Peripheral nerve injuries (PNIs) are stratified by severity according to Seddon’s and Sunderland’s systems. Seddon’s scheme distinguishes three types—neurapraxia (transient conduction block), axonotmesis (axon disruption with intact connective sheaths), and neurotmesis (complete nerve transection)—while Sunderland refines this into five grades, specifying the extent of damage to axons, endoneurium, perineurium, and epineurium [13, 14]. Etiologies range from blunt trauma and sharp lacerations to iatrogenic injury during surgery [15], tumor compression, and inflammatory neuropathies. The potential for regeneration diminishes as injury severity increases and as the interval to reinnervation lengthens; hence, prompt diagnosis and intervention are critical to maintain muscle responsiveness and prevent irreversible atrophic changes [16].

Following axonal transection, Wallerian degeneration ensues within hours: the distal axon and myelin sheath fragment and are cleared by Schwann cells and infiltrating macrophages [17]. Concurrently, Schwann cells adopt a reparative phenotype, upregulating neurotrophic factors such as NGF, GDNF, and BDNF, and aligning into Bands of Büngner that channel regrowing axonal sprouts [18]. Despite this orchestrated response, axonal regrowth proceeds slowly—typically 1–3 mm per day—and often fails to bridge long gaps, especially in proximal injuries or when surgery is delayed [19]. Such delays exacerbate target muscle atrophy and highlight the necessity of preserving the muscle niche to optimize reinnervation [20].

Denervated muscle fibers rapidly undergo atrophy driven by disrupted calcium homeostasis, oxidative stress, and activation of proteolytic systems such as the ubiquitin–proteasome pathway (via atrogin-1/MAFbx and MuRF1) [21]. Autophagy–lysosome activity is also upregulated early after denervation, facilitating removal of damaged organelles but becoming insufficient at later stages of atrophy [22]. FOXO3-mediated transcriptional upregulation of atrophy-related genes further amplifies catabolic signaling [23]. Concurrently, the IGF-1/Akt/mTOR axis is downregulated, reducing anabolic drive and protein synthesis [24]. Loss of neural stimuli also triggers sarcomere disassembly, shifts fiber-type composition toward fast-twitch phenotypes, and exacerbates mitochondrial dysfunction [25].

With prolonged denervation, myofibers are progressively replaced by fibrotic and adipose tissue [26]. Fibro‐adipogenic progenitors (FAPs), normally transiently activated after injury, proliferate unchecked in the absence of reinnervation, driving extracellular matrix (ECM) deposition through TGF-β signaling [27]. This process is exacerbated by a dysregulated balance of matrix metalloproteinases and their inhibitors (MMPs and TIMPs), leading to ECM stiffening that impedes myogenic repair and creates a physical barrier to regenerating axons [28]. Simultaneously, ectopic lipid accumulation from FAPadipogenic differentiation further degrades muscle architecture and metabolic homeostasis [29].

The NMJ, comprising the presynaptic motor terminal, the postsynaptic muscle membrane with clustered acetylcholine receptors (AChRs), and terminal Schwann cells, deteriorates rapidly after denervation [30]. AChR clusters disperse and synaptic folds flatten as presynaptic terminals retract; myonuclei drift from the subsynaptic region [31]. Initially, terminal Schwann cells extend processes that guide regenerating axons, but prolonged denervation leads to Schwann cell apoptosis, undermining reinnervation fidelity [32]. Restoring NMJ architecture thus requires coordinated nerve and muscle repair strategies [33].

PNI frequently precipitates neuropathic pain, marked by ectopic neuronal firing and central sensitization [34]. Denervated muscle contributes to pain through increased proinflammatory cytokines (e.g., IL-6, TNF-α), altered metabolic signaling, and heightened peripheral nociceptor sensitivity [35]. Effective management of sensory symptoms therefore demands both neurocentric interventions and strategies that modulate the denervated muscle environment, further supporting a role for muscle reprogramming in comprehensive PNI management [36].

Skeletal muscle reprogramming encompasses deliberate interventions that alter the fate or functional phenotype of muscle-resident or stromal cells to restore or enhance regeneration after injury. Unlike classical myogenesis, which relies solely on activation of endogenous satellite cells, reprogramming employs transcriptional, epigenetic, and metabolic cues to override impaired regenerative programs [37–39]. By reinforcing myogenic commitment in senescent MuSCs [40], converting fibro-adipogenic progenitors into myogenic-like cells [26, 41], or modulating the niche to support reinnervation, angiogenesis, and antifibrotic remodeling [42], this approach extends beyond mere muscle repair. In the setting of peripheral nerve injury (PNI), where denervated muscle rapidly atrophies and undergoes fibrosis and synaptic loss, skeletal muscle reprogramming serves as a critical adjunct to nerve-centered therapies.

