Modified nucleic acids encoding aspartoacylase (ASPA) and vectors 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 treating diseases and disorders associated with deficiency or dysfunction of ASPA, particularly Canavan disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 016,507, filed April 28, 2020, and U.S. Provisional Patent Application No. 63 / 077,144, filed September 11, 2020, the contents of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to modified nucleic acids encoding aspartoacylase (ASPA), methods of using modified nucleic acids encoding ASPA, vectors comprising modified nucleic acids encoding ASPA, and the use of the vectors in the treatment of diseases, disorders and conditions associated with reduced levels of functional ASPA, including diseases, disorders and conditions associated with reduced cellular catabolism of N-acetyl-L-aspartate, e.g., Canavan disease. [Background technology]

[0003] Canavan disease (CD) is associated with decreased expression and / or mutations from the ASPA gene, which encodes the enzyme aspartoacylase (ASPA) (also known as aminoacylase 2). Decreased aspartoacylase activity leads to the accumulation of N-acetylaspartate (NAA) (also known as N-acetyl-L-aspartic acid) due to reduced conversion of NAA to aspartate and acetate. The ASPA enzyme is involved in maintaining the metabolic integrity of myelin-forming cells. In the brain, ASPA gene expression is primarily restricted to white matter-producing oligodendrocytes. Accumulation of NAA in the brain is associated with oligodendrocyte dysfunction and interference with myelin sheath development and destruction of existing myelin sheaths associated with neurons.

[0004] CD is an autosomal recessive genetic disorder that primarily manifests in the neonatal / infantile form. Children affected by this form exhibit symptoms during infancy related to myelin degeneration in the brain and spinal cord. Symptoms include intellectual disability, loss of previously acquired motor skills, feeding difficulties, abnormal muscle tone, macrocephaly, paralysis, and seizures. Life expectancy is generally limited to the first decade of life for neonatal / infant children with CD. Individuals with milder / juvenile forms of CD may exhibit delayed speech and motor skill development and have an average life expectancy.

[0005] To date, there is no treatment to stop or delay the neurodegenerative effects of CD. Current treatment approaches in clinical use or under evaluation aim to alleviate symptoms and maximize quality of life. Physical therapy, feeding tubes, and anti-seizure medications may be used to treat some symptoms and improve quality of life. Therefore, there is a significant need for novel therapeutic approaches to treat CD. Summary of the Invention

[0006] Disclosed and exemplified herein are modified nucleic acids encoding aspartoacylase (ASPA) and vectors comprising the modified nucleic acids (e.g., rAAV vectors), as well as methods of treating diseases, disorders, or conditions mediated by reduced levels of ASPA protein by administering the modified nucleic acid or vectors comprising the modified nucleic acid to a patient in need thereof.

[0007] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by embodiment (E) below. E1. An isolated nucleic acid encoding an aspartoacyltransferase (ASPA), comprising 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:2. E2. An isolated nucleic acid encoding aspartoacyltransferase (ASPA), comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:2. E3. An isolated nucleic acid encoding an aspartoacyltransferase (ASPA), comprising 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:1. E4. An isolated nucleic acid encoding aspartoacyltransferase (ASPA), comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:1. E5. An isolated nucleic acid encoding an aspartoacyltransferase (ASPA), comprising 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:3. E6. An isolated nucleic acid encoding an aspartoacyltransferase (ASPA), comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:3. E7. A modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising 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:2. E8. A modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:2. E9. A modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising 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:1. E10. A modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:1. E11. A modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising 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:3. E12. A modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:3. E13. A recombinant nucleic acid comprising a modified nucleic acid encoding aspartoacyltransferase (ASPA), wherein the modified nucleic acid 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:2. E14. A recombinant nucleic acid comprising a modified nucleic acid encoding an aspartoacyltransferase (ASPA) comprising or consisting of the nucleic acid sequence of SEQ ID NO:2. E15. A recombinant nucleic acid comprising a modified nucleic acid encoding aspartoacyltransferase (ASPA), wherein the modified nucleic acid 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:1. E16. A recombinant nucleic acid comprising a modified nucleic acid encoding an aspartoacyltransferase (ASPA) comprising or consisting of the nucleic acid sequence of SEQ ID NO:1. E17. A recombinant nucleic acid comprising a modified nucleic acid encoding aspartoacyltransferase (ASPA), wherein the modified nucleic acid 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:3. E18. A recombinant nucleic acid comprising a modified nucleic acid encoding an aspartoacyltransferase (ASPA) comprising or consisting of the nucleic acid sequence of SEQ ID NO:3. E19. The recombinant nucleic acid of any one of E13 to E18, further comprising at least one element selected from the group consisting of an enhancer, a promoter, an exon, an intron, and a polyadenylation (polyA) signal sequence. E20. The recombinant nucleic acid of E19, wherein the enhancer comprises a nucleic acid sequence 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 SEQ ID NO:6, SEQ ID NO:17, or both. E21. The recombinant nucleic acid of E19 or E20, wherein the enhancer comprises or consists of the nucleic acid sequence of SEQ ID NO:6, SEQ ID NO:17, or both. E22. The recombinant nucleic acid of any one of E19 to E21, wherein the promoter is constitutive or regulated. E23. The recombinant nucleic acid of any one of E19 to E22, wherein the promoter is inducible or repressible. E24. The recombinant nucleic acid of any one of E19 to 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. E25. The recombinant nucleic acid of any one of E19 to E24, wherein the promoter comprises or consists of the nucleic acid sequence of SEQ ID NO:7. E26. The recombinant nucleic acid of any one of E19 to E25, wherein the exon 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:8, SEQ ID NO:18, or both. E27. The recombinant nucleic acid of any one of E19 to E26, wherein the exon comprises or consists of the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 18, or both. E28. The recombinant nucleic acid of any one of E19 to E27, wherein the intron 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:9, SEQ ID NO:10, or both. E29. The recombinant nucleic acid of any one of E19 to E28, wherein the intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or both. E30. A recombinant nucleic acid of any one of E19 to E29, wherein the polyA sequence 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:11. E31. The recombinant nucleic acid of any one of E19 to E30, wherein the polyA sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:11. E32. The recombinant nucleic acid of any one of E19-E31, wherein an enhancer is operably linked to the modified nucleic acid. E33. The recombinant nucleic acid of any one of E19 to E32, wherein a promoter is operably linked to the modified nucleic acid. E34. The recombinant nucleic acid of any one of E13 to E18, further comprising at least one element selected from the group consisting of a cytomegalovirus (CMV) enhancer, a hybrid form of the CBA promoter (CBh promoter), a chicken beta actin (CBA) exon, a CBA intron, a minute virus of mice (MVM) intron, and a bovine growth hormone (BGH) polyA. E35. The recombinant nucleic acid of any one of E13 to E18, further comprising at least one element selected from the group consisting of 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. E36. A vector genome comprising any one of the modified nucleic acids E7 to E12, or any one of the recombinant nucleic acids E13 to E35, further comprising at least one AAV ITR repeat sequence comprising a nucleic acid sequence 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:5, SEQ ID NO:12, or both. E37. The vector genome of E36, wherein at least one AAV ITR repeat sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof. E38. The vector genome of E36 or E37, comprising two AAV2 ITR sequences flanking the nucleic acid sequence encoding ASPA, and a CBh promoter upstream of the sequence encoding ASPA. E39. The vector genome of any one of E36 to E38, wherein the ASPA sequence comprises the nucleic acid sequence of SEQ ID NO: 2. E40. The vector genome of any one of E36 to E39, wherein at least one AAV2 ITR sequence comprises the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof. E41. The vector genome of any one of E36 to E40, wherein the CBh promoter comprises the nucleic acid sequence of SEQ ID NO: 7. E42. A vector genome comprising a nucleic acid, the nucleic acid comprising, 5' to 3': a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19; b) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17, preferably SEQ ID NO: 6; c) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO: 7; d) a CBA exon comprising the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 18, preferably SEQ ID NO: 18; e) a CBA intron comprising the nucleic acid sequence of SEQ ID NO: 9; f) an MMV intron comprising the nucleic acid sequence of SEQ ID NO: 10; g) a modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising any one of the nucleic acid sequences of SEQ ID NOs: 1 to 3; h) BGH polyA comprising the nucleic acid sequence of SEQ ID NO: 11, and i) A vector genome comprising AAV2 ITRs comprising the nucleic acid sequences of SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 19. E43. A vector genome comprising a nucleic acid, the nucleic acid comprising, 5' to 3': a) an AAV ITR comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19; b) an enhancer comprising the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17, preferably SEQ ID NO: 6; c) a promoter comprising the nucleic acid sequence of SEQ ID NO: 7; d) an exon comprising the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18, preferably SEQ ID NO: 18; e) an intron comprising the nucleic acid sequence of SEQ ID NO: 9; f) an intron comprising the nucleic acid sequence of SEQ ID NO: 10; g) a modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising any one of the nucleic acid sequences of SEQ ID NOs: 1 to 3; h) PolyA comprising the nucleic acid sequence of SEQ ID NO: 11, and i) A vector genome comprising AAV terminal repeats, comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12 or SEQ ID NO:19. E44. A vector genome of any one of E36 to E33, which is self-complementary. A recombinant adeno-associated virus (rAAV) vector comprising a vector genome of any one of E45, E36 to E44, and a capsid. E46. An rAAV vector, comprising a vector genome comprising a nucleic acid sequence that 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 100% identical to the nucleic acid sequence of SEQ ID NO:2. E47.Olig001, Olig002, Olig003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, A AV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15 rAAV vector of E46, comprising a capsid selected from the group consisting of capsids of AAV-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. E48. An rAAV vector, comprising a vector genome comprising a nucleic acid sequence that 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 100% identical to the nucleic acid sequence of SEQ ID NO:1. E49.Olig001, Olig002, Olig003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, A AV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15 rAAV vector of E48, comprising a capsid selected from the group consisting of capsids of AAV-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. E50. An rAAV vector, comprising a vector genome comprising a nucleic acid sequence that 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 100% identical to the nucleic acid sequence of SEQ ID NO:3. E51.Olig001, Olig002, Olig003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, A AV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15 rAAV vector E50, comprising a capsid selected from the group consisting of capsids of AAV-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. E52. The rAAV vector of any one of E45 to E51, wherein the capsid is selected from an Olig001, Olig002, and Olig003 capsid. E53. The rAAV vector of any one of E45 to E52, wherein the capsid is an Olig001 capsid comprising viral protein 1 (VP1), and VP1 comprises an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14. E54. The rAAV vector of any one of E45 to E53, wherein the capsid is an oligo001 capsid comprising viral protein 1 (VP1), and VP1 comprises the amino acid sequence of SEQ ID NO: 14. E55. The rAAV vector of any one of E45 to E52, wherein the capsid is an Olig002 capsid comprising viral protein 1 (VP1), and VP1 comprises an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. E56. The rAAV vector of any one of E45 to E52 and E55, wherein the capsid is an oligo002 capsid comprising viral protein 1 (VP1), and VP1 comprises the amino acid sequence of SEQ ID NO: 15. E57. The rAAV vector of any one of E45 to E52, wherein the capsid is an Olig003 capsid comprising viral protein 1 (VP1), and VP1 comprises an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. E58. The rAAV vector of any one of E46 to E52 and E57, wherein the capsid is an oligo003 capsid comprising viral protein 1 (VP1), and VP1 comprises the amino acid sequence of SEQ ID NO: 16. E59. The rAAV vector of any one of E45 to E58, wherein the vector genome is self-complementary. E60. The rAAV vector of any one of E46 to E59, wherein the vector genome comprises at least one element selected from the group consisting of at least one AAV inverted terminal repeat (ITR) sequence, enhancer, promoter, exon, intron, and polyadenylation (polyA) signal sequence. E61. The rAAV vector of E60, wherein the enhancer comprises a nucleic acid sequence 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. E62. The rAAV vector of E60 or E61, wherein the enhancer comprises or consists of the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17. E63. An rAAV vector of E60 or E62, wherein the promoter is constitutive or regulated. E64. The rAAV vector of any one of E60 to E63, wherein the promoter is inducible or repressible. E65. The rAAV vector of any one of E60 to E64, wherein the promoter comprises a nucleic acid sequence 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. E66. The rAAV vector of any one of E60 to E65, wherein the promoter comprises or consists of the nucleic acid sequence of SEQ ID NO: 7. E67. The rAAV vector of any one of E60 to E66, wherein the exon comprises 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:8 or SEQ ID NO:18. E68. The rAAV vector of any one of E60 to E67, wherein the exon comprises or consists of the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18. E69. The rAAV vector of any one of E60 to E68, wherein the intron comprises a nucleic acid sequence 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:9, SEQ ID NO:10, or both. E70. The rAAV vector of any one of E60 to E69, wherein the intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or both. E71. The rAAV vector of any one of E60 to E70, wherein the polyA sequence comprises 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: 11. E72. The rAAV vector of any one of E60 to E71, wherein the polyA sequence comprises or consists of the nucleic acid sequence of SEQ ID NO: 11. E73. The rAAV vector of any one of E60 to E72, wherein at least one AAV ITR repeat sequence comprises a nucleic acid sequence 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:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof. E74. The rAAV vector of any one of E60 to E73, wherein at least one AAV ITR repeat sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof. E75. The rAAV vector of any one of E46 to E59, wherein the vector genome further comprises at least one element selected from the group consisting of at least one AAV2 ITR sequence, a CMV enhancer, a CBh promoter, a CBA exon 1, a CBA intron 1, an MVM intron, and a BGH polyA. E76. The rAAV vector of any one of E46 to E59, wherein the vector genome further comprises at least one element selected from the group consisting of 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. E77. The rAAV vector of any one of E46 to E59, wherein the vector genome comprises two AAV2 ITR sequences flanking the ASPA-encoding sequence and a CBh promoter upstream of the ASPA-encoding sequence. E78. The rAAV vector of E77, wherein the ASPA sequence comprises the nucleic acid sequence of SEQ ID NO: 2. E79. The rAAV vector of E77 or E78, wherein the AAV ITR sequences comprise the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof. E80. Any one of the rAAV vectors E77 to E79, wherein the CBh promoter comprises the nucleic acid sequence of SEQ ID NO: 7. E81.5' to 3', a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof; b) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 16; c) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO: 7; d) CBA exon 1 comprising the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18; e) CBA intron 1 comprising the nucleic acid sequence of SEQ ID NO: 9; f) an MMV intron comprising the nucleic acid sequence of SEQ ID NO: 10; g) a modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising any one of the nucleic acid sequences of SEQ ID NOs: 1 to 3; h) BGH polyA comprising the nucleic acid sequence of SEQ ID NO: 11, and i) an rAAV vector comprising a vector genome comprising an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 19, or a combination thereof. E82.5' to 3', a) an AAV ITR comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof; b) an enhancer comprising the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17; c) a promoter comprising the nucleic acid sequence of SEQ ID NO: 7; d) an exon comprising the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18; e) an intron comprising the nucleic acid sequence of SEQ ID NO: 9; f) an intron comprising the nucleic acid sequence of SEQ ID NO: 10; g) a modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising any one of the nucleic acid sequences of SEQ ID NOs: 1 to 3; h) PolyA comprising the nucleic acid sequence of SEQ ID NO: 11, and i) an rAAV vector comprising an AAV terminal repeat comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof. E83. The rAAV vector of E81 or E82, wherein the vector genome is self-complementary. E84. The rAAV vector of any one of E81-E83, wherein the vector comprises an Olig001 capsid comprising a VP1 protein, wherein VP1 comprises an amino acid sequence 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. E85. The rAAV vector of any one of E81 to E83, wherein the vector comprises an Olig002 capsid containing a VP1 protein, wherein VP1 comprises an amino acid sequence 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. E86. The rAAV vector of any one of E81-E83, wherein the vector comprises an Olig003 capsid containing a VP1 protein, wherein VP1 comprises an amino acid sequence 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. E87.i) an Olig001 capsid comprising a VP1 protein, wherein VP1 comprises the amino acid sequence of SEQ ID NO: 14; and ii) from 5' to 3': a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof; b) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17; c) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO: 7; d) CBA exon 1 comprising the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18; e) CBA intron 1 comprising the nucleic acid sequence of SEQ ID NO: 9; f) an MMV intron comprising the nucleic acid sequence of SEQ ID NO: 10; g) a modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising any one of the nucleic acid sequences of SEQ ID NOs: 1 to 3; h) BGH polyA comprising the nucleic acid sequence of SEQ ID NO: 11, and i) a self-complementary vector genome comprising an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 12. E88.i) an Olig001 capsid comprising a VP1 protein, wherein VP1 comprises the amino acid sequence of SEQ ID NO: 14; and ii) from 5' to 3': a) an AAV ITR comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof; b) an enhancer comprising the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17; c) a promoter comprising the nucleic acid sequence of SEQ ID NO: 7; d) an exon comprising the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18; e) an intron comprising the nucleic acid sequence of SEQ ID NO: 9; f) an intron comprising the nucleic acid sequence of SEQ ID NO: 10; g) a modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising any one of the nucleic acid sequences of SEQ ID NOs: 1 to 3; h) PolyA comprising the nucleic acid sequence of SEQ ID NO: 11, and i) a self-complementary vector genome comprising AAV ITRs comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, or a combination thereof. E89. Any one of the rAAV vectors E45 to E88, which, when introduced into a cell, reduces the level of NAA in the cell. E90. The rAAV vector of E89, where the cells are brain cells. E91. The rAAV vector of E89 or E90, wherein the cells are oligodendrocytes. E92. The rAAV vector of any one of E45 to E91, wherein administration of the vector to a subject having an ASPA gene mutation increases balance, grip strength, and / or motor coordination in the subject compared to balance, grip strength, and / or motor coordination in the subject prior to administration of the vector. E93. An rAAV vector of any one of E45 to E92, wherein administration of the vector to a subject having an ASPA gene mutation increases the overall motor function in the subject compared to the overall motor function in the subject before administration of the vector. E94. An rAAV vector of any one of E45 to E93, wherein administration of the vector to a subject having an ASPA gene mutation reduces NAA levels in the subject compared to the NAA levels in the subject before administration of the vector. E95. An rAAV vector any one of E45 to E94, wherein administration of the vector to a subject having an ASPA gene mutation reduces the vacuolar volume ratio in the subject's thalamus compared to the vacuolar volume ratio in the subject's thalamus before administration of the vector. E96. Any one of the rAAV vectors E45 to E95, wherein administration of the vector to a subject having an ASPA gene mutation reduces the vacuolar volume percentage in the subject's cerebellar white matter / pons compared to the vacuolar volume percentage in the subject's cerebellar white matter / pons before administration of the vector. E97. Any one of the rAAV vectors of E45 to E96, wherein administration of the vector to a subject having an ASPA gene mutation increases the number of oligodendrocytes in the subject's thalamus compared to the number of oligodendrocytes in the subject's thalamus before administration of the vector. E98. Any one of E45 to E97 rAAV vectors, wherein administration of the vector to a subject having an ASPA gene mutation increases the number of oligodendrocytes in the subject's cerebral cortex compared to the number of oligodendrocytes in the subject's cerebral cortex before administration of the vector. E99. An rAAV vector of any one of E45 to E98, wherein administration of the vector to a subject having an ASPA gene mutation increases the number of neurons in the subject's thalamus compared to the number of neurons in the subject's thalamus before administration of the vector. E100. An rAAV vector of any one of E45 to E99, wherein administration of the vector to a subject having an ASPA gene mutation increases the number of neurons in the subject's cerebral cortex compared to the number of neurons in the subject's cerebral cortex before administration of the vector. E101. Any one of the rAAV vectors of E45 to E100, wherein administration of the vector to a subject having an ASPA gene mutation increases cortical myelination in the subject compared to cortical myelination in the subject prior to administration of the vector. E102. The rAAV vector of any one of E92 to E101, wherein the subject is a human patient. E103. The rAAV vector of any one of E92-E102, wherein the subject is a human patient having or at risk of developing Canavan disease. E104. The rAAV vector of any one of E92-E103, wherein the subject has at least one ASPA gene mutation. E105. A pharmaceutical composition comprising any one of the modified nucleic acids E7 to E12, any one of the recombinant nucleic acids E13 to E35, any one of the vector genomes E36 to E44, or any one of the rAAV vectors E45 to E104. A pharmaceutical composition comprising any one of the modified nucleic acids E106, E7 to E12, any one of the recombinant nucleic acids E13 to E35, any one of the vector genomes E36 to E44, or any one of the rAAV vectors E45 to E104, and a pharmaceutically acceptable carrier. E107. A method for treating and / or preventing a disease, disorder, or condition associated with ASPA deficiency or dysfunction, comprising administering a therapeutically effective amount of any one of modified nucleic acids E7 to E12, recombinant nucleic acids E13 to E35, vector genome E36 to E44, rAAV vector E45 to E104, or pharmaceutical composition E105 or E106 to a subject in need of treatment. E108. The method of E107, wherein the disease, disorder or condition associated with ASPA deficiency or dysfunction is Canavan disease. E109. The method of 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 a subject in need of treatment. 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 administered directly to the central nervous system of a subject in need of treatment. E111. The method of any one of E107 to E110, wherein the modified nucleic acid, recombinant nucleic acid, vector genome, rAAV vector, or pharmaceutical composition is administered to at least one region of the central nervous system selected from the group consisting of brain parenchyma, spinal canal, subarachnoid space, ventricles of the brain, cisterna magna, and any combination thereof. E112. Any one of the methods of E107 to 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 the group consisting of intraparenchymal administration, intrathecal administration, intraventricular administration, intracisternal administration, and any combination thereof. E113. The method of any one of E107-E112, wherein the subject is a human patient. E114. The method of any one of E107-E113, wherein the subject is a human patient having or at risk of developing Canavan disease. E115. The method of any one of E107-E114, wherein the subject has at least one mutation in the ASPA gene. E116. A method for treating or preventing Canavan disease, comprising the steps of: i) assessing whether a subject contains at least one ASPA gene mutation; and ii) administering to the subject a therapeutically effective amount of a modified nucleic acid of any one of E7 to E12, a recombinant nucleic acid of any one of E13 to E35, a vector genome of any one of E36 to E44, an rAAV vector of any one of E45 to E104, or a pharmaceutical composition of E105 or E106, thereby treating or preventing Canavan disease in the subject. E117. The method of EE116, wherein the subject is diagnosed with or at risk for developing Canavan disease. E118. A method for treating or preventing a disease associated with ASPA deficiency in a subject in need thereof, 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. E119. The method of E118, wherein the modified nucleic acid encoding ASPA comprises the nucleic acid sequence of SEQ ID NO:2. E120. The method of E118 or E119, wherein the modified nucleic acid encoding ASPA encodes an ASPA protein having the amino acid sequence of SEQ ID NO:4. E121. The method of any one of E118 to E120, wherein a modified nucleic acid encoding ASPA is expressed in a target cell, wherein the target cell is an oligodendrocyte. E122. The method of any one of E118 to E121, wherein the modified nucleic acid encoding ASPA is delivered to the target cell in a vector. E123. The method according to E122, wherein the vector is a viral vector or a non-viral vector. E124. The method of any one of E118-E123, wherein the vector is administered to the subject by systemic injection, by direct intracranial injection, or by direct spinal injection. A host cell comprising any one of the modified nucleic acids E125, E7 to E12, any one of the recombinant nucleic acids E13 to E35, any one of the vector genomes E36 to E44, or any one of the rAAV vectors E45 to E104. E126. The host cell of E125, selected from the group consisting of VERO, WI38, MRC5, A549, HEK293, B-50, or any other HeLa cell, HepG2, Saos-2, HuH7, and HT1080. E127. Host cells from E125-E126, which are HEK293 cells adapted to growth in suspension culture. E128. Host cells of any one of E125 to E127, which are HEK293 cells having American Type Culture Collection (ATCC) number PTA13274. E129. The host cell of any one of E125 to E128, comprising 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 early region 1A (E1a) protein, an E1b protein, an E2a protein, an E4 protein, and a virus-associated (VA) RNA. E130. A kit for treating Canavan disease (CD), comprising a therapeutically effective amount of i) an rAAV vector of any one of E45 to E104, or ii) a pharmaceutical composition of E105 or E106. E131. The kit of E130, further comprising a label or package insert containing instructions for using one or more of the kit components. E132. A modified nucleic acid of any one of E7 to E12, a recombinant nucleic acid of any one of E13 to E35, a vector genome of any one of E36-E44, an rAAV vector of any one of E45-E104, or a pharmaceutical composition of E105 or E106 for use in the treatment or prevention of a disease, disorder, or condition associated with ASPA deficiency or dysfunction. E133. The modified nucleic acid, recombinant nucleic acid, vector genome, rAAV vector, or pharmaceutical composition for use in E132, wherein the disease, disorder, or condition is Canavan disease. E134. Use of any one of modified nucleic acids E7 to E12, recombinant nucleic acids E13 to E35, vector genomes E36 to E44, rAAV vectors E45 to E104, or pharmaceutical compositions E105 or E106 in the manufacture of a medicament for treating and / or preventing diseases, conditions, or disorders associated with ASPA deficiency or dysfunction. E135. The use of E134, wherein the disease, disorder, or condition is Canavan disease. E136. A method for determining the biodistribution of a transgene delivered to the brain of a subject by an rAAV vector comprising an Olig001 capsid, wherein a protein encoded by the transgene is expressed, the method comprising: a) administering an rAAV vector to a subject; and b) Fixation of brain tissue; c) electrophoretic clearing of the brain; d) 3D microscopic imaging of brain tissue sections; e) detection of proteins; f) optionally quantifying the amount of protein present in the brain tissue. E137. The method of E136, wherein the administration is by intracerebroventricular (ICV) injection, intraparenchymal (IP) injection, intrathecal (IT) administration, intracisternal (ICM) injection, or a combination thereof. E138. The method of E136 or 137, wherein the brain tissue is fixed using, for example, paraformaldehyde or formalin. E139. Any one of the methods of E136 to E138, wherein the quantification includes volume rendering. E140. The method of any one of E136-E139, wherein the transgene encodes green fluorescent protein (GFP). E141. The method of any one of E136-E140, wherein the level of transgene expression detected in the tissue correlates with rAAV vector transduction efficiency. E142. The method of any one of E136 to E141, further comprising the step of (g) assessing cell type vector tropism by assessing cell morphology and spatial localization of GFP expression. E143. A modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising 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 any one of SEQ ID NOs: 1-3 and a promoter. E144. A modified nucleic acid encoding aspartoacyltransferase (ASPA), comprising a nucleic acid comprising or consisting of the sequence of SEQ ID NO: 2 and a promoter. 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 consists of the sequence of SEQ ID NO:2. E146. An isolated nucleic acid comprising a nucleic acid sequence specifying a promoter and further comprising a nucleic acid sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO:2. E147. The pharmaceutical composition of E105, further comprising 350 mM NaCl and 5% D-sorbitol in PBS. E148. The pharmaceutical composition of E106, wherein the pharmaceutically acceptable carrier comprises 350 mM NaCl and 5% D-sorbitol in PBS.

