] result

[0585] Macroscopic micrographs and full 3D imaging of each hemisphere revealed variable biodistribution patterns of GFP expression in the two ROAs (IP versus ICV); Figure 28 (and Figure 30). Additionally, cell type tropism was assessed visually by evaluating cell morphology and their determined spatial location. While these biodistribution patterns varied in the samples depending on the extent of vector spread, similarities in subregional transduction patterns remained consistent across the samples, such as high expression in Purkinje cells in the cerebellum. Then, for each hemisphere, the quantifications of “low” and “high” GFP expression, along with the overall intensity, were calculated and listed (Table 3). Consistent with the stereographic assessments in previous examples, the clear hemispheres exhibited superior vector spread within the subcortical white matter, a key region for Canavan disease, following ICV injection. Furthermore, although IP injection produced subregions with high GFP intensity, most of these subregions were concentrated near the injection site, supporting the conclusions drawn from the stereographic assessments.

[0586] Table 3. Quantitative analysis of four ICV-injected hemispheres.

[0587]

[0588] Conclusions and significance

[0589] Volumetric imaging of the fully transparent mouse brain provides a more comprehensive and holistic assessment of the AAV / Olig001 biodistribution. Customized algorithms enabling complete acquisition and quantification of the distribution support higher-resolution quantification from stereoscopic methods. Assessment using organ-level imaging provides a holistic evaluation of this biodistribution while preserving 3D spatial structure and regional connectivity. Finally, digital compilations of various ROAs can be used to generate a digital "library" for future reference when conducting additional assessments of AAV / Olig001 ROAs to evaluate optimal transduction efficiency and cell type specificity.

[0590] Example 7: Volumetric assessment of AAV biodistribution and pharmacodynamic effects based on CLARITY

[0591] In this embodiment, the CLARITY tissue transparency technique described in Example 6 above was used to evaluate and demonstrate the pharmacological effects of overall and local transgene-mediated demyelination reversal following injection of AAV / Olig001-ASPA into the brains of NUR7 mice.

[0592] In summary, Nur7 mice were divided into two groups and AAV / Olig001-ASPA (“Olig1” or “Olig1-ASPA”) or saline (“Nur7”) were administered via ICV or IP as described above. The brains of both groups were then analyzed as described above to quantify the vacuolar volume fraction in the thalamus and cerebellar white matter / pons. The brains of wild-type mice (“WT”) were also analyzed as a control group. The results are shown in Figure 31. More specifically, the arrows in Figure 31B indicate visible vacuolation in the thalamic regions of Nur7 mice, which was absent in WT and almost completely rescued in Olig1-ASPA-treated tissue. Additionally, as shown in Figure 31C, after one day of passive clearing, Nur7 mouse tissue achieved higher transparency than WT and Olig1-ASPA-treated tissues. These results demonstrate that AAV / Olig001-ASPA treatment reduces brain vacuolation and restores myelin formation in Nur7 mice.

[0593] Cell counting analysis was also performed on 2D single slices extracted from 3D images of all three groups with similar anatomical orientations (Fig. 32A). As shown in Figs. 32B and 32C, although the mean nuclear density (counts normalized according to segmented regions) showed very little overall difference in cell density within the cortical regions, mice in the Nur7 group had significantly lower overall nuclear density / nuclear area ratio in the thalamus. In contrast, the Olig1-ASPA and WT groups appeared to have similar overall nuclear density or nuclear area ratio in the thalamus. These results demonstrate that AAV / Olig001-ASPA treatment in Nur7 mice maintained or increased the number of cells in the thalamus to levels close to those observed in the WT group.

[0594] Brain tissues used for vacuolation analysis were treated as described above for immunofluorescence staining of MBP to identify oligodendrocytes. For this purpose, 3D volume analysis was performed to examine the pharmacodynamic therapeutic effect. The complete 3D volume of 2 mm tissue sections was measured, and the mean fluorescence intensity of SYTO (nuclear marker) and MBP was calculated. Tissues from mice in the Nur7 group showed lower mean MBP fluorescence values. In contrast, the Olig1-ASPA treatment group had increased overall MBP signal, almost reaching the level of the WT group (Figure 33B).

