GENETIC INSTABILITY OF A MOMLV-BASED ANTISENSE DOUBLE-COPY RETROVIRALVECTOR DESIGNED FOR HIV-1 GENE-THERAPY

We constructed a retroviral vector encoding a mutant tRNA,(met) gene f ollowed 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 hum an lymphoblastoid T-cell lines (CEM, Jurkat). In some clonal cell line s the expected short transcript initiated either from the 5' or 3' LTR tRNA-alpha rev gene was not detectable by Northern blot analyses of t ransduced, G418-resistant cells with an alpha rev-specific oligonucleo tide probe. In other clonal cells, neither the short polymerase III tr anscript nor the full-length genomic polymerase II transcript (contain ing the 3' LTR tRNA-alpha rev gene) was detectable when compared with the transduced cell pool. Southern blot and DNA-polymerase chain react ion (PCR) analyses specific for the tRNA-alpha rev cassette in the 5' or 3' LTR of the retroviral vector suggested hat 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 d emonstrating deletions and insertions. In summary, our results indicat e that this retroviral vector design with direct repeats flanking the polymerase III transcription unit plus the alpha rev insert is prone t o genetic rearrangements and consequently not useful for the developme nt of gene therapy protocols.