Ex vivo engineering of patient-derived regulatory T (Treg) cells holds promise as a safe and effective approach for treating type 1 diabetes (T1D). Despite promising preclinical studies, considerable biological and technical hurdles must be addressed before this therapeutic strategy can be realized. First, Treg cells must be engineered to optimally enhance their specificity, stability, and functionality. Second, scalability issues – or generating sufficient yield of patient-derived, ex vivo engineered cells to constitute a therapeutic dose – must be overcome. Indee. Inc. is addressing all of these challenges with their Hydropore™ platform. Hydropore™ uses a naturally occurring fluid dynamics phenomenon – vortex shedding – to enable Treg cell engineering that enhances the stability, functionality, and scalability issues in less time than the current gold standards that use virus-based methods and electroporation. HydroporeTM ultimately results in more effective engineered T cells such as Tregs. In this proposal, Indee. Inc. will demonstrate the technical performance of Hydropore™ in engineering the next generation of chimeric antigen receptor regulatory T cells (CAR-Tregs) for T1D. Studies will also focus on demonstrating the superior in vitro and in vivo biological activity of CAR-Tregs engineered using HydroporeTM relative to those engineered using traditional methods. Pending the successful completion of these objectives, CAR-Tregs will be engineered using Treg cells isolated from T1D patients and demonstrate clinical- and commercial-scale processing and enrichment of sufficient CAR-Tregs cells for human trials.