UNLABELLED: Lipid nanoparticles (LNPs) are a powerful emerging tool forT 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 bothand. We identify bispecific formulations that improve targeting and subsequent transfection of T cellsandrelative 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 ofgene delivery. in vivo in vitro in vivo in vitro in vivo in vivo
SIGNIFICANCE STATEMENT: A major challenge ingene 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 deliveryand. 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 enhancegene delivery that can be customized for different undruggable diseases. in vivo in vitro in vivo in vivo
