Orthogonal Design of Experiments for Optimization of Lipid Nanoparticles for mRNA Engineering of CAR T Cells

Abstract

Viral engineered chimeric antigen receptor (CAR) T cell therapies are potent, targeted cancer immunotherapies, but their permanent CAR expression can lead to severe adverse effects. Nonviral messenger RNA (mRNA) CAR T cells are being explored to overcome these drawbacks, but electroporation, the most common T cell transfection method, is limited by cytotoxicity. As a potentially safer nonviral delivery strategy, here, sequential libraries of ionizable lipid nanoparticle (LNP) formulations with varied excipient compositions were screened in comparison to a standard formulation for improved mRNA delivery to T cells with low cytotoxicity, revealing B10 as the top formulation with a 3-fold increase in mRNA delivery. When compared to electroporation in primary human T cells, B10 LNPs induced comparable CAR expression with reduced cytotoxicity while demonstrating potent cancer cell killing. These results demonstrate the impact of excipient optimization on LNP performance and support B10 LNPs as a potent mRNA delivery platform for T cell engineering.

Nonviral Delivery Systems of mRNA Vaccines for Cancer Gene Therapy

“…LNPs are prepared by mixing lipids in an ethanol phase and mRNA in an aqueous phase in a microfluidic mixing device [ 81 ]. Then, mRNA-loaded LNPs are injected into the body and subsequently enter targeted cells by interacting with negatively charged cell membrane components or specific proteins exposed on the cell membrane [ 82 ].…”

“…Compared with EP, the most common technique used for ex vivo transfection, LNPs are a promising alternative for mRNA delivery because they show low cytotoxicity, stable mRNA cargo, and enhanced intracellular delivery and can be used without the need for specialized equipment [ 81 ]. Currently, LNPs are widely used for in vivo delivery of mRNA vaccines.…”

Rational design of anti‐inflammatory lipid nanoparticles for mRNA delivery

“…HepG2 cells were seeded in a 96 well plate at a density of 1x10 4 cells/well and were allowed to grow for 24 h. LNPs with different cholesterol:dexamethasone (C:D) ratios (10:0, 9:1, 7:3, 5:5, 3:7, 0:10) were used to treat cells at a dose of 50 ng mRNA/well for 24 h. Afterwards, luciferase expression and cell viability were tested using a Luciferase Assay Kit (E4550, Promega) and a CellTiter-Glo ® Luminescent Cell Viability Assay Kit (G7572, Promega), respectively. RAW264.7 cells were seeded in a 12-well plate at a density of 2 Â 10 5 cells/well and were allowed to grow for 24 h. LNPs were used to treat cells at a dose of 500 ng mRNA/well for 24 h. The supernatant was collected for TNF-ɑ analysis.…”

“…Ionizable lipid nanoparticles (LNPs) are the most clinically advanced non‐viral delivery platform for RNA therapeutics, as illustrated by the clinical success of Onpattro and the Pfizer/BioNTech and Moderna COVID‐19 mRNA vaccines 1,2 . They are typically composed of ionizable lipids, cholesterol, polyethylene glycol (PEG)‐conjugated lipids, and phospholipids 3–5 . Ionizable lipids can electrostatically interact with RNA molecules and facilitate their intracellular delivery, phospholipids and cholesterol improve the overall membrane stability of LNPs, and PEG‐conjugated lipids reduce protein adsorption and prolong the circulation time of LNPs in vivo 6–8 .…”

“…ionizable lipids, cholesterol, polyethylene glycol (PEG)-conjugated lipids, and phospholipids. [3][4][5] Ionizable lipids can electrostatically interact with RNA molecules and facilitate their intracellular delivery, phospholipids and cholesterol improve the overall membrane stability of LNPs, and PEG-conjugated lipids reduce protein adsorption and prolong the circulation time of LNPs in vivo. [6][7][8] LNPs can protect and deliver mRNA therapeutics to target cells and tissues by overcoming biological barriers.…”