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

靶向外泌体相关 AAV 介导的基因治疗可消除转移性神经内分泌癌

• 批准号: 9907789
• 负责人: Joseph N Garner
• 金额: $ 24.31万
• 依托单位: TECHNOLOGY COMMERCIALIZATION PARTNERS, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907789
• 关键词: Affinity; Animal Model; Animals; Apoptosis; Binding; Biodistribution; Biological Markers; Biological Response Modifier Therapy; Bioluminescence; Biomanufacturing; Bioreactors; Bypass; Cancer Burden; Cancer Patient; Cancer cell line; Carcinoid Tumor; Cell Death; Cells; Cessation of life; Clinic; Clinical Trials; Cloning; Coupled; Cytosol; Data; Dependovirus; Diagnosis; Diarrhea; Drug Kinetics; Electron Microscope; Endocrine system; Energy Metabolism; Engineering; Enzymes; Excision; Flow Cytometry; Flushing; Future; Gene Delivery; Gene Proteins; Genes; Goals; Heart failure; Hormone secretion; Hormones; IgG1; Impairment; In Vitro; Inner mitochondrial membrane; Islet Cell Tumor; Islets of Langerhans; Legal patent; Light; Liver; Luciferases; Lung; Malignant - descriptor; Malignant Carcinoid Syndrome; Malignant Neoplasms; Maximum Tolerated Dose; Mediating; Membrane Potentials; Metastatic Neoplasm to the Liver; Mitochondria; Modeling; Monoclonal Antibodies; Morphology; Mus; Names; Neoplasm Metastasis; Neoplasms; Nervous system structure; Neuroendocrine Tumors; Neurosecretory Systems; Normal Cell; Octreotide; Operative Surgical Procedures; Pancreas; Patients; Peptide Receptor; Pharmaceutical Preparations; Play; Property; Proteins; Protons; Quality of life; Radiation therapy; Resistance; Rhodopsin; Role; SDZ RAD; SSTR2 gene; Safety; Scheme; Signal Transduction; Source; Surface; Survival Rate; System; Techniques; Technology; Therapeutic; Therapeutic Effect; Time; Translations; Treatment Efficacy; Western Blotting; Xenograft procedure; anti-cancer; base; cancer cell; cancer therapy; chemotherapy; clinical efficacy; confocal imaging; exosome; expression vector; gene therapy; imaging system; improved; in vivo; in vivo imaging system; innovation; light gated; luciferin; medullary thyroid carcinoma; mitochondrial membrane; mouse model; neuroendocrine cancer; novel; optogenetics; pre-clinical; promoter; protein expression; response; side effect; somatostatin receptor 2; subcutaneous; targeted treatment; therapeutic gene; transmission process; tumor; tumor growth

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.

摘要 神经内分泌(NE)恶性肿瘤是由内分泌和神经引起的荷尔蒙分泌肿瘤 系统。已诊断出多种NE肿瘤(Net)，如胰腺神经内分泌癌、 甲状腺髓样癌和肺神经内分泌类癌。大多数东北癌症患者是转移性的 在最初诊断的时候，这使得通过手术完全切除是不可能的。海流 化疗药物，包括奥曲肽、苏尼替尼、埃博利莫斯和多肽受体，疗效不明显。 好处和严重的副作用。因此，有效的靶向治疗对转移性NE患者至关重要。 癌症。 我们最近开发了一种新技术，名为“线粒体化学-光遗传学”，通过表达一种 癌细胞IMM中异源光门通道视紫红质蛋白及去极化IMM电位 以荧光素酶-荧光素生物发光为内源光源诱导细胞死亡。我们的 初步数据显示，这种新的线粒体基因疗法导致了大量NE癌细胞在 体外抑制NE肿瘤生长，甚至缩小荷瘤小鼠的肿瘤体积 模特。此外，我们已经建立了一个创新的NE癌症靶向基因传递平台，通过标记我们的新 抗生长抑素受体2(SSTR2)单抗。 然而，靶向基因治疗，如携带我们线粒体化学光遗传学的mAb-Exo-AAV 治疗性基因，是实现底物诱导线粒体去极化和选择性的迫切需要 体内消除癌细胞。此外，基因治疗对转移瘤a模型的治疗效果。 是必要的，因为大多数被诊断为NE癌的患者都是转移性的。此应用程序的特定目标 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，以选择性地 摧毁和消灭体内的网络。建议在12个月内实现以下两个具体目标。 目的1：开发、制备和鉴定以线粒体为靶向的NE-癌基因治疗。一款高品质的 抗SSTR2单抗-Exo-AAV将通过克隆肿瘤启动子(CFOS)和融合蓝光构建而成。 产生荧光素酶和光门控视紫质基因，即CFOS-NLuc-2A-ABCB-CoChR(~3.3kb)。 构建了pAAV-MCS无启动子表达载体，并以我们的搅拌槽生物反应器为基础生产 Exosome-AAV生物制造平台和表面标记技术。抗SSTR2单抗-Exo-AAV将 然后对其肿瘤特异性靶向性和体外抗癌效果进行评价。 目的2：评价临床前净转移线粒体基因治疗的价值。 动物模型。大多数东北癌症患者最初被诊断为转移，并已发展为 类癌综合征。因此，我们将评估最大耐受量(MTD)、药代动力学(PK)、抗肿瘤作用 净疗效，并使用转移模型开发的基因治疗减少肝转移。