Lysine-Derived Charge-Altering Releasable Transporters: Targeted Delivery of mRNA and siRNA to the Lungs

Abstract

Targeted delivery of nucleic acid therapeutics to the lungs could transform treatment options for pulmonary disease. We have previously developed oligomeric charge-altering releasable transporters (CARTs) for in vivo mRNA transfection and demonstrated their efficacy for use in mRNA-based cancer vaccination and local immunomodulatory therapies against murine tumors. While our previously reported glycine-based CART-mRNA complexes (G-CARTs/mRNA) show selective protein expression in the spleen (mouse, >99%), here, we report a new lysine-derived CART-mRNA complex (K-CART/mRNA) that, without additives or targeting ligands, shows selective protein expression in the lungs (mouse, >90%) following systemic IV administration. We further show that by delivering siRNA using the K-CART, we can significantly decrease expression of a lung-localized reporter protein. Blood chemistry and organ pathology studies demonstrate that K-CARTs are safe and well-tolerated. We report on the new step economical, organocatalytic synthesis (two steps) of functionalized polyesters and oligo-carbonate-co-α-aminoester K-CARTs from simple amino acid and lipid-based monomers. The ability to direct protein expression selectively in the spleen or lungs by simple, modular changes to the CART structure opens fundamentally new opportunities in research and gene therapy.

Figure 1.

CART/mRNA delivery concept and synthesis of new CARTs. (A) Amphiphilic CART oligomers complexed to mRNA at pH 5.5 release mRNA under physiological conditions (pH 7.4) by charge-altering degradation of the hydroxyethyl glycine repeat unit. (B) Organo-catalytic ring-opening polymerization (OROP) and deprotection strategy for the syntheses of G-CART (D₁₃:A₁₁) and novel K-CART (D₁₄:K₈).

Figure 2.

Amino acid-based synthesis of functionalized morpholinone monomers and homopolymers. (A) Synthetic pathway for amino acid-sourced monomer synthesis. (B) Polymerization and deprotection of cationic homopolymer 5. (C) Comparative kinetics and product distributions for the aqueous (pH 6.5) degradation of 6 and 5.

Figure 3.

CART/mRNA NP formulation and stability studies. (A) CART formulation of NPs via simple mixing of mRNA and CARTs in a 10:1 cation:anion charge ratio. (B) DLS sizing comparison over time between the G-CART and K-CART/mRNA NPs, open symbols are used to indicate uncertainty, and standard deviations are smaller than the symbol size. (C) Zeta potential measurements over time for G-CART and K-CART/mRNA NPs. All measurements are an average of three independent formulations; error bars represent standard deviation.

Figure 4.

Protein expression induced by G-CART/FLuc and K-CART/FLuc upon IV injection administration. (A) Representative bioluminescence images (units in radiance) of mice 8 h after IV injection with 5 μg FLuc mRNA (0.25 mg/kg) formulated with G-CART and K-CART at an overall cation:anion charge ratio of 10:1. (B) Overall BLI signal from FLuc/CART-treated mouse immediately before sacrifice (n = 3). (C) Representative organ BLI of mice sacrificed 8 h after mRNA delivery with G-CART (left) vs K-CART (right) (n = 1, see also Figure S6) (D) percent distribution of BLI signal from the organs (n = 3) as shown in panel C. (The percent organ distribution was determined by dividing the BLI signal for the heart, kidney, liver, lungs, spleen by the total signal of these organs combined.)

Figure 5.

Silencing of FLuc protein expression in tumor cells in the lung with K-CART/FLuc-siRNA. (A) 5 μg FLuc mRNA was administered to mice. After 6 h, BLI was measured; then, 10 μg of K-CART/FLuc-siRNA or K-CART/Scrambled-siRNA was administered IV and the FLuc signal was assayed again at t = 24 h (18 h after siRNA). Group 1(red) received K-CART/FLuc-siRNA, Group 2 (gray) received scrambled K-CART/Scrambled-siRNA, Group 3 (black) received free FLuc siRNA B). Measurement of BLI for siRNA treated vs control mice at T = 24 h compared to the levels at the time of administration of siRNA. (C) 12 BALB/c mice were IV injected with 5 × 10⁵ FLuc expressing CT26 colon carcinoma cells. Group 1 (red) received two doses of 10 μg of K-CART/FLuc-siRNA on day 7 and day 9 post tumor injection. Group 2 (black) received two doses of 10 μg control K-CART/Scrambled-siRNA on day 7 and day 9 post tumor injection. (D) Measurement of BLI for siRNA-treated mice was performed on day 7, 8, 9, and day 9 + 4 h. (arrows indicate injection timing) *Data representative of 2 indivudual experiments. Error bars are standard deviation; in panel D, open symbols are used to indicate uncertainty, where not visible standard deviations are smaller than the symbol size. Significance was calculated using the two-tailed T test with *p < 0.01.\B\C/D.