Polyplex Nanomicelle-Mediated Pgc-1α4 mRNA Delivery Via Hydrodynamic Limb Vein Injection Enhances Damage Resistance in Duchenne Muscular Dystrophy Mice

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, leading to the absence of dystrophin and progressive muscle degeneration. Current therapeutic strategies, such as exon-skipping and gene therapy, face limitations including truncated dystrophin production and safety concerns. To address these issues, a novel mRNA-based therapy is explored using polyplex nanomicelles to deliver mRNA encoding peroxisome proliferator-activated receptor gamma coactivator 1 alpha isoform 4 (PGC-1α4) via hydrodynamic limb vein (HLV) administration. Using an in vivo muscle torque measurement technique, it is observed that nanomicelle-delivered Pgc-1α4 mRNA significantly improved muscle damage resistance and mitochondrial activity in mdx mice. Specifically, HLV administration of Pgc-1α4 mRNA in dystrophic muscles significantly relieved the torque reduction and myofiber injury induced by eccentric contraction (ECC), boosted metabolic gene expression, and enhanced muscle oxidative capacity. In comparison, lipid nanoparticles (LNPs), a widely used mRNA delivery system, does not achieve similar protective effects, likely due to their intrinsic immunogenicity. This foundational proof-of-concept study highlights the potential of mRNA-based therapeutics for the treatment of neuromuscular diseases such as DMD and demonstrates the capability of polyplex nanomicelles as a safe and efficient mRNA delivery system for therapeutic applications.

Keywords: duchenne muscular dystrophy (DMD); lipid nanoparticle (LNP); mRNA therapeutics; peroxisome proliferator‐activated receptor gamma coactivator (PGC‐1α); polyplex nanomicelle.

Figure 1

Functional and histological evaluation of dystrophic muscles in response to eccentric contraction (ECC). A) Mean isometric torque of the plantar flexors upon supramaximal stimulation (45 V and 0.5 ms) at incremental frequencies. B) Isometric torque measured post‐ECC, relative to the baseline torque per mouse from (A). C) Torque produced during 100 cycles of ECC, normalized to the torque in the first cycle. D) Representative images of Evans blue dye (EBD) staining (top panel) and hematoxylin and eosin (H&E) staining (bottom panel). E–H) Quantitative histological analyses. (E) Percentage of EBD‐positive area relative to total muscle section area. (F) Percentage of centrally nucleated fibers. (G) Fiber diameter boxplot. (H) Fiber diameter distribution across different sizes. For F‐H, ≈500 fibers per mouse were analyzed. WT and mdx refer to wild‐type mice and mdx mice. EBD, evans blue dye. Data are presented as mean ± SEM (n = 6 mice), and Welch's t‐test or one‐way ANOVA followed by Tukey's post hoc test were used (^*** p < 0.001, ^** p < 0.01, and ^* p < 0.05).

Figure 2

Expression levels of PGC‐1α and associated genes in dystrophic muscles. A) Western blotting of PGC‐1α1 and PGC‐1α4 protein levels (n = 3 mice). B) Quantitative analysis of the western blot bands normalized to GAPDH. C) Relative mRNA expression levels of total Pgc‐1α (Pgc‐1α1,2,3, and 4), Pgc‐1α1, and Pgc‐1α4. D–K) Relative mRNA levels of genes associated with PGC‐1α in various aspects including muscle hypertrophy (D), myogenic induction (E), mitochondrial activity (F), angiogenesis (G), neuromuscular junction (NMJ) (H), myofiber type (I), metabolism (J), and inflammation (K). All analyses were performed using plantar flexor muscles. Data are presented as mean ± SEM (n = 6 mice), and Welch's t‐test was used (^*** p < 0.001, ^** p < 0.01, and ^* p < 0.05).