[0595] Further 3D volume analysis was performed, where MBP volume was calculated using signal thresholding. Thresholding was performed either more restrictively with a threshold set at fluorescence values ​​exceeding 2000 (Fig. 33C, left panel) or more inclusively with a threshold of 1000 (Fig. 33C, right panel). In both cases, MBP deficiency was observed in mice in the Nur7 group (Fig. 33D). In contrast, a significant increase in MBP volume was observed in the Olig1-ASPA group, and particularly when using lower thresholds, with total MBP volume values ​​approaching those of the WT group (Fig. 33D).

[0596] Region-based analysis was performed in 3D within the thalamus region. Manual segmentation of a portion of the region is shown in Figure 33E. The mean fluorescence intensity of nuclear (SYTO) and myelin (MBP) markers within this region is shown in Figure 33F. The SYTO and MBP levels in the Olig1-ASPA group were found to be almost equal to those in the WT group. In contrast, the Nur7 sample showed lower mean fluorescence values ​​for both markers. Region-based analysis was also performed on a portion of the cortex. Figures 33G and 33H show the mean fluorescence intensity levels of nuclear (SYTO) and myelin (MBP) markers within this cortical region. The overall trend is similar to that shown in Figure 33F. 3D cell concentrations (per 100 μm) in the cortical and thalamic regions were also obtained. 2 (The cell nucleus). For example... Figure 33I As shown, the overall nuclear concentration in both regions was lower in the Nur7 group mice. In contrast, the 3D cell concentration in the thalamic region of the Olig1-ASPA group mice showed levels close to those in the WT group.

[0597] These results demonstrate that administration of AAV / Olig001-ASPA can rescue or reverse demyelination and cell loss in the brains of NUR7 mice.

[0598] equivalent

[0599] The foregoing written description is considered sufficient to enable those skilled in the art to practice this disclosure. The foregoing description and examples detail certain exemplary embodiments of this disclosure. However, it should be understood that, however detailed the foregoing may be in textual form, this disclosure can be practiced in many ways and should be interpreted in accordance with the appended claims and any equivalents thereof.

[0600] All references cited in this article, including patents, patent applications, papers, textbooks, and the references cited therein, are incorporated herein by reference in their entirety, to the extent that they have not yet been cited.

