Method for generating replication defective viral vectors that are helper free
AssigneeThe Rockefeller University
InventorsMichael G. Kaplitt, Sergei Moussatov
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Abstract
Sequences are provided that are capable of directing circular adeno-associated virus replication, useful in vectors for providing therapeutic agents to a subject in need thereof. The vectors of the invention are particularly useful in the treatment of acute medical conditions requiring rapid gene expression. Further provided are methods for producing packaged defective viral vectors.
Claims42
A nucleotide sequence capable of directing circular adeno-associated virus replication, comprising a loop sequence TGGCCAA flanked on the 5′ and 3′ sides by complementary sequences, wherein a hairpin structure is formed between the complementary sequences.
A defective helper vector for use in the production of a packaged defective viral vector;
- wherein the defective helper vector:
- (a) requires the expression and/or transcription of one or more exogenous nucleic acid(s) to replicate;
- and
- (b) comprises one or more helper heterologous nucleic acid that aids in the replication and/or packaging of a defective viral vector.
A mammalian cell comprising a plasmid an Epstein-Barr viral origin of replication and AAV genes Rep and Cap.
A method for generating a production stock of packaged defective viral vectors (dvv) and packaged defective helper vectors (dhlpv), the method comprising placing a defective helper vector and a defective viral vector into a permissive cell, wherein the defective viral vector and the defective helper vector are replicated and packaged;
- wherein the dhlpv comprises one or more helper heterologous nucleic acid(s), the expression and/or transcription of which are necessary but not sufficient for the replication or packaging of the defective viral vector in the permissive cell;
- and wherein the dhlpv further requires the expression and/or transcription of one or more exogenous nucleic acid(s) to replicate and be packaged;
- wherein the permissive cell comprises the exogenous nucleic acid(s) required to replicate and package the dhlpv, and further comprises one or more ancillary heterologous nucleic acids, the expression and/or transcription of which in conjunction with the expression and/or transcription of the helper heterologous nucleic acid(s) enables the replication and/or packaging of the defective viral vector in the permissive cell;
- and wherein a production stock of packaged defective helper vector and packaged defective viral vector is generated.
A mammalian cell comprising:
- (i) nucleic acid sequences encoding Cap, E4orf6 and E2a under the control of a first inducible promoter;
- (ii) a nucleic acid sequence encoding Rep under the control of a second inducible promoter;
- and
- (iii) nucleic acid sequences encoding VAI RNA and E1A.
Dependencies are inferred from the claim text.