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  • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
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  • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
  • C12Y305/01015—Aspartoacylase (3.5.1.15)
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  • C12N2750/00011—Details
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

• the 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 use of the vectors in the treatment of diseases, disorders and conditions associated with a decreased level of functional ASPA including diseases, disorders and conditions associated with diminished cellular catabolism of N-acetyl-L- aspartic acid, for example Canavan disease.
• ASPA aspartoacylase
• Canavan disease is associated with reduction of expression from and/or mutation of the ASPA gene that encodes the enzyme aspartoacylase (ASPA) (also known as aminoacylase 2).
• ASPA enzyme aspartoacylase
• Decreased aspartoacylase activity results in accumulation of N- acetylaspartate (NAA) (also known as N-acetyl-L-aspartic acid) due to decreased conversion of NAA to aspartate and acetate.
• NAA N- acetylaspartate
• the ASPA enzyme has been implicated in maintenance of metabolic integrity of myelinating cells.
• ASPA gene expression is restricted primarily to white matter producing oligodendrocytes. Accumulation of NAA in the brain is associated with oligodendrocyte dysfunction and interference with development of the myelin sheath and destruction of existing myelin sheath associated with neurons.
• CD is an autosomal recessive genetic disease and manifests primarily in a neonatal/infantile form. Children who are affected with this form present in infancy with symptoms associated with degeneration of myelin 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 for children with the neonatal/infantile of CD. Individuals with the mild/juvenile form of CD may exhibit delayed development of speech and motor skills and have an average lifespan. [0005] To date, no treatment exists for stopping or slowing neurodegenerative effects of CD. Current therapeutic approaches in clinical use, or under evaluation, are directed to alleviating symptoms and maximizing quality of life. Physical therapy, feeding tubes and anti-seizure medication may be used to treat some symptoms and improve quality of life. Thus, there is an important need for a novel therapeutic approach to treat CD.
• modified nucleic acids encoding aspartoacylase (ASP A) and vectors (e.g., rAAV vector) comprising a modified nucleic acid and methods of treating a disease, disorder or condition mediated by a decreased level of ASPA protein by administering a modified nucleic acid, or a vector comprising a modified nucleic acid, to a patient in need thereof.
• vectors e.g., rAAV vector
• ASPA aspartoacyltransferase
• ASPA aspartoacyltransferase
• ASPA aspartoacyltransferase
• ASPA aspartoacyltransferase
• ASPA aspartoacyltransferase
• E6 An isolated nucleic acid encoding aspartoacyltransferase (ASPA) comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:3.
• a modified nucleic acid encoding aspartoacyltransferase (ASP A) 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:2.
• a modified nucleic acid encoding aspartoacyltransferase comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:2.
• a modified nucleic acid encoding aspartoacyltransferase 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: 1.
• a modified nucleic acid encoding aspartoacyltransferase comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:l.
• Ell. A modified nucleic acid encoding aspartoacyltransferase 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:3.
• a modified nucleic acid encoding aspartoacyltransferase comprising a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO:3.
• a recombinant nucleic comprising a modified nucleic acid encoding aspartoacyltransferase (ASP A) 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:2.
• ASP A aspartoacyltransferase
• a recombinant nucleic comprising a modified nucleic acid encoding aspartoacyltransferase (ASP A) comprising or consisting of the nucleic acid sequence of SEQ ID NO:2.
• a recombinant nucleic comprising a modified nucleic acid encoding aspartoacyltransferase (ASP A) 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:l.
• ASP A aspartoacyltransferase
• a recombinant nucleic comprising a modified nucleic acid encoding aspartoacyltransferase (ASP A) comprising or consisting of the nucleic acid sequence of SEQ ID NO:l.
• ASP A aspartoacyltransferase
• a recombinant nucleic comprising a modified nucleic acid encoding aspartoacyltransferase (ASP A) 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:3.
• ASP A aspartoacyltransferase
• a recombinant nucleic comprising a modified nucleic acid encoding aspartoacyltransferase (ASP A) comprising or consisting of the nucleic acid sequence of SEQ ID NO:3.
• ASP A aspartoacyltransferase
• E19 The recombinant nucleic of any one of E13-E18 further comprising at least one element selected from the group consisting of an enhancer, a promoter, an exon, an intron, and a poly-adenylation (polyA) signal sequence.
• E20 The recombinant nucleic 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 the nucleic acid sequence of SEQ ID NO: 6, SEQ ID NO: 17 or both.
• E21 The recombinant nucleic of any one of E19-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 of any one of E19-E21 wherein the promoter is constitutive or regulated.
• E23 The recombinant nucleic of any one of E19-E22 wherein the promoter is inducible or repressible.
• E24 The recombinant nucleic of any one of E19-E23 wherein the promoter 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 the nucleic acid sequence of SEQ ID NO:7.
• E25 The recombinant nucleic of any one of E19-E24 wherein the promoter comprises or consists of the nucleic acid sequence of SEQ ID NO:7.
• E26 The recombinant nucleic of any one of E19-E25 wherein the exon 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 the nucleic acid sequence of SEQ ID NO:8, SEQ ID NO: 18 or both.
• E27 The recombinant nucleic of any one of E19-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 of any one of E19-E27 wherein the intron 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 the nucleic acid sequence of SEQ ID NO:9, SEQ ID NO: 10 or both.
• E29 The recombinant nucleic of any one of E19-E28 wherein the intron comprises or consists of the nucleic acid sequence of SEQ ID NO:9, SEQ ID NO: 10 or both.
• E30. The recombinant nucleic of any one of E19-E29 wherein the polyA sequence 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 the nucleic acid sequence of SEQ ID NO: 11.
• E31 The recombinant nucleic of any one of E19-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 the enhancer is operably linked to the modified nucleic acid.
• E33 The recombinant nucleic acid of any one of E19-E32 wherein the promoter is operably linke to the modified nucleic acid.
• E34 The recombinant nucleic of any one of E13-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 b-actin (CBA) exon, a CBA intron, a minute virus of mice (MVM) intron and a bovine grown hormone (BGH) polyA.
• CMV cytomegalovirus
• CBh promoter hybrid form of the CBA promoter
• CBA chicken b-actin
• VMM minute virus of mice
• BGH bovine grown hormone
• E35 The recombinant nucleic of any one of E13-E18 further comprising a 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.
• 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:
• E36 A vector genome comprising a modified nucleic acid of any one of E7-E12 or a recombinant nucleic acid of any one of E13-E35 wherein the vector genome further comprises 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 the 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 a nucleic acid sequence encoding ASPA and a CBh promoter upstream of the sequence encoding the ASPA.
• E39 The vector genome of any one of E36-E38 wherein the ASPA sequence comprises the nucleic acid sequence of SEQ ID NO:2.
• E40 The vector genome of any one of E36-E39 wherein the 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-E40 wherein the CBh promoter comprises the nucleic acid sequence of SEQ ID NO:7.
• a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 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, preferabley 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 (ASP A) comprising the nucleic acid sequence
• a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 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 (ASP A) comprising the nucleic acid sequence of any one of SEQ ID NO: 1-3;
• E44 The vector genome of any one of E36-43, wherein the vector genome is self- complementary.
• E45 A recombinant adeno-associated virus (rAAV) vector comprising the vector genome of any one of E36-E44 and a capsid.
• rAAV adeno-associated virus
• An rAAV vector comprising a vector genome comprising a nucleic acid sequence 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.
• the rAAV vector of E46 comprising a capsid selected from the group consisting of a capsid of OligOOl, Olig002, Olig003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV Hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2- TT, AAV2-TT-S312N, AAV3B-S312N, and AAV-LK03.
• a capsid selected from the group consisting of a capsid of OligOOl, Olig002, Olig003, AAV
• An rAAV vector comprising a vector genome comprising a nucleic acid sequence 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.
• the rAAV vector of E48 comprising a capsid selected from the group consisting of a capsid of OligOOl, Olig002, Olig003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV Hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2- TT, AAV2-TT-S312N, AAV3B-S312N, and AAV-LK03.
• a capsid selected from the group consisting of a capsid of OligOOl, Olig002, Olig003, AAV
• An rAAV vector comprising a vector genome comprising a nucleic acid sequence 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.
• the rAAV vector of E50 comprising a capsid selected from the group consisting of a capsid of OligOOl, Olig002, Olig003, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV Hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2- TT, AAV2-TT-S312N, AAV3B-S312N, and AAV-LK03.
• a capsid selected from the group consisting of a capsid of OligOOl, Olig002, Olig003, AAV
• E52 The rAAV vector of any one of E45-E51 wherein the capsid is selected from an OligOOl, an Olig002 and an Olig003 capsid.
• E53 The rAAV vector of any one of E45-E52 wherein the capsid is an OligOOl capsid comprising a viral protein 1 (VPl) and wherein the VPl 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-E53 wherein the capsid is an OligoOOl capsid comprising a viral protein 1 (VP1) and wherein the VP1 comprises the amino acid sequence of SEQ ID NO: 14.
• E55 The rAAV vector of any one of E45-E52 wherein the capsid is an Olig002 capsid comprising a viral protein 1 (VP1) and wherein the 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.
• the capsid is an Olig002 capsid comprising a viral protein 1 (VP1) and wherein the 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-E52 and E55 wherein the capsid is an Oligo002 capsid comprising a viral protein 1 (VPl) and wherein the VP1 comprises the amino acid sequence of SEQ ID NO : 15.
• the capsid is an Oligo002 capsid comprising a viral protein 1 (VPl) and wherein the VP1 comprises the amino acid sequence of SEQ ID NO : 15.
• E57 The rAAV vector of any one of E45-E52 wherein the capsid is an Olig003 capsid comprising a viral protein 1 (VPl) and wherein the VPl 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.
• the capsid is an Olig003 capsid comprising a viral protein 1 (VPl) and wherein the VPl 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-E52 and E57 wherein the capsid is an Oligo003 capsid comprising a viral protein 1 (VPl) and wherein the VPl comprises the amino acid sequence of SEQ ID NO: 16.
• the capsid is an Oligo003 capsid comprising a viral protein 1 (VPl) and wherein the VPl comprises the amino acid sequence of SEQ ID NO: 16.
• E59 The rAAV vector of any one of E45-E58 wherein the vector genome is self complementary.
• E60 The rAAV vector of any one of E46-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, an enhancer, a promoter, an exon, an intron, and a poly-adenylation (poly A) signal sequence.
• ITR AAV inverted terminal repeat
• poly A poly-adenylation
• 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 The rAAV vector of any one of E60-E62 wherein the promoter is constitutive or regulated.
• E64 The rAAV vector of any one of E60-E63 wherein the promoter is inducible or repressible.
• E65 The rAAV vector of any one of E60-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-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-E66 wherein the exon 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: 8 or SEQ ID NO: 18.
• E68 The rAAV vector of any one of E60-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-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-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-E70 wherein the polyA 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: 11.
• E72 The rAAV vector of any one of E60-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-E72 wherein the 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-E73 wherein the 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-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.
• 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 The rAAV vector of any one of E77-E79 wherein the CBh promoter comprises the nucleic acid sequence of SEQ ID NO:7.
• An rAAV vector comprising a vector genome comprising 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: 16; c) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:7; d) a CBA exon 1 comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18; e) a 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 the nucleic acid sequence of any one of SEQ ID NO:
• An rAAV vector comprising a vector genome comprising 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 the nucleic acid sequence of any one of SEQ ID NO: 1-3; h) a PolyA comprising the nucleic acid sequence of SEQ ID
• E84 The rAAV vector of any one of E81-E83 wherein the vector comprises an OligOOl capsid comprising a VP1 protein wherein the 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-E83 wherein the vector comprises an Olig002 capsid comprising a VP1 proetin wherein the 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 comprising a VP1 protein wherein the 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.
• An rAAV vector comprising i) an OligOOl capsid comprising a VP1 protein wherein the VP1 comprises the amino acid sequence of SEQ ID NO: 14 and ii) a self-complementary vector genome comprising 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) a CBA exon 1 comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:18; e) a 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;
• An rAAV vector comprising i) an OligOOl capsid comprising a VP1 protein wherein the VP1 comprises the amino acid sequence of SEQ ID NO: 14 and ii) a self-complementary vector genome comprising 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 aspartoacyl
• E89 The rAAV vector of any one of E45-E88 wherein the vector, when introduced into a cell, decreases the level of NAA in the cell.
• E90 The rAAV vector of E89 wherein the cell is a brain cell.
• E91 The rAAV vector of E89 or E90 where the cell is an oligodendrocyte.
• E92 The rAAV vector of any one of E45-E91 wherein administration of the vector to a subject with an ASPA gene mutation increases balance, grip strength and/or motor coordination in the subject as compared to balance, grip strength and/or motor coordination in the subject before administration of the vector.
• E93 The rAAV vector of any one of E45-E92 wherein administration of the vector to a subject with an ASPA gene mutation increases generalized motor function in the subject as compared to generalized motor function in the subject before administration of the vector.
• E94 The rAAV vector of any one of E45-E93 wherein administration of the vector to a subject with an ASPA gene mutation decreases NAA levels in the subject as compared to NAA levels in the subject before administration of the vector.
• E95 The rAAV vector of any one of E45-E94 wherein administration of the vector to a subject with an ASPA gene mutation decreases vacuole volume fraction in the thalamus of the subject as compared to vacuole volume fraction in the thalamus of the subject before administration of the vector.
• E96 The rAAV vector of any one of E45-E95 wherein administration of the vector to a subject with an ASPA gene mutation decreases vacuole volume fraction in the cerebellar white matter/pons of the subject as compared to vacuole volume fraction in the cerebellar white matter/pons of the subject before administration of the vector.
• E97 The rAAV vector of any one of E45-E96 wherein administration of the vector to a subject with an ASPA gene mutation increases the number of oligodendrocytes in the thalamus of the subject as compared to the number of oligodendrocytes in the thalamus of the subject before administration of the vector.
• E98 The rAAV vector of any one of E45-E97 wherein administration of the vector to a subject with an ASPA gene mutation increases the number of oligodendrocytes in the brain cortex of the subject as compared to the number of oligodendrocytes in the brain cortex of the subject before administration of the vector.
• E99 The rAAV vector of any one of E45-E98 wherein administration of the vector to a subject with an ASP A gene mutation increases the number of neurons in the thalamus of the subject as compared to the number of neurons in the thalamus of the subject before administration of the vector.
• E100 The rAAV vector of any one of E45-E99 wherein administration of the vector to a subject with an ASP A gene mutation increases the number of neurons in the brain cortex of the subject as compared to the number of neurons in the brain cortex of the subject before administration of the vector.
• E101 The rAAV vector of any one of E45-E100 wherein administration of the vector to a subject with an ASP A gene mutation increases cortical myelination in the subject as compared to cortical myelination in the subject before administration of the vector.
• E102 The rAAV vector of any one of E92-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 with Canavan disease, 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.
• a pharmaceutical composition comprising the modified nucleic acid of any one of E7-E12, the recombinant nucleic acid of any one of E13-E35, the vector genome of any one of E36-E44 or the rAAV vector of any one of E45-E104.
• a pharmaceutical composition comprising the modified nucleic acid of any one of E7-E12, the recombinant nucleic acid of any one of E13-E35, the vector genome of any one of E36-E44 or the rAAV vector of any one of E45-E104 and a pharmaceutically acceptable carrier.
• E107 A method of treating and/or preventing a disease, disorder or condition associated with deficiency or dysfunction of ASPA, the method comprising administering a therapeutically effective amount of the modified nucleic acid of any one of E7-E12, the recombinant nucleic acid of any one of E13-E35, the vector genome of any one of E36-E44, the rAAV vector of any one of E45-E104 or the pharmaceutical composition of E105 or E106 to a subject in need of treatment.
• E108 The method of E107 wherein the disease, disorder or condition associated with deficiency or dysfunction of ASPA 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.
• El 11 The method of any one of E107-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 the brain parenchyma, spinal canal, subarachnoid space, a ventricle of the brain, cistema magna and any combination thereof.
• a method of treating or preventing Canavan disease comprising the steps of: i) assessing whether a subject comprises at least one ASPA gene mutation and ii) administering to the subject a therapeutically effective amount of the modified nucleic acid of any one of E7-E12, the recombinant nucleic acid of any one of E13-E35, the vector genome of any one of E36-E44, the rAAV vector of any one of E45-E104 or the pharmaceutical composition of E105 or E106, thereby treating or preventing Canavan disease in the subject.
• a method of 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 has been codon-optimized.
• E121 The method of any one of El 18-E120 wherein the modified modified nucleic acid encoding ASPA is expressed in a target cell and wherein the target cell is an oligodendrocyte.
• E122 The method of any one of El 18-E121 wherein the modified nucleic acid encoding ASPA is delivered in a vector to the target cell.
• El 23 The method of El 22, wherein the vector is a viral vector or a non-viral vector.
• El 24 The method of any one of El 18-El 23 wherein the vector is administered to the subject by systemic injection, by direct intracranial injection or by direct spinal canal injection.
• E125 A host cell comprising the modified nucleic acid of any one of the modified nucleic acid of any one of E7-E12, the recombinant nucleic acid of any one of E13-E35, the vector genome of any one of E36-E44 or the rAAV vector of any one of E45-E104.
• E126 The host cell of E125, wherein the cell is selected from the group consisting of VERO, WI38, MRC5, A549, HEK293, B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT 1080.
• E127 The host cell of E125-E126 wherein the cell is a HEK293 cell adapted to growth in suspension culture.
• E128 The host cell of any one of E125-E127 wherein the cell is a HEK293 cell having American Type Culture Collection (ATCC) No. PTA 13274.
• ATCC American Type Culture Collection
• E129 The host cell of any one of E125-E128 wherein the cell comprises at least one nucleic acid encoding at least one protein selected from the group consisting of an AAV Rep protein, an AAV capsid (Cap) protein, a adenovirus early region 1 A (Ela) protein, a Elb protein, an E2a protein, an E4 protein and a viral associated (VA) RNA.