[0601] Table 4

[0602] sequence

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609] sequence list <110> Pfizer Inc. <120> Modified nucleic acids and vectors encoding aspartate acylase (ASPA) for gene therapy <130> 323429.00101 <150> 63 / 016,507 <151> 2020-04-28 <150> 63 / 077,144 <151> 2020-09-11 <160> 20 <170> PatentIn Version 3.5 <210> 1 <211> 942 <212> DNA <213> Artificial <220> <223> Synthetic <400> 1 atgacctcct gtcatatagc cgaggagcac atccagaaag tggccatttt cggcgggaca 60 catgggaacg agctgactgg cgttttcctg gtcaagcact ggctcgaaaa tggcgcggaa 120 attcagagaa cgggcctgga ggtcaaacct tttattacta acccccgcgc ggtgaagaaa 180 tgtacccggt acatcgactg cgatcttaac cgaatctttg atctggaaaa tctgggaaaa 240 aaaatgagcg aggacctgcc ctacgaagtc cgcagagcac aggagattaa tcatctcttc 300 ggacccaagg actccgagga cagctacgat atcatcttcg acttgcacaa tactacttcc 360 aatatgggat gtaccttgat actggaggac tcacgaaata acttcttgat tcagatgttc 420 cattacatca aaacctctct cgctcctctc ccttgctacg tatatttgat cgagcaccct 480 agtctgaaat atgccactac acgaagcata gctaagtatc ccgttggtat tgaggtgggc 540 ccccagcccc agggagtgct gcgggctgac atccttgacc agatgagaaa aatgatcaaa 600 cacgcccttg acttcatcca ccactttaat gaaggcaaag agtttcctcc ctgtgccata 660 gaggtgtata aaatcatcga aaaagttgac tatccacggg atgagaacgg cgagatcgct 720 gccatcatcc atcccaattt gcaagatcag gattggaaac ctttgcaccc aggcgaccct 780 atgttcctga cattggatgg caagaccata cccctgggtg gtgattgcac tgtgtaccca 840 gttttcgtaa acgaggcagc gtactatgaa aagaaagagg catttgcaaa aaccactaag 900 ttgacactga atgccaagag cattagatgc tgtcttcatt aa 942 <210> 2 <211> 942 <212> DNA <213> Artificial <220> <223> Synthetic <400> 2 atgacctcct gtcatatagc cgaggagcac atccagaaag tggccatttt cggcgggaca 60 cacggaaacg aacttacagg agtgtttctg gtgaaacact ggcttgaaaa tggtgcggag 120 atccaaagga ccggcctgga ggtcaaacct tttattacaa atccccgggc ggtcaagaag 180 tgcacacggt acattgattg tgatcttaat cgcatattcg acctggagaa ccttgggaag aaaatgtctg aagatctgcc ctacgaagtg aggcgagcac aagagataaa ccacctgttc 360. ggaccgaaag acagtgaaga ctcctatgac atcattttcg acctgcacaa cactacgagt aacatggggt gtaccctgat cctcgaagac tcccgaaaca atttcctgat acagatgttt cattacatca aaactagtct ggcccctctc ccctgctcg tttatctgat cgaacaccct 540. tctctcaaat acgctaccac ccgctctatt gctaagtacc ccgtcgggat cgaggtcggc ccacaacctc aaggtgtgct ccgggccgat attttggacc agatgagaaa gatgattaaa 660. cacgctctcg acttcattca ccactttaac gaggggaagg aatttccccc ttgtgccatc gaggtttata agattatcga gaaggtggac tacccaagag acgaaaacgg ggagatagct gccatcatcc accctaattt gcaagatcag gactggaagc ccctgcaccc aggagacccc 780 atgtttctga ccttggatgg aaagacgatc cccctgggcg gtgattgtac agtgtaccca 840 gtctttgtca acgaggccgc ttactatgag aaaaaggagg cttttgcaaa gacaacaaag ctcactttga atgcaaagtc catcaggtgc tgtctgcact aa 942 <210> 3 <211> 942 <212> DNA <213> Homo sapiens <400> 3 atgacttctt gtcacattgc tgaagaacat atacaaaagg ttgctatctt tggaggaacc 60 catgggaatg agctaaccgg agtatttctg gttaagcatt ggctagagaa tggcgctgag 120 attcagagaa cagggctgga ggtaaaacca tttattacta accccagagc agtgaagaag 180 tgtaccagat atattgactg tgacctgaat cgcatttttg accttgaaaa tcttggcaaa 240 aaaatgtcag aagatttgcc atatgaagtg agaagggctc aagaaataaa tcatttattt 300 ggtccaaaag acagtgaaga ttcctatgac attatttttg accttcacaa caccacctct 360 aacatggggt gcactcttat tcttgaggat tccaggaata actttttaat tcagatgttt 420 cattacatta agacttctct ggctccacta ccctgctacg tttatctgat tgagcatcct 480 tccctcaaat atgcgaccac tcgttccata gccaagtatc ctgtgggtat agaagttggt 540 cctcagcctc aaggggttct gagagctgat atcttggatc aaatgagaaa aatgattaaa 600 catgctcttg attttataca tcatttcaat gaaggaaaag aatttcctcc ctgcgccatt 660 gaggtctata aaattataga gaaagttgat tacccccggg atgaaaatgg agaaattgct 720 gctatcatcc atcctaatct gcaggatcaa gactggaaac cactgcatcc tggggatccc 780 atgtttttaa ctcttgatgg gaagacgatc ccactgggcg gagactgtac cgtgtacccc 840 gtgtttgtga atgaggccgc atattacgaa aagaaagaag cttttgcaaa gacaactaaa 900 ctaacgctca atgcaaaaag tattcgctgc tgtttacatt ag 942 <210> 4 <211> 313 <212> PRT <213> Homo sapiens <400> 4 Met Thr Ser Cys His Ile Ala Glu Glu His Ile Gln Lys Val Ala Ile 1 5 10 15 Phe Gly Gly Thr His Gly Asn Glu Leu Thr Gly Val Phe Leu Val Lys 20 25 30 His Trp Leu Glu Asn