[0008] Other features and advantages of the invention will become apparent from the following detailed description, drawings, exemplary embodiments, and claims. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows an exemplary dose-response reduction of NAA as determined using HPLC in cells transfected with 1.0 μg of a plasmid expressing NAA synthase (Nat8L) and co-transfected with 1.0 μg, 0.5 μg, 0.2 μg, or 0.1 μg of a plasmid containing the wild-type human ASPA sequence (SEQ ID NO: 3) or a modified, e.g., codon-optimized ASPA sequence original (version 1) (SEQ ID NO: 1) or the codon-optimized ASPA sequence new (version 2) (SEQ ID NO: 2). [Figure 2] An exemplary sampling of GFP-positive cells transduced by rAAV vectors administered via the intraparenchymal (IP) route of administration (ROA) is shown. GFP-positive somatic cells (arrows) were scored in each region of interest to generate an estimate of the number of transduced cells (N). [Figure 3] Figure 1 shows an exemplary number (N) of GFP-positive cells in the cortex, subcortical white matter of the corpus callosum and external capsule, striatum, and cerebellum of 6-week-old nur7 mice after intraparenchymal (IP) administration of AAV / Olig001-GFP. Also shown is a representative image of native GFP fluorescence in a sagittal section of the brain from a mouse administered 1 x 10 AAV / Olig001-GFP vector genome via IP ROA, showing concentrated GFP expression adjacent to the injection site. Estimates of N were generated on 144 sections using an optical segmentor (k = 4). Means + / - standard errors for each group are presented (n = 5 animals). Significant differences in the number of GFP-positive cells between dose cohorts within each region of interest are indicated by asterisks. [Figure 4]Exemplary numbers of GFP-positive cells (N) within the cortex, subcortical white matter, striatum, and cerebellum of 6-week-old nur7 mice after intrathecal (IT) administration of AAV / Olig001-GFP are shown, as well as representative images of native GFP fluorescence in sagittal sections of the brain from mice administered 1 x 10 AAV / Olig001-GFP vector genomes via intrathecal (IT) ROA. These images show diffuse cortical marker expression, indicative of transduction by the vector, and moderate white matter tract cell expression, also indicative of transduction of cells within that region. The mean + / - standard error for each group is presented (n = 5 animals). Significant differences in the number of GFP-positive cells between dose cohorts within each region of interest are indicated by asterisks. [Figure 5] Exemplary numbers of GFP-positive cells (N) within the cortex, subcortical white matter, striatum, and cerebellum of 6-week-old nur7 mice after intracerebroventricular (ICV) administration of AAV / Olig001-GFP are shown, as well as representative images of native GFP fluorescence in sagittal sections of the brain of mice administered 1 x 10 AAV / Olig001-GFP vector genomes via ICV ROA, showing intense white matter tract GFP expression indicative of vector transduction of cells within that region. Means + / - standard error for each group are presented (n = 5 animals). Significant differences in the number of GFP-positive cells between dose cohorts within each region of interest are indicated by asterisks. [Figure 6] Exemplary numbers of GFP-positive cells (N) within the cortex, subcortical white matter, striatum, and cerebellum of 6-week-old nur7 mice after intracisternal administration (ICM) of AAV / Olig001-GFP are shown, as well as representative images of native GFP fluorescence in sagittal sections of the brain from mice administered 1 x 10 AAV / Olig001-GFP vector genomes via ICM ROA, demonstrating moderate white matter tract GFP marker expression, indicating transduction of cells within that region. Means + / - standard error for each group are presented (n = 5 animals). Significant differences in the number of GFP-positive cells between dose cohorts within each region of interest are indicated by asterisks. [Figure 7]Direct comparison of exemplary AAV / Olig001-GFP transduction efficiencies in four regions of interest (cortex, subcortical white matter, striatum, and cerebellum) for a 1 x 10 vg dose administered to each animal via four different routes of administration (IP, IT, ICV, and ICM) is shown, as well as representative images of native GFP fluorescence in sections outside the intraparenchymal and intraventricular injection sites. Both cortical and subcortical white matter tract transgene-positive cells were more numerous in sections outside the ICV brains. For each group, n = 5 animals, and means + / - standard error are shown. 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). [Figure 8] Figure 1 shows exemplary oligotropism of AAV / Olig001-GFP in the cortex of 6-week-old nur7 mice after intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV), and intracisternal (ICM) vector administration. Cortical sections were analyzed by IHC using Olig2 and NeuN antibodies. n = 5 animals per group, and the mean percentage of co-labeling with each indicated antigen + / - standard error is shown. Asterisks indicate significant differences between groups. [Figure 9] Figure 1 shows exemplary oligo-targeting of AAV / Olig001-GFP in the subcortical white matter of 6-week-old nur7 mice after intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV), and intracisternal (ICM) vector administration. Sections of subcortical white matter were analyzed by IHC using Olig2 and NeuN antibodies. For each group, n = 5 animals. The mean percentage of co-labeling with each indicated antigen + / - standard error is shown. [Figure 10] Figure 1 shows exemplary oligotropism of AAV / Olig001-GFP in the striatum of 6-week-old nur7 mice after intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV), and intracisternal (ICM) vector administration, with marker detection indicating cellular transduction by the vector. Striatal sections were analyzed by IHC using Olig2 and NeuN antibodies. For each group, n = 5 animals. The mean percentage of co-labeling with each indicated antigen + / - standard error is shown. [Figure 11] Figure 1 shows exemplary oligotropism of AAV / Olig001-GFP in the cerebellum of 6-week-old nur7 mice after intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV), and intracisternal (ICM) vector administration, with marker detection indicating vector transduction. Cerebellar sections were analyzed by IHC using Olig2 and NeuN antibodies. n = 5 animals per group, and the mean percentage of co-labeling with each indicated antigen + / - standard error is shown. [Figure 12] Figure 1 shows exemplary efficiencies of AAV / Olig001-GFP transduction in the cortical and subcortical white matter of age-matched wild-type (WT) and nur7 mouse brains 2 weeks after ICV administration of 1 x 10 vector genomes, as well as representative images of native GFP fluorescence in wild-type brains after administration of AAV / Olig001-GFP, demonstrating relatively restricted expression, thereby indicating transduction by the vector, particularly in the subcortical white matter. n = 5 animals per group. Shown are the mean number of GFP-positive cells per group + / - standard error; *p < 0.05, **p < 0.01. [Figure 13] 1 shows an expression plasmid encoding the codon-optimized ASPA coding sequence and regulatory elements. [Figure 14] Figure 1 shows rotarod fall latency over the in vivo study period for AAV / Olig001-ASPA-treated (at three dose levels), wild-type, and nur7 sham-treated mice. Data are presented as mean + / - standard error, with n=12 animals per group. [Figure 15] Illustrates exemplary open field activity over the course of an in vivo study for wild-type (WT) mice, AAV / Olig001-ASPA treated (at three dose levels), and sham-treated nur7 mice. Data are presented as mean + / - standard error, with n=12 animals per group. [Figure 16]Figure 1 shows exemplary NAA content in wild-type (WT), nur7 sham-treated, and AAV / Olig001-ASPA-treated (three dose levels) mouse brains. Data are expressed as mean + / - standard error. NAA is expressed as millimoles per gram of wet tissue weight (n = 6 animals per group). The dose of AAV / Olig001-ASPA is indicated on the x-axis. [Figure 17] Figure 1 shows exemplary mean vector genome copy numbers per mg of brain tissue (vg / mg) for nur7 mice treated with AAV / Olig001-ASPA at three different dose levels evaluated at 22 weeks of age. Mean vg / mg values ​​are presented as + / - standard error (n = 6 animals per dose cohort). [Figure 18] Representative H&E-stained brain sections from nur7 sham-treated, AAV / Olig001-ASPA-treated nur7 and wild-type mice showing areas of vacuolation are shown. [Figure 19] Exemplary vacuolar volume fractions as a percentage of thalamus and brain white matter / pons regions of interest (ROIs) from 22-week-old sham- and AAV / Olig001-ASPA-treated nur7 mice are shown. Asterisks indicate significant differences between groups. [Figure 20] Representative images of the thalamus and cortex of sham-treated and AAV / Olig001-ASPA-treated (2.5×10 11 vg dose) nur7 mice stained for Olig2, which indicates oligodendrocytes, are shown. [Figure 21] Figure 1 shows exemplary numbers of Olig2-positive cells in the thalamus and cortex of 22-week-old wild-type, sham-treated, and AAV / Olig001-ASPA-treated nur7 mice. Data are expressed as mean Olig2-positive cells + / - standard error (n = 6 animals per group). Asterisks indicate significant differences between groups. [Figure 22] Representative images of the thalamus and cortex of sham-treated and AAV / Olig001-ASPA-treated (2.5×10 11 vg dose) nur7 mice stained for NeuN are shown. [Figure 23]Figure 1 shows exemplary numbers of NeuN-positive cells in the thalamus and cortex of 22-week-old wild-type, sham-treated, and AAV / Olig001-ASPA-treated nur7 mice. Data are expressed as mean NeuN-positive cells + / - standard error (n = 6 animals per group). Asterisks indicate significant differences between groups. [Figure 24] Representative images of the cortex from sham-treated and AAV / Olig001-ASPA-treated (2.5×10 11 vg dose) nur7 mice stained for myelin basic protein (MBP) are shown. [Figure 25] Figure 1 shows representative myelin basic protein-positive fiber length density (MBP-LD) (μm / mm3) in the cortex of wild-type, sham-treated, and AAV / 001-ASPA-treated nur7 mice. Data are expressed as mean MBP-LD + / - standard error (n = 6 animals per group). Asterisks indicate significant differences between groups. [Figure 26] Shown are exemplary brain images from an ICV-injected mouse from an initial fixed pre-cleared sample, a sample after tissue clearing, a 3D GFP fluorescence image, a half-brain volume segmentation analysis, and an intensity heat map (left to right). [Figure 27] Intensity heatmaps from all four ICV-injected hemi-brains are shown. Total hemi-brain volume was calculated and is represented as the gray area. Calculated "low" GFP intensity is indicated by the gray area, and "high" GFP intensity is indicated by the white area. [Figure 28] Shown are 3D light-sheet GFP fluorescence microscopy images from the cleared brains of AAV / Oligo001-GFP administered animals administered via ICV versus IP administration routes. [Figure 29A] Representative high-magnification images showing the scoring of GFP-positive cells co-labeled with Olig2 or NeuN are shown. Total GFP cells were scored within each field, and the percentage of Olig2 and NeuN co-labeling was scored within the same field. [Figure 29B]Representative images of co-labeling of GFP with Olig2 in SCWM tract cells in the brain of an animal with AAV / Olig001-GFP given via ICV ROA are shown, demonstrating nearly 100% oligo-tropism and an almost complete absence of neuropathology. [Figure 29C] Representative images of cerebellar GFP transgene expression in large Purkinje neurons with sparse Olig2 co-labeling in the white matter (arrows) are shown. [Figure 29D] Representative images of GFP co-labeling with Olig2 in the striatum of ICV ROA brains are shown, demonstrating contrast with cerebellar targeting. [Figure 29E] Representative images of white matter tracts in 8-week nur7 and age-matched wild-type naive brains after BrdU labeling and Olig2 treatment are shown. [Figure 29F] Exemplary numbers of BrdU cells in wild-type and nur7 white matter tracts at 2 and 8 weeks are shown. Mean BrdU-positive cells per group + / - standard error is presented. For each group (genotype at each age), n=6. [Figure 29G] Representative images of BrdU / GFP co-labeled cells in the subcortical white matter of nur7 brains treated with AAV / Olig001-GFP via ICV ROA are shown. [Figures 30A-30C] Biodistribution volume analysis is shown. (A) Volume of tissue imaged both ICV and IP. (B) Mean and median GFP fluorescence intensity across two ROAs. (C) Percent volumetric GFP positivity representing low and high intensity across the ROAs. [Figures 31A-31C] Figure 1 shows the clarity and switch workflow for pharmacodynamic effect assessment. (A) Tissue clearing and labeling approach. Left to right: intact mouse brain, a central 2 mm section of the right hemibrain before clearing, the same tissue 1 day after passive clearing, and 3 days after passive clearing, as well as 3D images showing the fluorescent signal from previously labeled proteins (green: nuclei, red: myelin basic protein (MBP)). (B) Representative 2 mm sections of Nur7-, WT-, and Olig1-ASPA-treated tissue. The red arrow in each image indicates the thalamic region. (C) Tissue clarity 1 day after passive clearing. [Figures 32A-32C] Figure 1 shows 2D region-based cell counting of tissue. (A) Extracted 2D single slices of 3D images from all three groups with similar anatomical orientation. The red boxes mark the areas of the thalamus and cortical regions where cell counting was performed. (B) Zoomed-in image data from the red box in (A) and the respective cell segmentation. (C) Mean nuclear density (count normalized by segmentation area). [Figures 33A-33I] 3D volumetric analysis of pharmacodynamic treatment effects. (A) A complete 3D volume of a 2 mm tissue slice is measured. (B) Mean fluorescence intensity calculated within the 3D volume. (C) MBP characterization via a more restrictive threshold set at fluorescence values ​​above 2000 (left panel) or a more inclusive threshold at 1000 (left panel). (D) In ​​both cases, MBP depletion in Nur7 can be observed. The effect of the Olig1-ASPA group can be seen at lower thresholds, with overall values ​​approaching WT levels. (E) Region-based 3D analysis of the thalamic region, with a manual segmentation of a portion of the region shown in yellow. (F) Mean fluorescence within this region for both nuclear (SYTO) and myelin (MBP) markers. (G) Region-based analysis of a portion of the cortex, with a manual segmentation shown in yellow. (H) Mean fluorescence within this cortical region for both nuclear (SYTO) and myelin (MBP) markers. (I) 3D cell concentration (nuclei per 100 μm). DETAILED DESCRIPTION OF THE INVENTION

[0010] definition 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 belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. When used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. The following terms have the following meanings:

[0011] As used herein, the term "about" or "approximately" refers to a measurable value such as the amount of biological activity, the length of a polynucleotide or polypeptide sequence, the content of G and C nucleotides, the codon adaptation index, the number of CpG dinucleotides, dosage, time, temperature, etc., and is intended to encompass a variation 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 of the specified amount (greater than or less than), unless otherwise specified, apparent from the context, or unless such number exceeds 100% of the possible values.

[0012] As used herein, the term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and the lack of combinations when interpreted in the alternative ("or").

[0013] 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. The term encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise required. The wild-type genome contains 4681 bases (Berns and Bohenzky (1987) Advances in Virus Research 32:243-307) and contains terminal repeat sequences (e.g., inverted terminal repeats (ITRs)) at each end, which function in cis as origins of DNA replication and as viral packaging signals. The genome contains two large open reading frames known as the AAV replication ("AAV rep" or "rep") and capsid ("AAV cap" or "cap") genes, respectively. AAV rep and cap may also be referred to herein as AAV "packaging genes." These genes encode viral proteins involved in replication and packaging of the viral genome.

[0014] In wild-type AAV viruses, the three capsid genes VP1, VP2, and VP3 overlap each other within a single open reading frame, and 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 the expression of all three capsid proteins in a ratio of approximately 1:1:10 for VP1, VP2, and VP3, respectively, which complements AAV capsid production. More specifically, VP1 is a full-length protein, while VP2 and VP3 are increasingly truncated by increasing N-terminal truncations. A well-known example is the capsid of AAV9, described in U.S. Patent No. 7,906,111, in which VP1 comprises amino acid residues 1-736 of SEQ ID NO: 123, VP2 comprises amino acid residues 138-736 of SEQ ID NO: 123, and VP3 comprises amino acid residues 203-736 of SEQ ID NO: 123. As used herein, the term "AAV cap" or "cap" refers to the AAV capsid proteins VP1, VP2, and / or VP3, and variants and analogs thereof.

[0015] At least four viral proteins are synthesized from the AAV rep genes Rep 78, Rep 68, Rep 52, and Rep 40 and are named according to their apparent molecular weights. As used herein, "AAV rep" or "rep" refers to the AAV replication proteins Rep 78, Rep 68, Rep 52, and / or Rep 40, as well as mutants and analogs thereof. As used herein, rep and cap refer to both wild-type and recombinant (e.g., modified chimeric) rep and cap genes and the polypeptides they encode. In some embodiments, a nucleic acid encoding rep contains nucleotides from two or more AAV serotypes. For example, a nucleic acid encoding rep may contain nucleotides from the AAV2 serotype and nucleotides from the AAV3 serotype (Rabinowitz et al. (2002) J. Virology 76(2):791-801).

[0016] As used herein, "recombinant adeno-associated virus vector," "rAAV," and / or "rAAV vector" refer to an AAV comprising a vector genome, in which the polynucleotide sequence is not or is not entirely of AAV origin (e.g., a polynucleotide heterologous to AAV), and the rep and / or cap genes of the wild-type AAV viral genome have been removed from the viral genome. When the typical AAV rep and / or cap genes have been removed or absent (and the flanking ITRs are typically derived from a different serotype, for example, but not limited to, AAV2 ITRs, where the capsid is not AAV2), the nucleic acid within the AAV (including any ITRs and any nucleic acid therebetween) is referred to as the "vector genome." Thus, the term rAAV vector encompasses rAAV viral particles comprising a capsid and heterologous nucleic acid, i.e., nucleic acid not originally present in the capsid in nature, hereinafter referred to as the "vector genome." Thus, "rAAV vector genome" (or "vector genome") refers to a heterologous polynucleotide sequence (typically, but not necessarily, including at least one ITR that is not associated with the original nucleic acid present in the original AAV) that may, but is not necessarily, contained within an AAV capsid. rAAV vector genomes may be double-stranded (dsAAV), single-stranded (ssAAV), and / or self-complementary (scAAV).

[0017] As used herein, the terms "rAAV vector," "rAAV viral particle," and / or "rAAV vector particle" refer to an AAV capsid that contains at least one AAV capsid protein (although typically all of the AAV capsid proteins, e.g., VPI, VPS, and VP3, or variants thereof, are present) and contains a vector genome that includes a heterologous nucleic acid sequence not originally present in the original AAV capsid. These terms should be distinguished from "AAV viral particle" or "AAV virus," which are not recombinant; the capsid contains a viral genome encoding the rep and cap genes, and the AAV virus can replicate when present in a cell that also contains a helper virus, such as adenovirus and / or herpes simplex virus, and / or the necessary helper genes therefrom. Thus, production of rAAV vector particles necessarily involves the production of a recombinant vector genome using recombinant DNA technology, such that this vector genome is contained within a capsid to form an rAAV vector, rAAV viral particle, or rAAV vector particle.

[0018] The genomic sequences of various AAV serotypes, as well as the sequences of the inverted terminal repeats (ITRs), rep proteins, and capsid subunits, both naturally occurring and / or variants and mutations thereof, are known in the art and can be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. 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. 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. 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 Nos. 00 / 28061, WO99 / 61601, WO98 / 11244; WO2013 / 063379, WO2014 / 194132, WO2015 / 121501; and U.S. Patent Nos. 6,156,303 and 7,906,111.

[0019] As used herein, the term "amelioration" refers to a detectable or measurable improvement in a subject's disease, disorder, or condition, or its symptoms, or underlying cellular response. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limitation, or control of the occurrence, frequency, severity, progression, or duration of the disease, disorder, or condition, or the causes of complications due thereto or associated therewith, an improvement in the symptoms thereof, or a reversal thereof.

[0020] As used herein, the term "associated with" refers to being associated with one another when the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact directly or indirectly, such that they are in and / or remain in physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another, but are non-covalently associated by, for example, hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0021] As used herein, the term "cis-motif" or "cis-element" includes conserved sequences found at or near the ends of a genomic sequence, such as sequences recognized for the initiation of replication, codifying promoters or sequences at internal locations likely to be used for transcription initiation, splicing, or termination. A cis-motif or cis-element is present on the same nucleic acid molecule as those sequences with which it interacts. This should be distinguished from a "trans-motif" sequence, which acts "in trans" with other sequences that are not located on the same nucleic acid molecule.

[0022] As used herein, the terms "coding sequence" or "encoding nucleic acid" refer to a nucleic acid sequence that encodes a protein or polypeptide and is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control (operably linked) of appropriate regulatory sequences. The boundaries of the coding sequence are generally determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0023] As used herein, the term "chimera" refers to a viral capsid having capsid sequences 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 the sequence of an AAV2 capsid with the following mutations: 263Q to A, 265 insertion T, 705N to A, 708V to A, and 716T to N. The nucleotide sequence encoding such a capsid is defined as SEQ ID NO: 15 as described in WO 2006 / 066066. Other preferred chimeric AAV capsids include, but are not limited to, AAV2i8 described in WO2010 / 093784, AAV2G9 and AAV8G9 described in WO2014 / 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 WO2103 / 029030, RHM4-1 and RHM15-1 to RHM5-6 described in WO205 / 013313, and AAV-DJ, AAV-DJ / 8, and AAV-DJ / 9 described in WO2007 / 120542.

[0024] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with a biologically, chemically, or structurally similar residue. Biologically similar means that the substitution does not destroy biological activity. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine, and serine, or have similar sizes. Chemical similarity means that the residues have the same charge or are both hydrophilic and hydrophobic. Specific examples include the substitution of a hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, etc. Specific examples of conservative substitutions include the substitution of hydrophobic residues such as isoleucine, valine, leucine, or methionine for one another, or the substitution of a polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. Conservative amino acid substitutions typically include, for example, substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. "Conservative substitution" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0025] As used herein, the term "flanking" refers to a sequence adjacent to other elements and indicates the presence of one or more flanking elements upstream and / or downstream, i.e., 5' and / or 3', to the sequence. The term "flanking" is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and the flanking elements. A sequence (e.g., a transgene) "flanking" two other elements (e.g., ITRs) indicates that one element is located 5' to the sequence and the other element is located 3' to the sequence, although there may be intervening sequences between them.

[0026] As used herein, the term "fragment" refers to a material or entity having a structure that includes distinct portions of a whole, but lacks one or more portions found in the whole. In some embodiments, the fragment consists of distinct portions. In some embodiments, the fragment consists of or includes characteristic structural elements or portions found in the whole. In some embodiments, a 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.

[0027] As used herein, the term "functional" refers to a biomolecule in a form in which it exhibits the property and / or activity by which it is characterized. A biomolecule can have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).

[0028] As used herein, the term "gene" refers to a polynucleotide containing at least one open reading frame capable of encoding a particular polypeptide or protein after being transcribed and translated. "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting foreign DNA into a host cell. Such methods may result in the transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of a transferred replicon (e.g., an episome), and / or integration of the transferred genetic material into the genomic DNA of the host cell.

[0029] As used herein, the term "heterologous" 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 the nucleic acid into a cell. The heterologous nucleic acid is typically distinct from the vector (e.g., AAV) nucleic acid, i.e., the heterologous nucleic acid is non-native to the viral (e.g., AAV) nucleic acid found in a native AAV. Upon transfer (e.g., transduction) or delivery into a cell, the heterologous nucleic acid contained within the vector may be expressed (e.g., transcribed and translated, if appropriate). Alternatively, the transferred (transduced) or delivered heterologous nucleic acid contained within the vector need not be expressed. Although the term "heterologous" is not necessarily used herein with respect to a nucleic acid, reference to a nucleic acid, even in the absence of the modifier "heterologous," is intended to include a heterologous nucleic acid. For example, a heterologous nucleic acid may be a nucleic acid encoding an ASPA polypeptide, e.g., a codon-optimized nucleic acid encoding ASPA used to treat Canavan disease.

[0030] As used herein, the terms "homologous" or "homology" refer 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, if an amino acid position in two peptides is occupied by the same amino acid, the peptides are homologous at that position. In particular, homologous peptides retain an 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 a polypeptide, nucleic acid, or fragment thereof, "substantial homology" or "substantial similarity" means that there is at least about 95% to 99% sequence identity between the sequences 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 ascertained using computer programs or mathematical algorithms. Such algorithms that calculate percent sequence homology (or identity) generally account for sequence gaps and mismatches over the region or area of ​​comparison. Exemplary programs and algorithms are provided below.

[0031] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transfectants," "transformants," "transformed cells," and "transduced cells," including the primary transfected, transformed, or transduced cell and its derived progeny, regardless of the number of passages. In some embodiments, the host cell is a packaging cell for the production of rAAV vectors.

[0032] As used herein, the term "identity" or "identical" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be "substantially identical" to one another 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.

[0033] Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence aligned for comparison 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. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0034] To determine percent identity or homology, sequences can be aligned using methods and computer programs, including BLAST, available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, 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. Of particular interest are alignment programs that allow gaps in sequences. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70:173-187 (1997). Sequences can also be aligned using the GAP program using the Needleman and Wunsch alignment method. See J. Mol. Biol. 48:443-453 (1970).

