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Engineering resistance to Zika virus in Aedes aegypti for Cas9 driven population modification

Award Number: R01AI130085
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Project Summary. Zika virus (ZIKV; Flavivirus) is transmitted to humans by the mosquito vector Aedes aegypti and poses significant health risks to the Americas (1, 2).This phase I project from Colorado State University (CSU; Olson and Black), University of Missouri (MU; Franz) and University of Notre Dame (UND; Fraser) will apply gene drive technology to generate Ae. aegypti populations refractory to ZIKV and other arboviruses transmitted by this vector species. Our gene drive approach is based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 for rapid, effective, genetics-based population modification of Aedes aegypti. Our principal goals are: 1) apply two validated Anti-Viral Trans Gene (AVTG) strategies that target ZIKV to minimize or ablate ZIKV infection of Ae. aegypti, 2) generate transgenic Ae. aegypti that express the AVTGs in the context of a CRISPR/Cas9 (CC) gene drive system, 3) show that the genetically modified mosquitoes have a stable, refractory phenotype for ZIKV infection, and 4) validate gene drive (in indoor cage studies) by introgressing the AVTGs into an Ae. aegypti population to convert the population from a competent to a refractory infection phenotype. Previously, Drs. Olson and Franz have genetically modified Ae. aegypti (with no drive) that stably express DENV2-specific, inverted repeat (IR) RNAs forming dsRNA to trigger the small interfering (si)RNA pathway of RNAi in the mosquito (3, 4). Dr. Fraser has developed mosquito cell lines that express group I intron (GrpI) ribozymes targeted to a highly conserved region of arboviral genomes. He has already developed GrpI ribozymes that target all four DENV serotypes and chikungunya virus (CHIKV; Alphavirus) (6, 7). In this proposal, we will initially develop siRNA- and ribozyme-based anti-viral genes to target ZIKV in the context of CC gene drive. We will compare the two AVTG strategies with CC gene drive to see which is the most effective at suppressing mosquito infection. If the GrpI AVTG genes successfully suppress vector competence and spread the ZIKV AVTG into caged populations of Ae. aegypti, we will use other GrpI AVTGs with the CC drive system to additionally target DENVs and CHIKV. CC gene drive has already been used to efficiently modify cage-populations of Anopheles stephensi with anti- pathogen genes (8). We are confident we can adapt this approach to drive antiviral IR RNA and ribozyme AVTG genes to greatly reduce or ablate Ae. aegypti competence for transmitting ZIKV. The CC-AVTG system we propose will target two Ae. aegypti genome sites (TIMP P4 on chromosome aem 2q and 3'UTR polyadenylate binding protein (PABP on arm 3q). We use the ß2-tubulin promoter to express Cas9 to achieve gene drive through the male germ-line. This will maximize homology-dependent repair (HDR) to maintain drive over non-homologous end-joining (NHEJ) which impairs drive (9). This proposal represents a significant step towards developing and optimizing CC drive with AVTGs in Ae. aegypti. This effort will be foundational for using CC drive in Ae. aegypti for disease control We are confident the project greatly advances population replacement as a tool for potentially mitigating arbovirus transmission.


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: 2024 ( Subtotal = -$627 )
2024	2021	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	4	1/23/2024	NON-COMPETING CONTINUATION	-$627
														Subtotal = -$627

Issue Date FY: 2021 ( Subtotal = $574,502 )
2021	2021	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	4	3/5/2021	NON-COMPETING CONTINUATION	$574,502
														Subtotal = $574,502

Issue Date FY: 2020 ( Subtotal = $583,647 )
2020	2020	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	3	2/20/2020	NON-COMPETING CONTINUATION	$583,647
														Subtotal = $583,647

Issue Date FY: 2019 ( Subtotal = $735,512 )
2019	2019	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	2	3/8/2019	NON-COMPETING CONTINUATION	$735,512
														Subtotal = $735,512

Issue Date FY: 2018 ( Subtotal = $771,251 )
2018	2018	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	002	1	8/24/2018	NEW	$19,713
2018	2018	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	000	1	4/6/2018	NEW	$751,538
2018	2018	COLORADO STATE UNIVERSITY	601 S HOWES ST	FORT COLLINS	CO	80521	LARIMER	USA	Allergy and Infectious Diseases Research	001	1	4/16/2018	NEW	$0
														Subtotal = $771,251

Grand Total All Awards = $2,664,285

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Engineering resistance to Zika virus in Aedes aegypti for Cas9 driven population modification

Award Number: R01AI130085

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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