ApoE2-DARPin fusion proteins enable selective RNA transfer to CD8 T cells by lipid nanoparticles

Lipid nanoparticles (LNPs) have emerged as important tool to deliver therapeutic agents into cells. RNA-LNP vaccines demonstrated safety and efficacy during the COVID-19 pandemic and thereby boosted LNP technology for gene delivery. However, RNA-LNPs exhibit a broad cell tropism largely mediated by corona proteins using the low-density lipoprotein receptor (LDLR) for cell entry. Based on our experience with the retargeting of viral vectors we describe here RNA-LNPs targeted to CD8-positive T lymphocytes through the display of DARPins exhibiting high affinity for murine or human CD8. For particle display, DARPins were genetically fused to apolipoprotein E2 (ApoE2), expressed in bacteria and subsequently associated with RNA-LNPs. The resulting particles readily discriminated between CD8-positive and CD8-negative T cells in cell binding as well as reporter gene expression. This was demonstrated with mouse splenocytes as well as human peripheral blood mononuclear cells (PBMC). Notably, even when mixed into human donor blood or administered systemically into humanized mice, CD8-LNPs labeled a substantial fraction of CD8+ T cells, while cell binding was virtually absent from CD4+ T cells. The results suggest that ApoE2-DARPin proteins remain stably associated with RNA-LNPs even in the presence of serum making this approach a novel platform technology based on biological engineering instead of chemical coupling for precise targeting of RNA-LNPs to therapy-relevant cell types.