Liver-Tropic Helper Lipids (LTHL) for mRNA Therapeutics, Gene Therapy, and Gene Editing Platforms
Ashish Sarode, PhD, Associate Director, Formulation, and Delivery, Sanofi
Gene therapy or gene editing, characteristics, and the biodistribution of LNPs are key aspects when it comes to developing liver tropic LNPs for applications such as protein replacement, according to Ashish Sarode, PhD, Associate Director, Formulation, and Delivery, at Sanofi.
Sarode, who spoke at TIDES USA in San Diego, said that the main component is ionizable lipids, which determines endosomal escape and the efficiency of encapsulation and cellular uptake. Other components such as helper lipids and PEG lipids, composition, and process are also crucial in LNP formation and structure. Different structures of LNPs can be formed by optimizing the formulation. He underscored that structure dictates biodistribution and liver/spleen potency, which determines subsequent milestones such as dose ranging and duration of action.
Sarode and his team selected OTC deficiency where there are mutations in the gene and no OTC protein production, which is required for the metabolism of ammonia. He underlined that target protein requirement is usually higher for liver disorders. After screening 250 ionizable lipids and more than 500 formulations using different processes or different helper lipid combinations, they obtained topmost ionizable lipids. Then they optimized the process by including novel processes. They observed that novel processes lead to better protein production efficiency only for helper lipid 3 (HL-3) whereas the potency is independent of the process in the case of helper lipid 4.
Following that, they tried to develop the industry standard lipid DLin-MC3-DMA, which has shown great success in liver delivery. They tried two different compositions, which are industry standard and modified industry standard composition, where they increased the helper lipid amount in the composition. They found out that industry standard composition was better, and there was a significant boost in potency in the case of helper lipid 4. Cryo-TEM analysis showed that higher potency LNPs showed a mixture of core-like and multilamellar structures whereas lower potency LNPs showed more heterogeneity in terms of different structures including bleb-like, unilamellar, core-like, and multilamellar structures. Further experiments showed that the heterogeneity in the morphology of LNPs might lead to a decrease in potency.
They conducted experiments to make sure that the results they obtained in mice can be reproduced in rats. This time they utilized firefly luciferase and analyzed bioluminescence. Helper lipid 4 displayed the highest luminescence, while the liver/ spleen ratio reduced significantly in the industry standard and modified industry standard group.
For further biophysical characterization, they performed pKa and Zeta potential analysis. They did not see a correlation between pKa, Zeta potential, and potency. Cryo-TEM analysis using thionine which forms a complex with mRNA and enhances the contrast indicated that mRNA moved to the bleb structures at pH 7.5 for HL-3 whereas HL-4 is well distributed inside multilamellar structures. They also tested the route for administration and indicated a higher potency using HL-4 for intravenous delivery in contrast to intramuscular delivery.
To assess ApoE and LDLR mediated transport and uptake, they used ApoE and LDLR knockout mice. There was a significant boost in potency in WT, but it completely disappeared in ApoE knockout mice. In LDLR knockout mice with HL-4, there is still some potency where the mRNAs are being expressed. This suggests that once the LNPs are bound to ApoE and are taken up into the liver using HL-4, there are some other mechanisms playing a role to take those inside of the cell. He also shared major findings. HL-4 showed the highest potency. All three lipids (DLin-MC3-DMA, Lead Lipid C-2, Lead Lipid G-7) displayed strong dependence on ApoE mediated transport. HL-4 was relatively less dependent on LDLR mediated uptake as compared to HL-1 and HL-3. Lead ionizable lipid GL-7 was not dependent on LDLR mediated uptake. For further biophysical characterization, they measured the correlation of ApoE binding with QCMD and the results suggest that there is more binding between ApoE and LNPs when they used those helper lipids (HL-4> HL-3> HL-2> HL-1) and industry standard composition had lesser binding.
The next step is the assessment of dose ranging and duration of action. They established the dose-activity relationship. When they increased the dose, metabolism of ammonia was induced more. The repeat dosing in mice showed that sufficient activity was achieved in two to three weeks. Dose ranging studies in rats indicated that ionizable lipid G-7 showed sufficient potency at 0.3 mg/kg. ALT/ AST levels showed that it was tolerable up to 0.3 mg/kg; however, higher levels were observed at 1 mg/kg.