• AAV Rep protein an AAV capsid protein
• Ela adenovirus early region 1 A
• Elb Elb protein
• E2a protein an E4 protein
• VA viral associated
• kit of E130 wherein the kit further comprises a label or insert including instructions for using one or more of the kit components.
• E132 A modified nucleic acid of any one of E7-E12, a recombinant nucleic acid of any one of E13-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 treating or preventing a disease, disorder or condition associated with deficiency or dysfunction of ASPA.
• E133 The modified nucleic acid, the recombinant nucleci acid, the vector genome, the rAAV vector, or the pharmaceutical composition for use of El 32, wherein the disease, disorder or condition is Canavan disease.
• E134 Else of a modified nucleic acid of any one of E7-E12, a recombinant nucleic acid of any one of E13-E35, a vector genome of any one of E36-E44, an rAAV vector of any one of E45-E104 or a pharmaceutical composition of El 05 or El 06 in the manufacture of a medicament for treating and/ or preventing a disease, disorder of condition associated with deficiency or dysfunction of ASPA.
• E135. The use of E134 wherein the disease, disorder or condition is Canavan disease.
• E136 A method of determining biodistribution of a transgene delivered by an rAAV vector comprising an OligOOl capsid to the brain of a subject wherein a protein encoded by the transgene is expressed, the method comprising a) administration of the rAAV vector to the subject; b) fixation of the brain tissue; c) electrophoretic clearing of the brain; d) 3D microscopic imaging of a brain tissue section; e) detection of the protein; f) optionally, quantification of the amount of protein present in the brain tissue.
• E137 The method of E136 wherein the administration is by intracrebroventricular (ICV) injection, intraparenchymal (IP) injection, intrathecal (IT) administration, intracisternal magna (ICM) injection or a combination thereof.
• ICM intracisternal magna
• E138 The method of E136 or 137 wherein the brain tissue is fixed using, for example, paraformaldehyde or formalin.
• E139 The method of any one of E136-E138 wherein the quantification includes volumetric rendering.
• 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-E141, further comprising (g) the step of evaluation of cell-type vector tropism by assessment of cell morphology and spatial location determination of GFP expression.
• a modified nucleic acid encoding aspartoacyltransferase 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 any one of SEQ ID NO: 1-3 and a promoter.
• a modified nucleic acid encoding aspartoacyltransferase comprising a nucleic acid comprising or consisting of the sequence of SEQ ID NO:2 and a promoter.
• a nucleic acid comprising a nucleic acid sequence encoding a promoter, and further comprising a modified nucleic acid sequence encoding ASP A, 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.
• FIG. 1 depicts an exemplary dose response reduction in NAA as determined using HPLC in cells transfected with 1.0 pg plasmid expressing NAA synthase (Nat8L) and co transfected with 1.0 pg, 0.5 pg, 0.2 pg or 0.1 pg of a plasmid comprising 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:l) or codon-optimized ASPA sequence new (version 2) (SEQ ID NO:2).
• FIG. 2 depicts exemplary sampling of GFP positive cells transduced by rAAV vector administered via the intraparenchymal (IP) route of administration (ROA).
• IP intraparenchymal
• ROA route of administration
• FIG. 3 depicts an exemplary number of GFP-positive cells (N) in the cortex, subcortical white matter of the corpus callosum and external capsule, striatum and cerebellum of 6 week old nur7 mice following intraparenchymal (IP) administration of AAV/OligOOl- GFP and a representative image of native GFP fluorescence in a sagittal section of a brain from a mouse to which lx 10 11 AAV/OligOOl-GFP vector genomes were administered via the IP ROA showing concentrated GFP expression adjacent to injection sites.
• IP intraparenchymal
• FIG. 4 depicts an exemplary number of GFP-positive cells (N) in the cortex, subcortical white matter, striatum and cerebellum of 6 week old nur7 mice following intrathecal (IT) administration of AAV/OligOOl-GFP and a representative image of native GFP fluorescence in a sagittal section of a brain from a mouse to which lxlO 11 AAV/OligOOl-GFP vector genomes were administered via the intrathecal (IT) ROA showing diffuse cortical marker expression demonstrating transduction by the vector and modest white matter tract cell expression also demonstrating transduction of cells in that region.
• I intrathecal
• FIG. 5 depicts an exemplary number of GFP-positive cells (N) in the cortex, subcortical white matter, striatum and cerebellum in 6 week old nur7 mice following intracerebroventricular (ICV) administration of AAV/OligOOl-GFP and a representative image of native GFP fluorescence in a sagittal section of a brain of a mouse to which lxlO 11 AAV/OligOOl-GFP vector genomes were administered via the ICV ROA showing intense white matter tract GFP expression demonstrating transduction by the vector of cells in that region.
• ICV intracerebroventricular
• FIG. 6 depicts an exemplary number of GFP-positive cells (TV) in the cortex, subcortical white matter, striatum and cerebellum in 6 week old nur7 mice following intracistemal magna (ICM) administration of AAV/OligOOl-GFP and a representative image of native GFP fluorescence in a sagittal section of a brain from a mouse to which lxlO 11 AAV/OligOOl-GFP vector genomes were administered via the ICM ROA showing modest white matter tract GFP marker expression demonstrating transduction of cells in that region.
• Mean +/- sem for each group presented (n 5 animals). Significant differences in numbers of GFP-positive cells between dose cohorts within each region of interest are denoted by asterisks.
• FIG. 8 depicts exemplary oligotropism of AAV/OligOOl-GFP in the cortex of 6 week old nur7 mice following intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV) and intracistemal magna (ICM) vector administration.
• IP intraparenchymal
• IT intrathecal
• ICM intracerebroventricular
• ICM intracistemal magna
• FIG. 10 depicts exemplary oligotropism of AAV/OligOOl-GFP in the striatum matter of 6 week old nur7 mice following intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV) and intracistemal magna (ICM) vector administration where marker detection demonstrates transduction of cells by the vector.
• FIG. 11 depicts exemplary oligotropism of AAV/OligOOl-GFP in the cerebellum of 6 week old nur7 mice following intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV) and intraci sternal magna (ICM) vector administration where marker detection demonstrates transduction by the vector.
• FIG. 12 depicts exemplary efficiency of AAV/OligOOl-GFP transduction in the cortex and subcortical white matter of age-matched wild type (WT) and nur7 mouse brains 2 weeks-post ICV administration of lxlO 11 vector genomes and a representative image of native GFP fluorescence in a wild type brain following administration of AAV/OligOOl-GFP, showing relatively restricted expression, and thereby demonstrating transduction by the vector, particularly in subcortical white matter.
• WT age-matched wild type
• FIG. 12 depicts exemplary efficiency of AAV/OligOOl-GFP transduction in the cortex and subcortical white matter of age-matched wild type (WT) and nur7 mouse brains 2 weeks-post ICV administration of lxlO 11 vector genomes and a representative image of native GFP fluorescence in a wild type brain following administration of AAV/OligOOl-GFP, showing relatively restricted expression, and thereby demonstrating transduction by the vector, particularly in subcortical white matter.
• FIG. 13 depicts an expression plasmid encoding a codon-optimized ASPA coding sequence and regulatory elements.
• FIG. 18 depicts representative H&E stained brain sections from nur7 sham treated, AAV/OligOOl-ASPA treated nur7 and wild type mice demonstrating areas of vacuolation.
• FIG. 19 depicts exemplary vacuole volume fraction as a percentage of region of interest (ROI) of the thalamus and cerebral white matter/pons of brains from 22 week old sham treated and AAV/OligOOl-ASPA treated nur7 mice. Asterisks indicate a significant difference between groups.
• ROI region of interest
• FIG. 20 depicts representative images of sham treated and AAV/OligOOl-ASPA treated (2.5x10 11 vg dose) nur7 mouse thalamus and cortex stained for 01ig2 demonstrating oligodendrocytes.
• FIG. 22 depicts representative images of sham treated, and AAV/OligOOl-ASPA treated (2.5xlO u vg dose) nur7 mouse thalamus and cortex stained for NeuN.
• FIG. 24 depicts representative images of sham treated and AAV/OligOOl-ASPA treated (2.5xlO u vg dose) nur7 mouse cortex stained for myelin basic protein (MBP).
• MBP myelin basic protein
• MBP-LD myelin basic protein positive fiber length density
• FIG. 26 depicts exemplary brain images from an ICV injected mouse from an initial fixed, pre-cleared sample, a post-tissue cleared sample, a 3D GFP fluorescence image, a hemibrain volumetric segmentation analysis and an intensity heatmap (left to right).
• FIG. 27 depicts intensity heatmaps from all four ICV injected hemibrains. Full hemibrain volume is calculated and represented as gray areas. Calculated “low” GFP intensity is indicated in the gray areas; “high” GFP intensity is indicated in the white areas.
• FIG. 28 depicts 3D lightsheet GFP fluorescence microscopy images from cleared brains of animals administered AAV/OligoOOl-GFP via ICV versus IP routes of administration.
• FIG. 29A depicts representative high magnification images showing scoring of GFP-positive cells co-labelled with 01ig2 or NeuN. Total GFP cells were scored in each field of view, and the percentage of 01ig2 and NeuN co-labelling scored within the same field.
• FIG. 29B depicts representative images of co-labelling of GFP with 01ig2 in SCWM tract cells in the brain of an animal given AAV/OligOOl-GFP via the ICV ROA and demonstrating near 100% oligotropism and a near complete absence of neurotropism.
• FIG. 29C depicts a representative image of cerebellar GFP transgene expression in large purkinje neurons, with sparse 01ig2 co-labelling in white matter (arrow).
• FIG. 29D depicts a representative image of GFP co-labeling with 01ig2 in the striatum of an ICV ROA brain, showing contrast with cerebellar tropism.
• FIG. 29E depicts representative images of white matter tracts in 8-week nur7 and age-matched wild type naive brains after processing for BrdU labeling and 01ig2.
• FIG. 29G depicts representative images of BrdU/GFP co-labelled cells in subcortical white matter of a nur7 brain treated with AAV/OligOOl-GFP via the ICV ROA.
• FIGs. 30A, 30B, and 30C depict biodistribution volumetric analysis.
• FIGs. 31A, 31B, and 31C depict CLARITY and SWITCH workflow for pharmacodynamics effect evaluation.
• A Tissue clearing and labeling approach. From left to right: an intact mouse brain, a central 2-mm section of right hemibrain prior to clearing, the same tissue after 1 day of passive clearing and after 3 days of passive clearing, and a 3D image displaying fluorescence signal from previously labeled proteins (green: nuclei, red: myelin basic protein (MBP).
• B Representative 2-mm sections of Nur7, WT and Oligl- ASPA treated tissues. Red arrowhead in each image indicates the thalamic region.
• C Tissue transparency after one day of passive clearing.
• FIGs. 32A, 32B, and 32C depict 2D region-based cell counting of tissues.
• A Extracted 2D single slices of 3D images from all three groups with similar anatomical orientation. Red boxes mark areas in the thalamic and cortical region where cell counting was performed.
• B Image data enlarged from the red boxes in (A), and respective cell segmentation.
• C Average nuclei density (counts normalized by segmentation area).
• FIGs. 33 A, 33B, 33C, 33D, 33E, 33F, 33G, 33H, and 331 depict 3D volumetric analysis of pharmacodynamic treatment effect.
• A A full 3D volume of a 2-mm tissue slice is determined.
• B The average fluorescence intensity calculated within the 3D volume.
• C MBP characterization via a more restrictive threshold set at fluorescence value of over 2000 (left panel) or a more inclusive threshold at 1000 (left panel).
• D In both cases, MBP deficit in Nur7 can be observed. An effect of the Oligl-ASPA group can be seen in the lower threshold, where the overall value approaches WT levels.
• E Region-based 3D analyses in the thalamic region where a manual segmentation of a portion of the region is shown in yellow.
• F Average fluorescence within this region for both nuclei (SYTO) and myelin (MBP) markers.
• G Region-based analysis on a portion of the cortex where the manual segmentation is shown in yellow.
• H Average fluorescence within this cortical region for both nuclei (SYTO) and myelin (MBP) markers.
• I 3D cell concentration (nuclei per 100 um 2 ).
• the term “about,” or “approximately” refers to a measurable value such as an amount of the biological activity, length of a polynucleotide or polypeptide sequence, content of G and C nucleotides, codon adaptation index, number of CpG dinucleotides, dose, time, temperature, and the like, and is meant to encompass variations of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% 1%, 0.5% or even 0.1%, in either direction (greater than or less than) of the specified amount unless otherwise stated, otherwise evident from the context, or except where such number would exceed 100% of a possible value.
• the terms “adeno-associated virus” and/or “AAV” refer to a parvovirus with a linear single-stranded DNA genome and variants thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise.
• the wild-type genome comprises 4681 bases (Berns and Bohenzky (1987) Advances in Vims Research 32:243-307) and includes terminal repeat sequences (e.g., inverted terminal repeats (ITRs)) at each end which function in cis as origins of DNA replication and as packaging signals for the vims.
• ITRs inverted terminal repeats
• AAV rep and cap may also be referred to herein as AAV “packaging genes.” These genes code for the viral proteins involved in replication and packaging of the viral genome.
• VPI is the full- length protein, with VP2 and VP3 being increasingly shortened due to increasing tmncation of the N-terminus.
• a well-known example is the capsid of AAV9 as described in U.S. Patent No. 7,906,111, wherein VPI comprises amino acid residues 1 to 736 of SEQ ID NO: 123,
• VP2 comprises amino acid residues 138 to 736 of SEQ ID NO: 123
• VP3 comprises amino acid residues 203 to 736 of SEQ ID NO: 123.
• AAV Cap or “cap” refers to AAV capsid proteins VPI, VP2 and/or VP3, and variants and analogs thereof.
• At least four viral proteins are synthesized from the AAV rep gene, Rep 78, Rep 68, Rep 52 and Rep 40, and are named according to their apparent molecular weights.
• AAV rep” or “rep” means AAV replication proteins Rep 78, Rep 68, Rep 52 and/or Rep 40, as well as variants and analogs thereof.
• rep and cap refer to both wild type and recombinant (e.g., modified chimeric, and the like) rep and cap genes as well as the polypeptides they encode.
• a nucleic acid encoding a rep will comprise nucleotides from more than one AAV serotype.
• a nucleic acid encoding a rep may comprise nucleotides from an AAV2 serotype and nucleotides from an AA3 serotype (Rabinowitz et al. (2002) J. Virology 76(2):791-801).
• rAAV recombinant adeno-associated virus vector
• rAAV vector refers to an AAV comprising a vector genome wherein a polynucleotide sequence not of, or not entirely of, AAV origin (e.g., a polynucleotide heterologous to AAV), and wherein the rep and/or cap genes of the wild type AAV virus genome have been removed from the virus genome.
• the nucleic acid within the AAV is referred to as the “vector genome.”
• the term rAAV vector encompasses an rAAV viral particle that comprises a capsid and a heterologous nucleic acid, i.e., a nucleic acid not originally present in the capsid in nature, and hereinafter referred to as a “vector genome.”
• a “rAAV vector genome” refers to a heterologous polynucleotide sequence (including at least one ITR, typically, but not necessarily, an ITR not associated with the original nucleic acid present in the original AAV) that may, but need not, be
• an rAAV vector genome may be double-stranded (dsAAV), single-stranded (ssAAV) and/or self-complementary (scAAV).
• dsAAV double-stranded
• ssAAV single-stranded
• scAAV self-complementary
• the terms “rAAV vector,” “rAAV viral particle” and/or “rAAV vector particle” refer to an AAV capsid comprised of at least one AAV capsid protein (though typically all of the capsid proteins, e.g, VPI, VPS and VP3, or variant thereof, of an AAV are present) and containing a vector genome comprising a heterologous nucleic acid sequence not originally present in the original AAV capsid.
• AAV viral particle or “AAV virus” that is not recombinant wherein the capsid contains a virus genome encoding rep and cap genes and which AAV virus is capable of replicating if present in a cell also comprising a helper virus, such as an adenovirus and/or herpes simplex virus, and/or required helper genes therefrom.
• production of an rAAV vector particle necessarily includes production of a recombinant vector genome using recombinant DNA technologies, as such, which vector genome is contained within a capsid to form an rAAV vector, rAAV viral particle, or an rAAV vector particle.
• the term “ameliorate” means a detectable or measurable improvement in a subject’s disease, disorder or condition, or symptom thereof, or an underlying cellular response.
• a detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression or duration of, complication cause by or associated with, improvement in a symptom of, or a reversal of a disease, disorder or condition.
• the term “associated with” refers to with one another, if the presence, level and/or form of one is correlated with that of the other.
• a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
• two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
• two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example, by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and a combination thereof.
• cA-motif or “cA-element” includes conserved sequences such as those found at, or close to, the termini of the genomic sequence and recognized for initiation of replication; cryptic promoters or sequences at internal positions likely used for transcription initiation, splicing or termination.
• a c/.s-motif or c/.s-element is present on the same nucleic acid molecule as those sequences with which it interacts. This is to be distinguished from “trans- motif’ sequences that act “in trans ” with other sequences that are not located on the same nucleic acid molecule.
• coding sequence or “encoding nucleic acid” refers to a nucleic acid sequence which encodes a protein or polypeptide and denotes a sequence which 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 of (operably linked to) appropriate regulatory sequences. Boundaries of a 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.
• chimeric refers to a viral capsid, with capsid sequences from different parvoviruses, preferably different AAV serotypes, as described in Rabinowitz et ah, U.S. Patent No. 6,491,907, the disclosure of which is incorporated in its entirety herein by reference. See also Rabinowitz et al. (2004) J. Virol. 78(9):4421-4432.
• a chimeric viral capsid is an AAV2.5 capsid which has the sequence of the AAV2 capsid with the following mutations: 263 Q to A; 265 insertion T; 705 N to A; 708 V to A; and 716 T to N.
• nucleotide sequence encoding such 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 WO 2010/093784, AAV2G9 and AAV8G9 described in WO 2014/144229, and AAV9.45 (Pu Norwayla et al.
• the term “conservative substitution” refers to replacement of one amino acid by a biologically, chemically or structurally similar residue.
