Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters

Significance The transfection of lymphocytes with genetic material is a significant unmet need in immunotherapy and the treatment of many diseases. Current transfection strategies primarily rely on physical methods that must be conducted ex vivo and are often inefficient. We identified mixtures of lipid-varied mRNA delivery vehicles (charge-altering releasable transporters, CARTs) that show enhanced uptake into multiple lymphocyte cell types. The top performing lipid mixtures were rapidly identified using a combinatorial strategy and the resulting information was used to design single-delivery agents incorporating both lipid domains using our two-step organocatalytic ring-opening polymerization. Hybrid-lipid CARTs show >80% mRNA transfection efficiency in vitro and >1.5% lymphocyte transfection efficiency in mice, which is higher than previously reported systems. These materials should enable new immunotherapy strategies and applications. We report a strategy for generating a combinatorial library of oligonucleotide transporters with varied lipid domains and their use in the efficient transfection of lymphocytes with mRNA in vitro and in vivo. This library is based on amphiphilic charge-altering releasable transporters (CARTs) that contain a lipophilic block functionalized with various side-chain lipids and a polycationic α-amino ester mRNA-binding block that undergoes rearrangement to neutral small molecules, resulting in mRNA release. We show that certain binary mixtures of these lipid-varied CARTs provide up to a ninefold enhancement in mRNA translation in lymphocytes in vitro relative to either a single-lipid CART component alone or the commercial reagent Lipofectamine 2000, corresponding to a striking increase in percent transfection from 9–12% to 80%. Informed by the results with binary mixtures, we further show that CARTs consisting of optimized ratios of the two lead lipids incorporated into a single hybrid-lipid transporter molecule maintain the same delivery efficacy as the noncovalent mixture of two CARTs. The lead lipid CART mixtures and hybrid-lipid CARTs show enhanced lymphocyte transfection in primary T cells and in vivo in mice. This combinatorial approach for rapidly screening mRNA delivery vectors has provided lipid-varied CART mixtures and hybrid-lipid CARTs that exhibit significant improvement in mRNA delivery to lymphocytes, a finding of potentially broad value in research and clinical applications.