A Chimeric Integrase-Containing Expression Vector for Promoting Targeted Integration of Foreign DNA into a Host Genome
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Viruses are useful as vectors for the delivery and expression of genes because they enter cells efficiently and their genetic material is directed to a subcellular compartment where it is stable and can be efficiently expressed. However, when applied to the field of human gene therapy, several drawbacks to their use become apparent. They can stimulate strong immune responses, revert to replication competent viruses which are potentially pathogenic, and have been shown to cause fatal neoplasms in primates. Therefore, prohibitively expensive testing is required prior to therapeutic use. In addition, use of retroviral vectors is essentially limited to ex vivo gene therapy because their relatively low titre and sensitivity to human complement makes them impractical for in vivo therapy.
In non-viral gene delivery systems, DNA is introduced directly into cells. Non-viral gene delivery has been used to express various genes in animal tissues including muscle, lung, skin, CNS, liver, and others. For in vivo gene delivery, non-viral systems have the advantage of being more similar to pharmaceutical drugs in terms of safety, uniformity, and administration. However, the transduction frequency of the target cells is often barely adequate, and the persistence of cells expressing the transduced gene in vivo is poor. Further development of non-viral gene delivery technology will expand its application and will provide a viable alternative to the use of viral vectors.
At the Fox Chase Cancer Center, researchers are developing a non-viral gene delivery system that facilitates vector integration at specific sites in the recipient genome, thus increasing the efficiency of gene transfer. In vitro experiments have shown that synthesis of a chimeric integrase utilizing the ASV (avian sarcoma virus) integrase and the LEX A repressor DNA binding domain resulted in integration into the LEX operator region in a site specific manner. Studies utilizing other DNA binding domains to facilitate integration at different specific loci are ongoing. Candidate DNA binding domains include transcription factors, TATA box binding proteins, and other moieties that bind DNA in a sequence and gene specific manner. In addition, foreign genes can either be cloned into the vector carrying the chimeric integrase, or can be carried in a separate vector and co-transduced into recipient cells. Optimization of chimeric integrase vectors will contribute to resolving many of the problems associated with gene therapy, namely, promiscuous integration into random sites and activation or inactivation of unrelated genes.
In order to develop an efficient non-viral delivery system for use in gene therapy, Fox Chase researchers have engineered a novel expression vector that incorporates a DNA-binding moiety with a retroviral integrase moiety that allows for targeted integration of foreign genes into a host genome.
An opportunity for a research collaboration exists for further development of this technology into a tool for human gene therapy.
Katz, RA, Merkel, G, and Skalka, AM. (1996) Targeting of retroviral integrase by fusion to a heterologous DNA binding domain: in vitro activities and incorporation of a fusion protein into viral particles. Virology 217: 178-190.
US and foreign patent applications are pending. (May 30, 2000)