Gly Ala Glu Ile Gln Arg Thr Gly Leu Glu Val 35 40 45 Lys Pro Phe Ile Thr Asn Pro Arg Ala Val Lys Lys Cys Thr Arg Tyr 50 55 60 Ile Asp Cys Asp Leu Asn Arg Ile Phe Asp Leu Glu Asn Leu Gly Lys 65 70 75 80 Lys Met Ser Glu Asp Leu Pro Tyr Glu Val Arg Arg Ala Gln Glu Ile 85 90 95 Asn His Leu Phe Gly Pro Lys Asp Ser Glu Asp Ser Tyr Asp Ile Ile 100 105 110 Phe Asp Leu His Asn Thr Thr Ser Asn Met Gly Cys Thr Leu Ile Leu 115 120 125 Glu Asp Ser Arg Asn Asn Phe Leu Ile Gln Met Phe His Tyr Ile Lys 130 135 140 Thr Ser Leu Ala Pro Leu Pro Cys Tyr Val Tyr Leu Ile Glu His Pro 145 150 155 160 Ser Leu Lys Tyr Ala Thr Thr Arg Ser Ile Ala Lys Tyr Pro Val Gly 165 170 175 Ile Glu Val Gly Pro Gln Pro Gln Gly Val Leu Arg Ala Asp Ile Leu 180 185 190 Asp Gln Met Arg Lys Met Ile Lys His Ala Leu Asp Phe Ile His His 195 200 205 Phe Asn Glu Gly Lys Glu Phe Pro Pro Cys Ala Ile Glu Val Tyr Lys 210 215 220 Ile Ile Glu Lys Val Asp Tyr Pro Arg Asp Glu Asn Gly Glu Ile Ala 225 230 235 240 Ala Ile Ile His Pro Asn Leu Gln Asp Gln Asp Trp Lys Pro Leu His 245 250 255 Pro Gly Asp Pro Met Phe Leu Thr Leu Asp Gly Lys Thr Ile Pro Leu 260 265 270 Gly Gly Asp Cys Thr Val Tyr Pro Val Phe Val Asn Glu Ala Ala Tyr 275 280 285 Tyr Glu Lys Lys Glu Ala Phe Ala Lys Thr Thr Lys Leu Thr Leu Asn 290 295 300 Ala Lys Ser Ile Arg Cys Cys Leu His 305 310 <210> 5 <211> 106 <212> DNA <213> Artificial <220> <223> Synthetic <400> 5 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgg 106 <210> 6 <211> 284 <212> DNA <213> Artificial <220> <223> Synthetic <400> 6 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 120 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 120 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 180 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 180 cgtcaatgac ggtaaatggc ccgcctggca tttgcccagt acatgacctt atgggacttt 240 cgtcaatgac ggtaaatggc ccgcctggca tttgcccagt acatgacctt atgggacttt 240 cctacttggc agtacatcta cgtattagtc atcgctatta ccat 284 cctacttggc agtacatcta cgtattagtc atcgctatta ccat 284 <210> 7<210> 7 <211> 278<211> 278 <212> DNA<212> DNA <213> 人工<213> Artificial <220><220> <223> 合成<223> Synthetic <400> 7<400> 7 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 60 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 60 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg 120 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg 120 ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg 180 ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg 180 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc 240 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc 240 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcg 278 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcg 278 <210> 8 <210> 8 <211> 82 <211> 82 <212> DNA <212> DNA <213> Artificial <220> <223> Synthesis <400> 8 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg 60 ccccggctct gactgaccgc gt 82 <210> 9 <211> 43[[ID=;15]] <212> DNA <213> Artificial <220> <223> Synthesis <400> 9 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agc 43 <210> 10 <211> 92 <212> DNA <213> Artificial <220> <223> Synthesis <400> 10 》aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag 60 cacctgcctg aaatcacttt ttttcaggtt gg 92 <210> 11 <211> 225 <212> DNA <213> Artificial <220> <223> Synthesis <400> 11 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120 It should be noted that there may be some inaccuracies in the content due to the nature of the original text which seems to be a series of code-like sequences. If this is from a specific technical field, it may require more in-depth domain knowledge for a more accurate translation and understanding.tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180 gggaagacaa cagcaggcat gctggggatg cggtgggctc tatgg 225 <210> 12 <211> 113 <212> DNA <213> artificial <220> <223> synthetic <400> 12 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctggc 60 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctg 113 <210> 13 <211> 2214 <212> DNA <213> artificial <220> <223> synthetic <400> 