C14-4 (C14-494,Lipid B-4,Lipid B4)

C14-4 (C14-494, Lipid B-4, Lipid B4) is a novel ionizable lipid with the highest T-cell transfection efficiency and low cytotoxicity.The C14-4 ionizable lipid has been explored for CAR-T therapy.To screen the excellent formulations for mRNA delivery, alipid library of 24 ionizable lipids was constructed to makeiLNPs, which were used to deliver luciferase mRNA intoJurkat cells.[115] The optimal iLNPs formulation was C14-4iLNPs (C14-4 ionizable lipid, DOPE, chol, and PEG at a molarratio of 35%, 16%, 46.5%, and 2.5%) (Figure 6c). The optimaldose of luciferase mRNA for C14-4 iLNPs was 30 ng.Compared with electroporated CAR T cells, the CAR T cells engineeredvia C14-4 iLNPs showed potent cancer-killing activitywhen they were cocultured with Nalm-6 acute lymphoblastic leukemiacells. To obtain a safer and more effective CAR mRNAdelivery vehicle, the orthogonal design provided 256 potentialformulations, and 16 representative iLNPs formulations wereevaluated.Through evaluating the safety, delivery efficiency, and transfection efficiency of 16 iLNPs, the formulation B10(C14-4 ionizable lipid, DOPE, chol, PEG at a molar ratio of40%, 30%, 25%, and 2.5%) was screened out as the optimal performing formulation. The luciferase expression based on B10formulation was increased threefold than the initial formulation.Reducing the accumulation and clearance of iLNPs in the livercan increase the expression of CAR mRNA in T cells, furtherimproving the therapeutic effect of CAR-T. Studies have shownthat cholesterol analogs can alter the mechanisms of intracellularcirculation and enhance the delivery of mRNA, which may berelated to the reduced recognition of iLNPs by the NiemannPick C1 (NPC1) enzyme.The addition of a hydroxylgroup to various locations in the cholesterol molecule can alterthe binding kinetics between the modified cholesterol and NPC1, and reduced NPC1 recognition of cholesterol. The resultsshowed that replacement of 25% and 50% 7 α-hydroxycholesterolfor cholesterol in iLNPs improved mRNA delivery toprimary human T cells in vitro by 1.8-fold and twofold, respectively.C14-4 is one of the ionizable lipids to efficiently deliver mRNAto Jurkat cells or primary human T cells. It will effectively promotethe development of mRNA delivery by iLNPs for CAR-Ttherapy.