Extracellular vesicle-derived MicroRNAs as potential therapies for spinal cord and peripheral nerve injuries

ABSTRACT

Complete nerve regeneration is limited in current therapeutic approaches for spinal cord injuries (SCIs) and peripheral nerve injuries (PNIs). Extracellular vesicles (EVs) and microRNAs (miRNAs) play a pivotal role in intercellular communication by transporting various biomolecules, including miRNAs, to the recipient cells. Thus, they are promising targets for novel neural regeneration drugs. This comprehensive study examined the roles of EV-derived miRNAs in facilitating neural rejuvenation after SCI and PNI. It also explored the mechanisms by which they augment neuroprotection and promote cell viability. It also discusses their translational potential for treating nerve injury and evaluates their potential impact on advancements in nerve resurrection and prospective research in regenerative medicine. The findings may provide effective treatments and improve outcomes, as well as contribute to addressing the direction for the next studies, for the pathologies of SCI and PNI.

Introduction

Nerve damage is a significant medical condition that debilitates millions of people worldwide [1]. Spinal cord and peripheral nerve damage can cause severe functional impairment, which remarkably reduces the quality of life. Spinal cord injuries (SCIs) are mainly caused by traffic accidents, falls, and sports mishaps, resulting in motor and sensory function loss below the injured area [1]. Peripheral nerve injury (PNI) occurs due to trauma, surgery, or disease; complete recovery is rare because of the limited regenerative capacity of the damaged nerves. The development of effective treatment methods for SCI and PNI is a major challenge in neuroscience and regenerative medicine because of their unique characteristics and renewal mechanisms.

Various approaches have been employed to promote spinal cord and peripheral nerve regeneration. Current therapies for SCI and PNI primarily focus on reducing secondary damage to the injured area or restoring function. Nerve cell regeneration is limited, particularly in SCI, mainly due to factors such as an inflammatory response and scar formation at the injury site [2]. In contrast, peripheral nerves have a relatively enhanced self-regeneration ability [3]. However, as full functional recovery remains elusive, novel treatment strategies are required urgently.

Extracellular vesicles (EVs) are crucial for nerve regeneration [4]. EVs comprise a natural bio-delivery system that is more biocompatible than artificially produced nanoparticles and can minimize immune responses [5–7]. Additionally, they can cross the blood – brain barrier (BBB) and are promising tools for treating central nervous system (CNS) diseases [8,9]. In particular, miRNAs within EV employ various pathways to suppress inflammatory responses, promote neuronal regeneration, and aid damaged nerve repair at the injury site. MiRNAs are small, noncoding (nc) RNAs, ~22 nucleotides long that post-transcriptionally regulate gene expression. They play critical roles in various physiological and pathological processes, such as regulating neuronal survival, growth, differentiation, inflammatory responses, and nerve regeneration [10–12]. MiRNAs are also vital regulators of the regenerative processes post-nerve injury.

This review comprehensively examined the effects of miRNAs and EV-derived miRNAs on spinal cord and peripheral nerve regeneration. Specifically, it focused on the roles of miRNAs in healing processes following SCI and PNI, the functions of EV-derived miRNAs, miRNA-based therapeutic strategies, and their clinical applicability. It explored the possibility of novel miRNA-based treatment strategies and suggested directions for future research.

EV and EV-derived miRNAs as therapeutic agents for neural regeneration

Stem cells are a major source of cells for tissue regeneration. However, as they can differentiate into varied cell types, their therapeutic application has certain significant limitations, particularly in developing off-the-shelf products, as well as storage and transport. To overcome these drawbacks, novel materials have been developed for use in regenerative medicine. EVs emerged as sources for regenerative medicine in the 1990s, demonstrating strengths in various aspects, such as developing biomarkers, specific material carriers, and therapeutic agents. EVs consist of a stable, lipid bilayer membrane with transmembrane proteins such as tetraspanin, integrins, and immunomodulators [13,14]. EVs contain several biological materials, including nucleic acids (DNA, RNA, and miRNA), functional proteins, biological lipids, cytokines, and other vital factors. Thus, EVs play a major role in transporting biological factors or their cargo.

