Genetically Modified Conditional Sexing A. stephensi Line for PfSPZ Manufacture

用于 PfSPZ 生产的转基因条件性别鉴定 A.stephensi 品系

• 批准号: 9889027
• 负责人: Peter F. Billingsley
• 金额: $ 29.76万
• 依托单位: SANARIA, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-07 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9889027
• 关键词: Adult; African; Anopheles Genus; Antigens; Antimalarials; Attenuated; Automobile Driving; Binding; Bioreactors; Blood; CRISPR/Cas technology; Cessation of life; Chemoprophylaxis; Chromosomes; Consumption; Country; Cryopreservation; Culicidae; DNA cassette; Development; Disease; Docking; Dominant-Negative Mutation; Dose; Doxycycline; Embryo; European; Falciparum Malaria; Family; Female; Gene Deletion; Genes; Genetic; Genome; Geographic Locations; Grant; Human; Immunity; Immunization; Impairment; Infection; Infection prevention; Integrase; Lethal Genes; Link; Location; Maintenance; Malaria; Methods; Modification; Morphology; Nutrient; Parasites; Partner in relationship; Pharmaceutical Preparations; Plasmodium falciparum; Plasmodium falciparum vaccine; Prevalence; Prevention; Process; Production; Pupa; Radiation; Regimen; Reproducibility; Response Elements; Site; Small Business Innovation Research Grant; Specificity; Sporozoite vaccine; Sporozoites; Stains; Sterility; System; Technology; Testing; Tetanus Helper Peptide; Tetracycline Control; Trans-Activators; Transgenes; Transgenic Organisms; Vaccines; Vial device; Y Chromosome; base; cost; early embryonic stage; egg; feeding; in vivo; malaria infection; male; prevent; progenitor; promoter; scaffold; sex; success; tool; transgenic insect; transmission process

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.

迫切需要预防、控制和消除疟疾的新工具：“后 在全球疟疾控制取得前所未有的成功的时期，进展已经停滞。2016年，有一个 估计有2.16亿例疟疾病例，比2015年增加约500万例。死亡人数达到445人 000”，比2015年增加1.5万人。恶性疟原虫（Pf）子孢子（SPZ）是唯一的免疫原， 在>90%的人类受体中预防了Pf感染。Sanaria® PfSPZ疫苗，由辐射组成 减毒的、无菌的、纯化的、冷冻保存的PfSPZ反复赋予高水平的抗 控制的人疟疾感染（CHMI）和自然传播的异质Pf. PfSPZ生长在 无菌饲养的斯氏按蚊。在这个项目中，我们计划使无菌蚊子 通过将雄蚊从系统中移除，饲养过程的效率提高100%，而无需额外成本 在胚胎阶段使用致命的基因插入，可以通过应用药物强力霉素来控制。 在我们的四个具体目标中，我们将：1）在A.史蒂芬西使用 CRISPR/Cas9基因插入，我们将在Y（男性）染色体上创建一个位点， 可以插入盒; 2）建立A.携带Y连锁显性-负性Relish 2基因的stephensi （dnRel 2）在Tet-on反式激活因子的控制下。dnRel 2在A.斯蒂芬西原因 胚胎致死率Y-连接的对接菌株将用于整合基因驱动盒，所述基因驱动盒含有 Tet-response element（TRE），一个最小启动子和dnRel 2。在此，仅当两个参数都满足时，才表示dnRel 2。 3）建立转基因A.斯蒂芬西线 携带Tet-On反式激活因子转基因株系中dnRel 2的表达需要Tet-On的结合 反式激活因子为TRE。因此，通过插入Tet-On反式激活因子，将建立另一个转基因株系 在bZP 1的控制下，A. stephensi早期胚胎基因启动子在这一行中， 反式激活因子将受到bZP 1启动子的严格控制，bZP 1启动子表达至产蛋后36小时。一 将建立该转基因的纯合系;和4）产生并测试转基因条件 雄性致死的A.史蒂芬西dnRel 2和反式激活因子将通过杂交结合在一起， dnRel 2雄性与Tet-On反式激活因子雌性。在祖先雄性卵子中，Tet-On反式激活因子将 在多西环素存在的情况下与TRE结合，驱动dnRel 2的表达，从而导致对 转基因雄性在没有强力霉素的情况下，两性都能存活，因为Tet-On反式激活因子是 不能与TRE结合。来自该品系的雌性蚊子将被评估其具有高繁殖力的能力。 本项目中产生的所有品系和菌株的卵将被冷冻保存。