| Gene therapy has long been a promising approach for treating inherited genetic disorders. The LAGLIDADG homing endonucleases (LHEs) show great potential as effective tools for targeted gene therapy. These DNA-cutting enzymes recognize long DNA sequences with extremely high specificity; yet also maintain some flexibility in target site recognition. By taking advantage of these properties we may be able to engineer LHEs with tailored specificities to stimulate site-specific gene correction. The studies described here first focus on a basic understanding of the structure and biophysical properties of the LHE I-AniI. The structure of I-AniI bound to its endogenous DNA target site was refined to 2.4A resolution, and the enzyme was found to bind with greater affinity to a hypercleavable target site than to its endogenous target. Information from this study was then used to engineer a variant of I-AniI that nicks, rather than cleaves, its cognate DNA target site. The I-AniI nickase maintains the specificity of the wild-type enzyme and is more efficient at cutting its DNA target. The I-AniI nickase was also shown to be capable of stimulating homologous recombination at its DNA target, in vivo, though at one-quarter the efficiency of the wild-type enzyme. These results suggest that the nicking I-AniI may be a safer alternative to double-strand break (DSB) inducing enzymes for gene therapy applications; nicks are inherently less mutagenic than DSBs. A novel method of directed evolution was then deployed, combining iterative rounds of somatic hypermutation and cell surface display with FACS, to successfully identify an I-AniI variant that preferentially binds a DNA sequence proximal to the CFTR DeltaF508 deletion site, the most common mutation that causes cystic fibrosis. The AniCF variant displays high specificity, both with respect to binding and cleavage, for the CFTR target. Finally, current gene therapy strategies are summarized, and various tools for targeted gene repair are discussed. LHEs are among the most promising targeted gene therapy reagents under development, and the research presented in this study will contribute to the body of LHE knowledge and facilitate future attempts to generate enzymes for targeted gene correction. |
