Method for generating replication defective viral vectors that are helper free

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.

1 . 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. 
     
     
         2 - 7 . (canceled) 
     
     
         8 . A defective circular adeno-associated virus-derived vector comprising (i) at least one of the nucleotide sequence of  claim 1 , and (ii) a heterologous nucleic acid sequence encoding a protein of interest. 
     
     
         9 - 12 . (canceled) 
     
     
         13 . The vector of  claim 8 , wherein the protein of interest is a therapeutic protein consisting of a protein, an enzyme, or a growth factor. 
     
     
         14 . The vector of  claim 13 , wherein the therapeutic protein is insulin, β-globin, p53, or ARF-P19. 
     
     
         15 . The vector of  claim 13 , wherein the therapeutic enzyme is selected from the group consisting of adenosine deaminase, α-antitrypsin, 5-α reductase, 17-α reductase, hypoxanthine guanine phosphoribosyl transferase, ornithine transcarbamolase, tyrosine hydroxylase, hexosamindase A, and acid cholesterylester hydrolase. 
     
     
         16 . A method of treating an acute medical condition in a subject in need thereof, comprising administering a circular adeno-associated virus (cAAV)-derived vector comprising the nucleotide sequence of  claim 1 , and a nucleic acid sequence encoding a therapeutic protein of interest operably linked to a promoter sequence, wherein the therapeutic protein is expressed within 1 day after administration of the cAAV-derived vector. 
     
     
         17 . The method of  claim 16 , wherein expression is achieved within 8-24 hours after administration. 
     
     
         18 . The method of  claim 17 , wherein expression is achieved within 8-12 hours. 
     
     
         19 . The method of  claim 18 , wherein expression is increased 10 fold within 48 hours. 
     
     
         20 . 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.   
     
     
         21 - 30 . (canceled) 
     
     
         31 . 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.   
     
     
         32 . The method of  claim 31 , wherein the dvv further comprises a heterologous nucleic acid of interest. 
     
     
         33 . A production stock generated by the method of  claim 31 . 
     
     
         34 . The method of  claim 31 , wherein the defective helper vector is a herpes simplex virus (HSV); the exogenous nucleic acid is a HSV ICP4 gene, and the helper heterologous nucleic acids are adenoviral genes E1A, E2a, E4orf6, and VAI RNA. 
     
     
         35 . The method of  claim 31 , wherein the permissive cell comprises a plasmid that has an Epstein-Barr Viral origin of replication and encodes ancillary heterologous nucleic acids AAV Rep and Cap; and wherein the cell expresses the exogenous nucleic acid, HSV ICP4. 
     
     
         36 . A method of producing a helper-free defective viral vector comprising co-infecting the production stock of packaged dhlpv and dvv of  claim 33  into a non-permissive cell; wherein the nonpermissive cell 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 non-permissive cell; but wherein the replication and/or packaging of the dhlpv is prevented because the non-permissive cell does not comprise the exogenous nucleic acid(s). 
     
     
         37 . The method of  claim 34 , wherein the defective helper vector is a herpes simplex virus (HSV); the exogenous nucleic acid is the HSV ICP4 gene; the helper heterologous nucleic acids are the adenoviral genes E1A, E2a, E4orf6, and VAI RNA; the permissive cell comprises a plasmid that has an Epstein-Barr Viral origin of replication and encodes the ancillary heterologous nucleic acids AAV Rep and Cap. 
     
     
         38 . The helper-free defective viral vector produced by the method of  claim 36 . 
     
     
         39 . A non-human mammalian host transformed with the vector of  claim 38 . 
     
     
         40 . A method of delivering a gene of interest to a target tissue of an animal subject, comprising administering the vector of  claim 38  to the tissue of the animal subject. 
     
     
         41 . (canceled) 
     
     
         41 . (canceled)