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Various methods of dystrophin gene transfer have been applied for treatment of muscular dystrophy. Several viral vectors, notably the adenoviral vector, have been used, and modifications have been made to accommodate the large dystrophin gene. Various routes of administration have been tested; a clinical trial of intramuscular plasmid is currently in progress and appears to be promising. Other approaches are cell therapy, antisense approaches, and nonviral vectors for gene therapy of muscular dystrophy.
• There are several methods of gene therapy for muscular dystrophy including viral vector-mediated gene transfer, cell/gene therapy, gene editing, antisense approaches, and nonviral vectors.
• Viral vectors such as adenoviral vectors have been used and several modifications have been made to accommodate the large dystrophin gene including microgene versions.
• A few exon-skipping antisense oligonucleotides have been approved for treatment of Duchenne muscular dystrophy.
This article will discuss gene therapy for Duchenne muscular dystrophy, the most common of the various genetic muscular disorders. The mutated gene that causes Duchenne muscular dystrophy was discovered in 1986 (25). Dystrophin, the protein product of the Duchenne muscular dystrophy gene that forms the basis of future gene therapy of this disorder, was identified in 1987 (18). In 1992, correction of myopathy was carried out in a transgenic mouse model of Duchenne muscular dystrophy by germline gene transfer of human dystrophin using a retroviral vector (35). Other muscular dystrophies are also being investigated for feasibility of gene therapy. Considerable progress was made during 2004 to 2006 in the efficient systemic delivery of viral and nonviral gene transfer agents and antisense oligonucleotides for gene therapy of Duchenne muscular dystrophy, which are in clinical trials. Eteplirsen, an antisense-mediated exon skipping therapy for mutation suppression, has been approved by the FDA for Duchenne muscular dystrophy, followed by other therapies in this category.
• Several viral vectors have been used for transfer of dystrophin gene including adenoviral vectors.
• Nonviral vector approaches include intramuscular DNA plasmid injection.
• Antisense approaches include exon-skipping oligonucleotides.
• Gene editing includes CRISPR-Cas.
This article describes various approaches to gene therapy for muscular dystrophy. Prognosis of the disease is discussed in the clinical summaries that address the various muscular dystrophies. Various gene therapy approaches to Duchenne muscular dystrophy are shown in Table 1.
Viral vector-mediated gene transfer
Nonviral gene therapy
• Direct injection of plasmid DNA into the muscles
- injection of antisense oligonucleotides to generate an in-frame transcript by induced skipping of mutated exons
- administration of oral drugs to suppress nonsense mutations by translational read through
• Injection of chimeric oligonucleotides to correct single base mutations
The replacement of defective genes is the goal of gene therapy. Because of the large size of the defective genes and difficulties in delivering a gene to all muscle groups, complete correction cannot be accomplished with the current techniques. Endogenous gene expression of dystrophin should be restored to above 20% of normal levels for improvement of muscular dystrophy symptoms.
Indications of gene therapy include: Duchenne muscular dystrophy and Becker muscular dystrophy.
No contraindications have yet been identified.
Much has been accomplished since the discovery of the dystrophin gene. Adenoviral vectors have been improved and are the method of choice for delivery of dystrophin to the muscles in mdx mice. Adenoviral vectors devoid of viral genes appear to be the best candidates for filling the role of the ideal vector. Plasmid DNA has many advantages that include ease of manufacturing, low toxicity, immune innocuousness and accommodation of the large 11 kb dystrophin cDNA. This nonviral preparation has been successfully tested in a clinical trial. The first viral-mediated phase 1 clinical trial of gene transfer for Duchenne muscular dystrophy involved intramuscular injection of adeno-associated virus into a single muscle with safety as the primary endpoint (31). Adeno-associated, virus-based vectors provide efficient systemic gene delivery to skeletal muscle in vivo, and lentivirus-based vectors show promise of combining ex vivo gene modification with cell therapies. Gene therapy by means of gene transfer holds the promise of more long-lasting effects. Gene therapy trials have used viral vectors to deliver a minidystrophin gene. Animal studies suggest that it may be possible to overcome the main challenges currently facing gene therapy, ie, immunogenicity of the vector and wide-spread systemic delivery in the body (33).