• Biologically similar means that the substitution does not destroy a biological activity.
• Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine or are of a similar size.
• Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic.
• Particular examples include the substitution of a hydrophobic residue, such as isoleucine, valine, leucine or methionine with another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, and the like.
• Particular examples of conservative substitutions include the substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for one another, 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, and the like.
• 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.
• a “conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.
• the term “flanked,” refers to a sequence that is flanked by other elements and indicates the presence of one or more flanking elements upstream and/or downstream, i.e., 5' and/or 3', relative to the sequence.
• flanking element a sequence (e.g., a transgene) that is “flanked” by two other elements (e.g., ITRs), indicates that one element is located 5' to the sequence and the other is located 3' to the sequence; however, there may be intervening sequences there between.
• fragment refers to a material or entity that has a structure that includes a discrete portion of the whole but lacks one or more moieties found in the whole.
• a fragment consists of a discrete portion.
• a fragment consists of or comprises a characteristic structural element or moiety found in the whole.
• 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,
• the term “functional” refers to a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
• a biological molecule may have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).
• the term “gene” refers to a polynucleotide containing at least one open reading frame that is 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 host cells. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g. episomes), and/or integration of transferred genetic material into the genomic DNA of host cells.
• heterologous nucleic acid refers to a nucleic acid inserted into a vector (e.g., rAAV vector) for purposes of vector mediated transfer/delivery of the nucleic acid into a cell.
• Heterologous nucleic acids are typically distinct from the vector (e.g., AAV) nucleic acid, that is, the heterologous nucleic acid is non native with respect to the viral (e.g., AAV) nucleic acid found in the AAV in nature.
• a heterologous nucleic acid contained within a vector, can be expressed (e.g., transcribed and translated if appropriate). Alternatively, a transferred (transduced) or delivered heterologous nucleic acid in a cell, contained within the vector, need not be expressed.
• heterologous is not always used herein in reference 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.
• a heterologous nucleic acid would be a nucleic acid encoding an ASPA polypeptide, for example a codon optimized nucleic acid encoding ASPA used in the treatment of Canavan disease.
• homologous refers to two or more reference entities (e.g., a nucleic acid or polypeptide sequence) that share at least partial identity over a given region or portion. For example, when an amino acid position in two peptides is occupied by identical amino acids, the peptides are homologous at that position. Notably, a homologous peptide will retain activity or function associated with the unmodified or reference peptide and the modified peptide will generally have an amino acid sequence “substantially homologous” with the amino acid sequence of the unmodified sequence.
• nucleic acid or fragment thereof “substantial homology” or “substantial similarity,” means that when optimally aligned with appropriate insertions or deletions with another polypeptide, nucleic acid (or its complementary strand) or fragment thereof, there is sequence identity in at least about 95% to 99% of the sequence.
• sequence identity in at least about 95% to 99% of the sequence.
• the extent of homology (identity) between two sequences can be ascertained using computer program or mathematical algorithm. Such algorithms that calculate percent sequence homology (or identity) generally account for sequence gaps and mismatches over the comparison region or area. Exemplary programs and algorithms are provided below.
• a host cell As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refers to a cell into which an exogenous nucleic acid has been introduced, and includes the progeny of such a cell.
• a host cell includes a “transfectant,” “transformant,” “transformed cell,” and “transduced cell,” which includes the primary transfected, transformed or transduced cell, and progeny derived therefrom, without regard to the number of passages.
• a host cell is a packaging cell for production of an rAAV vector.
• the term “identity” or “identical to” 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.
• 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.
• Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
• the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of a reference sequence. Nucleotides at corresponding positions are then compared.
• 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, which need to be introduced for optimal alignment of the two sequences.
• the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
• sequences can be aligned using the methods and computer programs, including BLAST, available over 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.
• GCG Genetics Computing Group
• Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc.
• GCG Genetics Computing Group
• Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc.
• alignment programs that permit gaps in the sequence. Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173- 187 (1997).
• the BestFit program using the local homology algorithm of Smith and Waterman (1981, Advances in Applied Mathematics 2: 482-489) to determine sequence identity.
• the gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in some embodiments will be 3.
• the gap extension penalty will generally range from about 0.01 to 0.20 and in some instances will be 0.10.
• the program has default parameters determined by the sequences inputted to be compared.
• the sequence identity is determined using the default parameters determined by the program.
• This program is available also from Genetics Computing Group (GCG) package, from Madison, WI, USA.
• GCG Genetics Computing Group
• Another program of interest is the FastDB algorithm.
• FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.
• the terms “increase,” improve” or “reduce” indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein.
• a “control individual” is an individual afflicted with the same form of disease or injury as an individual being treated.
• the terms “inverted terminal repeat,” “FTR,” “terminal repeat,” and “TR” refer to palindromic terminal repeat sequences at or near the ends of the AAV genome, comprising mostly complementary, symmetrically arranged sequences. These ITRs can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into host genome, for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in cis for vector genome replication and its packaging into viral particles. “5’ FTR” refer to the ITR at the 5’ end of the AAV genome and/or 5’ to a recombinant transgene.
• “3’ ITR” refers to the ITR at the 3’ end of the AAV genome and/or 3’ to a recombinant transgene. Wild-type ITRs are approximately 145 bp in length. A modified, or recombinant ITR, may comprise a fragment or portion of a wild-type AAV ITR sequence. One of ordinary skill in the art will appreciate that during successive rounds of DNA replication ITR sequences may swap such that the 5’ ITR becomes the 3’ ITR, and vice versa.
• At least one ITR is present at the 5’ and/or 3’ end of a recombinant vector genome such that the vector genome can be packaged into a capsid to produce an rAAV vector (also referred to herein as “rAAV vector particle” or “rAAV viral particle”) comprising the vector genome.
• rAAV vector particle also referred to herein as “rAAV vector particle” or “rAAV viral particle”
• isolated refers to a substance or composition that is 1) designed, produced, prepared, and or manufactured by the hand of man and/or 2) separated from at least one of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting).
• isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate and/or cell membrane.
• isolated does not exclude man-made combinations, for example, a recombinant nucleic acid, a recombinant vector genome (e.g., rAAV vector genome), an rAAV vector particle (e.g., such as, but not limited to, an rAAV vector particle comprising an AAV/OligOOl capsid) that packages, e.g., encapsi dates, a vector genome and a pharmaceutical formulation.
• a recombinant nucleic acid e.g., rAAV vector genome
• an rAAV vector particle e.g., such as, but not limited to, an rAAV vector particle comprising an AAV/OligOOl capsid
• packages e.g., encapsi dates
• a vector genome and a pharmaceutical formulation e.g., encapsi dates
• isolated also does not exclude alternative physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation), variants or derivatized forms, or forms expressed in host cells that are man-made.
• 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 more than about 99% of the other components with which they were initially associated.
• 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 more than about 99% pure.
• a substance is “pure” if it is substantially free of other components.
• a substance may still be considered “isolated” or even “pure,” after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.
• carriers or excipients e.g., buffer, solvent, water, etc.
• nucleic acid sequence As used herein, the terms “nucleic acid sequence,” “nucleotide sequence,” and
• polynucleotide refer interchangeably to any molecule composed of or comprising monomeric nucleotides connected by phosphodiester linkages.
• a nucleic acid may be an oligonucleotide or a polynucleotide. Nucleic acid sequences are presented herein in the direction from the 5’ to the 3’ direction.
• a nucleic acid sequence (i.e., a polynucleotide) of the present disclosure can be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule and refers to all forms of a nucleic acid such as, double stranded molecules, single stranded molecules, small or short hairpin RNA (shRNA), micro RNA, small or short interfering RNA (siRNA), trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA, ribosomal RNA.
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• a polynucleotide is a DNA molecule
• that molecule can be a gene, a cDNA, an antisense molecule or a fragment of any of the foregoing molecules.
• Nucleotides are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U).
• a nucleotide sequence may be chemically modified or artificial.
• Nucleotide sequences include peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA).
• Each of these sequences is distinguished from naturally- occurring DNA or RNA by changes to the backbone of the molecule.
• phosphorothioate nucleotides may be used.
• Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3’-P5’-phosphoramidates, and oligoribonucleotide phosphorothioates and their 2’-0-allyl analogs and 2’-0-methylribonucleotide methylphosphonates which may be used in a nucleotide sequence of the disclosure.
• nucleic acid construct refers to a non-naturally occurring nucleic acid molecule resulting from the use of recombinant DNA technology (e.g., a recombinant nucleic acid).
• a nucleic acid construct is a nucleic acid molecule, either single or double stranded, which has been modified to contain segments of nucleic acid sequences, which are combined and arranged in a manner not found in nature.
• a nucleic acid construct may be a “vector” (e.g., a plasmid, an rAAV vector genome, an expression vector, etc.), that is, a nucleic acid molecule designed to deliver exogenously created DNA into a host cell.
• operably linked refers to a 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.
• a promoter or other transcription regulatory sequence e.g., an enhancer
• operably linked means that nucleic acid sequences being linked are contiguous.
• operably linked does not mean that nucleic acid sequences are contiguously linked, rather intervening sequences are between those nucleic acid sequences that are linked.
• the term “pharmaceutically acceptable” and “physiologically acceptable” refers to a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact.
• polypeptide As used herein, the terms “polypeptide,” “protein,” “peptide” or “encoded by a nucleic acid sequence” (i.e., encode by a polynucleotide sequence, encoded by a nucleotide sequence) refer to full-length native sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retains some degree of functionality of the native full-length protein.
• polypeptides, proteins and peptides encoded by the nucleic acid sequences can be but are not required to be identical to the endogenous protein that is defective, or whose expression is insufficient, or deficient in a subject treated with gene therapy.
• prevention refers to delay of onset, and/or reduction in frequency and/or severity of one or more sign or symptom 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 decrease in the development, frequency and/or intensity of one or more sign or symptom of the disease, disorder or condition is observed in a population susceptible to the disease, disorder or condition. Prevention may be considered complete when onset of disease, disorder or condition has been delayed for a predefined period of time.
• the term “recombinant,” refers to a vector, polynucleotide (e.g., a recombinant nucleic acid), polypeptide or cell that is the product of various combinations of cloning, restriction or ligation steps (e.g. relating to a polynucleotide or polypeptide comprised therein), and/or other procedure that results in a construct that is distinct from a product found in nature.
• polynucleotide e.g., a recombinant nucleic acid
• polypeptide or cell that is the product of various combinations of cloning, restriction or ligation steps (e.g. relating to a polynucleotide or polypeptide comprised therein), and/or other procedure that results in a construct that is distinct from a product found in nature.
• a recombinant virus or vector comprises a vector genome comprising a recombinant nucleic acid (e.g., a nucleic acid comprising a transgene and one or more regulatory elements, e.g., a codon optimized nucleic acid encoding ASPA and a CBh promoter).
• a recombinant nucleic acid e.g., a nucleic acid comprising a transgene and one or more regulatory elements, e.g., a codon optimized nucleic acid encoding ASPA and a CBh promoter.
• the terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
• the term “subject” refers to an organism, for example, 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).
• a subject is a nur7 mouse.
• a human subject is an adult, adolescent, or pediatric subject.
• a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein.
• a subject is suffering from a disease, disorder or condition associated with deficient or dysfunctional aspartoacylase activity, e.g., Canavan disease.
• a subject is susceptible to a disease, disorder, or condition.
• a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing a disease, disorder or condition.
• a subject displays one or more symptoms of a disease, disorder or condition.
• a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition.
• a subject does not display any symptom or characteristic of a disease, disorder, or condition.
• a subject is a human patient.
• a subject is an individual to whom diagnosis and/or therapy is and/or has been administered (e.g., gene therapy for Canavan disease).
• a subject is a human patient with Canavan disease.
• the term “substantially” refers to the qualitative condition of exhibition of total or near-total extent or degree of a characteristic or property of interest.
• One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or an absolute result.
• the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
• symptoms are reduced” or “reduce symptoms” refers to when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity etc.) and/or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.
• therapeutic polypeptide is a peptide, polypeptide or protein (e.g., enzyme, structural protein, transmembrane protein, transport protein) that may alleviate or reduce symptoms that result from an absence or defect in 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 related to expression or function.
• a “therapeutic transgene” is the transgene that encodes the therapeutic polypeptide.
• a therapeutic polypeptide, expressed in a host cell is an enzyme expressed from a transgene (i.e., an exogenous nucleic acid that has been introduced into the host cell).
• a therapeutic polypeptide is an ASP A protein expressed from a therapeutic transgene transduced into a cerebral cortical cell (e.g., an oligodendrocyte).
• 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 that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
• transgene is used to mean any heterologous polynucleotide for delivery to and/or expression in a host cell, target cell or organism (e.g., a subject). Such “transgene” may be delivered to a host cell, target cell or organism using a vector (e.g., rAAV vector). A transgene may be operably linked to a control sequence, such as a promoter. It will be appreciated by those of skill in the art that expression control sequences can be selected based on ability to promote expression of the transgene in a host cell, target cell or organism.
• a transgene may be operably linked to an endogenous promoter associated with the transgene in nature, but more typically, the transgene is operably linked to a promoter with which the transgene is not associated in nature.
• An example of a transgene is a nucleic acid encoding a therapeutic polypeptide, for example an ASPA polypeptide, and an exemplary promoter is one not operable linked to a nucleotide encoding ASPA in nature.
• Such a non-endogenous promoter can include a CBh promoter, among many others known in the art.
• a nucleic acid of interest can be introduced into a host cell by a wide variety of techniquest that are well-known in the art, including transfection and transduction.
• Transfection is generally known as a technique for introducing an exogenous nucleic acid into a cell without the use of a viral vector.
• the term “transfection” refers to transfer of a recombinant nucleic acid (e.g., an expression plasmid) into a cell (e.g., a host cell) without use of a viral vector.
• a cell into which a recombinant nucleic acid has been introduced is referred to as a “transfected cell.”
• a transfected cell may be a host cell (e.g., a CHO cell, ProlO cell, HEK293 cell) comprising an expression plasmid/vector for producing a recombinant AAV vector.
• a transfected cell may comprise a plasmid comprising a transgene (e.g., an ASPA transgene), a plasmid comprising an AAV rep gene and an AAV cap gene and a plasmid comprising a helper gene.
• transgene e.g., an ASPA transgene
• AAV rep gene e.g., an AAV rep gene
• AAV cap gene e.g., an AAV rep gene
• helper gene e.g., a helper gene.
• transfection techniques include, but are not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal.
• a gene therapy for Canavan disease includes transducing a vector genome comprising a modified nucleic acid encoding ASPA into an oligodendrocyte.
• a cell into which a transgene has been introduced by a virus or a viral vector is referred to as a “transduced cell.”
• a transduced cell is an isolated cell and transduction occurs ex vivo.
• a 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 which 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).
• a polynucleotide e.g., a transgene, e.g., a modified nucleic acid encoding ASPA
• Cells that may be transduced include a cell of any tissue or organ type, or any origin (e.g., mesoderm, ectoderm or endoderm).
• 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., neural or ependymal cells, oligodendrocytes) or spine, kidney, eye (e.g., retinal), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts, myocytes), synoviocytes, chondrocytes, osteoclasts, epithelial cells, end
• 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, such as brain (e.g., neural or ependymal cells, oligodendrocytes) or spine, kidney, eye (e.g., retinal), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblast, myocytes), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nervous cells or hematopoietic (e.g.
• liver
• cells present within particular areas of a tissue or organ may be transduced by an rAAV vector (e.g., an rAAV comprising an ASPA transgene) that is administered to the tissue or organ.
• an rAAV vector e.g., an rAAV comprising an ASPA transgene
• a brain cell is transduced with an rAAV comprising an ASPA transgene.
• a cell of the cortex of the brain is transduced with an rAAV comprising an ASPA transgene.
• a cell of the striatum of the brain is transduced with an rAAV comprising an ASPA transgene.
• a subcortical white matter cell of the brain is transduced with an rAAV comprising an ASPA transgene.
• a cell of the cerebellum of the brain is transduced with rAAV comprising an ASPA transgene.
• the terms “treat,” “treating” or treatment refer to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
• the term “vector” refers to a plasmid, virus (e.g., an rAAV), cosmid, or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid (e.g., a recombinant nucleic acid).
• a vector can be used for various purposes including, e.g., genetic manipulation (e.g., cloning vector), to introduce/transfer a nucleic acid into a cell, to transcribe or translate an inserted nucleic acid in a cell.
• a vector nucleic acid sequence contains at least an origin of replication for propagation in a cell.
• a vector nucleic acid includes a heterologous nucleic acid sequence, an expression control element(s) (e.g., promoter, enhancer), a selectable marker (e.g., antibiotic resistance), a poly-adenosine (poly A) sequence and/or an ITR.
• an expression control element(s) e.g., promoter, enhancer
• a selectable marker e.g., antibiotic resistance
• poly A sequence poly-adenosine sequence and/or an ITR.
• the nucleic acid sequence when delivered to a host cell, the nucleic acid sequence is propagated.
• the cell when delivered to a host cell, either in vitro or in vivo , the cell expresses the polypeptide encoded by the heterologous nucleic acid sequence.
• the nucleic acid sequene, or a portion of the nucleic acid sequence is packaged into a capsid.
• a host cell may be an isolated cell or a cell within a host organism.
• additional sequences e.g., regulatory sequences
• regulatory sequences may be present within the same vector (i.e., in cis to the gene) and flank the gene.
• regulatory sequences may be present on a separate (e.g., a second) vector which acts in trans to regulate the expression of the gene. Plasmid vectors may be referred to herein as “expression vectors.”
• vector genome refers to a recombinant nucleic acid sequence that is packaged or encapsidated to form an rAAV vector.
• a vector genome includes a heterologous polynucleotide sequence, e.g., a transgene, regulatory elements, ITRs not originally present in the capsid.
• a recombinant plasmid is used to construct or manufacture a recombinant vector (e.g., rAAV vector)
• the vector genome does not include the entire plasmid but rather only the sequence intended for delivery by the viral vector.
