Combine mitochondrial gene therapy and synthetic lethal chemotherapy to treat triple-negative breast cancer

总结 三阴性乳腺癌（TNBC）是高度侵袭性的和标准的细胞毒性化疗（例如， 蒽环类-紫杉烷类）是临床上的主要治疗策略。我们以前的研究和文献 证明聚（ADP-核糖）聚合酶抑制剂（PARPi）作为单一药剂或与 表皮生长因子受体抑制剂（EGFRi）诱导了背景合成致死性，并降低了TNBC 通过抑制DNA损伤的修复来抑制细胞凋亡。我们的临床试验表明，维利帕尼（PARPi）/拉帕替尼 在具有野生型BRCA 1/2的TNBC患者中，EGFRi（表皮生长因子受体i）实现了24%的应答率。尽管取得了这些成就， TNBC细胞通常对化疗产生耐药性，患者反应率低，并且在化疗后再生。 初级治疗。迫切需要能够有效治疗TNBC的新策略。线粒体是 细胞的发电站，并在调节细胞功能中发挥关键作用，使其成为一个有前途的肿瘤学 目标破坏线粒体功能，如直接使线粒体内膜（IMM）去极化 潜在的通过合成异源基因，可以绕过修复信号转导通路，触发 一个不归点癌细胞死亡，并随后防止耐药性的发展。我们 最近开发了一种线粒体发光遗传学（mLumiOpto）技术， IMM中的异源光门控通道视紫红质和细胞质中的工程化荧光素酶，其诱导 IMM去极化通过开放线粒体通道视紫红质与发射的辅酶A酶 内源性蓝色生物发光初步研究表明，mLumiOpto有效地去极化 代表多种亚型的TNBC细胞系中的线粒体，导致持续的DNA损伤， 在三种TNBC异种移植物模型中显著降低肿瘤负荷。应用我们的双重目标运载工具， 即EGFR/CD 276单克隆抗体标记的外泌体相关腺相关病毒（mAb-Exo-AAV）， 和癌症特异性启动子（cfos）在mLumiOpto中实现了高TNBC特异性、功能性表达， 最小的不良全身毒性。该项目的目标是利用有针对性的 mLumiOpto，其与mAb-Exo-AAV和PARPi一起递送以在体内消除TNBC细胞。这个假设是 组合的mLumiOpto/PARPi整合了多种抗癌机制，即，IMM去极化，DNA 损伤和抑制修复，以及肿瘤免疫。具体来说，大规模双靶向mLumiOpto将在 生成和表征;治疗剂量和策略将得到优化;抗癌疗效将得到优化。 在TNBC原发性异种移植物模型和远处转移模型中评估（Aim 1）。此外，协同 将评估mLumiOpto/PARPi的效果，并研究其潜在机制， 免疫活性模型（目的2）。最后，转移减少和异质性TNBC治疗 将在转移性和患者来源的异种移植物（PDX）模型中充分测试功效，并且还将 研究（目标3）。该项目的成功完成将为治疗TNBC提供新的策略。

SUMMARY Triple-negative breast cancers (TNBCs) are highly aggressive and standard cytotoxic chemotherapies (e.g. anthracycline-taxane) are the main treatment strategies in clinics. Our previous research and literature demonstrated that poly (ADP-Ribose) polymerase inhibitors (PARPi) as single agents or in combination with epidermal growth factor receptor inhibitor (EGFRi) induced a contextual synthetic lethality and reduced TNBC metastsis by inhibiting the repair of DNA damage. Our clinical trial showed that veliparib (PARPi)/lapatinib (EGFRi) achieved a 24% response rate in TNBC patients with wildtype BRCA1/2. Despite these achievements, TNBC cells often develop drug resistance to chemotherapies, have low patient response rate, and regrow after primary treatment. Novel strategies that can effectively treat TNBCs are urgently needed. Mitochondria are the powerhouse of cells and play a pivotal role in regulating cell functions, rendering them a promising oncological target. Destroying mitochondrial function, such as directly depolarizing the inner mitochondrial membrane (IMM) potential via synthesized heterologous genes, can bypass the repair of signaling transduction pathways, trigger a point-of-no-return cancer cell death, and subsequently prevent the development of drug resistance. We recently developed a mitochondrial luminoptogenetics (mLumiOpto) technology by synthesizing the heterologous light-gated channelrhodopsin in IMM and an engineered luciferase in cytoplasm, which induces IMM depolarization through opening mitochondrial channelrhodopsin with luciferase-luciferin emitted endogenous blue bioluminescence. Preliminary studies showed that mLumiOpto effectively depolarized mitochondria in TNBC cell lines representing multiple subtypes, resulted in persistent DNA damage, and significantly reduced tumor burden in three TNBC xenograft models. Applying our dual-targeted delivery vehicle, i.e. EGFR/CD276 monoclonal antibodies tagged exosome-associated adeno-associated virus (mAb-Exo-AAV), and cancer-specific promoter (cfos) in mLumiOpto achieved high TNBC specificity, functional expression, and minimal undesirable systemic toxicity. The objective of this project is to harness the combination of targeted mLumiOpto that is delivered with mAb-Exo-AAV and PARPi to eliminate TNBC cells in vivo. The hypothesis is that the combined mLumiOpto/PARPi integrates multiple anti-cancer mechanisms, i.e., IMM depolarization, DNA damage and inhibition of repair, and tumoral immunity. Specifically, large-scale dual-targeted mLumiOpto will be generated and characterized; treatment dosage and strategy will be optimized; and anti-cancer efficacy will be evaluated in TNBC primary xenograft model and distant metastatic model (Aim 1). Furthermore, the synergistic effects of mLumiOpto/PARPi will be assessed and the underlying mechanisms will be investigated in immunocompetent models (Aim 2). Finally, the metastasis reduction and heterogeneous TNBC treatment efficacy will be fully tested in metastatic and patient-derived xenograft (PDX) models, and toxicology will also be investigated (Aim 3). Successful completion of this project will provide a new strategy to treat TNBCs.