Use of utrophin rather than dystrophin gene to avert immunological reactions is worth serious consideration. Successful experimental dystrophin gene transfer has also been demonstrated in the golden retriever dog model of muscular dystrophy. Two main issues need to be resolved before this therapy is applied to Duchenne muscular dystrophy patients:
• Development of methods for long-term expression of dystrophin in most of the muscle fibers.
• A suitable method for delivery of dystrophin not only to limb muscles but also to respiratory and cardiac muscles. At present, the use of an arterial route by left ventricular catheterization appears to be the most promising.
Cell transfer therapy, drug design to up-regulate utrophin, and strategy to repair the mutation in vivo are cell and gene-based (nonviral vector) treatments that need to be considered for future development. Intra-arterial administration of the autologous stem cells transduced by antisense oligonucleotide technology is a promising approach for muscular dystrophy. Another promising approach to the treatment of Duchenne muscular dystrophy is a combination of gene and stem cell therapies.
Personalized gene therapy of muscular dystrophy. Because numerous mutations have been found in muscular dystrophy leading to the investigation of a variety of methods of treatment, a mutation-focused approach, selected according to the nature of the gene defect, offers the opportunity for personalized gene therapy for muscular dystrophy (22).
Monitoring of dystrophin levels after therapy by use of monoclonal antibodies is required to determine if new dystrophin is present, and whether it is correlated with effects of gene therapy. More than 150 “exon-specific” monoclonal antibodies are available, which have been mapped to different regions of dystrophin gene and are available for research as well as clinical trials tailored to individual patients (26).
Although successful in animal models, some gene therapies show only modest efficacy in human clinical trials. Variable effects are observed in patients carrying the same mutation, indicating that several factors (such as genetic modifiers and environmental factors) can affect treatment outcomes. New approaches for gene and cell therapies for muscular dystrophies may require tailoring for individual patients according to variable factors (03).
Gene therapy has been studied extensively in animal experiments and human clinical trials. The record of safety has been well maintained. The following are some of the adverse effects that have been observed:
Retroviral vectors. Although the viruses are made replication-defective, the possibility of production of a replication-competent virus remains. Stable transformation of target cells makes it is difficult to reverse the treatment if appearance of undesirable side effects requires that the treatment be stopped.
Adenoviral vectors. Most of the adverse effects of viral vectors are associated with adenoviruses and some of these are:
Inflammatory effects. These have been observed mostly in the respiratory tract after local administration of adenoviral vectors.
Induction of immune response. One major concern about using adenoviral vectors for repetitive gene delivery is the induction of immune response to the vector that impedes effective gene transduction. Alternate use of adenovirus vectors from different stereotypes within the same subgroup can circumvent anti-adenovirus humoral immunity to permit effective gene transfer after repeat administration although the chronicity of the expression is limited by cellular immune processes directed both against the transgene and the viral gene products expressed by the vector. Animal experimental data show the potential of immunosuppression in the management of immune reactions triggered by adenoviral vectors by use of monoclonal antibodies.
Potential toxicity of gene therapy of muscular dystrophy. One theoretical concern is that overexpression of dystrophin to superaphysiological levels may cause cardiotoxicity. A study in transgenic mouse models of muscular dystrophy that overpress minidystrophin suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity (37).
Newborns. Most of the clinical trials of gene therapy for Duchenne muscular dystrophy have been conducted in patients aged 6 to 15 years. With the commercial availability of an FDA-approved Duchenne muscular dystrophy newborn screening immunoassay for muscle-specific creatine kinase isoform, newborn screening will identify affected infants early in the disease course, enabling systematic testing of earlier interventions with the hope of better clinical outcomes (09).
• Simple replacement of a defective gene is unlikely to work in muscular dystrophy unless blocking of normal gene expression is prevented by an antisense approach.
• Ex vivo engineering of autologous stem cells is used to develop into a myogenic lineage that can be implanted into the patient.
• Intramuscular injection of plasmid containing a full-length dystrophin cDNA can lead to expression of dystrophin.
• Because of the large size of the dystrophin gene, a truncated minigene is used, which provides adequate expression of dystrophin.
• Antisense oligonucleotides induce specific exon 51 skipping from the dystrophin gene’s premessenger RNA, restoring expression of dystrophin.
