Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

Important note: Information in this article was accurate in 1999. The state of the art may have changed since the publication date.

Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

Hum Gene Ther. 1998 Nov 20;9(17):2459-68. Unique Identifier : AIDSLINE MED/99068402
Fassati A; Bardoni A; Sironi M; Wells DJ; Bresolin N; Scarlato G; Hatanaka M; Yamaoka S; Dickson G; Department of Biochemistry, Royal Holloway College, University of; London, Egham, Surrey, United Kingdom.

Abstract: The use of retroviral vectors (RVs) derived from the murine oncoretroviruses for gene therapy is associated with the risk of malignant transformation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would increase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette containing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resulting retroviral vectors could be transcriptionally trans-activated by tax. In the absence of Tax, however, the viruses showed muscle-specific expression. Trans-complementing packaging and indicator cells stably expressing Tax were used to isolate high-titer producer cell clones (10(6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-expressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expression of the vectors and the endogenous muscle creatine kinase gene was similarly dependent on the maturity of the muscle cultures. One vector with modified LTRs was also tested in vivo in regenerating muscle and showed a delayed pattern of expression in myofibers compared with the vector containing the wild-type LTRs. These vectors can be easily modified to contain different tissue-specific enhancer and promoter elements and the availability of complementing packaging and indicator cells expressing Tax should allow their application in a variety of gene therapy settings.

Keywords: JOURNAL ARTICLE Animal Base Sequence Cell Line DNA Primers *Enhancer Elements (Genetics) Gene Products, tax/GENETICS Genes, Reporter Genetic Complementation Test *Genetic Vectors HTLV-I/GENETICS Kanamycin Kinase/GENETICS Lac Operon Luciferase/GENETICS Mice Mice, Nude Plasmids *Repetitive Sequences, Nucleic Acid Retroviridae/*GENETICS Support, Non-U.S. Gov't 3T3 Cells
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