       Document 0731
 DOCN  M9640731
 TI    Genetic instability of a MoMLV-based antisense double-copy retroviral
       vector designed for HIV-1 gene therapy.
 DT    9604
 AU    Junker U; Bohnlein E; Veres G; Progenesys, Department of Molecular
       Therapy, Palo Alto, CA 94304,; USA.
 SO    Gene Ther. 1995 Nov;2(9):639-46. Unique Identifier : AIDSLINE
       MED/96129243
 AB    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.
 DE    Animal  Base Sequence  Cell Line  DNA Mutational Analysis  DNA
       Polymerase III/GENETICS  DNA, Antisense/GENETICS  DNA,
       Recombinant/GENETICS  Gene Products, rev/*GENETICS  Gene
       Therapy/*METHODS  *Gene Transfer  Genetic Vectors/*GENETICS  Human  HIV
       Infections/THERAPY  HIV Long Terminal Repeat/GENETICS  HIV-1/*GENETICS
       Mice  Molecular Sequence Data  Moloney Leukemia Virus/*GENETICS
       Proviruses/GENETICS  RNA, Messenger/ANALYSIS  RNA, Transfer,
       Met/GENETICS  RNA, Viral/ANALYSIS  Sequence Deletion  T-Lymphocytes
       Transcription, Genetic  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

