Precise gene inactivation, gene addition, and gene repair in patient cells are all part of a new paradigm in gene therapy. However, the full potential of these gene editing therapies can only be realized when it becomes possible to treat each tissue directly in the body of the patient. This goal requires novel gene editing delivery vehicles and routes of administration where uptake kinetics, penetration, and distribution are well established. A first step to achieve this goal is to develop model animals that mimic size, physiology, and aging of human patients. Swine models meet these criteria to enable in utero, pediatric, and adult gene editing therapies. Accordingly, Recombinetics has been using gene editing to create a suite of swine models with humanized disease alleles for the past 6 years to augment preclinical research and development. While these models are valued for efficacy studies, findings from these animals are not broadly applicable for optimization in vivo editing. To address this limitation, we propose to develop a suite of swine models and vectors capable of reporting gene-editing outcomes with a combination of in vivo (whole animal) and single cell readouts. Our modular design and production pipeline will enable rapid and reliable modeling of most human disease alleles within a single reporter system. The proposed models will report outcomes of two types of gene editing tools: 1) those that cut or nick DNA to stimulate DNA repair, ie. CRISPR/Cas9, TALENs, ZFNs, and 2) base-editors that convert selected DNA bases to another base at the target site without creating a double strand break. The validated reporter constructs will be integrated into the swine genome at one of three safe-harbor loci prior to animal production by somatic cell nuclear transfer (cloning). Reporter activity in these founder animals and their offspring will be characterized by whole body imaging and single cell analysis. The validated reporters, associated data, and animal models will then be provided to the Somatic Cell Gene Editing consortium for distribution. These novel models will enable new discoveries to characterize therapeutic delivery, DNA repair preferences, and off-target risk for any tissue or cell type in the body. 0
This project will fund development of broadly applicable swine reporters for in vivo somatic cell gene editing. Reporters for gene editing tools that either cut DNA or edit bases directly will be developed and validated in vitro prior to integration into the pig genome at one of three safe harbor loci. Reporter activity in founder animals and their offspring will be characterized by whole body imaging and single cell analysis. The validated reporters, associated data, and animal models will be provided to the Somatic Cell Gene Editing consortium for distribution. These novel, human sized models, will drive innovation for in vivo gene therapies based on gene editing. 0
