Novel In Vivo CAR platform for autoimmune disease therapy

Abstract Background: Autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS) arise from a breakdown in immune tolerance, leading to immune-mediated destruction of the body's own tissues. This loss of self-tolerance results in chronic inflammation, progressive tissue damage, and debilitating symptoms. Current therapies rely on broad immunosuppression, which may alleviate symptoms but rarely induce lasting remission, and often carry significant side effects, including increased risk of infection and organ toxicity. There is a critical unmet need for therapies that restore immune balance with greater specificity, without compromising overall immune surveillance. Recent advances in cellular immunotherapies, particularly chimeric antigen receptor (CAR) technology, have enabled more precise immune modulation. Early-phase trials using ex vivo engineered, CD19-directed CAR-T cells have demonstrated rapid and sustained remission in refractory SLE, characterized by depletion of autoreactive B cells and restoration of immune homeostasis. However, ex vivo CAR-T therapy remains complex, resource-intensive, and logistically challenging. In contrast, in vivo CAR strategies offer a scalable, off-the-shelf alternative by directly engineering immune cells within the patient. Velvet Therapeutics is developing a next-generation, non-viral in vivo CAR platform that uses biodegradable nanoparticles and minicircle DNA (mcDNA) to reprogram endogenous immune cells. This approach aims to safely and reproducibly eliminate pathogenic immune subsets and reestablish immune tolerance. Methods and Results: Our in vivo STAR-CAR platform utilizes star-shaped multi-arm polyaspartamide nanoparticles to deliver mcDNA constructs encoding CAR payloads directly into immune cells. The polypeptides were synthesized using multi-arm amine initiators via a ring-opening polymerization of N-carboxyanhydrides and validated through nuclear magnetic resonance (NMR) spectroscopy. CAR-encoding mcDNA was produced using a dual-plasmid bacterial system in E. coli DH5α, incorporating an inducible recombinase. Cells were co-transformed with donor and recombinase plasmids. Clones were selected to optimize mcDNA yield and verified by gel electrophoresis, liquid chromatography, and sequencing. Polymer synthesis parameters were tuned to control the mean hydrodynamic radius and zeta potential, aiming to reduce hepatic delivery and enhance cellular uptake. Polyplexes were formed by combining mcDNA with STAR polymer at varying nitrogen-to-phosphate (N:P) ratios. Successful complex formation was confirmed by a decrease in particle size to 100–200 nm and a zeta potential >10 mV, plateauing at an N:P ratio of 10:1. To identify the uptake mechanism, cultured HEK293 cells were pretreated with endocytic pathway inhibitors prior to transfection. Only inhibition of clathrin-mediated endocytosis significantly reduced transfection efficiency, confirming it as the primary route of cellular uptake. In vivo biodistribution studies demonstrated selective delivery and expression of CAR constructs in immune cells, with minimal off-target accumulation in the liver. To assess functional activity, we administered CD19-CAR encoding polyplexes intravenously to C57BL/6 mice at 1.6 mg/kg, twice weekly. This regimen induced robust depletion of peripheral and splenic B cells by day 7, with effects sustained through day 21. Flow cytometry revealed modest T cell expansion, consistent with systemic immune activation. Treated mice maintained normal behavior and body weight, and serum chemistry showed no abnormalities in liver (ALT, AST, GGT) or kidney (BUN) function, indicating a favorable safety profile. Conclusions: Velvet Therapeutics' in vivo STAR-CAR platform is a novel, non-viral strategy for directly engineering immune cells within the body, representing a potential breakthrough for autoimmune disease therapy. This approach enables selective B cell depletion without the need for ex vivo manipulation. Our data establish proof-of-concept for systemic immune modulation via repeat-dosed mcDNA encoding CARs. Ongoing studies aim to optimize dosing, evaluate redosability, and enhance efficacy in syngeneic autoimmune models. With the potential for durable CAR expression through repeat dosing, this platform offers a promising path toward safe, scalable, and long-lasting immune resetting across multiple autoimmune indications.