Gene editing: Triplex DNA provokes DNA repair and stimulates site-specific gene editing.

In the early 1990's, triplex-forming oligonucleotides (TFOs) were being touted as tools to suppress gene expression by binding to promoter sites to block transcription factor access. However, I realized that the site-specific binding properties of TFOs could also be used to mediate sequence-specific gene editing. We discovered that triplex formation, itself, constitutes a helical alteration sufficient to induce DNA repair at the site of the triplex (Wang, Science 1996 and Vasquez, Science 2000), via the nucleotide excision repair (NER) pathway. This activates the target site for recombination with "donor DNAs" via homology-dependent repair. After a systematic evaluation of DNA analogs for improved triple helix formation in cells, we have focused on peptide nucleic acids (PNAs), which have a neutral polyamide backbone and bind DNA with high affinity. Using PNAs, we demonstrated successful editing of the beta-globin gene in human primary hematopoietic stem cells. In collaboration with the Saltzman lab, we developed a strategy to encapsulate the PNAs and donor DNAs in polymer-based, biocompatible nanoparticles to achieve effective in vivo delivery in mice with minimal toxicity. Recent work with the Egan lab has demonstrated the ability of nanoparticles containing PNAs and donor DNAs to mediate editing of the F508del CFTR gene mutation in airway epithelia in vivo in a mouse model of cystic fibrosis (McNeer, Nature Communications 2015) and to mediate substantial correction of anemia and in mice with thalassemia by simple intravenous injection of PNA and DNA containing nanoparticles in adult mice (Bahal, Nature Communications 2016) and in fetal mice via in utero injection (Ricciardi, Nature Communications, 2018).