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. 2020 Mar 11;20(3):1578-1589.
doi: 10.1021/acs.nanolett.9b04246. Epub 2020 Feb 5.

Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering

Margaret M Billingsley  1 , Nathan Singh  2   3 , Pranali Ravikumar  2 , Rui Zhang  1 , Carl H June  4   5 , Michael J Mitchell  1   4   6   7   8
Affiliations

Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering

Margaret M Billingsley et al. Nano Lett. .

Abstract

Chimeric antigen receptor (CAR) T cell therapy relies on the ex vivo manipulation of patient T cells to create potent, cancer-targeting therapies, shown to be capable of inducing remission in patients with acute lymphoblastic leukemia and large B cell lymphoma. However, current CAR T cell engineering methods use viral delivery vectors, which induce permanent CAR expression and could lead to severe adverse effects. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral vectors, but it most commonly requires electroporation for T cell mRNA delivery, which can be cytotoxic. Here, ionizable lipid nanoparticles (LNPs) were designed for ex vivo mRNA delivery to human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for luciferase mRNA delivery to Jurkat cells, revealing seven formulations capable of enhanced mRNA delivery over lipofectamine. The top-performing LNP formulation, C14-4, was selected for CAR mRNA delivery to primary human T cells. This platform induced CAR expression at levels equivalent to electroporation, with substantially reduced cytotoxicity. CAR T cells engineered via C14-4 LNP treatment were then compared to electroporated CAR T cells in a coculture assay with Nalm-6 acute lymphoblastic leukemia cells, and both CAR T cell engineering methods elicited potent cancer-killing activity. These results demonstrate the ability of LNPs to deliver mRNA to primary human T cells to induce functional protein expression, and indicate the potential of LNPs to enhance mRNA-based CAR T cell engineering methods.

Keywords: CAR T; Lipid nanoparticles; T cell engineering; mRNA delivery.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
(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.
Figure 2.
Figure 2.
Structures of the alkyl chains (A) and polyamine cores (B) used to generate the ionizable lipid library and a reaction scheme (C) of the Michael addition chemistry used to synthesize the ionizable lipids by reacting an excess of alkyl chains with the polyamine cores. C14—4 is used here as a representative reaction.
Figure 3.
Figure 3.
(A) Luciferase expression in Jurkat cells after treatment with the LNP library and lipofectamine for 48 h at a dose of 30 ng/60 000 cells identifies top-performing LNPs. Results were normalized to untreated cells, and the background luminescence was subtracted. *: p < 0.05 in paired student t test to lipofectamine. n = 4 biological replicates. (B) Luciferase expression of Jurkat cells treated with the top five performing LNP formulations to determine the top-performing LNP formulation. Results were normalized to untreated cells, and the averaged luminescent background was subtracted. *: p < 0.05 in Tukey’s multiple comparison test between C14—4 and each formulation. n = 3 biological replicates. (C) Table reporting the diameters (z-average), polydispersity index, and mRNA concentration (± standard deviation) of the top five performing LNP formulations. n = 3. (D) Luciferase expression over time in Jurkat cells treated with 30 ng/60 000 cells of C14—4 for 24 h confirms transient expression of the protein. Results normalized to expression at 24 h with the background subtracted. n = 3 biological replicates. (E) Viability of Jurkat cells treated with 30 ng mRNA/60 000 cells for 48 h using lipofectamine or C14—4, showing minimal cytotoxicity associated with C14—4 LNP treatment. Results normalized to untreated cells with the background subtracted. n = 3 biological replicates.
Figure 4.
Figure 4.
(A) Luciferase expression and viability of primary T cells treated with crude C14—4 LNPs for 24 h. n = 3 biological replicates. (B) Results of the TNS assay to determine LNP pKa for the crude and purified C14—4 LNPs encapsulating luciferase mRNA. pKa is calculated as the pH corresponding to half of the maximum TNS fluorescence value. (C) Luciferase expression and viability of primary T cells treated with either crude or purified C14—4, showing increased luciferase expression with no increase in cytotoxicity. *: p < 0.05 in paired student t test comparison of the crude and purified LNPs. n = 3 biological replicates. For panels A and C, luciferase expression was normalized to the lowest treatment (75 ng/60 000 cells), and the viability was normalized to no treatment with the background subtracted.
Figure 5.
Figure 5.
(A) Surface expression of CAR on primary T cells assessed using flow cytometry, indicating increased CAR surface expression, evaluated as the mean fluorescent intensity (MFI), in purified C14—4 LNP and EP treated groups compared to those treated with crude C14—4 LNPs. (B) DLS measurments of crude and purified C14—4 LNPs to indicate their respective sizes. Error bars represent the standard deviation across three samples. (C) Table reporting the mRNA concentration, diameters (z-average), and polydispersity index (±standard deviation) of the crude and purified C14—4 LNPs encapsulating mRNA. (D) Viability of primary T cells treated with each group normalized to treatment with the background subtracted, n = 3 biological replicates, *: p < 0.05 in paired t test to EP. (E) Results of Nalm6 and CAR T cell coplating at different effector-to-target ratios for 48 h normalized to Nalm6 cells co-plated with untreated T cells as the control group. n = 3 wells. *: p < 0.05, **: p < 0.01 in the paired t test to control.
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