Recessive conditions, such as muscular dystrophies and metabolic myopathies, are amenable to gene therapy and require the replacement of defective genes. The autosomal dominant conditions are difficult to treat because a straightforward complementation of the defective gene is unlikely to work. It is possible to block expression of both chromosomal copies of the defective native gene by an antisense approach. Normal protein can be expressed by a normal gene construct that is introduced and contains divergent codons to prevent blocking by the antisense compound. Description of various methods including scientific basis is as follows:
Cell therapy. Studies in the muscular dystrophy model mouse, an animal model of Duchenne muscular dystrophy, indicate that the intravenous injection of either normal hematopoietic stem cells or a novel population of muscle-derived stem cells into irradiated animals leads to incorporation of donor-derived nuclei into muscle and the partial restoration of dystrophin expression in the affected muscle. After intra-arterial injection in experimental animals, muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage. Another approach to improving therapy of muscular dystrophy is to limit differentiation of the embryonic stem cells to the muscle lineage by transducing them with an adenovirus encoding for MyoD, a transcription factor used for muscle cell differentiation.
The ability to isolate and expand a patient’s own stem cells that can be developed into myogenic lineage provides the opportunity to establish cell lines that can be used for transplantation following ex vivo manipulation and expansion (05).
Myoblast-based gene transfer. Myoblast transfer therapy is a cell-mediated gene transfer method aimed at the restoration of normal dystrophin expression in Duchenne muscular dystrophy. Myoblasts are mononuclear, fusion-competent muscle precursor cells that can be generated in pure culture from satellite cells of muscle. These cells from patients can be transfected with functional dystrophin gene constructs. When these myoblasts are injected into the muscles of patients (autologous myoblast transfer), sufficient dystrophin may be produced to protect the myofibers from necrosis. This technique has the advantage over heterologous myoblast transfer in that the autologous myoblasts do not evoke an immunological reaction and the use of immunosuppressant drugs is not necessary. One drawback is that satellite cells in Duchenne muscular dystrophy have already spent a great deal of their proliferative capacity by participating in repeated cycles of regeneration. The myoblasts that are produced age after relatively few doublings.
This problem may be overcome by using nonmuscle cells such as fibroblasts that are converted into myoblasts by transfection with a myo-D construct. In vivo conversion of these nonmyogenic cells to myogenic pathway results in the formation of dystrophin-positive muscle fibers in the deficient host. It is feasible to introduce functional copies of dystrophin into dermal fibroblasts before they are implanted into muscle cells.
Muscle biopsies of patients in clinical trials of myoblast transfer have been examined using fluorescent in situ hybridization-based methods to demonstrate the persistence of donor cells and their production of dystrophin. The clinical trials, however, have not shown any significant benefit because most of the donor cells fail to survive beyond the first week.
An improvement on this approach is the implantation of genetically modified, marrow-derived myogenic progenitors as an alternative to myoblasts.
In vivo targeting of muscle progenitor cells, notably satellite cells, with a pseudotyped lentiviral vector encoding the minidystrophin restores dystrophin expression and provides functional correction in skeletal muscle of the mouse model of muscular dystrophy. Human muscle precursor cells have been genetically modified with a plasmid coding for the full-length human dystrophin gene fused with a small transgene, eGFP, and transplanted in severe combined immuno-deficient mice leading to the expression of eGFP dystrophin in muscle fibers (28). This is a possible approach to treat Duchenne muscular dystrophy.
Nonviral vectors. These include plasmid-mediated gene delivery, antisense-mediated exon skipping, and oligonucleotide-mediated gene editing. Compared to viral vectors, nonviral vectors are relatively simple, safe, and cost-effective, with great promise for future gene therapy approaches to the treatment of Duchenne muscular dystrophy.
Dystrophin gene plasmid. The first gene transfer trial in patients with Duchenne muscular dystrophy was aimed at providing evidence on transgene expression and safety of the intramuscular administration of a plasmid containing a full-length dystrophin cDNA (32). Dystrophin expression was found in 6 of the 9 patients treated even though the level of expression was low. There were no adverse effects.
Liposome-mediated gene transfer. As a gene transfer system in vivo, liposomes hold promise for Duchenne muscular dystrophy gene therapy because of the lack of any limit for DNA size and low antigenicity. Liposome-mediated gene transfer into normal and dystrophin-deficient mouse muscle cells, however, is inadequate by this method. A combination of liposome with hemagglutinating virus of Japan has been used for the expression of full-length cDNA of Duchenne muscular dystrophy.
