(provide by applicant): We have developed new technology for site-specific integration in mammalian cells. The technology involves use of novel phage integrases that mediate efficient integration at compact recognition sites. Sites recognized by the enzymes occur at low frequency in mammalian genomes and can be used to target integration. We have validated this technology in mammalian tissue culture cells, and it is ready for transfer to animals. In vivo experiments are now essential for application of the technology to improve existing methodologies for gene therapy and for construction of transgenic animals. The proposed experiments include the generation of transgenic mice that carry recognition sites for the integrases. These animals will be used for gene therapy studies and to improve basic technology for creation of transgenic mammals. We will carry out gene therapy studies in wild-type, transgenic, and disease model mice by introducing plasmid DNA to the liver by the hydrodynamic tail vein injection method. We will deliver a plasmid carrying the gene for a phage integrase along with a plasmid carrying the therapeutic gene and a recognition site for the integrase. The enzyme will mediate site-specific integration of the therapeutic gene into the genome at sites recognized by the enzyme. We will use this technology to develop effective permanent gene therapy treatments for emphysema and hemophilia B by introducing the a1-antitrypsin and factor IX genes. Success in these experiments will lead to application of this innovative technology to other gene therapy settings and progress toward clinical trials in patients.
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