Muscle satellite cells (MuSCs), marked by Pax7, are the principal drivers of myofiber regeneration. Under chronic denervation or aging, MuSCs enter senescence or apoptosis, depleting the progenitor pool [43]. Reprogramming strategies restore MuSC function bysuppressing oxidative stress [44], inhibiting NF-κB–mediated inflammatory suppression [45], and remodeling histone marks (H3K27ac) to revive stemness [46]. Fibro-adipogenic progenitors (FAPs) normally support acute repair but drive fibrosis and fatty infiltration under chronic insult. Redirecting FAP fate through TGF-β/Smad blockade and IL-4/STAT6 activation promotes secretion of pro-myogenic factors such as follistatin while suppressing adipogenesis [8, 47]. Moreover, epigenetic remodeling ofFAP-derived extracellular vesicles with miR-206/133 cargo enhances MuSC activity and niche rewiring via M2 macrophage polarization or GDNF-mediated innervation further restrains FAP dysregulation. Fibroblasts and myofibroblasts, which contribute to ECM deposition and scarring after PNI, can be transdifferentiated into induced myogenic progenitor cells (iMPCs) using MyoD overexpression, small-molecule cocktails, and HDAC inhibition; yet this conversion is limited by TGF-β–driven epigenetic barriers, matrix stiffness, and inflammation [48]. Although not direct reprogramming targets, immune and vascular cells are reshaped by muscle-centric interventions, with M2 macrophages and neovessels forming supportive niches [49]. Lastly, mature myofibers exhibit metabolic and structural plasticity: interventions such as AMPK activation or NAD⁺ supplementation stimulate mitochondrial biogenesis and shift fiber-type profiles, creating a substrate more conducive to reinnervation [50].

Transcriptional reprogramming via overexpression of myogenic factors like MyoD or Pax7 can restart myogenesis in fibroblasts or senescent MuSCs; however, the balance between these factors is critical, as Pax7 overexpression may inhibit MyoD-driven differentiation [51]. Epigenetic modulation uses HDAC inhibitors and DNA methylation inhibitors such as 5-azacytidine to relax chromatin, facilitating activation of muscle-specific genes [52]. Chemical cocktails that recapitulate developmental signals—combining forskolin, CHIR99021, FGF2, and IGF-1—activate Wnt/β-catenin and PI3K/AKT pathways to induce reprogramming without genetic manipulation [53]. CRISPR-mediated activation (CRISPRa) employs dCas9 fused to transcriptional activators to upregulate endogenous Myod1 and Myf5 with spatial and temporal precision [54]. Finally, metabolic and mechanical cues such as hypoxia-induced ROS, ECM stiffness, and cyclic stretch modulate mechanotransduction pathways (e.g., YAP/TAZ) to influence cell fate decisions [55].

Reprogramming approaches can be categorized mechanistically into four classes (Table 1). Transcriptional reprogramming involves forced expression or activation of myogenic transcription factors, exemplified by MyoD-driven conversion of fibroblasts [56]. Epigenetic reprogramming modulates chromatin accessibility via HDAC inhibitors, DNA demethylases like 5-aza, or CRISPRa/dCas9 tethered to epigenetic effectors [57, 58]. Metabolic reprogramming alters bioenergetic states through AMPK activation or NAD⁺ supplementation, enhancing mitochondrial function and oxidative capacity [50]. Pharmacological reprogramming utilizes cytokines and small molecules—such as IGF-1, Wnt agonists, and TGF-β inhibitors—to shift cell fate trajectories [53, 59]. This framework informs tailored therapy design for specific PNI-related pathologies, whether combating atrophy, fibrosis, or NMJ instability.

To maximize efficacy, emerging studies advocate for integrated, staged interventions. In a murine denervation model, sequential administration of CHIR99021 and HDAC inhibitors followed by MyoD-CRISPRa achieved superior myofiber regeneration compared to any single modality, illustrating the synergism of metabolic priming, epigenetic opening, and transcriptional activation [60]. Such combinatorial regimens may be delivered via smart biomaterials that release agents in response to enzymatic or pH changes in the injured niche, providing temporal control aligned with endogenous repair phases [61, 62].

Transcription factors (TFs) are primary regulators of cellular identity and lineage reprogramming. In skeletal muscle, the core myogenic regulatory factors (MRFs)— MyoD, Myf5, Myogenin, and MRF4—coordinate muscle-specific gene expression and lineage commitment. MyoD, in particular, can convert non-myogenic cells, such as fibroblasts, into myoblast-like cells by binding to E-box elements and recruiting epigenetic coactivators [63]. Pax7 is another key TF that maintains the self-renewal and quiescence of satellite cells; its acetylation status regulates muscle stem cell function and differentiation potential [64]. TF networks are further modulated by context-specific cofactors such as MEF2, TEADs, and YAP/TAZ, which integrate developmental cues and mechanical signals to fine-tune myogenic outputs [65, 66].

