Engineering liposomal DNA-driven membrane fusion for efficient protein, nucleic acid, and lipid delivery

The National Institute of General Medical Sciences awarded the Regents of the University of Michigan $995,204 under the Biomedical Research and Research Training program (CFDA 93.859) on April 1, 2026, to develop DNA-mediated membrane fusion technology for delivery of proteins, nucleic acids, and lipids into cells.

The project aims to engineer liposomal DNA-driven membrane fusion as a generalizable delivery platform addressing a gap in current therapeutics: while lipid nanoparticles and cationic polymers excel at nucleic acid delivery and mRNA-based vaccines, efficient cytoplasmic delivery of protein biologics remains challenging. Existing protein-delivery approaches via engineered bacteria, fusion-peptide-decorated liposomes, or endocytosis-dependent methods face significant efficiency or escape limitations. The funded research will establish DNA-mediated fusion of small synthetic vesicles with cell membranes across multiple cell types to deliver various cargo types (Aim 1) and develop direct membrane protein reconstitution into living cells via cell-free expression combined with this delivery platform (Aim 2). The work integrates the recipient's existing expertise in cell-free membrane protein expression with the new fusion technology to create a versatile delivery system currently unavailable.

The University of Michigan, through its Office of Research and Sponsored Projects Division, is the recipient. Work is performed in Ann Arbor, Michigan, with a period of performance from April 1, 2026, through March 31, 2029. This is a Project Grant, the standard research grant mechanism under NIGMS's portfolio supporting basic research that advances understanding of biological processes and underpins diagnostic, therapeutic, and preventive advances.

Abstract

Intracellular delivery of protein- or nucleic acid-based biologics is critical for many of the current therapeutic strategies as well as for basic research in cell biology. Various strategies have been reported for gene delivery, including viral and non-viral approaches, with notable successes of lipid nanoparticles and cationic polymers in mrna-based vaccines. However, delivering protein biologics into the cytoplasm is more challenging due to limited efficiency in crossing cell membranes. Current strategies for delivering protein biologics to interfere with intracellular signaling pathways include engineered bacteria or liposomes decorated with membrane fusion peptides. Other approaches based on endocytosis often face limited endosomal escape into the cytoplasm. Direct delivery of transmembrane proteins or lipid molecules to cell membranes is under-explored but offers opportunities for introducing receptors or signaling lipids, expanding intracellular delivery beyond nucleic acid or protein biologics. Presently, a versatile delivery technology for nucleic acid, soluble or transmembrane proteins, and lipids does not yet exist. We propose a generalizable strategy for the delivery of various cargos to cellular membranes by leveraging DNA-mediated membrane fusion via small synthetic vesicles. Combined with cell-free expression of membrane proteins - a strategy our lab excels in - this platform may enable the direct reconstitution of membrane proteins into living cells. To achieve these goals, we propose the following aims in this technology development proposal: in aim 1, we will develop and optimize DNA-mediated membrane fusion of small vesicles with different cargos to cell membranes in various cell types, thereby establishing the strategy to deliver proteins, nucleic acids, and lipids into cells. Aim 2 will establish direct membrane protein reconstitution into cellular membranes by combining cell-free expression and DNA-mediated membrane fusion. Various types of transmembrane proteins will be reconstituted into small vesicles before allowing them to fuse with cellular membranes. Finally, in aim 3, we will demonstrate this new membrane protein reconstitution approach in a vertebrate tissue model system using xenopus laevis embryo explants. We will introduce a soluble nanobody to interfere with actin cytoskeleton dynamics as well as the transmembrane tight junction protein claudin-6 into xenopus explants in situ using our proposed methodology. The technology developed through this project will have broad utility for direct intracellular delivery of proteins and lipids, providing a novel research tool for delivering exogenous biomolecules in situ without the need for genetic manipulations in recipient cells.