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

C14-4 (C14-494,Lipid B-4) 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, a lipid library of 24 ionizable lipids was constructed to make iLNPs, which were used to deliver luciferase mRNA into Jurkat cells.[115] The optimal iLNPs formulation was C14-4 iLNPs (C14-4 ionizable lipid, DOPE, chol, and PEG at a molar ratio of 35%, 16%, 46.5%, and 2.5%) (Figure 6c). The optimal dose of luciferase mRNA for C14-4 iLNPs was 30 ng. Compared with electroporated CAR T cells, the CAR T cells engineered via C14-4 iLNPs showed potent cancer-killing activity when they were cocultured with Nalm-6 acute lymphoblastic leukemia cells. To obtain a safer and more effective CAR mRNA delivery vehicle, the orthogonal design provided 256 potential formulations, and 16 representative iLNPs formulations were evaluated.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 of 40%, 30%, 25%, and 2.5%) was screened out as the optimal performing formulation. The luciferase expression based on B10 formulation was increased threefold than the initial formulation. Reducing the accumulation and clearance of iLNPs in the liver can increase the expression of CAR mRNA in T cells, further improving the therapeutic effect of CAR-T. Studies have shown that cholesterol analogs can alter the mechanisms of intracellular circulation and enhance the delivery of mRNA, which may be related to the reduced recognition of iLNPs by the Niemann Pick C1 (NPC1) enzyme.The addition of a hydroxyl group to various locations in the cholesterol molecule can alter the binding kinetics between the modified cholesterol and NPC1, and reduced NPC1 recognition of cholesterol. The results showed that replacement of 25% and 50% 7 α-hydroxycholesterol for cholesterol in iLNPs improved mRNA delivery to primary human T cells in vitro by 1.8-fold and twofold, respectively.C14-4 is one of the ionizable lipids to efficiently deliver mRNA to Jurkat cells or primary human T cells. It will effectively promote the development of mRNA delivery by iLNPs for CAR-T therapy.