Viral vectors. Recombinant replication deficient retroviral vectors are used for myoblast-based gene transfer, but they are unsuitable for gene therapy of Duchenne muscular dystrophy for the following reasons:
• The capacity of retroviral vectors is too small (about 7 kb) for the large dystrophin gene (2.4 mb).
• Integration of the transferred gene with long-term expression is not required for postmitotic nuclei of muscle fibers.
• The transcription rate of the transferred gene in vivo tends to be low.
Herpes simplex virus is a possible candidate with its large capacity (30 kb) and long latency in vivo. Although it exhibits neurotropisms, there is no information available about its myotropism.
Adenoviral vectors. Adenoviral vectors have a significant potential utility for muscle gene transfer because of their relative safety, high titer, and efficient transduction of postmitotic cells. Adenovirus may enhance efficiency of gene transfer not only as a vector but also as a facilitator. The facilitating role of adenovirus in gene uptake may be due to several factors, including a facilitation of uptake through the cell membrane, release from the endosomes, and facilitating uptake into nuclei. One of the drawbacks of adenovirus vector for gene transfer into muscle is that free viral receptors at mature muscle fiber surface may be sparse. This disadvantage may be overcome by causing abundant fiber regeneration in the recipient muscle prior to injection of recombinant adenovirus. The other disadvantage is cellular and immune attack directed against adenoviral transduced muscle fibers. This can be counteracted with immunosuppressant agents.
Full expression of the gene is not necessary, and about 20% of normal dystrophin is effective in preventing muscle necrosis and dystrophy as judged by reduction of creatine kinase levels that are elevated secondary to muscle damage. Experiments in mdx mice have shown that expression of high levels of dystrophin, up to 50 times the normal amount, is not toxic.
Injection of high-titer suspensions of adenoviral recombinants containing dystrophin cDNA (truncated 200 kd gene) into muscles produces many dystrophin-positive muscle fibers initially but the prevalence of dystrophin-positive fibers declines in 2 months, probably due to elimination of transduced muscle fibers by the immune response of the host mediated by cytotoxic CD8+ lymphocytes. This points out the need for suppression of host immune response if adenoviral vector-mediated gene transfer is to become a practical method of gene replacement therapy. FK506 (an immunosuppressant agent) treatment of experimental animals improves the efficiency of adenoviral-mediated gene transfer and permits a sustained high-level dystrophin expression.
Gene expression in muscles is monitored by immunocytochemical staining for the detecting dystrophin. False-positive staining can be due to reverting fibers seen both in mdx mice and humans. In the case of human trials, this can be controlled by selecting patients with dystrophin deletions and using antibodies that recognize epitomes internal to the deletion indicating that staining is due to reversion.
There is a fiber-to-fiber variation in the level of gene expression in Duchenne muscular dystrophy as seen by staining, giving rise to the phenomenon described as mosaicism. It is anticipated that this would disappear following gene therapy and uniform expression of dystrophin in the muscles.
Another method for the assessment of recovery of muscle following dystrophin gene transfer is measurement of nitric oxide synthase in the muscle fibers. Nitric oxide synthase is absent in patients with Duchenne muscular dystrophy as well as in mdx mice. Half of the dystrophin-positive fibers also show significant levels of nitric oxide synthase, indicating biochemical recovery. These observations indicate that nNOS is a useful marker for complete restoration of the dystrophin associated complex and should be used as one of the criteria for selecting the recombinant molecule to be used for gene therapy in Duchenne muscular dystrophy.
Modifications of adenoviral vectors. Adenoviral vectors used for gene therapy have deletion of the E-1 region and are replication-defective and driven by a suitable promoter. The maximum capacity of first- and second-generation adenoviral vectors is 8 kb.
Because of the large size of the dystrophin gene (DNA 14 kb), most of the studies have involved the use of a truncated minigene. Adenovirus-mediated transfer of a dystrophin minigene has been shown to provide functional protection of mdx mouse muscles.
Various modifications of adenoviral vectors have been made to accommodate the full-length dystrophin gene. This has been achieved by deleting all viral genes from adenoviral vectors. Such a deleted vector carrying full-length dystrophin gene can transduce muscle fibers when injected directly in skeletal muscles of murine models of Duchenne muscular dystrophy. This vector also minimizes the destructive immune response to adenoviral vectors.
