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Novel method to create knockout rats using endonucleases and spermatogonial stem

Award Number: R44GM099206
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/05/2011
PERIOD OF PERFORMANCE END DATE: 08/31/2017

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Novel method to create knockout rats using endonucleases and spermatogonial stem - The laboratory rat is the preferred rodent model in pre-clinical drug studies and encompasses the bulk of accumulated knowledge in drug development. Their larger size facilitates procedures otherwise difficult in mice, including studies using instrumentation, blood sampling, and surgeries. Although rats are 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 2012. The proposed studies will address the unmet need for rat models for drug discovery applications. Creating knockout models for enzymes involved in drug metabolism, which are often arranged as 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 with humanizing 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 will eliminate whole gene families from the genome. To optimize the experimental approach, we will outline studies to delete the 2-gene MATE complex which is located within a 70-kb segment of Chromosome 10 and 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 a Cyp2d null rat for CYP2D6 humanization studies. To create humanized rats, we will clone the wild type CYP2D6 gene (including its promoter) as well as four other common CYP2D6* variant genes into individual piggyBacTM (PB) vectors. The five PB-CYP2D6 constructs will be used to create five SSC lines that carry an average of one copy of the transgenic vector per cell, and founder animals will be generated from each SSC line. Each PB-CYP2D6 transgenic allele will be crossed into the Cyp2d cluster mutant background for phenotypic 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 will develop multiple protocols for genome engineering in the rat so that the most appropriate strategy can be executed to engineer the desired mutation. The success of this project will promote cost-effective technologies that can be used to create humanized models of choice in the appropriate null background for drug testing.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2018 ( Subtotal = $0 )
2018	2016	HERA TESTING LABORATORIES INC.	535 W SECOND ST L10	LEXINGTON	KY	40508	FAYETTE	USA	Biomedical Research and Research Training	000	4	10/19/2017	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2016 ( Subtotal = $774,736 )
2016	2016	HERA TESTING LABORATORIES INC	511 WEST THIRD STREET	LEXINGTON	KY	40508	FAYETTE	USA	Biomedical Research and Research Training	000	4	8/16/2016	NON-COMPETING CONTINUATION	$774,736
														Subtotal = $774,736

Issue Date FY: 2015 ( Subtotal = -$260,355 )
2015	2014	TRANSPOSAGEN BIOPHARMACEUTICALS INC	3701 Market St FL 4	Philadelphia	PA	19104	PHILADELPHIA	USA	Biomedical Research and Research Training	001	2	4/3/2015	COMPETING CONTINUATION	-$687,732
2015	2014	HERA TESTING LABORATORIES INC	511 WEST THIRD STREET	LEXINGTON	KY	40508	FAYETTE	USA	Biomedical Research and Research Training	003	3	9/18/2015	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$0
2015	2014	HERA TESTING LABORATORIES INC	511 WEST THIRD STREET	LEXINGTON	KY	40508	FAYETTE	USA	Biomedical Research and Research Training	002	3	4/6/2015	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$687,732
2015	2014	TRANSPOSAGEN BIOPHARMACEUTICALS INC	3701 Market St FL 4	Philadelphia	PA	19104	PHILADELPHIA	USA	Biomedical Research and Research Training	000	2	11/20/2014	COMPETING CONTINUATION	-$260,355
														Subtotal = -$260,355

Issue Date FY: 2014 ( Subtotal = $948,087 )
2014	2014	TRANSPOSAGEN BIOPHARMACEUTICALS INC	3701 Market St FL 4	Philadelphia	PA	19104	PHILADELPHIA	USA	Biomedical Research and Research Training	000	2	9/24/2014	COMPETING CONTINUATION	$948,087
														Subtotal = $948,087

Grand Total All Awards = $1,462,468

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Novel method to create knockout rats using endonucleases and spermatogonial stem

Award Number: R44GM099206

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/05/2011

PERIOD OF PERFORMANCE END DATE: 08/31/2017

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