[0035] Also of interest is the BestFit program, which uses the local homology algorithm of Smith and Waterman (1981, Advances in Applied Mathematics 2:482-489) to determine sequence identity. The gap creation penalty generally ranges from 1 to 5, usually from 2 to 4, and in some embodiments is 3. The gap extension penalty generally ranges from about 0.01 to 0.20, and in some cases is 0.10. This program has default parameters determined by the sequences entered to be compared. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package from Madison, WI, USA.

[0036] Another interesting program 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. Percent 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 joining penalty: 30.0.

[0037] As used herein, the terms "increase," "improve," or "reduce" refer to a value that is relative to a baseline measurement, such as a measurement in the same individual before initiation of a treatment described herein, or a measurement in a control individual (or control individuals) in the absence of a treatment described herein. In some embodiments, a "control individual" is an individual suffering from the same form of disease or disorder as the individual being treated.

[0038] As used herein, the terms "inverted terminal repeat," "ITR," "terminal repeat," and "TR" refer to palindromic terminal repeat sequences at or near the ends of the AAV genome, containing approximately complementary and symmetrically arranged sequences. These ITRs can fold to form T-shaped hairpin structures that function as primers during the initiation of DNA replication. They are also required for viral genome integration into the host genome, rescue from the host genome, and encapsidation of viral nucleic acids into mature virions. ITRs are required in cis for vector genome replication and its packaging into viral particles. "5' ITR" refers to the ITR at the 5' end of the AAV genome and / or 5' to the recombinant transgene. "3' ITR" refers to the ITR at the 3' end of the AAV genome and / or 3' to the recombinant transgene. The length of a wild-type ITR is approximately 145 bp. Modified or recombinant ITRs may comprise fragments or portions of the wild-type AAV ITR sequence. Those skilled in the art will understand that the ITR sequences of successive rounds of DNA replication may be swapped, such that the 5' ITR becomes the 3' ITR, or vice versa. In some embodiments, 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 an "rAAV vector particle" or "rAAV viral particle").

[0039] As used herein, the term "isolated" refers to a substance or composition that: 1) has been designed, produced, prepared, and / or manufactured by the hand of man; and / or 2) has been separated (in natural and / or experimental settings) from at least one of the components with which it was associated when it was originally produced. Generally, an isolated composition is substantially free of one or more materials with which it is normally associated in nature, e.g., one or more proteins, nucleic acids, lipids, carbohydrates, and / or cellular membranes. The term "isolated" does not exclude artificial combinations that package, e.g., encapsidate, a vector genome and a pharmaceutical formulation, such as recombinant nucleic acids, recombinant vector genomes (e.g., rAAV vector genomes), rAAV vector particles (e.g., rAAV vector particles comprising an AAV / Olig001 capsid, etc.). The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrid / chimeric, multimeric / oligomeric, modified (e.g., phosphorylated, glycosylated, lipidated), mutant or derivatized forms, or forms expressed in artificial host cells.

[0040] Isolated substances or compositions 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 greater than about 99% of other components with which they were initially associated. In some embodiments, isolated agents are 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 greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by one of skill in the art, a substance may still be considered "isolated" or "pure" after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the percent isolation or purity of the substance is calculated without including such carriers or excipients.

[0041] As used herein, the terms "nucleic acid sequence," "nucleotide sequence," and "polynucleotide" refer interchangeably to any molecule composed of or comprising monomeric nucleotides linked by phosphodiester bonds. A nucleic acid can be an oligonucleotide or a polynucleotide. Nucleic acid sequences are presented herein in a 5' to 3' orientation. A nucleic acid sequence (i.e., a polynucleotide) of the present disclosure can be a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule and refers to all forms of nucleic acid, such as double-stranded molecules, single-stranded molecules, short or short-stranded hairpin RNA (shRNA), microRNA, short or short-stranded interfering RNA (siRNA), trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA, and ribosomal RNA. When a polynucleotide is a DNA molecule, the molecule can be a gene, cDNA, antisense molecule, or a fragment of any of the foregoing molecules. Nucleotides are referred to herein by the single-letter code: 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 (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Each of these sequences is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule. Phosphorothioate nucleotides may also be used. Other deoxynucleotide analogs include methyl phosphonates, phosphoramidates, phosphorodithioates, N3'-P5'-phosphoramidates, and oligoribonucleotide phosphorothioates, as well as their 2'-0-allyl analogs and 2'-0-methylribonucleotide methylphosphonates, which may be used in the nucleotide sequences of the present disclosure.

[0042] As used herein, the term "nucleic acid construct" refers to a non-naturally occurring nucleic acid molecule resulting from the use of recombinant DNA technology (e.g., recombinant nucleic acid). A nucleic acid construct is a nucleic acid molecule, either single-stranded or double-stranded, that has been modified to contain segments of nucleic acid sequences joined and arranged in a manner not found in nature. A nucleic acid construct may be a "vector" (e.g., a plasmid, a rAAV vector genome, an expression vector, etc.), i.e., a nucleic acid molecule designed to deliver exogenously formed DNA into a host cell.

[0043] As used herein, the term "operably linked" refers to the linkage of nucleic acid sequence (or polypeptide) elements in a functional relationship. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or other transcriptional regulatory sequence (e.g., an enhancer) is operably linked to a coding sequence if it affects the transcription of 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 linked contiguously, but rather that there are intervening sequences between the linked nucleic acid sequences.

[0044] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to a biologically acceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration for in vivo delivery or contact.

[0045] As used herein, the terms "polypeptide," "protein," "peptide," or "encoded by a nucleic acid sequence" (i.e., encoded by a polynucleotide sequence that is encoded by a nucleotide sequence) refer to the full-length native sequence, as well as to functional subsequences, modified forms, or sequence variants, as well as naturally occurring proteins, so long as the subsequence, modified form, or variant retains some functionality of the native, full-length protein. In the methods and uses of the present disclosure, such polypeptides, proteins, and peptides encoded by nucleic acid sequences may, but need not, be identical to the endogenous protein that is defective, or whose expression is insufficient or absent, in the subject being treated by gene therapy.

[0046] As used herein, the term "prevent" or "prevention" refers 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, disorder, or condition (e.g., Canavan disease). In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the onset, frequency, and / or intensity of one or more signs or symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is delayed for a predetermined period of time.

[0047] 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 cloning, restriction, or ligation steps, and / or other procedures that result in a construct that differs from the product found in nature. A recombinant virus or vector (e.g., an rAAV vector) comprises a vector genome that includes a recombinant nucleic acid (e.g., a nucleic acid that includes a transgene and one or more regulatory elements, e.g., codon-optimized nucleic acids encoding the ASPA and CBh promoters). The term includes copies of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0048] As used herein, the term "subject" refers to an organism, e.g., a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, the subject is a nur7 mouse. In some embodiments, the human subject is an adult, an adolescent, or a pediatric subject. In some embodiments, the subject is suffering from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, the subject is suffering from a disease, disorder, or condition associated with a deficiency or dysfunction of aspartoacylase activity, e.g., Canavan disease. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, a susceptible subject is prone to and / or exhibits an increased risk (compared to the average risk observed in a reference subject or population) of developing the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit specific symptoms (e.g., clinical symptoms of the disease) or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a human patient. In some embodiments, the subject is an individual who is being treated with and / or has been treated with a diagnosis and / or therapy (e.g., gene therapy for Canavan disease). In some embodiments, the subject is a human patient with Canavan disease.

[0049] As used herein, the term "substantially" refers to the qualitative condition of expression of all or nearly all degrees or extents of a characteristic or property of interest. Those skilled in the art will understand that biological and chemical phenomena rarely, if ever, reach perfection and / or proceed perfectly or achieve absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0050] As used herein, the terms "symptoms are reduced" or "reducing symptoms" refer to when the magnitude (e.g., intensity, severity, etc.) and / or frequency of one or more symptoms of a particular disease, disorder, or condition is reduced. For clarity, delaying the onset of a particular symptom is considered a form of reducing the frequency of that symptom.

[0051] As used herein, the term "therapeutic polypeptide" refers to a peptide, polypeptide, or protein (e.g., an enzyme, structural protein, transmembrane protein, transport protein) that can alleviate or reduce symptoms resulting from the absence or deficiency of a protein in a target cell (e.g., an isolated cell) or organism (e.g., a subject). A therapeutic polypeptide or protein encoded by a transgene is one that confers a benefit to a subject, e.g., to correct a genetic defect, to correct a deficiency in a gene associated with expression or function. Similarly, a "therapeutic transgene" is a transgene that encodes a therapeutic polypeptide. In some embodiments, a therapeutic polypeptide expressed in a host cell is an enzyme expressed from a transgene (i.e., an exogenous nucleic acid introduced into a host cell). In some embodiments, a therapeutic polypeptide is ASPA protein expressed from a therapeutic transgene transferred into cerebral cortical cells (e.g., oligodendrocytes).

[0052] As used herein, the term "therapeutically effective amount" refers to an amount that produces the desired therapeutic effect for which it is administered. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, or condition when administered according to a therapeutic regimen to a population suffering from or susceptible to the disease, disorder, or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of, and / or delays the onset of, one or more symptoms of the disease, disorder, and / or condition. Those skilled in the art will understand that the term "therapeutically effective amount" does not actually require that successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount can be an amount that, when administered to patients in need of such treatment, provides a specific, desired pharmacological response in a significant number of subjects.

[0053] As used herein, the term "transgene" refers to any heterologous polynucleotide for delivery to and / or expression in a host cell, target cell, or organism (e.g., a subject). Such a "transgene" can be delivered to a host cell, target cell, or organism using a vector (e.g., an rAAV vector). The transgene can be operably linked to a control sequence, such as a promoter. Those skilled in the art will understand that expression control sequences can be selected based on their ability to promote expression of the transgene in a host cell, target cell, or organism. Generally, a transgene can be operably linked to an endogenous promoter naturally associated with the transgene, but more typically, the transgene is operably linked to a promoter with which the transgene is not naturally associated. An example of a transgene is a nucleic acid encoding a therapeutic polypeptide, e.g., an ASPA polypeptide, and an exemplary promoter is one that is not operably linked to a nucleotide encoding ASPA in nature. Such non-endogenous promoters can include the CBh promoter, among many others known in the art.

[0054] Nucleic acids of interest can be introduced into host cells by a variety of techniques well known in the art, including transfection and transduction.

[0055] "Transfection" is generally known as a technique for introducing exogenous nucleic acid into cells without the use of a viral vector. As used herein, the term "transfection" refers to the transfer of a recombinant nucleic acid (e.g., an expression plasmid) into a cell (e.g., a host cell) without the use of a viral vector. A cell into which a recombinant nucleic acid has been introduced is referred to as a "transfected cell." The transfected cell may be a host cell (e.g., a CHO cell, Pro10 cell, or HEK293 cell) containing an expression plasmid / vector for producing a recombinant AAV vector. In some embodiments, the transfected cell (e.g., a packaging cell) may contain a plasmid containing a transgene (e.g., an ASPA transgene), a plasmid containing the AAV rep gene and the AAV cap gene, and a plasmid containing a helper gene. 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 a nuclear localization signal.

[0056] As used herein, the term "transduction" refers to the transfer of a nucleic acid (e.g., a vector genome) into a cell (e.g., a target cell, including, but not limited to, an oligodendrocyte) by a viral vector (e.g., an rAAV vector). In some embodiments, gene therapy for Canavan disease involves transducing an oligodendrocyte with a vector genome comprising a modified nucleic acid encoding ASPA. A cell into which a transgene has been introduced by a virus or viral vector is referred to as a "transduced cell." In some embodiments, the transduced cell is an isolated cell, and transduction occurs ex vivo. In some embodiments, the transduced cell is a cell within an organism (e.g., a subject), and transduction occurs in vivo. A transduced cell may be a target cell of an organism that has been transduced by a recombinant AAV vector such that the target cell of the organism expresses a polynucleotide (e.g., a transgene, e.g., a modified nucleic acid encoding ASPA).

[0057] Cells that can be transduced include cells of any tissue or organ type, or of any origin (e.g., mesodermal, ectodermal, or endodermal). Non-limiting examples of cells include liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells, exocrine), lung, central or peripheral nervous system, such as brain (e.g., nerve or ependymal cells, oligodendrocytes) or spine, kidney, eye (e.g., retina), spleen, skin, thymus, testis, lung, diaphragm, heart (cardiac), muscle or lumbar, or intestine (e.g., endocrine), adipose tissue (white, brown, or beige), muscle (e.g., fibroblasts, myocytes), synoviocytes, chondrocytes, osteocytes, epithelial cells, endothelial cells, salivary gland cells, inner ear neurons, or hematopoietic cells (e.g., blood or lymphocytes). Additional examples include stem cells such as pluripotent or multipotent progenitor cells that develop or differentiate into liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells, exocrine cells), lung, central or peripheral nervous system, e.g., brain (e.g., neural or ependymal cells, oligodendrocytes) or spine, kidney, eye (e.g., retina), spleen, skin, thymus, testis, lung, diaphragm, heart (cardiac), muscle or lumbar, or intestine (e.g., endocrine), adipose tissue (white, brown, or beige), muscle (e.g., fibroblasts, myocytes), synoviocytes, chondrocytes, osteocytes, epithelial cells, endothelial cells, salivary gland cells, inner ear neurons, or hematopoietic cells (e.g., blood or lymphocytes).

[0058] In some embodiments, cells present within a specific area of ​​a tissue or organ (e.g., the brain) can be transduced by an rAAV vector (e.g., an rAAV containing an ASPA transgene) administered to the tissue or organ. In some embodiments, brain cells are transduced with an rAAV containing an ASPA transgene. In some embodiments, cells of the cortex of the brain are transduced with an rAAV containing an ASPA transgene. In some embodiments, cells of the striatum of the brain are transduced with an rAAV containing an ASPA transgene. In some embodiments, subcortical white matter cells of the brain are transduced with an rAAV containing an ASPA transgene. In some embodiments, cells of the cerebellum of the brain are transduced with an rAAV containing an ASPA transgene.

[0059] As used herein, the terms "treat," "treating," or treatment refer to the administration of a therapy that partially or completely relieves, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition.

[0060] As used herein, the term "vector" refers to a plasmid, virus (e.g., rAAV), cosmid, or other vehicle that can be manipulated by the insertion or incorporation of a nucleic acid (e.g., a recombinant nucleic acid). Vectors can be used for a variety of purposes, including, for example, genetic engineering (e.g., cloning vectors), to introduce / transfect a nucleic acid into a cell and transcribe or translate the inserted nucleic acid within the cell. In some embodiments, the vector nucleic acid sequence contains at least an origin of replication for propagation in a cell. In some embodiments, the vector nucleic acid comprises a heterologous nucleic acid sequence, expression control elements (e.g., promoter, enhancer), a selectable marker (e.g., antibiotic resistance), a polyadenosine (polyA) sequence, and / or ITRs. In some embodiments, the nucleic acid sequence propagates when delivered to a host cell. In some embodiments, when delivered to a host cell, either in vitro or in vivo, the cell expresses a polypeptide encoded by the heterologous nucleic acid sequence. In some embodiments, when delivered to a host cell, the nucleic acid sequence or a portion of the nucleic acid sequence is packaged into a capsid. The host cell can be an isolated cell or a cell within a host organism. In addition to a nucleic acid sequence encoding a polypeptide or protein (e.g., a transgene), additional sequences (e.g., regulatory sequences) may be present within the same vector (i.e., in cis with respect to the gene) and adjacent to the gene. In some embodiments, regulatory sequences may be present on a separate (e.g., second) vector that acts in trans to regulate expression of the gene. Plasmid vectors may be referred to herein as "expression vectors."

[0061] As used herein, the term "vector genome" refers to a recombinant nucleic acid sequence that is packaged or encapsidated to form a rAAV vector. Typically, a vector genome includes heterologous polynucleotide sequences, such as transgenes, regulatory elements, and ITRs not originally present in the capsid. When a recombinant plasmid is used to construct or produce a recombinant vector (e.g., an rAAV vector), the vector genome does not include the entire plasmid, but rather only the sequences intended for delivery by the viral vector. This non-vector genome portion of the recombinant plasmid is typically referred to as the "plasmid backbone," and is important for the cloning, selection, and amplification of the plasmid, processes required for the propagation of recombinant viral vector production, but is not itself packaged or encapsidated into the rAAV vector.

[0062] As used herein, the term "viral vector" generally refers to a viral particle that functions as a nucleic acid delivery vehicle and contains a vector genome (e.g., containing a transgene in place of nucleic acids encoding AAV rep and cap) packaged within the viral particle (i.e., capsid), including, for example, lentiviruses and parvoviruses, including AAV serotypes and variants (e.g., rAAV vectors). Recombinant viral vectors do not contain a vector genome containing the rep and / or cap genes.

[0063] The present disclosure provides modified nucleic acids comprising a modified ASPA coding sequence and uses thereof in gene therapy pharmaceutical compositions. As used herein, "modified" refers, in one embodiment, to alteration of a naturally occurring polypeptide-encoding nucleic acid sequence such that the modified nucleic acid sequence drives a higher level of protein expression in a cell compared to the level of protein expression in an otherwise identical cell of an unmodified, i.e., naturally occurring (including a mutated form of the gene) nucleic acid sequence. The present disclosure also provides recombinant nucleic acids comprising vector genomes that include the modified ASPA coding sequence as part of their sequence. Furthermore, the present disclosure provides packaged gene delivery vehicles, such as rAAV vectors, that include the modified ASPA coding sequence. The present disclosure also includes methods of intracellular delivery, preferably expression, of the modified ASPA coding sequence. The present disclosure also provides gene therapy methods in which the modified ASPA coding sequence is administered to a subject, for example, as a component of a vector and / or packaged as a component of a viral gene delivery vehicle (e.g., an rAAV vector). Treatment can be performed, for example, to increase ASPA levels in a subject and treat ASPA deficiency in a subject. Each of these aspects of the disclosure is further described in the following sections.

[0064] AAV and rAAV vectors AAV As mentioned above, the terms "adeno-associated virus" and / or "AAV" refer to parvoviruses and their variants that have a linear, single-stranded DNA genome. The term encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise required. Parvoviruses, including AAV, are useful as gene therapy vectors because they can enter cells and deliver nucleic acids (e.g., transgenes) to the nucleus. In some embodiments, the delivered nucleic acid (e.g., rAAV vector genome) forms circular concatemers that persist episomally in the nucleus of transduced cells. In some embodiments, the transgene is inserted into a specific site within the host cell genome, e.g., a site on human chromosome 19. Site-specific integration, as opposed to random integration, is believed to be more likely to result in a predictable long-term expression profile. The insertion site of AAV into the human genome is designated AAVS1. Once delivered to a cell, the polypeptide 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 by AAV can be used to express therapeutic polypeptides for the treatment of diseases, disorders, and / or conditions in human subjects.

[0065] Multiple serotypes of AAV exist in nature, and at least 15 wild-type serotypes have been identified in humans to date (i.e., AAV1 through AAV15). Naturally occurring and variant serotypes are distinguished by possessing serologically distinct protein capsids from other AAV serotypes. These serotypes include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), including AAV type 3A (AAV3A), and AAV type 3B (AAV3B), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), and AAV type 12 (AAV12). These include AAVrh10, AAVrh74 (see WO2016 / 210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, as well as recombinantly produced variants (e.g., capsid variants with insertions, deletions, and substitutions), such as AAV type 2i8 (AAV2i8), NP4, NP22, NP66, DJ, DJ / 8, DJ / 9, LK3, and RHM4-1, among many other variants. For example, "primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals. Serotype identity is determined based on the lack of cross-reactivity between antibodies against one AAV compared to another. Such differences in cross-reactivity are usually 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 variants (e.g., recombinant AAVs) may not exhibit serological differences from any of the currently known serotypes. These viruses may then be considered subgroups of the corresponding type, or more simply, mutant AAVs. Thus, as used herein, the term "serotype" refers to both serologically distinct viruses, e.g., AAVs, as well as viruses, e.g., AAVs, that are not serologically distinct but may be subgroups or variants of a given serotype.

[0066] 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, particularly in Supplementary Figure 1.

[0067] The genomic sequences of various AAV serotypes, as well as the sequences of the native terminal repeats (ITRs), rep proteins, and capsid subunits, are known in the art and can be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. 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 disclosures of which are incorporated herein by reference. 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. 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 Publications WO 00 / 28061, 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. By way of example only, wild-type AAV2 contains a small (20-25 nm) icosahedral viral capsid of AAV, which is composed of three proteins (VP1, VP2, and VP3; a total of 60 capsid proteins make up 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. That is, for AAV, VP1 is the full-length protein, while VP2 and VP3 are progressively shorter versions of VP1 with increased N-terminal truncations compared to VP1.

[0068] Recombinant AAV As mentioned above, "recombinant adeno-associated virus" or "rAAV" is distinguished from wild-type AAV by replacing all or part of the endogenous viral genome with non-native sequences. The incorporation of non-native sequences into the virus defines the viral vector as a "recombinant" vector, and therefore an "rAAV vector." rAAV vectors can contain a heterologous polynucleotide encoding a desired protein or polypeptide (e.g., an ASPA polypeptide). Recombinant vector sequences may be encapsidated or packaged into an AAV capsid and are referred to as "rAAV vectors," "rAAV vector particles," "rAAV viral particles," or simply "rAAV."

[0069] For rAAV vector production, 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. The desired ratio of VP1:VP2 is 1:1, although the range of VP1:VP2 ratios can vary from 1:50 to 50:1.

[0070] The present disclosure provides rAAV vectors containing polynucleotide sequences not of AAV origin (e.g., polynucleotides heterologous to AAV). The heterologous polynucleotide may be flanked by at least one, and optionally two, AAV terminal repeat sequences (e.g., inverted terminal repeats (ITRs)). The heterologous polynucleotide flanked by the ITRs, also referred to herein as the "vector genome," typically encodes a polypeptide or gene of interest ("GOI"), such as a target for therapeutic treatment (e.g., a nucleic acid encoding ASPA for the treatment of Canavan disease). Delivery or administration of the rAAV vector to a subject (e.g., a patient) provides the encoded protein or peptide to the subject. Thus, rAAV vectors can be used to transcribe / deliver heterologous polynucleotides for expression, for example, to treat various diseases, disorders, and conditions.

[0071] rAAV vector genomes generally retain 145-base ITRs in cis with the heterologous nucleic acid sequences that replace the viral rep and cap genes. Such ITRs are necessary for producing recombinant AAV vectors, although modified AAV ITRs and non-AAV terminal repeats containing partially or completely synthetic sequences can also serve this purpose. The ITRs form hairpin structures and function, for example, as primers for host cell-mediated synthesis of complementary DNA strands after infection. ITRs also play a role in viral packaging, integration, and the like. ITRs are the only AAV viral elements required in cis for AAV genome replication and packaging into rAAV vectors. rAAV vector genomes optionally contain two ITRs, typically located at the 5' and 3' ends of the vector genome, that contain heterologous sequences (e.g., a transgene encoding a gene of interest, or a nucleic acid sequence of interest, including, but not limited to, antisense and siRNA, and CRISPR molecules, among many others). The 5' and 3' ITRs may both contain the same sequence, or each may contain a different sequence. The AAV ITRs can be derived from any AAV, including but not limited to serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or any other AAV.

[0072] The rAAV vectors of the present disclosure may contain ITRs from an AAV serotype (e.g., wild-type AAV2, a fragment, or a variant thereof) that is different from the capsid serotype (e.g., AAV8, Olig001). Such rAAV vectors that contain at least one ITR from one serotype but a capsid from a different serotype may be referred to as hybrid viral vectors (see U.S. Patent No. 7,172,893). The AAV ITRs may include the entire wild-type ITR sequence or may be a variant, fragment, or modification thereof, while retaining functionality.

[0073] In some embodiments, the heterologous polypeptide comprises ITRs (e.g., ITRs from AAV2, but may include ITRs from any wild-type AAV serotype, or variants thereof) located at the left and right ends (i.e., the 5' and 3' ends, respectively) of the vector genome. In some embodiments, the left (e.g., 5') ITR comprises or consists of the 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 a nucleic acid sequence that is about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical to SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19. In some embodiments, the right (e.g., 3') ITR comprises or consists of 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 a nucleic acid sequence that is about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical to SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19. Each ITR may be in cis with, but separated from, each other or other elements in the vector genome by a nucleic acid sequence of variable length, such as a recombinant nucleic acid comprising a modified nucleic acid encoding ASPA and a regulatory element. In some embodiments, the ITRs are AAV2 ITRs, or variants thereof, and flank the ASPA transgene. In some embodiments, the rAAV comprises an ASPA transgene (e.g., comprising the nucleic acid sequence of SEQ ID NO:2) flanked by AAV2 ITRs (e.g., an ITR having the sequence set forth in SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19).

[0074] In some embodiments, the rAAV vector genome is linear, single-stranded, and flanked by AAV ITRs. Prior to transcription and translation of the heterologous gene, the free 3'-OH of one of the self-priming ITRs must be used by a DNA polymerase (e.g., a DNA polymerase within the transduced cell) to convert the approximately 4700 nucleotide single-stranded DNA genome into a double-stranded form and prime second-strand synthesis. In some embodiments, full-length single-stranded vector genomes (i.e., sense and antisense) anneal to generate full-length double-stranded vector genomes. This can occur when multiple rAAV vectors carrying genomes of opposite polarity (i.e., sense or antisense) simultaneously transduce the same cell. Regardless of how they are produced, once the double-stranded vector genomes are formed, the cell can transcribe and translate the double-stranded DNA and express the heterologous gene.

