(T1130-01-05) CARTs - A New Vehicle for Nucleic Acid Delivery with a Unique Controllable Release Mechanism

Purpose: CARTs are biodegradable, amphiphilic oligomers suitable for the formulation and delivery of nucleic acids, which undergo a pH-dependent self-degradation mechanism. The presentation introduces this novel platform and gives an overview about the underlying concepts and early application data.

Methods: CARTs have a carbonate/aminoester backbone and are prepared by ring-opening polymerization of corresponding monomers. Monomer synthesis is conducted via reactive ester chemistry. Due to their cationic net charge, CARTs readily self-assemble with nucleic acid into nanoparticles. The nanoparticles are characterized by dynamic and electrophoretic light scattering, gel electrophoresis and Ribogreen assay. Biocompatibility of CART nanoparticles is assessed by MTS assay and inflammation assay in THP-1 cells. Finally, transfection efficiency compared to various relevant benchmark formulations is evaluated by luciferase assay.

Results: After synthesis of CART monomers using EDC, purification is achieved by column chromatography as demonstrated by ¹H NMR. Polymerization and subsequent deprotection yields oligomers with a block length of ~10 units. CART mRNA nanoparticles are around 100 nm in size and can be prepared by a nanoprecipitation method through manual mixing as well as with microfluidic approaches. Moreover, particles maintain colloidal stability over a freeze-thaw cycle as well as after lyophilization. CARTs don’t show in vitro toxicity as verified by MTS assay and don’t trigger any cytokine production. They exhibit high transfection efficiency in various cell lines and are also active in hard to transfect cells, such as primary T cells, outperforming benchmark formulations in some cases.

Conclusion: CARTs are a promising new delivery platform for mRNA and other types of nucleic acids showing encouraging results from first PoC studies. Although there are still several development steps ahead, they could become a novel solution for nucleic acid delivery challenges complementary to the established systems based on Lipid Nanoparticles.

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