Genetic instability of a MoMLV-based antisense double-copy retroviral vector designed for HIV-1 gene therapy.
We constructed a retroviral vector encoding a mutant tRNA(imet) gene followed by a HIV-1 rev-specific antisense sequence in the U3 region of the 3' long terminal repeat (LTR). This Moloney murine leukemia virus (MoMLV)-based double-copy retroviral vector was used to transduce human lymphoblastoid T-cell lines (CEM, Jurkat). In some clonal cell lines the expected short transcript initiated either from the 5' or 3' LTR tRNA-alpha rev gene was not detectable by Northern blot analyses of transduced, G418-resistant cells with an alpha rev-specific oligonucleotide probe. In other clonal cells, neither the short polymerase III transcript nor the full-length genomic polymerase II transcript (containing the 3' LTR tRNA-alpha rev gene) was detectable when compared with the transduced cell pool. Southern blot and DNA-polymerase chain reaction (PCR) analyses specific for the tRNA-alpha rev cassette in the 5' or 3' LTR of the retroviral vector suggested that the transfer of the 3' LTR U3 region to the 5' LTR was incorrect in most proviruses. These data were confirmed by DNA sequence analyses of several clonal lines demonstrating deletions and insertions. In summary, our results indicate that this retroviral vector design with direct repeats flanking the polymerase III transcription unit plus the alpha rev insert is prone to genetic rearrangements and consequently not useful for the development of gene therapy protocols.
Progenesys, Department of Molecular Therapy, Palo Alto, CA 94304, USA.
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Junker
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Böhnlein
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Veres
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D004258:DNA Polymerase III / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D015694:Gene Products, rev / Q000235:genetics*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D015658:HIV Infections / Q000628:therapy; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D011533:Proviruses / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012358:RNA, Transfer, Met / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis; D017384:Sequence Deletion; D013601:T-Lymphocytes; D014158:Transcription, Genetic; D054321:rev Gene Products, Human Immunodeficiency Virus
D016373:DNA, Antisense; D004274:DNA, Recombinant; D015694:Gene Products, rev; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D004258:DNA Polymerase III