Stability of transgene expression in vivo: Extrachromosomal vs. integration.

摘要

The success of a gene therapy treatment requires a regulated, therapeutic level of transgene expression that is stable for the duration of the treatment, which may equal the life of the individual. This work presents two different approaches to achieving such persistent expression in vivo.; The use of extrachromosomal vectors is advantageous because they do not integrate into the genome, thereby avoiding any potential insertional mutageneses that may have deleterious effects. We show here that, in non-dividing tissue, such extrachromosomal vectors can provide long-term, high-levels of transgene expression in vivo. More importantly, this study demonstrates that full mammalian genomic sequences are able to provide more prolonged and higher levels of expression in vivo than their cDNA counterparts.; Permanence of a therapeutic vector can also be achieved by integration into the human genome, a feature that may be necessary in actively dividing tissues. The use of a site-specific integration system is advantageous in that integration can be directed toward innocuous sites in the genome, thereby avoiding the aforementioned insertional mutageneses that may result from randomly integrating viral and nonviral vectors. Bacteriophage integrases, such as those of &phis;C31 and R4, show great potential as site-specific integration tools for the mammalian genome, and the related integrase of phage TP901-1 may prove equally useful. Here, we characterize the recombination activity of the TP901-1 integrase and demonstrate that this enzyme does possess the potential to act as a tool for mammalian genomic manipulations. Furthermore, we show here that these three integrases also show utility as tools for creating mammalian chromosomal excision events, making them useful enzymes not just for gene therapy, but also for any genomic research in which precise manipulations of the genome are required.; The decision to use either extrachromosomal or site-specific integrating vectors in a genetic treatment may depend on the delivery method, the target tissue, or the length of time that expression is required. The work we present here demonstrates that both types of vectors have the potential to be successful tools for gene therapy.