Among the EV cargoes, certain nucleic acids, particularly miRNAs, have been identified as regenerative factors for the targeted treatment of pathologies or diseases. Specific miRNAs play critical roles in disease progression, regression, or both; including cancer [15–17]. Some miRNAs are related to oncogenesis; the therapeutic targets and mechanisms of action for miRNA – target interactions have been discovered. Thus, miRNAs from EVs may also be used for regeneration post-neural damage. This possible scenario is crucial as the therapeutic material and medication developed do not focus on nerve injury. Specifically, as EV-related research has gained popularity in regenerative medicine, studies regarding the role or significance of EV-derived miRNAs in the rejuvenation of spinal cord and peripheral nerves are of particular interest [18,19] EVs also have a prognostic and diagnostic role [20,21] besides their therapeutic function, which is the main topic of this review (Figure 1). EVs for both SCIs and PNIs are injected intravitreally, intranasally, intravenously, and locally. EVs can be delivered accurately to the target tissue by intravitreal injections, but being local cause great discomfort to patients. Intranasal injections have high patient compliance because they can be administered repeatedly without distress. However, their absorption rate may vary depending on the size and formulation of the EV. Intravenous injections are easy to deliver anywhere in the body and are advantageous for multiple injuries such as SCIs and PNIs. However, they pass the BBB with difficulty and the available concentrations may be low. Local injections allow the direct delivery of EVs but are limited to non-extensive damage because the EVs do not diffuse into the surrounding tissues [22–30].

Figure 1.

The preparation, administration route, and functions of extracellular vesicles (EVs).

EVs are isolated from biofluid using several methods such as ultracentrifuge and TFF. Isolated EVs were characterized using methods such as PCR, FACS, and Western blotting. EVs play prognostic, therapeutic, and diagnostic roles.

Neuroprotective impacts of EV-derived miRNAs in SCIs

Clinically, SCI severely damages the nervous tissues, resulting in motor, sensory, and autonomic function loss. This damage is exacerbated not only by the initial physical damage but also by the subsequent secondary damage-associated mechanisms. These include inflammatory responses, excitotoxicity, and apoptosis, which fatally impact nerve cells and impede axon survival in the damaged areas. EVs play a crucial role in promoting neuroprotection and regeneration. In particular, EV-miRNAs can help protect and renew damaged neural tissues by regulating various cell signalling pathways. They play a crucial role in suppressing the secondary damage-related pathways in SCIs and exert local neuroprotective effects. Thus, they play a vital role in suppressing pathological SCI progression and promoting nerve regeneration, emerging as a crucial element of future SCI treatment strategies.

Enhancement of neuronal survival by EV-derived miRNAs in SCI

EV-derived miRNAs, such as miR-124, play a crucial role in enhancing neuronal survival after SCI. It promotes anti-inflammatory microglial activation but suppresses inflammatory cytokine expression, reducing neural tissue inflammation. Additionally, it can help rejuvenate damaged nerve tissues by promoting neuronal growth and differentiation [31–33], maximizing the neuroprotective impact and minimizing neural tissue damage post-SCI.

Another crucial miRNA is miR-133b, which plays a vital role in regulating the RhoA, PI3K, and AKT pathways involved in nerve revival. Administering EVs containing miR-133b to SCI models improves the survival rate of damaged nerve cells and promotes nerve regeneration [34,35], serving as a vital factor for the survival and recovery of neuronal function post-SCI. These miRNAs may be critical components of therapeutic strategies for promoting nerve renewal. miR-9 also has marked effects on neuronal survival after SCI [36]. It regulates neuronal survival by targeting the monocyte chemotactic protein-induced protein 1 (MCPIP1) [37]. It also coordinates intracellular autophagy, enhancing nerve cell survivability after injury [38]. It enables nerve tissue regeneration by removing the damaged cells and promoting cytogenesis. These findings highlight the applicability of miR-9 in SCI treatment.

Therapeutics utilizing EV-derived miRNAs have great potential to improve neuronal survival post-SCI. They promote the regeneration of damaged neural tissues, suppress inflammation responses, and support neuronal survival (Table 1), thereby promoting functional recovery following SCI. The optimization of EV isolation, miRNA loading, and delivery methods is essential. Although various EV isolation methods exist, additional technological developments are imperative to secure simultaneous high purity and enhanced yield. In particular, fabricating a cost-effective process that maintains the biological activity of EVs during mass production is a key task.

Table 1.

Brief description of the roles of miRNA during SCIs and PNIs.

