The laboratory rat is used extensively by the pharmaceutical industry for preclinical animal efficacy and toxicology studies. In addition, the rat has been a valuable animal model for physiology, behavior, and pharmacology research programs, due to ease of experimental manipulation. Their larger size facilitates procedures otherwise difficult in mice, including studies using instrumentation, blood sampling, nerve conduction studies, and surgeries. Generating inbred immunodeficient rat strains that can maintain functional human xenografts would provide unique and valuable resources for drug discovery and therapeutic research programs. This project will investigate the use of a novel rat strain carrying a transposon-mediated knockout (KO) insertional mutation within the adenosine deaminase (Ada) locus in the inbred Fischer line. This mutation is an underlying cause of severe combined immunodeficiency (SCID) in humans. In mice, Ada KO mutants have no discernable thymus, two-to-three-fold lower cell numbers in the spleen, and livers that exhibit severe histological defects. This latter defect is the underlying cause of perinatal lethality in Ada KO mice. Each of these defects is also observed in Ada KO rats. Studies in Ada KO mice have shown that low levels of ADA activity can rescue the liver dysfunction, as well as other non-immune defects associated with Ada deficiencies, while maintaining the SCID phenotype. These results indicate that it may be feasible to create a conditional inbred SCID Ada KO rat line suitable for human cell transplantation studies. To achieve this goal, we will assess the feasibility of using doxycycline-inducible systems to conditionally complement the non- immune defects while maintaining the SCID phenotype. In Phase I, we will generate transgenic founders that co-segregate two expression modules: (a) a ubiquitously expressed Tet-repressor;and (b) an Ada mini-gene under the control of a CMV/Tet operator transcriptional element. The Tet-repressor will inhibit the expression of the Ada mini-gene;expression can be rapidly induced by providing doxycycline (dox) in the drinking water of the animals. We will identify founder animals that exhibit dox dose-dependent activation of the Ada mini-gene (referred to as conditional Ada transgenic rats;AdacTG), and determine whether Ada -/- /AdacTG rats are viable and exhibit no overt phenotypes except for immune deficiency. In Phase II, we will transplant primary human cells (e.g., hepatocytes) into the transgenic animals, in proof-of-principal studies to demonstrate the long-term viability and function of human cells in the new SCID rat models. In the long term, we will develop Ada -/- /AdacTG rats with humanized livers for drug discovery and therapeutic applications. This project should provide, for the first time, an inbred rat SCID Ada model for human cell transplantation studies, enabling broad applications for assessing the function of human cells in the context of the rat, which is amenable to a wide variety of experimental manipulations not possible in mice.
The development of a rat strain that can support efficient human cell engraftment would represent a major advance for the production of humanized tissues for drug discovery and therapeutic research applications. In the application """"""""Novel SCID rat models for human cell transplantation studies,"""""""" studies are aimed at examining the feasibility of using a novel knockout immunodefective (SCID) rat line to generate animals that can serve as hosts to study the function of normal and diseased human tissues. The proposed innovations would further increase the utility of rat models for drug discovery and would aid in the development of pre-clinical animal models that represent more predicative indicators of efficacy and toxicity associated with drug candidates. Being able to generate rats that can permit the long-term propagation of human cells and tissues would provide unique and valuable resources for drug discovery and therapeutic research. In the rat, such transplants would enable a wide variety of experimental manipulations that would not be possible in mice. There is a compelling need for an abundant and accessible supply of animals to screen for new therapeutic agents. Thus, if successful, this project would benefit many goals of public health.