Epigenetic modifications govern the accessibility of myogenic loci, influencing a cell’s responsiveness to reprogramming signals. Histone acetylation, such as H3K9ac mediated by p300/CBP, facilitates open chromatin at myogenic enhancers, while histone deacetylation by HDACs represses myogenic genes. HDAC inhibitors, like trichostatin A, have been shown to enhance MyoD-induced conversion efficiency [67]. DNA methylation also plays a crucial role; hypermethylation of muscle-specific promoters (e.g., Myf5) can block myogenic differentiation, whereas inhibitors like 5-azacytidine demethylate these regions and restore transcriptional competency [68]. Chromatin regulators, such as the Polycomb and Trithorax complexes, dynamically repress or activate gene loci. EZH2, a methyltransferase in PRC2, represses Pax7 and MyoD; its inhibition reactivates satellite cell proliferation in aged or denervated muscle [69]. CRISPRa/i systems, employing engineered dCas9-fused activators or repressors, offer locus-specific epigenetic modulation, enabling fine control over endogenous gene expression without genome editing [69].

Muscle regeneration and cell fate decisions are tightly coupled to metabolic states. Oxidative metabolism favors MuSC quiescence and self-renewal, whereas glycolytic flux supports proliferation and differentiation [70]. Denervation leads to mitochondrial fragmentation and oxidative stress, impairing regenerative potential [71]. Boosting NAD⁺ levels through nicotinamide riboside or SIRT1 activation improves mitochondrial function and enhances myogenic capacity in aging and injury [72]. The AMPK and mTOR pathways are also pivotal; AMPK promotes autophagy and metabolic resilience, while mTORC1 drives protein synthesis and muscle hypertrophy [73, 74]. Their balanced modulation is essential in reprogramming strategies. Reactive oxygen species (ROS) play dual roles; while excessive ROS is damaging, moderate levels act as signaling molecules to stimulate regeneration via p38 MAPK and NRF2 pathways [75].

The extracellular matrix (ECM), vasculature, and immune system shape the reprogramming landscape via paracrine and mechanical cues. TGF-β signaling is a central inhibitor of regeneration, promoting FAP differentiation into fibrotic and adipogenic lineages; its blockade with agents such as losartan or TGF-β–neutralizing antibodies reduces fibrosis and enhances muscle regeneration in injury models [76, 77]. The Wnt/β-catenin pathway plays a dual role: canonical Wnt signaling promotes MuSC activation, whereas non-canonical ligands (e.g., Wnt5a) can impair myogenic reprogramming by inducing pro-inflammatory cascades [78]. Notch signaling maintains satellite cell quiescence and is essential during early regeneration; modulating Notch–Delta interactions (for example, via γ-secretase inhibitors or Delta-like ligand mimetics) improves functional recovery after PNI [79]. Finally, macrophage polarization from M1 to M2 phenotypes supports regenerative myogenesis—delivery of IL-4 or IL-10 to injured muscle skews macrophages toward an M2 profile, creating a pro-regenerative microenvironment that synergizes with reprogramming cues [80].

Recent findings suggest that reprogrammed muscle can exert retrograde effects on the regenerating nerve. Muscle‐restricted overexpression of neurotrophic factors—including IGF-1, BDNF and GDNF—enhances neuronal survival and axonal elongation in models of peripheral nerve injury [81]. Myogenic reprogramming also restores the expression of key synaptic organizers, such as Rapsyn, Agrin and MuSK, which are essential for neuromuscular junction integrity [82]. Additionally, axon‐guidance molecules—including Netrin-1, Semaphorins and Slit-Robo ligands—are modulated in reprogrammed muscle, influencing reinnervation fidelity and guiding regenerating axons to their targets [83].

Integrating these molecular mechanisms provides a comprehensive framework for developing targeted interventions in peripheral nerve repair. Combinatorial regimens that couple transcriptional activation with epigenetic modulation and metabolic priming— delivered via advanced vector systems or smart biomaterials—have demonstrated synergistic restoration of muscle mass and function in preclinical models [10]. However, clinical translation must overcome challenges in delivery, including achieving tissue-specific targeting, minimizing immunogenicity, and avoiding off-target effects inherent to viral vectors and genome editors [84, 85]. Furthermore, patient-specific variables such as age-related declines in MuSC responsiveness and heterogeneity in injury severity necessitate personalized dosing and modality selection to optimize therapeutic outcomes [86].

One of the earliest and most severe consequences of PNI is muscle atrophy, driven by a disruption in neuromuscular signaling and sustained inactivity. Prompt initiation of reprogramming strategies is essential, as delayed intervention may lead to irreversible myofiber degeneration. By reactivating myogenic programs through overexpression of transcription factors such as MyoD and Pax7, satellite cells can be re-engaged to promote tissue regeneration even in non-myogenic compartments [87, 88]. In parallel, reprogramming approaches that inhibit catabolic pathways—including FOXO3, atrogin-1, and MuRF1— have been shown to effectively reduce proteolysis, thereby preserving muscle mass [89]. Enhancing mitochondrial function through NAD⁺ supplementation and AMPK activation further supports oxidative metabolism and delays disuse atrophy [90, 91]. Preclinical studies in sciatic nerve transection models treated with small molecule cocktails have demonstrated sustained preservation of fiber cross-sectional area and contractile protein expression, even after prolonged denervation [92].

