# Genetically Modified Conditional Sexing A. stephensi Line for PfSPZ Manufacture

> **NIH NIH R43** · SANARIA, INC. · 2020 · $297,557

## Abstract

There is an the urgent need for new tools for prevention, control, and elimination of malaria: “..after an
unprecedented period of success in global malaria control, progress has stalled. In 2016, there were an
estimated 216 million cases of malaria, an increase of about 5 million cases over 2015. Deaths reached 445
000”, 15,000 more than in 2015. Plasmodium falciparum (Pf) sporozoites (SPZ) are the only immunogens that
have prevented Pf infection in >90% of human recipients. Sanaria® PfSPZ Vaccine, composed of radiation
attenuated, aseptic, purified, cryopreserved PfSPZ has repeatedly conferred high level protection against
controlled human malaria infection (CHMI) and naturally transmitted heterogeneous Pf. PfSPZ are grown in
aseptically reared Anopheles stephensi mosquitoes. In this project we plan to make the aseptic mosquito
rearing process up to 100% more efficient at no additional cost by removing male mosquitoes from the system
at the embryonic stage using a lethal gene insert that can be controlled by application of the drug, doxycycline.
In our four specific aims we will: 1) Establish a Y-linked docking strain in A. stephensi. Using
CRISPR/Cas9 gene insertion we will create a site in the Y (male) chromosome into which larger gene drive
cassettes can be inserted; 2) Establish A. stephensi carrying Y-linked dominant-negative form of relish2
(dnRel2) under the control of Tet-on transactivator. Expression of dnRel2 in A. stephensi causes
embryonic lethality. The Y-linked docking strain will be used to integrate a gene drive cassette containing a
Tet-response element (TRE), a minimal promoter and dnRel2. Here, dnRel2 will be expressed only when both
doxycycline and the Tet-On transactivator are available; 3) Establish a transgenic A. stephensi line
carrying Tet-On transactivator. Expression of dnRel2 in the transgenic line requires the binding of Tet-On
transactivator to TRE. Therefore, another transgenic line will be established by inserting Tet-On transactivator
under the control of bZP1, an A. stephensi early embryonic gene promoter. In this line, the expression of the
transactivator will be tightly controlled by the bZP1 promoter which is expressed until 36 h after egg laying. A
homozygous line for this transgene will be established; and 4) Generate and test a transgenic conditional
male-lethal sexing strain of A. stephensi. dnRel2 and the transactivator will be brought together by crossing
dnRel2 males with Tet-On transactivator females. In the progenitor male eggs, the Tet-On transactivator will
bind to TRE in the presence of doxycycline, driving the expression of dnRel2, thus causing lethality to the
transgenic males. In the absence of doxycycline both sexes will survive because the Tet-On transactivator is
not able to bind to TRE. Female mosquitoes from this strain will be assessed for their capacity to harbor high
intensity infections of Pf. Eggs from all lines and strains generated in this project will be cryopreserved.

## Key facts

- **NIH application ID:** 9889027
- **Project number:** 5R43AI143077-02
- **Recipient organization:** SANARIA, INC.
- **Principal Investigator:** Peter F. Billingsley
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $297,557
- **Award type:** 5
- **Project period:** 2019-03-07 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9889027

## Citation

> US National Institutes of Health, RePORTER application 9889027, Genetically Modified Conditional Sexing A. stephensi Line for PfSPZ Manufacture (5R43AI143077-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9889027. Licensed CC0.

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