Engineering Targeted Gene Delivery Systems for Primary Hereditary Skeletal Myopathies: Current Strategies and Future Perspectives

Abstract

Skeletal muscle, constituting ~40% of body mass, serves as a primary effector for movement and a key metabolic regulator through myokine secretion. Hereditary myopathies, including dystrophinopathies (DMD/BMD), limb-girdle muscular dystrophies (LGMD), and metabolic disorders like Pompe disease, arise from pathogenic mutations in structural, metabolic, or ion channel genes, leading to progressive weakness and multi-organ dysfunction. Gene therapy has emerged as a transformative strategy, leveraging viral and non-viral vectors to deliver therapeutic nucleic acids. Adeno-associated virus (AAV) vectors dominate clinical applications due to their efficient transduction of post-mitotic myofibers and sustained transgene expression. Innovations in AAV engineering, such as capsid modification (chemical conjugation, rational design, directed evolution), self-complementary genomes, and tissue-specific promoters (e.g., MHCK7), enhance muscle tropism while mitigating immunogenicity and off-target effects. Non-viral vectors (liposomes, polymers, exosomes) offer advantages in cargo capacity (delivering full-length dystrophin), biocompatibility, and scalable production but face challenges in transduction efficiency and endosomal escape. Clinically, AAV-based therapies (e.g., Elevidys^® for DMD, Zolgensma^® for SMA) demonstrate functional improvements, though immune responses and hepatotoxicity remain concerns. Future directions focus on AI-driven vector design, hybrid systems (AAV-exosomes), and standardized manufacturing to achieve "single-dose, lifelong cure" paradigms for muscular disorders.

Keywords: AAV vector engineering; gene therapy; skeletal muscle disease; skeletal muscle target.

Figure 1

Schematic structure of recombinant adeno-associated virus (rAAV). The viral capsid encloses a single-stranded DNA genome flanked by inverted terminal repeats (ITRs). The key genetic cassette includes a promoter driving expression, the therapeutic transgene cassette, and a polyadenylation (polyA) signal sequence.

Figure 2

Engineering strategies for adeno-associated virus (AAV) vector optimization. This schematic summarizes five principal approaches to enhancing AAV vectors for gene therapy. Chemical modification: Covalent conjugation of functional moieties (e.g., polymers, peptides) to capsid surfaces, redirecting tropism and reducing immunogenicity. Rational design: Structure-guided genetic engineering of capsid proteins (e.g., peptide insertions, site-directed mutagenesis) to enhance tissue specificity and transduction efficiency. Directed evolution: Iterative selection of capsid variants from combinatorial libraries under physiological pressure to isolate mutants with improved muscle tropism and reduced off-targeting. AI-assisted engineering: Computational prediction of capsid–receptor interactions using machine learning (e.g., AlphaFold) and generative algorithms to design de novo capsids with tailored properties. Self-complementary AAV (scAAV): Genome engineering to enable double-stranded DNA packaging, bypassing second-strand synthesis and accelerating transgene expression kinetics.

Figure 3

Engineering of AAV gene expression cassettes for muscle-specific therapy. This schematic illustrates key strategies to optimize AAV transgene cassettes for skeletal-muscle-targeted gene delivery. Muscle-specific promoters: MCK-derived promoters (e.g., full-length MCK, tMCK etc.) drive high-fidelity expression in striated muscle. Alternative promoters (e.g., HSA for human α-actin, SPc5-12 synthetic promoter) enable tailored transcriptional activity and reduced off-targeting. Skeletal-muscle-specific enhancers: E-box motifs (CANNTG) bound by myogenic transcription factors (e.g., MyoD) enhance spatial specificity, and MEF2-binding sequences recruit regulators of muscle development, amplifying transgene expression while maintaining tissue restriction. Transgene payload engineering, microprotein variants: Design of functional miniaturized proteins (e.g., microdystrophin ΔR4-R23/ΔCT) to overcome AAV packaging limits (~4.7 kb). Split intein systems: Co-delivery of split-intein-fused protein fragments that undergo in vivo reconstitution into full-length functional proteins (e.g., POI: protein of interest).

Figure 4

Non-viral delivery strategies for skeletal-muscle-targeted gene therapy. Functionalized nanocarriers, liposomes/polymeric nanoparticles/exosomes: Surface conjugated with muscle-targeting ligands (e.g., integrin αVβ6-binding peptides, myosin-specific antibodies) to enable receptor-mediated uptake. Physical enhancement methods, electroporation: Transient electrical pulses increase sarcolemmal permeability, boosting plasmid/nucleic acid uptake in myofibers. Ultrasound-mediated targeted delivery: Microbubble cavitation under focused ultrasound disrupts vascular endothelia, enhancing the extravasation and penetration of nanoparticles (e.g., LNP-ASOs) into deep muscle groups. Antibody–oligonucleotide conjugates (AOCs): Covalent linkage of antisense oligonucleotides (ASOs) or siRNAs to skeletal-muscle-specific antibodies enables antibody-directed endocytosis and precise intracellular delivery.

Figure 5

Therapeutic strategies for genetic therapy in hereditary myopathies. This schematic delineates core gene therapy approaches to address pathogenic mutations causing functional protein deficiency. Wild-type state: Biallelic expression yields sufficient functional protein to maintain cellular homeostasis. Loss-of-function (LOF) variation: Pathogenic variants (e.g., nonsense, frameshift) cause haploinsufficiency, resulting in insufficient functional protein production. Gene replacement therapy: Delivery of exogenous functional genes (e.g., via AAV or non-viral vectors) to restore physiological protein levels and compensate for endogenous deficiency. Gene editing; Precise correction of variations using CRISPR-Cas9 or base editors to reconstitute native gene function and enable endogenous production of full-length functional proteins. Exon skipping: Antisense oligonucleotides (ASOs) mediate the exclusion of variation-harboring exons during pre-mRNA splicing, restoring an open reading frame to generate truncated yet functional proteins.