L1 retrotransposons are mobile DNA elements which move by a copy and paste mechanism and show no strong insertion site preference. Preliminary studies have shown that Lis can deliver attached reporter genes into the DNA of human cells in culture and that these genes can be expressed. Such studies suggest that the L1 might be utilized as a gene transfer vector having several advantages over other gene therapy vectors currently in preclinical and clinical trials. This proposal is designed to explore strategies for modifying the L1 retrotransposon to produce a gene delivery vector which can target to specific sequences in the human genome. This may be accomplished by replacing the L1 endonuclease domain with endonuclease domains from site-specific retrotransposons of other species, including the R1 element of insects and the Tx1L element from Xenopus, or by linking the L1 endonuclease domain to site-specific binding motifs (eg. GAL4 binding domain, zinc finger domains). I also will test the ability of wild-type R1, R2, and Tx1L elements to retrotranspose in human cells and will consider their direct application as gene delivery vectors. In addition, I will assay in cell culture the efficiency of the L1 at integrating increasing lengths of exogenous DNA.