Enhancing Immunological Memory Using Aptamertargeted siRNA Delivery to T Cells

描述(申请人提供)：最近对小鼠、非人类灵长类动物的研究和对人类患者的临床试验都强调了疫苗诱导的免疫反应、免疫记忆在调节针对传染病和癌症的保护性免疫中的持久性的重要性。值得注意的是，利用遗传手段或只要有可用的药物，抑制mTOR、GSK3b、Blimp-1或T-bet等效应器分化的介体，不仅可以防止短暂效应器的积累，还可以将激活的T细胞重新定向到沿记忆途径分化，并增强疫苗诱导的保护性免疫。尽管如此，药物制剂，如用于抑制mTOR的雷帕霉素，往往表现出不良的免疫抑制作用，反映出其靶标的广泛分布。在这里，我们建议开发一种通用的、广泛的 可应用的和临床可行的方法，以促进记忆T细胞反应的产生，解决了药理学药物的主要限制。我们建议使用RNAi通过与寡核苷酸适体配体的结合来下调针对CD8T细胞的效应分化的细胞内介质。适体和适体-siRNA结合物在合成、结合和降低免疫原性方面具有潜在的重要优势。这项研究的中心假设是，针对适体靶向的siRNA抑制疫苗诱导的CD8 T细胞中的细胞内介质将增强其向长期记忆T细胞的分化，并增强抗肿瘤免疫，这将在降低毒性、提高疗效和适用于“不可用药”的细胞内靶点方面优于药物。初步研究表明，4-1BB适配子靶向的猛禽siRNA抑制激活的CD8T细胞中的mTORC1功能导致了强大的记忆反应，并增强了疫苗诱导的荷瘤小鼠的保护性免疫，其效果优于雷帕霉素。本申请中提出的研究的具体目标是确定一种最佳的I类适体-siRNA结合物，以增强小鼠肿瘤模型(AIMS#1和AIMS#2)中确定的疫苗诱导的保护性免疫，这将指导能够促进抗原激活的T细胞在体外的持久性的人结合物的开发(AIMS#3)。成功实现拟议研究的目标将为临床试验奠定基础，以加强疫苗诱导的癌症患者的保护性免疫，使用该提案中开发的试剂。通过使用siRNAs来抑制细胞内介质来促进记忆分化，siRNAs通过与寡核苷酸适配子配基结合而针对激活的CD8T细胞，可以说是新颖的。将siRNA靶向循环免疫或造血细胞的特定亚群的能力将为出于研究和治疗目的而操纵免疫和造血系统提供一种新的工具。

DESCRIPTION (provided by applicant): Recent studies in mice, nonhuman primates, and clinical trials in human patients have emphasized the importance of the persistence of the vaccine-induced immune response, immunological memory, in mediating protective immunity against infectious diseases and cancer. Notably, inhibition of mediators of effector differentiatio like mTOR, GSK3b, Blimp-1 or T-bet, using genetic means or whenever available pharmacological agents, not only prevented the accumulation of the short-lived effectors but also redirected the activated T cells to differentiate along the memory pathway, and potentiated vaccine-induced protective immunity in mice. Notwithstanding, pharmacological agents, like rapamycin that was used to inhibit mTOR, often exhibit undesirable immune suppressive effects reflecting the broad distribution of their targets. Here we propose to develop a versatile, broadly applicable, and clinically feasible approach to promote the generation of memory T cell responses that addresses the main limitations of pharmacological agents. We propose to use RNAi to downregulate intracellular mediators of effector differentiation that will be targeted to CD8+ T cells by conjugation to oligonucleotide aptamer ligands. Aptamer and aptamer-siRNA conjugates offer potentially important advantages in terms of synthesis, conjugation, and reduced immunogenicity. The central hypothesis of the proposed studies is that aptamer-targeted siRNA inhibition of intracellular mediators in vaccine-induced CD8+ T cells will enhance their differentiation into long-lasting memory T cells and potentiate antitumor immunity that will be superior to pharmacological agents in terms of reduced toxicity, increased efficacy, and applicability to "nondrugable" intracellular targets. The proposed approach is supported by preliminary studies showing that 4-1BB aptamer-targeted raptor siRNA inhibition of mTORC1 function in activated CD8+ T cells led to the generation of a potent memory response and enhanced vaccine-induced protective immunity in tumor-bearing mice that was superior to that of rapamycin. The specific goal of the studies proposed in this application is to identify a best-i-class aptamer-siRNA conjugate to potentiate vaccine-induced protective immunity as determined in murine tumor models (Aims #1 and #2), that will guide the development of human conjugates capable of promoting the persistence of antigen- activated T cells in vitro (Aim #3). Successful accomplishment of the goals of the proposed studies will set the stage for clinical trials to potentiate vaccine-induced protective immunity in cancer patients using the agents developed in this proposal. Promoting memory differentiation by inhibition of intracellular mediators using siRNAs that are targeted to activated CD8+ T cells by conjugation to oligonucleotide aptamer ligands is arguably novel. The ability to target siRNAs to specific subsets of circulating immune or hematopoietic cells will provide a novel tool to manipulate the immune and hematopoietic systems for both investigational and therapeutic purposes.