Targeted Exosome-Associated AAV-Mediated Gene Therapy to Eliminate Metastatic Neuroendocrine Cancers

摘要 神经内分泌（NE）恶性肿瘤是由内分泌和神经系统引起的激素分泌肿瘤。 系统已经诊断出多种NE肿瘤（NET），例如胰腺神经内分泌癌， 甲状腺髓样癌和肺神经内分泌类癌。大多数NE癌症患者是转移性的 这使得通过手术完全切除是不可能的。当前 化疗，包括奥曲肽，舒尼替尼，依维莫司和肽受体， 好处和严重的副作用。因此，有效的靶向治疗对于转移性NE患者至关重要 癌的 我们最近开发了一种新的技术，命名为“线粒体化学光遗传学”，通过表达一个 异源光门控通道视紫红质蛋白在癌细胞IMM中的作用，以及去极化IMM电位 以生物发光中的荧光素酶为内源性光源诱导细胞死亡。我们 初步数据显示，这种新的线粒体基因疗法导致大量NE癌细胞死亡， 在皮下NE癌异种移植小鼠中， 模型此外，我们已经建立了一个创新的NE癌症靶向基因递送平台， 抗生长抑素受体2（SSTR 2）单克隆抗体（mAb）。 然而，靶向基因治疗，例如携带我们的线粒体化学光遗传学的mAb-Exo-AAV， 治疗基因，迫切需要实现底物诱导的线粒体去极化和选择性 在体内消除癌细胞。此外，在转移性a模型中，基因治疗的治疗效果 是必不可少的，因为大多数诊断的NE癌症患者是转移性的。本申请的具体目的 是开发，生产和评估一种创新的NE癌症靶向线粒体基因疗法， 破坏和消除体内的NET。建议在12个月期间实现以下两个具体目标。 目的1：开发、生产和鉴定NE-肿瘤靶向线粒体基因治疗药物。高质量 抗SSTR 2 mAb-Exo-AAV将通过克隆癌启动子（cfos）和融合蓝光- 产生荧光素酶和光门控视紫红质基因，即cfos-NLuc-2A-ABCB-CoChR（~3.3 kb）， 工程化的pAAV-MCS无启动子表达载体，并使用我们的基于搅拌罐生物反应器的 外泌体-AAV生物制造平台和表面标记技术。抗SSTR 2 mAb-Exo-AAV将 然后评价其癌症特异性靶向和体外抗癌功效。 目的2：探讨线粒体基因治疗对临床前转移性NET的治疗价值 动物模型大多数NE癌症患者最初被诊断为转移， 类癌综合征因此，我们将评估最大耐受剂量（MTD）、药代动力学（PK）、抗肿瘤活性（TNF-α）和抗肿瘤活性（TNF-α）。 NET的疗效，以及使用转移模型开发的基因疗法的肝转移减少。

ABSTRACT Neuroendocrine (NE) malignancies are hormone secreting neoplasms that arise from endocrine and nervous system. Multiple NE tumors (NETs) have been diagnosed, such as pancreatic neuroendocrine cancers, medullary thyroid cancers, and pulmonary neuroendocrine carcinoids. Most NE cancer patients are metastatic at the time of initial diagnosis which makes the complete resections via surgery impossible. The current chemotherapies, including Octreotide, Sunitinib, Everolimus and peptide receptor, have marginal curative benefits and severe side effects. Thus, an effective targeted therapy is critical for patients with metastatic NE cancers. We have recently developed a novel technique, named “mitochondrial chemo-optogenetics”, by expressing a heterologous light-gated channelrhodopsin protein in the IMM of cancer cells, and depolarizing IMM potentials and inducing cell death by using luciferase-luciferin bioluminescence as the endogenous light source. Our preliminary data showed that this new mitochondrial gene therapy caused substantial NE cancer cell death in vitro and stopped NE tumor growth and even reduced tumor size in a subcutaneous NE cancer xenograft mouse model. Additionally, we have built an innovative NE cancer-targeted gene delivery platform by tagging our new anti-somatostatin receptor 2 (SSTR2) monoclonal antibody (mAb) to the surface of exosome. However, a targeted gene therapy, such as mAb-Exo-AAV carrying our mitochondrial chemo-optogenetics therapeutic gene, is urgently needed to achieve substrate-induced mitochondrial depolarization and selective elimination of cancer cells in vivo. Moreover, the therapeutic efficacy of the gene therapy in metastatic a model is essential because most diagnosed NE cancer patients are metastatic. The specific objective of this application is to develop, produce and evaluate an innovative NE cancer-targeted mitochondrial gene therapy to selectively destroy and eliminate NETs in vivo. The following two specific aims over a 12-month period are propose. Aim 1: To develop, produce and characterize the NE-cancer targeted mitochondrial gene therapy. A high-quality anti-SSTR2 mAb-Exo-AAV will be constructed by cloning a cancer promoter (cfos) and the fused blue light- producing luciferase and light-gated rhodopsin gene, i.e. cfos-NLuc-2A-ABCB-CoChR (~3.3 kb), into the engineered pAAV-MCS promoterless expression vector, and produced using our stirred-tank bioreactor-based exosome-AAV biomanufacturing platform and surface tagging technology. The anti-SSTR2 mAb-Exo-AAV will then be evaluated for its cancer specific targeting and in vitro anti-cancer efficacy. Aim 2: To evaluate the therapeutic values of the mitochondrial gene therapy using preclinical NET metastatic animal model. Most NE cancer patients are initially diagnosed with metastases and have already developed carcinoid syndrome. Therefore we will evaluate the maximal tolerated dose (MTD), pharmacokinetics (PK), anti- NET efficacy, and liver metastases reduction of the developed gene therapy using metastatic model.