Following chronic denervation, skeletal muscle undergoes fibrotic and adipogenic remodeling, compromising both tissue function and its capacity for reinnervation [93]. Fibro-adipogenic progenitors (FAPs), which initially support regeneration, become pathological if not properly regulated. Reprogramming during the early post-injury period takes advantage of a critical window in which FAPs remain plastic and responsive [94]. Targeted inhibition of TGF-β signaling in FAPs has been shown to redirect their differentiation toward a quiescent or pro-myogenic state, thereby mitigating fibrotic scarring [95]. Simultaneously, pharmacological agents that normalize extracellular matrix composition—such as inhibitors of collagen synthesis or enhancers of matrix metalloproteinase activity—contribute to structural recovery and tissue elasticity [11]. The use ofWnt agonists and HDAC inhibitors further blocks adipogenic transcription factors like PPARγ and C/EBPα, limiting ectopic fat deposition [96]. Collectively, these effects preserve structural integrity and create a more permissive environment for axonal regrowth and synapse formation [97].

Following spinal cord injury, the neuromuscular junction (NMJ) is particularly vulnerable to denervation, leading to rapid disassembly characterized by fragmentation of acetylcholine receptor (AChR) clusters and degeneration of synaptic architecture [98]. However, reprogramming interventions, if implemented early—ideally within days of injury—can restore NMJ integrity [99]. This restoration involves the upregulation of synaptic scaffolding proteins such as Rapsyn and Dok-7 [100, 101], which stabilize AChR distribution across the postsynaptic membrane. Additionally, the restoration of agrin- MuSK signaling is crucial for accurate synaptogenesis, enabling regenerating axons to reform functional NMJs [102]. Furthermore, reprogramming indirectly benefits terminal Schwann cells, whose reactivation is essential for guiding axons back to their target zones [98]. Improved NMJ integrity, as demonstrated in multiple animal models, corresponds with better functional outcomes, including enhanced muscle responsiveness and coordination (Figure 3) [103].

While axonal regeneration has been primarily attributed to neuron-intrinsic mechanisms, recent studies demonstrate that reprogrammed muscle actively participates via retrograd signaling [11]. Muscle fibers and satellite cells subjected to reprogramming secrete neurotrophic myokines—including IGF-1, CNTF, and BDNF—which collectively enhance neuronal survival and axonal elongation [11, 104, 105]. In addition, muscle-derived axon guidance molecules such as netrin-1, semaphorins, and ephrin ligands orchestrate axonal pathfinding, ensuring precise reinnervation of target muscle territories [106]. The release of synaptic attractants, notably agrin and laminins, further strengthens axon–muscle connectivity and promotes accurate synapse formation [107]. These insights reposition muscle tissue from a passive recipient to a dynamic signaling hub that supports and directs nerve regeneration.

Ultimately, the success of any intervention for PNI hinges on its ability to restore motor function [14]. Skeletal muscle reprogramming improves functional outcomes by enhancing both structural and metabolic properties of the muscle. This approach upregulates key sarcomeric proteins, including myosin heavy chain and titin [108], which are essential for efficient force generation. It also counteracts denervation-induced fiber-type transitions by promoting a shift from fast glycolytic to oxidative phenotypes, thereby augmenting fatigue resistance and endurance [109]. Moreover, muscle reprogramming stabilizes the tendon–muscle interface and drives targeted ECM remodeling to restore effective force transmission across the myotendinous junction [110]. Functional benefits in rodent models—evidenced by enhanced EMG compound muscle action potentials, increased grip strength, and improved gait parameters—underscore the translational promise of these strategies [111].

Although skeletal muscle reprogramming is not intended to replace conventional nerve repair, it functions as a powerful adjunct when combined with nerve-centered therapies [112]. Nerve-centered therapies encompass a range of strategies designed to restore or reconstruct damaged nerves, including nerve grafting, neurotrophic scaffolds, and cell-based interventions, such as the transplantation of induced pluripotent stem cell (iPSC)-derived motor neurons [113, 114]. These interventions primarily aim to promote axonal regrowth, restore neural continuity, and reestablish functional synapses with target muscles. Despite their utility, the clinical success of these therapies is often limited by challenges such as poor receptivity of target muscles, misdirected axonal growth, and insufficient trophic or structural support for regenerating neurons, particularly in cases involving long-gap peripheral nerve injuries or delayed surgical repair [115]. Integrating skeletal muscle-targeted reprogramming with these nerve-centered modalities can overcome many of these limitations, yielding regenerative outcomes superior to those achieved by either approach alone. For example, simultaneous nerve grafting and localized delivery of muscle reprogramming factors not only enhances the intrinsi regenerative capacity of grafts but also improves axonal targeting and synaptic specificity within reinnervated muscles [116]. By optimizing the distal muscle environment, reprogramming strategies facilitate the precise alignment of regenerating axons with neuromuscular junctions, thereby promoting stable and functional connectivity [117]. Additionally, co-delivery of reprogramming factors with iPSC-derived motor neurons has been shown to establish a supportive regenerative niche, enriched with trophic cues and extracellular matrix components, which promotes neuronal survival, integration, and synapse formation [118]. Beyond cellular co-therapies, bioengineered scaffolds seeded with reprogrammed muscle cells can serve as hybrid conduits that bridge the muscle–nerve interface, offering both physical alignment for axonal growth and biochemical conditioning of the muscle to sustain neural input. These constructs enable synchronized regeneration of nerve and muscle tissues, addressing one of the major barriers to full functional recovery [119]. Such integrated approaches hold particular promise for long-gap injuries and delayed repair contexts, where temporal and spatial coordination of proximal axonal outgrowth with distal muscle preservation is critical. By simultaneously supporting nerve regeneration and maintaining a receptive, functional muscle substrate, skeletal muscle reprogramming amplifies the therapeutic impact of nerve-centered interventions, ultimately driving more robust and durable functional recovery [120].

