Hematologic disorders such as sickle cell disease, ?-thalassemia, and hereditary spherocytosis are caused by hundreds of characterized monogenetic mutations and affect hundreds of thousands of newborn infants each year. The consequences of such diseases result in billions in yearly healthcare costs and an estimated yearly loss of over 15 million disability adjusted life years (DALYs) attributed to hemoglobinopathies alone. Gene editing technologies hold tremendous potential in the curative treatment of hematologic, monogenetic disease via site-directed mutation correction of hematopoietic stem and progenitor cells (HSPCs). While initially promising, CRISPR/Cas9-mediated approaches to treatment have proven difficult due to low engraftment frequencies after ex vivo treatment and safety concerns related to double strand break (DSB) induction by the Cas9 nuclease. As an alternative to nuclease-based editing approaches, triplex-forming peptide nucleic acids (PNAs) have been shown to promote site-directed corrective recombination using co- delivered short ssDNA templates. Further, when co-encapsulated in polymeric nanoparticles, and in contrast to Cas9 technologies, PNAs and ssDNA donors can be safely delivered directly in vivo or in utero to create heritable, disease-ameliorating sequence correction in a murine model of ?-thalassemia. To further develop this technology with the goal of eventual translation to human therapies, it is critical to determine the precise repair mechanism by which PNAs promote corrective recombination. Our hypothesis is that PNA-mediated editing takes advantage of non-mutagenic DNA repair pathways that target a single strand of genomic DNA, thus yielding an efficient and safe mechanistic basis for eventual in vivo therapeutic use in humans.
SPECIFIC AIMS : In order to test our hypothesis and characterize the mechanism of PNA-mediated editing we have set out two specific aims. First, using an RNAi screen of known DNA repair factors in the genome and a fluorescent reporter of gene editing, we will elucidate the key repair factors responsible for PNA editing. The results of aim 1 will reveal implicated repair pathways and allow us to develop a model describing this phenomenon. Next, in aim 2, I will manipulate DNA repair targets in order to bias pathways of recombination and improve PNA editing efficiency. I will test small molecule inhibitors, biological inhibitors such as antibodies, and co-delivered siRNAs against repair targets to rationally improve PNA editing frequency. All results from both aims will be validated in human CD34+ HSCs.
Monogenetic, hematologic disorders such as ?-thalassemia, sickle cell disease, and hereditary spherocytosis represent a significant worldwide burden in terms of healthcare cost and life years lost. Gene editing-based correction of pathologic mutations holds tremendous promise as a means to cure such diseases, and triplex- forming peptide nucleic acids (PNAs) co-delivered with ssDNA templates are an example of this approach. In a crucial step towards eventual translation as a human therapeutic, this project seeks to elucidate the repair mechanisms by which PNA-mediated editing occurs and take advantage of this information to manipulate repair pathways to rationally improve this technology.