[0075] The efficiency of transgene expression from rAAV vectors can be hindered by the need to convert single-stranded rAAV genomes (ssAAV) to double-stranded DNA prior to expression. This step is circumvented by using self-complementary AAV genomes (scAAV), which can package inverted repeat genomes that 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). A limitation of scAAV vectors is that the size of the unique transgene, regulatory elements, and IRT packaged into the capsid is approximately half the size (i.e., approximately 2,500 nucleotides, of which 2,200 nucleotides may be the transgene and regulatory elements, plus two copies of the approximately 145 nucleotide ITRs) of the ssAAV vector genome (i.e., approximately 4,900 nucleotides including the two ITRs).

[0076] scAAV vector genomes are generated by using nucleic acids that do not contain a terminal resolution site (TRS) or by modifying the TRS from one rAAV ITR of a vector, such as a plasmid, containing the vector genome, thereby preventing replication initiation from that end (see U.S. Patent No. 8,784,799). AAV replication in a host cell initiates at the wild-type ITR of the scAAV vector genome, continues through the ITR lacking a terminal resolution site or containing a modified terminal resolution site, and then back through the genome to form a complementary strand. The resulting complementary single nucleic acid molecule is thus a self-complementary nucleic acid molecule, resulting in a vector genome with a mutated (uncleaved) ITR in the middle and wild-type ITRs at both ends. In some embodiments, the mutated ITR lacking a TRS or containing a modified TRS is at the 5' end of the vector genome. In some embodiments, the mutated ITR lacking a TRS or containing a modified TRS that is not degraded (cleaved) is at the 3' end of the vector genome. In some embodiments, the mutated ITR comprises the nucleic acid of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19.

[0077] Without wishing to be bound by theory, it is believed that although the two halves of the scAAV genome are complementary, substantial base pairing within the capsid is unlikely because many bases contact amino acid residues in the inner capsid shell and the phosphate backbone is sequestered toward the center (McCarty, Molec. Therapy (2008) 16(10):1648-1656). Upon uncoating, the two halves of the scAAV genome likely anneal to form a dsDNA hairpin molecule with a covalently closed ITR at one end and two open ITRs at the other. The ITRs flank the double-stranded region that encodes, among other things, the transgene and its associated regulatory elements in cis.

[0078] The viral capsid of the rAAV vector may be a wild-type AAV or a mutant AAV, such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAVrh74 (see WO2016 / 210170), AAV12, AAV2i8, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO: 5 in WO2015 / 013313), RHM15-1, RHM15-2, RHM15-3, RHM15-4, RHM15-5, RHM15-6, RHM15-7, RHM15-8, RHM15-9, RHM15-10, RHM15-11, RHM15-12, RHM15-13, RHM15-14, RHM15-15, RHM15-16, RHM15-17, RHM15-18, RHM15-19, RHM15-20, RHM15-21, RHM15-22, RHM15-23, RHM15-24, RHM15-25, RHM15-26, RHM15-27, RHM15-28, RHM15-29, RHM15-30, RHM15-31, RHM15-32, RHM15-33, RHM15-34, RHM15-35, RHM15-36, RHM15-37, RHM15-38 The AAV vector may be derived from avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, snake AAV, caprine AAV, shrimp AAV, ovine AAV, and variants thereof (see, e.g., Fields et al., VIROLOGY, volume 2, chapter 69 (4 th(See, e.g., Lippincott-Raven Publishers). Capsids may be derived from any of the AAV serotypes disclosed in U.S. Pat. No. 7,906,111; Gao et al. (2004) J. Virol. 78:6381; Morris et al. (2004) Virol. 33:375; WO 2013 / 063379; WO 2014 / 194132, including the true type AAV (AAV-TT) variant disclosed in WO 2015 / 121501, and RHM4-1, RHM15-1 through RHM15-6, and variants thereof, disclosed in WO 2015 / 013313. Those skilled in the art will recognize that there are likely other AAV variants, as yet unidentified, that perform the same or similar functions. The full complement of AAV cap proteins includes VP1, VP2, and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein may contain less than the full complement of AAV Cap proteins, or the full complement of AAV Cap proteins may be provided.

[0079] In another embodiment, the present disclosure provides the use of ancestral AAV vectors for use in therapeutic in vivo gene therapy. Specifically, computationally derived sequences can be synthesized de novo and characterized for biological activity. Prediction and synthesis of ancestral sequences, along with assembly into rAAV vectors, can be achieved using methods described in WO2015 / 054653, the contents of which are incorporated herein by reference. In particular, rAAV vectors assembled from ancestral viral sequences can have reduced susceptibility to pre-existing immunity in the human population compared to contemporary viruses or portions thereof.

[0080] In some embodiments, rAAV vectors comprising capsid proteins encoded by nucleotide sequences from two or more AAV serotypes (e.g., wild-type AAV serotype, mutant AAV serotypes) are referred to as "chimeric vectors" or "chimeric capsids" (see U.S. Patent No. 6,491,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, the chimeric capsid proteins are encoded by nucleic acid sequences from two, three, four, five, six, seven, eight, nine, ten, or more AAV serotypes. In some embodiments, the recombinant AAV vector comprises capsid sequences derived from, for example, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10, AAV2i8, or variants thereof, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the aforementioned AAV serotypes (see Rabinowitz et al. (2002) J. Virology 76(2):791-801). Alternatively, the chimeric capsid may comprise a VP1 from one serotype, a VP2 from a different serotype, a VP3 from yet another serotype, and a mixture of combinations thereof. For example, the chimeric viral capsid may comprise an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit. The chimeric capsid can include, for example, an AAV capsid having one or more B19 cap subunits, e.g., the AAV cap protein or subunit can be replaced by a B19 cap protein or subunit. For example, in one embodiment, the VP3 subunit of an AAV capsid can be replaced by the VP2 subunit of B19. In some embodiments, the chimeric capsid is an Olig001 capsid, as described in WO2014052789, incorporated herein by reference.

[0081] In some embodiments, the chimeric vector is engineered to exhibit altered tropism or tropism for specific tissues or cell types. The term "tropism" refers to the preferential entry of a virus into a particular cell or tissue type and / or preferential interactions with cell surfaces that facilitate entry into a particular cell or tissue type. AAV tropism is generally determined by the specific interactions between different viral capsid proteins and their cognate cellular receptors (Lykken et al. (2018) J. Neurodev. Disord. 10:16). Preferably, upon entry of the virus or viral vector into a cell, sequences (e.g., heterologous sequences such as transgenes) carried by the vector genome (e.g., rAAV vector genome) are expressed.

[0082] A "tropism profile" refers to the pattern of transduction of one or more target cells in various tissues and / or organs. For example, a chimeric AAV capsid may have a tropism profile characterized by efficient transduction of oligodendrocytes with only low transduction of neurons, astrocytes, and other CNS cells. See WO 2014 / 052789, incorporated herein by reference. Such chimeric capsids may be considered "oligodendrocyte-specific," exhibiting tropism for oligodendrocytes, and are referred to herein as "oligotropic" if they preferentially transduce oligodendrocytes over neurons, astrocytes, and other CNS cell types when administered directly to the CNS. In some embodiments, at least about 80% of cells transduced by an oligodendrocyte-specific capsid are oligodendrocytes, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the transduced cells are oligodendrocytes.

[0083] In some embodiments, the rAAV vectors are useful for treating or preventing "disorders associated with oligodendrocyte dysfunction." As used herein, the term "associated with oligodendrocyte dysfunction" refers to a disease, disorder, or condition in which oligodendrocytes are damaged, lost, or function inappropriately compared to otherwise identical normal oligodendrocytes. This term includes diseases, disorders, and conditions in which oligodendrocytes are directly affected, as well as diseases, disorders, or conditions in which oligodendrocytes malfunction secondary to damage to other cells. In some embodiments, the disorder associated with oligodendrocyte dysfunction is Canavan disease (CD).

[0084] In some embodiments, the chimeric AAV capsid with oligodendrocyte tropism is Olig001 (also known as BNP61), which includes sequences from AAV1, AAV2, AAV6, AAV8, and AAV9 (see WO2014 / 052789). In some embodiments, Oligo001 capsid VP1 is encoded by a nucleic acid sequence that includes or consists of the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, Olig001 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.

[0085] The nucleic acid sequences encode overlapping AAV capsid proteins, VP1, VP2, and VP3. The amino acid sequences of the Olig001 capsid proteins are set forth in SEQ ID NO:14, with VP1 beginning at amino acid residue 1 (methionine) of SEQ ID NO:14, VP2 beginning at amino acid residue 148 (threonine), and VP3 beginning at amino acid residue 203 (methionine).

[0086] In some embodiments, the chimeric AAV capsid with tropism for oligodendrocytes is Olig002 (also known as BNP62) or Olig003 (also known as BNP63) (see WO2014 / 052789). In some embodiments, the Oligo002 capsid VP1 comprises or consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the Olig002 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 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 consists 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 that encodes the amino acid sequence of SEQ ID NO: 16.

[0087] In some embodiments, rAAV vectors comprising a chimeric AAV capsid (e.g., Olig001) and a therapeutic transgene can be used to treat diseases, disorders, or conditions associated with oligodendrocyte dysfunction. In such diseases, disorders, or conditions, oligodendrocytes are damaged, lost, or function improperly. This can be the result of a direct effect on oligodendrocytes, or can occur when oligodendrocytes malfunction secondary to damage to other cells. In some embodiments, rAAV vectors comprising an AAV / Olig001 capsid and a modified ASPA nucleic acid are used to treat Canavan disease.

[0088] Recombinant nucleic acids Recombinant nucleic acids of the present disclosure include modified nucleic acids, as well as plasmids and vector genomes comprising the modified nucleic acids. The recombinant nucleic acid, plasmid, or vector genome may comprise regulatory sequences for regulating propagation (e.g., of the plasmid) and / or controlling expression of the modified nucleic acid (e.g., a transgene). The recombinant nucleic acid may also be provided as a component of a viral vector (e.g., an rAAV vector). Generally, a viral vector comprises a vector genome comprising the recombinant nucleic acid packaged in a capsid.

[0089] modified nucleic acid A modified or mutant form of a gene, nucleic acid, or polynucleotide (e.g., a transgene) refers to a nucleic acid that deviates from a reference sequence. The reference sequence may be a naturally occurring wild-type sequence (e.g., a gene) or may 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). A modified / variant nucleic acid may have substantially the same, greater, or lesser activity, function, or expression compared to the reference sequence. Preferably, a modified or mutant nucleic acid, as used interchangeably herein, exhibits improved protein expression, e.g., the protein encoded thereby is expressed at a detectably higher level in a cell compared to the protein expression level provided by an endogenous gene (e.g., a wild-type gene, a mutant gene) in an otherwise identical cell. In some embodiments, a modified nucleic acid or variant nucleic acid, as used interchangeably herein (e.g., a modified nucleic acid encoding ASPA), exhibits improved protein expression, e.g., the protein encoded thereby is expressed at a detectably higher level in a cell compared to the expression level of the protein provided by an endogenous gene containing the mutation in an otherwise identical cell.

[0090] Modifications to nucleic acids include substitutions of one or more nucleotides of a reference sequence (e.g., substitutions of 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 100 or more nucleotides), additions (e.g., insertions of 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 100 or more nucleotides), and deletions (e.g., deletions of 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 100 or more nucleotides, deletions of motifs, domains, fragments, etc.). The modified nucleic acid can be about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the reference sequence.

[0091] Modified nucleic acids can encode polypeptides having about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identity to a reference polypeptide. In some embodiments, a modified nucleic acid encoding ASPA (e.g., SEQ ID NO: 2) encodes a polypeptide having 100% identity to a reference polypeptide (e.g., SEQ ID NO: 4).

[0092] In some embodiments, the modified nucleic acid (e.g., a transgene) encodes a wild-type protein. Such modified nucleic acids may be codon-optimized. "Optimized" or "codon-optimized," referred to interchangeably herein, refers to a coding sequence that has been optimized relative to a wild-type coding sequence or a reference sequence (e.g., a coding sequence for an ASPA polypeptide) to increase expression of the polypeptide, for example, by minimizing rare codon usage, reducing the number of CpG dinucleotides, removing cryptic splice donor or acceptor sites, removing Kozak sequences, removing ribosome entry sites, etc. In some embodiments, the expression level of a protein from a codon-optimized sequence (e.g., a modified nucleic acid encoding ASPA) is increased compared to the expression level of the protein from a wild-type gene in an otherwise identical cell. In some embodiments, the expression level of a protein from a codon-optimized sequence (e.g., a modified nucleic acid encoding ASPA) is not increased compared to the expression level of the protein from a wild-type gene in an otherwise identical cell (e.g., expression is substantially similar). In some embodiments, the expression level of protein from a codon-optimized sequence (e.g., a modified nucleic acid encoding ASPA) is increased compared to the expression level of protein from a mutated gene in an otherwise identical cell.

[0093] Examples of modifications include the elimination of one or more cis-acting motifs and the introduction of one or more Kozak sequences, hi some embodiments, one or more cis-acting motifs are eliminated and one or more Kozak sequences are introduced.

[0094] Examples of cis-acting motifs that may be excluded include internal TATA boxes; Chi sites; ribosome entry sites; ARE, INS, and / or CRS sequence elements; repeat sequences and / or RNA secondary structures; (potential) splice donor and / or acceptor sites, branch points; and restriction sites.

[0095] 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 coding sequence or reference sequence. In some embodiments, the modified polypeptide has one or more non-conservative or conservative amino acid changes. In some embodiments, certain domains that have been shown to play limited or no role in polypeptide function are absent from the modified polypeptide (e.g., certain binding domains) (e.g., WO2016 / 097219). The modified nucleic acid present in the rAAV vector may contain fewer nucleotides than the wild-type coding or reference sequence due to the packaging capabilities of the rAAV capsid (e.g., a truncated mini-dystrophin transgene, see WO 2001 / 83695; a B-domain deleted human Factor VIII transgene, see WO 2017 / 074526), ​​or may comprise a truncated transgene that has been truncated and codon optimized (e.g., the codon-optimized mini-dystrophin transgene described in WO 2017 / 221145). In some embodiments, the polypeptide encoded by the modified nucleic acid has less than, the same as, or greater than, but at least a portion of, the function or activity of the polypeptide encoded by the reference sequence.

[0096] A modified nucleic acid may have a modified GC content (e.g., the number of G and C nucleotides present in a nucleic acid sequence), a modified (e.g., increased or decreased) CpG dinucleotide content, and / or a modified (e.g., increased or decreased) codon adaptation index (CAI) relative to a reference and / or wild-type sequence (e.g., a wild-type ASPA coding sequence). See, e.g., WO2017 / 077451 (discussing various considerations well known in the art for codon optimization of a nucleic acid sequence of interest, including publicly available software for analyzing nucleic acid sequences for optimization). As used herein, modification refers to a decrease or increase in a particular value, amount, or effect.

[0097] In some embodiments, the GC content of modified nucleic acid sequences of the present 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% greater than the GC content of the wild-type coding sequence (e.g., SEQ ID NO: 3). In some embodiments, GC content is expressed as the percentage of G (guanine) and C (cytosine) nucleotides in the sequence.

[0098] In some embodiments, the codon adaptation index of the modified nucleic acid sequences of the present 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.

[0099] In some embodiments, modified nucleic acid sequences of the present disclosure have reduced levels of CpG dinucleotides compared to a wild-type or reference nucleic acid sequence that are about 10%, 20%, 30%, 50% or more reduced. In some embodiments, modified nucleic acids have 1 to 5 fewer, 5 to 10 fewer, 10 to 15 fewer, 15 to 20 fewer, 20 to 25 fewer, 25 to 30 fewer, 30 to 40 fewer, 40 to 45 fewer, or 45 to 50 fewer, or even fewer dinucleotides than a reference sequence (e.g., a wild-type sequence).

[0100] It is known that the methylation of CpG dinucleotide plays an important role in the regulation of gene expression in eukaryotes.Specifically, the methylation of CpG dinucleotide in eukaryotes essentially plays a role in silencing gene expression by interfering with the transcriptional machinery.Therefore, due to the gene silencing induced by the methylation of CpG motifs, nucleic acids and vectors with reduced number of CpG dinucleotides will result in high and long-lasting transgene expression levels.

[0101] The modified nucleic acid sequence may contain flanking restriction sites to facilitate subcloning into an expression vector. Many such restriction sites are well known in the art and include, but are not limited to, those shown in Figure 13, e.g., AvaI, XmaI, and XmaI.

[0102] The present disclosure includes fragments of any one of the sequences set forth in SEQ ID NOS: 1-3 that encode functionally active fragments of ASPA polypeptides. "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 a functional fragment thereof also encompasses reversing or preventing the neurodegenerative phenotype associated with Canavan disease, as demonstrated elsewhere herein and in nur7 mice.

[0103] The present disclosure provides modified ASPA nucleic acid sequences (e.g., SEQ ID NO: 3; GenBank accession numbers NM_000049.4 or NM_001128085.1) that encode ASPA polypeptides and contain at least one modification compared to the wild-type nucleic acid sequence, with an alternative 5'UTR but encoding the same ASPA protein (SEQ ID NO: 4).

[0104] In some embodiments, the modified nucleic acid encoding ASPA is a codon-optimized nucleic acid that encodes a wild-type ASPA polypeptide (e.g., SEQ ID NO: 4) and comprises 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 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 a 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 SEQ ID NO: 1 or SEQ ID NO: 2.

[0105] In some embodiments, cells containing a modified nucleic acid encoding ASPA exhibit increased protein expression, e.g., the protein encoded thereby is expressed at a detectably higher level in the cell compared to the expression level of the protein in an otherwise identical cell containing a wild-type ASPA nucleic acid or an otherwise identical cell containing a mutant nucleic acid encoding ASPA. In some embodiments, the level of ASPA protein expression in a cell containing a modified nucleic acid encoding ASPA (e.g., comprising the nucleic acid sequence of SEQ ID NO: 2) is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 150%, 200%, 300%, 400%, or more compared to the level of ASPA protein expression in an otherwise identical cell containing a wild-type ASPA nucleic acid. In some embodiments, the level of ASPA protein expression in a cell comprising a modified nucleic acid encoding ASPA (e.g., comprising the nucleic acid sequence of 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 level of ASPA protein expression in an otherwise identical cell comprising a mutant nucleic acid encoding ASPA.

[0106] In some embodiments, this may be referred to as an "expression-optimized" or "expression-enhanced" nucleic acid, or simply a "modified nucleic acid."

[0107] Those skilled in the art will understand that the polypeptides encoded by the modified nucleic acids and variants thereof (e.g., SEQ ID NO: 1, SEQ ID NO: 2) of the present disclosure are "functional ASPA polypeptides" that provide the same or similar biological functions and / or activities as the ASPA polypeptides encoded by wild-type nucleic acids encoding ASPA (e.g., SEQ ID NO: 3). That is, the 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 include reversal or prevention of neurodegenerative phenotypes associated with Canavan disease, as demonstrated elsewhere herein in nur7 mice, including, but not limited to, improved rotarod fall latency performance, improved open field distance traversed, 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.

[0108] Regulatory elements The present 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 influence expression of an operably linked polynucleotide. The exact nature of regulatory elements useful for gene expression varies between organisms and cell types, including, for example, promoters, enhancers, introns, etc., intended to facilitate proper heterologous polynucleotide transcription and translation. Regulatory control can be affected at the level of transcription, translation, splicing, message stability, etc. Typically, regulatory control elements that regulate transcription are juxtaposed near the 5' end (i.e., upstream) of the transcribed polynucleotide. Regulatory control elements may also be located at the 3' end (i.e., downstream) of the transcribed sequence or within the transcript (e.g., within an intron). Regulatory control elements can be located at a distance from the transcribed sequence (e.g., 1-100, 100-500, 500-10000, 1000-5000, 5000-10000, or more nucleotides). However, due to the length of the AAV vector genome, the regulatory control elements are typically within 1-1000 nucleotides of the polynucleotide.

[0109] promoter As used herein, the term "promoter," such as "eukaryotic promoter," refers to a nucleotide sequence that initiates transcription of a particular gene or one or more coding sequences (e.g., the ASPA coding sequence) in a eukaryotic cell (e.g., an oligodendrocyte). A promoter can cooperate with other regulatory elements or regions to direct the level of transcription of a gene or coding sequence. These regulatory elements include, for example, transcription binding sites, repressor and activator protein binding sites, and other nucleotide sequences known to act directly or indirectly to regulate the amount of transcription from the promoter, including, for example, attenuators, enhancers, and silencers. A promoter is often located on the same strand and 5' to the transcription start site of the gene or coding sequence to which it is operably linked. Promoters are generally 100 to 1000 nucleotides in length. A promoter typically increases gene expression compared to expression of the same gene in the absence of the promoter.

[0110] As used herein, "core promoter" or "minimal promoter" refers to the minimum portion of a promoter sequence required to properly initiate transcription. This may include a transcription initiation site, an RNA polymerase binding site, and any of the general transcription factor binding sites. A promoter may also include a proximal promoter sequence (5' of the core promoter) that contains other primary regulatory elements (e.g., enhancers, silencers, boundary elements, insulators) and a distal promoter sequence (3' of the core promoter).

[0111] 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 promoter; Rous sarcoma virus (RSV) promoter; albumin promoter; inducible promoters such as the mouse mammary tumor virus (MMTV) promoter; metallothionein promoter; heat shock promoter; alpha-1 antitrypsin promoter; hepatitis B surface antigen promoter; transferrin promoter; apolipoprotein A-1 promoter; chicken beta actin (CBA) promoter; elongation factor 1a promoter (EF1a), a hybrid form of the CBA promoter (CBh promoter), and the CAG promoter (cytomegalovirus early enhancer element and promoter), as well as the first exon and first intron of the bird beta actin gene and the splice acceptor of the rabbit beta globin gene (Alexopoulou et al. (2008) BioMed. Central Cell Biol. 9:2); and the human ASPA gene promoter. In some embodiments, the promoter is a fragment or variant of the CBh promoter and comprises or consists of the nucleic acid sequence of SEQ ID NO:7.

[0112] In some embodiments of the present disclosure, a eukaryotic promoter sequence (e.g., a CBh promoter) is operably linked to a modified nucleic acid encoding ASPA. In some embodiments, a promoter (e.g., a CBh promoter) comprising the nucleic acid sequence of SEQ ID NO: 7 is operably linked to a modified nucleic acid encoding ASPA. In some embodiments, a promoter comprising or consisting of a nucleic acid sequence 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 operably linked to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, a promoter comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 7 is operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2 and directs expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 2 in oligodendrocytes. In some embodiments, expression of a polypeptide encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 2 operably linked to a promoter comprising a nucleic acid comprising SEQ ID NO: 7 is at a detectably higher level in a cell compared to the expression level in an otherwise identical cell of a polypeptide encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 2 that is not operably linked to a promoter comprising the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 7 operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2, and which directs expression of the polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 2 in an oligodendrocyte.

[0113] Promoters can be constitutive, tissue-specific, or regulated. A constitutive promoter is one that causes an operably linked gene to be expressed essentially all the time. In some embodiments, a constitutive promoter is active in most eukaryotic tissues under most physiological and developmental conditions.

[0114] A regulated promoter is one that can be activated or deactivated. Regulated promoters include inducible promoters, which are normally "off" but can be induced to be "on," and "repressible" promoters, which are normally "on" but can be turned "off." Many different regulators are known, including temperature, hormones, cytokines, heavy metals, and regulatory proteins. The distinction is not absolute, and constitutive promoters can often be regulated to some degree. In some cases, endogenous pathways can be utilized to provide regulation of transgene expression, for example, using promoters that are naturally down-regulated when a pathological condition improves.

[0115] A tissue-specific promoter is a promoter that is active only in a specific type of tissue, cell, or organ. Typically, a tissue-specific promoter is recognized by a transcriptional activation element specific to that tissue, cell, and / or organ. For example, a tissue-specific promoter may be more active in one or more specific tissues (e.g., two, three, or four) than in other tissues. In some embodiments, expression of a gene regulated by a tissue-specific promoter is much higher in the tissue for which the promoter is specific than in other tissues. In some embodiments, a promoter may have little or no activity in any tissue other than the tissue for which it is specific. A promoter may be a tissue-specific promoter such as the mouse albumin promoter or transthyretin promoter (TTR), which are active in hepatocytes. Other examples of tissue-specific promoters include promoters from genes encoding skeletal alpha actin, myosin light chain 2A, dystrophin, and muscle creatine kinase, which direct expression in skeletal muscle (Li et al. (1999) Nat. Biotech. 17:241-245). Liver-specific expression can be induced using promoters 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 alpha-fetoprotein (Arbuthnot et al., (1996) Hum. Gene. Ther. 7:1503-1514).

[0116] Enhancer In another embodiment, the modified nucleic acid encoding a therapeutic polypeptide further comprises an enhancer to increase expression of the therapeutic polypeptide (e.g., ASPA protein). Typically, the enhancer element is located upstream of the promoter element, but may 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 expression of the modified nucleic acid (e.g., encoding a therapeutic polypeptide, e.g., encoding ASPA) beyond the increased expression provided by the promoter element alone.

[0117] Many enhancers are known in the art, including, but not limited to, the cytomegalovirus major immediate-early enhancer. More specifically, the cytomegalovirus (CMV) MIE promoter contains three regions: a modulator, 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 hybrid promoter to further increase expression of a nucleic acid operably linked thereto. For example, the chicken β-actin (CBA) promoter, or a portion thereof, can be combined with a CMV promoter / enhancer, or a portion thereof, to create a version of CBA called the "CBh" promoter, which stands for chicken β-actin hybrid promoter, as described by Gray et al. (2011, Human Gene Therapy 22:1143-1153). Like promoters, enhancers can be constitutive, tissue-specific, or regulated.