Despite encouraging preclinical outcomes, the translation of skeletal muscle reprogramming into clinical therapies faces multiple challenges [10]. Foremost are regulatory considerations for gene-modification and cell-transplantation approaches, including vector safety, manufacturing consistency, and long-term follow-up requirements [121]. Scaling protocols from rodent models to human patients necessitates optimizing dosage, delivery volumes, and accounting for slower regeneration kinetics and complex biomechanics in larger muscles [122]. Efficient, targeted delivery of reprogramming agents remains a pivotal hurdle, driving the development of minimally invasive modalities such as ultrasound-mediated injection and nanoparticle carriers [123]. Immune compatibility, particularly with viral vectors or allogeneic cells, further complicates clinical deployment and underscores the need for non-immunogenic delivery platforms [124]. Looking ahead, advances in biomaterials design, synthetic biology, and non-viral gene-delivery vectors hold promise to overcome these barriers. Rigorous clinical trials will be essential to evaluate the safety, efficacy, and durability of reprogramming-based interventions in diverse PNI patient populations.

Recent single-cell RNA sequencing (scRNA-seq) and single-nucleus ATAC sequencing (snATAC-seq) studies have revealed extensive cellular heterogeneity and identified reprogramming-responsive subpopulations in injured muscle [126]. Fate-mapping analyses distinguish regenerative FAP subsets from those driving fibrosis, while integrated transcriptome–accessibility profiling uncovers master regulators of myogenic reprogramming [127]. Crucially, patient-derived single-cell signatures can guide personalized small-molecule cocktails or CRISPR targets by pinpointing dominant fibrotic or senescent pathways in individual samples.

Defined small-molecule combinations now enable genetic-free induction of myogenic progenitors. For example, the “chemical iMPC” cocktail—CHIR99021 (GSK-3β inhibitor), forskolin (adenylate cyclase activator), and RepSox (TGF-β inhibitor)— efficiently converts mouse fibroblasts into induced myogenic progenitor cells capable of myotube formation and muscle engraftment [128]. Concurrent application of chromatin-modifying agents, such as trichostatin A (HDAC inhibitor) and JQ1 (BET inhibitor), further enhances accessibility at myogenic loci. Inhibition ofTGF-β and ROCK signaling reduces fibrosis and skews aged or chronically denervated tissues toward a regenerative phenotype. Because these molecules are reversible, titratable, and compatible with injectable hydrogels or nanoparticles, they represent a highly translatable approach.

CRISPR/Cas systems now enable not only gene knockout but also precise activation (CRISPRa) or repression (CRISPRi) of endogenous myogenic regulators. Activation platforms—dCas9-VP64 and dCas9-p300—have been applied to upregulate MyoD, Pax7, and Myf5 in fibroblasts and FAPs, driving high-fidelity myogenic conversion without introducing exogenous sequences [129]. In contrast, CRISPRi targeting of anti-myogenic factors (e.g., Pparg, Tgfbr1) attenuates fibrotic signaling and shifts the stromal niche toward regeneration. Emerging epigenome editors, which fuse dCas9 to histone methyltransferases or demethylases such as LSD1 and TET1, permit locus-specific chromatin remodeling, thereby affording spatially and temporally precise lineage control in complex neuromuscular environments.

Three-dimensional culture platforms better recapitulate native muscle biomechanics and architecture than traditional 2D systems [105]. Myospheres and myobundles—engineered from reprogrammed progenitors—exhibit contractile activity and sustain functional neuromuscular junctions in vitro. Neuromuscular organoids, generated by co-culturing these constructs with motor neurons, enable dynamic studies of synaptogenesis and reinnervation. Furthermore, 3D bioprinting of cell-laden bioinks produces grafts tailored to complex nerve-injury geometries, facilitating high-throughput optimization of reprogramming parameters prior to in vivo application.

Effective clinical translation requires tissue-specific, efficient delivery of reprogramming tools. Adeno-associated viruses—especially serotypes AAV9 and AAVrh74—have successfully delivered MyoD or CRISPRa components directly into skeletal muscle [130]. As a non-viral alternative, lipid nanoparticles carrying mRNA or small molecules offer minimal immunogenicity and enable repeat dosing. Injectable hydrogel scaffolds, responsive to muscle-specific enzymatic cues, further improve local retention and controlled release, collectively mitigating off-target expression and systemic toxicity.

