Engineered long terminal repeats of retroviral vectors enhance transgene expression in hepatocytes in vitro and in vivo.
To analyze the important elements for retroviral expression in hepatocytes, cis-acting elements in the U3 region of the long terminal repeat (LTR) of the polycythemic strain of spleen focus-forming virus (SFFVp) were analyzed in a hepatocellular carcinoma cell line. Two cis-acting elements located within the upstream region of the direct repeat, which positively regulated retroviral expression, were identified. Transcription factors NFAT5 and Sp1, which are ubiquitously expressed in a variety of tissues, bound to these elements. To increase specificity without lowering the potency of retroviral expression in hepatocytes, these elements were replaced by a sequence derived from the hepatitis B virus enhancer II region. Novel vectors, SF-Hep3 and SF-Hep5 (SFFVp-based vector for hepatocytes 3 and 5), were developed with these engineered LTRs. The engineered LTRs of these vectors enhanced the retroviral expression only in hepatocellular carcinoma cell lines in vitro. These vectors also increased transgene expression 4- to 9-fold or 3.5- to 5-fold in comparison with a Moloney murine leukemia virus-based vector or a vector containing the wild-type LTR of SFFVp, respectively, in murine hepatocytes in vivo.
Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, 606-8507, Kyoto, Japan.
Kanji
K
Yamaguchi
Katsuhiko
K
Itoh
Naoki
N
Ohnishi
Yoshito
Y
Itoh
Christopher
C
Baum
Takashi
T
Tsuji
Toshikazu
T
Nagao
Hiroaki
H
Higashitsuji
Takeshi
T
Okanoue
Jun
J
Fujita
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics; D013155:Spleen Focus-Forming Viruses / Q000235:genetics; D020079:Terminal Repeat Sequences; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D019076:Transgenes / Q000235:genetics
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase