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

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.