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

项目总结： 主要疾病，如心力衰竭、帕金森氏病(PD)、1型糖尿病和老年性黄斑变性- 代，是由特定细胞类型的损害引起的器官和全身衰竭的例子。一种可能 治疗方案是用体外工程的、具有生理功能的细胞来取代受损的细胞 缓解临床症状。然而，昂贵、耗时且低效的单元重编程方法 为退行性疾病产生可移植的治疗细胞阻碍发育和临床翻译 潜在的变革性疗法。我们的目标是开发一种高效的电池生产工艺。 能够可靠地规模化生产的替代疗法，用于治疗目前难以治愈的疾病，如 帕金森氏症。这一目标将建立在我们获得专利和经过验证的Cell Squze®技术基础上，该技术可以 将包括信使核糖核酸、蛋白质和多肽在内的物质输送到敏感的原代细胞。对于此第二阶段SBIR 提案中，我们的总体目标是证明使用Cell Squeeze®技术，我们可以引入转录- 提高外周血细胞转分化效率的因子临床应用 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、剂量和 转录因子的组合，以创造更多的高质量的功能细胞产品 时间比目前的方法更短，而且没有与病毒基因传递相关的风险。支持- 为了达到这个目标，我们已经证明了挤压处理本身并不显著影响基因。 表达，我们可以高效地从IPSCs产生神经元，我们可以引入多种转录因子 转化为PBMC，上调关键神经元信号通路的表达。理由是我们的非病毒式病毒 传递转录因子来驱动细胞命运的方法可以显著提高效率和疗效 与其他转分化方法相比，生产的细胞具有更少的安全性和监管顾虑。 此外，与同种异体IPSC衍生产品相比，自体细胞不需要慢性 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 Squze®技术将基于mRNA的转录因子传递到PBMC以提高效率的方法 向多巴胺能神经元的有效转分化。生成的dns将在 并与使用现有方法从IPSC产生的糖尿病肾病进行比较。在目标2中，这些域名将在功能上 在活体小鼠帕金森病模型中进行评估以支持进一步发展为潜在的转化细胞 心理治疗。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 正在寻找神经退行性变的差异化细胞疗法的公司。

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.