A Combinatorial Library of Lipid Nanoparticles for RNA Delivery to Leukocytes

TLDR

Lipid nanoparticles (LNPs) are the most advanced clinically approved nonviral platforms for siRNA delivery, primarily targeting the liver, and recent work shows that variations in ionizable lipid linker moieties can markedly influence in‑vivo gene silencing efficiency. The authors designed and synthesized novel ionizable amino lipids incorporating hydrazine, hydroxylamine, and ethanolamine linkers to create LNPs capable of delivering siRNAs to leukocytes. These lipids were formulated into LNPs, screened for leukocyte transfection efficiency, and evaluated in mice for biodistribution, gene silencing, liver toxicity, and immune activation, including testing with a β7 integrin–targeted LNP in primary lymphocytes. Two lipids exhibited potent in‑vitro gene silencing, and one formulated with a pan‑leukocyte targeting agent achieved robust silencing in primary lymphocytes, indicating promise for RNA delivery to leukocytes.

Abstract

Lipid nanoparticles (LNPs) are the most advanced nonviral platforms for small interfering RNA (siRNA) delivery that are clinically approved. These LNPs, based on ionizable lipids, are found in the liver and are now gaining much attention in the field of RNA therapeutics. The previous generation of ionizable lipids varies in linker moieties, which greatly influences in vivo gene silencing efficiency. Here novel ionizable amino lipids based on the linker moieties such as hydrazine, hydroxylamine, and ethanolamine are designed and synthesized. These lipids are formulated into LNPs and screened for their efficiency to deliver siRNAs into leukocytes, which are among the hardest to transfect cell types. Two potent lipids based on their in vitro gene silencing efficiencies are also identified. These lipids are further evaluated for their biodistribution profile, efficient gene silencing, liver toxicity, and potential immune activation in mice. A robust gene silencing is also found in primary lymphocytes when one of these lipids is formulated into LNPs with a pan leukocyte selective targeting agent (β7 integrin). Taken together, these lipids have the potential to open new avenues in delivering RNAs into leukocytes.