[0118] In some embodiments of the present disclosure, an enhancer sequence (e.g., a CMV enhancer) is operably linked to a modified nucleic acid encoding ASPA. In some embodiments, an enhancer (e.g., a CMV enhancer) comprising or consisting of the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17 is operably linked to a modified nucleic acid encoding ASPA. In some embodiments, an enhancer comprising a nucleic acid sequence 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 operably linked to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:2, and optionally, is operably linked to a promoter comprising the nucleic acid sequence of SEQ ID NO:7. In some embodiments, an enhancer comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17 is operably linked to a nucleic acid sequence 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 at least 95% identical to the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17 is operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO:7, and is operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO:2, and together with the nucleic acid sequences of SEQ ID NO:6 (or SEQ ID NO:17) and SEQ ID NO:7, induces expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 in oligodendrocytes. In some embodiments, expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 operably linked to an enhancer comprising the nucleic acid sequence of SEQ ID NO:6 (or SEQ ID NO:17) is at a detectably higher level in a cell compared to the expression level of a polypeptide encoded by SEQ ID NO:2 not operably linked to an enhancer comprising the nucleic acid sequence of SEQ ID NO:5 in an otherwise identical cell.In some embodiments, the recombinant nucleic acid comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17, operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: ...2, and comprises an enhancer that, together with the nucleic acid sequences of SEQ ID NO:6 (or SEQ ID NO:17) and SEQ ID NO:7, induces expression of the polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 in oligodendrocytes.

[0119] Fillers, spacers and stuffers As disclosed herein, recombinant nucleic acids intended for use in rAAV vectors may contain additional nucleic acid elements to adjust the length of the nucleic acid to or near the normal size (e.g., about 4.7 to 4.9 kilobases) of viral genomic sequences acceptable for AAV packaging into rAAV vectors (Grieger and Samulski (2005) J. Virol. 79(15):9933-9944). Such sequences may be synonymously referred to as fillers, spacers, or stuffers. In some embodiments, filler DNA is a non-translated (non-protein-coding) segment of nucleic acid. In some embodiments, the filler or stuffer polynucleotide sequences are 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 more in length.

[0120] AAV vectors typically accept DNA inserts with sizes ranging from about 4 kb to about 5.2 kb or about 4.1 to 4.9 kb for optimal packaging of nucleic acids into AAV capsids. In some embodiments, rAAV vectors comprise a vector genome with a total length of 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, rAAV vectors comprise a vector genome with a total length of about 4.7 kb. In some embodiments, rAAV vectors comprise a self-complementary vector genome. The total length of the self-complementary (sc) vector genome in an rAAV vector is equal to that of the single-stranded (ss) vector genome (i.e., about 4 kb to about 5.2 kb), but the nucleic acid sequence encoding the sc vector genome (i.e., including the transgene, regulatory elements, and ITRs) must be half the length of the nucleic acid sequence encoding the ss vector genome in order for the sc vector to be packaged into capsids.

[0121] Introns and Exons In some embodiments, the recombinant nucleic acid comprises, for example, introns, exons, and / or portions thereof. Introns can function as filler or stuffer polynucleotide sequences to achieve the appropriate length for vector genome packaging into an rAAV vector. Intron and / or exon sequences can also enhance expression of a polypeptide (e.g., a transgene) compared to expression in the absence of the intron and / or exon elements (Kurachi et al. (1995) J. Biol. Chem. 270(10):576-5281; WO2017 / 074526). Additionally, filler / stuffer polynucleotide sequences (also referred to as "insulators") are well known in the art and include, but are not limited to, those described in WO2014 / 144486 and WO2017 / 074526.

[0122] The intron element may be derived from the same gene as the heterologous polynucleotide, or may be derived from an entirely different gene or other DNA sequence (e.g., chicken β-actin gene, minute virus of mice (MVM)). In some embodiments, the recombinant nucleic acid comprises at least one element selected from an intron and an exon derived from a non-cognate 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, e.g., comprising or consisting of the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the intron comprises a nucleic acid sequence that is 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, e.g., comprising or consisting of the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the intron comprises a nucleic acid sequence that is 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 a chicken beta-actin gene, e.g., comprising or consisting of the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the exon comprises a nucleic acid sequence that is 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 optionally regulates expression of a heterologous polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 2.In some embodiments, expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 2 operably linked to a regulatory region comprising 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) is detectably higher in a cell compared to the expression level of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 2 that is not operably linked to such regulatory elements in an otherwise identical cell.

[0123] In some embodiments, the recombinant nucleic acid comprises a modified nucleic acid of SEQ ID NO: 2 operably linked to a regulatory element comprising 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).

[0124] Polyadenylation signal sequence (polyA) Additional regulatory elements may include, but are not limited to, stop codons, termination sequences, and polyadenylation (polyA) signal sequences, such as the bovine growth hormone polyA signal sequence (BHG polyA). The polyA signal sequence drives the efficient addition of a polyadenosine "tail" at the 3' end of eukaryotic mRNA, which leads to the termination of gene transcription (see, e.g., Goodwin and Rottman J. Biol. Chem. (1992) 267(23):16330-16334). The polyA signal functions as a signal for endonucleolytic cleavage of the newly formed precursor mRNA at its 3' end and the addition of an RNA stretch consisting only of adenine bases to this 3' end. The polyA tail is important for the nuclear export, translation, and stability of mRNA. In some embodiments, the polyA is an SV40 early polyadenylation signal, an SV40 late polyadenylation signal, an HSV thymidine kinase polyadenylation signal, a protamine gene polyadenylation signal, an adenovirus 5E1b polyadenylation signal, a growth hormone polyadenylation signal, a PBGD polyadenylation signal, or an in silico designed polyadenylation signal.

[0125] In some embodiments, the polyA signal sequence of the recombinant nucleic acid is a polyA signal capable of directing and effecting intranuclear cleavage and polyadenylation of a precursor mRNA resulting from transcription of a modified nucleic acid encoding ASPA (e.g., SEQ ID NO:2), optionally in combination with one or more other regulatory elements described herein. In some embodiments, the polyA sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:11. In some embodiments, the polyA sequence comprises a nucleic acid sequence that is 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), optionally modulating expression of a heterologous polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2.

[0126] In some embodiments, the rAAV vector (e.g., AAV / Olig001-ASPA) has tropism for oligodendrocytes and contains a self-complementary vector genome comprising AAV ITRs (e.g., AAV2 ITRs) and a recombinant nucleic acid comprising a modified (i.e., codon-optimized) nucleic acid encoding ASPA and at least one of the following regulatory elements: an enhancer (e.g., CMV enhancer), a promoter (e.g., CBh promoter), an exon (e.g., CBA exon 1), an intron (e.g., CBA intron, MVM intron), and a polyA (e.g., BHG polyA).

[0127] In some embodiments, an rAAV vector with tropism for oligodendrocytes (e.g., AAV / Olig001-ASPA) contains a self-complementary genome comprising AAV ITRs (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).

[0128] In some embodiments, an rAAV vector with tropism for oligodendrocytes (e.g., AAV / Olig001-ASPA) contains a self-complementary genome comprising SEQ ID NO:20.

[0129] Biological Activity of the rAAV Vectors of the Disclosure In some embodiments, the rAAV vectors of the present disclosure (e.g., comprising an ASPA transgene) transduce target cells (e.g., oligodendrocytes) and mediate a biological activity. In some embodiments, the rAAV vectors (e.g., AAV / Olig001-ASPA) transduce target cells (e.g., oligodendrocytes) and mediate at least one detectable activity selected from the group consisting of: (i) reducing intracellular NAA levels in vitro; (ii) improving, increasing, and / or enhancing balance, grip strength, and / or motor coordination; (iii) improving, increasing, and / or enhancing fall latency (seconds); (iv) improving, increasing, and / or enhancing general motor function; (v) reducing, inhibiting, and / or neutralizing the accumulation of NAA levels in vivo; (vi) reducing, inhibiting, and / or neutralizing vacuolar volume fraction in the thalamus; (vii) reducing, inhibiting, and / or neutralizing cerebellar white matter / pontine vacuolar volume fraction; (viii) improving, increasing, and / or enhancing the number of oligodendrocytes in the thalamus; (ix) improving, increasing, and / or enhancing the number of oligodendrocytes in the cortex; (x) improving, increasing, and / or enhancing the number of neurons in the thalamus; (xi) improving, increasing, and / or enhancing the number of neurons in the cortex; and (xii) improving, enhancing, and / or increasing cortical myelination;

[0130] 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.

[0131] In some embodiments, cells transduced with an rAAV vector (e.g., AAV / Olig001-ASPA) have reduced levels of NAA compared to the level of NAA in otherwise identical cells transduced with an rAAV containing a wild-type nucleic acid sequence encoding ASPA (e.g., SEQ ID NO: 3). In some embodiments, cells transduced with an rAAV vector (e.g., AAV / Olig001-ASPA) have reduced levels of NAA compared to the level of NAA in otherwise identical cells transduced with an rAAV containing an alternative codon-optimized nucleic acid encoding ASPA (e.g., SEQ ID NO: 1). In some embodiments, cells transduced with an rAAV vector (e.g., AAV / Olig001-ASPA) have reduced levels of NAA compared to the level of NAA in cells containing a mutant nucleic acid encoding ASPA that was not transduced.

[0132] In some embodiments, cells transduced in vivo with an rAAV vector (e.g., AAV / Olig001-ASPA) have reduced levels of NAA compared to the level of NAA in otherwise identical cells transduced in vivo with an rAAV containing a wild-type nucleic acid encoding ASPA (e.g., SEQ ID NO: 3). In some embodiments, cells transduced in vivo with an rAAV vector (e.g., AAV / Olig001-ASPA) have reduced levels of NAA compared to the level of NAA in otherwise identical cells transduced in vivo with an rAAV containing an alternative codon-optimized nucleic acid encoding ASPA (e.g., SEQ ID NO: 1). In some embodiments, cells transduced in vivo with an rAAV vector (e.g., AAV / Olig001-ASPA) have reduced levels of NAA compared to the level of NAA in otherwise identical cells containing a mutant ASPA gene that was not transduced.

[0133] In some embodiments, the balance, grip strength and / or motor coordination in a subject with an ASPA gene mutation who has been administered an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly improved, as measured, for example, by rotarod performance, compared to the balance, grip strength and / or motor coordination of an otherwise similar subject with an ASPA gene mutation who has not been administered the rAAV vector, or compared to the same subject prior to administration of the rAAV vector.

[0134] In some embodiments, the balance, grip strength, and / or motor coordination in subjects with an ASPA gene mutation who have been administered an rAAV vector (e.g., AAV / Olig001-ASPA) is indistinguishable from the balance, grip strength, and / or motor coordination in otherwise similar subjects who do not have an ASPA gene mutation and who have not been administered the rAAV vector, as measured, for example, by rotarod performance. In some embodiments, the rAAV vector (e.g., AAV / Olig001-ASPA) is administered via the intracerebroventricular (ICV) route of administration. In some embodiments, rotarod performance is measured as fall latency in seconds.

[0135] In some embodiments, the general motor function of a subject with an ASPA gene mutation who has been administered an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly improved, e.g., as measured by open field activity, compared to the general motor function of an otherwise similar subject with an ASPA gene mutation who has not been administered the rAAV vector, or compared to the function in the subject prior to administration of the rAAV vector. In some embodiments, the rAAV vector (e.g., AAV / Olig001-ASPA) is administered via the intracerebroventricular (ICV) route of administration.

[0136] In some embodiments, the overall motor function in a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) is indistinguishable from the overall motor function in an otherwise similar subject who does not have an ASPA gene mutation and who is not administered rAAV, as measured, for example, by open field activity. In some embodiments, the rAAV vector (e.g., AAV / Olig001-ASPA) is administered via the intracerebroventricular (ICV) route of administration.

[0137] In some embodiments, NAA levels in the brain of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) are significantly reduced compared to NAA levels in the brain of an otherwise similar subject with an ASPA gene mutation who is not administered the rAAV vector, or compared to NAA levels in the subject prior to administration of the rAAV vector. In some embodiments, NAA levels in the brain of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) are reduced or indistinguishable from NAA levels in the brain of an otherwise similar subject who does not have an ASPA gene mutation and who is not administered the rAAV vector.

[0138] In some embodiments, the percentage vacuolar volume in the thalamus of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly reduced compared to the percentage vacuolar volume in the thalamus of an otherwise similar subject with an ASPA gene mutation who is not administered the rAAV vector, or compared to the subject before administration of the rAAV vector, as measured, for example, by unbiased stereology. In some embodiments, the percentage vacuolar volume in the cerebellar white matter / pons of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly reduced compared to the percentage vacuolar volume in the cerebellar white matter / pons of an otherwise similar subject with an ASPA gene mutation who is not administered the rAAV vector, or compared to the subject before administration of the rAAV vector, as measured, for example, by unbiased stereology.

[0139] In some embodiments, the number of oligodendrocytes in the thalamus of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly increased compared to the number of oligodendrocytes in the thalamus of an otherwise similar subject with an ASPA gene mutation who is not administered the vector, or compared to the subject before the vector was administered, where the number of oligodendrocytes in the thalamus is measured, for example, by IHC using an Olig2 antibody and unbiased stereology. In some embodiments, the number of oligodendrocytes in the cerebral cortex of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly increased compared to the number of oligodendrocytes in the cerebral cortex of an otherwise similar subject with an ASPA gene mutation who is not administered the rAAV vector, or compared to the same subject before the vector was administered, where the number of oligodendrocytes in the cerebral cortex is measured, for example, by IHC using an Olig2 antibody and unbiased stereology. In some embodiments, the number of oligodendrocytes in the cerebral cortex of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) is indistinguishable from the number of oligodendrocytes in the cerebral cortex of an otherwise similar subject who does not have an ASPA gene mutation and who is not administered the rAAV vector, as measured by, for example, IHC using an Olig2 antibody and unbiased stereology.

[0140] In some embodiments, the number of neurons in the thalamus of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly increased compared to the number of neurons in the thalamus of a subject with an ASPA gene mutation who is not administered the rAAV vector, or compared to the number of neurons in the thalamus of the subject prior to administration of the vector, as measured by, for example, IHC using a NeuN antibody and unbiased stereology. In some embodiments, the number of neurons in the cerebral cortex of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) is significantly increased compared to the number of neurons in the cerebral cortex of an otherwise similar subject with an ASPA gene mutation who is not administered the rAAV vector, or compared to the number of neurons in the cerebral cortex of the subject prior to administration of the vector, as measured by, for example, IHC using a NeuN antibody and unbiased stereology. In some embodiments, the number of neurons in the brain cortex of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Olig001-ASPA) is indistinguishable from the number of neurons in the brain cortex of an otherwise similar subject who does not have an ASPA gene mutation and who is not administered the rAAV vector, as measured, for example, by IHC using a NeuN antibody and unbiased stereology.

[0141] In some embodiments, cortical myelination in the brain of a subject with an ASPA gene mutation who is administered an rAAV vector (e.g., AAV / Oligo001-ASPA) is significantly increased compared to cortical myelination in the brain of an otherwise similar subject with an ASPA gene mutation who is not administered the rAAV vector, or compared to cortical myelination in the brain of the subject prior to administration of the vector, where cortical myelination is measured, for example, by cortical myelin basic protein-positive fiber length density (MBP-LD).

[0142] Assembly of viral vectors Viral vectors (e.g., rAAV vectors) carrying a transgene (e.g., ASPA) are assembled from polynucleotides encoding the transgene, appropriate 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, adenoviral, retroviral, lentiviral, herpesviral, and adeno-associated viral (AAV) vectors, particularly rAAV vectors (as discussed above).

[0143] The vector genome component of the rAAV vector produced according to the methods of the present disclosure includes at least one transgene, e.g., a modified nucleic acid encoding ASPA, and associated expression control sequences for controlling expression of the modified nucleic acid encoding ASPA.

[0144] In a preferred embodiment, the vector genome comprises a portion of a parvovirus genome, such as an AAV genome, in which rep and cap have been deleted and / or replaced with a modified nucleic acid (e.g., a transgene, e.g., a modified nucleic acid encoding ASPA) and its associated expression control sequences. The modified nucleic acid encoding ASPA is typically inserted adjacent to one or two (i.e., flanked by) AAV ITRs or ITR elements appropriate for viral replication (Xiao et al. (1997) J. Virol. 71(2):941-948). Other regulatory sequences suitable for use in promoting tissue-specific expression of the modified nucleic acid encoding ASPA in target cells (e.g., oligodendrocytes) may also be included.

[0145] Packaging cells Those skilled in the art will understand that an rAAV vector containing a transgene and lacking viral proteins necessary for viral replication (e.g., cap and rep) will be unable to replicate because such proteins are required for viral replication and packaging. The cap and rep genes may be provided to a cell (e.g., a host cell, e.g., a packaging cell) as part of a plasmid separate from the plasmid providing the transgene in the vector genome.

[0146] "Packaging cell" or "producer cell" refers to a cell or cell line that can be transfected with a vector, plasmid, or DNA construct to provide in trans the missing functions required for complete replication and packaging of the viral vector. Genes required 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 helper genes derived from other viruses (e.g., adenovirus). Those skilled in the art will understand that the genes required for AAV production can be introduced into packaging cells by various techniques, including, for example, transfection of one or more plasmids. However, in some embodiments, some genes (e.g., rep, cap, helper) may already be present in the packaging cell, integrated into the genome, or carried on an episome. In some embodiments, the packaging cell expresses one or more missing viral functions in a constitutive or inducible manner.

[0147] Any suitable packaging cell known in the art can be used to produce packaged viral vectors. Mammalian or insect cells are preferred. Examples of cells useful for producing packaging cells in the practice of the present disclosure include, for example, PER.C6, WI38, MRC5, A549, HEK293 cells (expressing functional adenovirus E1 under the control of a constitutive promoter), B-50, or any other human cell line, such as HeLa, 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.

[0148] In some embodiments, the packaging cells can be grown in suspension culture. In some embodiments, the packaging cells can be grown in serum-free medium. For example, HEK293 cells are grown in suspension in serum-free medium. In another embodiment, the packaging cells are HEK293 cells as described in U.S. Pat. No. 9,441,206 and deposited as American Type Culture Collection (ATCC) No. PTA13274. Numerous rAAV packaging cell lines are known in the art, including, but not limited to, those disclosed in WO2002 / 46359.

[0149] Cell lines suitable for use as packaging cells include insect cell lines. Any insect cell that allows AAV replication and can be maintained in culture can be used in accordance with the present disclosure. For example, Spodoptera frugiperda, such as Sf9 or Sf21 cell lines, Drosophila cell lines, or mosquito cell lines, such as Aedes albopictus cell lines, are included. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for their teachings regarding the use of insect cells for the expression of heterologous polypeptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, 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. al. (2000) Virol. 272:382-393; and U.S. Patent No. 6,204,059.

[0150] As a further alternative, the viral vectors of the present disclosure can be produced in insect cells using a baculovirus vector to deliver the rep / cap genes and rAAV template, as described, for example, by Urabe et al. (2002) Human Gene Therapy 13:1935-1943. When using baculovirus production for AAV, 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 acid encoding baculovirus helper functions, thereby facilitating production of viral capsids.

[0151] Packaging cells generally contain one or more viral vector functions, along with helper functions and packaging functions sufficient to effect replication and packaging of the viral vector. These various functions can be provided to the packaging cells together or separately using genetic constructs, such as plasmids or amplicons, which can be extrachromosomal within the cell line or integrated into the host cell chromosome. In some embodiments, packaging cells are transfected with at least i) a plasmid containing a vector genome including a codon-optimized human ASPA transgene (e.g., SEQ ID NO:2) and AAV ITRs (e.g., SEQ ID NO:5 and SEQ ID NO:12), and further including at least one of an enhancer (e.g., SEQ ID NO:6), a promoter (e.g., SEQ ID NO:7), an exon (e.g., CBA exon SEQ ID NO:8), an intron (e.g., SEQ ID NO:9 and SEQ ID NO:10), and a polyA (e.g., SEQ ID NO:11), and ii) a plasmid containing a rep gene (e.g., AAV2 rep) and a cap gene (e.g., Olig001 cap).

[0152] In some embodiments, the host cell is supplied with one or more packaging or helper functions, e.g., integrated into a host cell line with one or more vector functions that are integrated extrachromosomally or integrated into the chromosomal DNA of the cell.

[0153] Helper Functions AAV is a dependent virus in that it cannot replicate intracellularly without co-infection of the cell with a helper virus. Helper functions include helper virus elements necessary to establish active infection of the packaging cell, which is necessary to initiate packaging of the viral vector. Helper viruses typically include adenovirus or herpes simplex virus. Adenoviral helper functions typically include the adenoviral components adenovirus 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 the packaging cell by transfecting the cell with one or more nucleic acids encoding various helper elements. Alternatively, the host cell (e.g., packaging cell) can contain nucleic acids encoding helper proteins. For example, HEK293 cells were generated by transforming human cells with adenovirus 5 DNA and now express several adenovirus genes, including, but not limited to, E1 and E3 (see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59-72). Thus, these helper functions can be provided by the HEK293 packaging cells without the need to supply them to the cells, e.g., by plasmids encoding them. In some embodiments, the packaging cells are transfected with at least i) a plasmid comprising a vector genome including a codon-optimized human ASPA transgene (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 an enhancer (e.g., SEQ ID NO: 6), a promoter (e.g., SEQ ID NO: 7), an exon (e.g., CBA exon SEQ ID NO: 8), an intron (e.g., SEQ ID NO: 9 and SEQ ID NO: 10), and a polyA (e.g., SEQ ID NO: 11); ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and a cap gene (e.g., Olig001 cap); and iii) a plasmid comprising helper functions.

[0154] Any method for introducing nucleotide sequences with helper functions into a cellular host for replication and packaging can be used, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with nuclear localization signals. In some embodiments, helper functions are provided by transfection using a viral vector or by infection using a helper virus, and standard methods for producing viral infections can be used.

[0155] The vector genome may be any suitable recombinant nucleic acid, such as a DNA or RNA construct, and may be single-stranded, double-stranded, or duplex (i.e., self-complementary as described in WO2001 / 92551).

[0156] Generation of packaged viral vectors Viral vectors can be produced by several methods known to those skilled in the art (see, e.g., WO2013 / 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. Briefly, efficient transfection of HEK293 cells is used as a starting point. Adherent HEK293 cell lines from qualified clinical master cell banks are grown in animal-component-free suspension conditions in shaker flasks and WAVE bioreactors, enabling rapid and scalable rAAV production. Using the triple transfection method (e.g., WO96 / 40240), HEK293 cell line suspensions yielded 1 x 10 cells when harvested 48 hours post-transfection. 5 particles containing vector genome (vg) / cell, or 1 x 10 14More than 1000 vg / L (of cell culture) can be produced. 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 encodes various helper functions (e.g., adenovirus or HSV proteins such as E1a, E1b, E2a, E4, and VA RNA), and another encodes a transgene (e.g., ASPA) and various elements that control transgene expression.

[0157] The single-stranded vector genome is packaged into capsids in approximately equal proportions as either the positive or negative strand. In some embodiments of rAAV vectors, the vector genome is in positive strand polarity (i.e., the sense or coding sequence of the DNA strand). In some embodiments of rAAV vectors, the vector is in negative strand polarity (i.e., the antisense or template DNA strand). Given the nucleotide sequence of the positive strand in the 5' to 3' direction, the nucleotide sequence of the negative strand in the 5' to 3' direction can be determined as the reverse complement of the nucleotide sequence of the positive strand.

[0158] To achieve the desired yield, several variables are optimized, including the selection of a suitable serum-free suspension medium that supports both growth and transfection, the choice of transfection reagent, transfection conditions, and cell density.

[0159] rAAV vectors can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. 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 No. 6,566,118; and WO 98 / 09657.

[0160] A universal purification strategy based on ion-exchange chromatography can be used to generate highly pure vector preparations of AAV serotypes 1-6, 8, 9, and various chimeric capsids (e.g., Olig001). In some embodiments, this process can be completed within a week, resulting in high total-to-empty capsid ratios (>90% total capsids), and post-purification yields (>1 x 10) suitable for clinical use. 13 vg / L) and purity. In some embodiments, such methods are universal for all serotypes and chimeric capsids. Scalable manufacturing techniques can be utilized to produce GMP clinical and commercial grade rAAV vectors (e.g., for the treatment of Canavan disease).

[0161] After the rAAV vectors of the present disclosure have been produced and purified, they can be titered (e.g., the amount of rAAV vector in a sample can be quantified) to prepare a composition for administration to a subject, such as a human subject with Canavan disease. Titering of rAAV vectors can be accomplished using methods known in the art.

[0162] In some embodiments, the number of viral particles, including particles containing vector genomes and "empty" capsids that do not contain a vector genome, can be determined by electron microscopy, such as transmission electron microscopy (TEM). Such TEM-based methods can provide the number of vector particles (or viral particles in the case of wild-type AAV) in a sample.

[0163] In some embodiments, rAAV vector genomes can be titrated using quantitative PCR (qPCR) using primers directed to sequences within 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 within the transgene (e.g., SEQ ID NO:2) or regulatory elements. By performing qPCR in parallel on dilutions of a standard of known concentration, such as a plasmid containing the vector genome sequence, a standard curve can be generated that allows for the calculation of the rAAV vector concentration as the number of vector genomes (vg) per unit volume, such as microliters or milliliters. For example, the number of vector particles measured by electron microscopy can be compared to the number of vector genomes in the sample to determine the number of empty capsids. Because the vector genome contains the therapeutic transgene, the vg / kg or vg / ml of the vector sample can indicate the therapeutic dose a subject will receive rather than the number of vector particles, some of which may be empty and contain no vector genomes. Once the concentration of the rAAV vector genome in the stock solution is determined, it can be diluted or dialyzed into a buffer suitable for use in preparing a composition for administration to a subject (e.g., a subject with Canavan disease).

