E- vectors: development of novel self-inactivating and self-activating retroviral vectors for safer gene therapy.
We have developed novel self-inactivating and self-activating retroviral vectors based on the previously observed high-frequency deletion of direct repeats. We constructed spleen necrosis virus (SNV)-based viral vectors that contained large direct repeats flanking the viral encapsidation sequence (E). A large proportion of the proviruses in the target cells had E and one copy of the direct repeat deleted. Direct repeats of 1,333 and 788 bp were deleted at frequencies of 93 and 85%, respectively. To achieve a 100% deletion efficiency in target cells after ex vivo infection and drug selection, we constructed a self-activating vector that simultaneously deleted E and reconstituted the neomycin phosphotransferase gene. Selection of the target cells for resistance to G418 (a neomycin analog) ensured that all integrated proviruses had E deleted. The proviruses with E deleted were mobilized by a replication-competent virus 267,000-fold less efficiently than proviruses with E. We named these self-inactivating vectors E- (E-minus) vectors. These vectors should increase the safety of retroviral vector-mediated gene therapy by preventing the spread of vector sequences to nontarget cells in the event of coinfection with helper virus. We propose that direct-repeat deletions occur during RNA-dependent DNA synthesis and suggest that template switches occur without a requirement for RNA breaks. The minimum template dissociation frequency was estimated as 8%/100 bp per replication cycle. These vectors demonstrate that large direct repeats and template-switching properties of reverse transcriptase can be utilized to delete any sequence or reconstitute genes during retroviral replication.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015316:Genetic Therapy / Q000379:methods* / Q000592:standards; D005822:Genetic Vectors; D006801:Humans; D019868:Kanamycin Kinase; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis; D011533:Proviruses / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012190:Retroviridae / Q000235:genetics; D012449:Safety; D017384:Sequence Deletion; D014162:Transfection; D016662:Virus Integration
D012367:RNA, Viral; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase