Click‐Chemistry Functionalized Lipid Nanoparticles for In Vitro and In Vivo Transfection of Primary Human CD4 + T Cells

ABSTRACT CD4 + T cells orchestrate adaptive immunity and strategies that enable their genetic reprogramming hold promise for treating cancer, immune dysregulation and infectious diseases such as HIV. However, current T cell engineering approaches rely heavily on ex vivo manipulation, which is labor‐intensive, costly, and can alter cell phenotypes. Lipid nanoparticles (LNPs) offer a scalable nonviral alternative, yet conventional formulations show minimal interaction with lymphocytes and are rapidly cleared by the liver. In this study, we develop a modular DBCO‐azide click‐chemistry approach to functionalize LNPs with a high‐affinity CD4 nanobody, enabling precise CD4 receptor‐directed delivery to human CD4 + T cells. Optimized CD4‐targeted LNPs transfect up to 48% of non‐activated CD4 + T cells within a PBMC co‐culture, the highest efficiency reported so far through LNP transfection. Moreover, they successfully deliver CRISPR‐Cas9 components to activated CD4 + T cells and achieve up to 32% gene editing. In humanized NSG‐SGM3 mice, CD4‐targeted LNPs selectively associate with and transfect circulating and bone marrow–resident human CD4 + T cells while showing minimal off‐target activity in murine cells. These results establish CD4‐nanobody LNPs as a versatile platform for targeted mRNA and gene‐editing delivery, with broad potential for cancer immunotherapy, T cell reprogramming, and HIV cure strategies.