An adenoviral vector in which all the viral genes are deleted has a capacity to carry 30 kb of foreign cDNA and can accommodate a full-length dystrophin gene. Because viral genes are eliminated, no viral proteins are produced. Various studies demonstrate that, in the absence of an immune response induced by beta-galactosidase expression, an adenoviral vector lacking all viral genes is stably maintained in muscle.
Adeno-associated virus vectors (AAV). AAV vectors are now also being used. When injected into the muscles of mdx mice, a recombinant AAV (rAAV) type 2 vector expressing microdystrophin produces extensive and long-term expression of microdystrophin with significant improvement in force muscle contraction. Of the many approaches being pursued to treat muscular dystrophy, gene therapy based on AAV-mediated delivery of microdystrophin is the most direct and promising method to treat the cause of the disorder (30). A study in the dog model of Duchenne muscular dystrophy has shown that circulating myostatin is a reliable quantitative biomarker for measuring the therapeutic response to gene therapy by rise of low myostatin levels as well as a for noninvasive follow-up of a therapeutic effect (23). Moreover, a 2-year follow-up also suggests that myostatin could be a longitudinal monitoring tool to follow maintenance or decrease of the therapeutic effect.
Although AAV-mediated gene therapy is limited by the small viral packaging capacity of the vector, trans-splicing AAV vectors can package twice the size of the vector genome. Several advances in the areas of AAV serotype analysis, transgene engineering, and vector delivery to muscle, together with novel means of rescuing mutant mRNA transcripts, have yielded impressive results in animal models of Duchenne muscular dystrophy. Functional dystrophin transgenes larger than that typically carried by a single rAAV genome can be reconstituted in vivo by homologous recombination following intravascular co-delivery with rAAV6 (27). Among the few viral vectors available, rAAV has emerged as the most promising for safe as well as effective in vivo gene delivery that can be regulated, but immune response following rAAV administration poses a significant barrier to gene transfer in humans (34). Alternate serotype vectors (AAV1-10), specific tissue targeting, immune-suppression, and engineered capsid variants are some of the approaches that have been proposed to minimize immune stimulation by rAAV (16). Based on success in achieving global muscle transduction in rodents following intravenous injection of AAV-9, the first systemic therapy has been initiated in patients (12).
Dual AAV technology was developed to double the packaging capacity of the AAV vector, and the proof of principle was demonstrated in various mouse models. Because large animal models are more relevant to human disease, a dual AAV-9 vector packaging a tyrosine-modified 7-kb mini-dystrophin gene was tested in a DMD canine model and resulted in widespread mini-dystrophin expression 2 months after gene transfer, which restored the missing dystrophin-associated glycoprotein complex (19). Treatment also reduced muscle degeneration, improved myofiber size distribution, and protected the muscle from eccentric contraction-induced force loss. Dual AAV gene therapy is expected to be more effective in human patients with Duchenne muscle dystrophy therapy.
Lentiviral vectors. Lentiviral vectors are considered for gene therapy of Duchenne muscular dystrophy as they have a relatively large transgene carrying capacity and can integrate into nondividing cells. The demonstration that lentiviral vectors can transduce myogenic progenitors using a minidystrophin cassette regulated by a muscle-specific promoter suggests that this system could be useful for ex vivo gene therapy of muscular dystrophy (21). In experimental studies, myoblasts of a Duchenne muscular dystrophy patient, deleted for dystrophin exons 49 and 50, were infected with a lentivirus expressing a small nuclear RNA containing antisense sequences against exon 51, leading to correct exon skipping. Expression of dystrophin was demonstrated in vitro and in vivo, indicating the feasibility of lentiviral-based ex vivo gene therapy for Duchenne muscular dystrophy (29).
Electrotransfer of naked DNA in the skeletal muscles. Naked DNA electrotransfer has been accomplished in animal models of muscular dystrophies; despite some limitations, it needs to be further explored as a potential avenue for treatment of muscular dystrophies.
Routes of administration. Various methods of gene delivery have been investigated during the past few years. Advantages and disadvantages of these approaches are as follows:
Intramuscular injection. Direct intramuscular injection of an adenovirus suspension results in infection of most of the myofibers near the site of injection. For a widespread dispersal, multiple injections are required over a wide area of the body, which is inconvenient.
