Highly Specific Nucleases for Gene Therapy

The goal of gene therapy is to repair a specific genetic defect without additional modification of the genome. One of the most promising strategies is based on homologous recombination (HR), in which the DNA sequence needed to correct the gene of interest is supplied in trans. A major obstacle for the HR-driven approach is the low efficiency of natural recombination between an introduced DNA and the chromosomal target. Remarkably, it was discovered that making a specific double-strand break (DSB) in the target sequence increases the frequency of HR by several orders of magnitude. To stimulate DSB-induced recombination, zinc finger nucleases, engineered homing endonucleases and chimeric restriction endonucleases have been used in experimental systems [1]. Endonucleases with tailored specificities hold great potential for gene therapy as they allow cleaving DNA at a chosen target sequence. Unfortunately, they also cleave at off-target sites, resulting in toxicity. The goal of this project is to engineer new nucleases that are highly specific and whose activity can be regulated, thus making them safe for in vivo applications. One of our strategies is to create hybrid nucleases consisting of a cleavage module (restriction enzyme) and transcription activator-like effector (TALE) proteins as DNA binding modules. For this purpose, we engineered a fusion enzyme consisting of the restriction enzyme PvuII and the AvrBs3 TALE protein. The characterization of the fusion protein will be reported.