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

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 (10⁶ 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.

Overview summary

Fassati et al. describe a new approach to obtain transcriptionally targeted retroviral vectors. The viral enhancer and promoter have been substituted with a small transcriptional cassette containing two distinct enhancer elements. The first one is the Tax-responsive element of the human T cell leukemia virus type I and the second is a muscle-specific enhancer/promoter element. This design allowed transcriptional activation of the viral LTRs by Tax to obtain high-titer vectors. In the absence of Tax the vectors showed muscle-specific activity. Activation in trans of the vector LTRs in cells stably expressing Tax allowed the selectable marker to be expressed from an internal ribosome entry site element, avoiding the use of a nonspecific internal promoter. The possibility of substituting the muscle-specific enhancer with other tissue-specific enhancers should make these vectors, along with the complementing packaging and indicator cells, suitable for various gene therapy studies.