Another problem is that artificial gene constructs may end up in the interstitial space of the muscles following intramuscular injection. From there the gene must reach the myoneural space in a functionally intact form to be expressed. Breakdown of a substantial amount of DNA in the interstitial space by the enzyme DNAse could markedly reduce the number of intact gene constructs available for reuptake into muscle cells. Spread of the injected gene constructs in the interstitial space may be hindered by endomysial fibrosis, which occurs in Duchenne muscular dystrophy patients. Expression of transforming growth factor beta 1 in early stages of Duchenne muscular dystrophy may be critical in initiating muscle fibrosis. An approach to reducing this fibrosis should be developed and would facilitate gene therapy of Duchenne muscular dystrophy.
Substantially high levels of dystrophin expression are achieved after injection into the ventricular wall of the heart but such a potentially hazardous method of delivery is appropriate only in animal models. Injection into diaphragmatic and cardiac muscles, which are desirable targets in Duchenne muscular dystrophy patients, would be a problem.
Intraperitoneal injection. About 25% of diaphragmatic muscle fibers in neonatal rats can be transduced following an intraperitoneal injection of an adenoviral vector containing dystrophin gene. This method is unlikely to be applicable to humans.
Intravenous injection. Simple intravenous injections of adenovirus suspensions give rise to marked gene expression in muscle cells but the level of expression is extremely low. Gene delivery by this route is not satisfactory, as the gene constructs could end up expressing in tissues other than muscles unless a muscle-specific promoter restricts its expression to skeletal and cardiac muscle cells. Eventually, the gene constructs, whether delivered intramuscularly or via circulation, end up in the interstitial space of the muscle. From there the gene must reach the myoneural space in a functionally intact form to be expressed. Several obstacles could destroy the gene during this journey. DNA degradation may occur due to action of DNAse, which is released by histiocytes and fibroblasts. Even if the gene construct survives this passage, there are further barriers; it does not easily reach the myonuclear space. To address these problems of delivery, a highly abbreviated micro-dystrophin gene has been developed for body-wide systemic gene transfer with AAV vector. Numerous microgene configurations and various AAV serotypes have been investigated in animal models, and preclinical data suggest that intravascular AAV micro-dystrophin delivery can significantly alleviate muscle pathology, enhance muscle force, and attenuate dystrophic cardiomyopathy in animals (13). Several clinical trials have been initiated to test the safety and tolerability of this promising therapy in Duchenne muscular dystrophy patients.
Regional intra-arterial injection. This is a conceivable way of disseminating artificial genes into muscles, but a possible complication is obstruction of larger blood vessels, which could cause infarcts.
Regional vascular perfusion. A promising technique involves regional vascular isolation to achieve high rates of adenoviral transduction of hind limb muscles of mice. The blood from the regional vascular bed is drained and replaced with adenoviral infusion and blood flow is re-established following a short period of incubation. Transfection rates of over 25% can be achieved in the muscles with little escape of virus into the systemic circulation. The application of this method in humans is questionable.
Intracardiac injection. Systemic administration of adenoviral vector into the left ventricle of the heart in mice has led to a high number of dystrophin positive fibers in heart, diaphragm, and intercostal muscles but not in the limb muscles. A catheter can be introduced into the left ventricle of the heart by the standard procedure of cardiac catheterization.
Clinical trials of gene therapy of muscular dystrophies. As of August 2021, 50 clinical trials are registered on the U.S. government clinical trials database: U.S. government clinical trials database. The experimental designs of these trials were based on different rationales, including immunomodulation, readthrough strategies, exon skipping, gene therapy, and cell therapy. Trials include the following:
• A phase 3 open-label study of eteplirsen, an approved drug, is ongoing to provide confirmatory evidence of its efficacy in Duchenne muscular dystrophy patients who are amenable to skipping exon 51 (NCT02255552). Additional objectives of this trial include evaluation of safety, biomarkers, and the long-term effects of eteplirsen up to 96 weeks.
• Ataluren is a molecule that binds with ribosomes and allows the insertion of an amino acid in the premature termination codon as well as exon-skipping, which binds with RNA and excludes specific sites of RNA splicing, producing a dystrophin that is smaller but functional (06). Ataluren showed activity and safety in a phase IIb, double-blind, long-term study in nonsense mutation DMD (14). It has completed phase 3 trials and was approved by the European Medicines Agency in 2014 but not by the FDA.
