Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

Abstract

Functional delivery of mRNA to tissues in the body is key to implementing fundamentally new and potentially transformative strategies for vaccination, protein replacement therapy, and genome editing, collectively affecting approaches for the prevention, detection, and treatment of disease. Broadly applicable tools for the efficient delivery of mRNA into cultured cells would advance many areas of research, and effective and safe in vivo mRNA delivery could fundamentally transform clinical practice. Here we report the step-economical synthesis and evaluation of a tunable and effective class of synthetic biodegradable materials: charge-altering releasable transporters (CARTs) for mRNA delivery into cells. CARTs are structurally unique and operate through an unprecedented mechanism, serving initially as oligo(α-amino ester) cations that complex, protect, and deliver mRNA and then change physical properties through a degradative, charge-neutralizing intramolecular rearrangement, leading to intracellular release of functional mRNA and highly efficient protein translation. With demonstrated utility in both cultured cells and animals, this mRNA delivery technology should be broadly applicable to numerous research and therapeutic applications.

Keywords: cell-penetrating; gene therapy; nanoparticle; organocatalysis; stimuli-responsive.

Fig. 1.

CARTs effect the complexation (1), intracellular delivery (2), and cytosolic release (3) of mRNA transcripts, resulting in induction of protein expression (4).

Fig. 2.

Oligo(carbonate-b-α-amino ester) CARTs designed for mRNA delivery. (A) Proposed rearrangement mechanism for oligo(α-amino ester)s through tandem five-membered then six-membered transition states. (B) Two-step synthesis of amphipathic oligo(carbonate-b-α-amino ester) CARTs via OROP of cyclic carbonate and ester monomers. (C) Nonimmolative oligo(carbonate) control compounds synthesized via previously-reported OROP methodology (45, 46).

Fig. 3.

Exploration of the CART rearrangement mechanism. (A) Self-immolative rearrangement of the α-amino ester portion of a block cooligomer yields the intact oligocarbonate block and small molecule 2. (B) GPC traces of D₁₅ homooligomer 10 (blue), protected block cooligomer D₁₅:A₁₂ 11a (red), and the product of deprotection and rearrangement 11c (black). R denotes 4.

Fig. 4.

Evaluation of CARTs for EGFP mRNA delivery. (A) Flow cytometry-determined mean EGFP fluorescence intensity from HeLa cells treated with naked mRNA, a Lipo/mRNA complex, and mRNA complexes of transporters 7–13. (B) Representative flow cytometry histograms of EGFP fluorescence showing percent transfection in HeLa cells treated with EGFP mRNA complexes. (C) The effect of theoretical cation:anion charge ratio on EGFP expression using D₁₃:A₁₁ 7 complexes. (D) Epifluorescence microscopy images showing EGFP fluorescence alone and a bright-field overlay of HeLa cells treated with mRNA either alone, complexed with Lipo, or complexed with 7. All data shown are for HeLa cells treated with mRNA concentrations of 125 ng per well in 24-well plates for 8 h. All error bars expressed as ± SD, n = 3.

Fig. 5.

Functional delivery of mRNA is due to the charge-altering, self-immolative mechanism that drives mRNA release and endosomal escape by CART D₁₃:A₁₁ 7. (A) Uptake of CART/Cy5-mRNA complexes at 4 °C, a condition that inhibits endocytosis. (B) Relative uptake and expression of Cy5-EGFP mRNA following treatment with complexes formed with degrading and nondegrading transporter systems. Filled bars represent EGFP expression and open bars represent Cy5 fluorescence. (C) Effect of endosomal acidification inhibitor (Con A) or endosomolytic agent (Chl) on EGFP expression following CART/mRNA delivery. (D) Confocal microscopy of HeLa cells treated with Cy5-mRNA complexes using CART 7 or nonimmolative oligomer 13 after 4 h. Cells were cotreated TRITC-Dextran₄₄₀₀. All error bars expressed as ± SD, n = 3. (Scale bar, 10 μm.)

Fig. 6.

Applications of mRNA delivery using CARTs in multiple cell lines and mice. (A) Transfection efficiencies of EGFP mRNA delivery by D₁₃:A₁₁ 7 compared with Lipo in HeLa (blue), J774 (red), HEK 293 (gray), CHO (yellow), and HepG2 (green) cell lines and primary CD1-derived mesenchymal stem cells (purple). Error bars expressed as ± SD, n = 3. (B) Delivery of Fluc mRNA with CART 7 follows the same trend in charge ratio as EGFP. Charges reported as theoretical (cation:anion) ratios. Representative bioluminescent images for treatment conditions are shown above their corresponding bars. Error bars expressed as ± SD, n = 3. (C) In vivo BLI following i.m. injection of naked Fluc mRNA (○) and CART/mRNA complexes using 7 (●). Bars represent average of all animals (n = 3 at 1, 4, and 7 h; n = 5 for 24 and 48 h). (D) Representative bioluminescence images following i.m. injection of naked mRNA (left flank) or CART/mRNA complexes (right flank). (E) In vivo BLI following i.v. (tail vein) injection of naked mRNA (○) and CART/mRNA complexes (●). Bars represent average of all animals (n = 2 for 1 and 7 h; n = 4 for 4, 24, and 48 h). Dotted lines are background BLI signals from an animal that had not been injected with d-luciferin. (F) Representative BLI images of mice treated with CART/mRNA complexes via i.v. injection.