• This non-vector genome portion of the recombinant plasmid is typically referred to as the “plasmid backbone,” which is important for cloning selection and amplification of the plasmid, a process that is needed for propagation of recombinant viral vector production, but which is not itself packaged or encapsidated into an rAAV vector.
• viral vector generally refers to a viral particle that functions as a nucleic acid delivery vehicle and which comprises a vector genome (e.g., comprising a transgene instead of a nucleic acid encoding an AAV rep and cap) packaged within the viral particle (i.e., capsid) and includes, for example, lenti- and parvo- viruses, including AAV serotypes and variants (e.g., rAAV vectors).
• a recombinant viral vector does not comprise a vector genome comprising a rep and/or a cap gene.
• modified nucleic acids comprising a modified ASPA coding sequence, and use thereof, in gene therapy pharmaceutical compositions.
• modified is meant that the nucleic acid sequence encoding a polypeptide that exists in nature has been altered such that, in one embodiment, the modified nucleic acid sequence drives a higher level of expression of the protein in a cell compared with the level of expression of the protein from the unmodified, i.e., occurring in nature (including mutant forms of a gene), nucleic acid sequence in an otherwise identical cell.
• the disclosure also provides recombinant nucleic acids, including vector genomes, which include as part of their sequence, a modified ASPA coding sequence.
• the disclosure provides for packaged gene delivery vehicles, such as an rAAV vector, which includes the modified ASPA coding sequence.
• the disclosure also includes methods of delivery and, preferably, expression of the modified ASPA coding sequence in a cell.
• the disclosure also provides gene therapy methods in which the modified ASPA coding sequence is administered to a subject, e.g., as a component of a vector and/or packaged as a component of a viral gene delivery vehicle (e.g., an rAAV vector). Treatment may, for example, be effected to increase levels of ASPA in a subject and to treat an ASPA deficiency in a subject.
• the terms “adeno-associated virus” and/or “AAV” refer to parvoviruses with a linear single-stranded DNA genome and variants thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. Parvoviruses, including AAV, are useful as gene therapy vectors as they can penetrate a cell and introduce a nucleic acid (e.g., transgene) into the nucleus. In some embodiments, the introduced nucleic acid (e.g, rAAV vector genome) forms circular concatemers that persist as episomes in the nucleus of transduced cells.
• a nucleic acid e.g., transgene
• a transgene is inserted in specific sites in the host cell genome, for example at a site on human chromosome 19. Site-specific integration, as opposed to random integration, is believed to likely result in a predictable long-term expression profile.
• the insertion site of AAV into the human genome is referred to as AAVS1.
• polypeptides encoded by the nucleic acid can be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, a nucleic acid delivered by AAV can be used to express a therapeutic polypeptide for the treatment of a disease, disorder and/or condition in a human subject.
• AAV1-AAV15 Multiple serotypes of AAV exist in nature with at least fifteen wild type serotypes having been identified from humans thus far (i.e., AAV1-AAV15). Naturally occurring and variant serotypes are distinguished by having a protein capsid that is serologically distinct from other AAV serotypes.
• AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3) including AAV type 3 A (AAV3 A) 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), AAV type 12 (AAV 12), AAVrhlO, AAVrh74 (see WO 2016/210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non primate AAV, and ovine AAV, and recombinantly produced variants (e.g., capsid variants with insertions, deletions and substitutions, etc.), such as variants referred to as AAV type 2i8 (AAV2i8), NP4, NP22, NP66, DJ, DJ
• Prime AAV refers to AAV that infect primates
• non-primate AAV refers to AAV that infect non primate mammals
• bovine AAV refers to AAV that infect bovine mammals, and so on.
• Serotype distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences and antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).
• serotype refers to both serologically distinct viruses, e.g., AAV, as well as viruses, e.g., AAV, that are not serologically distinct but that may be within a subgroup or a variant of a given serotype.
• Genomic sequences of various serotypes of AAV, as well as sequences of the native terminal repeats (ITRs), rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank.
• wild type AAV2 comprises a small (20-25 nm) icosahedral virus capsid of AAV composed of three proteins (VPl, VP2, and VP3; a total of 60 capsid proteins compose the AAV capsid) with overlapping sequences.
• the proteins VPl (735 aa; Genbank Accession No. AAC03780), VP2 (598 aa; Genbank Accession No. AAC03778) and VP3 (533 aa; Genbank Accession No. AAC03779) exist in a 1:1:10 ratio in the capsid. That is, for AAVs, VPl is the full length protein and VP2 and VP3 are progressively shorter versions of VPl, with increasing truncation of the N-terminus relative to VPl.
• a “recombinant adeno-associated virus” or “rAAV” is distinguished from a wild-type AAV by replacement of all or part of the endogenous viral genome with a non-native sequence. Incorporation of a non-native sequence within the virus defines the viral vector as a “recombinant” vector, and hence a “rAAV vector.”
• An rAAV vector can include a heterologous polynucleotide encoding a desired protein or polypeptide (e.g., ASPA polypeptide).
• a recombinant vector sequence may be encapsidated or packaged into an AAV capsid and referred to as an “rAAV vector,” an “rAAV vector particle,” “rAAV viral particle” or simply a “rAAV.”
• 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, more preferably in the range of about 1 : 1 :5 to about 1 : 1 :20.
• the desired ratio of VPl:VP2 is 1:1, the ratio range ofVPl:VP2 could vary from 1:50 to 50:1.
• the present disclosure provides for an rAAV vector comprising a polynucleotide sequence not of AAV origin (e.g., a polynucleotide heterologous to AAV).
• the heterologous polynucleotide may be flanked by at least one, and sometimes by two, AAV terminal repeat sequences (e.g., inverted terminal repeats (ITRs)).
• ITRs inverted terminal repeats
• the heterologous polynucleotide flanked by ITRs also referred to herein as a “vector genome,” typically encodes a polypeptide of interest, or a gene of interest (“GO I”), such as a target for therapeutic treatment (e.g., a nucleic acid encoding ASPA for the treatment of Canavan disease).
• an rAAV vector can be used to transfer/deliver a heterologous polynucleotide for expression for, e.g., treating a variety of diseases, disorders and conditions.
• rAAV vector genomes generally retain 145 base ITRs in cis to the heterologous nucleic acid sesquence that replaced the viral rep and cap genes. Such ITRs are necessary to produce a recombinant AAV vector; however, modified AAV ITRs and non-AAV terminal repeats including partially or completely synthetic sequences can also serve this purpose.
• ITRs form hairpin structures and function to, for example, serve as primers for host-cell- mediated synthesis of the complementary DNA strand after infection. ITRs also play a role in viral packaging, integration, etc. ITRs are the only AAV viral elements which are required in cis for AAV genome replication and packaging into rAAV vectors.
• An rAAV vector genome optionally comprises two ITRs which are generally at the 5’ and 3’ ends of the vector genome comprising a heterologous sequence (e.g., a transgene encoding a gene of interest, or a nucleic acid sequence of interest including, but not limited to, an antisense, and siRNA, a CRISPR molecule, among many others).
• a 5’ and a 3’ ITR may both comprise the same sequence, or each may comprise a different sequence.
• An AAV ITR may be from any AAV including by not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV.
• An rAAV vector of the disclosure may comprise an ITR from an AAV serotype (e.g., wild-type AAV2, a fragment or variant thereof) that differs from the serotype of the capsid (e.g., AAV8, OligOOl).
• an rAAV vector comprising at least one ITR from one serotype, but comprising a capsid from a different serotype may be referred to as a hybrid viral vector (see U.S. Patent No. 7,172,893).
• An AAV ITR may include the entire wild type ITR sequence, or be a variant, fragment, or modification thereof, but will retain functionality.
• a heterologous polypeptide comprises an ITR (e.g., an ITR from AAV2, but can comprise an ITR from any wild type AAV serotype, or a variant thereof) positioned at the left and right ends (i.e., 5’ and 3’ termini, respectively) of a vector genome.
• a 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.
• a 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.
• a right (e.g., 3’) ITR comprises or consists of a nucleic acid sequence of SEQ ID NO:5, SEQ ID NO: 12 or SEQ ID NO: 19.
• a 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 is in cis with but may be 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 regulatory elements.
• ITRs are AAV2 ITRs, or variants thereof, and flank an ASPA transgene.
• an rAAV comprises an ASPA transgene (e.g., comprising the nucleic acid sequence of SEQ ID NO:2) flanked by AAV2 ITRs (e.g., ITRs having the sequence as set forth in SEQ ID NO:5, SEQ ID NO: 12 or SEQ ID NO: 19).
• ASPA transgene e.g., comprising the nucleic acid sequence of SEQ ID NO:2
• AAV2 ITRs e.g., ITRs having the sequence as set forth in SEQ ID NO:5, SEQ ID NO: 12 or SEQ ID NO: 19.
• an rAAV vector genome is linear, single-stranded and flanked by AAV ITRs.
• a single stranded DNA genome of approximately 4700 nucleotides Prior to transcription and translation of the heterologous gene, a single stranded DNA genome of approximately 4700 nucleotides must be converted to a double- stranded form by DNA polymerases (e.g., DNA polymerases within the transduced cell) using the free 3’ -OH of one of the self-priming ITRs to initiate second-strand synthesis.
• DNA polymerases e.g., DNA polymerases within the transduced cell
• full length-single stranded vector genomes i.e., sense and anti-sense
• This step is circumvented by using a self-complementary AAV genome (scAAV) that can package an inverted repeat genome 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).
• scAAV self-complementary AAV genome
• a limitation of a scAAV vector is that size of the unique transgene, regulatory elements and IRTs to be package in the capsid is about half the size (i.e., -2,500 nucleotides of which 2,200 nucleotides may be be a transgene and regulatory elements, plus two copies of the -145 nucleotide ITRs) of a ssAAV vector genome (i.e., - 4,900 nucleotides including two ITRs).
• scAAV vector genomes are made by using a nucleic acid not comprising the terminal resolution site (TRS), or by altering the TRS, from one rAAV ITR of a vector, e.g, a plasmid, comprising the vector genome thereby preventing initiation of replication from that end (see U.S. Patent No. 8,784,799).
• TRS terminal resolution site
• AAV replication within a host cell is initiated at the wild type ITR of the scAAV vector genome and continues through the ITR lacking or comprising an altered terminal resolution site and then back across the genome to create a complementary strand.
• the resulting complementary single nucleic acid molecule is thus a self-complementary nucleic acid molecule that results in a vector genome with a mutated (is not resolved) ITR in the middle, and wild-type ITRs at each end.
• a mutant ITR lacking a TRS or comprising an altered TRS is at the 5’ end of the vector genome.
• a mutant ITR lacking a TRS or comprising an altered TRS that is not resolved (cleaved) is at the 3’ end of the vector genome.
• a mutant ITR comprises the nucleic acid of SEQ ID NO:5, SEQ ID NO: 12 or SEQ ID NO: 19.
• a viral capsid of an rAAV vector may be from a wild type AAV or a variant AAV such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAVrh74 (see W02016/210170), AAV 12, AAV2i8, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO: 5 of WO 2015/013313), RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312
• Capsids may be derived from a number of AAV serotypes disclosed in U.S. Patent 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; and include true type AAV (AAV-TT) variants disclosed in WO 2015/121501, and RHM4-1, RHM15-1 through RHM15-6, and variants thereof, disclosed in WO 2015/013313.
• a full complement of AAV cap proteins includes VPl, VP2, and VP3.
• the ORF comprising nucleotide sequences encoding AAV VP capsid proteins may comprise less than a full complement AAV Cap proteins or the full complement of AAV cap proteins may be provided.
• the present disclosure provides for the use of ancestral AAV vectors for use in therapeutic in vivo gene therapy.
• in silico-derived sequences may be synthesized de novo and characterized for biological activities.
• Prediction and synthesis of ancestral sequences, in addition to assembly into an rAAV vector may be accomplished using methods described in WO 2015/054653, the contents of which are incorporated by reference herein.
• rAAV vectors assembled from ancestral viral sequences may exhibit reduced susceptibility to pre-existing immunity in human populations as compared to contemporary viruses or portions thereof.
• an rAAV vector comprising a capsid protein encoded by a nucleotide sequence derived from more than one AAV serotype (e.g., wild type AAV serotypes, variant AAV serotypes) is referred to as a “chimeric vector” or “chimeric capsid” (See U.S. Patent No. 6,491,907, the entire disclosure of which is incorporated herein by reference).
• a chimeric capsid protein is encoded by a nucleic acid sequence derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes.
• a recombinant AAV vector includes a capsid sequence derived from e.g., AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrhlO, AAV2i8, or variant thereof, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the foregoing AAV serotypes (see, Rabinowitz et al. (2002) J. Virology 76(2):791-801).
• a chimeric capsid can comprise a mixture of a VP1 from one serotype, a VP2 from a different serotype, a VP3 from yet a different serotype, and a combination thereof.
• a chimeric virus capsid may include an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit.
• a chimeric capsid can, for example include an AAV capsid with one or more B19 cap subunits, e.g., an AAV cap protein or submint can be replaced by a B19 cap protein or subunit.
• a VP3 subunit of an AAV capsid can be replaced by a VP2 subunit of B19.
• a chimeric capsid is an OligOOl capsid as described in WO2014052789 and incorporated herein by reference.
• chimeric vectors have been engineered to exhibit altered tropism or tropism for a particular tissue or cell type.
• the term “tropism” refers to preferential entry of the virus into certain cell or tissue types and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types.
• AAV tropism is generally determined by the specific interaction between distinct viral capsid proteins and their cognate cellular receptors (Lykken et al. (2016) J. Neurodev. Disord. 10:16).
• sequences e.g., heterologous sequences such as a transgene carried by the vector genome (e.g., an rAAV vector genome) are expressed.
• a “tropism profile” refers to a pattern of transduction of one or more target cells in various tissues and/or organs.
• 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 WO2014/052789, incorporated herein by reference.
• Such a chimeric capsid may be considered “specific for oligodendrocytes” exhibiting tropism for oligodendrocytes, and referred to herein as “oligotropism,” if when administered directly into the CNS, preferentially transduces oligodendrocytes over neurons, astrocytes and other CNS cell types.
• oligotropism if when administered directly into the CNS, preferentially transduces oligodendrocytes over neurons, astrocytes and other CNS cell types.
• at least about 80% of cells that are transduced by a capsid specific for oligodendrocytes are oligodendrocytes, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98% 99% or more of the transduced cells are oligodendrocytes.
• an rAAV vector is useful for treating or preventing a “disorder associated with oligodendrocyte dysfunction.”
• the term “associated with oligodendrocyte dysfunction” refers to a disease, disorder or condition in which oligodendrocytes are damaged, lost or function improperly compared to otherwise identical normal oligodendrocytes.
• the term includes diseases, disorders and conditions in which oligodendrocytes are directly affected as well as diseases, disorders or conditions in which oligodendrocytes become dysfunctional secondary to damage to other cells.
• a disorder associated with oligodendrocyte dysfunction is Canavan disease (CD).
• a chimeric AAV capsid with tropism for oligodendrocytes is OligOOl (also known as BNP61) and comprises sequences from AAV1, AAV2, AAV6, AAV8 and AAV9 (see WO 2014/052789).
• the OligoOOl capsid VP1 is encoded by a nucleic acid sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 13.
• the OligOOl capsid VP1 is encoded by a nucleic acid sequence 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.
• Nucleic acid sequences encode overlapping AAV capsid proteins, VP1, VP2 and VP3.
• the amino acid sequence of the OligOOl capsid proteins is set forth in SEQ ID NO: 14 with VP1 starting at amino acid residue 1 (methionine), VP2 starting at amino acid residue 148 (threonine) and VP3 starting at amino acid residue 203 (methionine) of SEQ ID NO: 14.
• a chimeric AAV capsid with tropism for oligodendrocytes is Olig002 (also known as BNP62) or Olig003 (also known as BNP63) (see WO 2014/052789).
• the Oligo002 capsid VP1 comprises or consists of the amino acid sequence of SEQ ID NO: 15.
• 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.
• a nucleic acid comprises a sequence encoding the amino acid sequence of SEQ ID NO: 15.
• the Oligo003 capsid comprises or consists of the amino acid sequence of SEQ ID NO: 16.
• 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.
• a nucleic acid comprises a sequence encoding the amino acid sequence of SEQ ID NO: 16.
• an rAAV vector comprising a chimeric AAV capsid (e.g., OligOOl) and a therapeutic transgene may be used to treat a disease, disorder or condition associated with oligodendrocyte dysfunction.
• oligodendrocytes are damaged, lost or function improperly. This may be the result of a direct effect on the oligodendrocyte or result when oligodendrocytes become dysfunctional secondary to damage to other cells.
• an rAAV vector comprising an AAV/OligOOl capsid and a modified ASPA nucleic acid is used to treat Canavan disease.
• Recombinant nucleic acids of the present disclosure include modified nucleic acids as well as plasmids and vector genomes that comprise a modified nucleic acid.
• a recombinant nucleic acid, plasmid or vector genome may comprise regulatory sequences to modulate propagation (e.g., of a plasmid) and/or control expression of a modified nucleic acid (e.g., a transgene).
• Recombinant nucleic acids may also be provided as a component of a viral vector (e.g., an rAAV vector).
• a viral vector includes a vector genome comprising a recombinant nucleic acid packaged in a capsid.
• a modified, or variant form, of a gene, nucleic acid or polynucleotide refers to a nucleic acid that deviates from a reference sequence.
• a reference sequence may be a naturally occurring, wild type sequence (e.g., a gene) and may include naturally occurring variants (e.g., splice variants, alternative reading frames).
• reference sequences can be found in publicly available databases such as GenBank (ncbi.nlm.nih.gov/genbank).
• Modified/variant nucleic acids may have substantially the same, greater or lesser activity, function or expression as compared to a reference sequence.
• a modified, or variant nucleic acid exhibits improved protein expression, e.g., a protein encoded thereby is expressed at a detectably greater level in a cell compared with the level of expression of a protein provided by an endogenous gene (e.g., a wild type gene, a mutant gene) in an otherwise identical cell.