[0164] Treatment method Modified nucleic acids, such as those encoding ASPA, as disclosed herein, can be used for gene therapy treatment and / or prevention of diseases, disorders, or conditions associated with a deficiency or dysfunction of ASPA polypeptide (e.g., Canavan disease), as well as any other condition and / or disease in which upregulation of the ASPA gene can result in a therapeutic benefit or improvement mediated by or associated with a decrease in the level or function of ASPA polypeptide compared to the level or function of ASPA polypeptide in an otherwise healthy individual.

[0165] Vector genomes and / or rAAV vectors comprising modified nucleic acids encoding ASPA, as disclosed herein, can be used for gene therapy treatment and / or prevention of diseases, disorders, or conditions associated with or caused by ASPA enzyme deficiency or dysfunction (e.g., Canavan disease), as well as any other conditions and / or diseases in which upregulation of ASPA enzyme may provide a therapeutic benefit or improvement. In some embodiments, the methods of the disclosure include use of an rAAV vector or a pharmaceutical composition thereof in treating Canavan disease in a subject. In some embodiments, the methods of the 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 in need thereof.

[0166] The modified nucleic acids encoding ASPA of the present disclosure, vector genomes comprising modified nucleic acids encoding ASPA, and / or rAAV vectors comprising modified nucleic acids encoding ASPA (e.g., AAV / Oligo001-ASPA) can be used in the preparation of medicaments for use in the treatment and / or prevention of diseases, disorders, or conditions associated with or caused by ASPA deficiency or dysfunction (e.g., reduced levels of functional ASPA enzyme, such as Canavan disease), and any other conditions or diseases in which upregulation of ASPA can provide a therapeutic benefit or improvement.

[0167] In some embodiments, gene therapy treatment and / or prevention of diseases, disorders, or conditions associated with ASPA enzyme deficiency or dysfunction (e.g., Canavan disease), and any other conditions and / or diseases in which upregulation of ASPA gene expression and / or increased expression of functional ASPA enzyme may result in therapeutic benefit or improvement, comprises administering to a subject (e.g., patient) in need of treatment a therapeutically effective amount of a modified nucleic acid encoding ASPA, a vector genome comprising a modified nucleic acid encoding ASPA, and / or an rAAV vector comprising a modified nucleic acid encoding ASPA (e.g., AAV / Oligo001-ASPA).

[0168] Treatment of a subject (e.g., patient) with a therapeutically effective amount of a modified ASPA-encoding nucleic acid of the present disclosure, a vector genome comprising a modified ASPA-encoding nucleic acid, and / or an rAAV vector comprising the modified nucleic acid ASPA (e.g., AAV / Oligo001-ASPA) can alleviate, ameliorate, treat, prevent, or reduce the severity of one or more symptoms of Canavan disease compared to a baseline measurement, such as a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control individual (or control individuals thereby establishing levels for comparison) in the absence of a treatment described herein. In some embodiments, a "control individual" is an individual suffering from the same form of disease or disorder as the individual being treated, but who is not currently being treated but may be treated in the future.

[0169] For example, treatment of a subject with a therapeutically effective amount of a modified nucleic acid encoding ASPA, a vector genome comprising a modified nucleic acid encoding ASPA, and / or an rAAV vector (e.g., AAV / Oligo001-ASPA) may reduce NAA accumulation compared to NAA accumulation in a control individual or compared to NAA accumulation in the same individual prior to treatment. In some embodiments, NAA accumulation may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% in a treated subject compared to a control individual or compared to the same individual prior to treatment.

[0170] In some embodiments, treatment of a subject with a therapeutically effective amount of a modified nucleic acid encoding ASPA, a vector genome comprising a modified nucleic acid encoding ASPA, and / or an rAAV vector (e.g., AAV / Oligo001-ASPA) may increase aspartate and / or acetate levels compared to aspartate and / or acetate levels in a control individual or compared to aspartate and / or acetate levels in the same individual before treatment. In some embodiments, aspartate and / or acetate levels are increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% in a treated subject compared to a control individual or compared to the same individual before treatment.

[0171] In some embodiments, treatment may also alleviate, ameliorate, treat, prevent, or reduce the severity of myelin degeneration in the brain and spinal cord, intellectual disability, loss of previously acquired motor skills, feeding difficulties, abnormal muscle tone, macrocephaly, paralysis, and seizures, and / or delayed development of speech and motor skills compared to that of a control individual or a subject prior to treatment. In some embodiments, treatment of a subject (e.g., a patient) with a therapeutically effective amount of a modified ASPA-encoding nucleic acid of the present disclosure, a vector genome comprising a modified ASPA-encoding nucleic acid, and / or an rAAV vector comprising a modified ASPA-encoding nucleic acid may increase, improve, prevent further loss of, or enhance balance, grip, strength, and / or motor coordination, and general motor function compared to that of a control individual or compared to the same subject prior to treatment. In some embodiments, treatment of a subject (e.g., a patient) with a therapeutically effective amount of a modified nucleic acid encoding ASPA of the present disclosure, a vector genome comprising a modified nucleic acid encoding ASPA, and / or an rAAV comprising a modified nucleic acid encoding ASPA may reduce the percentage vacuolar volume 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 same in a control individual or compared to the same subject before treatment.

[0172] Subjects suitable for treatment include any subject who has or is at risk of producing insufficient amounts of a functional gene product (protein), or who produces an abnormal, partially functional, or non-functional gene product (protein, e.g., an enzyme) that may result in disease. In some embodiments, a patient is treated with a vector or pharmaceutical composition of the disclosure before exhibiting any symptoms of a disease, disorder, or condition (e.g., Canavan disease). In some embodiments, a patient diagnosed by genetic analysis as being at risk for a disease, disorder, or condition (e.g., Canavan disease) is treated with a rAAV vector or composition of the disclosure before exhibiting symptoms.

[0173] In some embodiments, the subject to be treated may be a mammal, and in particular, the subject may be a human patient, such as a patient with Canavan disease. The subject may need treatment because, as a result of one or more mutations in the coding sequence of the ASPA gene, the ASPA protein has an incorrect amino acid sequence, thereby having reduced or no function, being expressed in the wrong tissue, being expressed at the wrong time, or not being expressed at all. The modified nucleic acid encoding ASPA of the present invention may be administered to enhance, improve, or provide the production of functional ASPA enzyme, which, in turn, may catalyze the breakdown of NAA into aspartate and acetate, among other biological functions discussed elsewhere herein.

[0174] Target cells for the rAAV vectors of the present invention are cells, particularly oligodendrocytes, which are normally capable of endogenously expressing the ASPA enzyme, such as those in the mammalian brain.

[0175] In embodiments that refer to methods of treatment described herein, such embodiments are also further embodiments for use in that treatment, or alternatively, for the manufacture of a medicament for use in that treatment.

[0176] Pharmaceutical Composition In certain embodiments, the present disclosure provides pharmaceutical compositions or medicaments for preventing or treating a disease, disorder, or condition mediated by or associated with decreased 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 an rAAV vector, and a pharmaceutically acceptable carrier.

[0177] 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 comprising a modified nucleic acid encoding ASPA that can increase the expression level and / or activity level of ASPA in the cell.

[0178] In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of a vector (e.g., a viral vector genome, an expression vector, a rAAV vector) or host cell (e.g., for ex vivo gene therapy) comprising a modified nucleic acid encoding ASPA, the composition further comprising a pharmaceutically acceptable carrier, adjuvant, diluent, excipient, and / or other agent. A pharmaceutically acceptable carrier, adjuvant, diluent, excipient, or other agent is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing undesirable biological effects that outweigh the beneficial biological effects of the material.

[0179] Any suitable pharmaceutically acceptable carrier or excipient may be used to prepare a pharmaceutical composition according to the present invention (see, for example, Remington The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor), Mack Publishing Company, April 1997).

[0180] Pharmaceutical compositions are typically sterile, pyrogen-free, and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as solutions (e.g., water, saline, dextrose solution, buffered solution, or other pharmaceutically sterile fluids), microemulsions, liposomes, or other ordered structures compatible with high product (e.g., viral vector particle, microparticle, or nanoparticle) concentrations. In some embodiments, pharmaceutical compositions comprising the modified nucleic acids of the present disclosure, vector genomes comprising modified nucleic acids, host cells, or rAAV vectors are formulated in water or buffered saline. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. In some embodiments, the nucleic acids, vectors, and / or host cells of the present disclosure may be administered in compositions that include a slow release polymer or other carrier that protects the product against rapid release, including a controlled release formulation, for example, implants and microencapsulated delivery systems.

[0181] In some embodiments, pharmaceutical compositions of the present disclosure are parenteral pharmaceutical compositions, including compositions suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV), and / or intracisternal (ICM) administration. In some embodiments, pharmaceutical compositions comprising an rAAV vector comprising a modified nucleic acid encoding ASPA are formulated for administration by ICV injection.

[0182] In some embodiments, the rAAV vector (e.g., AAV / Olig001 ASPA) is formulated in 350 mM NaCl and 5% D-sorbitol in PBS.

[0183] Administration method A modified nucleic acid encoding a transgene (e.g., ASPA) of the present disclosure, or a vector (e.g., vector genome, rAAV vector) comprising the modified nucleic acid, 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 thereof can be by any means known in the art for administering vectors. Target cells for the vectors of the present disclosure include cells of the CNS, preferably oligodendrocytes.

[0184] The vectors can be administered in addition to and as an adjunct to standard care treatments. That is, the vectors can be co-administered with another agent, compound, drug, treatment, or treatment regimen at the same time, contemporaneously, or at predetermined dosing intervals, as determined by one of skill in the art using routine methods. The uses disclosed herein include administering the rAAV vectors of the present disclosure according to dosing schedules in addition to and / or concurrently with standard treatments for Canavan disease known in the art.

[0185] In some embodiments, the combination composition comprises one or more immunosuppressants. In some embodiments, the combination composition comprises an rAAV vector comprising a transgene (e.g., a modified nucleic acid encoding ASPA) and one or more immunosuppressants. In some embodiments, the method comprises administering or delivering to a subject an rAAV vector comprising a transgene (e.g., a modified nucleic acid encoding ASPA), and administering to the subject an immunosuppressant prophylactically prior to administration of the vector or after administration of the vector (i.e., before or after symptoms of a response to the vector and / or the protein provided thereby become apparent).

[0186] In some embodiments, the rAAV of the present invention can be co-administered with empty capsids (i.e., viral capsids that do not contain a nucleic acid molecule or vector genome) that contain the same or different capsid proteins as the rAAV vector containing the modified nucleic acid (e.g., encoding ASPA). One skilled in the art will understand that co-administration of empty capsids can reduce immune responses, such as neutralizing responses, to the rAAV of the present disclosure. Without wishing to be bound by any particular theory, the empty capsids may function as immune decoys, allowing the rAAV vector containing the modified nucleic acid (e.g., encoding ASPA) to avoid neutralizing antibody (Nab) immune responses, for example, as described in WO2015 / 013313.

[0187] In one embodiment, a vector of the present disclosure (e.g., an rAAV vector comprising a modified nucleic acid encoding ASPA) is administered systemically. Exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intra-arterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic, and intramuscular administration, as well as direct tissue or organ injection. One skilled in the art will appreciate that systemic administration can deliver a 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 an area directly affected by ASPA deficiency (e.g., the brain and / or central nervous system). In some embodiments, the vectors of the present disclosure and pharmaceutical compositions thereof are administered to the brain parenchyma (i.e., by intraparenchymal administration), to the spinal canal or subarachnoid space to reach the cerebrospinal fluid (CSF) (i.e., by intrathecal administration), to the ventricles of the brain (i.e., by intraventricular administration), and / or to the cisterna magna of the brain (i.e., by intracisternal administration).

[0188] Thus, in some embodiments, vectors of the present disclosure comprising modified nucleic acids encoding ASPA are 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. Target cells of vectors of the present disclosure include cells located in the cortex, subcortical white matter of the corpus callosum, striatum, and / or cerebellum. In some embodiments, target cells of vectors of the present disclosure are oligodendrocytes. Additional routes of administration can also include local application of the vector under direct visualization, e.g., superficial cortical application, or other non-stereotactic application.

[0189] In some embodiments, the vectors of the present disclosure are administered by at least two routes, for example, the vectors are administered systemically and also directly to the brain. When administered via at least two routes, the administration of the vectors can be, but need not be, simultaneous or contemporaneous. Instead, the administration via the different routes can be performed separately with a time interval between each administration.

[0190] The modified nucleic acids encoding ASPA of the present disclosure, vector genomes comprising modified nucleic acids encoding ASPA, and / or rAAV vectors comprising modified nucleic acids encoding ASPA can be used for ex vivo transduction of cells or administered directly to a subject (e.g., directly to the CNS of a patient with Canavan disease). In some embodiments, the transduced cells (e.g., host cells) are administered to a subject to treat or prevent a disease, disorder, or condition (e.g., cell therapy for Canavan disease). The rAAV vector comprising a modified therapeutic nucleic acid (e.g., encoding ASPA) is preferably administered to cells in a biologically effective amount. In some embodiments, a biologically effective amount of vector is an amount sufficient to result in transduction and expression of the modified nucleic acid encoding ASPA (i.e., the transgene) in the target cell.

[0191] In some embodiments, the present disclosure includes methods for increasing the level and / or activity of ASPA in a cell by administering to the cell (in vivo, in vitro, or ex vivo) a modified nucleic acid encoding ASPA, either alone or in a vector (including a plasmid, viral vector, nanoparticle, liposome, or any known method for delivering nucleic acids to a cell).

[0192] The dosage of the rAAV vector depends, for example, on the mode of administration, the disease or condition being treated, the stage and / or aggressiveness of the disease, the condition of the individual subject (age, sex, weight, etc.), the particular viral vector, the stability of the expressed protein, the host immune response to the vector, and / or the gene being delivered. Generally, the dose should be at least 1 x 10 per kg of subject body weight to achieve a therapeutic effect. 8 or more, e.g., 1 x 10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 or greater range of vector genomes (vg).

[0193] In some embodiments, modified nucleic acids encoding ASPA can be administered as components of a DNA molecule (e.g., recombinant nucleic acid) having appropriate regulatory elements (e.g., promoters) for expression in target cells (e.g., oligodendrocytes). Modified nucleic acids encoding ASPA can be administered as components of a plasmid or viral vector, such as an rAAV vector. The rAAV vector can be administered in vivo to a patient in need of treatment (e.g., a Canavan patient) by delivering the vector directly (e.g., directly to the CNS). The rAAV vector can also be administered ex vivo to a patient by in vitro administration of the vector to cells from a donor patient in need of treatment, followed by reintroduction of the transduced cells into the donor (e.g., cell therapy).

[0194] The present disclosure includes administration methods that result in levels of mRNA encoding ASPA, levels of ASPA protein expression, and / or levels of ASPA activity that are detectably higher than the levels of ASPA expression (mRNA and / or protein) or ASPA activity in otherwise identical cells that are not administered a modified nucleic acid (e.g., a modified nucleic acid encoding ASPA).

[0195] In another embodiment, the present disclosure includes methods of administration that result in levels of mRNA encoding functional ASPA and / or levels of functional (e.g., biologically active) ASPA protein expression that are detectably higher than the levels of functional ASPA (mRNA and / or protein) present in otherwise identical cells that are not administered the modified nucleic acid (e.g., a modified nucleic acid encoding ASPA). That is, the present disclosure includes methods of increasing the level of functional ASPA in cells in which the cells produce normal levels of ASPA, but in which the ASPA protein lacks activity or exhibits reduced activity compared to normal, wild-type ASPA.

[0196] Those skilled in the art will understand that the cells can be cultured or grown in vitro or can reside within an organism (i.e., in vivo). Furthermore, the cells may express endogenous ASPA such that the level of ASPA within the cells is increased, and / or the cells may express endogenous ASPA that is a mutation or variant of wild-type ASPA, e.g., ASPA having the sequence of SEQ ID NO: 3; in particular, there may be two or more wild-type alleles of human ASPA. Thus, the level of ASPA is increased compared to the level of ASPA expressed in otherwise identical but untreated cells.

[0197] kit The present disclosure provides kits comprising packaging materials and one or more components. The kits typically include a label or package insert containing a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein. The kits may contain a collection of such components, such as modified nucleic acids, recombinant nucleic acids, vector genomes, rAAV vectors, rAAV, and optionally a second active agent, such as a compound, therapeutic agent, drug, or composition.

[0198] A kit refers to a physical structure containing one or more components of the kit. The packaging material can maintain the sterility of the components and can be made of materials commonly used for such purposes (e.g., paper, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0199] The label or package insert may include identification of one or more components therein, dosage, mechanism of action, clinical pharmacology of the active ingredients, including pharmacokinetics and pharmacodynamics. The label or package insert may include information identifying manufacture, lot number, location and date of manufacture, expiration date. The label or package insert may include information regarding the disease for which the kit components may be used (e.g., Canavan disease). The label or package insert may include instructions for a clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or regimen. The instructions may include dosage, frequency of duration, and instructions for practicing any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0200] The label or package insert may include information about potential side effects, complications or reactions, for example, warnings to the subject or clinician about situations in which it is not appropriate to use a particular composition.

[0201] equivalent The foregoing written specification is deemed sufficient to enable one skilled in the art to practice the present disclosure. The foregoing description and examples set forth certain exemplary embodiments of the present disclosure in detail. However, no matter how detailed the foregoing appears in text, it should be understood that the present disclosure can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0202] All references cited herein, including patents, patent applications, articles, textbooks, and the like, and the references cited therein, to the extent they have not already been introduced, are hereby incorporated by reference in their entirety.

[0203] Illustrative Embodiments The present invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any variations that become evident as a result of the teachings provided herein. [Example]

[0204] Example 1: Dose-responsive reduction in NAA using rAAV vectors containing codon-optimized nucleic acids encoding ASPA Human embryonic kidney (HEK) cells were transfected with 1.0 μg of a plasmid expressing NAA synthase (Nat8L) and cotransfected with 0.1, 0.2, 0.5, or 1.0 μg of a plasmid containing either the wild-type human ASPA nucleic acid sequence (SEQ ID NO: 3), a codon-optimized nucleic acid encoding ASPA (comprising the nucleic acid sequence of SEQ ID NO: 1; see Francis et al. (2016) Neurobiol. Dis. 96:323-334), or a codon-optimized nucleic acid encoding ASPA comprising the nucleic acid sequence of SEQ ID NO: 2. NAA concentrations were measured by HPLC (n=4 / group). A dose-responsive reduction 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 either the wild-type nucleic acid encoding ASPA or the codon-optimized nucleic acid encoding ASPA of SEQ ID NO: 1 (Figure 1).

[0205] Example 2: Biodistribution of oligo-directed AAV / Olig001 This study was conducted to define the most effective dose and route of administration (ROA) of an oligotropic AAV (AAV / Olig001; (WO2014 / 052789; Powell et al. (2016) Gen. Ther. 23:807-814)) capsid mutant in promoting widespread CNS oligodendrocyte transduction in a mouse model of the inherited human leukocyte dystrophy, Canavan disease. Three doses of AAV / Olig001 delivered via four different ROAs were tested in adult symptomatic Canavan mice (nur7). Vector spread and transduction were quantified 2 weeks posttransduction by generating stereological estimates of reporter green fluorescent protein (GFP)-positive cells in four anatomical regions of interest. The tropism of AAV / Olig001 delivered via each ROA was assessed by validating oligotropism by scoring the incidence of lineage-specific antigen colabeling with GFP in these same regions. The ROAs used were intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV), and intracisternal (ICM). Via each route, 1 × 10 10 , 1×10 11, and 5 × 10 11 Three doses of total vector genome (VG) were administered, with the volume of delivered material remaining constant across all treated cohorts. Direct pairwise comparisons were performed to define the optimal combination of dose and ROA for AAV / Olig001 treatment of Canavan disease. Six-week-old aspartoacylase-deficient nur7 mice exhibiting the acute symptomatic phase of Canavan disease (Traka et al. (2008) J. Neurosci 28:11537-11549) were used. The results generated by this study formed the basis for subsequent preclinical efficacy testing to support the clinical application of AAV / Olig001 for currently intractable white matter disorders such as Canavan disease.

[0206] material AAV / Olig001 vector Two lots of AAV / Olig001 vectors containing a constitutive expression cassette for the GFP reporter gene were generated (Lot No. 7660 and Lot No. LAV38A). All vectors generated contained the GFP reporter gene driven by a hybrid CMV / chicken β-actin promoter (CBh) flanked by self-complementary AAV ITRs. 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). Vector concentration was defined as the total number of viral vector genomes (vg) determined by qPCR quantification of DNAse-resistant AAV inverted terminal repeat (ITR) sequences in the original preparation.

[0207] animal All animals used in this study were obtained from a colony maintained at the Rowan School of Osteopathic Medicine animal facility in accordance with approved institutional protocols. Founder animals were derived from a commercial source (Jackson Laboratories). nur7 mice are a well-characterized model of Canavan disease, which harbors an inactivating point mutation in the gene encoding the glial hydrolase aspartoacylase (aspa), rendering the protein nonfunctional (Traka et al. J. Neuroscience (2008) 28(45) 11537-11549). Homozygous nur7 mutant animals were obtained by pairing heterozygous carrier animals and genotyped using an in-house customized SNP assay and real-time PCR.

[0208] AAV / Olig001-GFP diluted to the appropriate concentration in 0.9% saline was delivered to 6-week-old nur7 mutant mice by stereotactic injection under inhalation anesthesia (4% saline, effectively measuring induction and maintenance titers). Four treatment cohorts, each distinguished by a different route of administration (ROA), were generated: intrathecal (IT), intraparenchymal (IP), intracerebroventricular (ICV), and intracisternomagnathus (ICM). Within each ROA cohort, subgroups of animals were established, defined by the vector dose administered by each ROA (1 × 10). 10 , 1×10 11 , and 5 × 10 11 total vector genome).

[0209] Therefore, for each ROA, three subgroups defined by dose were generated, with n = 5 animals for each dose at each ROA, yielding a total of 60 nur7 mice for the study. AAV / Olig001-GFP was administered to anesthetized mice, and the total delivery volume of 5 μL remained constant for all surgeries, regardless of dose or ROA. IP ROA required five injections of 1 μL of vector at five stereotaxic coordinates: two injections into the anterior and posterior subcortical white matter in each hemisphere (i.e., four injections in the cingulum) and one injection into the cerebellar white matter (five injections total), with additional injections at a rate of 0.1 μL / min using a digital pump. IT ROA animals received a single 5 μL injection of vector into the subarachnoid space accessed via lumbar puncture between L5 and L6. ICV ROA animals received two 2.5 μL vector injections, one into each lateral ventricle at a rate of 0.1 μL / min. ICM ROA animals received 5 μL of vector delivered directly into the CSF via the cisterna magna at a rate of 0.1 μL / min. All animals received 0.5 mL of 20% mannitol (ip) 20 minutes before surgery. All animals were housed in groups for 2 weeks after AAV / Olig001-GFP delivery and then sacrificed for postmortem analysis.

[0210] Groups of 2- and 8-week-old wild-type and nur7 mice received systemic BrdU (50 mg / kg, i.p.) twice daily for two consecutive days and were sacrificed on day 3. Animals were administered BrdU at a concentration of 50 mg / kg. Brain tissue sections were processed for BrdU staining after DNA hydrolysis in 1 M HCl using a commercially available antibody (Millipore-Sigma).

[0211] method Quantifying vector biodistribution by unbiased stereology Two weeks after vector surgery, animals were deeply anesthetized and the brain was prepared by transcardial perfusion with 0.9% saline followed by freshly prepared buffered 4% paraformaldehyde. The perfused brains were removed and fixed in 4% PFA overnight at 4°C. Fixed brains were cryopreserved, flash frozen in a dry ice / isopentane bath, and stored at -80°C prior to immunohistochemical processing. Serial 40-μm sagittal sections were made for each brain (total of 144 sections), and every fourth section was stained for GFP using commercially available antibodies (Sigma / Millipore). GFP-positive somatic cells in the cortex, subcortical white matter, striatum, and cerebellum were scored by unbiased stereology using the optical dissector method (Figure 2) (West et al. Anat Rec. (1991) 231:482-97). Stereological software (Stereologer, Stereology Resource Center) coupled to an upright brightfield microscope with a motorized stage was used to generate counts of GFP-positive somata within four different regions of interest, namely the cerebral cortex, subcortical white matter of the corpus callosum and external capsule, striatum, and cerebellum. GFP-positive cells of the sampling fraction were re-converted to absolute estimates across each region of interest using the formula ΣQ*(t / h)*(1 / asf)*(1 / ssf). Where ΣQ = number of particles, t = section thickness, h = counting frame height, asf = area sampling fraction, and ssf = section sampling fraction). For all datasets generated in this way, the variation within samples was monitored by calculation of the coefficient of error (CE) that meets a contribution of less than 15% to the total variance (CV) threshold in order to reduce the technical noise masking the true biological variance between samples. The significant difference in the mean group mean estimates of N was determined by Student's unpaired two-sided t-test with a threshold significance of <p0.05.

[0212] Quantification of vector orientation Vector tropism in AAV / Olig001-GFP-transduced brains was quantified by scoring for lineage-specific antigen co-labeling with GFP fluorescence. Alternate sections were processed for immunohistochemistry for NeuN (present in most CNS and PNS neuronal cell types in vertebrates), GFAP (glial fibrillary acidic protein), or Olig2 (oligodendrocyte lineage transcription factor 2) using commercially available antibodies (Sigma / Millipore) to label neurons, astrocytes, and oligodendrocytes, respectively. Scanning confocal microscopy was used to generate multipoint image stacks across each intercellular region. NIS-Elements Advanced Research software (Nikon) was used to count the total GFP-positive cells in each stack and the number of GFP-positive cells co-labeling with each lineage-specific antigen (Olig2 and NeuN). Numerical values ​​were collated for each individual brain (a total of eight serial sections were sampled from each brain with a sampling interval of four). To score both GFP immunofluorescent somas and GFP / Olig2 or NeuN positive somas, ROIs in individual sections were outlined by the software and individual points sampled at high magnification every 200 μm. The total number of GFP positive somatic co-labeling with either Olig2 or NeuN was calculated by dividing the number of GFP positive somatic cells by the lineage-specific co-labeling in each serial section. The mean of each ROA was calculated (n = 5 animals).

