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Lipid nanoparticle-based mRNA delivery for CAR T-cell engineering

A library of ionizable lipid nanoparticles (LNPs) was screened for ability to deliver mRNA to T-cells, leading to an optimized LNP formulation for delivery of mRNA to T-cells. This thereby enhances the ability to generate transient chimeric antigen receptor (CAR)-expressing T-cells for cancer immunotherapy.

Problem:

Chimeric antigen receptor (CAR) T-cells are T-cells that have been genetically engineered to express a synthetic T-cell receptor that gives them the ability to initiate immune responses against specific proteins.

By engineering T-cells to target proteins expressed specifically by cancer cells, CAR T-cells are a highly effective cancer immunotherapy. The current manufacturing process of generating CAR T-cells utilizes viruses to permanently express the CAR on the T-cell surface. This permanent CAR expression is associated with adverse side effects such as cytokine storm and neurotoxicity long after the CAR T-cells have performed their therapeutic function.

Solution:

This technology overcomes the challenges associated with virally engineered CAR T-cell therapy is by utilizing LNPs to transfect T-cells with CAR-encoding mRNA. Because mRNA is translated without genomic integration, this results in transient CAR expression.

Technology Overview:

This technology is a novel ionizable lipid (C14-4) and a method of synthesizing (C14-4)-based LNPs. A library of novel ionizable lipids was generated and screened for their ability to transduce T-cells with mRNA when combined into LNPs. The novel ionizable lipid with the highest T-cell transfection efficiency was C14-4 and this was subsequently shown to be more effective than lipofectamine electroporation at generating CAR T-cells via delivery of CAR mRNA. CAR T-cells generated with (C14-4)-based LNPs are effective at killing cancer in vitro.

Advantages:

C14-4 LNP-based delivery of CAR mRNA to T-cells results in transient CAR expression, thereby decreasing the likelihood of adverse side effects caused by permeant CAR expression resulting from viral T-cell engineering.
Enhanced mRNA delivery to T-cells relative to electroporation with lipofectamine.
Less toxic to T-cells than electroporation with lipofectamine.

(A) Schematic of the components used to generate LNPs via microfluidic mixing and the expected structure of the resulting LNPs. (B) The size (z-average) distribution of a representative sample of C14–4 LNPs, revealing a diameter of approximately 70 nm using dynamic light scattering. Error bars represent the standard deviation across three samples. (C) Schematic of CAR mRNA loaded LNPs inducing CAR expressionin T cells, resulting in tumor cell targeting and killing.