• an endogenous gene e.g., a wild type gene, a mutant gene
• a modified, or variant nucleic acid exhibits improved protein expression, e.g., a protein encoded thereby is expressed at a detectably greater level in a cell compared with the level of expression of a protein provided by an endogenous gene comprising a mutation in an otherwise identical cell.
• Modifications to nucleic acids include one or more nucleotide substitutions (e.g., substitution of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides), additions (e.g., insertion of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides), deletions (e.g., deletion of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides, deletion of a motif, domain, fragment, etc.) of a reference sequence.
• nucleotide substitutions e.g., substitution of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides
• additions e.g., insertion of 1-3, 3-5, 5-10, 10-15, 15-20, 20
• a modified nucleic acid may 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 a reference sequence.
• a modified nucleic acid may encode a polypeptide with 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.
• a modified nucleic acid encoding ASPA (e.g., SEQ ID NO:2) encodes a polypeptide with 100% identify to a reference polypeptide (e.g., SEQ ID NO:4).
• a modified nucleic acid encodes a wild- type protein.
• Such modified nucleic acid may be codon optimized.
• “Optimized” or “codon- optimized,” as referred to interchangeably herein, refers to a coding sequence that has been optimized relative to a wild type coding sequence or reference sequence (e.g., a coding sequence for ASPA polypeptide) to increase expression of the polypeptide, e.g, by minimizing usage of rare codons, decreasing the number of CpG dinucleotides, removing cryptic splice donor or acceptor sites, removing Kozak sequences, removing ribosomal entry sites, and the like.
• a level of expression of a protein from a codon- optimized sequence is increased as compared to a level of expression of a protein from a wild type gene in an otherwise identical cell.
• a level of expression of a protein from a codon-optimized sequence is not increased (e.g., expression is substantially similar) as compared to a level of expression of a protein from a wild-type gene in an otherwise identical cell.
• a level of expression of a protein from a codon-optimized sequence is increased as compared to a level of expression of a protein from a mutant gene in an otherwise identical cell.
• modifications include elimination of one or more cis- acting motifs and introduction of one or more Kozak sequences. In some embodiments, one or more ex acting motifs are eliminated and one or more Kozak sequences are introduced.
• Examples of cis- acting motifs that may be eliminated include internal TATA- boxes; chi-sites; ribosomal entry sites; ARE, INS, and/or CRS sequence elements; repeat sequences and/or RNA secondary structures; (cryptic) splice donor and/or acceptor sites, branch points; and restriction sites.
• a modified nucleic acid encodes a modified or variant polypeptide.
• a modified polypeptide encoded by a modified nucleic acid may retain all or a part of the function or activity of a polypeptide encoded by a wild type coding or reference sequence.
• a modified polypeptide has one or more non-conservative or conservative amino acid changes.
• certain domains that have been demonstrated to play a limited or no role in a function of a polypeptide are not present in a modified polypeptide (e.g., certain binding domains) (e.g., WO 2016/097219).
• Modified nucleic acids present in rAAV vectors may comprise fewer nucleotides than the wild type coding, or reference sequence, due to the packaging capacity of an rAAV capsid (e.g., shortened minidystrophin transgene, see WO 2001/83695; a B-domain deleted human Factor VIII transgene, see WO 2017/074526), and also include shortened transgenes that are both truncated and codon-optimized (e.g., a codon optimized mini-dystrophin transgene described in WO 2017/221145).
• a polypeptide encoded by a modified nucleic acid has less than, the same, or greater, but at least a part of, a function or activity of a polypeptide encoded by a reference sequence.
• Modified nucleic acids 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).
• CAI codon adaptation index
• modified refers to a decrease or an increase in a particular value, amount or effect.
• a GC content of a modified nucleic acid sequence of the present disclosure is increased relative to a reference and/or a wild-type gene or coding sequence.
• the GC content of a 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%, at least 70% greater than GC content of a wild type coding sequence (e.g., SEQ ID NO:3).
• GC content is expressed as a percentage of G (guanine) and C (cytosine) nucleotides in the sequence.
• a codon adaptation index of a modified nucleic acid sequence 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.
• a modified nucleic acid sequence of the present disclosure has a reduced level of CpG dinucleotides, that being a reduction of about 10%, 20%, 30%, 50% or more, as compared to a wild type or reference nucleic acid sequence.
• a modified nucleic acid has 1-5 fewer, 5-10 fewer, 10-15 fewer, 15-20 fewer, 20-25 fewer, 25-30 fewer, 30-40 fewer, 40-45 fewer or 45-50 fewer, or even fewer di nucleotides than a reference sequence (e.g., a wild type sequence).
• methylation of CpG dinucleotides plays an important role in the regulation of gene expression in eukaryotes. Specifically, methylation of CpG dinucleotides in eukaryotes essentially serves to silence gene expression through interfering with the transcriptional machinery. As such, because of the gene silencing evoked by methylation of CpG motifs, nucleic acids and vectors having a reduced number of CpG dinucleotides will provide for high and longer-lasting transgene expression level.
• Modified nucleic acid sequences may include 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 FIG. 13, such as, Aval, Xmal and Xmal.
• the present disclosure includes fragments of any one of the sequences set forth in SEQ ID NOs: 1-3 and which encode a functionally active fragment of the ASPA polypeptide.
• a “functionally active” or “functional ASPA polypeptide” indicates that the fragment provides the same or similar biological function and/or activity as a 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.
• the present disclosure provides for modified ASPA nucleic acid sequences that encode an ASPA polypeptide and which comprise at least one modification as compared with a wild type nucleic acid sequence (e.g. SEQ ID NO:3; GenBank Accession Number NM_000049.4 or NM_001128085.1, having an alternate 5’UTR but encoding for the same ASPA protein (SEQ ID NO:4)).
• a wild type nucleic acid sequence e.g. SEQ ID NO:3; GenBank Accession Number NM_000049.4 or NM_001128085.1, having an alternate 5’UTR but encoding for the same ASPA protein (SEQ ID NO:4).
• a modified nucleic acid encoding ASPA is a codon- optimized nucleic acid encoding a wild-type ASPA polypeptide (e.g., SEQ ID NO:4) and comprises the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
• a modified nucleic acid encoding ASPA is a codon-optimized nucleic acid and consists of the sequence of SEQ ID NO:l or SEQ ID NO:2.
• a modified nucleic acid encoding ASPA is a codon-optimized nucleic acid and comprises a 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 sequence of SEQ ID NO: 1 or SEQ ID NO:2.
• a cell comprising a modified nucleic acid encoding ASPA exhibits increased protein expression, e.g., the protein encoded thereby is expressed at a detectably greater level in a cell as compared with the level of expression of the protein in an otherwise identical cell comprising a wild type ASPA nucleic acid, or an otherwise identical cell comprising a mutant nucleic acid encoding ASPA.
• a level of ASPA protein expression in a cell comprising a modified nucleic acid encoding ASPA 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 as compared to the level of ASPA protein expression in an otherwise identical cell comprising a wild-type ASPA nucleic acid.
• the level of ASPA protein expression in a cell comprising a modified nucleic acid encoding ASPA 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 as compared to the level of ASPA protein expression in an otherwise identical cell comprising a mutant nucleic acid encoding ASPA.
• this can be referred to as an “expression optimized” or “enhanced expression” nucleic acid, or simply, as a “modified nucleic acid.”
• a polypeptide encoded by a modified nucleic acid, and variants thereof, of the disclosure is a “functional ASPA polypeptide” that provides the same or similar biological function and/or activity as a ASPA polypeptide encoded by a wild-type nucleic acid encoding ASPA (e.g., SEQ ID NO:3). That is, an ASPA polypeptide encoded by a modified nucleic acid encoding ASPA provides the same activity including, but not limited to, the ability to convert NAA to acetate and aspartate.
• ASPA encompasses reversing or preventing the neurodegenerative phenotype associated with Canavan disease as demonstrated elsewhere herein in nur7 mice including, but not limited to, improved performance of rotarod latency to fall, improved open field distance traversed, decreased NAA in brain tissue, decreased vacuole volume in the brain (e.g., thalamus, cerebellar white matter/pons), an increase in 01ig2 positive cells in the brain (e.g., thalamus, cortex), and/or an increase in cortical myelination. Regulatory elements
• the present disclosure includes a recombinant nucleic acid including a modified nucleic acid encoding ASPA and various regulatory or control elements.
• regulatory elements are nucleic acid sequence(s) that influence expression of an operably linked polynucleotide.
• the precise nature of regulatory elements useful for gene expression will vary from organism to organism and from cell type to cell type including, for example, a promoter, enhancer, intron etc., with the intent to facilitate proper heterologous polynucleotide transcription and translation. Regulatory control can be affected at the level of transcription, translation, splicing, message stability, etc.
• a regulatory control element that modulates transcription is juxtaposed near the 5’ end of the transcribed polynucleotide (i.e., upstream). Regulatory control elements may also be located at the 3’ end of the transcribed sequence (i.e., downstream) or within the transcript (e.g., in an intron). Regulatory control elements can be located at a distance away from the transcribed sequence (e.g., 1 to 100, 100 to 500, 500 to 1000, 1000 to 5000, 5000 to 10,000 or more nucleotides). However, due to the length of an AAV vector genome, regulatory control elements are typically within 1 to 1000 nucleotides from the polynucleotide.
• promoter refers to a nucleotide sequence that initiates transcription of a particular gene, or one or more coding sequences (e.g., an ASPA coding sequence), in eukaryotic cells (e.g., an oligodendrocyte).
• a promoter can work with other regulatory elements or regions to direct the level of transcription of the gene or coding sequence(s). 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, enhances and silencers.
• the promoter is most often located on the same strand and near the transcription start site, 5’ of the gene or coding sequence to which it is operably linked.
• a promoter is generally 100 - 1000 nucleotides in length.
• a promoter typically increases gene expression relative to expression of the same gene in the absence of a promoter.
• a “core promoter” or “minimal promoter” refers to the minimal portion of a promoter sequence required to properly initiate transcription. It may include any of the following: a transcription start site, a binding site for RNA polymerase and a general transcription factor binding site.
• a promoter may also comprise a proximal promoter sequence (5’ of a core promoter) that contains other primary regulatory elements (e.g., enhancer, silencer, boundary element, insulator) as well as a distal promoter sequence (3’ of a core promoter).
• adenoviral promoters such as the adenoviral major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus promoter; the Rous Sarcoma Virus (RSV) promoter; the albumin promoter; inducible promoters, such as the Mouse Mammary Tumor Virus (MMTV) promoter; the metallothionein promoter; heat shock promoters; the a- 1 -antitrypsin promoter; the hepatitis B surface antigen promoter; the transferrin promoter; the apolipoprotein A-l promoter; chicken b-actin (CBA) promoter, the elongation factor la promoter (EFla), the hybrid form of the CBA promoter (CBh promoter), and the CAG promoter (cytomegalovirus early enhancer element and the promoter, the first exon, and the first intron of chicken beta-
• CMV cytomegalovirus
• a eukaryotic promoter sequence (e.g., a CBh promoter) is operably linked to a modified nucleic acid encoding ASPA.
• a promoter comprising the nucleic acid sequence of SEQ ID NO:7 e.g., a CBh promoter
• a modified nucleic acid encoding ASPA e.g., a CBh promoter
• 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.
• 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 induces expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 in oligodendrocytes.
• 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 greater level in a cell compared with the level of expression of a polypeptide encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:2, not operably linked to a promoter comprising the nucleic acid of SEQ ID NO:7, in an otherwise identical cell.
• a 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 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.
• a promoter may be constitutive, tissue-specific or regulated. Constitutive promoters are those which cause an operably linked gene to be expressed essentially at all times. In some embodiments, a constitutive promoter is active in most eukaryotic tissues under most physiological and developmental conditions.
• Regulated promoters are those which can be activated or deactivated. Regulated promoters include inducible promoters, which are usually “off’ but which may be induced to turn “on,” and “repressible” promoters, which are usually “on” but may be turned “off.” Many different regulators are known, including temperature, hormones, cytokines, heavy metals and regulatory proteins. The distinctions are not absolute; a constitutive promoter may often be regulated to some degree. In some cases, an endogenous pathway may be utilized to provide regulation of the transgene expression, e.g., using a promoter that is naturally downregulated when the pathological condition improves.
• a tissue-specific promoter is a promoter that is active in only specific types of tissues, cells or organs.
• a tissue-specific promoter is recognized by transcriptional activator elements that are specific to a particular tissue, cell and/or organ.
• a tissue-specific promoter may be more active in one or several particular tissues (e.g., two, three or four) than in other tissues.
• expression of a gene modulated by a tissue-specific promoter is much higher in the tissue for which the promoter is specific than in other tissues.
• a promoter may be a tissue-specific promoter, such as the mouse albumin promoter, or the transthyretin promoter (TTR), which are active in liver cells.
• tissue specific promoters include promoters from genes encoding skeletal a-actin, myosin light chain 2A, dystrophin, muscle creatine kinase which induce expression in skeletal muscle (Li et al. (1999) Nat. Biotech. 17:241- 245).
• Liver specific expression may be induced using promoters from the albumin gene (Miyatake et al. (1997) J. Virol. 71:5124-5132), 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).
• a modified nucleic acid encoding a therapeutic polypeptide further comprises an enhancer to increase expression of the therapeutic polypeptide (e.g., a ASPA protein).
• an enhancer element is located upstream of a promoter element but may also be located downstream or within another sequence (e.g., a transgene).
• An enhancer may be located 100 nucleotides, 200 nucleotides, 300 nucleotides or more upstream or downstream of a modified nucleic acid.
• An enhancer typically increases expression of a modified nucleic acid (e.g., encoding a therapeutic polypeptide, e.g., encoding ASPA) beyond the increased expression provided by a promoter element alone.
• CMV MIE promoter comprises three regions: the modulator, the unique region and the 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.
• CB A chicken b-actin
• CBh chicken beta-actin hybrid promoter
• enhancers may be constitutive, tissue-specific or regulated.
• an enhancer sequence (e.g., a CMV enhancer) is operably linked to a modified nucleic acid encoding ASPA.
• an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO: 17 (e.g., a CMV enhancer) is operably linked to a modified nucleic acid encoding ASPA.
• 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 operably linked to a promoter comprising the nucleic acid sequence of SEQ ID NO:7.
• 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.
• 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 the nucleic acid sequences of SEQ ID NO:6 (or SEQ ID NO: 17) and SEQ ID NO:7 induce expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 in oligodendrocytes.
• 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 greater level in a cell compared with the level of expression of a polypeptide encoded by SEQ ID NO:2, not operably linked to an enhancer comprising the nucleic acid of SEQ ID NO:5, in an otherwise identical cell.
• a recombinant nucleic acid comprises 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, operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 7, and operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO:2, and together the nucleic acid sequences of SEQ ID NO:6 (or SEQ ID NO: 17) and SEQ ID NO: 7 induce expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:2 in oligodendrocytes.
• a recombinant nucleic acid intended for use in an rAAV vector may include an additional nucleic acid element to adjust the length of the nucleic acid to near, or at the normal size (e.g., approximately 4.7 to 4.9 kilobases), of the viral genomic sequence acceptable for AAV packaging into an rAAV vector (Grieger and Samulski (2005)
• filler DNA is an untranslated (non-protein coding) segment of nucleic acid.
• a filler or stuffer polynucleotide sequence is a sequence between about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90-90- 100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1000, 1000- 1500, 1500-2000, 2000-3000 or more in length.
• AAV vectors typically accept inserts of DNA having a size ranging from about 4 kb to about 5.2 kb or about 4.1 to 4.9 kb for optimal packaging of the nucleic acid into the AAV capsid.
• an rAAV vector comprises a vector genome having a total length between about 3.0 kb to about 3.5 kb, about 3.5 kb to about 4.0 kb, about 4.0 kb to about 4.5kb, about 4.5 kb to about 5.0 kb or about 5.0 kb to about 5.2 kb.
• an rAAV vector comprises a vector genome having a total length of about 4.7 kb.
• an rAAV vector comprises a vector genome that is self complementary. While the total length of a self-complementary (sc) vector genome in an rAAV vector is equivalent to a single-stranded (ss) vector genome (i.e., from about 4 kb to about 5.2 kb), the nucleic acid sequence (i.e., comprising the transgene, regulatory elements and ITRs) encoding the sc vector genome must be only half as long as a nucleic acid sequence encoding a ss vector genome in order for the sc vector genome to be packaged in the capsid.
• sc self-complementary
• a recombinant nucleic acid includes, for example, an intron, exon and/or a portion thereof.
• An intron may function as a filler or stuffer polynucleotide sequence to achieve an appropriate length for vector genome packaging into an rAAV vector.
• An intron and/or an exon sequence can also enhance expression of a polypeptide (e.g., a transgene) as compared to expression in the absence of the intron and/or exon element (Kurachi et al. (1995) J. Biol. Chem. 270 (10):576-5281; WO 2017/074526).
• 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 WO 2014/144486 and WO 2017/074526.
• An intron element may be derived from the same gene as a heterologous polynucleotide, or derived from a completely different gene or other DNA sequence (e.g., chicken b-actin gene, minute virus of mice (MVM)).
• a recombinant nucleic acid includes at least one element selected from an intron and an exon derived from a non-cognate gene (i.e., not derived from the modified nucleic acid, e.g., transgene).
• an intron is derived from a chicken b-actin gene, for example comprising or consisting of the nucleic acid sequence of SEQ ID NO: 9.
• an intron comprises a nucleic acid sequence 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.
• an intron is derived from a MVM, for example comprising or consisting of the nucleic acid sequence of SEQ ID NO: 10.
• an intron comprises a nucleic acid sequence 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.
• an exon is derived from a chicken b-actin gene, for example comprising or consisting of the nucleic acid sequence of SEQ ID NO:8.
• an exon comprises a nucleic acid sequence 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.
• a recombinant nucleic acid is comprised of 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 modulates expression of a heterologous polypeptide, optionally encoded by the nucleic acid sequence of SEQ ID NO:2.
