Modulation of Immune responses to gene therapy by Creation of AAV-specific Chimeric antigen receptor regulatory T cells (CAR-Tregs)

Project 4 - Abstract In Project 4, we will use our novel adeno-associated virus (AAV) directed chimeric antigen receptor (CAR) T-cells and T regulatory cells (Tregs) to modulate and mitigate immune responses to AAV gene therapy for alpha-1-antitrypsin (AAT) disease. We have shown that our AAV CARs can clear AAV transduced cells similar to what was observed in the early

Hemophilia trials and our AAV-CAR Tregs are able to allow modulate anti- capsid and anti- transgene immune responses allowing for stable and persistent transgene expression. We will optimize our current AAV CAR Treg construct and protocols to make the most suppressive and functional CAR Tregs. Then we will further characterize the suppressive function of our AAV

CAR T-regs in response to the immunogenic rh32.33 capsid in mouse models. We will further determine if they are able to overcome pre-existing immunity to capsid which is a large issue for clinical gene therapy due to the large number of patients who have pre-existing immunity to AAV. And, we will study if AAV CAR T-regs can overcome pre-existing immunity to transgenes

like those involved in CRISPR genome editing, as again most patients have immune response to CRISPR proteins causing the likelihood of survival of edited cells to be limited. We will also determine if AAV CAR T-regs can allow for re-dosing of AAV vectors, allowing for multiple doses of AAV gene therapy to be delivered to reach optimal therapeutic expression. In addition,

we will further characterize the modulation of the immune system to immunogenic transgenes by AAV CAR Tregs allowing for stable immunogenic transgene expression. Finally, we will characterize the immune responses to AAV delivery by in ferrets, a novel model in the AAV field regarding immune responses. We will investigate how well the ferret model recapitulates the

observations in the clinic and whether capsid specific cytotoxic and/or T-regulatory cells are generated after systemic or intramuscular injection of ferrets. Overall this project will allow us to learn considerably about the immune responses generated after AAV gene therapy and allow us to develop powerful tools to modulate and mitigate immune responses that are relevant for

clinical gene therapy development.