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Novel reporter cell lines for neurotoxicant assays

Award Number: R43ES023718
ORGANIZATION: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/18/2014
PERIOD OF PERFORMANCE END DATE: 08/31/2016

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Novel reporter cell lines for neurotoxicant assays - DESCRIPTION (provided by applicant): While it has been demonstrated that gene mutations are associated with an increasing number of neurodegenerative syndromes, environmental neurotoxicants remain a key factor in the etiology of many such diseases. For example, exposure to pesticides such as maneb and paraquat increase the risk of developing Parkinson's disease. Unfortunately, there is no optimal in vitro model system to assess the neurotoxic potential of compounds. As result, exposure to poorly characterized compounds represents a significant contributor to the development of environmentally-induced diseases. There is a compelling unmet need for in vitro models and endpoint assays that are cost-effective, accurate, predictive, and sensitive that would also be amenable to high throughput screening. In particular, there is a need to develop homogeneous in vitro screens that can be used in quantitative high-throughput screening to query large libraries of chemical compounds such as the 'Tox21 10K' chemical library. We will take advantage of our core strengths in site-specific nuclease genome engineering and Footprint-Free" Gene Editing to produce reporter cell lines in which we will insert reporter genes into the endogenous toxicant-responsive genes SERPINE1 and DDIT3 by homologous recombination (i.e., create knockin mutations) in order to create a cost- effective, rapid system for neurotoxicity testing. Briefly, we engineer a SERPINE1 exon 2-specific site-specific nuclease in-house to drive the targeted insertion of firefly luciferase T2A-renilla luciferase (lux) reporter immediately downstream of the SERPINE1 initiator ATG so that the lux gene will be placed under the control of SERPINE1 promoter within the native genomic context; similarly we will knock in a TEM1-beta lactamase reporter behind the ATG of DDIT3. The targeting vectors will contain a piggyBac selection module containing the puromycin resistance and thymidine kinase genes, to allow selection for candidate targeted clones via puromycin resistance and to select for complete removal of the expression module upon transient expression of excision-only piggyBac transposase via ganciclovir resistance. As a result, the endogenous SERPINE1 and DDIT3 loci will contain their respective reporters without any other foreign DNA sequences will be created. To validate the function of the reporter cells, we will compare the induction properties of the targeted lux and BLA reporters with that of their corresponding untargeted (wild type) alleles using reporter (luciferase or beta-lactamase) and qRT-PCR assays, respectively. Success will be achieved if the magnitude of induction for the reporters falls within 20% of the induction observed for each reporter's corresponding endogenous gene for each test compound assayed. Validation of the cell lines will provide an initial tool for screening high-throughput screening for neurotoxicants and will represent the development of a powerful platform technology for the creation of cell lines for neurotoxicology screening. In the long term, Phase II studies would be aimed at creating a bank of reporter cell lines that sample the responses of multiple loci in a variety of lines that we will be able to markt as catalog items; we would also be able to market custom creation of knock-in, toxicant-responsive reporter lines and the use of either catalog or custom cell lines in fee-for-service toxicity testing as a service to academic and industry investigators, and open new opportunities for examining the impact of agents on specific pathways in a wide variety of cellular contexts.


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 = -$216,348 )
2018	2014	HERA TESTING LABORATORIES INC.	535 W SECOND ST L10	LEXINGTON	KY	40508	FAYETTE	USA	Environmental Health	000	2	10/18/2017	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	-$216,348
														Subtotal = -$216,348

Issue Date FY: 2015 ( Subtotal = $0 )
2015	2014	HERA TESTING LABORATORIES INC	511 WEST THIRD STREET	LEXINGTON	KY	40508	FAYETTE	USA	Environmental Health	001	2	3/25/2015	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$225,000
2015	2014	TRANSPOSAGEN BIOPHARMACEUTICALS INC	3701 Market St FL 4	Philadelphia	PA	19104	PHILADELPHIA	USA	Environmental Health	000	1	3/24/2015	NEW	-$225,000
														Subtotal = $0

Issue Date FY: 2014 ( Subtotal = $225,000 )
2014	2014	TRANSPOSAGEN BIOPHARMACEUTICALS INC	3701 Market St FL 4	Philadelphia	PA	19104	PHILADELPHIA	USA	Environmental Health	000	1	9/18/2014	NEW	$225,000
														Subtotal = $225,000

Grand Total All Awards = $8,652

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Novel reporter cell lines for neurotoxicant assays

Award Number: R43ES023718

ORGANIZATION: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 09/18/2014

PERIOD OF PERFORMANCE END DATE: 08/31/2016

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