	Micro RNA	Biological Targets	Effects	References
SCI	miRNA-21	FasL/PTEN/PDCD4	Regulation of apoptosis	[39–41]
	miRNA-133b	RhoA/PI3K/AKT	Neural regeneration following a spinal cord injury	[42,43]
	miRNA-17–92 miRNA-221	PTEN/AKT/FOXO1		[44–47]
	miRNA-132	p250GAP	Neurite outgrowth	[48]
	miRNA-29c	ECM proteins	Axonal growth	[49,50]
PNI	miRNA-214	PTEN/JAK2/STAT3	Nerve regeneration	[37]
	miRNA-181	IL-1, IL-6 COX-2 TNF-α	Reduction in neuropathic pain	[38]
		Nrf2

The process of loading miRNAs into EVs is also an important research area. Various efficient and stable approaches such as electroporation and chemical- or nanotechnology-based loading have been applied. However, their impact on the structural stability of EVs and strategies to improve the loading efficiency should be studied continuously. Furthermore, the parallel functionalization or modification of the EV surface is imperative to deliver selective miRNAs to the target cells or tissues.

For the clinical success of EV-derived miRNAs, a deeper understanding of the long-term impacts and in vivo biodistribution of therapeutics is required. For example, evaluating the EV stability duration within specific tissues, the pathways eliminated during metabolism, or the likely and unexpected immune responses or adverse effects is crucial. These studies provide baseline data that can help ensure the safety and efficacy of EV-based therapeutics.

Promotion of axonal regeneration and synaptic plasticity in PNIs

PNIs often disrupt axonal continuity, leading to sensory and motor function loss. Axon regeneration is crucial for recovery from PNIs. EV-derived miRNAs hold promise in promoting axonal regeneration, for example, by modulating the gene expression profiles that favour neurite extension and cell repair mechanisms [51–53]. Several miRNAs induce an environment conducive to nerve regeneration by targeting the related pathways, which play a pivotal role in cell survival and axonal growth. As shown in Figure 2, miRNAs influence PNI regeneration through multiple pathways.

Figure 2.

Mechanism of EV-miRNAs in peripheral neural injury (PNI).

miRNAs regulate functions in processes such as nerve regeneration and reduction in neuropathic pain.

Synaptic plasticity, the ability of synapses to strengthen or weaken over time in response to activation or suppression, respectively, is essential for functional recovery post-nerve injury (Figure 3). EV-miRNAs play a vital role in enhancing synaptic plasticity by regulating the expression of synaptic proteins, such as synapsin and PSD-95, which are crucial for synapse formation and stabilization. Additionally, EV-delivered miRNAs enhance dendritic spine density and synaptic plasticity by targeting the Rho family GTPase signalling pathway. These miRNAs collectively contribute to synaptic connection reorganization and neural circuit restoration after PNI.

Figure 3.

Mechanism of EV-miRNAs in spinal cord injury (SCI).

miRNAs influence processes such as apoptosis, neural regeneration in spinal cord injury, and proinflammatory cytokines. In particular, miRNAs are essential for neuroprotection because they induce nerve regeneration by targeting multiple molecular pathways.

Axonal growth regulation

In the USA, 2.3% of individuals who have experienced a traumatic event are diagnosed with PNIs [54]. The annual incidence of PNI ranges from 9.9 to 12.3 cases, with a mean incidence of 11.2 cases per 100,000 population [55]. The self-repair capacity of the PNS is limited; however, this process may be only partially or completely halted in the event of severe damage. This effect results in severe motor and sensory function loss, necessitating the involvement of effective regenerative processes to restore neurological function. EVs originate from cells; harbour abundant proteins, lipids, and nucleic acids; and crucially facilitate intercellular communication. Research on axonal growth is necessary and advances are to be continuous. However, no solution has optimally addressed this problem.

Thus, EV therapy is viable for peripheral nerve regeneration following PNI (Table 1). EVs enhance axial growth [56] and are effective in promoting axonal growth and regrowth. After the pro-regenerative role of glial exosomes was demonstrated initially, EV-mediated recruitment of ncRNAs and proteins to neurons was also identified. Asymmetrical expression of miRNAs and mRNAs between EVs and other cell types has also been reported [42,43]. Many miRNAs have been implicated in axon guidance regulation, neurite outgrowth promotion, and axon regeneration in the PNS and CNS.