• 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
• 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
• a 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).
• 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
• Further regulatory elements may include a stop codon, a termination sequence, and a polyadenylation (polyA) signal sequence, such as, but not limited to a bovine growth hormone poly A signal sequence (BHG polyA).
• a polyA signal sequence drives efficient addition of a poly-adenosine “tail” at the 3’ end of a eukaryotic mRNA which guides termination of gene transcription (see, e.g., Goodwin and Rottman J. Biol. Chem. (1992) 267(23): 16330-16334).
• a polyA signal acts as a signal for the endonucleolytic cleavage of the newly formed precursor mRNA at its 3’ end and for addition to this 3’ end of an RNA stretch consisting only of adenine bases.
• a polyA tail is important for the nuclear export, translation and stability of mRNA.
• a poly A is a SV40 early polyadenylation signal, a SV40 late polyadenylation signal, an HSV thymidine kinase polyadenylation signal, a protamine gene polyadenylation signal, an adenovirus 5 Elb polyadenylation signal, a growth hormone polyadenylation signal, a PBGD polyadenylation signal or an in silico designed polyadenylation signal.
• a polyA signal sequence of a recombinant nucleic acid is a polyA signal that is capable of directing and effecting the endonucleolytic cleavage and polyadenylation of the precursor mRNA resulting from the transcription of a modified nucleic acid encoding ASPA (e.g., SEQ ID NO:2).
• a polyA sequence comprises or consists of the nucleic acid sequence of SEQ ID NO: 11.
• a polyA sequence comprises a nucleic acid sequence 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.
• a 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 a polyA (SEQ ID NO: 11) and modulates the expression of a heterologous polypeptide, optionally encoded by the nucleic acid sequence of SEQ ID NO:2.
• 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
• an rAAV vector (e.g., AAV/OligOOl-ASPA), with tropism for oligodendrocytes, 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., a CMV enhancer), a promoter (e.g., a CBh promoter), an exon (e.g., a CBA exon 1), an intron (e.g., CBA intron, MVM intron) and a poly A (e.g., a BHG polyA).
• AAV ITRs e.g., AAV2 ITRs
• a recombinant nucleic acid comprising a modified (i.e., codon- optimized) nucleic acid
• an rAAV vector e.g., AAV/OligOOl-ASPA
• AAV ITRs e.g., SEQ ID NO:5, SEQ ID NO: 12 and/or SEQ ID NO: 19
• an rAAV vector e.g., AAV/OligOOl-ASPA
• a self-complementary genome comprising SEQ ID NO:20.
• an rAAV vector of the present disclosure transduces a target cell (e.g., an oligodendrocyte) and mediates a biological activity.
• a target cell e.g., an oligodendrocyte
• an rAAV vector e.g., AAV/OligOOl-ASPA
• transduces a target cell e.g., an oligodendrocyte
• mediates at least one detectable activity selected from the group consisting of:
• an rAAV vector which transduces a target cell e.g., an oligodendrocyte
• mediates at least one detectable activity of (i) through (xii) is AAV/OligoOOl-ASPA.
• a cell transduced with an rAAV vector (e.g., AAV/OligOOl-ASPA) has a reduced level of NAA as compared to a level of NAA in an otherwise identical cell transduced with an rAAV comprising a wild-type nucleic acid sequence encoding ASPA (e.g., SEQ ID NO:3).
• a cell transduced with an rAAV vector has a reduced level of NAA as compared to a level of NAA in an otherwise identical cell transduced with an rAAV comprising an alternative codon-optimized nucleic acid encoding ASPA (e.g., SEQ ID NO:l).
• a cell transduced with an rAAV vector e.g., AAV/OligOOl-ASPA
• a cell transduced in vivo with an rAAV vector e.g., AAV/OligOOl-ASPA
• an rAAV vector e.g., AAV/OligOOl-ASPA
• a cell transduced in vivo with an rAAV vector has a reduced level of NAA as compared to a level of NAA in an otherwise identical cell transduced in vivo with an rAAV comprising an alternative codon-optimized nucleic acid encoding ASPA (e.g., SEQ ID NO:l).
• a cell transduced in vivo with an rAAV vector e.g., AAV/OligOOl-ASPA
• balance, grip strength and/or motor coordination in a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) has been administered is significantly improved as compared to balance, grip strength and/or motor coordination of an otherwise similar subject with an ASPA gene mutation to whom the rAAV vector has not been administered, or compared to the same subject prior to administration of the rAAV vector, as measured by, e.g., rotarod performance.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• balance, grip strength and/or motor coordination in a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) has been administred is indistinguishable from balance, grip strength and/or motor coordination in an otherwise similar subject without an ASPA gene mutation, and to whom the rAAV vector has not been administered, as measured by, e.g., rotarod performance.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• ICV intracerebroventricular
• rotarod performance is measured as latency to fall in seconds.
• generalized motor function of a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) has been administered is significantly improved as compared to generalized motor function of an otherwise similar subject with an ASPA gene mutation to whom the rAAV vector is not administered, or compared to the function in the subject prior to administration of the rAAV vector, as measured by, e.g., open field activity.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• ICV intracerebroventricular
• generalized motor function in a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is indistinguishable from generalized motor function in an otherwise similar subject without an ASPA gene mutation, and to whom the rAAV has not been administered, as measured by, e.g., open field activity.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• ICV intracerebroventricular
• an NAA level in the brain of subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is significantly reduced as compared to a NAA level in the brain of an otherwise similar subject with an ASPA gene mutation to whom the rAAV vector is not administered, or as comparted with the NAA level in the subject prior to administration of the rAAV vector.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• an NAA level in the brain of a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is reduced or indistinguishable as compared to an NAA level in the brain of an otherwise similar subject without a ASPA gene mutation, and to whom the rAAV vector has not been administered.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• vacuole volume fraction in the thalamus of a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is significantly reduced as compared to vacuole fraction in the thalamus of an otherwise similar subject with an ASPA gene mutation to whom the rAAV vector is not administered, or compared with the subject prior to administration of the rAAV vector, wherein the vacuole fraction is measured by, e.g., unbiased stereology.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• vacuole volume fraction in the cerebellar white matter/pons of a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is significantly reduced as compared to vacuole fraction in the cerebellar white matter/pons of an otherwise similar subject with an ASPA gene mutation to whom the rAAV vector is not administered, or compared with the subject prior to administration of the rAAV vector, wherein the vacuole fraction is measured by, e.g., unbiased stereology.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• the number of oligodendrocytes in the thalamus of a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is significantly increased as compared to the number of oligodendrocytes in the thalamus of an otherwise similar subject with an ASPA gene mutation to whom the vector is not administered, or compared with the subject before the vector is administered, wherein the number of oligodendrocytes in the thalamus is measured by, e.g., IHC using 01ig2 antibody and unbiased stereology.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• the number of oligodendrocytes in the brain cortex of a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is significantly increased as compared to the number of oligodendrocytes in the brain cortex of an otherwise similar subject with an ASPA gene mutation to whom the rAAV vector is not administered, or compared with the same subject before the vector is administered, wherein the number of oligodendrocytes in the brain cortex is measured by, e.g., IHC using 01ig2 antibody and unbiased stereology.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• the number of oligodendrocytes in the brain cortex of a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is indistinguishable from the number of oligodendrocytes in the brain cortex of an otherwise similar subject without a ASPA gene mutation, and to whom the rAAV vector is not administered, wherein the number of oligodendrocytes in the brain cortex is measured by, e.g., IHC using 01ig2 antibody and unbiased stereology.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• the number of neurons in the thalamus of a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is significantly increased as compared to the number of neurons in the thalamus of an otherwise identical subject with an ASPA gene mutation to whom the rAAV vector is not administered, or compared with the number of neurons in the thalamus of the subject prior to administration of the vector, wherein the number of neurons in the thalamus is measured by, e.g., IHC using NeuN antibody and unbiased stereology.
• an rAAV vector e.g., AAV/OligOOl-ASPA
• the number of neurons in the brain cortex of a subject with an ASPA gene mutation to whom an rAAV vector is administered is significantly increased as compared to the number of neurons in the brain cortex of an otherwise similar subject with an ASPA gene mutation to whom the rAAV vector is not administered, or as compared with the number of neurons in the brain cortex of the subject prior to administration of the vector, wherein the number of neurons in the brain cortex is measured by, e.g., IHC using NeuN antibody and unbiased stereology.
• the number of neurons in the brain cortex of a subject with an ASPA gene mutation to whom an rAAV vector (e.g., AAV/OligOOl-ASPA) is administered is indistinguishable from the number of neurons in the brain cortex of an otherwise similar subject without an ASPA gene mutation, and to whom the rAAV vector is not administered, wherein the number of neurons in the brain cortex is measured by, e.g.,
• cortical myelination in the brain of a subject with an ASPA gene mutation to whom an rAAV vector is adminitereed is significantly increased as compared to cortical myelination in the brain of an otherwise similar subject with an ASPA gene mutation to whom the rAAV vector is not administered, or compared with the cortical myelination in the brain of the subject prior to administration of the vector, wherein the cortical myelination is measured by, e.g., cortical myelin basic protein-positive fiber length density (MBP-LD).
• MBP-LD cortical myelin basic protein-positive fiber length density
• a viral vector e.g., rAAV vector
• a transgene e.g., ASPA
• a viral vector carrying a transgene (e.g., ASPA) is assembled from a polynucleotide encoding a transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.
• a viral vector include but are not limited to adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors, and in particular rAAV vector (as discussed, supra).
• a vector genome component of an rAAV vector produced according to the methods of the disclosure include 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.
• a vector genome includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and/or replaced by a modified nucleic acid (e.g., transgene, e.g., modified nucleic acid encoding ASPA) and its associated expression control sequences.
• a modified nucleic acid encoding ASPA is typically inserted adjacent to one or two (i.e., is flanked by) AAV ITRs or ITR elements adequate for viral replication (Xiao et al. (1997) J. Virol. 71(2): 941-948), in place of the nucleic acid encoding viral rep and cap proteins.
• Other regulatory sequences suitable for use in facilitating tissue- specific expression of a modified nucleic acid encoding ASPA in the target cell may also be included.
• Cap and rep genes may be supplied to a cell (e.g., a host cell, e.g., a packaging cell) as part of a plasmid that is separate from a plasmid supplying the vector genome with the transgene.
• Packaging cell or “producer cell” means a cell or cell line which may be transfected with a vector, plasmid or DNA construct, and provides in trans all the missing functions which are required for the complete replication and packaging of a viral vector.
• the required genes for rAAV vector assembly include the vector genome (e.g., a modified nucleic acid encoding ASPA, regulatory elements, and ITRs), AAV rep gene, AAV cap gene, and certain helper genes from other viruses such as, e.g., adenovirus.
• a packaging cell expresses, in a constitutive or inducible manner, one or more missing viral functions.
• Any suitable packaging cell known in the art may be employed in the production of a packaged viral vector. Mammalian cells or insect cells are preferred.
• Examples of cells useful for the production of a packaging cell in the practice of the disclosure include, for example, human cell lines, such as PER.C6, WI38, MRC5, A549, HEK293 cells (which express functional adenoviral El under the control of a constitutive promoter), B-50 or any other HeLa cell, 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.
• a packaging cell is capable of growing in suspension culture.
• a packaging cell is capable of growing in serum-free media.
• HEK293 cells are grow in suspension in serum free medium.
• a packaging cell is a HEK293 cell as described in U.S. Patent No. 9,441,206 and deposited as American Type Culture Collection (ATCC) No. PTA 13274. Numerous rAAV packaging cell lines are known in the art, including, but not limited to, those disclosed in WO 2002/46359.
• a cell line for use as a packaging cell includes insect cell lines. Any insect cell which allows for replication of AAV and which can be maintained in culture can be used in accordance with the present disclosure. Examples include Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line.
• the following references are incorporated herein for their teachings concerning use of insect cells for expression of heterologous polypeptides, methods of introducing nucleic acids into such cells, and methods of maintaining such cells in culture: Methods in Molecular Biology, ed.
• viral vectors of the disclosure may be produced in insect cells using baculovirus vectors to deliver the rep/cap genes and rAAV template as described, for example, by Urabe et al. (2002) Human Gene Therapy 13:1935-1943.
• a vector genome is self- complementary.
• a host cell is a baculovirus-infected cell (e.g., an insect cell) comprising, optionally, additional nucleic acids encoding baculovirus helper functions, thereby facilitating production of a viral capsid.
• a packaging cell generally includes one or more viral vector functions along with helper functions and packaging functions sufficient to result in replication and packaging of the viral vector. These various functions may be supplied together, or separately, to the packaging cell using a genetic construct such as a plasmid or an amplicon, and they may exist extrachromosomally within the cell line, or integrated into the host cell’s chromosomes.
• a packaging cell is transfected with at least i) a plasmid comprising a vector genome comprising 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 the following regulatory elements: an enhancer (e.g., SEQ ID NO:6), a promoter (e.g.,
• SEQ ID NO:7 an exon (e.g., a CBA exon SEQ ID NO:8), an intron (e.g., SEQ ID NO:9 and SEQ ID NO: 10) and a poly A (e.g., SEQ ID NO: 11) and ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and a cap gene (e.g., OligOOl cap).
• a rep gene e.g., AAV2 rep
• cap gene e.g., OligOOl cap
• a host cell is supplied with one or more of the packaging or helper functions incorporated within, e.g., a host cell line with one or more vector functions incorporated extrachromosomally or integrated into the cell’s chromosomal DNA.
• AAV is a Dependovirus in that it cannot replicate in a cell without co-infection of the cell by a helper virus.
• Helper functions include helper virus elements needed for establishing active infection of a packaging cell, which is required to initiate packaging of the viral vector.
• Helper viruses include, typically, adenovirus or herpes simplex virus.
• Adenovirus helper functions typically include adenovirus components adenovirus early region 1 A (Ela), Elb, E2a, E4, and viral associated (VA) RNA.
• Helper functions e.g., Ela, Elb, E2a, E4, and VA RNA
• a host cell e.g., a packaging cell
• HEK293 cells were generated by transforming human cells with adenovirus 5 DNA and now express a number of adenoviral genes, including, but not limited to El and E3 (see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59-72).
• those helper functions can be provided by the HEK 293 packaging cell without the need of supplying them to the cell by, e.g., a plasmid encoding them.
• a packaging cell is transfected with at least i) a plasmid comprising a vector genome comprising 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 the following regulatory elements: an enhancer (e.g., SEQ ID NO:6), a promoter (e.g., SEQ ID NO:7), an exon (e.g., a CBA exon SEQ ID NO:8), an intron (e.g., SEQ ID NO:9 and SEQ ID NO: 10) and a poly A (e.g., SEQ ID NO: 11), ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and a cap gene (e.g., OligOOl cap) and iii) a plasmid comprising a helper function
• any method of introducing a nucleotide sequence carrying a helper function into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal.
• helper functions are provided by transfection using a virus vector, or by infection using a helper virus, standard methods for producing viral infection may be used.
• 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 duplexed (i.e., self complementary as described in WO 2001/92551).
• Viral vectors can be made by several methods known to skilled artisans (see, e.g., WO 2013/063379). A preferred method is described in Grieger, et al. (2015) Molecular Therapy 24(2):287-297, the contents of which are incorporated by reference herein for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, wherein an adherent HEK293 cell line from a qualified clinical master cell bank is used to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allow for rapid and scalable rAAV production.
• a HEK293 cell line suspension can generate greater than lxlO 5 vector genome containing particles (vg)/cell, or greater than lxlO 14 vg/L of cell culture, when harvested 48 hours post-transfection.
• triple transfection refers a method whereby a packaging cell is transfected with three plasmids: one plasmid encodes the AAV rep and cap (e.g., OligOOl cap) genes, another plasmid encodes various helper functions (e.g., adenovirus or HSV proteins such as Ela, Elb, E2a, E4, and VA RNA, and another plasmid encodes a transgene (e.g., ASP A) and various elements to control expression of the transgene.
• AAV rep and cap e.g., OligOOl cap
• helper functions e.g., adenovirus or HSV proteins such as Ela, Elb, E2a, E4, and VA RNA
• transgene e.g., ASP A
• Single-stranded vector genomes are packaged into capsids as the plus strand or minus strand in about equal proportions.
• a vector genome is in the plus strand polarity (i.e., the sense or coding sequence of the DNA strand).
• a vector is in the minus strand polarity (i.e., the antisense or template DNA strand). Given the nucleotide sequence of a plus strand in its 5’ to 3’ orientation, the nucleotide sequence of a minus strand in its 5’to 3’ orientation can be determined as the reverse-complement of the nucleotide sequence of the plus strand.
• An rAAV vector may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art and include methods 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
• a universal purification strategy may be used to generate high purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids (e.g., OligOOl). In some embodiment, this process can be completed within one week, result in high full to empty capsid ratios (>90% full capsids), provide post purification yields (>lxl0 13 vg/L) and purity suitable for clinical applications. In some embodiments, such a method is universal with respect to all serotypes and chimeric capsids. Scalable manufacturing technology may be utilized to manufacture GMP clinical and commercial grade rAAV vectors (e.g., for the treatment of Canavan disease).
• rAAV vectors of the present disclosure After rAAV vectors of the present disclosure have been produced and purified, they can be titered (e.g., the amout of rAAV vector in a sample can be quantified) to prepare compositions for administration to subjects, such as human subjects with Canavan disease. rAAV vector titering can be accomplished using methods know in the art.
• the number of viral particles can be determined by electron microscopy, e.g., transmission electron microscopy (TEM).
• TEM transmission electron microscopy
• Such a TEM-based method can provide the number of vector particles (or virus particles in the case of wild type AAV) in a sample.
• rAAV vector genomes can be titered using quantitative PCR (qPCR) using primers against sequences in the vector genome, for example ITR sequences (e.g, SEQ ID NO:5, SEQ ID NO: 12 or SEQ ID NO: 19), and/or sequences in the transgene (e.g., SEQ ID NO:2) or regulatory elements.
