Background In Vivo immune cells reprogramming is a novel therapeutic field that aims to reduce the manufacturing burden, improve the safety profile and simultaneously engage collaborating immune cell types to treat variety of non-oncology and oncology indications. Strategies for in vivo reprogramming have been proposed in the hope to recapitulate the success of ex vivo engineered chimeric antigen receptor T cells (CAR-T). Current viral approaches^1–3 for in vivo reprogramming applications are been attempted, but the tissue delivery specificity and safety concerns continue to persist. Recently, mRNA based therapies have been tested as well.^{4 5} Despite improved safety profile, the transient and chemically vulnerable nature of the mRNA molecule introduces an additional roadblock to achieve a clinically viable alternative to the conventional ex vivo engineered cell therapies.

Methods We deployed supercoiled DNA encoding murine CD19-CAR, formulated with multi-arm polyasparagine nanoparticles (STAR). The polyasparagine nanoparticle was synthesized as an amino acid polymer on a multi-amine initiator. NMR, PAGE gel and DLS were used to characterize the nanoparticle and its stability. The mixture of the nanoparticle and DNA (polyplex) was evaluated using fluorescence correlation spectroscopy techniques to characterize the molecular dynamics. The biodistribution of the polyplex was evaluated with in DNA encoding GFP and murine CD19 CAR expression models. Blood, liver, lymph nodes, spleen and lungs were analyzed for the payload expression.

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. The durability of the payload expression way outlives the half-life of the biodegradable STAR. In combination, the platform promises an advantageous alternative to mRNA or lentivirus for in vivo reprogramming.

References

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Ethics Approval All animal studies were conducted in accordance with the Animal Welfare Act and Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at K2bio.