Transgenic Mosquitoes for Improved Malaria Sporozoite Vaccine Manufacture

描述（由申请人提供）：消除恶性疟原虫（Pf）的理想工具是一种高效疫苗，可预防血液期感染，从而预防疾病和传播，恶性疟原虫是99%疟疾死亡的病原体。Sanaria的目标是开发和商业化Pf子孢子（SPZ）疫苗，其在> 90%的接受者中预防Pf血液期感染。Sanaria PfSPZ疫苗由减毒、纯化、无菌、冷冻保存的PfSPZ组成。为生产疫苗而开发的平台技术还允许生产PfSPZ，用于攻毒感染以检测疫苗和药物（PfSPZ攻毒），以及用于氯喹保护下的攻毒疫苗接种（PfSPZ-CVac）。这些产品组合正处于商业化的积极时间轴上。这些产品中使用的PfSPZ是从无菌饲养的蚊子中提取的。增加每只蚊子的SPZ数量直接降低了商品成本。该项目将开发一种对PfSPZ感染高度敏感的转基因斯氏按蚊。通过沉默蚊子免疫效应基因LRIM 1（LRR基因家族的一员），我们可重复地增加了A.史蒂芬西我们还证实了转基因A.在一个实施方案中，本发明提供了在发夹构建体中表达SRPN 6双链RNA（dsRNA）序列的stephensi菌株，所述发夹构建体完全整合到基因组中。这些主要结果证明了第二阶段项目的合理性，该项目将创建GM A。本发明提供了一种稳定且高效的SPZ生产平台，用于Sanaria的生产工艺。计划了两种策略用于沉默LRIM 1：1）通过基因特异性dsRNA的表达直接沉默基因，以引发针对LRIM 1或APL 1转录物的内源性RNA干扰反应; 2）通过负调控蛋白Caspar或转录因子Rel 2的显性负变体的过表达破坏相关调控信号传导途径来间接沉默基因。我们成功的I期策略是通过驱动基因特异性dsRNA的表达以引发内源性RNA干扰来直接沉默基因;这种方法将被改进用于GM A的产生。史蒂芬西我们将通过整合GAL 4/UAS二元表达系统和“双靶点”基因沉默技术来创建基因沉默转基因。这种策略将使我们能够限制昂贵和耗时的感染表型评估，因为它能够在感染性喂养之前有效地确定基因沉默。这种方法将提高我们生产具有最佳基因型和表型的菌株的能力。我们将筛选GM品系以鉴定那些与目前的A生产菌株相比持续产生2倍以上PfSPZ的品系。史蒂芬西将在标准昆虫饲养条件和GMP合规生产所用的无菌条件下研究选定菌株。我们将把最佳的转基因菌株A。史蒂芬西进入圣利亚的生产流程

DESCRIPTION (provided by applicant): An ideal tool for eliminating Plasmodium falciparum (Pf), the causative agent of 99% of all malaria deaths, would be a highly effective vaccine that prevents blood stage infection and thereby prevents both disease and transmission. Sanaria's goal is to develop and commercialize a Pf sporozoite (SPZ) vaccine that prevents Pf blood stage infection in > 90% of recipients. The Sanaria(R) PfSPZ Vaccine is composed of attenuated, purified, aseptic, cryopreserved PfSPZ. The platform technology developed to manufacture the vaccine has also allowed the manufacture of PfSPZ for challenge infections to test vaccines and drugs (PfSPZ Challenge) and for vaccination by challenge under chloroquine protection (PfSPZ-CVac). These comprise a portfolio of products that are on an aggressive timeline to commercialization. The PfSPZ used in these products are extracted from aseptically reared mosquitoes. Increasing the number of SPZ per mosquito directly reduces the cost of goods. This project will develop a strain of genetically modified (GM) Anopheles stephensi mosquitoes highly susceptible to PfSPZ infections. By silencing a mosquito immune effector gene, LRIM1 (a member of the LRR gene family), we have reproducibly increased SPZ loads in A. stephensi. We have also demonstrated a phenotype of increased oocyst burdens in a transgenic A. stephensi strain expressing a SRPN6 double- stranded RNA (dsRNA) sequence in a hairpin construct that was fully integrated into the genome. These major results justify this Phase II project which will create GM A. stephensi lines in which expression of LRIM1 or APL1 is reduced or eliminated, providing a stable and efficient SPZ-production platform for Sanaria's manufacturing process. Two strategies are planned for silencing LRIM1: 1) directly silencing the genes through the expression of gene-specific dsRNA to elicit an endogenous RNA interference response against LRIM1 or APL1 transcripts; 2) indirectly silencing the genes by disrupting the relevant regulatory signaling pathway through either the over-expression of the negative-regulatory protein Caspar or a dominant-negative variant of the transcription factor Rel2. Our successful Phase I strategy was to directly silence genes by driving the expression of gene-specific dsRNA to elicit endogenous RNA interference; this approach will be improved upon for the creation of GM A. stephensi. We will create gene-silencing transgenes by incorporating the GAL4/UAS binary expression system and a 'two-target' gene silencing technology. This strategy will enable us to limit the costly and time consuming assessments of infection phenotype as it enables efficient determination of gene silencing prior to infectious feeding. This approach will enhance our capacity to produce strains with the optimal genotypes and phenotypes. We will screen GM lines to identify those that consistently produce e 2 fold more PfSPZ compared to the current production strain of A. stephensi. Selected strains will be studied under standard insectary conditions, and under the aseptic conditions used for GMP compliant manufacturing. We will incorporate the optimal GM strain of A. stephensi into Sanaria's manufacturing process.