Application and engineering of phage integrases for gene therapy.

摘要

The field of gene therapy, and more broadly, genetics, relies upon efficient tools for both basic and translational research. A variety of methods have been developed or discovered that aid in the transfer of exogenous genetic material into cells. There has been a long struggle in gene therapy to provide stable, long-term, and site-specific genomic integration of therapeutic genes. To date, the most successful means to deliver therapeutic genes is through the use of retroviruses. This is an approach that cannot be sustained, as insertional mutagenesis, prohibitive costs, and immunological risks continue to hamper the virus-dominated field of gene therapy. To overcome these obstacles, non-viral alternatives have been explored and are demonstrating feasible and efficient gene transfer. Amongst this group is the large serine recombinase family that is capable of unidirectional and site-specific integration of donor DNA. One member in particular, the bacteriophage phiC31 integrase, has demonstrated higher efficiency than many other non-viral strategies. It is capable of recombining two attachment sites, termed attP and attB, whereby recombination destroys the original target site, thus making it refractory to excision events. PhiC31 attP sites have been found throughout a wide range of mammalian and lower eukaryotic genomes. This allows for a donor plasmid bearing the reciprocal attB site to be co-delivered with phiC31 and integrated site-specifically into nonidentical, but functional, attP sites, termed "pseudo attP.";In this work, phiC31 integrase is compared to several recently discovered members of the serine recombinase family. It is demonstrated that phiC31 is more efficient, but leaves room for other recombinases to be utilized in certain situations. Also demonstrated is the application of phiC31 integrase for use in mouse hematopoietic stem cells, which represents the first documentation of a recombinase for this cell type. By engineering phiC31-zinc finger DNA binding domains, a method to improve upon the most site-specific strategy to date is demonstrated. Finally, a directed evolution approach was used to evolve phiC31 toward a single location in the human genome. Evidence is provided to suggest that both positive selection toward the chromosome Xg22.1 site and negative selection away from the natural attP target site have occurred. The results documented here signify that there are viable alternatives to virus-based therapeutics and that phiC31 can be easily engineered to target predetermined sites.