Engineering Clinical Trials on a Chip for Dystrophin-Deficient Muscular Dystrophy

项目摘要 目标：我们将开发和验证3D工程肌肉组织（EMT），作为一种使能的“临床试验”， “芯片”平台，以确定人类Duchenne和Becker肌的心脏和骨骼肌缺陷 营养不良（DMD/BMD），并测试新疗法的功效。我们利用最先进的技术 我们的团队开发的：（1）分化和成熟iPSC衍生的心肌细胞和骨骼肌细胞的方法 成肌细胞(2)（3）使用3D组织工程技术， 脱细胞的细胞外基质（dECM）（4）构建多细胞结构的方案（5）非侵入性， 高通量筛选系统，允许并行电生理和收缩评估。(6)小说 阳离子通道-TRPC 6的拮抗剂，其在严重的DMD小鼠模型中显示出体内潜力。我们 将这些技术和方法整合到概括DMD主要标志的测定中， 实时评估治疗效果。为了证明我们的EMT检测作为“临床试验-对- 新的TRPC 6阻滞剂通过I期安全性/毒性，II期剂量/疗效在EMT中进行了测试， 少数DMD患者，以及来自较大DMD/BMD异质性人群的EMT的III期结局 患者重点/目标：UG 3阶段建立了设计优化的hiPSC衍生心脏和 骨骼肌组织使用我们的磁感应平台，并将该平台与我们的高- 吞吐量成像能力。开发的平台将用于表征功能表型 从hiPSC衍生的心肌细胞和骨骼肌成肌细胞产生的工程化肌肉组织， 营养不良患者或健康对照。这将验证“芯片上临床试验”测定具有足够的 对概括DMD表型、分层疾病严重程度和定义收缩性的敏感性， 电生理结果可用于告知UH 3阶段的治疗有效性测试。UH3 阶段将使用EMT测定来模拟用于运行3阶段临床试验的方案。治疗方法是 测试的是BI 749327，一种新型的有前途的TRPC 6（瞬时受体电位- Canonical Channel 6）。该药物是第一种口服生物可利用的TRPC 6阻滞剂，我们已经报道了 在体内压力负荷和肾纤维化模型中的功效。新数据显示了DMD的疗效。在目标1中， 在健康EMT中测定BI 749327的毒性特征和剂量范围。在目标2中， 将BI 749327在一定剂量范围内的效应应用于DMD衍生的EMT，以确定最佳剂量， 药效学特征，以进行更广泛的测试。在目标3中，我们将使用先验信息， 进行一项3期研究，该研究将涉及来自具有不同突变的DMD患者的iPSC衍生的EMT 导致总的肌营养不良蛋白缺失，以及来自表达突变型肌营养不良蛋白的BMD患者，导致不同的 临床表型目标是建立临床试验芯片，为人类DMD临床提供信息和支持 试验设计，优化剂量和个性化治疗的患者。

PROJECT SUMMARY Goal: We will develop and validate 3D engineered muscular tissues (EMTs) as an enabling “clinical trial-on-a- chip” platform to determine cardiac and skeletal muscle deficiencies in human Duchenne and Becker muscular dystrophy (DMD/BMD), and test the efficacy of novel therapeutics. We leverage state-of-art techniques developed by our team: (1) a method to differentiate and mature iPSC-derived cardiomyocytes and skeletal myoblasts. (2) phenotype-confirmed hiPSCs from DMD patients (3) 3D-tissue engineering technique using decellularized extracellular matrix (dECM) (4) Protocols to construct a multicellular architecture (5) non-invasive, high-throughput screening system allowing parallel electrophysiological and contractile assessment. (6) Novel antagonist of the cation channel – TRPC6 that displays in vivo potential in a severe mouse model of DMD. We integrate these techniques and methods into an assay that recapitulates the major hallmarks of DMD, enabling real-time assessment of treatment efficacy. To demonstrate the utility of our EMT assay as a “clinical trial-on-a- chip,” the new TRPC6 blocker is tested through Phase I safety/toxicity, Phase II dosing/ efficacy in EMT from a few DMD patients, and Phase III outcomes in EMTs from a larger heterogenous population of DMD/BMD patients. Focus/Aim: The UG3 phase establishes protocols to engineer optimized, hiPSC-derived cardiac and skeletal muscle tissues using our magnetic sensing platform and integrating this platform with our high- throughput imaging capabilities. The developed platform will be used to characterize the functional phenotypes of engineered muscle tissues generated from hiPSC-derived cardiomyocytes and skeletal myoblasts from dystrophic patients or healthy controls. This will verify that the ‘clinical trial-on-a-chip’ assay possesses sufficient sensitivity to recapitulate DMD phenotypes, stratify disease severity, and define contractility and electrophysiological outcomes that can be used to inform therapy efficacy testing in the UH3 phase. The UH3 phase will use the EMT assay to simulate protocols for running a 3-phase clinical trial. The therapeutic to be tested is BI 749327, a novel and promising selective and potent inhibitor of TRPC6 (Transient Receptor Potential- Canonical channel 6). The drug is the first orally bioavailable TRPC6 blocker, and we have already reported efficacy in pressure-load and renal fibrosis models in vivo. New data shows efficacy in DMD. In Aim 1, the toxicity profile and dose range of BI 749327 is determined in healthy EMTs. In Aim 2, mechanical and electrical effects of BI 749327 over a range of doses is applied to DMD-derived EMTs to identify an optimal dose and pharmacodynamic profile to move forward to broader testing. In Aim 3, we will use the prior information to perform a Phase 3-style study that will involve iPSC-derived EMTs from DMD patients with varying mutations causing total dystrophin deletion, and from BMD patients that express mutant dystrophin resulting in varying clinical phenotypes. The goal is to establish the clinical trial-on-a-chip to inform and support human DMD clinical trial design, optimizing dosing and personalizing therapy for patients.