Most reprogramming interventions remain at the preclinical stage, yet murine sciatic-nerve transection models demonstrate restored electromyographic activity, increased muscle force, and enhanced NMJ density [131]. Large-animal studies in canine and porcine models are underway to evaluate reprogrammed muscle grafts within nerve conduits [132]. Early-phase human trials—initially targeting Duchenne muscular dystrophy—have established the safety and feasibility of AAV-mediated MyoD or IGF-1 delivery, providing valuable insights for PNI applications.

As muscle reprogramming advances toward the clinic, robust safety frameworks are essential. Key risks include oncogenic transformation of proliferative progenitors, immune rejection of viral vectors or allogeneic cells, and epigenetic instability that may compromise long-term function [133]. Future efforts should standardize manufacturing protocols, integrate reversible “kill-switch” mechanisms, and implement extended monitoring to ensure clinical safety and efficacy [134].

Denervation drives excessive deposition of extracellular matrix proteins—mainly collagen I and III—that stiffen tissue, impair mechanotransduction, and reduce contractile efficiency. Reprogramming strategies redirect fibro-adipogenic progenitors away from fibrogenic differentiation via transient TGF-β inhibition and locus-specific epigenetic editing of profibrotic genes [8, 135]. Concurrent modulation of matrix metalloproteinases and their inhibitors rebalances ECM turnover, improves compliance, and facilitates regenerating axons’ penetration into the muscle scaffold.

Chronic denervation skews fibro-adipogenic progenitors toward adipocyte formation, leading to lipid accumulation that disrupts contractile architecture. Small-molecule reprogramming—using PPARγ antagonists with Wnt/β-catenin agonists—rebalances lineage choice in favor of myogenesis [96]. Activation ofthe IGF-1/Akt axis further suppresses adipogenic networks, promoting myofiber hypertrophy and metabolic resilience. In rodent models, this combinatorial approach reduced intramuscular fat by 60% and improved grip strength by 30% compared to controls [136].

Persistent neuropathic pain following PNI arises from ectopic neural firing, central sensitization, and elevated proinflammatory cytokines in denervated muscle. Muscle-centric reprogramming that shifts cytokine milieus—decreasing IL-6 and TNF-α while boosting IL-10—has attenuated mechanical allodynia in sciatic-transected rats [137]. Restoration of NMJ integrity through agrin mimetics further normalizes aberrant afferent signaling and reduces dorsal-horn hyperexcitability.

Denervation disrupts the agrin–MuSK–LRP4 signaling axis, dispersing acetylcholine receptor clusters and retracting presynaptic terminals. Epigenetic upregulation of Rapsyn and Dok-7 in reprogrammed myofibers enhances AChR clustering and stabilizes terminal Schwann cell processes, preserving NMJ architecture during prolonged denervation [138]. This extended window enables more effective nerve reconnection and improves post-repair synaptic transmission.

Sustained denervation drives satellite cells into senescence, depleting the myogenic progenitor pool. Targeted CRISPRa activation of Pax7 and MyoD revives quiescent MuSCs and reinstates youthful chromatin landscapes—evidenced by telomere elongation and reduced β-galactosidase activity [139]. Concurrent modulation of the niche with anti-inflammatory cytokines creates a supportive microenvironment that sustains long-term progenitor function.

When both nerve and muscle regeneration fail, inducible reservoirs of myogenic cells can be generated from interstitial stromal populations. Secretion of neurotrophic factors such as GDNF and BDNF by reprogrammed muscle grafts attracts axonal sprouts and forms functional neuromuscular interfaces when combined with exogenous stem cell therapies [140]. In porcine models, these hybrid constructs bridge critical-sized nerve gaps and recover up to 70% of baseline muscle force.

Effective reinnervation also depends on adequate vascular support and immune balance. Co-delivery of VEGF with myogenic reprogramming factors enhances angiogenesis in denervated muscle, improving nutrient delivery and waste clearance [141]. Simultaneous polarization of macrophages toward an M2 phenotype reduces chronic inflammation and provides additional pro-regenerative cytokines, further facilitating axonal regrowth. By integrating these multifaceted muscle reprogramming strategies—targeting fibrosis, adipogenesis, pain, NMJ integrity, satellite cell rejuvenation, regenerative salvage, vascular support, and immune modulation—we can comprehensively address PNI- induced complications and extend the therapeutic window for successful nerve repair.

Despite encouraging preclinical outcomes, translating skeletal muscle reprogramming for peripheral nerve injury (PNI) into clinical therapies remains hindered by biological constraints, delivery inefficiencies, safety concerns, and complex regulatory and economic landscapes [142]. Overcoming these barriers is essential to move reprogramming approaches from laboratory prototypes into standardized treatments. The injured muscle niche contains satellite cells, fibro-adipogenic progenitors (FAPs), endothelial cells, immune infiltrates, and resident fibroblasts—any of which may inadvertently respond to reprogramming cues. Unintended modulation of non-myogenic populations risks aberrant remodeling and inflammation [143]. Moreover, most vectors use ubiquitous promoters lacking cell-type specificity. Recent strategies employ microRNA-regulated expression cassettes that remain inert in off-target cells but activate transgenes selectively in FAPs or myoblasts [144].

