HER2-targeted exosomal delivery of therapeutic mRNA for enzyme pro-drug therapy

描述（由申请人提供）：响应NIH RFA：RM-12-014，提出工程化外泌体，身体的“天然抗原递送系统”，以增强其天然能力来活化还原性前药并附着至其表面配体，所述表面配体特异性靶向HER 2阳性乳腺癌，其中许多病例对当前疗法具有抗性（例如，曲妥珠单抗）。这将用于P.I.新发现的酶激活前药疗法。（A.C. Matin，Ph.D.）。前药为6-氯-9-硝基-5-氧代-5H-苯并-（a）-吩恶嗪（hocB），PI改良的人源化酶为hChrR 43。该方案的一个非常有用的特征是hocB的活化细胞毒性产物9-氨基-6-氯-5H-苯并[a]吩恶嗪-5-酮（MCHB）具有强荧光，并且可以在活小鼠中可视化。这一特性使得“观察方法”成为可能，并将最大限度地减少对小鼠牺牲的需要和使用更多涉及的测试，例如，LC/MS/MS，免疫组织化学，Western。将从小鼠树突细胞中分离外泌体，并使用外泌体表面蛋白Lamp 2b将HER 2靶向配体KCCYSL添加到其表面。治疗方案将由通过尾静脉注射施用的两种类型的外泌体组成，负载有稳定的hChrR 43 mRNA的外泌体和负载有hocB的外泌体。mRNA而不是DNA将用于基因递送，因为它被证明更好地用于该目的;它将通过电穿孔和/或使用选择性地将细胞mRNA引导到外泌体中的“邮政编码”序列被加载到外泌体中。hocB将通过先前证明的技术通过将其与外来体一起在PBS中孵育来加载。还将构建几种类型的报告基因外泌体，以优化HER 2阳性乳腺癌特异性靶向的条件，确保mRNA对癌细胞的必要转染水平及其翻译效率。UH 2阶段将涉及定向和负载的外泌体的构建;其靶向特异性的优化和体外HER 2阳性乳腺癌细胞杀伤，然后是在免疫受损小鼠中使用人HER 2阳性人乳腺癌细胞系的植入肿瘤进行体内优化。后者研究还将包括过表达HER 2+受体的患者源性乳腺癌肿瘤异种移植小鼠模型，这是一种更现实的测试治疗疗效的系统。UH 3阶段将涉及在大鼠和犬中进行的单次给药药代动力学和毒性研究，随后是在大鼠和犬中进行的重复剂量范围确定毒性研究，以根据无明显不良作用水平（NOAEL）和毒性靶器官选择GLP亚慢性研究的剂量水平;并寻求FDA的IND前反馈。这项拟议中的研究拥有所需的专业知识，涉及5名斯坦福大学科学家，一名附近的外泌体专家和一个主要的药理学研究组织SRI。

DESCRIPTION (provided by applicant): In response to NIH RFA: RM-12-014, it is proposed to engineer exosomes, the body's "natural antigen delivery system," in order to enhance their natural capacity to activate reductive prodrugs and attach to their surface ligands that specifically target HER2-positive breast cancer, many cases of which are resistant to the current therapies (e.g., trastuzumab) . This will be done for an enzyme-activated prodrug therapy newly discovered by the P.I. (A.C. Matin, Ph.D.). The prodrug is 6-chloro-9-nitro-5-oxo-5H-benzo-(a)-phenoxazine (CNOB), and the humanized enzyme improved by the PI is hChrR43. A highly helpful feature of this regimen is that the activated cytotoxic product of CNOB, 9-amino-6-chloro-5H-benzo[a]phenoxazine-5-one (MCHB), is strongly fluorescent and can be visualized in living mice. This property makes an 'observational approach' possible, and will minimize the need for mouse sacrifice and the use of more involved tests, e.g., LC/MS/MS, immunohistochemical, Westerns. Exosomes will be isolated from mouse dendritic cells, and the HER2- targeting ligand KCCYSL will be added to their surface using the exosome surface protein Lamp2b. The therapeutic regimen will consist of two types of exosomes administered by tail vein injection, those loaded with stabilized hChrR43 mRNA and those loaded with CNOB. mRNA instead of DNA will be used for gene delivery as it is proving to be better for this purpose; it will be loaded into the exosomes by electroporation and/or using the "zip code" sequence that selectively directs cellular mRNA into the exosomes. CNOB will be loaded by previously proven techniques by incubating it in PBS with the exosomes. Several types of reporter exosomes will also be constructed to optimize the conditions for specific targeting of HER2-positive breast cancer, ensuring the necessary transfection levels of cancer cells by the mRNA and its translational efficiency. The UH2 phase will concern with the construction of the directed and loaded exosomes; the optimization of their targeting specificity and HER2-positive breast cancer cell killing in vitro, followed by this optimization in vivo using implanted tumors o human HER2-positive human breast cancer cell lines in immunocompromised mice. The latter studies will also include a patient-derived breast cancer tumor xenograft mouse model that overexpress the HER2+ receptor, which is a more realistic system for testing the efficacy of the therapy. The UH3 phase will concern single dose pharmacokinetics and toxicity studies in rats and dogs, followed by repeat dose range finding toxicity studies in rats and dogs to select dose levels for the GLP subchronic studies, based on the no observed adverse effects level (NOAEL) and target organs for toxicity; and seeking pre-IND feedback from the FDA. The proposed research possesses the required mix of expertise and involves 5 Stanford scientists, a nearby exosome expert, and a premier organization, SRI for pharmacological studies.