Harnessing the Immunologic Capacity of RNA-nanoparticle Vaccines Targeting Glioblastoma

摘要： 我现在是我的第一个教职，是大学神经外科和儿科学助理教授 来自佛罗里达州。我在杜克大学完成了美国国立卫生研究院癌症生物学和发育专业的T32奖学金 在接受佛罗里达大学的初级教职之前，我和我的 导师杜安·米切尔博士。米切尔医生和我已经选择了一个咨询委员会来确保我的 智力和职业成长。我们咨询委员会的非重叠专业知识将是 让我在肿瘤免疫学、分子生物学、纳米技术和 翻译肿瘤学。这份提案将充分利用这些经验，勾勒出一份职业发展蓝图 计划让我成为一名独立调查员，探索新型RNA纳米颗粒疫苗，这种疫苗可以重新- 引导免疫系统对抗恶性脑瘤。 背景：胶质母细胞瘤(GBM)几乎都是致死性的，中位生存期不到15 因此，有必要开发更有效和更有针对性的治疗方法。当我们有了 一项随机/盲法试验表明，RNA冲击的树突状细胞(DC)疫苗可显著延长存活率 虽然这些疗法对GBM患者有好处，但仍受到成本和复杂性的制约。或者，RNA- 纳米粒子(RNA-NPs)可以传递总的肿瘤RNA(TTRNA)，从500个样本中提取和扩增 经活检的肿瘤细胞，至内源性抗原提呈细胞(APC)，诱导有效、无毒的抗肿瘤 豁免权。由于这些纳米脂质体已被用于临床级别的药物，毒性有限，因此 在溶液中稳定几个小时，保护核酸不被降解，并且可以被设计成调节 关于免疫反应，我们探索了负载TTRNA的NPs作为一种有吸引力的、现成的 治疗平台，以重新定向宿主免疫对抗颅内肿瘤。虽然我们已经证明了 静脉注射RNA-NPs介导抗颅内恶性肿瘤抗原特异性T细胞反应 与DC疫苗相比，这些制剂被证明在APC上诱导不同的表型 脾和肝。 假设：RNA-NPs在脾和肝中导入不同的APC，诱导不同的免疫反应 可以进行调制，以支持增强的效应器功能。 具体目标： 1)确定关键的APC亚群，并评价它们在RNA-NP介导的免疫应答中的作用。 2)确定参与RNA-NP介导的免疫的调控途径，并调查靶向能力 这些途径是通过将免疫调节RNA纳入疫苗配方而实现的。 3)评价最有希望的RNA-NP制剂治疗恶性鼠脑胶质瘤的安全性和有效性 模特。 研究设计：我们建议确定与RNA-NP介导的免疫有关的关键APC，靶向 确定疫苗接种后的调控途径，并评估RNA-NPs在小鼠体内的安全性和有效性 侵袭性临床前小鼠恶性胶质瘤模型。由于这个平台可以提供组合疗法 使用单一传递平台，我们将调查RNA-NP联合传递RNAs(即小干扰RNAs 或编码单抗的RNA)靶向调控通路(即程序性死亡配体1) 可以增强我们疫苗已经很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，因此服从中央 分发，并可以在肿瘤切除后几天内制成，这些制剂取代了DC疫苗 提供近乎即时的免疫诱导，对抗煽动的恶性肿瘤。通过使用脂质体RNA-NPs 编码肿瘤RNA和免疫调节分子，作为一种创新和多功能的平台 通过单一的治疗模式提供组合疗法，我们可以快速筛选策略以 增强我们疫苗平台的效力。 潜在影响：尽管采用积极和剧毒的多模式治疗，但GBM始终顽固不化。 RNA-NP疫苗可以提供一种更有效和更特异的治疗方法，对于改善临床结果至关重要 受GBMS影响的患者，而不会在现有治疗中增加进一步的毒性。这一新颖的治疗平台 有可能更好地了解RNA-NPs的免疫学潜力，并包含广泛的临床 应用手术切除实体获得的TTRNA靶向所有恶性肿瘤 肿瘤。

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.