Converting stromal cells into early myogenic progenitors does not guarantee their maturation into fully contractile fibers. A significant fraction of induced cells stall at the MyoD-positive stage and fail to upregulate late structural genes such as myosin heavy chain [145]. Even when new fibers form, misalignment with regenerating axons can produce unstable or non-functional neuromuscular junctions. Beyond structural assembly, a significant “translational value gap” exists; traditional functional assessments in rodent PNI models, such as simple gait analysis, often fail to capture the complex neuromuscular coordination and fine motor control essential for human clinical recovery [146]. Engineering biomaterial scaffolds with aligned topography guides both myofiber orientation and axonal trajectories, improving integration.

Reprogramming—especially using viral vectors, synthetic mRNAs, or chromatin modifiers—can provoke innate and adaptive immunity. Repeat AAV administrations frequently elicit neutralizing antibodies, reducing transduction efficacy in large-animal and human studies. Off-target epigenetic remodeling may activate cGAS–STING signaling, triggering proinflammatory cascades that impair regeneration [147].

Immunomodulatory co-therapies, such as transient corticosteroids or localized macrophage reprogramming, are under investigation to mitigate these responses.

Reprogramming protocols often transiently suppress tumor suppressors (e.g., p53) to enhance plasticity, inadvertently increasing genomic instability. Sustained overexpression of myogenic transcription factors has been linked to rhabdomyosarcoma-like lesions in rodent models [148]. To reduce oncogenic risk, inducible “suicide switch” constructs—such as drug-activated caspase-9 systems—allow selective ablation of aberrantly proliferating cells [149]. While previous studies focused on macroscopic tumor formation, a more critical analytical framework is now required to monitor dynamic genomic stability. Recent 2025 clinical evaluations of human iPSC technologies emphasize that establishing rigorous standards for genomic integrity is a prerequisite for any clinical transition of reprogramming therapies [150]. The efficacy of reprogramming diminishes as denervation becomes chronic: satellite cells senesce, ECM cross-links accumulate, and synaptic basal lamina degrades [68]. Experimental data indicate a critical therapeutic window of approximately two to 4 weeks post-denervation for optimal responsiveness [151]. Early injury detection via advanced imaging or biomarker panels is therefore crucial to enable timely intervention. Scaling delivery from rodents to humans introduces logistical challenges: achieving uniform vector or small-molecule distribution across large, deep muscle compartments is difficult, and repeated intramuscular injections are impractical [152]. This “translational disconnect” is exacerbated by the vast anatomical differences between species; human muscles present severe physical diffusion barriers and varying tissue densities that prevent the uniform distribution of reprogramming factors, a challenge often oversimplified in standardized laboratory settings [125]. Non-invasive methods—such as focused ultrasound–mediated vascular delivery—transiently increase vascular permeability, enabling systemic agents to penetrate target tissues [153].

Reprogramming therapies often combine gene editing, drug delivery, and celltransplantation, falling into ambiguous regulatory categories that vary by region [154]. Ethical concerns over germline editing and off-target CRISPR effects continue to provoke public debate. Furthermore, while small-molecule “molecular time machines” offer a promising route to reverse senescence without genetic modification, their potential systemic side effects and disruption of long-term genomic stability in humans necessitate rigorous critical scrutiny beyond initial experimental success [155]. Additionally, the high cost of GMP-grade vectors, personalized cell products, and prolonged rehabilitation regimens presents an economic burden that may limit patient access [156].

The field of skeletal muscle reprogramming in the context of peripheral nerve injury (PNI) has achieved significant milestones, yet key questions remain unanswered. As efforts shift toward clinical translation, it is imperative to identify synergistic strategies, resolve outstanding biological uncertainties, and adopt standardized protocols. The followingsections outline six priority areas where concerted research can accelerate therapeutic innovation.

The integration of muscle and nerve reprogramming is crucial for achieving functional recovery after PNI, as current therapies often target these systems in isolation. Recent studies suggest that coordinated repair is essential, with evidence indicating that reprogramming skeletal muscle can enhance its ability to accept innervation. For instance, a 2024 study by Mehrotra et al. demonstrated that transient expression of NANOG in skeletal muscle improves neuromuscular junction formation and functional recovery in murine models of sciatic nerve injury [10]. This approach upregulates genes involved in muscle development and axon guidance, supporting synchronized regeneration. Additionally, in vitro models like neuromuscular organoids offer insights into the molecular interactions between muscle and nerve cells. A 2021 study in Nature Communications developed human sensorimotor organoids from induced pluripotent stem cells (iPSCs), revealing physiologically functional neuromuscular junctions and providing a platform to study disease mechanisms [157]. These models highlight retrograde signaling loops, such as those involving glial cell line-derived neurotrophic factor (GDNF), which may promote Schwann cell maturation. Advanced techniques like single-cell multi-omics and spatial transcriptomics are increasingly applied to delineate bidirectional molecular dialogues, though specific studies directly linking these to PNI are still emerging.

