In Vivo CAR Delivery As Supercoiled DNA with Polyasparagine Nanoparticles

Background

Strategies for in vivo reprogramming of immune effector cells have been proposed in the hope to recapitulate the success of ex vivo engineered chimeric antigen receptor t cells (CAR-T) while reducing the manufacturing burden, improving the safety profile and recruiting collaborating immune cell types to expand the appliable indications. Viral and non-viral vectors with tissue tropism selected for in vivo reprogramming applications have been attempted, and the efforts to fully address the off-tissue delivery safety concerns for viral vector and a clinically viable dosing scheme to demonstrate efficacy with the brief durability of mRNA in non-viral vectors continue.

Methods

We deploy CAR encoded in supercoiled DNA formulated with multi-arm polyasparagine nanoparticles (STAR). The physical characteristics, the stability, the molecular dynamics and the biodistribution of the DNA/polyasparagine polyplex were characterized. Anti-murine CD19 CAR expression, its durability and the efficacy as the result of the polyplex transfection were evaluated in vitro and in vivo in blood, livers, lymph nodes, spleen and lungs.

Results

The cationic STAR was found to have half-life of less than 24 hours in PBS, while the expression of the DNA payload sustained over one week. 40ug of DNA delivered by 125ug of STAR was tolerable in mice. The polyplex was found to transfect lymphatic tissue, primarily t-cells, following tail vein injection. The DNA encoding anti-murine CD19 CAR was able to achieve b cell ablation in mouse PBMC and in vivo.

Conclusions

We demonstrated a novel technology platform for in vivo CAR delivery.

Zhang:Velvet Therapeutics: Current Employment. Coker:Velvet Therapeutics: Current Employment.