2011 |
Fahrenkrug, Scott Christopher |
R41Activity Code Description: To support cooperative R&D projects between small business concerns and research institutions, limited in time and amount, to establish the technical merit and feasibility of ideas that have potential for commercialization. Awards are made to small business concerns only. |
Development of Porcine Genetic Models of Atherosclerosis
DESCRIPTION (provided by applicant): Because the cardiovascular anatomy of swine is similar to humans with regard to size, morphology, relative collateral arterial supply, and the presence of a well-developed vasa vasorum, the porcine coronary arterial model is the standard for the preclinical evaluation of endovascular devices. Indeed, a consensus report concerning FDA approval of the stents recommends the use of the pig model to assess these devices. However, a marked deficiency in current pig models is that they do not present with metabolic syndrome and significant atherosclerosis, underlying conditions that can drastically alter restenosis and thrombosis. Thus, the development of efficacious and safe stents continues to be confounded by the absence of a sufficient large animal model of atherosclerosis. The ideal pig model would be of manageable size and manifest both metabolic syndrome and atherosclerosis at an early age. Like mice, the pig genome can be efficiently manipulated to create hypomorphic and null alleles by genome engineering. In this Phase 1 STTR we propose to generate a pig model of metabolic syndrome and rapid atherosclerosis by knocking out the low density lipoprotein receptor gene in a strain of minipigs (Ossabaw) with a genetic propensity for Type 2 diabetes. A strategy employing sequential gene targeting by homologous recombination using adeno-associated virus, and iterative cloning by somatic cell nuclear transfer will result in the generation of LDLR-null minipigs during this Phase I STTR. Successful creation of OssabawLDLR -/- minipigs with evidence of enhanced dyslipidemia would justify submission of a Phase II STTR proposal focused on herd expansion, in-depth physiological characterization, and potentially commercialization of the model for sales to the biomedical research community. PUBLIC HEALTH RELEVANCE: This STTR proposes to create a pig model of metabolic syndrome and rapid atherosclerosis by knocking out the low density lipoprotein receptor (LDLR) gene in the Ossabaw minipig. Using a novel accelerated approach to gene inactivation, pigs without the LDLR gene will be generated during this Phase 1 STTR and their lipid profile will be examined for evidence of disrupted lipid processing. These pigs will be further characterized on the basis of subsequent funding, and if evidence of accelerated atherosclerosis is found, a population of pigs will be generated and provided for sale to the biomedical research community.
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0.903 |
2012 |
Fahrenkrug, Scott Christopher |
R43Activity Code Description: To support projects, limited in time and amount, to establish the technical merit and feasibility of R&D ideas which may ultimately lead to a commercial product(s) or service(s). |
Artiodactyl Gene Modification With Tal Effector Nucleases
DESCRIPTION (provided by applicant): Large animal models of human disease are indispensible for the development of relevant preclinical therapeutic protocols. Gene inactivation and gene conversion are powerful approaches for creating genocopies of alleles that cause disease in patients and for the modification of genes for biomedical materials development. A DNA double strand break (DSB) at a desired genome location is a flexible substrate for genome engineering, enabling; mutation of a target sequence, targeted integration of transgenes, or the stimulation of gene conversion by homologous recombination (HR) with a repair template. Consequently, the tools for genome engineering are rapidly converging on the concept of DSB induction using a variety of technologies, including the widely publicized Zinc-Finger Nucleases (ZFN) and Meganuclease (MGN) platforms. While both MGN and ZFN are efficient and reasonably precise, significant limitations in targetable sites, difficulty in developing new enzymes, and a constrained IP landscape limit their widespread commercial application. The recently described TAL effector nucleases (TALENs) are a novel DSB-inducing platform that appears to be easier to deploy and with a greater flexibility in target site selection, since their modular DNA binding motifs enable localization of nuclease activity to a broad range of targets. Recombinetics proposes to investigate enablement of TALENs for gene inactivation and, in combination with RCI technologies to stimulate gene conversion in livestock cells and embryos. The studies proposed here will establish a new paradigm for the genetic modification of artiodactyls (pigs, sheep, and cattle) for use as large animal models of human disease and the production of biomedical materials. PUBLIC HEALTH RELEVANCE: Large animal models of human disease are indispensible for the development of relevant preclinical therapeutic protocols. We propose to investigate enablement of TALENs for gene inactivation in livestock cells and, in combination with RCI technologies to stimulate gene conversion in livestock cells and embryos. The studies proposed here will establish a new paradigm for the genetic modification of artiodactyls (pigs, sheep, and cattle) for use as large animal models of human disease.
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0.903 |
2014 |
Carlson, Daniel Fred Fahrenkrug, Scott Christopher |
R43Activity Code Description: To support projects, limited in time and amount, to establish the technical merit and feasibility of R&D ideas which may ultimately lead to a commercial product(s) or service(s). |
Modeling Disease in Swine by Transplantation of Gene Targeted Germ Cells.
DESCRIPTION (provided by applicant): Swine models of human disease will become increasingly important for the development of relevant, preclinical therapeutics. However, the creation of swine congenital disease models is plagued by inefficiencies related to animal development and reproduction. The contemporary method for development relies on somatic cell nuclear transfer (SCNT) of genetically modified primary cells. While effective for the development of founders, cloning is not suitable for the creation of enough animals to meet the biomedical research market, requiring instead a production breeding program. The phenotype of affected animals may be inconsistent with long-term viability and/or sexual reproduction, so disease alleles need to be propagated as heterozygotes. However, a breeding program based on heterozygotes is slow and results in the only 1/4 of the production being affected offspring, dramatically increasing the cost of swine model production. As a solution we propose to develop and propagate congenital disease alleles by transplantation of germline stem cells (GSC) to surrogate testes. Germline stem cell transplantation (GST) provides an alternative to cloning for founder creation because GSC can be genetically modified in tissue culture before transplanting to surrogates. Furthermore, GSC can be isolated from affected juvenile males and transplanted to the gonads of healthy surrogates as a method to produce more affected animals without breeding from heterozygotes. GST would be greatly facilitated by the development of surrogate males unable to produce their own gametes, which would eliminate competition for the spermatogenic niche and result in the transmission of exclusively donor genetics. A single herd of male gamete-deficient swine could provide a platform for the development and propagation of many different congenital disease alleles. We therefore propose to generate gamete-deficient male swine as surrogates for GSC transplantation by knocking out (KO) the Deleted-in- Azoospermia-like gene (DAZL), a locus that has effectively enabled GST in rodents. We have used Recombinetics' proprietary TALEN technology and SCNT to generate homozygous DAZL-KO boars. Over the course of this Phase I proposal, we will characterize the genetic disruption of spermatogenesis in DAZL-KO boars in collaboration with Dr. Ina Dobrinski, the recognized world leader in swine GST. Validation that DAZL- KO boars are indeed sterile will justify propagation of heterozygous DAZL-KO animals. These animals provide a resource for Phase II studies to evaluate GST transplantation into DAZL deficient swine, and to develop a commercialization platform for sale of these animals and to implement their use as a platform technology for production and distribution of swine disease models.
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0.903 |