TRANSPOSAGEN BIOPHARMACEUTICALS, INC. — Department of Health and Human Services SBIR Phase II: 200

Description

DESCRIPTION provided by applicant The laboratory rat is a preferred rodent model in pre clinical drug studies Their larger size facilitates procedures otherwise difficult in mice includng studies using instrumentation blood sampling and surgeries and allows for ten times the amount of tissue collection Although rats are often more suitable than mice for pharmacological toxicological physiological and many other biological assays the ease of genetic engineering technologies has made the mouse the preeminent rodent model However the recent emergence of new and more precise gene targeting techniques for the rat has resulted in significant growth in the production of genetically modified rats In Phase I we demonstrated the feasibility of combining custom site specific Xanthomonas TAL Nuclease XTNTM a k a TALEN technology with spermatogonial stem cells SSCs for rapid cost effective and precise genome engineering in the rat Indeed the Phase I studies enabled Transposagen to launch custom XTNTM and knockout rat production services in and we have successfully created and delivered custom knockout rats to both industry and academic investigators In work outside the Phase I project we paired XTN TALEN technology with piggyBacTM technology to create the Footprint FreeTM Gene Editing System the only commercially available system that can engineer as little as a single nucleotide without leaving unwanted mutations and allowing for selection of rare events For Phase II studies we propose to use SSC and Footprint FreeTM Gene Editing technology to create a suite of rats that would express a specific human CP D allele in the absence of the homologous rat Cyp d genes i e humanized CYP D rat models Humans carry a single but highly polymorphic p CYP D gene CYP D metabolizes nearly of current drugs and is represented by over variants in the population which possess a wide range of enzyme activities As a consequence adverse drug effects or lack of drug effect depend on the specific allele s an individual is carrying Humanized CYP D animal models would be of great value for drug testing To accomplish this task we will optimize strategies to simultaneously delete a kb segment of the rat genome that contains the gene Cyp d cluster and knock in a specific human CYP D allele so that it would be placed under transcriptional control of the rat Cyp d promoter to ensure a physiological pattern of expression Not only would the humanized rat be useful for assessing the consequences of CYP D specific metabolites in the whole animal but hepatocytes isolated from these animals would provide a reliable and consistent source of CYP D hepatocytes for in vitro drug metabolism testing More generally successful optimization of Footprint FreeTM Gene Editing protocols for the creation of large scale humanizing knock in mutations would enable for the first time the possibility of sophisticated and cost effective genome engineering in many mammalian organisms PUBLIC HEALTH RELEVANCE The laboratory rat has been a valuable animal model for biomedical research due to its similarity to human physiology Pharmaceutical companies currently rely primarily on animal or transformed cell models for pre clinical metabolism and toxicity testing during drug discovery Thus there is a compelling need for animal models that are more predictive for ADME properties in humans However the ease and lower costs associated with generating mutations in mice has lead to a greater reliance on genetically engineered mouse models despite the inability of many of these models to mimic human pathways We outline a strategy that integrates our expertise in spermatogonial stem cells SSCs with a site specific enzyme technology to create knockout and humanized rat models for drug discovery applications Thus this project would benefit many goals of public health by making the production of mutations in the rat that better model human physiology readily accessible to the research community