Peptide Nucleic Acid-Mediated Recombination for Targeted Genomic Repair and Modification

The ability to directly manipulate the human genome to correct a disease-related mutation, introduce a sequence change that would lead to site-specific gene knockout, or increase gene expression is a very powerful tool with tremendous clinical value. Triplex formation by synthetic DNA-binding molecules such as peptide nucleic acids (PNAs) has been studied for over 20 years and much of the work in the last 10 years has shown its great promise in its use to direct site-specific gene modification for the use in gene therapy. In this chapter, detailed protocols are described for the design and use of triplex-forming PNAs to bind and mediate gene modification at specific chromosomal targets. Target site identification, PNA and donor oligonucleotide design, in vitro characterization of binding, optimization with reporter systems, as well as various methods to assess gene modification and isolate modified cells are described.