13 atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60 cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120 gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180 aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240 cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300 caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420 ggaaaagaa ggcctgtaga gcagtctcct caggaaccgg actcctcctc gggcatcggc 480 aagacaggcc agcagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540 tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600 cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660 gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720 accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780 tccaacggga catcgggagg agccaccaac gacaacacct acttcggcta cagcacccc 840 tggggtatt ttgactttaa cagattccac tgccactttt caccacgtga ctggcagcga 900 ctcatcaaca acaactgggg attccggccc aagagactca gcttcaagct cttcaacatc 960 caggtcaagg aggtcacgca gaatgaaggc accaagacca tcgccaataa ccttaccagc 1020 acggtccagg tcttcacgga ctcggagtac cagctgccgt acgttctcgg ctctgcccac 1080 cagggctgcc tgcctccgtt cccggcggac gtgttcatga ttccccagta cggctaccta 1140 acactcaaca acggtagtca ggccgtggga cgctcctcct tctactgcct ggaatacttt 1200 ccttcgcaga tgctgagaac cggcaacaac ttccagttta cttacacctt cgaggacgtg 1260 cctttccaca gcagctacgc ccacagccag agcttggacc ggctgatgaa tcctctgatt 1320 gaccagtacc tgtactactt gtctcggact caaaacag gaggcacggc aaatacgcag 1380 actctgggct tcagccaagg tgggcctaat acaatggcca atcaggcaaa gaactggctg 1440 ccaggaccct gttaccgcca acaacgcgtc tcaacgacaa ccgggcaaaa caacaatagc 1500 aactttgcct ggactgctgg gaccaaatac catctgaatg gaagaaattc attggctaat 1560 cctggcatcg ctatggcaac acacaaagac gacaaggagc gtttttttcc cagtaacggg 1620 atcctgattt ttggcaaaca aaatgctgcc aagacaatg cggattacag cgatgtcatg 1680 1740 gtggcagata acttgcagca gcaaaacacg gctcctcaaa ttggaactgt caacagccag 1800 ggggccttac ccggtatggt ttggcagaac cgggacgtgt acctgcaggg tcccatctgg 1860 gccaagattc ctcacacgga cggcaacttc cacccgtctc cgctgatggg cggctttggc 1920 ctgaaacatc ctccgcctca gatcctgatc aagaacacgc ctgtacctgc ggatcctccg 1980 accaccttca accagtcaaa gctgaactct ttcatcacgc aatacagcac cggacaggtc 2040 agcgtggaaa ttgaatggga gctgcagaag gaaaacagca agcgctggaa ccccgagatc 2100 cagtacacct ccaactacta caaatctaca agtgtggact ttgctgttaa tacagaaggc 2160 gtgtactctg aaccccaccc cattggcacc cgttacctca cccgtcccct gtaa 2214 <210> 14 <211> 737 <212> PRT <213> Artificial <220> <223> Synthetic <400> 14 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp Asn 260 265 270 Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr Thr 405 410 415 Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr Leu Gly Phe 450 455 460 Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp Leu 465 470 475 480 Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly Gln 485 490 495 Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His Leu 500 505 510 Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr His 515 520 525 Lys Asp Asp Lys Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile Phe 530 535 540 Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val Met 545 550 555 560 Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu 565 570 575 Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala Pro 580 585 590 Gln Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp 595 600 605 Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro 610 615 620 His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly 625 630 635 640 Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro 645 650 655 Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe Ile 660 665 670 Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu 675 680 685 Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser 690 695 700 Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu Gly 705 710 715 720 Val Tyr Ser Glu Pro His Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro 725 730 735 Leu <210> 15 <211> 736 <212> PRT <213> Artificial <220> <223> Synthetic <400> 15 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Le...

