Enhancement of gametocytogenesis in Plasmodium falciparum by genetic engineering for improved PfSPZ Vaccine Manufacture

通过基因工程增强恶性疟原虫配子细胞发生以改进 PfSPZ 疫苗生产

• 批准号: 10239239
• 负责人: STEPHEN Lev HOFFMAN
• 金额: $ 30万
• 依托单位: SANARIA, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-17 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10239239
• 关键词: Africa; Africa South of the Sahara; Antigenic Variation; Area; Attenuated; Authorization documentation; Blood; Brazil; C10; CRISPR/Cas technology; Calmodulin; Cambodia; Cessation of life; Child; Clinical; Clinical Trials; Communities; Country; Cryopreservation; Culicidae; Development; Disease; Elements; Engineering; Epigenetic Process; Europe; Evaluation; Falciparum Malaria; Future; GTP-Binding Proteins; Gene Deletion; Gene Expression; Generations; Genes; Genetic Engineering; Geographic Locations; Geography; Growth; High Prevalence; Human; Immunize; In Vitro; Individual; Infection; Infection prevention; Investments; Licensure; Ligands; Liver; Malaria; Malaria Vaccines; Marketing; Measures; Medical; Modification; Molecular; Nature; Nucleotides; Oocysts; Parasites; Phase; Plasmodium; Plasmodium falciparum; Plasmodium falciparum vaccine; Prevalence; Process; Production; Proteins; Quality Control; Radiation; Regulatory Element; Seeds; Sporozoite vaccine; Sporozoites; System; Tacrolimus Binding Proteins; Transgenic Organisms; Untranslated RNA; Untranslated Regions; Vaccines; base; cost; epigenetic silencing; improved; large scale production; next generation; overexpression; prevent; promoter; research clinical testing; tool; transcription factor; transmission process

The WHO estimates that in 2018, malaria caused ~228M clinical episodes and ~405,000 deaths worldwide. Despite an annual investment of >$3 billion for control measures, 2018 was the 3rd consecutive year in which there was no decrease in malaria cases, indicating a saturation of capacity to implement further impact with currently available strategies. There is an urgent unmet medical need for a highly efficacious malaria vaccine that prevents infection and disease. Sanaria’s 1st generation vaccine based on radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ) PfSPZ Vaccine has been assessed in 19 clinical trials in 6 countries in Africa, 2 countries in Europe, and the US, and received Fast Track designation from the FDA. Clinical trials with Phase 3 compliant vaccine will begin in mid 2020, and licensure (marketing authorization) in the US (FDA) and in Europe (EMA) is planned for 2022. PfSPZ Vaccine is targeted to prevent malaria in travelers to and residents of Africa, and for immunizing the entire community to halt transmission and eliminate malaria from geographically focused areas of Africa. During the next 5-10 years, we aim to significantly increase potency and decrease cost of goods (COGs) of PfSPZ-based vaccines so they can be optimally used to prevent Pf malaria. One of our strategies to improve the breadth of protection in our vaccines is to include additional strains of Pf from geographically diverse regions such as Pf7G8 (Brazil) and PfNF135.C10 (Cambodia). Other than PfNF54 (West Africa strain), all other Pf strains assessed in humans are poor gametocyte producers. An approach to improving the efficiency of large-scale production of PfSPZs from different geographic regions and decreasing COGs would be generating increased numbers of fertile gametocytes per unit of culture for production of PfSPZ in mosquitoes. In nature, gametocytogenesis occurs only in a small subset of blood stage parasites due to epigenetic suppression of gametocytogenesis-related genes. Control of gametocyte commitment would provide a powerful tool for improving production of PfSPZ. A key molecule in this process is the master switch transcription factor, PfAP2-G, the expression of which correlates directly with the percent of gametocytes produced by a given Pf strain. Deletion of this gene completely abolishes gametocyte production. We propose to increase the gametocyte production capacity of Sanaria’s vaccine strains and decrease PfSPZ manufacturing COGs by over-expressing this gene by using CRISPR-Cas9 gene editing to modify the pairing elements (PE) in the 3’-UTR of pfap2-g. As an alternative, we will also relieve epigenetic silencing of PfAP2-G by replacing its promoter with the constitutive calmodulin promoter in a conditionally regulatable gametocyte induction (on/off) system we have developed. We will generate enhanced gametocyte-producing lines of Pf from different geographic regions, compare and evaluate clones with enhanced gametocytogenesis and select those with optimal in vitro growth that maintain high prevalence and intensities of infectious PfSPZs in aseptic mosquitoes.

世卫组织估计，2018年，疟疾在全球造成约22800万个临床发作，死亡约405,000人。 尽管每年投资30亿美元用于控制措施，但2018年是连续第三年 疟疾病例没有减少，表明实施进一步影响的能力饱和 当前有可用的策略。紧急未满足的医疗需求需要高效的疟疾疫苗 这可以防止感染和疾病。 Sanaria的第一代疫苗基于辐射衰减，无菌性， 已评估了纯化的冷冻保存恶性疟原虫（PF）孢子岩（SPZ）PFSPZ疫苗 非洲6个国家，欧洲和美国的2个国家 /地区的19个临床试验，并获得了快速的道路 FDA的名称。符合3期疫苗的临床试验将于2020年中期开始 （营销授权）在美国（FDA）和欧洲（EMA）计划在2022年进行。PFSPZ疫苗针对 为了防止旅行者和非洲居民的疟疾，并使整个社区免疫停止 从非洲地理位置的地区传播和消除疟疾。在接下来的5 - 10年中，我们 旨在显着增加效力并降低基于PFSPZ的疫苗的商品成本（COG），以便它们 可以最佳地用于预防PF疟疾。我们改善保护广度的策略之一 疫苗将包括来自地理上不同地区的其他PF菌株，例如PF7G8（巴西）和 PFNF135.C10（柬埔寨）。除了PFNF54（西非菌株）以外，在人类中评估的所有其他PF菌株都是 可怜的配子细胞生产商。一种提高PFSPZ大规模生产效率的方法 不同的地理区域和齿轮减少将导致肥沃的数量增加 每单位培养物的配子细胞用于蚊子中PFSPZ的生产。在自然界中，配子细胞发生发生 仅在一小部分血液阶段寄生虫中，由于表观遗传学抑制了与配子细胞相关的 基因。对配子细胞承诺的控制将为改善PFSPZ的生产提供强大的工具。一个 在此过程中，关键分子是主开关转录因子PFAP2-G，其表达 与给定的PF菌株产生的配子细胞百分比直接相关。删除该基因 完全废除了配子细胞的生产。我们建议提高配子细胞的生产能力 Sanaria的疫苗菌株并通过使用过度表达该基因来减少PFSPZ制造COG CRISPR-Cas9基因编辑以修改PFAP2-G 3'-UTR中的配对元件（PE）。作为替代方案，我们 还将通过用构型钙调蛋白代替其启动子来缓解PFAP2-G的表观遗传沉默 我们已经开发的有条件调节的配子细胞诱导（ON/OFF）系统中的启动子。我们将 从不同地理区域生成增强的PF的配子细胞生产线，比较和评估 具有增强的配子细胞生成的克隆，并选择具有最佳体外生长的克隆 无菌蚊子中感染性PFSPZ的患病率和强度。