Harnessing the Immunologic Capacity of RNA-nanoparticle Vaccines Targeting Glioblastoma

摘要： 我现在在我的第一个教师职位作为一个助理教授神经外科和儿科在大学 来自佛罗里达。我在杜克大学完成了T32 NIH奖学金， 在接受佛罗里达大学的一个初级教师职位之前，我和我的朋友一起搬到了那里。 导师杜安·米切尔博士米切尔博士和我已经选择了一个咨询委员会，以确保我的收益 智力和专业成长。我们的咨询委员会的专业知识不重叠， 途径，我发展新的知识，肿瘤免疫学，分子生物学，纳米技术和 转化肿瘤学该提案将充分利用这些经验，因为它概述了职业发展 我计划成为一名独立的研究人员，探索新的RNA纳米颗粒疫苗，可以重新 引导免疫系统对抗恶性脑肿瘤。 背景：胶质母细胞瘤（GBM）几乎是一致致死的，中位生存期小于15 因此需要开发更有效和更有针对性的治疗方法。虽然我们已经 在随机/盲法试验中显示，RNA脉冲的树突状细胞（DC）疫苗引起显著的存活 尽管这些治疗方法在GBM患者中具有显著的益处，但这些治疗方法仍然受到成本和复杂性的阻碍。或者，RNA- 纳米颗粒（RNA-NPs）可以递送总肿瘤RNA（TTRNA），从少至500个 活组织检查的肿瘤细胞，内源性抗原呈递细胞（APC）诱导有效的，无毒的抗肿瘤 免疫力由于这些纳米脂质体已在临床级药物中以有限的毒性使用， 在溶液中稳定数小时，保护核酸免于降解，并可被工程化以调节 免疫反应，我们已经探索了使用TTRNA负载的纳米颗粒作为一种有吸引力的，“现成的” 治疗平台，以重新引导针对颅内肿瘤的宿主免疫。虽然我们已经证明， 静脉内递送RNA-NPs介导抗颅内恶性肿瘤抗原特异性T细胞应答 与DC疫苗相比，这些制剂显示在小鼠中诱导APC上的差异表型。 脾和肝。 假设：RNA-NPs在脾和肝中转染不同的APC，诱导不同的免疫应答 其可以被调节以有利于增强效应器功能。 具体目标： 1)确定关键的APC亚群并评估它们在RNA-NP介导的免疫应答中的作用。 2)确定参与RNA-NP介导的免疫的调节途径，并研究靶向 这些途径通过将免疫调节RNA掺入疫苗制剂中。 3)评价最有前途的RNA-NP制剂在恶性鼠胶质瘤中的安全性和有效性 模型 研究设计：我们建议鉴定参与RNA-NP介导的免疫的关键APC，靶向 疫苗接种后确定的调控途径，并评估RNA-NP在免疫后的安全性和有效性。 侵袭性临床前鼠恶性胶质瘤模型。因为这个平台可以提供组合疗法 使用单一递送平台，我们将研究RNA（即小干扰RNA）的RNA-NP共递送 或编码单克隆抗体的RNA）靶向调节途径（即程序性死亡配体1） 可以增强我们的疫苗已经有希望的抗肿瘤免疫力。 创新：由于RNA-NPs绕过了细胞治疗的复杂性， 分布，并且可以在肿瘤切除后的几天内制成，这些制剂取代了DC疫苗 提供针对诱发恶性肿瘤的近即时免疫诱导。通过使用脂质体RNA-NP 编码肿瘤RNA和免疫调节分子，作为一种创新的多功能平台， 通过单一治疗方式提供组合疗法，我们可以快速筛选策略， 增强我们疫苗平台的效力。 潜在影响：尽管有积极的和高毒性的多模式治疗，GBM仍然总是不稳定的。 RNA-NP疫苗可以提供一种更有效和特异性的治疗方法，这对改善临床结果至关重要。 受GBM影响的患者，而不对现有治疗增加进一步的毒性。这个新的治疗平台 具有更好地理解RNA-NPs的免疫学潜力的潜力，并包含广泛的临床应用。 应用于可使用从实体瘤的手术切除获得的TTRNA靶向的所有恶性肿瘤 肿瘤的

Abstract: I am now in my first faculty position as an Assistant Professor of Neurosurgery and Pediatrics at the University of Florida. I completed a T32 NIH fellowship at Duke University in Cancer Biology and Developmental Therapeutics before accepting a junior faculty position at the University of Florida where I moved with my mentor, Dr. Duane Mitchell. Dr. Mitchell and I have selected an advisory committee to ensure accrual of my intellectual and professional growth. The non-overlapping expertise of our advisory committee will be an avenue for me to develop new knowledge in tumor immunology, molecular biology, nanotechnology and translational oncology. This proposal will leverage much of this experience as it outlines a career development plan for me to become an independent investigator exploring novel RNA-nanoparticle vaccines that can re- direct the immune system against malignant brain tumors. Background: Glioblastoma (GBM) remains almost uniformly lethal with a median survival of less than 15 months thus necessitating the development of more efficacious and targeted therapeutics. While we have shown in a randomized/blinded trial that RNA-pulsed dendritic cell (DC) vaccines elicit significant survival benefits in GBM patients, these therapies remain encumbered by cost and complexity. Alternatively, RNA- nanoparticles (RNA-NPs) can deliver total tumor RNA (TTRNA), extracted and amplified from as few as 500 biopsied tumor cells, to endogenous antigen presenting cells (APCs) inducing potent, nontoxic anti-tumor immunity. Since these nanoliposomes have been used with limited toxicity in clinical-grade medicine, are stable for several hours in solution, protect nucleic acids from degradation, and can be engineered to modulate immune responses, we have explored the use of TTRNA-loaded NPs as an attractive, “off-the-shelf” therapeutic platform to re-direct host-immunity against intracranial tumors. While we have demonstrated that intravenous delivery of RNA-NPs mediate antigen specific T cell responses against intracranial malignancies comparable to DC vaccines, these formulations were shown to induce differential phenotypes on APCs in the spleen and liver. Hypothesis: RNA-NPs transfect distinct APCs in the spleen and liver inducing differential immune responses that can be modulated in favor of enhanced effector functions. Specific Aims: 1) Determine critical APC subsets and evaluate their role in RNA-NP mediated immune responses. 2) Identify regulatory pathways involved in RNA-NP mediated immunity and investigate capacity to target these pathways through incorporation of immunomodulatory RNAs into vaccine formulations. 3) Evaluate the safety and efficacy of the most promising RNA-NP formulation in a malignant murine glioma model. Research Design: We propose to identify critical APCs involved in RNA-NP mediated immunity, target regulatory pathways identified after vaccination, and evaluate the safety and efficacy of RNA-NPs in an invasive preclinical murine malignant glioma model. Since this platform can deliver combinatorial therapies using a single delivery platform, we will investigate if RNA-NP co-delivery of RNAs (i.e. small interfering RNAs or RNAs encoding for monoclonal antibodies) targeting regulatory pathways (i.e. programmed death-ligand 1) can potentiate our vaccine’s already promising anti-tumor immunity. Innovation: Since RNA-NPs bypass the complexity of cellular therapeutics, are amenable to central distribution, and can be made within days of tumor resection, these formulations supplant DC vaccines providing near immediate immune induction against inciting malignancies. By employing liposomal RNA-NPs encoding for both tumor RNAs and immunomodulatory molecules, as an innovative and versatile platform for delivering combinatorial therapeutics via a single treatment modality, we can rapidly screen strategies to enhance the efficacy of our vaccine platform. Potential Impact: Despite aggressive and highly toxic multi-modal therapy, GBM remains invariably recalcitrant. RNA-NP vaccines can provide a more effective and specific therapy critical in improving clinical outcomes for patients affected by GBMs without adding further toxicity to existing treatments. This novel therapeutic platform has potential to better understand the immunologic potential of RNA-NPs and contains a wide range of clinical application for all malignancies that can be targeting using TTRNA obtained from surgical resection of solid tumors.