Claims

1. An isolated nucleic acid encoding aspartoacyltransferase (ASPA) consisting of the nucleic acid sequence of SEQ ID NO:

2.

2. A vector genome comprising a modified nucleic acid encoding aspartoacyltransferase (ASPA) consisting of the nucleic acid sequence of SEQ ID NO:

2.

3. The vector genome of claim 2, wherein the vector genome is a recombinant adeno-associated viral (rAAV) vector genome.

4. The vector genome of claim 2 or 3, wherein the vector genome is self-complementary.

5. A recombinant adeno-associated viral (rAAV) vector comprising: a vector genome comprising a modified nucleic acid consisting of the nucleic acid sequence of SEQ ID NO: 2; and a capsid selected from the group consisting of OligOOl, Olig002, Olig003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAVhu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, and AAV-LK03.

6. The rAAV vector of claim 5, wherein the capsid is an OligOOl, Olig002, or Olig003 capsid.

7. The rAAV vector of claim 6, wherein the capsid is an OligOOl capsid comprising a viral protein 1 (VP1) and wherein the VP1 consists of the amino acid sequence of SEQ ID NO:

14.

8. The rAAV vector of claim 5, wherein the vector genome is self-complementary.

9. The rAAV vector of any one of claims 5-8, wherein the vector genome further comprises at least one element selected from the group consisting of at least one AAV inverted terminal repeat (ITR) sequence, an enhancer, a promoter, an exon, an intron, and a polyadenylation (polyA) signal sequence.

10. The rAAV vector of any one of claims 5-8, wherein the vector genome further comprises at least one element selected from the group consisting of at least one AAV2 ITR, a cytomegalovirus (CMV) enhancer, a CBA promoter in a hybrid form (CBh promoter), a chicken beta-actin (CBA) exon, a CBA intron, a mouse minute virus (MVM) intron, and a bovine growth hormone (BGH) polyA.

11. The rAAV vector of any one of claims 5-8, wherein the vector genome further comprises at least one element selected from the group consisting of: at least one ITR consisting of the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 12, or SEQ ID NO: 19; an enhancer consisting of the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17; a promoter consisting of the nucleic acid sequence of SEQ ID NO: 7; an exon consisting of the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18; an intron consisting of the nucleic acid sequence of SEQ ID NO: 9; an intron consisting of the nucleic acid sequence of SEQ ID NO: 10; and a polyA consisting of the nucleic acid sequence of SEQ ID NO:

11.

