DNA-directed assembly of multivalent lipid nanoparticles for targeted T cell gene delivery

Abstract Lipid nanoparticles (LNPs) are a powerful emerging tool for in vivo T cell engineering with applications ranging from B cell lymphomas to other cancers and autoimmune diseases. Key challenges in designing these therapeutics include achieving both precise cell targeting and efficient mRNA translation. While single-targeted LNPs have been extensively studied, bispecific LNPs have only been briefly explored. Engagement of multiple T cell receptors offers the opportunity for enhanced mRNA delivery, expression, and T cell targeting. Here, a DNA-tethering method enables rapid modification of lipid nanoparticles with commercial antibodies. Using this strategy, we evaluated a variety of bispecific LNPs for targeted mRNA delivery to T cells both in vitro and in vivo . We identify bispecific formulations that improve targeting and subsequent transfection of T cells in vitro and in vivo relative to monotargeted LNPs. Additionally, we find that targeting molecules can alter LNP biodistribution to the spleen and liver. This fast and efficient approach to assembling antibody-targeted LNPs should enable high-throughput screening of diverse antibody combinations for improved specificity and efficiency of in vivo gene delivery. Significance Statement A major challenge in in vivo gene delivery is achieving both precise targeting and efficient mRNA translation. Multitargeted LNPs offer a potential solution to this challenge; however, their rapid assembly remains difficult, necessitating the development of new methods to construct and evaluate targeted LNPs. Here, we use a DNA-tethering method to enable rapid antibody modification of LNPs and evaluate bispecific formulations for targeted T cell mRNA delivery in vitro and in vivo . We find bispecific LNPs improve T cell targeting and expression compared to single-targeted particles. To the best of our knowledge, this study represents the first systematic screening and comparison of bispecific LNPs. Our method provides a modular approach for identifying effective antibody combinations to enhance in vivo gene delivery that can be customized for different undruggable diseases.