• qPCR quantitative PCR
• a standard curve can be generated permitting the concentration of the rAAV vector to be calculated as the number of vector genomes (vg) per unit volume such as microliters or milliliters.
• the number of empty capsids can be determined. Because the vector genome contains the therapeutic transgene, vg/kg or vg/ml of a vector sample may be more indicative of the therapeutic amount of the vector that a subject will receive than the number of vector particles, some of which may be empty and not contain a vector genome.
• the concentration of rAAV vector genomes in the stock solution is determined, it can be diluted into or dialyzed against suitable buffers for use in preparing a composition for administration to subjects (e.g., subjects with Canavan disease).
• a modified nucleic acid such as a modified nucleic acid encoding ASP A, as disclosed herein, may be used for gene therapy treatment and/or prevention of a disease, disorder or condition associated with deficiency or dysfunction of an ASPA polypeptide (e.g., Canavan disease), and of any other condition and or illness in which an upregulation of an ASPA gene may produce a therapeutic benefit or improvement, e.g., a disease, disorder or condition mediated by, or associated with, a decrease in the level or function of an ASPA polypeptide compared with the level or function of an ASPA polypeptide in an otherwise healthy individual.
• a disease, disorder or condition associated with deficiency or dysfunction of an ASPA polypeptide e.g., Canavan disease
• an upregulation of an ASPA gene may produce a therapeutic benefit or improvement, e.g., a disease, disorder or condition mediated by, or associated with, a decrease in the level or function of an ASPA polypeptide compared with the level or function of an A
• a vector genome and/or an rAAV vector comprising a modified nucleic acid encoding ASPA may be used for gene therapy treatment and/or prevention of a disease, disorder or condition associated with or caused by deficiency or dysfunction of an ASPA enzyme (e.g., Canavan disease), and of any other condition and/or illness in which an upregulation of an ASPA enzyme may produce a therapeutic benefit or improvement.
• methods of the disclosure include use of an rAAV vector, or a pharmaceutical composition thereof, in the treatment of Canavan disease in a subject.
• methods of the disclosure include use of an rAAV vector (e.g., AAV/OligoOOl-ASPA), or pharmaceutical composition thereof, to increase the level of ASPA in a subject in need thereof.
• a modified nucleic encoding ASPA may be used in the preparation of a medicament for use in the treatment and/or prevention of a disease, disorder or condition associated with or caused by deficiency or dysfunction of ASPA (e.g., a decreased level of functional ASPA enzyme such as in Canavan disease) and of any other condition or illness in which an upregulation of ASPA may produce a therapeutic benefit or improvement.
• a disease, disorder or condition associated with or caused by deficiency or dysfunction of ASPA e.g., a decreased level of functional ASPA enzyme such as in Canavan disease
• any other condition or illness in which an upregulation of ASPA may produce a therapeutic benefit or improvement.
• gene therapy treatment and/or prevention of a disease, disorder or condition associated with deficiency or dysfunction of an ASPA enzyme comprises administration of 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/OligoOOl-ASPA) comprising a modified nucleic acid encoding ASPA of the disclosure to a subject (e.g., a patient) in need of treatment.
• 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/OligoOOl-ASPA) comprising a modified nucleic acid ASPA of the disclosure may alleviate, ameliorate, treat, prevent or reduce the severity of one or more symptoms of Canavan disease as compared to a baseline measurement, such as a measurement in the same individual prior to initiation of treatment described herein, or a measurement in a control individual (or multiple control individuals thereby establishing a level for comparision) in the absence of the treatment described herein.
• a “control individual” is an individual afflicted with the same form of disease or injury as an individual being treated, but who is not currently being treated, but may receive treatment in the future.
• 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 may reduce NAA accumulation as compared to NAA accumulation in a control individual, or as compared to NAA accumulation in the same individual prior to treatment.
• NAA accumulation is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or by about 100% in a subject who is treated as compared to a control individual, or as compared with the same individual prior to treatment.
• 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 may increase aspartate and/or increase acetate levels as compared to aspartate and/or acetate levels in a control individual, or as compared to aspartate and/or acetate levels in the same individual prior to treatment.
• 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 by about 100% in a subject who is treated as compared to a control individual, or as compared with the same individual prior to treatment.
• treatment may also alleviate, ameliorate, treat, prevent or reduce the severity of degeneration of myelin 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 a delay in development of speech and motor skills as compared to the same in a control individual, or in a subject prior to treatment.
• 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 comprising a modified nucleic acid encoding ASPA of the disclosure may also increase, improve, prevent further loss of, or enhance balance, grip, strength and or motor coordination and generalized motor function as compared to the same in a control individual, or as compared to the same subject prior to treatment.
• 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 comprising a modified nucleic acid encoding ASPA of the disclosure may reduce vacuole volume fraction in the brain (e.g., thalamus, cerebellar white matter/pons), increase the number of oligodendrocytes in the brain (e.g., thalamus, cortex), increase the number of neurons in the brain (e.g., thalamus, cortex) and/or increase cortical myelination as compared to the same in a control individual, or as compared to the same subject prior to treatment.
• vacuole volume fraction in the brain e.g., thalamus, cerebellar white matter/pons
• oligodendrocytes in the brain e.g., thalamus, cortex
• neurons in the brain e.g., thal
• a subject appropriate for treatment includes any subject having, or at risk of, producing an insufficient amount, or having a deficiency of, a functional gene product (protein), or that produces an aberrant, partially functional or non-function gene product (protein, e.g., an enzyme), which can lead to disease.
• a patient is treated with a vector or pharmaceutical composition of the present disclosure prior to exhibiting any symptoms of a disease, disorder or condition (e.g., Canavan disease).
• a patient who has been diagnosed as at-risk for a disease, disorder or condition (e.g., Canavan disease) by genetic analysis is treated with an rAAV vector or composition of the present disclosure prior to exhibiting symptoms.
• a subject to be treated may be mammal, and in particular a subject is a human patient, for example, a patient with Canavan disease.
• a subject may be in need of 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, and thereby has decreased or no function, is expressed in the wrong tissues or at the wrong time, is under expressed or not expressed at all.
• a modified nucleic acid encoding ASPA of the present invention may be administered to enhance, improve or provide production of a functional ASPA enzyme which can, in turn, catalyze the breakdown of NAA to aspartate and acetate, among other biological functions as discussed elsewhere herein.
• a target cell of the rAAV vector of the instant invention is a cell, in particular an oligodendrocyte, this is normally, endogenously capable of expressing the ASPA enzyme, such as those of in the brain of a mammal.
• a pharmaceutical composition for preventing or treating a disease, disorder or condition mediated by or associated with decreased expression and/or activity of ASPA, e.g., Canavan disease.
• a 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.
• a pharmaceutical composition comprises a therapeutically effective amount of a vector (e.g., viral vector genome, expression vector, rAAV vector) or host cell comprising a modified nucleic acid encoding ASPA which can increase the level of expression and/or the level of activity of ASPA in a cell.
• a vector e.g., viral vector genome, expression vector, rAAV vector
• host cell comprising a modified nucleic acid encoding ASPA which can increase the level of expression and/or the level of activity of ASPA in a cell.
• a pharmaceutical composition comprises a therapeutically effective amount of a vector (e.g., viral vector genome, expression vector, rAAV vector) or host cell (e.g., for ex vivo gene therapy) comprising a modified, nucleic acid encoding ASPA wherein the composition further comprises a pharmaceutically-acceptable carrier, adjuvant, diluent, excipient and/or other medicinal agents.
• a pharmaceutically acceptable carrier, adjuvant, diluent, excipient or other medicinal agent is one that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing undesirable biological effects which outweigh the advantageous biological effects of the material.
• Any suitable pharmaceutically acceptable carrier or excipient can be used in the preparation of a pharmaceutical composition according to the invention (See e.g., Remington The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997).
• a pharmaceutical composition is typically sterile, pyrogen-free and stable under the conditions of manufacture and storage.
• a pharmaceutical composition may be formulated as a solution (e.g., water, saline, dextrose solution, buffered solution, or other pharmaceutically sterile fluid), microemulsion, liposome, or other ordered structure suitable to accommodate a high product (e.g., viral vector particles, microparticles or nanoparticles) concentration.
• a pharmaceutical composition comprising a modified nucleic acid, vector genome comprising the modified nucleic acid, host cell or rAAV vector of the disclosure is formulated in water or a buffered saline solution.
• a carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• Proper fluidity can be maintained, for example, by use of a coating such as lecithin, by maintenance of a required particle size, in the case of dispersion, and by the use of surfactants.
• Prolonged adsorption of an injectable composition can be brought about by including, in the composition, an agent which delays absorption, e.g., a monostearate salt and gelatin.
• a nucleic acid, vector and/or host cell of the disclosure may be administered in a controlled release formulation, for example, in a composition which includes a slow release polymer or other carrier that protects the product against rapid release, including an implant and microencapsulated delivery system.
• a pharmaceutical composition of the disclosure is a parenteral pharmaceutical composition, including a composition suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV) and/or intraci sternal magna (ICM) administration.
• a pharmaceutical composition comprising an rAAV vector comprising a modified nucleic acid encoding ASPA is formulated for administration by ICV injection.
• an rAAV vector (e.g., AAV/OligOOl ASPA) is formulated in 350 mM NaCl and 5% D-sorbitol in PBS.
• a modified nucleic acid encoding a transgene e.g., ASPA
• a vector e.g., vector genome, rAAV vector
• a target cell of a vector of the present disclosure includes cells of the CNS, preferably oligodendrocytes.
• a vector can be administered in addition to, and as an adjunct to, the standard of care treatment. That is, the vector can be co-administered with another agent, compound, drug, treatment or therapeutic regimen, either simultaneously, contemporaneously, or at a determined dosing interval as would be determined by one skilled in the art using routine methods. Uses disclosed herein include administration of an rAAV vector of the disclosure at the same time, in addition to and/or on a dosing schedule concurrent with, the standard of care for Canavan disease as known in the art.
• a combination composition includes one or more immunosuppressive agents.
• a combination composition includes an rAAV vector comprising a transgene (e.g., a modified nucleic acid encoding ASPA) and one or more immunosuppressive agents.
• a method includes administering or delivering an rAAV vector comprising a transgene (e.g., a modified nucleic acid encoding ASPA) to a subject and administering an immunosuppressive agent to the subject either prophylactically prior to administration of the vector, or after administration of the vector (i.e., either before or after symptoms of a response against the vector and/or the protein provided thereby are evident).
• an rAAV of the invention can be co-administered with empty capsids (i.e., a virus capsid that does not contain a nucleic acid molecule or vector genome) comprising the same, or a different, capsid protein as an rAAV vector comprising a modified nucleic acid (e.g., encoding ASPA).
• empty capsids i.e., a virus capsid that does not contain a nucleic acid molecule or vector genome
• a modified nucleic acid e.g., encoding ASPA
• an empty capsid may serve as an immune decoy allowing an rAAV vector comprising a modified nucleic acid (e.g., encoding ASP A) to avoid a neutralizing antibody (Nab) immune response as discussed in, e.g., WO 2015/013313.
• a modified nucleic acid e.g., encoding ASP A
• Nab neutralizing antibody
• a vector of the disclosure is administered systemically.
• exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intraarterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic and intramuscular administration, and the like, as well as direct tissue or organ injection.
• intravenous e.g., portal vein
• intraarterial e.g., femoral artery, hepatic artery
• intravascular subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic and intramuscular administration, and the like, as well as direct tissue or organ injection.
• systemic administration can deliver a modified nucleic acid (e.g., a modified nucleic acid encoding ASP A) to all tissues.
• direct tissue or organ administration includes administration to the liver.
• direct tissue or organ administration includes administration to areas directly affected by ASPA deficiency (e.g., brain and/or central nervous system).
• vectors of the disclosure, and pharmaceutical compositions thereof are administered to the brain parenchyma (i.e., by intraparenchymal administration), to the spinal canal or the subarachnoid space so that it reaches the cerebrospinal fluid (CSF) (i.e., by intrathecal administration), to a ventricle of the brain (i.e., by intracerebroventricular administration) and/or to the cistema magna of the brain (i.e., by intraci sternal magna administration).
• CSF cerebrospinal fluid
• a vector of the present disclosure comprising a modified nucleic acid encoding ASPA is administered by direct injection into the brain (e.g., into the parenchyma, ventricle, cisterna magna, etc.) and/or into the CSF (e.g., into the spinal canal or subarachnoid space) to treat a neurodegenerative aspect of Canavan disease.
• a target cell of a vector of the present disclosure includes a cell located in the cortex, subcortical white matter of the corpus callosum, striatum and/or cerebellum.
• a target cell of a vector of the present disclosure is an oligodendrocyte.
• Additional routes of administration may also comprise local application of a vector under direct visualization, e.g., superficial cortical application, or other nonstereotaxic application.
• a vector of the disclosure is administered by at least two routes.
• a vector is administered systemically and also directly into the brain.
• a modified nucleic acid encoding ASP A a vector genome comprising a modified nucleic acid encoding ASPA and/or an rAAV vector comprising a modified nucleic acid encoding ASPA of the disclosure, may be used for transduction of a cell ex vivo or for administration directly to a subject (e.g., directly to the CNS of a patient with Canavan disease).
• a transduced cell e.g., a host cell
• a disease, disorder or condition e.g., cell therapy for Canavan disease
• An rAAV vector comprising a modified therapeutic nucleic acid e.g., encoding ASPA
• a biologically-effective amount of a vector is an amount that is sufficient to result in transduction and expression of a modified nucleic acid encoding ASPA (i.e., a transgene) in a target cell.
• the disclosure includes a method of increasing the level and/or activity of ASPA in a cell by administering to a cell (in vivo , in vitro or ex vivo) a modified nucleic acid encoding ASPA, either alone or in a vector (including a plasmid, a virus vector, a nanoparticle, a liposome, or any known method for providing a nucleic acid to a cell).
• a cell in vivo , in vitro or ex vivo
• a modified nucleic acid encoding ASPA either alone or in a vector (including a plasmid, a virus vector, a nanoparticle, a liposome, or any known method for providing a nucleic acid to a cell).
• the dosage amount of an rAAV vector depends upon, e.g., the mode of administration, disease or condition to be treated, the stage and/or aggressiveness of the disease, individual subject's condition (age, sex, weight, etc.), particular viral vector, stability of protein to be expressed, host immune response to the vector, and/or gene to be delivered.
• doses range from at least 1 x 10 8 , or more, e.g., 1 x 10 9 , l x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 or more vector genomes (vg) per kilogram (kg) of body weight of the subject to achieve a therapeutic effect.
• a modified nucleic acid encoding ASPA may be administered as a component of a DNA molecule (e.g., a recombinant nucleic acid) having a regulatory element (e.g, a promoter) appropriate for expression in a target cell (e.g., oligodendrocytes).
• the modified nucleic acid encoding ASPA may be administered as a component of a plasmid or a viral vector, such as an rAAV vector.
• An rAAV vector may be administered in vivo by direct delivery of the vector (e.g., directly to the CNS) to a patient (e.g., a Canavan patient) in need of treatment.
• An rAAV vector may be administered to a patient ex vivo by administration of the vector in vitro to a cell from a donor patient in need of treatment, followed by introduction of the transduced cell back into the donor (e.g., cell therapy).
• the present disclosure includes a method of administration that results in a level of mRNA encoding ASP A, a level of ASPA protein expression, and/or a level of ASPA activity that is detectably greater than the level of ASPA expression (mRNA and/or protein) or ASPA activity in an otherwise identical cell that is not administered a modified nucleic acid (e.g., a modified nucleic acid encoding ASPA).
• the present disclosure includes a method of administration that results in a level of mRNA encoding functional ASPA, and/or a level of functional (e.g., biologically active) ASPA protein expression, that is detectably greater than the level of functional ASPA (mRNA and/or protein) present in an otherwise identical cell that is not administered the modified nucleic acid (e.g., a modified nucleic acid encoding ASPA). That is, the present invention includes method of increasing the level of functional ASPA in a cell where the cell produces a normal level of ASPA but the ASPA protein lacks activity or demonstrates decreased activity compared with normal wild type ASPA.
• a level of functional ASPA protein expression that is detectably greater than the level of functional ASPA (mRNA and/or protein) present in an otherwise identical cell that is not administered the modified nucleic acid (e.g., a modified nucleic acid encoding ASPA). That is, the present invention includes method of increasing the level of functional ASPA in a cell where the cell produces a normal level of ASPA
• a cell can be cultured or grown in vitro, or can be present in an organism ( i.e ., in vivo). Further, a cell may express endogenous ASPA such that the level of ASPA in the cell is increased, and/or the cell expresses an endogenous ASPA that is a mutant or variant of wild type ASPA, e.g., ASPA having the sequence of SEQ ID NO:3, especially as there may be more than one wild type allele for human ASPA. Thus, the level of ASPA is increased as compared with the level of ASPA expressed in an otherwise identical, but untreated cell.
• kits typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo or ex vivo, of the components therein.
• a kit can contain a collection of such components, e.g., a modified nucleic acid, a recombinant nucleic acid, a vector genome, an rAAV vector an rAAV, and optionally a second active agent such as a compound, therapeutic agent, drug or composition.
• a kit refers to a physical structure that contains one or more components of the kit.
• Packaging material can maintain the components in a sterile manner and can be made of material commonly used for such purposes (e.g., paper, glass, plastic, foil, ampules, vials, tubes, etc).
• a label or insert can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredients(s) including mechanism of action, pharmacokinetics and pharmacodynamics.
• a label or insert can include information identifying manufacture, lot numbers, manufacture location and date, expiration dates.
• a label or insert can include information on a disease (e.g., Canavan disease) for which a kit component may be used.
• a label or insert can include instructions for a clinician or subject for using one or more of the kit components in a method, use or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency of duration and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimens described herein.
• a label or insert can include information on potential adverse side effects, complications or reaction, such as a warning to a subject or clinician regarding situations where it would not be appropriate to use a particular composition.