12. An rAAV vector comprising a vector genome comprising, from 5’ to 3’: a) an AAV inverted terminal repeat (ITR) consisting of the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 12, or SEQ ID NO: 19; b) an enhancer consisting of the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17; c) a promoter consisting of the nucleic acid sequence of SEQ ID NO: 7; d) an exon consisting of the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18; e) an intron consisting of the nucleic acid sequence of SEQ ID NO: 9; f) an intron consisting of the nucleic acid sequence of SEQ ID NO: 10; g) a modified nucleic acid encoding aspartoacylase (ASPA) consisting of the nucleic acid sequence of SEQ ID NO: 2; h) a polyA consisting of the nucleic acid sequence of SEQ ID NO: 11; and i) an AAV ITR consisting of the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 12, or SEQ ID NO:

19.

13. The rAAV vector of claim 12, wherein the nucleic acid is self-complementary.

14. The rAAV vector of claim 12 or 13, wherein the vector comprises an Olig001 capsid comprising viral protein 1 (VP1) and wherein the VP1 consists of the amino acid sequence of SEQ ID NO:

14.

15. An rAAV vector comprising: an Olig001 capsid comprising viral protein 1 (VP1) and wherein the VP1 consists of the amino acid sequence of SEQ ID NO: 14; and a self-complementary nucleic acid comprising, from 5’ to 3’: a) an AAV2 inverted terminal repeat (ITR) consisting of the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 12, or SEQ ID NO: 19; b) a CMV enhancer consisting of the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17; c) a CBh promoter consisting of the nucleic acid sequence of SEQ ID NO: 7; d) a CBA exon 1 consisting of the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18; e) a CBA intron 1 consisting of the nucleic acid sequence of SEQ ID NO: 9; f) a MMV intron consisting of the nucleic acid sequence of SEQ ID NO: 10; g) a modified nucleic acid encoding aspartoacyltransferase (ASPA) consisting of the nucleic acid sequence of SEQ ID NO: 2; h) a BGH poly A consisting of the nucleic acid sequence of SEQ ID NO: 11; and i) an AAV2 ITR consisting of the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 12, or SEQ ID NO:

19.

16. A pharmaceutical composition comprising the rAAV vector of any one of claims 5-15.

17. A recombinant nucleic acid or expression cassette comprising: a modified nucleic acid encoding ASPA and consisting of SEQ ID NO: 2, and a regulatory element.

18. A host cell comprising the isolated nucleic acid of claim 1, the vector genome of any one of claims 2-4, the rAAV vector of any one of claims 5-15, or the recombinant nucleic acid or expression cassette of claim 17.

19. The host cell of claim 18, wherein the cell is selected from the group consisting of: VERO, WI38, MRC5, A549, HEK293, B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT1080.

20. The host cell of claim 19, wherein the cell is a HEK293 cell adapted for growth in suspension culture.

21. The host cell of claim 20, wherein the cell is a HEK293 cell having American Type Culture Collection (ATCC) number PTA 13274.

22. The host cell of any one of claims 19-21, wherein the cell comprises at least one nucleic acid encoding at least one protein selected from the group consisting of: an AAV rep protein, an AAV capsid (Cap) protein, an adenovirus (Ad) early region 1A (E1a) protein, an Ad E1b protein, an Ad E2a protein, an Ad E4 protein, and a virus-associated (VA) RNA.

23. A kit for treating Canavan Disease (CD) comprising a therapeutically effective amount of the isolated nucleic acid of claim 1, the vector genome of any one of claims 2-4, the rAAV vector of any one of claims 5-15, the pharmaceutical composition of claim 16, or the recombinant nucleic acid or expression cassette of claim 17.

24. The kit of claim 23, wherein the kit further comprises a label or insert comprising instructions for using one or more of the kit components.

25. Use of the isolated nucleic acid of claim 1, the vector of any one of claims 2-4, the rAAV vector of any one of claims 5-15, or the recombinant nucleic acid or expression cassette of claim 17 in the manufacture of a medicament for the treatment and / or prevention of Canavan disease.

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