Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters

Abstract

We report a strategy for generating a combinatorial library of oligonucleotide transporters with varied lipid domains and their use in the efficient transfection of lymphocytes with mRNA in vitro and in vivo. This library is based on amphiphilic charge-altering releasable transporters (CARTs) that contain a lipophilic block functionalized with various side-chain lipids and a polycationic α-amino ester mRNA-binding block that undergoes rearrangement to neutral small molecules, resulting in mRNA release. We show that certain binary mixtures of these lipid-varied CARTs provide up to a ninefold enhancement in mRNA translation in lymphocytes in vitro relative to either a single-lipid CART component alone or the commercial reagent Lipofectamine 2000, corresponding to a striking increase in percent transfection from 9-12% to 80%. Informed by the results with binary mixtures, we further show that CARTs consisting of optimized ratios of the two lead lipids incorporated into a single hybrid-lipid transporter molecule maintain the same delivery efficacy as the noncovalent mixture of two CARTs. The lead lipid CART mixtures and hybrid-lipid CARTs show enhanced lymphocyte transfection in primary T cells and in vivo in mice. This combinatorial approach for rapidly screening mRNA delivery vectors has provided lipid-varied CART mixtures and hybrid-lipid CARTs that exhibit significant improvement in mRNA delivery to lymphocytes, a finding of potentially broad value in research and clinical applications.

Keywords: combinatorial; gene therapy; immunotherapy; nanoparticle; polyplex.

Fig. 1.

(A) Structure of previously reported CART D₁₃:A₁₁ and neutral products of the charge-altering mechanism. (B) Transfection efficiencies of EGFP mRNA alone, lipofectamine-formulated EGFP mRNA, and D₁₃:A₁₁-complexed EGFP mRNA into HeLa cells versus Jurkat cells (T lymphocytes). Error bars represent the SD over three experiments in triplicate.

Fig. 2.

Structures of CARTs with varying lipid blocks used in this study.

Fig. 3.

High-throughput evaluation of mixtures of amphipathic CARTs for the delivery of luciferase (Fluc) mRNA. For mixture matrices, CARTs in columns and rows correspond to 66% and 33% of the total cationic charge in the mixture, respectively. Each grid image is three-color-weighted to designate the relative expression levels of the given mixture, with white representing no expression, blue representing the expression of CART D₁₃:A₁₁ 2, and orange being the highest-performing mixture. The scale bar below each matrix gives the bioluminescence (in photons⋅s⁻¹⋅cm⁻²⋅sr⁻¹) of each experiment. (A) Expression in nonadherent lymphocytes (Jurkat cells, EL4 cells, A20 cells, and activated primary T cells). (B) Expression in adherent epithelial cells and monocytes (HeLa cells, DC 2.4 cells, and A549 cells). All values represent the average bioluminescence of three separate experiments. (C) Normalized bioluminescence intensity of cells treated with the highest-performing mixture of CART 3 and 7 demonstrating the fold improvement over D₁₃:A₁₁ 2 in all cell lines tested. Horizontal dashed line corresponds to expression from unmixed D₁₃:A₁₁ 2. Data are the average of three separate experiments, each in triplicate, with error bars representing the SD.

Fig. 4.

Performance of binary mixtures of varying ratios of O₁₁:A₉ (3) and N₁₀:A₁₀ (7) for the delivery of Fluc mRNA into Jurkat cells. All values represent the average bioluminescence of three separate experiments with error bars corresponding to the SD.

Fig. 5.

Synthesis of CARTs containing mixtures of lipophilic blocks in either block (b) or statistical (stat) architectures. Monomer ratios were selected based on the optimized performance of a 50:50 mixture of O₁₁:A₉ (3) and N₁₀:A₁₀ (7).

Fig. 6.

Performance of noncovalent (3 + 7) and hybrid-lipid CARTs (9, 10, and 11) for the delivery of EGFP mRNA to Jurkat cells, as well as baseline performance of Lipo and single-lipid CARTs (2, 3, and 7). All values represent the average percent of EGFP-positive cells from three separate experiments with error bars corresponding to the SD. Unpaired Student’s t test: *P < 0.0005, **P < 0.0001.

Fig. 7.

Evaluating the mechanism of enhanced mRNA delivery by lipid mixtures. (A) Percent encapsulation of free mRNA by CART complexes. Encapsulation is normalized to free mRNA (0%) and Qubit-mRNA dye alone (100%). (B) CART–mRNA complex particle sizes as determined by DLS. (C) Comparison of the uptake of a fluorescently labeled Cy5-mRNA by single-CART oligomer complexes versus lipid CART mixtures and hybrid-lipid CARTs in Jurkat cells. Data are reported as the mean Cy5 fluorescence of all cells measured (bars, left axis) or percentage of Cy5-positive cells (markers, right axis). All values are expressed as the average of three separate experiments with error bars corresponding to the SD.

Fig. 8.

In vivo transfection of lymphocytes using mixed-lipid CARTs. (A) Structure of fluorescently labeled single-lipid CART 12 and hybrid-lipid CART 13. (B) Representative bioluminescence images following in vivo delivery of luciferase mRNA in BALB/c mice via tail vein injection by hybrid-lipid CART 13. (C) Transfection of lymphocytes and monocytes in the spleen following i.v. injection of CART 12–mRNA and CART 13–mRNA complexes. All values are expressed as the average of separate experiments (CART 12: n = 5; CART 13: n = 6 mice) with error bars corresponding to the SD (*P < 0.001, **P < 0.05, ***P < 0.01, unpaired Student’s t test).