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 neurodegeneratio...