• Example 1 Dose-Responsive Reduction in NAA Using an rAAV Vector Comprising a
• HEK Human embryonic kidney cells were transfected with 1.0 ug of plasmid expressing NAA synthase (Nat8L) and co-transfected with 0.1, 0.2, 0.5 or 1.0 pg of a plasmid comprising 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:l, 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.
• a dose-responsive reduction in NAA was observed in cultures transfected using the codon-optimized nucleic acid encoding ASPA of SEQ ID NO:2 relative to the cultures transfected with either the wild-type nucleic acid encoding ASPA or the codon-optimized nucleic acid encoding ASPA of SEQ ID NO: 1 (FIG. 1) .
• Example 2 Biodistribution of an Oligotropic AAV/OligOOl [0248] This study was undertaken to define the most effective dose and route of administration (ROA) of an oligotropic AAV (AAV/OligOOl; (WO2014/052789; Powell et al. (2016) Gen. Ther.23:807-814)) capsid variant in promoting widespread CNS oligodendrocyte transduction in a mouse model of the inherited human leukodystrophy, Canavan disease.
• ROA dose and route of administration
• AAV/OligOOl Three doses of AAV/OligOOl, delivered via four distinct ROA were tested in adult, symptomatic Canavan mice (nur7), and vector spread and transduction quantified two weeks post-transduction by generating stereological estimates of reporter green fluorescent protein (GFP) positive cells in four anatomical regions of interest.
• GFP reporter green fluorescent protein
• ROA employed were intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV), and intracistema magna (ICM).
• AAV/OligOOl vector containing a constitutive expression cassette for a GFP reporter gene were produced (Lot# 7660 and Lot# LAV38A). All vector produced contained a GFP reporter gene driven by a hybrid CMV/chicken B-actin promoter (CBh) flanked by self-complimentary AAV ITRs. Vector was produced by transient transfection of HEK293 cells followed by iodixanol gradient centrifugation and ion-exchange chromatography (Gray et ak, (2013) Gene Ther. 20:450-9). Concentration of vector was defined as total numbers of viral vector genomes (vg), determined by qPCR quantification of DNAse-resistant AAV inverted terminal repeat (ITR) sequence in the stock preparation. [0251] Animals
• AAV/OligOOl -GFP diluted to the appropriate concentration in 0.9% saline, was delivered by stereotaxic injection to 6-week old nur7 mutant mice under inhalation anesthesia (4% induction and maintenance titered to effect.
• ROA route of administration
• ICM intracisterna magna
• IP ROA required 5x injections of 1 pL of vector at 5 stereotaxic coordinates, two in each hemisphere to anterior and posterior subcortical white matter (i.e., 4 injections total to the cingulum) and 1 additional injection in cerebellar white matter (to give a total of 5) at a rate of O.lpL/min using a digital pump.
• IT ROA animals received a single 5 pL infusion of vector into the subarachnoid space accessed via lumbar puncture between L5 and L6.
• ICV ROA animals received two 2.5 pL injections of vector, one in each lateral ventricle at a rate of O.lpL/min.
• ICM ROA animals received 5 pL of vector delivered directly to the CSF via the cistema magna at a rate of O.lpL/min. All animals received 0.5 mL 20% mannitol (ip) 20 minutes prior to surgery. All animals were group-housed for two weeks following AAV/OligOOl-GFP delivery then sacrificed for post mortem analyses.
• GFP-positive soma in the cortex, subcortical white matter, striatum, and cerebellum were scored by unbiased stereology using the optical fractionator method (FIG. 2) (West et al. Anat. Rec. (1991) 231:482-97).
• Stereology software (Stereologer, Stereology Resource Center) coupled to an upright bright field microscope fitted with a motorized stage was used to generate counts of GFP-positive soma within four different regions of interest, namely, the cerebral cortex, subcortical white matter of the corpus callosum and external capsule, striatum, and cerebellum.
• CE coefficient of error
• CV total variance
• Vector tropism in AAV/OligOOl-GFP transduced brains was quantified by scoring for lineage-specific antigen colabeling with GFP fluorescence. Alternate sections were processed for NeuN (which is present in most CNS and PNS neuronal cell types of vertebrates), GFAP (glial fibrillary acidic protein), or 01ig2 (oligodentrocyte lineage transcription factor 2) immunohistochemistry using commercially available antibodies (Sigma/Millipore) to label neurons, astrocytes, and oligodendrocytes, respectively. Scanning confocal microscopy was employed to generate multipoint image stacks throughout each region of interest.
• IP Intraparenchymal
• IP ROA animals were given 5 individual injections targeting subcortical white matter in both hemispheres and the cerebellum.
• Treated animals were sacrificed 2-weeks post-vector administration (8 weeks of age) and brains were processed for GFP immunohistochemistry and GFP-positive soma in the cortex, subcortical white matter, striatum and cerebellum were scored using unbiased stereology using the optical fractionator to provide absolute estimates of transduced cells in each region of interest. All three doses of AAV/OligOOl-GFP administered resulted in significant levels of transduction of cells throughout the brain. (FIG.
• the cortex presented the highest numbers of transduced cells (513,477), followed by subcortical white matter (178,362), cerebellum (86,820), and finally striatum (62,706).
• GFAP co-labeling was less than 2%.
• ICV administered AAV/OligOOl-GFP resulted in prominent transgene expression throughout all areas of interest, with particularly robust transduction of subcortical white matter notable (FIG. 5). All regions of interest displayed a dose responsive increase in numbers of transduced cells when the dose was increased from lxlO 10 to lxlO 11 , although there were subtle non-significant increases in most regions at the 5xl0 u dose compared to the lxlO 11 dose, except for the cerebellum.
• the cerebellum was the only region that presented with a further appreciable increase in GFP-positive cells in ICV ROA brains.
• a prominent point of difference between IP and ICV ROA brains that was evident throughout the sampling process was the greater distribution of vector in the ICV groups. Transgene expression at injection sites was more intense in IP brains but diluted rapidly from the site. By contrast, ICV transgene expression was relatively evenly distributed over a far greater area of the brain.
• ICM administration of AAV/OligOOl-GFP resulted in relatively widespread transgene yet modest transgene expression in the cortex, striatum and cerebellum.
• ICM brains like IT ROA brains, there was an absence of significant transgene expression in subcortical white matter of ICM brains (FIG. 6).
• ROA Routes of Administration
• IP and ICV ROA brains were comparable in absolute numbers of cells transduced by AAV/OligOOl-GFP in specific regions, the bulk of positive cell counts in IP brains were the product of sections immediately adjacent to injection sites, while positive cells in ICV brains were relatively evenly distributed.
• Systemic non-random stereological sampling allows for the identification of variance between sections sampled from individual brains (intrasample variance), and is represented as the coefficient of error (CE) in a dataset, calculated by the standard error of the mean of repeated estimates divided by the mean.
• CE is one half of total variance in a sampled population, with true biological variance (CV), or difference in the mean between individual brains, constituting the other half.
• the mean CE for individual IP brains was calculated as -12% of total variance, while that for ICV brains was -3%, meaning GFP-positive cells were more evenly distributed across all sections sampled in ICV brains.
• actual numbers of positive cells in individual sections sampled became fewer the further laterally from injection sites the sampled section was, while positive cells numbers in ICV brains were consistently closer to the intrasample mean in all sections sampled.
• the net result of this difference was a greater spread of vector in ICV ROA brains relative to IP brains, particularly in the cortex and subcortical white matter (FIG.
• a distinguishing characteristic of AAV/OligOOl is its clear oligotropism as compared to other AAV capsid variants (Powell et al. (2016) Gen. Ther. 23:807-814; Francis et al. (2016) Neurobiol. Dis. 96:323-334).
• AAV/OligOOl vectors must be capable of exhibiting this tropism when applied by different ROA. Oligotropism may vary due to variables such as age of intervention (Gholizadeh et al. Hum. Gene Ther. Methods (2013) 24:205-13; Foust et al. Nature Biotech.
• the cortex, subcortical white matter, striatum, and cerebellum used for the generation of absolute numbers of GFP-positive cells were analyzed for co-labeling of GFP transgene with the lineage specific antigens 01ig2 (i.e., target specific labeling for oligodendrocytes) and NeuN (i.e., target specific labeling for neurons).
• lineage specific antigens 01ig2 i.e., target specific labeling for oligodendrocytes
• NeuN i.e., target specific labeling for neurons
• IP ROA did not affect neurotropism at the expense of oligotropism.
• IP ROA did not affect neuronal transduction in this ROA.
• ICM cohort When assessed against the ROA cohort presenting the poorest levels of GFP transgene expression, the ICM cohort, ICV administration resulted in an increase in AAV/OligOOl -transduced oligodendrocytes of over 200,000 cells per brain.
• a group of healthy wild type animals, age matched to nur7 ROA cohorts (i.e. 6 weeks of age) were transduced with lxlO 11 vg of AAV/OligOOl-GFP via the ICV ROA, and sacrificed 2 weeks later for generation of stereological estimates of GFP-positive cells within the cortex and subcortical white matter tracts (FIG.8).
• Subcortical white matter GFP transgene expression in wild type brains was very much restricted to regions immediately surrounding the lateral ventricles in wild type brains, while cortical expression, although reasonably diffuse, was very modest in absolute number of transduced cells.
• Examples 2 and 3 demonstrate that intracerebroventricular (ICV) route of administration of the AAV/OligOOl GFP vector provided the best combination of vector spread and oligodendrocyte tropism.
• ICV intracerebroventricular
• this ROA appears well suited to the transduction of subcortical white matter, the tissue impacted by Canavan disease pathology.
• the ability to transduce hundreds of thousands of cells, and maintain a near 100% tropism for oligodendrocytes confers a significant advantage to AAV/OligOOl over other AAV capsids.
• CSF-targeted ROA namely IT and ICM
• IP brains approached comparable levels of transduction in terms of numbers of cells transduced, but the majority of these cells were concentrated about injection sites. Cells at these sites likely had a greater vector genome copy number/cell of any other ROA, but vector spread away from these sites was markedly lower as compared to the ICV ROA.
• the broader distribution of the GFP transduction by ICV administration is advantageous in the appropriate balance may be achieved between the number of cells transduced and the number of copies of the vector per transduced cell.
• Vector tropism in all regions of interest was 75-90% oligotropic, with the exception of the cerebellum. This region presented with >80% neurotropism in all ROA groups. Particularly strong transgene expression was seen in granule layer purkinje neurons. The reason for this apparent reversal of tropism is not readily apparent, but the cerebellum is clearly a distinct anatomical entity with respect to resident cell types. Purkinje cells within the cerebellum express 01ig2 at low but appreciable levels, and it is possible that the AAV/OligOOl capsid has a markedly different interaction with the Purkinje neurons surface than the surface of other neurons in other regions of the brain.
• Example 4 Difference in Efficiency of AAV/OligOOl-GFP Transduction between Wild Type and nur7 Brains.
• the nur7 mouse model of Canavan disease manifests symptoms of gross motor dysfunction at 2 weeks of age.
• the nur7 brain has suffered significant cell loss, loss of white matter and is extensively vacuolated.
• the 6-week nur7 brain is therefore a markedly different microenvironment than a healthy brain, possibly influencing AAV/OligOOl -GFP spread and transduction.
• AAV/OligOOl-ASPA comprising the codon-optimized ASPA sequence of SEQ ID NO:2.
• the expression plasmid encoding the codon-optimized ASPA and regulatory elements is shown in FIG. 13.
• a total dose of 2.5xlO u , 7.5xl0 10 or 2.5xl0 10 vg was administered via the intracerebroventricular (ICV) route of administration (ROA).
• ROI intracerebroventricular
• Vector for all dose cohorts was delivered in a total volume of 5pl, with 2.5m1 injected in the lateral ventricle of each hemisphere of the brain.
• a control cohort of age-matched nur7 animals was generated by injection of an equivalent volume of physiological saline via the same ROA.
• Age-matched naive wild type animals were used as a calibration reference for all motor function testing. Two weeks after administration of vector, animals were tested once a month for four months for latency to fall from an accelerating rotarod and for generalized activity using open field activity chambers. All behavioral tests were performed by individuals blinded to treatment.
• AAV/OligOOl-ASPA rescued progressively deteriorating balance, grip strength and/or motor coordination as measured by rotarod performance in nur7 mice to a level indistinguishable from age-matched wild type animals and highly significantly improved over sham nur7 controls.
• AAV/OligOOl-ASPA was effective at promoting improved rotarod performance in the last two time points tested only (18 and 22 months).
• Table 1 provides mean latency to fall measured in seconds for each treatment group (with standard deviation). For each group 12 mice (6 male and 6 female) were tested.
• Table 2 provides p- values for unpaired t-test comparisons between AAV/OligOOl-ASPA treated and sham nur7 mice at each age. Statistically significant improvements were observed in all groups except for mice administered 2.5 x 10 10 vs. sham treated mice at 10 and 14 weeks.
• FIG. 14 shows plotted rotarod mean latency to fall over the course of in-life study period for each AAV/OligOOl-ASPA nur7 dose cohort, sham nur7, and naive wild type controls. Latency to fall was increased in all 3 dose cohorts, with the highest dose being significant over the whole study period by repeat measures ANOVA (*).
• the lowest (2.5xl0 10 ) dose of AAV/OligOOl-ASPA did not significantly normalize pathological hyper- activity and more closely resembled sham nur7 controls than wild type references.
• NAA accumulation and vector genome (vg) copy number [0305] Following rotarod testing at 22 weeks, mice were sacrificed, and brain tissue was isolated. One hemisphere of each brain was processed for the HPLC analysis of NAA, and the remaining hemisphere processed for analysis of vector genome (vg) copy number by quantitative PCR.
• the hemispheres remaining from brains analyzed for NAA were used to quantify vector genome (vg) copy number by quantitative PCR using a custom TaqMan probe/primer set targeted to the bovine growth hormone (BGH) polyadenylation sequence of the recombinant AAV/OligOOl-ASPA expression cassette.
• BGH bovine growth hormone
• Total DNA content of hemispheres was isolated using commercially available DNA purification columns and kits (Qiagen) and samples of DNA thus generated run against a purified plasmid standard curve to generate vg/wet tissue weight for each sample.
• VG/mg of tissue values generated reflected the dose of AAV/OligOOl-ASPA administered (FIG. 17), consistent with the response of NAA to vector dose.
• sham treated nur7 mouse brains In the cortex (motor and somatosensory), neuronal loss in sham treated nur7 mouse brains relative to age-matched wild type mouse brains was less profound, but significant.
• Successive doses of AAV/OligOOl-ASPA resulted in a stable 1.2-fold increase in cortical neurons relative to sham treated.
• At the lowest 2.5xl0 10 dose, AAV/OligOOl-ASPA treated mice maintained a significant 1.2-fold increase in cortical neurons over sham treated controls (p 0.05).
• MBP-LD cortical myelin basic protein-positive fiber length density
• Example 6 CLARITY-aided biodistribution for Canavan sene therapy
• OligOOl oligodendrocyte-tropic rAAV vector
• GFP Green Fluorescent Protein
• Intracerebroventricular (ICV) and intraparenchymal (IP) ROAs were compared for biodistribution efficacy and this method was used to supplement conventional stereology data obtained from traditional, two-dimensional (2D) histological evaluation.
• This example demonstrates the applicability of the 3D method, and its significance in assessing AAV/OligOOl-GFP biodistribution, in adult murine hemibrains of Canavan disease mouse models. Results are presented as visual qualitative and quantitative representations of 3D cleared brain images of lightsheet microscopy data and tabulated parameters of biodistribution estimations.
• the raw dataset was preprocessed and reconstructed into a full, seamless 3D image using an in-house custom designed algorithm for each hemibrain.
• Final images each contained one hemibrain and were imported into a commercial 3D image processing and analysis program (Imaris, Bitplane) for a global, quantitative biodistribution analysis.
• GFP global average and median
• two GFP intensity thresholds were chosen to designate “low” or “high” GFP expression (FIG. 27). These thresholds were then kept constant across all samples for consistency. The volumes of these classified intensity regions were then determined and compared to the full hemibrain volume to give rise to the “vol% high/low expression” (Table 3).
• volumetric imaging of intact, tissue clarified, murine brains provide a more comprehensive and holistic assessment of AAV/OligOOl biodistribution.
• the custom algorithms to enable full acquisition and quantification of the distribution supports higher- resolution quantification obtained from stereology methods.
• Assessment of organ-level imaging provides global evaluation of this biodistribution retaining 3D spatial structural and regional connectivity.
• the digital compilation of various ROAs can be used to generate a digital ‘library’ to be used for future references when performing additional assessments into AAV/OligOOl ROAs to assess optimal transduction efficiencies and cell- type specific tropism.
• Example 7 CLARITY-based volumetric assessment of AAV biodistribution and pharmacodynamic effect
• Example 6 the CLARITY tissue clearing technique described in Example 6 above was utilized to assess and demonstrate the global and local transgene-mediated pharmacological effect of reversal in demyelination after injection with AAV/OligOOl -ASP A in nur7 mouse brains.
• FIG. 32A Cell counting analysis was also carried out in extracted 2D single slices of 3D images from all three groups with similar anatomical orientation (FIG. 32A). As shown in FIGs. 32B and 32C, although average nuclei density (counts normalized by segmentation area) showed overall little difference in cell density within the cortical region, mice of the Nur7 group had a significantly lower overall nuclei density/nuclei area in the thalamic region. In contrast, the Oligl-ASPA group and the WT group appeared to have similar overall nuclei densities or nuclei areas in the thalamic regions.
• Region-based analyses were performed in 3D in the thalamic region. A manual segmentation of a portion of the region was shown in FIG. 33E. Average fluorescence intensities within this region for both nuclei (SYTO) and myelin (MBP) markers were shown in FIG. 33F. It was found that the SYTO and MBP levels of the Oligl-ASPA group almost reached to the levels of the WT group. In contrast, the Nur7 samples exhibited lower average fluorescence values in both markers. Region-based analyses were also performed on a portion of the cortex. Shown in FIG. 33G and 33H were average fluorescence intensity levels within this cortical region for both nuclei (SYTO) and myelin (MBP) markers.

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