EV-miRNAs promote axon regeneration via the RhoA signalling pathway, which inhibits axon growth and affects cytoskeletal reorganization [39,40]. Additionally, EV-miR-21 enhances axonal growth by suppressing PTEN expression [41], a negative regulator of the PI3K/AKT pathway [44,45], activating the AKT signalling cascade, essential for neuronal survival and axonal regeneration. Moreover, the miR-17–92 cluster promotes axonal outgrowth by targeting multiple PTEN/mTOR pathway components, thereby inducing mTOR signalling and protein synthesis essential for axonal growth [46]. EV-derived miRNAs are pivotal in orchestrating the molecular events that drive axonal regeneration post-PNI through these intricate regulatory mechanisms. Furthermore, miR-221 and −222, encapsulated within EVs, contribute markedly to axonal growth by downregulating the expression of p27kip1, a cyclin-dependent kinase inhibitor of cell cycle progression [47]. This downregulation induces Schwann cell proliferation and axonal regrowth, facilitating nerve regeneration [48,49]. Additionally, miR-132 promotes axonal growth by targeting p250GAP, a Rho GTPase-activating protein, which enhances neurite outgrowth and synaptic plasticity [50]. EV-derived miRNAs modulate the extracellular matrix (ECM), contributing to axonal growth. For instance, miR-29c targets ECM proteins, such as laminin and collagen IV, thereby suppressing ECM deposition and inducing a permissive environment for axonal growth [57,58]. Such modulation of the ECM by EV-derived miRNAs underscores their multifaceted role in enhancing axonal regeneration and regulating axonal growth through various mechanisms such as modulating signalling pathways, targeting apoptotic mediators, and altering the ECM. Thus, these miRNAs are promising therapeutics for enhancing nerve regeneration and restoring nerve function.

Synaptic remodelling facilitation through EV-derived miRNAs in the PNI

Synaptic remodelling is a vital aspect of nerve repair and regeneration, particularly post-PNI. EV-derived miRNAs play an important role in these processes by regulating synaptic plasticity, ensuring the reorganization of the functional synaptic connections, and facilitating synaptic remodelling. MiR-146a improves synaptic plasticity by targeting inflammation-related pathways [59]. It can downregulate the expression of proinflammation cytokines and other inflammatory mediators, creating an environment favourable for synaptic growth and remodelling [60]. It facilitates the maintenance and formation of synaptic connections vital for cognitive function and recovery from injury by reducing neuroinflammation. In addition, miR-134 regulates the synaptic strength by suppressing the expression of LIMK1, a key regulator of intrasynaptic actin dynamics [61], promoting synaptic growth and plasticity, thereby contributing to neural circuit recovery and regeneration after PNI [62]. MiR-181c also found within EVs, regulates synaptic plasticity by modulating mitochondrial function and energy-related metabolism. It targets the genes involved in mitochondrial dynamics and function, ensuring an adequate energy supply to neurons for synaptic activity and remodelling. It can enhance synaptic efficacy by promoting mitochondrial health, crucial for sustaining the elevated energy demands for synaptic remodelling. In addition, it contributes to neuronal survival and synaptic stability during nerve injury recovery [63].

MiR-29a is a crucial regulator of synaptic plasticity and the expression of genes related to spinal morphology and synaptic strength. Its delivery via EVs may aid neuro-functional recovery post-injury [64]. Current research focuses on delivery optimization to maximize therapeutic effectiveness while minimizing any potential side effects. Clinical trials are required to evaluate the safety and efficacy of miRNA-based therapies, and understanding their long-term impacts is essential for their successful implementation as medical treatments.

Emerging therapeutic implications

The neuroprotective effects of EV-derived miRNAs present profound therapeutic potential in treating SCIs and PNIs [65,66]. As natural carriers of those miRNAs involved in intercellular communication, EVs possess unique biological properties that make them ideal vehicles for efficient miRNA delivery to specific tissue or cell types [67]. Various strategies can enhance the delivery efficiency and target specificity of EVs to maximize their therapeutic utility. Surface modification of EVs can improve their selective delivery to target tissues while maintaining biocompatibility by conjugating specific ligands targeting the receptors of interest. For instance, attaching specific peptides or antibodies to the EV surface can promote the targeted binding to neurons or regions requiring regeneration.