[0213] result Intraparenchymal (IP) ROA dose response IP ROA animals received five separate injections targeting the subcortical white matter in both hemispheres and the cerebellum. Treated animals were sacrificed 2 weeks after vector administration (8 weeks of age), and brains were processed for GFP immunohistochemistry. GFP-positive somatic cells in the cortex, subcortical white matter, striatum, and cerebellum were scored using unbiased stereology with an optical segmentor to obtain absolute estimates of transduced cells in each region of interest. All three doses of AAV / Olig001-GFP administered resulted in significant levels of transduction of cells throughout the brain. (Figure 3) Within the cortex, an increase in the number of transduced cells was observed, exceeding 1 x 10 10vg dose and 1 × 10 11 The effect was significant between the 5 × 10 vg dose (+1.6-fold, p = 0.0096), but not between the 5 × 10 11 No further increase with dose was evident (p = 0.659), suggesting saturation (Figure 3). High levels of transduction were evident in the subcortical white matter of the corpus callosum and external capsule, with positive cells concentrated immediately adjacent to the four injection sites. Subcortical white matter GFP-positive cells also increased in a dose-dependent manner, reaching 1 × 10 10 From 1×10 11 The 2.2-fold increase in dose was statistically significant (p=0.0144), but the 1×10 11 From 5 x 10 11 A 1.3-fold increase in striatal activity from 1 × 10 to 1 × 10 did not reach statistical significance (p = 0.283). Moderate striatal transduction was evident. 10 From 1×10 11 The 3.4-fold increase to -5 ) but 5×10 11 No further increase in transduction was evident with the dose (p=0.706). Transgene expression in the cerebellum was significantly higher at 1×10 11 and 5 x 10 11 Both doses were confined to the area immediately surrounding a single injection site and resulted in a significant increase from the previous dose (1 × 10 11 : 1.5-fold increase [p=0.0016], 5x10 11 (1.4-fold increase [p=0.0019]). The cortex had the highest number of transduced cells (513,477), followed by the subcortical white matter (178,362), the cerebellum (86,820), and finally the striatum (62,706). Co-labeling of GFAP was less than 2%.

[0214] Intrathecal (IT) ROA dose response IT administration of AAV / Olig001-GFP resulted in excellent distribution of transgene expression throughout the brain, with the exception of the subcortical white matter of the corpus callosum and external capsule (Figure 4). 10 From 1×10 11 A highly significant increase in vg was evident (6.1-fold increase, p=0.000026), with a 5×10 11There was no significant increase with the vg dose (p=0.273). The distribution of GFP expression in the IT ROA cortex was excellent, but the intensity of expression was somewhat reduced compared to the IP brain. Not surprisingly, noticeable GFP expression was observed in the lumbar region of the spinal cord, suggesting some dilution of the vector by spinal cord tissue on its way to the brain. The most striking observation in the IT ROA brain was the paucity of transgene expression in the corpus callosum and external capsule. At a dose of 1 × 10 10 From 1×10 11 Although there was a highly significant increase in GFP-expressing white matter tract cells when the concentration of GFP was increased to 1.5 (6.3-fold increase, p=0.00021), the absolute number of transduced white matter cells in IT ROA brains was relatively modest. The mean number of positive cells in this white matter tract region of the brain was 1 x 10 11 At the same dose, the IT ROA brain dose was 64,970 compared to 178,362 in the IP brain at the same dose. Similar to the cortical ROA, the IT ROA brain dose was 1 x 10 11 From 5 x 10 11 Further increase in the concentration of α-glucan did not significantly increase the number of transduced white matter furnace cells (p=0.203).

[0215] The striatum showed a dose-responsive increase in transduced cells at each successive dose. 10 From 1×10 11 Increasing the concentration of 5 × 10 11 Increasing the dose resulted in a 3.2-fold increase in positive cells (p=0.000037), which resulted in a number comparable to IP ROA brain striatal transduction (5 × 10 11 IT averaged 79,444, 5 x 10 11 The IP yielded an average of 65,203.

[0216] IT ROA resulted in strong cerebellar transgene expression, with 1 × 10 10 From 1×10 11 A significant 1.5-fold increase was observed when the dose was increased to 5 × 10 (p = 0.0064). 11No further increase was observed with dose. Cerebellar transduction was comparable to IP ROA brain, with 1 × 10 11 and 5 x 10 11 The dose was slightly higher, but not significantly so.

[0217] Intracerebroventricular (ICV) ROA dose response ICV administration of AAV / Olig001-GFP resulted in significant transgene expression across all regions of interest, with particularly robust transduction of the subcortical white matter (Figure 5). All regions of interest were transduced at a dose of 1 × 10 10 From 1×10 11 showed a dose-responsive increase in the number of transduced cells when the concentration was increased to 5 × 10, except for the cerebellum. 11 The dose is 1 x 10 11 There was a small, non-significant increase in most regions compared to the 1 × 10 dose. Cortical transgene expression was comparable to IP ROA brain, with a dose of 1 × 10 10 From 1×10 11 When the concentration of transgene-positive cells was increased to 5 × 10, the number of transgene-positive cells increased by 2-fold (p = 0.00029). 11 There was a further 1.2-fold increase after administration of the dose, but this did not reach statistical significance (p=0.123).

[0218] Subcortical white matter transduction in ICV brains was substantial, with doses of 1 × 10 10 From 1×10 11 When the concentration of GFP-positive white matter tract cells was increased to 5 × 10, a two-fold increase in GFP-positive white matter tract cells was observed (p = 0.00052). 11 A modest, non-significant increase was observed with dose (p=0.334). 11 Subcortical white matter transduction in ICV brains at doses significantly increased 1.5-fold compared to IP brains (p=0.041) and significantly increased 4.2-fold compared to IT ROA brains (p=0.0001).

[0219] A very similar pattern of transgene expression was seen in the striatum of the ICV dose cohorts, with doses of 1 × 10 10 From 1×10 11A significant 2-fold increase in GFP-positive cells was observed when the concentration was increased to 5 × 10 (p = 0.000043), but 11 No significant further increase was seen with dose (p=0.537). Robust striatal transgene expression was evident in ICV brains, with a 2.5-fold increase in GFP-positive cells in this region over IP brains (p=0.00004).

[0220] Cerebellar ICV transduction was robust, and a dose-dependent increase in GFP-positive cells was observed at both successively higher doses (1 × 10 11 +2 times, p=9.56×10 6-6 ;5×10 11 There was a 1.7-fold increase in GFP-positive cells compared to IP brains (p=0.0001), with a 1x10 11 There was a 1.4-fold increase in GFP-positive cells at this dose compared to the IT dose (p=0.0013). The cerebellum was the only region to show a further significant increase in GFP-positive cells in ICV ROA brains.

[0221] A notable difference between IP and ICV ROA brains that was evident throughout the sampling process was the greater distribution of vector in the ICV group. Transgene expression at the injection site was more intense in IP brains but rapidly diluted away from that site. In contrast, ICV transgene expression was relatively evenly distributed over a much larger area of ​​the brain.

[0222] Intracisternal magna (ICM) ROA dose response ICM administration of AAV / Olig001-GFP resulted in relatively widespread yet modest transgene expression in the cortex, striatum, and cerebellum. However, similar to IT ROA brains, there was no significant transgene expression in the subcortical white matter of ICM brains (Figure 6). Cortical transgene expression was dose-responsive, with each successively higher dose resulting in a significant increase in GFP-positive cells (1 x 10 11 , 2.2-fold increase, p=0.018; 5×10 11, 1.3-fold increase, p=0.043). In both the striatum and cerebellum, 1 × 10 11 A significant increase in GFP-positive cells was observed at the 20 dose (p = 2.49 × 10 in the striatum and cerebellum, respectively). -6 and p = 0.0062), the highest 5 × 10 11 The dose of 1 × 10 resulted in a further increase in positive cells only in the cerebellum (p = 0.061). Transduction of subcortical white matter tracts by ICM ROA was crucially modest. 10 From 1×10 11 Although increasing the concentration of GFP-positive cells to 100 mg / ml resulted in a significant increase in GFP-positive cells (p=0.00086), the actual number of transgene-positive cells present was relatively negligible.

[0223] Compared to ICV brains, ICM subcortical white matter GFP-positive cells were reduced 14.2-fold (ICV mean 271,274; ICM mean 18,996, p = 0.00002), and IT was reduced 3.4-fold compared to animals in the next lowest subcortical white matter transduction ROA group (IT mean 64,970), making ICM the lowest effective ROA for white matter transduction. Distribution across other regions of interest was comparable to other ROA treatment groups, and no significant differences were evident in cortical transduction compared to all three other ROAs. Striatal transduction via ICM was slightly reduced compared to ICV ROA (p = 0.043). Striatal ICM GFP expression was significantly higher in animals receiving 1 x 10 11 The transduction rate was significantly greater than both IP (+2.0-fold, p=0.00005) and IT (+5.1-fold, p=0.0000005) at doses of 1 x 10. ICM brains showed the highest number of transduced cerebellar cells of any of the four ROAs examined. 11 dose increased transduction of cerebellar ICM 1.5-fold over ICV (ICM mean 228,282; ICV mean 157,203), 2.6-fold over IP, and 2.2-fold over IT.

[0224] Compared routes of administration (ROA) For all ROAs explored herein, vector doses of 1 × 10 were used in all regions of interest. 10 From 1×10 11Increasing the concentration to 5 × 10 increased the number of transduced cells by 2–3 fold, whereas increasing the concentration to 5 × 10 11 Further dose escalation to 1 × 10 resulted in a negligible increase in overall transduced cells. 11 Direct comparison of all four ROIs in dose revealed clear differences in the absolute number of transduced cells in all four ROIs (Figure 7). 11 The number of transduced cells in the vector genome-transduced brain cortex did not differ significantly between ROAs, all resulting in an average of 44,000–50,000 positive cell bodies. In contrast, there was a clear advantage to ICV ROAs in the subcortical white matter, with transduced cell numbers significantly higher in ICV brains than in any other group. ICV- and IP-transduced brains exhibited the highest and second-highest number of transduced white matter tract cells, respectively. 1 × 10 transduced cells were obtained via ICV ROAs. 11 An average of 2.7 × 10 in brain white matter tracts transduced with the AAV / Olig001-GFP vector genome 5 An average of 1.8 x 10 positive cells were present in IP brains exposed to the same dose. 5 The number of positive cells was significantly higher than that of negative cells (p=0.041).

[0225] Both IT and ICM ROA were inefficient in transducing subcortical white matter cells, with a mean of 2.7 × 10 in the ICV group. 5 positive cells in the ICM group (p=0.000083) than in the control group (p=0.000083). 4 There were significantly 14-fold fewer cells in the IL-16 group than in the IT group, compared with 4-fold fewer in the IL-16 group (p=0.0001), which may be of concern in disease model systems that exhibit a lack of myelin.

[0226] The ICV route also results in efficient transduction of cells in the striatum with a higher number of GFP-positive cells in ICV brains than all other ROAs (ICV vs. IP p=3.68 × 10 -5 ICV vs IT p=1.61×10 -5;ICV vs. ICM p=0.043). Cerebellar transduction efficiency was comparable across IP, IT, and ICV ROAs, but ICM brains had the highest number of transduced cerebellar cells (ICM vs. ICV p=0.045).

[0227] Although IP and ICV ROA brains had comparable absolute numbers of cells transduced by AAV / Olig001-GFP in a given region, the majority of the positive cell count in IP brains was a product of sections directly adjacent to the injection site, whereas positive cells in ICV brains were relatively evenly distributed. Systematic nonrandom stereological sampling allowed for the identification of variance between sections sampled from individual brains (intra-sample variance), expressed as the coefficient of error (CE) within a dataset and calculated by dividing the standard error of the mean of repeated estimates by the mean. CE represents half of the total variance in the sampled population; true biological variance (CV), or the difference in means between individual brains, constitutes the other half. The average CE for individual IP brains was calculated as approximately 12% of the total variance, while the average CE for ICV brains was approximately 3%, indicating that GFP-positive cells were more evenly distributed across all sections sampled in ICV brains. In IP brains, the actual number of positive cells in each sampled section decreased as the sampled section decreased further laterally from the injection site, whereas the number of positive cells in ICV brains was consistently close to the within-sample mean across all sampled sections. The net result of this difference was greater vector diffusion in ICV ROA brains compared to IP brains, particularly in the cortical and subcortical white matter (Figure 7).

[0228] conclusion Four different ROAs were used to define dose and ROA combinations that promote global CNS oligodendrocyte transduction in acutely symptomatic animals, closely modeling the Canavan brain at diagnosis. Administration of the AAV / Olig001-GFP vector resulted in greater than 70% oligotropism in all regions of interest except the cerebellum, without the need for lineage-specific expression elements. A dose-dependent increase in transgene-positive oligodendrocytes was evident at all ROAs, and intraventricular ROAs promoted greater numbers of transduced white matter tract cells while maintaining greater than 90% oligotropism in this critical region of interest. These data highlight capsid-cell surface interactions as a key determinant of oligotropism, which is most relevant for clinical applications in oligodendrocyte-specific disorders such as Canavan disease. These data also demonstrate that the Olig001 capsid harbors potential therapeutic capsids for the treatment of oligodendrocyte-related diseases, disorders, and / or conditions, including Canavan disease.

[0229] Example 3: Vector tropism by route of administration (ROA) A distinctive feature of AAV / Olig001 is its distinct oligotropism 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 to Canavan disease, a defining white matter disorder, AAV / Olig001 vectors must be able to exhibit this tropism when applied with a different ROA. Oligotropism can vary due to variables such as age at intervention (Gholizadeh et al. Hum. Gene Ther. Methods (2013) 24:205-13; Foust et al. Nature Biotech. (2009) 27:59-65), and while previous studies have documented the potential oligotropism of AAV / Olig001 in neonatal nur7 mice (Francis et al. Neurobiol. Dis. (2016) 96:323-334), the translation of this potential tropism to older symptomatic animals has not yet been tested. Accordingly, the 1 × 10 11 All four ROA doses were evaluated for their potential effect on vector tropism in 6-week-old animals. The cortex, subcortical white matter, striatum, and cerebellum used to generate absolute counts of GFP-positive cells were analyzed for co-labeling of the GFP transgene with the lineage-specific antigens Olig2 (i.e., target-specific labeling for oligodendrocytes) and NeuN (i.e., target-specific labeling for neurons).

[0230] result All four ROAs produced comparable results, with intact oligotargeting. Non-oligodendrocyte transgene expression was attributed to neurons, with few GFP-expressing astrocytes observed in all four ROA cohorts (<5%).

[0231] Cortical co-labeling of Olig2 with GFP was comparable among IT, ICV, and ICM ROAs, with the percentage of total GFP-positive cells co-labeled with Olig2 consistently being approximately 75%. In IP-transduced brains, approximately 62.3% of GFP-positive cells co-labeled with Olig2, a small but significant reduction (Figure 8). GFP-positive cells in these same brains co-labeled with NeuN essentially accounted for the remaining transduced cortical population (35.1%). All three of the IT, ICV, and ICM ROAs showed approximately 20% NeuN co-labeling. In IT ROA brains, 75.5% of cortical GFP-positive cells co-labeled with Olig2 and 20.2% of cortical GFP-positive cells co-labeled with NeuN. ICV ROA brains exhibited 70.8% oligotropism and 23.6% neurotropism in the cortex, while ICM brains expressed 76% GFP co-labeled with Olig2 and 17.4% GFP co-labeled with NeuN in the cortex. Although the difference in oligotropism expression among the four different ROAs was small, IP ROA showed a significant increase in NeuN co-labeling (p = 0.0043 vs. IT; p = 0.0119 vs. ICV; p = 0.00059 vs. ICM), which coincided with a slight but significant reduction in Olig2 co-labeling compared to the other three ROAs (p = 0.026 vs. IT; p = 0.048 vs. ICV; p = 0.0085 vs. ICM), suggesting that IP ROA promoted a slight increase in neurotropism at the expense of oligotropism. Again, the IP ROA was significant for increased NeuN colabeling (+1.5-fold, p=0.012), suggesting that the reduced Olig2 colabeling is explained by increased neuronal transduction in this ROA. Most of the GFP-NeuN colabeling in the IP ROA brain was clustered immediately around the injection site, indicating saturating amounts of AAV / Olig001-GFP adjacent to the injection site.

[0232] Subcortical white matter co-labeling of Olig2 with GFP was greater than 90% in all four ROAs (Figure 9). Co-labeling of NeuN with GFP was less than 6% in all four ROAs. 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, regardless of ROA. ICV transduction resulted in nearly ubiquitous Olig2 co-labeling in the corpus callosum, with no NeuN co-labeling.

[0233] Striatal colabeling of Olig2 with GFP was comparable to the percentage of total GFP-positive cells colabeling with Olig2 (>80%) among all ROAs (Fig. 10). The remaining GFP-positive cells in the striatum (<20%) colabeled with NeuN.

[0234] Co-labeling of the cerebellum showed opposite ratios of Olig2 and NeuN in all four ROAs compared to other brain regions studied. The percentage of Olig2 co-labeling was 10% of total GFP-positive cells in all four ROAs (Figure 11). Transgene expression was dominated by neurons within the cerebellum, accounting for over 80% of GFP-expressing cells. No significant differences in the percentage of co-labeling with either antigen were observed between ROA cohorts within the cerebellum. Large Purkinje neurons in the granule cell layer were strongly GFP-positive, with only sporadic Olig2 / GFP co-labeling within the cerebellar white matter tracts (Figure 29C). This contrasted with the nearly 100% oligotropism observed in the subcortical white matter (Figure 29B) and the 70%-80% oligotropism observed in relatively neuron-dense regions such as the cortex and striatum (Figure 29D). 1 × 10 11 Total GFP-positive cells scored for each ROA at dose were ranked from highest mean to lowest mean of total GFP-positive cells (+ / - sd), n=5: ICV 1104256.4 (106816.96); IP 841365.6 (121722.7); ICM 815486.9 (106979.7); IT 742143.1 (79496.5).

[0235] ICV ROA yielded the highest number of total GFP-positive cells (sum of all ROA counts in an individual brain), 1.3-fold more than the next-ranked ROA, IP (p = 0.0067). Total counts in ICV brains were significantly increased across all ROAs, including ICM (p = 0.0027) and IT (p = 0.0003). Cell counts in IP ROA brains were not significantly increased compared to either ICM (p = 0.730) or IT counts (p = 0.165), marking a clear superiority of ICV ROA in total transduced cells. Approximately 75% of the difference in overall GFP-positive cell counts between the ICV and IP cohorts (approximately 262,891) was accounted for by ROIs in the subcortical white matter (35%) and striatum (36%), which exhibited over 80% oligotropism in both ROA cohorts. This means that the ICV brains contained at least 210,000 more transduced oligodendrocytes somewhere in that region than the IP brains. If this analysis were limited to the subcortical white matter, ROIs showing >90% oligotropism by all ROAs, then at least 83,000 more transduced oligodendrocytes per brain would be expected if AAV / Olig001 were administered via ICV ROA. When evaluated against the ROA cohort showing the worst levels of GFP transgene expression, the ICM cohort, ICV administration resulted in an increase of AAV / Olig001-transduced oligodendrocytes of over 200,000 cells per brain.

[0236] The adult mammalian CNS is known to harbor substantial numbers of oligodendrocyte precursor cells in its white matter (Dawson et al. Mol. Cell Neurosci. (2003) 24:476-488), and evidence of attempted remyelination in juvenile nur7 neurons in the form of increased turnover of immature oligodendrocytes has previously been shown (Francis et al. J. Cerebral Blood Flow Metabolism (2012) 32:1725-36). Given that white matter possesses a remarkable capacity for remyelination, even in the adult brain, the persistence of a resident population of immature oligodendrocytes in adult nur7 white matter should be considered an ideal target for oligodendrocyte gene delivery vectors.

[0237] To assess the relative numbers of proliferative oligodendrocyte precursors / immature oligodendrocytes, both nur7 and wild-type mice were given systemic BrdU twice daily for 2 days and sacrificed on day 3 for the process of BrdU / Olig2 colabeling (Figure 29E-G). BrdU administration began in both 2- and 8-week-old cohorts to quantify the possible persistence of proliferative oligodendrocytes in the juvenile and adult brains. The number of BrdU-positive cells in the corpus callosum and external capsule of genotype cohorts at each age revealed a significant 1.8-fold increase (p = 0.029) and a 1.6-fold increase (p = 0.034) in BrdU-positive cells in 2-week-old nur7 brains and 8-week-old nur7 brains, respectively, compared with wild-type mice (Figure 29F). The majority of BrdU-labeled cells in the nur7 white matter at both ages co-labeled with Olig2, indicating the persistence of proliferating precursor / immature oligodendrocytes in the white matter of adult symptomatic nur7 mice. A subset of three 6-week-old nur7 mice received 1 × 10 11 Animals were given systemic BrdU for 2 days before transduction with AAV / Olig001-GFP in vg mice, and sacrificed 2 weeks after transduction for evidence of transduction of proliferating cells within the white matter tracts. Numerous BrdU / GFP co-labeled cells were observed within the white matter tracts of these animals, indicating successful transduction of resident progenitor / immature cells.

[0238] A group of healthy wild-type animals matched to the nur7 ROA cohort (i.e., 6 weeks old) were injected with 1x10 mice via ICV ROA. 11 vg mice were transduced with AAV / Olig001-GFP and sacrificed 2 weeks later for stereological estimation of GFP-positive cells within the cortical and subcortical white matter tracts (Figure 8). Estimates of GFP-positive cells revealed a significant 2-fold reduction in both the cortex and subcortical white matter of wild-type brains compared to nur7 brains (p = 0.00032 and p = 0.0116 for the respective ROIs). Subcortical white matter GFP transgene expression in wild-type brains was highly restricted to the region immediately surrounding the lateral ventricles, whereas cortical expression, although reasonably diffuse, was very modest in absolute number of transduced cells.

[0239] conclusion Examples 2 and 3 demonstrate that the intracerebroventricular (ICV) administration route of the AAV / Olig001GFP vector provided the best combination of vector spread and oligodendrocyte tropism. Importantly, this ROA appears well suited for transduction of subcortical white matter, the tissue affected by Canavan disease pathology. Thus, the ability to transduce hundreds of thousands of cells and maintain nearly 100% tropism for oligodendrocytes confers a significant advantage to AAV / Olig001 over other AAV capsids. A 4-6 week old nur7 corpus callosum / external capsule contains approximately 1,500,000 Olig2-positive cells, so transduction of 1 x 10 6 cells via an ICV ROA is feasible. 11 Administration of a dose of AAV / Olig001 vector could transduce approximately 20% of the resident oligodendrocyte population. It should be noted that the white matter tracts of nur7 mice, with evidence of attempted remyelination, contain substantial numbers of proliferating oligodendrocyte precursors. Given that a single oligodendrocyte can myelinate multiple axons, the potential for remyelination after transduction of white matter with therapeutic AAV / Olig001 vectors is significant.

[0240] Other CSF-targeted ROAs, i.e., intrathecal and intrathecal tracts, showed relatively poor transduction of white matter tracts and would not be the first choice for consideration as therapeutic ROAs. While IP brain administration approached comparable transduction levels in terms of the number of transduced cells, the majority of these cells were concentrated around the injection site. While cells at these sites likely had a greater vector genome copy number per cell than any other ROA, vector diffusion away from these sites was significantly lower compared to ICV ROAs. The broader distribution of GFP transduction with ICV administration is advantageous in that an appropriate balance between the number of transduced cells and the number of vector copies per transduced cell can be achieved.

[0241] Indeed, the strong concentration of transgene expression in IP brains in Examples 2 and 3 was associated with a small but significant reduction in oligodendrocyte targeting and a balanced increase in neuronal targeting within the cortex. This indicates that saturating a region with AAV / Olig001 can result in reduced oligodendrocyte specificity. Furthermore, cortical oligodendrocytes in nur7 mice from the animal group used in this study were reduced in number compared to wild-type mice and showed evidence of stress and apoptosis (Francis et al. (2012) J. Cereb. Blood Fl. Metab. 32:1725-1736), which may be expected to affect transduction efficiency.

[0242] Vector tropism in all regions of interest was 75-90% oligotropic, except for the cerebellum, which showed greater than 80% neurotropism in all ROA groups. Particularly strong transgene expression was observed in granule layer Purkinje neurons. While the reason for this apparent reversal of tropism is not readily apparent, the cerebellum is clearly a distinct anatomical entity with respect to resident cell types. Purkinje cells within the cerebellum express low but appreciable levels of Olig2, suggesting that AAV / Olig001 capsids may have interactions with Purkinje neurons that are significantly different from the surface of other neurons in other brain regions.

[0243] The current example demonstrates that AAV / Olig001 drives robust oligodendrocyte transgene expression throughout the brain of nur7 Canavan disease mice, with the notable exception of the cerebellum. In all other brain regions, greater than 70% oligotropism was achieved without the need for lineage-specific promoters. The inherent affinity of the AAV / Olig001 capsid for the oligodendrocyte surface is a significant advantage over the use of selective promoters in other non-oligotropic serotypes, as it ensures that as close as possible to the total dose of delivered vector is expressed in target cells. These data identify the benefits of a distinct ROA for targeting white matter in the brain and demonstrate the applicability of ICV ROA for preclinical efficacy studies in symptomatic adult nur7 mice as a model for the treatment of Canavan disease.