• In a phase 1 trial of limb girdle muscular dystrophy 2D (alpha-sarcoglycan deficiency), the transgene was transferred using AAV vector expressing the human gamma-sarcoglycan gene under the control of the desmin promoter by local injection into the extensor carpi radialis muscle (17). Expression of gamma-sarcoglycan protein can be induced in patients with no serious adverse effects.
• A phase 1 clinical trial of rAAV2.5-CMV-mini-dystrophin gene vector in Duchenne muscular dystrophy has been completed (NCT00428935). This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective, eg, limb infusion gene delivery, and should usher in the next generation of viral delivery systems for human gene transfer (07).
Most of the approaches to gene therapy of muscular dystrophy are based on animal models of the disease.
Animal models. Various animal models, with naturally occurring dystrophin deficiency to study the feasibility of gene therapy, are available. In the muscular dystrophy model mouse, mdx mouse, dystrophin is missing due to a single base missense mutation of the dystrophin gene. It is a good model for investigating the beneficial effects and duration of gene expression following gene transfer in limb muscles, but extrapolation of the results to humans is difficult because of the small size of the animals, and transitory nature of the muscle weakness. Several transgenic muscular dystrophy model mouse lines have been developed to study the protective effects of full-length or partial dystrophin cDNA constructs.
Muscular dystrophy in mixed breed golden retriever dogs closely resembles the clinical course and pathology of Duchenne muscular dystrophy. This animal is optimal for preclinical gene therapy studies, but the difficulty and expense of maintaining such a colony are limiting factors.
Earlier experiments of gene transfer in muscular dystrophy model mice involved untargeted or direct naked DNA transfer. Such techniques produced sarcolemmal dystrophin in about 1% to 2% of muscle fibers after direct intramuscular injection. Attempts to target by injecting lacZ reporter genes driven by human creatine kinase promoter in adenoviral vector into mouse muscle were not more effective than naked DNA injection. More recent efforts have improved the expression to 5% to 50% dystrophin-positive fibers in muscular dystrophy model mice by use of adenoviral vectors.
For full optimization of gene therapy, the majority of myonuclei must acquire at least 1 adequate dystrophin construct that is expressed in muscle cells only and remains transcriptionally active for long periods of time. To attain this goal, the following issues need to be attended to:
• Selection of dystrophin construct
Gene targeting. In direct gene transfer, the gene is probably taken up by muscle cell via constitutive bulk-fluid-phase endocytosis. This is a process by which extracellular fluid and molecules are indiscriminately interiorized by the cell. Uptake of the DNA into cells can be enhanced by the following methods:
• Use of a gene gun
Types of dystrophin constructs. Because the full-length dystrophin cDNA exceeds the carrying capacity of most currently used viral vectors, the efficacy of dystrophin minigenes was investigated. Truncated dystrophin gene (producing 200 kd protein) from some patients with mild Becker muscular dystrophy is the basis of many minigene constructs used for experiments. Half the dystrophin gene is apparently enough for a mild clinical course. The transfection of human primary myoblasts with large plasmids can only be achieved when the DNA is linked to an adenovirus with the use of polyethylenimine, with efficiencies ranging between 3% and 5% of transitory transfection. Muscles of transgenic mdx mice expressing this construct show improvement in structure and function. Modified versions of this micro-dystrophin gene may further improve the function. Nevertheless, the currently available truncated dystrophin microgenes are sufficiently effective to be tested in clinical trials. Apart from the small size, another advantage of minigenes is the short transcription time (20 minutes), as compared with about 24 hours for a full gene. An open-label phase 1/2a clinical trial (NCT03375164) showed rAAVrh74.MHCK7.micro-dystrophin (SRP-9001) was well tolerated for delivery of micro-dystrophin transgene and robust expression and correct localization of micro-dystrophin protein with improvements in creatine kinase levels and functional outcomes measured by North Star Ambulatory Assessment scores (24). MRI biomarker data indicate marked sparing and minimal fat infiltration in boys with Duchenne muscular dystrophy who received SRP-9001 compared with an age-matched natural history cohort (36).
Several options are available for promoters for dystrophin constructs. Constitutive promoters have been tried including Roux sarcoma virus promoter or cytomegalic virus promoter. These are effective for driving the transferred dystrophin constructs in vitro and in vivo, but they lack tissue specificity. This would pose a problem if the gene constructs were administered intravascularly because dystrophin expression may occur in other cell types in the body and can be harmful. Muscle-specific promoters, such as those of creatine kinase, have been shown to be effective in driving the dystrophin minigene in transgenic mice after adenovirus-mediated transfer.
