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.

Public Health Relevance

This SBIR proposes to create a line of azoosperic swine for use as recipient animals for germline stem cell transplantation. Using our proprietary TALEN technology for gene inactivation, pigs heterozygous and homozygous for knockout of the Deleted in Azoospermia-like (DAZL) gene will be generated during this Phase 1 SBIR and their capacity to generate mature spermatozoa will be assessed. Germline stem cell transplantation to these pigs will be conducted on the basis of subsequent funding, and if they prove to be a suitable recipient for germline stem cell transplantation, animals and services utilizing them will be provided for sale to the biomedical research community.

Agency
National Institute of Health (NIH)
Type
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
Project #
1R43GM108150-01A1
Application #
8711710
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Maas, Stefan
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Recombinetics, Inc.
Department
Type
DUNS #
City
Saint Paul
State
MN
Country
United States
Zip Code
55104