Large Scale Nanochannel Electroporation (NEP) for Cell Reprogramming

描述（由申请人提供）：细胞重编程在许多医学和生物学应用中具有巨大的前景，包括再生/修复医学和细胞疾病模型。自从引入诱导多能干细胞（iPSC）以及随后开发定向核重编程（即转分化）方法以来，该领域已经取得了重大进展。然而，由于多种障碍，核重编程技术迄今为止尚未用于治疗患者，其中重编程细胞群的高度异质性和有时固有的不可预测性，这在很大程度上是由于无法控制转染的重编程因子（基于DNA或mRNA）的数量和组合。已经开发了多种转染方法，生物转染方法（例如病毒载体）、化学转染方法（例如脂质复合物、聚阳离子）和物理转染方法（例如显微注射、电穿孔）；然而，除显微注射外，这些技术中的绝大多数都是基于随机过程，导致随机细胞转染，并具有显着的细胞间差异。另一方面，显微注射仅适用于相对较大的细胞，并且产量较低。该领域显然需要能够在单细胞水平上以受控（即时间和剂量）、安全和有效的方式提供重编程因子的新技术，以成功地从实验室工作台过渡到临床。我们最近开发的基于纳米通道的电穿孔 (NEP) 技术满足这些标准，因此有可能使其成为实现此目的的强大工具。在这里，我们建议利用我们在 NEP 方面的独特专业知识来开发更强大、更通用的 3D 系统，该系统可以在广泛的细胞重编程应用中实施。我们将首先实施建模和微/纳米技术来开发最佳的3D NEP平台，该平台可以支持大细胞数量（106）的顺序转染，然后我们将使用诱导多能性和直接神经元转分化作为核重编程模型来测试该平台。最后，我们将使用我们的 NEP 技术系统地研究细胞重编程过程的许多方面，这些方面是使用传统转染技术无法解决的。

DESCRIPTION (provided by applicant): Cell reprogramming holds great promise for a number of medical and biological applications, including regenerative/reparative medicine and cellular disease models. Significant progress has been made in this field since the introduction of induced pluripotent stem cells (iPSCs) and the subsequent development of directed nuclear reprogramming (i.e., transdifferentiation) approaches. However, nuclear reprogramming technologies have not been used to date to treat patients due to several obstacles, among them, the high heterogeneity and sometimes inherent unpredictability of the reprogrammed cell population, which is largely due in part to the inability to control the quantity and combination o the transfected reprogramming factors (DNA- or mRNA- based). A number of transfection methods, biological (e.g., viral vectors), chemical (e.g., lipoplexes, polycations) and physical (e.g., microinjection, electroporation), have been developed; however, the great majority of these techniques, with the exception of microinjection, are based on stochastic processes that lead to random cell transfection with significant cell-to-cell variations. Microinjection on the oter hand is only compatible with relatively large cells, and has low yields. New technologies capable of delivering reprogramming factors in a controlled (i.e., timing and dosage), safe, and efficient manner, at the single cell level, are clearly needed in this field for successful transition from te lab bench to the clinic. Our recently developed nanochannel-based electroporation (NEP) technology meets these criteria, thus potentially making it a powerful tool for this purpose. Here we propose to build upon our unique expertise on NEP to develop a more robust and versatile 3D system that could be implemented in a wide range of cell reprogramming applications. We will first implement modeling and micro/nanoscale technologies to develop an optimum 3D NEP platform that can support sequential transfection of large cell numbers (¿106), and then we will test this platform using induced pluripotency and direct neuronal transdifferentiation as nuclear reprogramming models. Finally, we will use our NEP technology to methodically study a number of aspects of the cell reprogramming process that cannot be addressed using conventional transfection technologies.