Gene therapy will allow for the treatment of both acquired and genetic diseases at the most fundamental level by introduction of therapeutic genes. Despite advances in delivery of foreign genes, achieving high and stable levels of expression has been problematic. The use of plasmid DNA for gene therapy has many advantages, including safety, accommodation of large genes, ease of production, and low cost. Plasmid DNA is lost, however, as cells divide. Viral integration of a transgene into the host genome prevents loss of the gene. Integration is hypothesized to be responsible for the long-term expression of some transgenes delivered through viral vectors. Disadvantages of viral vectors include safety concerns, cost, and difficulties in production. In order to achieve nonviral integration of a transgene and then expression at therapeutically useful levels, we propose to deliver the gene in pre-formed synaptic complexes of hyperactive Tn5 transposase dimers bound to DNA elements flanking the transgene to be integrated. This integration requires no specific sequence in the target DNA (the genome). During Phase I, we will verify that these synaptic complexes can be delivered into mammalian cells in culture and that the bacterial transposase can indeed effect integration in mammalian cells. In Phase II, the use of pre-formed Tn5 transposase-DNA complexes will be optimized and incorporated into gene therapy approaches such as the transplantation of cells genetically modified ex vivo (e.g., fibroblasts, keratinocytes, and myoblasts) or the direct gene transfer into cells in vivo (e.g., hepatocytes). These studies will form the basis for the commercial development of Tn5 transposase-mediated integration of transgenes into mammalian cells in vitro as a research tool, and for gene therapy ex vivo and in vivo.
A gene integration system could have immediate application for ex vivo gene therapy (e.g., transplantation of genetically modified fibroblasts, keratinocytes, hemopoietic stem cells, and myoblasts). Mirus will immediately commercialize (on the basis of the Phase I studies) reagents for enabling the more efficient integration of cells in culture (in conjunction with our line of transfection reagents) and cells in vivo (on the basis of Phase II studies). Its commercial development will be accomplished by initiation of pre-clinical trials using model disorders such as hemophilia and licensing to larger pharmaceutical and biotechnology companies.
