Microfluidic cell squeezing platform for the transdifferentiation of somatic cells for efficient generation of a cell replacement therapy for Parkinsons Disease

项目概要： 主要疾病，如心力衰竭、帕金森病（PD）、1型糖尿病和年龄相关性黄斑变性， 这些疾病是由对特定细胞类型的损伤驱动的器官和系统衰竭的例子。一个可能 治疗方案是用离体工程化的生理功能细胞替换受损细胞， 缓解临床症状。然而，昂贵的、时间密集的和低效的细胞重编程方法， 产生用于退行性疾病的可移植治疗细胞阻碍了发展和临床移植， 潜在的变革性疗法。我们的目标是开发一种高效的生产细胞的方法， 替代疗法，可以可靠地大规模生产，以治疗目前难治性疾病， 帕金森氏症这一目标将建立在我们的专利和成熟的细胞挤压®技术， 将包括mRNA、蛋白质和肽的材料递送到敏感的原代细胞中。对于本阶段II SBIR 我们的总体目标是证明，通过Cell Squeeze®技术，我们可以引入转录- 可以增加外周血细胞（PBMC）转分化为临床上可接受的细胞的效率的因子。 相关的多巴胺能神经元。我们的中心假设是，我们可以精确地控制时间，剂量， 转录因子的组合，以产生更大量的高质量的功能性细胞产物， 比现有方法可能的时间更短，并且没有与病毒基因递送相关的风险。支持- 为了实现这一目标，我们已经证明，单独的挤压处理不会显著影响基因表达。 表达，我们可以有效地从iPSC产生神经元，我们可以引入多种转录因子， 进入PBMC以上调关键神经元信号传导途径的表达。其原理是，我们的非病毒 一种传递转录因子以驱动细胞命运的方法可以显著提高效率和功效 与其他转分化方法相比，产生的细胞具有更少的安全性和监管问题。 此外，与同种异体iPSC衍生产品相比，自体细胞不需要长期使用 免疫抑制-确保患者长期健康的关键因素。在目标1中，我们寻求优化 Cell Squeeze®技术将基于mRNA的转录因子递送至PBMC以驱动效率的方法。 快速转分化为多巴胺能神经元（DN）。生成的DN将在 体外并与使用现有方法从iPSC产生的DN进行比较。在目标2中，这些DN将在功能上 在体内鼠PD模型中评估，以支持进一步发育为潜在的转化细胞 疗法成功完成这些目标可能会支持与其他生物制药公司的合作机会 正在寻求神经退行性疾病的差异化细胞治疗方法的公司。

PROJECT SUMMARY: Major diseases, such as heart failure, Parkinson’s Disease (PD), type 1 diabetes, and age-related macular de- generation, are examples of organ and systemic failure driven by damage to specific cell types. One possible therapeutic solution is to replace the damaged cells with ex vivo engineered, physiologically functional cells to alleviate clinical symptoms. However, expensive, time-intensive, and inefficient cell reprogramming methods for generating transplantable therapeutic cells for degenerative disorders hinders development and clinical transla- tion of potentially transformative therapies. Our goal is to develop a highly efficient process for producing cell replacement therapies that can be reliably manufactured at-scale to treat currently intractable diseases such as Parkinson’s Disease. This goal will build upon our patented and proven Cell Squeeze® technology that can deliver materials including mRNA, proteins, and peptides into sensitive primary cells. For this Phase II SBIR proposal, our overall objective is to demonstrate that with Cell Squeeze® technology we can introduce transcrip- tion factors that can increase the efficiency of transdifferentiating peripheral blood cells (PBMCs) into clinically relevant dopaminergic neurons. Our central hypothesis is that we can precisely control the timing, dose, and combinations of transcription factors to create high quality, functional cell products in greater quantities in a shorter time than is possible with current methods and free of risks associated with viral gene delivery. Support- ing this goal, we have already demonstrated that the squeeze treatment alone does not significantly affect gene expression, we can efficiently generate neurons from iPSCs, and we can introduce multiple transcription factors into PBMCs to upregulate expression of key neuronal signaling pathways. The rationale is that our non-viral method of delivering transcription factors to drive cell fate could significantly improve the efficiency and efficacy of cells produced with fewer safety and regulatory concerns as compared to other methods of transdifferentiation. Furthermore, in comparison to allogeneic iPSC derived products, autologous cells would not require chronic immunosuppression – a key factor to ensure long term health of the patient. In Aim 1, we seek to optimize methods for the Cell Squeeze® technology to deliver mRNA-based transcription factors to PBMCs to drive effi- cient transdifferentiation into dopaminergic neurons (DNs). Resultant DNs will be thoroughly characterized in vitro and compared to DN generated from iPSC using existing methods. In Aim 2, these DNs will be functionally assessed in an in vivo murine PD model to support the further development into a potentially transformative cell therapy. Successful completion of these aims may support partnering opportunities with other biopharmaceutical companies who are seeking differentiated cell therapy approaches in neurodegeneration.