[0244] Example 4: Differences in AAV / Olig001-GFP transduction efficiency between wild-type and nur7 brains. The nur7 mouse model of Canavan disease exhibits symptoms of aortic dysfunction at 2 weeks of age. By 6 weeks of age, nur7 brains have significant cell loss, white matter loss, and extensive vacuolization. Thus, the 6-week-old nur7 brain has a significantly different microenvironment from that of healthy brains, which may affect the spread and transduction of AAV / Olig001-GFP. Indeed, in a cohort of 6-week-old wild-type mice, 1 × 10 11 Administration of the 200 mg / kg dose resulted in a significant reduction in transduction levels in the cortical and subcortical white matter compared to the brains of nur7 mice (Figure 12) (n=5 animals per group, mean + / - standard error shown, *p≦0.05, **p≦0.01).

[0245] Stereological estimation of GFP-positive cells in the cortex and subcortical white matter demonstrated a significant reduction (at least 50%) in the incidence of transgene expression in wild-type brains. Strong GFP fluorescence was restricted to the area directly adjacent to the lateral ventricles, with moderate cortical and subcortical white matter GFP fluorescence signal in wild-type brains. Transgene expression in the cerebellum was poor. These data indicate genotype-specific effects on AAV / Olig001 spread and transduction efficiency. Furthermore, because nur7 brains, like human Canavan brains, have severe vacuolation, excessively large ventricles, and markedly elevated NAA, these signs and symptoms may affect vector spread and biodistribution of human AAV / Olig001 therapeutics.

[0246] Example 5: In vivo administration of AAV / Olig001-ASPA to nur7 mice improves rotarod performance method Six-week-old nur7 mice were administered a dose of AAV / Olig001-ASPA containing the codon-optimized ASPA sequence of SEQ ID NO: 2. The expression plasmid encoding the codon-optimized ASPA and regulatory elements is shown in Figure 13.11 , 7.5×10 10 or 2.5 x 10 10 A total dose of 100 mg / kg was administered via the intracerebroventricular (ICV) route of administration (ROA). All dose cohorts of vector were delivered in a total volume of 5 μl, injected into the lateral ventricles of each hemisphere of the brain in 2.5 μl volumes. A control cohort of age-matched nur7 animals was generated by injecting an equal volume of saline via the same ROA. Age-matched naive wild-type animals served as a calibration standard for all motor function tests. Two weeks after vector administration, animals were tested monthly for four months for latency to fall from an accelerating rotarod and for general activity using an open-field activity chamber. All behavioral tests were performed by individuals blinded to treatment.

[0247] result Rotarod performance AAV / Olig001-ASPA was the highest dose administered (2.5x10 11 At a dose of 100 mg / kg (vg), gradually deteriorating balance, grip strength, and / or motor coordination, as measured by rotarod performance, in nur7 mice was restored to levels indistinguishable from age-matched wild-type animals and significantly improved compared to sham nur7 controls. At this dose, the increase in rotarod performance in AAV / Olig001-ASPA-treated animals was significant over the entire study period, as determined by repeated measures ANOVA (p = 0.028), and significantly higher at each individual time point, as determined by unpaired Student's t-test. A mid-range dose (7.5 x 10 10 At the lowest dose administered (2.5 × 10 vg), AAV / Olig001-ASPA also promoted significant improvements in rotarod performance in nur7 mice at each time point tested, although this improvement was not significant over the entire study period (repeated measures ANOVA p = 0.19). 10vg), AAV / Olig001-ASPA was effective in promoting improved rotarod performance only at the last two time points tested (18 and 22 months). Table 1 shows the mean fall latency measured in seconds for each treatment group (with standard deviation). For each group, 12 mice (6 males and 6 females) were tested. Table 2 shows the p-values ​​for unpaired t-test comparisons between AAV / Olig001-ASPA-treated and sham nur7 mice at each age. 2.5 × 10 10 Statistically significant improvements were observed in all groups except for mice treated with α-glucan at 10 and 14 weeks compared to sham-treated mice. [Table 1] [Table 2]

[0248] Figure 14 shows the mean rotarod latency plotted over the lifetime study period for each AAV / Olig001-ASPA nur7 dose cohort, sham nur7, and naive wild-type controls. Fall latency was increased in all three dose cohorts, with the highest dose being significant over the entire study period by repeated measures ANOVA ( * ).

[0249] Open field activity At each age when the animals were run on the rotarod, they were also assessed for general motor function in an open-field activity chamber (Figure 15). Each time, animals were given a single 20-minute session, and the total distance traveled per session was recorded. Compared to age-matched wild-type animals, sham nur7 mice were significantly more hyperactive at all ages, especially at later time points. At 22 weeks of age, sham nur7 animals showed a significant 3-fold increase in activity (distance traveled; p=0.0202) over wild-types. In contrast, 2.5x10 11The lower 7.5 x 10 dose of AAV / Olig001-ASPA resulted in normalized activity levels in nur7 mice that were statistically significant compared to sham controls (p = 0.0312) and indistinguishable from age-matched wild-type. 10 The lowest dose of AAV / Olig001-ASPA (2.5 × 10) resulted in an activity pattern closer to wild-type than to the sham nur7 pattern, but was just below the threshold for statistical significance versus sham at 22 weeks of age (p = 0.1181). 10 ) dose did not significantly normalize pathological overactivity, which more closely resembled sham nur7 controls than wild-type references.

[0250] Assessment of open field activity in these same animals demonstrated a dose-dependent normalization of hyperactivity in AAV / Olig001-ASPA-treated nur7 animals. Data are presented as mean + / - standard error with n=6 animals per group.

[0251] NAA accumulation and vector genome (vg) copy number At 22 weeks after the rotarod test, mice were sacrificed and brain tissue was isolated. One hemisphere of each brain was processed for HPLC analysis of NAA, and the other hemisphere was processed for analysis of vector genome (vg) copy number by quantitative PCR.

[0252] Sham-saline-treated nur7 mouse brains typically contained elevated 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-treated cohort, with a highest reduction of 2.5 x 10 11 The dose was significantly reduced by 2.6-fold (p=5.06×10 -6 ), resulting in the most intermediate 7.5×10 10 The dose was reduced by 1.6 times (p=5.17×10 -5 ) yielding the lowest 2.5×10 10The dose resulted in a 1.4-fold reduction (p=0.001). NAA in nur7 brains treated with the highest dose of AAV / Olig001-ASPA was actually significantly lower than that in age-matched wild-type brains (p=0.0012).

[0253] Using the remaining hemispheres from the brains analyzed for NAA, vector genome (vg) copy number was quantified by quantitative PCR using a custom TaqMan probe / primer set targeting the bovine growth hormone (BGH) polyadenylation sequence of the recombinant AAV / Olig001-ASPA expression cassette. The total DNA content of the hemispheres was isolated using a commercially available DNA purification column and kit (Qiagen). Samples of DNA thus generated were tested against a purified plasmid standard curve to generate a vg / wet tissue weight for each sample. The resulting VG / mg tissue value reflected the administered AAV / Olig001-ASPA dose, consistent with the NAA response to vector dose (Figure 17).

[0254] Vacuolization analysis Brains from nur7 mice treated with AAV / Olig001-ASPA were analyzed by unbiased stereology to quantify vacuolar volume fraction in the thalamus and cerebellar white matter / pons as a function of vector dose (Figure 18). The area within each region of interest occupied by empty space was defined as vacuole and expressed as a percentage of the total volume of the region of interest. At each dose, AAV / Olig001-ASPA treatment significantly reduced thalamic vacuolar volume fraction (2.5 x 10) compared to sham-treated mice. 11 , p=4.6×10 -8 ;7.5×10 10 , p=6.4×10 -8 ; and 2.5 x 10 10 , p=6.2×10 -8 ) compared to sham-treated mice (Figure 19). Vacuolation in the cerebellar white matter / pons was also significantly reversed at all doses (2.5 x 10 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 extent of rescue was proportional to the dose of vector administered. 10 Dose cohorts were the highest 2.5x10 11 Dose (p=5.74×10 -6 ), but still showed a significantly decreased vacuole volume fraction compared to the sham-treated controls (p=0.0009) (FIG. 19).

[0255] Oligodendrocyte recovery The same brains analyzed for vacuolation were processed for Olig2 immunohistochemistry to identify oligodendrocytes. Both the thalamus and cortex were sampled by unbiased stereochemistry for Olig2-positive cells, identifying significant differences in resident white matter-producing cells in both areas affected and unaffected by vacuolation, respectively (Figure 20). Sham nur7 brains showed a 4.6-fold greater loss of Olig2-positive cells compared to age-matched wild-type brains, resulting in only 21% of the normal wild-type content (p=4.9x10). -7 Olig2 counts in the thalamus of AAV / Olig001-ASPA-treated and sham-treated nur7 mice (Fig. 21) were significantly higher than those in sham controls (2.5 x 10 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 (2.5 × 10) revealed a significant increase in oligodendrocytes in all three AAV / Olig001-ASPA-treated nur7 cohorts compared to wild-type mice. Olig2 loss in the cortical region was less dramatic but significant (1.7-fold reduction in sham-treated nur7 mice versus wild-type mice, p = 0.0025). 11 Olig2 content in the cortex of vg-treated nur7 brains (FIG. 21) was also significantly increased compared to sham-treated nur7 control mice (p=0.0002), but not in the brains of the two lower dose cohorts.

[0256] Neuronal recovery The thalamus and cortex were scored for NeuN-positive neurons in the same 22-week-old brains used for Olig2 staining (Figure 22). Sham-treated nur7 animals showed significantly higher levels of NeuN-positive neurons than age-matched wild-type animals (p=2.8×10 -5 ) showed a number of thalamic neurons that was approximately 35% of the 2.5 × 10 11 Nur7 mice treated with AAV / Olig001-ASPA contained a 2.3-fold increased number of thalamic neurons over sham-treated control mice (p=0.0009) and approximately 84% of the thalamic neurons observed in wild-type mice (7.5 × 10 10 and 2.5 x 10 10 At the two lower doses, AAV / Olig001-ASPA promoted an increase in thalamic NeuN-positive cells that was 1.8-fold and 1.6-fold greater than sham-treated control mice, respectively (p=0.012, p=0.042). In the cortex (motor and somatosensory), neuronal loss in sham-treated nur7 mouse brains was less severe but still significant compared to age-matched wild-type mouse brains. Cortices from 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). 11 Nur7 mice treated with AAV / Olig001-ASPA contained a number of cortical neurons that was approximately 98% of that observed in wild-type mice, a 1.2-fold increase over that observed in sham-treated nur7 mice (p=0.013). Successive doses of AAV / Olig001-ASPA resulted in a stable 1.2-fold increase in cortical neurons compared to sham treatment. 7.5 x 10 10 For dose, this increase was not significant (p=0.113) due to the high variance of the sampling data. 10 At this dose, AAV / Olig001-ASPA treated mice maintained a significant 1.2-fold increase in cortical neurons over sham-treated controls (p=0.05).

[0257] Improved myelination Using unbiased stereochemistry, we quantified cortical myelin basic protein-positive fiber length density (MBP-LD) throughout the cortex of sham-treated and AAV / Olig001-ASPA-treated 22-week-old nur7 brains, providing an index of the degree of myelination recovery after treatment with AAV / Olig001-ASPA. The motor and somatosensory cortices were sampled for MBP-positive fibers using computer-generated probes and scored for isotropic probe-fiber interactions in 3D tissue space. The sum of MBP-positive fiber lengths within the cortex was divided by the volume of sampled tissue to obtain a final MBP length density (mm). 3 The results showed that the number of fibers per μm was significantly reduced (Figure 24). When compared to age-matched wild-type brains, sham nur7 brains showed a highly significant 2-fold reduction in cortical MBPLD (p=0.0001). Treatment with AAV / Olig001-ASPA at all three doses significantly increased cortical MBP-LD compared to sham controls, and the degree of improvement was dose-proportional (2.5x10 11 p=0.0014; 7.5×10 10 p=0.003, 2.5×10 10 p=0.016). Sham-treated and AAV / Olig001-ASPA-treated nur7 mouse brains were stained with anti-myelin basic protein (MBP) (FIG. 25).

[0258] These data demonstrate that AAV / Olig001-ASPA treatment of 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 vacuolization, increases the number of Olig2- and NeuN-positive cells, and restores myelination.

[0259] Example 6: CLARITY-assisted adult distribution for Canavan gene therapy The biodistribution of an oligodendrocyte-tropic rAAV vector (Olig001) carrying a green fluorescent protein (GFP) transgene in Nur7 mouse brains exhibiting a Canavan disease phenotype was evaluated using a three-dimensional (3D) tissue sharpening and imaging method. This allowed for global depiction and volumetric measurement of vector biodistribution within the Nur7 mouse hemibrain administered via alternative routes of administration (ROA). Intracerebroventricular (ICV) and intraparenchymal (IP) ROA were compared for biodistribution efficacy, and this method was used to supplement traditional stereological data obtained from conventional two-dimensional (2D) histological evaluation.

[0260] This example demonstrates the applicability of the 3D method and its significance in assessing AAV / Olig001-GFP biodistribution in the adult mouse hemibrain of a Canavan disease mouse model. Results are presented as visual qualitative and quantitative depictions of 3D sharpened brain images from light-sheet microscopy data and tabulated parameters of biodistribution estimation.

[0261] Sample preparation and imaging Four adult mice per ROA (8 mice total) were given 5 × 10 11 Animals were given vector genomes (vg) per group and sacrificed two weeks after dosing. PFA-fixed brains were received and prepared for 3D tissue sharpening and volumetric light-sheet microscopy imaging. Each brain was bisected sagittally, and the right hemisphere was subjected to tissue sharpening using CLARITY (Chung et al., Nature, 2013). Each sample was prepared identically by hydrogel embedding and polymerization, followed by electrophoretic tissue sharpening, using commercially available reagents (Logos Biosystems) and a commercially available device (X-Clarity, Logos Biosystems). Macroscopic micrographs were taken at key steps during sample handling to document the condition of the samples (Figure 26).

[0262] Complete 3D microscopic imaging of each sharpened hemibrain was performed using a Zeiss Z.1 light-sheet microscope, utilizing a 5x magnification objective and tiling-based acquisition covering the entirety of each hemibrain. Imaging parameters were adjusted to detect GFP expression and kept constant across all samples to ensure consistency and allow for relative comparison across samples. All samples were processed and imaged under identical conditions, from tissue sharpening to image acquisition and analysis.

[0263] Image Processing and Analysis The raw datasets were preprocessed and reconstructed into complete, seamless 3D images using an in-house custom-designed algorithm for each hemibrain. The final images, each containing one hemibrain, were imported into commercial 3D image processing and analysis programs (Imaris, Bitplane) for global quantitative biodistribution analysis. First, the overall mean and median (GFP) signal values ​​within the entire hemibrain volume were calculated. Furthermore, two GFP intensity thresholds were selected to designate "low" or "high" GFP expression (Figure 27). These thresholds were then kept constant across all samples for consistency. The volumes of these classified intensity regions were then determined and compared with the entire hemibrain volume to generate the "volume % high / low expression" (Table 3).

[0264] result Macroscopic micrographs and full 3D imaging of each hemibrain revealed variable biodistribution patterns of GFP expression across the two ROAs (IP vs. ICV, Figures 28 and 30). Furthermore, cell type targeting was assessed by visual assessment of cell morphology and determining their spatial location. These biodistribution patterns differed between samples depending on the degree of vector diffusion, but similarities in subdivided transduction patterns remained consistent across samples, such as high expression in Purkinje cells within the cerebellum. Quantification of "low" and "high" GFP expression, along with overall intensity, was then calculated and tabulated for each hemibrain (Table 3). Consistent with the stereological assessment in the previous example, the enhanced hemibrain demonstrated excellent vector diffusion within the subcortical white matter after ICV injection, a critical region for Canavan disease. Furthermore, although IP injection resulted in subregions of high GFP intensity, the majority of these subregions were concentrated around the injection site, supporting the conclusions drawn from the stereological assessment. [Table 3]

[0265] Conclusions and Significance Volumetric imaging of intact, tissue-cleared mouse brains provides a more comprehensive and holistic assessment of AAV / Olig001 biodistribution. Custom algorithms that enable complete acquisition and quantification of distribution support the higher-resolution quantification obtained from stereology. Organ-level imaging assessment provides a global assessment of this biodistribution while preserving 3D spatial structural and regional connectivity. Finally, using digital compilation of various ROAs, additional evaluation can be performed on AAV / Olig001 ROAs to generate a digital "library" for future reference when assessing optimal transduction efficiency and cell-type-specific tropism.

[0266] Example 7: CLARITY-based volumetric assessment of AAV biodistribution and pharmacodynamic effects In this example, the CLARITY tissue sharpening technology described in Example 6 above was utilized to evaluate and demonstrate the global and local transgene-mediated pharmacological effects of reversal of demyelination following injection of AAV / Olig001-ASPA in the nur7 mouse brain.

[0267] Briefly, nur7 mice were divided into two groups and administered AAV / Olig001-ASPA ("Olig1" or "Olig1-ASPA") or saline ("Nur7") via ICV or IP routes in the manner described above. The brains of the two groups of mice were then analyzed to quantify the vacuolar volume fraction in the thalamus and cerebellar white matter / pons in the manner described above. Brains from wild-type mice ("WT") were also analyzed as a control. The results are shown in Figure 31. More specifically, the arrows in Figure 31B indicate that the thalamic region of nur7 mice showed visible vacuolation, which was absent in WT mice and was almost completely recaptured in Olig1-ASPA-treated tissue. Furthermore, as shown in Figure 31C, after 1 day of passive clearing, nur7 mouse tissue reached a higher transparency than both WT and Olig1-ASPA-treated tissue. These results demonstrate that AAV / Olig001-ASPA treatment reduced brain vacuolation and restored myelination in nur7 mice.

[0268] Cell counting analysis was also performed on extracted 2D single slices of 3D images from all three groups with similar anatomical orientations (Figure 32A). As shown in Figures 32B and 32C, mean nuclear density (counts normalized by segmentation area) showed little overall difference in cell density within cortical regions, but mice in the Nur7 group had significantly lower overall nuclear density / area in the thalamic region. In contrast, the Olig1-ASPA and WT groups appeared to have similar overall nuclear density or area in the thalamic region. These results demonstrate that AAV / Olig001-ASPA treatment of nur7 mice maintained or increased the number of cells in the thalamic region to levels similar to those seen in the WT group.

[0269] Brains analyzed for vacuolation were processed for immunofluorescence staining of MBP to identify oligodendrocytes in the manner described above. To this end, 3D volumetric analysis was performed to examine the pharmacodynamic treatment effects. The complete 3D volume of 2 mm tissue slices was determined, and mean fluorescence intensities were calculated for SYTO (a nuclear marker) as well as MBP. Tissues from mice in the Nur7 group were found to exhibit lower mean MBP fluorescence values. In contrast, the Olig1-ASPA-treated group had an overall increase in MBP signal, which was nearly comparable to the level of the WT group (Figure 33B).

[0270] Additional 3D volumetric analyses were performed to calculate MBP volume by signal thresholding. Thresholding was performed more restrictively, with a threshold set at a fluorescence value above 2000 (Fig. 33C, left panel), or more comprehensively, with a threshold of 1000 (Fig. 33C, right panel). In both cases, MBP deficiency was observed in Nur7 mice (Fig. 33D). In contrast, an increase in MBP volume was clearly observed in the Olig1-ASPA group, especially when a lower threshold was used, with overall MBP volume values ​​approaching the level of the WT group (Fig. 33D).

[0271] Region-based analysis was performed in 3D in the thalamic region. A manual segmentation of a portion of the region is shown in Figure 33E. The mean fluorescence intensities within this region for both nuclear (SYTO) and myelin (MBP) markers are shown in Figure 33F. SYTO and MBP levels in the Olig1-ASPA group were found to have nearly reached the levels of the WT group. In contrast, Nur7 samples showed lower mean fluorescence values ​​for both markers. Region-based analysis was also performed on a portion of the cortex. The mean fluorescence intensity levels within this cortical region for both nuclear (SYTO) and myelin (MBP) markers are shown in Figures 33G and 33H. The overall trend was similar to that shown in Figure 33F. The 3D cell concentrations (100 μm) in the cortical and thalamic regions were significantly higher than those in the WT group. 2 The 3D cell density in the thalamus region of the Olig1-ASPA mice was also obtained (nuclei per 1000 cells / 1000 cells). As shown in Figure 33I, the overall nuclear density in both regions of the Nur7 mice was lower. In contrast, the 3D cell density in the thalamus region of the Olig1-ASPA mice was close to that of the WT group.

[0272] These results demonstrate that administration of AAV / Olig001-ASPA rescued or reversed demyelination and cell loss in the nur7 mouse brain.

[0273] equivalent The foregoing written specification is deemed sufficient to enable one skilled in the art to practice the present disclosure. The foregoing description and examples set forth certain exemplary embodiments of the present disclosure in detail. However, no matter how detailed the foregoing appears in text, it should be understood that the present disclosure can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0274] All references cited herein, including patents, patent applications, articles, textbooks, and the like, and the references cited therein, to the extent they have not already been introduced, are hereby incorporated by reference in their entirety. [Table 4] TIFF0007821742000005.tif177170TIFF0007821742000006.tif233170TIFF0007821742000007.tif23317 0TIFF0007821742000008.tif28170TIFF0007821742000009.tif232170TIFF0007821742000010.tif181170 TIFF0007821742000011.tif137170TIFF0007821742000012.tif138170TIFF0007821742000013.tif21317 0TIFF0007821742000014.tif28170TIFF0007821742000015.tif232170TIFF0007821742000016.tif201170

Claims

1. 1. An isolated or modified nucleic acid encoding an aspartoacyltransferase (ASPA), comprising the nucleic acid sequence of SEQ ID NO: 2, wherein said ASPA has an enzymatic function.

2. A vector genome comprising the modified nucleic acid of claim 1.

3. 3. The vector genome of claim 2, wherein the vector genome is a recombinant adeno-associated virus (rAAV) vector genome, self-complementary, or both.

4. A vector genome according to claim 2 or 3, and BNP61, BNP62, BNP63, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAVhu. and a capsid selected from the group consisting of capsids of AAV26, 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.

5. The capsid is an amino acid sequence that is at least 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14 (BNP61); an amino acid sequence that is at least 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15 (BNP62), and An amino acid sequence that is at least 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16 (BNP63) 5. The rAAV vector of claim 4, comprising an amino acid sequence selected from the group consisting of:

6. Features include: the capsid is a BNP61, BNP62, or BNP63 capsid; the vector genome is self-complementary; 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; 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 hybrid form of the CBA promoter (CBh promoter), a chicken B-actin (CBA) exon, a CBA intron, a minute virus of mice (MVM) intron, and a bovine growth hormone (BGH) polyA; and the vector genome further comprises at least one element selected from the group consisting of at least one ITR comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12 or SEQ ID NO:19, an enhancer comprising the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:17, a promoter comprising the nucleic acid sequence of SEQ ID NO:7, an exon comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18, an intron comprising the nucleic acid sequence of SEQ ID NO:9, an intron comprising the nucleic acid sequence of SEQ ID NO:10, and a polyA comprising the nucleic acid sequence of SEQ ID NO:11; 6. The rAAV vector of claim 5, comprising one or more of:

7. The rAAV vector of claim 6, wherein the capsid is a BNP61 capsid comprising VP1, and the VP1 comprises the amino acid sequence of SEQ ID NO:

14.

8. From 5' to 3', a) an AAV inverted terminal repeat (ITR) comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19; b) an enhancer comprising the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17; c) a promoter comprising the nucleic acid sequence of SEQ ID NO: 7; d) an exon comprising the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 18; e) an intron comprising the nucleic acid sequence of SEQ ID NO: 9; f) an intron comprising the nucleic acid sequence of SEQ ID NO: 10; g) a modified nucleic acid encoding aspartoacyltransferase (ASPA) comprising the nucleic acid sequence of SEQ ID NO: 2; h) polyA comprising the nucleic acid sequence of SEQ ID NO: 11, and i) an AAV ITR comprising the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:12, or SEQ ID NO:19; 1. An rAAV vector comprising a vector genome comprising:

9. The rAAV vector described in Claim 8, wherein the vector comprises a BNP61 capsid containing viral protein 1 (VP1), and the VP1 comprises the amino acid sequence of SEQ ID NO:

14.

10. A pharmaceutical composition comprising the rAAV vector of any one of claims 4 to 9.

11. A host cell comprising the isolated or modified nucleic acid of claim 1, the vector genome of claim 2 or 3, or the rAAV vector of any one of claims 4 to 9.

12. Features include: The cells are selected from the group consisting of VERO, WI38, MRC5, A549, HEK293, B-50, HeLa cells, HepG2, Saos-2, HuH7, and HT1080; and the cell comprises at least one nucleic acid encoding at least one protein selected from the group consisting of AAV rep protein, AAV capsid (Cap) protein, adenovirus (Ad) early region 1A (E1a) protein, Ad E1b protein, Ad E2a protein, Ad E4 protein, and virus-associated (VA) RNA; The host cell of claim 11 , comprising:

13. The host cell of claim 12, wherein the cell is a HEK293 cell.

14. 14. The host cell of claim 13, wherein the cell is a HEK293 cell having American Type Culture Collection (ATCC) number PTA13274.

15. 11. A kit for the treatment of Canavan disease (CD), comprising a therapeutically effective amount of the isolated or modified nucleic acid of claim 1, the vector genome of claim 2 or 3, the rAAV vector of any one of claims 4 to 9, or the pharmaceutical composition of claim 10, and optionally further comprising a label or package insert containing instructions for using one or more of the kit components.

16. 11. The isolated or modified nucleic acid of claim 1, the vector genome of claim 2 or 3, the rAAV vector of any one of claims 4 to 9, or the pharmaceutical composition of claim 10, for use in the treatment or prevention of a disease, disorder, or condition associated with ASPA deficiency or dysfunction.

17. 17. The isolated nucleic acid, modified nucleic acid, vector genome, rAAV vector, or pharmaceutical composition for use according to claim 16, wherein the disease, disorder, or condition is Canavan disease.

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