Retroviral RLI immunomodulatory gene therapy for glioblastoma

项目摘要/摘要 抗肿瘤免疫反应需要天然免疫细胞和获得性免疫细胞的功能。胶质母细胞瘤 然而，(GBM)存在着严重的免疫抑制微环境，特别是它的T细胞无知 由骨髓隔离引起；T细胞耗竭由免疫检查点分子在 抑制T细胞功能的T细胞表面；以及受损的记忆T细胞反应。不幸的是，努力 以免疫抑制的GBM微环境为靶点进行系统免疫治疗尚未产生 在临床试验中产生重大影响。针对GBM的本地化病毒治疗也进行了研究，同时 这些病毒引发了抗肿瘤免疫反应，这些治疗也未能影响患者的存活率 临床试验。为了解决这些局限性，我们研究了复制型逆转录病毒在肿瘤内的传递。 表达RLI，它编码白介素15融合蛋白，增强CD8+和CD4+幼稚和 记忆T细胞增殖，作为一种治疗策略，没有全身治疗的毒副作用 肿瘤微环境。我们证明了复制的逆转录病毒载体RLI延长了小鼠的存活时间。 免疫功能正常的小鼠使用多种不同的脑胶质瘤模型。在这里，我们将在我们的 数据通过研究我们的中心假设，即肿瘤内RLI免疫调节基因治疗可以 通过添加其他免疫调节策略、结合免疫原性细胞死亡或靶向来增强 抗性机制。我们将通过四个具体目标来检验我们的假设：(1)增强RLI 通过增强T细胞动员、共刺激和记忆进行免疫调节基因治疗；(2)确定 如果靶向检查点通路加强逆转录病毒RLI免疫调节基因治疗；(3)增强RLI 通过结合免疫原性细胞死亡进行免疫调节基因治疗；以及(4)识别和靶向 胶质母细胞瘤-表达的蛋白质对抗逆转录病毒RLI免疫调节基因治疗。我们追求的是 这些目标将利用我们实验室开发的新技术，如我们的二元逆转录病毒系统，以提供 大量免疫调节基因和我们的逆转录病毒载体Cas13d RNA靶向CRISPR 抗性机制。我们将把这些创新方法与尖端技术相结合，例如 CyTOF研究基于RLI的逆转录病毒疗法对先天和获得性全队列的影响 免疫反应；定制CRISPRi文库；配对免疫缺陷和免疫活性小鼠品系 分离免疫耐药机制；以及单细胞测序以分析由 这些疗法。这些研究将开发我们的新型局部RLI逆转录病毒免疫疗法 通过记述引起GBM局部和系统性免疫缺陷的各种机制 对于T细胞的无知和疲惫，以及识别和靶向肿瘤细胞和免疫细胞驱动的抵抗 机制在它们进化之前。在此过程中，我们将验证我们关于基于RLI的影响的假设 免疫调节性基因治疗GBM，一种具有显著翻译潜力的新策略。

PROJECT SUMMARY/ABSTRACT Antitumor immune responses require a functional repertoire of innate and adaptive immune cells. Glioblastoma (GBM), however, harbors a profoundly immunosuppressed microenvironment, particularly its T cell ignorance caused by bone marrow sequestration; T cell exhaustion caused by immune checkpoint molecules on the surface of T cells that suppress T cell function; and impaired memory T-cell responses. Unfortunately, efforts to target the immunosuppressed GBM microenvironment with systemic immunotherapies have not produced meaningful impact in clinical trials. Localized viral treatments have also been investigated for GBM and, while these viruses elicit an anti-tumoral immune response, these treatments have also failed to impact survival in clinical trials. To address these limitations, we have investigated intratumoral delivery of a replicating retrovirus expressing RLI, which encodes an interleukin-15 fusion protein that enhances CD8+ and CD4+ naïve and memory T-cell proliferation, as a therapeutic strategy free of the toxicities of systemic treatments targeting the tumor microenvironment. We demonstrated that replicating retroviral delivery of RLI prolonged survival of immunocompetent mice with intracranial gliomas using multiple different models. Here, we will build upon our data by investigating our central hypothesis that intratumoral RLI immunomodulatory gene therapy can be potentiated by adding other immunomodulatory strategies, incorporating immunogenic cell death, or targeting resistance mechanisms. We will investigate our hypothesis through four specific aims: (1) Potentiate RLI immunomodulatory gene therapy by enhancing T-cell mobilization, co-stimulation, and memory; (2) Determine if targeting checkpoint pathways potentiates retroviral RLI immunomodulatory gene therapy; (3) Enhance RLI immunomodulatory gene therapy by incorporating immunogenic cell death; and (4) Identify and target glioblastoma-expressed proteins that counteract retroviral RLI immunomodulatory gene therapy. Our pursuit of these aims will utilize novel technologies developed by our lab such as our binary retroviral system to deliver a large payload of immunomodulatory genes and our retroviral compact Cas13d RNA-targeting CRISPR to target resistance mechanisms. We will combine these innovative approaches with cutting-edge technologies such as CyTOF to characterize the effects of RLI-based retroviral therapies on the full cohort of innate and adaptive immune responses; customized CRISPRi libraries; paired immunodeficient and immunocompetent mice strains to isolate immunologic resistance mechanisms; and single cell sequencing to profile T-cell subsets altered by these therapies. These studies will develop our novel localized RLI retroviral immunotherapy in a manner that addresses the spectrum of mechanisms creating local and systemic immunodeficiency in GBM by accounting for T-cell ignorance and exhaustion, and identifying and targeting tumor cell and immune cell-driven resistance mechanisms before they evolve. In doing so, we will validate our hypothesis regarding the impact of RLI-based immunomodulatory gene therapy on GBM, a novel strategy with significant translational potential.