A novel mRNA delivery system offers organ-specific targeting

More effective and selective delivery systems are required for messenger RNA (mRNA) therapeutics to realize their full potential. To address this need, researchers at Stanford University have developed a novel class of charge-altering releasable transporters (CARTs) that differ from previous CARTs through their beta amido carbonate backbone (bAC) and side chain spacing. These modifications provide enhanced mRNA transfection of primary T lymphocytes in vitro and achieve highly selective organ tropism in vivo, suggesting bAC CARTs to have broad utility for therapeutic applications. TriLink’s CleanCap® mRNA products facilitated this research, which was recently published in Nature Communications. 

CARTs represent unique alternatives to lipid nanoparticles 

While lipid nanoparticles (LNPs) have demonstrated considerable success in delivering mRNA therapeutics, their use is currently limited to intramuscular administration and intravascular targeting of liver hepatocytes. CARTs, first reported in 2016, are alternative tools for mRNA delivery that promise improved cell and organ tropism. To function, CARTs initially serve as cations that complex, protect, and deliver their mRNA cargo. They then undergo a unique charge-neutralizing intramolecular rearrangement (from charged amine to neutral amide) to trigger its intracellular release. 

CART modifications improve T lymphocyte transfection 

T lymphocytes are important therapeutic targets, but their transfection with genetic material presents a significant challenge. To tackle this problem, the Stanford University researchers previously adapted the CART lipid composition to form oleyl-nonenyl amino CARTs (ONA CARTs), which enhanced the mRNA transfection efficiency of Jurkat cells, a T lymphocyte line. However, the ONA CARTs were less effective in transfecting primary T cells, indicating a need for further modulation. This latest study describes the impact of replacing the CART methyl-trimethylene carbonate (MTC) backbone with a bAC backbone featuring variable side chain spacing, both through in vitro and in vivo testing.  

bAC CARTs enhance mRNA transfection efficacy in Jurkat cells 

A library of 24 bAC CARTs was used to transfect Jurkat cells with eGFP mRNA (TriLink). Flow cytometric evaluation showed the best performing bAC CART (bAC-7c) to have a transfection efficiency of 78%, compared with just 46% for the ONA CART control, which equated to increased protein expression. 

bAC CARTs enable ex vivo generation of highly cytotoxic CAR-T cells 

To demonstrate the therapeutic potential of bAC CARTs, activated CD8+ T cells (isolated from healthy human donors) were transfected with mRNA encoding anti-human CD19 using either bAC-7c or ONA CART. The resultant anti-hCD19 CAR-T cells were then co-cultured with a B-cell precursor leukemia cell line to assess the impact of anti-hCD19 CAR on CD8+ T cell function. mCherry mRNA (TriLink) served as a control. Key findings included significantly higher levels of anti-hCD19 CAR expression using bAC-7c compared to ONA CART, which resulted in specific targeting of leukemia cells through CD19-CAR interaction. 

bAC CARTs efficiently transfect T lymphocytes in vivo 

Having observed efficient transfection of primary T lymphocytes in vitro, the Stanford University researchers sought to achieve similar results in vivo. This included complexing bAC CARTs with luciferase mRNA (TriLink) for intravenous delivery to BALB/c mice and measuring luciferase expression by whole body imaging and flow cytometric analysis of isolated organs. Data showed bAC-7c to out-perform the other bAC CARTs that were tested, as well as exhibit ten-fold higher luciferase expression than its MTC analog and be selective for spleen uptake (>90 %). Subsequently, the intravenous administration of Cre mRNA (TriLink) to a Cre recombinase murine model found most subsets of CD45+ leukocytes in the spleen (including CD4+ and CD8+ T cells) to be involved in mRNA internalization. Critically, the ability of bAC CARTs to achieve organ tropism without the need for a targeting ligand opens up new opportunities for RNA delivery. 

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Article reference: Li, Z., Amaya, L., Pi, R. et al. Charge-altering releasable transporters enhance mRNA delivery in vitro and exhibit in vivo tropism. Nat Commun 14, 6983 (2023) https://www.nature.com/articles/s41467-023-42672-x