Achieving high-fidelity reprogramming requires tools that operate selectively in target cells and adapt to changing tissue states, minimizing off-target effects. Research suggests that pH-responsive hydrogels can release reprogramming factors in the acidic microenvironment of denervated muscle, enhancing specificity. A review in PMC highlighted the potential of pH-sensitive hydrogels for drug delivery, which could be adapted for muscle reprogramming [158]. This approach reduces unintended effects on healthy tissue, aligning with the need for context-sensitive strategies. MicroRNA-based regulation is another promising avenue, with microRNA-206 (miR-206) playing a significant role in muscle differentiation and regeneration. Studies show that miR-206 promotes myoblast differentiation and regulates satellite cell proliferation by repressing Pax7, confining transgene expression to myogenic progenitors [105]. Timing the delivery of epigenetic modulators to match tissue repair phases, such as the transition from inflammation to proliferation, is also critical, with evidence suggesting improved efficiency and functional integration, though specific studies on PNI are limited.

Robust in vitro platforms that recapitulate neuromuscular complexity are essential for preclinical validation, reducing reliance on animal models. Recent advances in 3D bioprinting have facilitated the creation of muscle-nerve constructs, incorporating aligned extracellular matrix (ECM) fibers and reprogrammed progenitors. A study demonstrated that neural cell integration into 3D bioprinted skeletal muscle constructs accelerates functional muscle regeneration in vivo, allowing precise assessment of fiber orientation and axonal targeting [159]. These constructs can be enhanced with optogenetic stimulation and high-resolution calcium imaging for real-time quantification of contractile kinetics and synaptic transmission, standard techniques in neuroscience research. To improve translational relevance, microfluidic “body on a chip” devices using human iPSC-derived fibro-adipogenic progenitors and motor neurons are being explored. A 2023 study in eLife developed human skeletal muscle organoids that sustain uncommitted Pax7-positive myogenic progenitors, offering a model for personalized assessment of reprogramming cocktails [160]. These platforms pave the way for tailoring therapies to individual genetic backgrounds, though challenges remain in scaling and standardization.

Translating findings from rodents to larger, anatomically and immunologically comparable species is a critical step for clinical application. Studies in large animal models, such as canines and porcines, have shown the feasibility of AAV-mediated gene delivery for muscle-related disorders. For example, a 2011 study in PMC demonstrated whole-body skeletal muscle transduction in neonatal dogs using AAV-9, leading to functional improvements in muscle strength [161]. While specific studies on PNI using muscle reprogramming in large animals are limited, the general approach of gene therapy for nerve regeneration is actively explored, with evidence suggesting potential for restoring gait and muscle fiber cross-sectional area. Human trials for gene therapy in PNI are still in early stages, with no widely reported clinical trials identified in recent literature.

Long-term outcomes and safety profiling are essential for clinical translation, yet longitudinal data remain scarce. Preliminary studies in rodent models suggest that reprogrammed muscles can maintain stable transgene expression without evidence of tumorigenesis. A 2024 study in Nature Communications on skeletal muscle reprogramming for PNI monitored outcomes over extended periods, showing preserved contractile strength under chronic exercise, though specific long-term safety data are limited [10]. Lineage tracing studies, a standard approach in gene therapy, have been used to ensure no clonal expansions or aberrant growths, supporting the safety of epigenetic reprogramming approaches, but further research in larger models is needed. Functional assessments, such as subjecting reprogrammed muscles to mechanical stress, indicate resilience, with a 2022 meta-analysis in Journal ofScience in Sport and Exercise suggesting long-lasting stretching induces muscle hypertrophy, relevant for maintaining function [162]. However, the field requires more comprehensive data to address safety concerns, particularly in humans, to ensure long-term efficacy and minimize risks like immune responses or unintended cellular changes.

Breakthroughs will emerge at the interface of biology, engineering, and computation. Park et al. (2023) showed that cyclic mechanical stretch, applied via bespoke bioreactors, enhances MyoD-mediated reprogramming efficiency by over 30 percent through mechanotransductive activation of YAP signaling [163]. Wang et al. (2021) developed in silico models that integrate diffusion dynamics of reprogramming factors with cellular signaling networks, accurately predicting optimal dosing schedules later confirmed in vivo [164]. To foster collaboration and reproducibility, Thompson et al. (2024) launched the open‐access NeuroMuscle Atlas, a repository cataloging reprogramming outcomes, vector designs, and safety metrics from multiple laboratories. By pursuing integrated muscle–nerve reprogramming, refining cell-specific tools, engineering advanced in vitro platforms, validating in large mammals, establishing long-term safety, and embracing interdisciplinary approaches, the field can transition muscle reprogramming from experimental promise to clinical reality.