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
| Autor: | Dominic J. Wells, Guglielmo Scarlato, George Dickson, Manuela Sironi, Nereo Bresolin, Masakazu Hatanaka, Shoji Yamaoka, Ariberto Fassati, Alessandra Bardoni |
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| Rok vydání: | 1998 |
| Předmět: |
Genetic enhancement
Genetic Vectors Mice Nude Biology Cell Line Malignant transformation Mice Genes Reporter Genetics Animals Tissue specific High titer Vector (molecular biology) Luciferases Enhancer Molecular Biology DNA Primers Repetitive Sequences Nucleic Acid Human T-lymphotropic virus 1 Base Sequence Kanamycin Kinase Genetic Complementation Test 3T3 Cells Gene Products tax Virology Molecular biology Long terminal repeat Enhancer Elements Genetic Retroviridae Lac Operon Molecular Medicine Plasmids |
| Zdroj: | Human Gene Therapy. 9:2459-2468 |
| ISSN: | 1557-7422 1043-0342 |
| DOI: | 10.1089/hum.1998.9.17-2459 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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