Gene

Cell biology

Biotechnology

Biology

Cell culture

Gene delivery

Biochemistry

Transfection

Immunology

Genetics

In vivo

Transporter

Immune system

Ex vivo

In vitro

Lipid raft

Chemistry

Cell

Immunotherapy

Proceedings of the National Academy of Sciences

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 &gt;80% mRNA transfection efficiency in vitro and &gt;1.5% lymphocyte transfection efficiency in mice, which is higher than previously reported systems. These materials should enable new immunotherapy strategies and applications.

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