The laboratory rat is the preferred rodent model in pre-clinical drug studies and encompasses the bulk ofaccumulated knowledge in drug development. Their larger size facilitates procedures otherwise difficult inmice, including studies using instrumentation, blood sampling, and surgeries. Although rats are moresuitable than mice for pharmacological, toxicological, physiological, and many other biological assays, theease 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 insignificant growth in the production of genetically-modified rats. In Phase I, we demonstrated the feasibilityof combining custom site-specific Xanthomonas TAL Nuclease (XTNTM) [a.k.a. TALEN] technology withspermatogonial 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 productionservices in 2012. The proposed studies will address the unmet need for rat models for drug discoveryapplications. Creating knockout models for enzymes involved in drug metabolism, which are often arrangedas closely linked members of multi-gene families, presents a new production challenge. In this proposal,we will develop strategies for creating large-scale deletions of multi-gene families combined withhumanizing transgenic engineering to create novel and more predictive models for drug discovery.We will develop protocols to use XTNTMs to generate large-scale chromosomal deletions in SSCs that willeliminate whole gene families from the genome. To optimize the experimental approach, we will outlinestudies to delete the 2-gene MATE complex which is located within a 70-kb segment of Chromosome 10and the 5-gene Cyp2d cluster located within a 60-kb segment of Chromosome 7. SSCs that harbor a 45-kb deletion that knocks out the entire Cyp2d complex will be transplanted into recipient males to create aCyp2d null rat for CYP2D6 humanization studies. To create humanized rats, we will clone the wild typeCYP2D6 gene (including its promoter) as well as four other common CYP2D6* variant genes into individualpiggyBacTM (PB) vectors. The five PB-CYP2D6 constructs will be used to create five SSC lines that carryan average of one copy of the transgenic vector per cell, and founder animals will be generated from eachSSC line. Each PB-CYP2D6 transgenic allele will be crossed into the Cyp2d cluster mutant background forphenotypic analysis. In the future, humanized animal models in which various combinations of CYP2D6*alleles would be of great value for testing the effect of drug candidates. The proposed Phase II studies willdevelop multiple protocols for genome engineering in the rat so that the most appropriate strategy can beexecuted to engineer the desired mutation. The success of this project will promote cost-effectivetechnologies that can be used to create humanized models of choice in the appropriate null background fordrug testing.
The laboratory rat has been a valuable animal model for biomedical research due to its similarity to humanphysiology. 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 animalmodels that are more predictive for ADME properties in humans. However; the ease and lower costsassociated with generating mutations in mice has lead to a greater reliance on genetically engineered mousemodels despite the inability of many of these models to mimic human pathways. We outline a strategy thatintegrates our expertise in spermatogonial stem cells (SSCs) with a site-specific enzyme technology to createknockout and humanized rat models for drug discovery applications. Thus; this project would benefit manygoals of public health by making the production of mutations in the rat that better model human physiologyreadily accessible to the research community.
| Noto, Fallon K; Adjan-Steffey, Valeriya; Tong, Min et al. (2018) Sprague Dawley Rag2-Null Rats Created from Engineered Spermatogonial Stem Cells Are Immunodeficient and Permissive to Human Xenografts. Mol Cancer Ther 17:2481-2489 |
