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.

迫切需要新工具来预防，控制和消除疟疾：“ .. 全球疟疾控制成功的前所未有的时期，进步已经停滞不前。在2016年，有一个 估计有2.16亿例疟疾病例，2015年增加了约500万例。 000英寸，比2015年多15,000。 > 90％的人的受体中阻止了PF感染。 Sanaria®PFSPZ疫苗，由辐射组成 衰减，无菌，纯化，冷冻保存的PFSPZ反复提供高水平的保护 受控的人类疟疾感染（CHMI）和自然传播的异质PF。 PFSPZ生长 无菌饲养的围绕蚊子蚊子。在这个项目中，我们计划制作无菌蚊子 通过从系统中取出雄性蚊子，饲养过程效率高达100％，无需额外的费用 在胚胎阶段，使用致命基因插入物可以通过使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. Stephensi中建立Y连锁的对接菌株。使用 CRISPR/CAS9基因插入我们将在Y（雄性）染色体中创建一个位点，其中较大的基因驱动 可以插入盒式磁带； 2）建立A. Stephensi带有Y连锁的主导性形式的刷新形式2 （DNREL2）在Tet-On反式激活器的控制下。 dnrel2在A. stephensi原因中的表达 胚胎致死性。 Y连锁的对接菌株将用于整合包含A的基因驱动盒 TET-RESPONSE元件（TRE），最小启动子和DNREL2。在这里，DNREL2仅在两者时才表示 有强力霉素和Tet-On反式激活器可用； 3）建立转基因A. stephensi系 携带Tet-On反式激活器。 DNREL2在转基因线中的表达需要Tet-On的结合 反式激活器到TRE。因此，将通过插入Tet-On反式激活器来建立另一个转基因线 在BZP1的控制下，A. stephensi早期胚胎基因启动子。在这一行中， 反式激活器将由BZP1启动子严格控制，该启动子在产卵后直到36小时表示。一个 将建立这种转换的纯合线； 4）生成和测试转基因条件 A. stephensi的男性致命性菌株。 DNREL2和反式激活器将通过交叉聚集在一起 DNREL2雄性具有Tet-on denslactivator雌性。在祖细胞雄性卵中，Tet-On反式激活器将 在强力霉素存在下与TRE结合，驱动DNREL2的表达，从而导致致命性 转基因男性。在没有强力霉素的情况下 无法与TRE结合。该菌株中的女性蚊子的能力将被评估 PF的强度感染。该项目中产生的所有线条和应变的鸡蛋将是冷冻保存的。