Regulatory T cells (Tregs) are key players in the maintenance of peripheral tolerance in healthy individuals. Numerous reports suggest that inherent defects in Tregs play a critical role in T1D development. Thus, Tregs have been heavily investigated as a therapeutic target in T1D, with the aim to re-establish islet tolerance. Antigen-specific Tregs are more superior to polyclonal Tregs in their migration to and persistence in target tissue, and prevention of unwanted widespread suppression. However, they are rare in peripheral blood, requiring significant expansion for therapeutic quantities, which can be costly and time-consuming. Therefore, this project aims to utilise chimeric antigen receptors (CARs) to confer antigen-specificity to Tregs. Method: We have generated CAR Tregs specific for the Glutamic Acid Decarboxylase (GAD65), a key autoantigen expressed in islets using lentiviral transduction. The spacer domain, a region between the antigen binding and transmembrane domain, which is important for optimal CAR binding, was optimised in CD3+ T cells. Naïve Tregs were then isolated from human peripheral blood and used to generate CAR Tregs expressing the optimised spacer domain. An additional intracellular signalling domain was also introduced into these CAR- Tregs to screen for its benefit. These CAR Tregs were then assayed for functional marker expression and suppressive capacity.  Results: Following expansion, 80%-96% of CAR Tregs remain FOXP3 positive, and express high level of the functional markers CTLA4 and CD25, showing >80 % and 80-90% positivity respectively.  Moreover, their suppressive capacity, when stimulated via their TCR, was not altered by their expression of the CAR when compared to a donor matched un-transduced (CAR negative) control.  In addition, unlike their donor matched un-transduced control, these CAR Tregs are able to bind and proliferate in response to GAD65 over a 5- and 7-day period both in the presence and absence of IL-2.  Conclusion: We have developed a GAD65-CAR Treg which can be further tested in preclinical studies in Tregs as a method for overcoming GAD65 specific autoimmunity in T1D.