Figure 3

Characterization of protein expression after hydrodynamic limb vein (HLV) injection of mRNA‐loaded nanomicelles or LNPs. A) Schematic illustration of the formation of polyplex nanomicelles resulting from the interaction of mRNA and the block copolymer PEG‐PAsp(DET). B) Graphic representation of the HLV administration technique. C–G) Evaluation of luciferase expression from Fluc mRNA‐loaded nanomicelles or LNPs following HLV administration in mdx mice. (C) Experimental design of (D,E). (D) Representative bioluminescence images. Color bar represents the radiance intensity. (E) Time course of luciferase expression was observed at multiple time points by IVIS. (F) Experimental design of (G). (G) Luciferase activity was measured at different time points by luciferase assay. IVIS, in vivo imaging system. HEPES was used as a control injection in the contralateral limb. Data are presented as mean ± SEM (n = 3 mice), and log transformation was performed in (F) and (G). One‐way ANOVA followed by Tukey's post hoc test was used (^** p < 0.01 and ^* p < 0.05, micelle + Fluc vs. LNP + Fluc; ns indicates non‐significant).

Figure 4

Improvement of muscle damage resistance in dystrophic mice following nanomicelle‐delivevred Pgc‐1α4 mRNA treatment. A) Schematic illustration of the experiment design. B–D), Assessment of plantar flexor muscle performance after injection. (B) Mean isometric torque of the plantar flexors upon supramaximal stimulation at incremental frequencies (n = 5 for the saline group; n = 6 for all other groups). (C) Isometric torque measured post‐ECC relative to the baseline torque per mouse from (B). (D) Torque produced during 100 cycles of ECC normalized to the torque in the first cycle (n = 5 for the saline and micelle + Fluc groups; n = 6 for all other groups). Data are presented as mean ± SEM, and one‐way ANOVA followed by Tukey's post hoc test was used (^*** p < 0.001, ^** p < 0.01, and ^* p < 0.05).

Figure 5

Reduction of myofiber damage in dystrophic mice following nanomicelle‐delivevred Pgc‐1α4 mRNA treatment. A) Representative images of EBD staining (upper panel) and H&E staining (lower panel) post‐treatment. B–E), Quantitative histological analyses. (B) Percentage of EBD‐positive area. (C) Percentage of centrally nucleated fibers. (D) Fiber diameter boxplot. (E) Fiber diameter distribution across different sizes. Data are presented as mean ± SEM (n = 5 for the saline and LNP + α4 groups; n = 6 for all other groups), and one‐way ANOVA followed by Tukey's post hoc test was used (^*** p < 0.001, ^** p < 0.01, and ^* p < 0.05). For (B–E), 300–500 fibers per mouse were analyzed.

Figure 6

General upregulation of dysregulated genes after nanomicelle‐delivered Pgc‐1α4 mRNA treatment. A,B) Western blotting images of PGC‐1α4 protein expression on day 2 (A) and day 7 (B). C) Quantification of western blotting bands normalized to GAPDH (for day 2, n = 5 for the LNP + α4 group, n = 6 for all other groups; for day 7, n = 4 for the LNP + α4 group, n = 5 for the micelle + Fluc group, and n = 6 for the micelle + α4 group). D–L) Relative mRNA levels of genes associated with PGC‐1α on day 2 and day 7. All analyses were performed using plantar flexor muscle samples. Data are presented as mean ± SEM (n = 6 mice), and one‐way ANOVA followed by Tukey's post hoc test was used (^*** p < 0.001, ^** p < 0.01, and ^* p < 0.05).

Figure 7

Enhancement of mitochondrial activity by nanomicelle‐deliverd Pgc‐1α4 mRNA treatment. A) Representative succinate dehydrogenase (SDH) assay images. B) Quantitative analysis of the percentage of SDH‐positive fibers. Data are presented as mean ± SEM (n = 5 for the saline group; n = 6 for all other groups), and one‐way ANOVA followed by Tukey's post hoc test was used (^* p < 0.05 and ^** p < 0.01).