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 lentivirus expressing various CARs specific for the Glutamic Acid Decarboxylase (GAD65), a key auto-antigen expressed in islets. These CARs differ in their spacer domain length (small, medium and large), a region between the antigen binding and transmembrane domain which is important for optimal CAR binding. CD3+ T cells isolated from human peripheral blood were transduced with this lentivirus and screened for CAR expression and antigen-specific proliferation. Results: We have generated GAD65-specific CAR T cells, with a high (70-90%) transduction efficiency. In addition, GAD65 CAR T cells with small and large spacer domains were highly viable with 88% and 73% proliferation respectively upon exposure to native GAD65 protein over a 5-day period, which was comparable with bead stimulation. Conversely, medium length spacer domain conferred <20% proliferation and significantly lower viability. Moreover, when exposed to BSA, no proliferation was observed. Conclusion: We have developed a GAD65-CAR which can be further tested in preclinical studies in Tregs as a method for overcoming GAD65 specific autoimmunity in T1D.