In vivo engineering of T cells with AAV

In vivo T-cell engineering has the potential to transform cellular immunotherapy by eliminating complex ex vivo manufacturing and avoiding lymphodepleting pre-conditioning regimens, thereby enabling rapid, scalable, and broadly accessible treatments. Here, we present a versatile platform in which the modality of CAR expression is tailored to the biological requirements of distinct disease settings and is based on adeno-associated virus (AAV), a clinically validated gene delivery platform with an established safety profile and multiple approved gene therapies.
For autoimmune diseases, we developed an AAV-mediated approach that enables in vivo T-cell engineering, resulting in transient expression of a CD19-directed CAR. In a preclinical model of systemic lupus erythematosus (SLE), a single AAV injection induced robust and transient B-cell depletion, reduced autoantibody production, ameliorated disease pathology, and prolonged survival. These findings demonstrate that transient CAR expression is sufficient to induce a therapeutic immune reset, followed by immune reconstitution and sustained disease control.
We extended this concept to allergic diseases through Q-CAR, a CAR designed to selectively target IgE-producing B cells while avoiding interference from circulating soluble IgE. Importantly, using AAV-mediated in vivo T-cell engineering, we demonstrated that elimination of IgE-secreting cells led to protection against food allergy-induced anaphylaxis in mice. These results further support the concept that transient CAR expression can effectively remodel pathogenic humoral immune responses.
In contrast, cancer requires continuous immune surveillance and long-term persistence of engineered lymphocytes. To address this need, we developed an in vivo genome-engineering strategy that combines AAV-mediated delivery of a CAR transgene with genome-editing machinery to achieve targeted integration into the TRAC locus, enabling stable and physiologically regulated CAR expression. Using this approach, we achieved site-specific in vivo generation of anti-CD19 CAR T cells following a single AAV administration in an acute lymphoblastic leukemia (ALL) model.
Together, our studies demonstrate the feasibility and versatility of in vivo T-cell engineering as a platform for disease-tailored cellular immunotherapy. This approach overcomes many of the logistical and manufacturing challenges associated with current ex vivo strategies and expands the potential application of CAR-based therapies across a broad spectrum of diseases, thereby increasing accessibility to a wider patient population.