Additionally, encapsulating EVs within nanoparticles or leveraging highly advanced nanotechnology platforms, can not only enhance the in vivo stability and half-life of EV-derived miRNAs but also enable the controlled release of the therapeutic payload, ensuring sustained and uniform impacts [68–70]. Nanotechnology-engineered EVs are effective at crossing the BBB, markedly expanding their potential for delivering therapeutics to the CNS injury sites [71,72].

The pathophysiology of SCI- and PNI-associated secondary neural damage is highly complex, necessitating multifaceted approaches for effective management. EV-derived miRNAs play pivotal roles across various mechanisms, including anti-inflammatory, anti-apoptotic, neuroregenerative, and immunomodulatory effects, making them particularly suited for addressing the multifactorial nature of neural injury. Notably, specific miRNAs promoted cell survival, suppressed inflammation responses, and induced axonal regeneration, further underscoring the therapeutic promise of EV-derived miRNA-based therapies.

The use of EVs and miRNA for treating spinal and peripheral nerve injuries holds great promise for promoting nerve regeneration and modulating inflammation. However, standardized EV isolation techniques and optimized miRNA delivery systems are essential for clinical translation. EV isolation methods, including ultracentrifugation, size-exclusion chromatography, and immunoaffinity-based techniques, exhibit varied yield, purity, and reproducibility. Additionally, miRNA therapy faces challenges related to target tissue specificity, stability, and delivery efficiency. Since miRNAs are rapidly degraded in vivo and may trigger immune responses, various delivery approaches such as liposomes, nanoparticles, and EV-based systems are being explored, although further optimization of the loading efficiency and release kinetics is required. Compared with conventional gene therapy utilizing viral vectors, EV-based therapy offers advantages such as lower immunogenicity, reduced risk of insertional mutagenesis, and enhanced biocompatibility, making it a more safe and effective alternative. Several pre-clinical and early-phase clinical trials have demonstrated the therapeutic potential of EVs derived from mesenchymal stem cells (MSCs) in neural revival, improving functional recovery, and reducing inflammation in SCI models. However, for widespread clinical applicability, scalability and manufacturing challenges must be addressed. These include the development of standardized, large-scale EV production methods, quality control measures, and efficient storage solutions to maintain stability and bioactivity. Ensuring consistency in the manufacturing process, minimizing immunogenicity, and controlling in vivo half-life remains critical, alongside large-scale clinical trials to validate safety and long-term therapeutic efficacy.

Future research must focus on standardizing the production and large-scale manufacturing processes for EV-derived miRNA-based therapeutics to enhance their clinical applicability. Moreover, developing personalized therapeutic strategies tailored to the specific severity and biological differences of injuries could usher in a new era of precision medicine. These advancements are anticipated to fundamentally transform the paradigm of SCI and PNI management, driving groundbreaking innovations in rehabilitation medicine and neurology.

Conclusions

There are several implications of EV-derived miRNA-based neural regeneration therapy. Current miRNA research focuses on maximizing therapeutic efficacy while minimizing potential side effects. This therapeutic strategy may effectively treat SCIs, PNIs, and neural injuries. EV-derived miRNA-21, 133b, 17–92, 221, and 146a can prevent neuronal cell death and restore damaged neural structures. Additionally, synaptic remodelling may help reorganize damaged synapses and restore neural function. Similarly, some miRNAs from EVs facilitate the regeneration of damaged neural structures, synaptic reorganization, and sensory and motor function restoration in PNI. Such strategies can help advance research and technology for nerve regeneration and provide better treatment options for SCI and PNI patients. These strategies can not only improve clinical outcomes but also represent a vital step towards personalized medicine, where treatments are tailored to the individual needs of patients based on their specific neural damage profiles.

Funding Statement

Dr. Lee GW was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [No. 2022R1C1C1005410], grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), Ministry of Health & Welfare, Republic of Korea [RS-2023-00305198], and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [No.00219725]. Dr. Park S was supported by the NIH R01AR083086.

Acknowledgments

Lim YJ, Seo MS, Park WT, Park S, and Lee GW developed the idea, Lim YJ and Seo MS wrote the manuscript, Park WT and Park S helped in writing and revising the manuscript, and Lee GW revised and finalized the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).