Alternative approaches. Due to limitations of conventional gene therapy, several alternative approaches have been considered.
Regulation of dystrophin. One alternative approach to treatment would be to upregulate the closely related protein utrophin, which might be able to compensate for the dystrophin deficiency in relevant muscles. Utrophin transgene can be expressed at a high level in dystrophin-deficient mdx mice suggesting that systemic upregulation of utrophin in Duchenne muscular dystrophy patients may lead to development of an effective treatment. This might be accomplished by small molecules. Furthermore, as utrophin is normally expressed in all tissues including muscles, the use of this utrophin transgene, rather than the dystrophin transgene in conventional gene therapy approaches using viruses or liposomes, may avert any potential immunological responses against the transgene. Upregulation of utrophin by a nontoxic pharmacological agent would be an elegant and convenient way of treating Duchenne muscular dystrophy and Becker muscular dystrophy. It is presently impossible to predict if or when such an agent will be identified.
Blockage of myostatin. Myostatin, a member of the transforming growth factor-beta superfamily is a negative regulator of skeletal muscle growth. Inhibition of the myostatin gene product is predicted to increase muscle mass and improve the disease phenotype in a variety of primary and secondary myopathies. Inhibition of endogenous myostatin by blocking antibodies results in an increase in muscle size and muscle strength in the mdx mouse model of Duchenne muscular dystrophy. It may be possible to develop an antisense approach to inhibit the myostatin gene.
Antisense approaches. Antisense-mediated exon skipping therapy is being developed for muscular dystrophy. Small molecules for exon skipping and mutation suppression and gene transfer to replace or provide surrogate genes as tools for molecular-based approaches for the treatment of muscular dystrophies were studied in clinical trials. They are chemically distinct oligonucleotides inducing specific exon 51 skipping from the dystrophin gene's premessenger RNA. They are gene medicines because of their mode of action by antisense-mediated exon skipping. As of 2021, 4 exon-skipping antisense oligonucleotides – eteplirsen, golodirsen, vitolarsen, and casimersen – are approved by the FDA for clinical use and they are collectively applicable to approximately 30% of the Duchenne muscular dystrophy patients owing to the variant-specific mechanisms of action (10; 15).
In clinical trials, eteplirsen was shown to increase dystrophin expression after weekly intravenous infusions. The increase in dystrophin is effective for slowing or preventing the progression of Duchenne muscular dystrophy. Despite some of the controversy surrounding the approval of this drug and its excessive cost, eteplirsen is an excellent example of clinical application of science-based progress in gene therapy (11).
A single injection of AAV9-U7 exon 51 was shown to induce widespread and sustained levels of exon 51 skipping, leading to significant restoration of dystrophin and improvement of the dystrophic phenotype in the mdx52 mouse, but levels of dystrophin re-expression were lower than the skipping levels (02). These findings are in contrast to previously reported results in the mdx mouse indicating that efficacy of exon skipping varies depending on the targeted exon.
Gene editing. Correction of the genetic defect using oligonucleotides or engineered nucleases holds great potential for the treatment of Duchenne muscular dystrophy. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for Duchenne muscular dystrophy. To restore the dystrophin protein, 3 correction methods -- exon skipping, frameshifting, and exon knockin -- were performed in Duchenne muscular dystrophy patient-derived iPSCs, and exon knockin was found to be the most effective approach (20). The corrected iPSCs were differentiated toward skeletal muscle cells, and successful expression of full-length dystrophin protein was detected.
Multiple approaches have been developed for editing the mutation in dystrophic mdx4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation (04). Muscle-restricted Cas9 expression enables direct editing of the mutation, multiexon deletion, or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. These results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.
Genome editing using CRISPR/Cas9 system can induce permanent corrections of the DMD gene with restoration of dystrophin expression but efficient delivery to cardiac and skeletal muscles presents inherent challenges (01). One study has used an adenine base editor to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 in cardiomyocytes derived from human-induced pluripotent stem cells, restoring dystrophin expression (08). The results show the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with Duchenne muscular dystrophy.
All contributors' financial relationships have been reviewed and mitigated to ensure that this and every other article is free from commercial bias.
K K Jain MD†
Dr. Jain was a consultant in neurology and had no relevant financial relationships to disclose.See Profile
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