Monogenic blood disorders such as ?-thalassemia and sickle cell anemia are attractive targets for genome engineering as the root cause of the disease is dysfunction in a single gene. New techniques can catalyze correction of the disease-causing mutation at the associated site in the genome by homologous recombination (HR); for example, engineered nucleases including CRISPR/Cas9 systems have shown promise and entered clinical trials. However, nuclease-based platforms also exhibit frequent off-target effects due to the inability to restrict nuclease activity to the intended genomic DNA site. As an alternative non-nuclease-based technology, triplex-forming peptide nucleic acids (PNAs) offer significant advantages. PNAs have no intrinsic nuclease activity and enable activation of endogenous DNA repair activity when bound adjacent to the target site and co- delivered with a donor DNA strand containing the corrected sequence. PNA-mediated gene editing occurs via a combination of nucleotide excision repair (NER) and HR pathways and exhibits low off-target effects. Moreover, PNA and donor DNA can be readily encapsulated into poly(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles (NPs) for delivery and have already been shown to successfully correct ?-thalassemia after simple intravenous injection. While PNA editing technology has been successful thus far, important challenges remain before translation to the clinic. PLGA NPs have low encapsulation efficiencies and are relatively inefficient transfection agents, highlighting the need for better delivery vehicles. Further, improvement of gene editing frequency is necessary; editing could be enhanced by co-delivery of PNA/DNA and siRNA against key DNA repair factors such as RAD51, the knockdown of which significantly increased editing in preliminary studies. Moreover, the 2D cell culture assays typically used to screen delivery formulations are poor predictors of in vivo treatment response. The goal of this project is to improve PNA/DNA technology as a treatment for hereditary ?-thalassemia by 1) developing effective poly(amine-co-ester) (PACE) polymeric vehicles for encapsulating and delivering multiple nucleic acids for gene editing and 2) developing a more physiologically relevant primary bone marrow (BM) 3D screening platform to accurately assess the efficacy of delivery, toxicity, and frequency of gene editing and off-target mutagenesis in target cells and serve as a bridge between 2D in vitro studies and in vivo models of ??thalassemia. I hypothesize that even higher gene editing frequencies and maintained low off-target effects will be achievable by improved polymeric delivery systems and co-delivery of PNA/DNA with siRNA against RAD51. I also expect that successful 3D culture of BM will yield unique NP screening systems that correlate with in vivo treatment response and provide valuable information about the dynamics of local delivery and mechanisms of editing. Overall, the proposed interdisciplinary research is highly clinically relevant, furthering the translation of promising gene editing therapeutics for ?-thalassemia and other diseases resulting from single gene mutations.
The proposed research is directly relevant to public health in that it is aimed at developing a cure for hereditary blood disorders such as ?-thalassemia with the possibility of being translated to the clinic. The work has the potential to contribute to the treatment of devastating genetic diseases, significantly enhancing the health of patients and reducing the burden of illness. Moreover, the research will enhance fundamental knowledge of disease progression as well as drug delivery mechanisms in the bone marrow.
