Project Summary Hereditary disorders such as β-thalassemia and cystic fibrosis are attractive targets for genome engineering as these maladies are curable upon correction of the disease-causing mutation. New technologies can catalyze correction at the associated genomic site by homologous recombination (HR); for example, engineered nucleases including CRISPR/Cas9 systems have shown promise and entered clinical trials. Alternative non- nuclease-based triplex-forming peptide nucleic acids (PNAs) have also been successful in vivo. 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 nucleotide excision repair (NER) and HR pathways and exhibits low off-target effects. While these editing technologies have been successful thus far, important challenges remain before translation to the clinic. The development of safe and effective delivery vehicles that are able to efficiently encapsulate gene editing agents and target disease-relevant cells/tissues is necessary for the advancement of these therapeutics. The goal of this research is to further the translation of genome engineering technologies by developing biodegradable poly(amine-co-ester) (PACE) into polymeric vehicles that efficiently encapsulate and deliver gene editing agents to target cells in the bone marrow and the lung upon systemic intravenous (IV) administration. PACE is structurally diverse, allowing us to generate libraries of vehicles and identify compositions for targeting bone marrow or lung. In preliminary work, we have observed efficient encapsulation and delivery of gene editing agents using PACE. Further, specific PACE formulations have exhibited favorable biodistribution to the bone marrow and lung. The project will proceed in two phases: a development phase (UG3) and a demonstration phase (UH3). In the UG3 phase, a library of PACE polymers with unique characteristics will be synthesized and tested for their ability to encapsulate PNA- and CRISPR/Cas9-based editing reagents, deliver them to target cells, and promote efficient editing in vitro and in vivo. Cell-type targeting and editing will be quantified using an innovative high-throughput single cell RNA sequencing (scRNA-seq) screen. Candidate formulations targeting bone marrow and lung will be administered to murine disease models of β-thalassemia and cystic fibrosis, respectively, to confirm their editing capabilities and determine their ability to ameliorate disease symptoms. In the UH3 phase, candidate formulations and gene editing agents will be scaled up to accommodate large animal studies, primarily in pigs, but also in non- human primates in collaboration with other investigators in the SCGE program. These studies are designed to confirm cell-type targeting, using quantitative measures of gene editing, and disease improvement, enabling key steps towards clinical trials. This interdisciplinary research will yield a platform of targeted delivery vehicles, furthering the translation of gene editing therapeutics for diseases resulting from genetic mutations.

Project Narrative The proposed research is directly relevant to public health in that it seeks to develop new methods for targeted gene editing to the bone marrow and lung. Safe and effective gene editing at these targeted sites could open the door to cures for hereditary genetic diseases such as!β-thalassemia and cystic fibrosis. Further, the project is designed to incorporate key steps towards clinical translation of a therapeutic gene editing platform, which has the potential to contribute to the treatment of devastating genetic diseases, significantly enhancing the health of patients and reducing the burden of illness.