Authors: Robynne Braun, MD, PhD Zejing Wang, MD, PhD Gene Therapy David L. Mack, PhD Martin K. Childers, DO, PhD

Affiliations: From the Department of Rehabilitation SUPPLEMENT Medicine and Institute for Stem Cell and Regenerative Medicine, University of Washington (RB, DLM, MKC); and Fred Hutchinson Cancer Research Center (ZW), Seattle, Washington. Gene Therapy for Inherited

Correspondence: Muscle Diseases All correspondence and requests for Where Genetics Meets Rehabilitation Medicine reprints should be addressed to: Martin K. Childers, DO, PhD, University of Washington, Campus Box 358056, S184, South Research Building, ABSTRACT 950 Republican St, Seattle, WA 98109. Braun R, Wang Z, Mack DL, Childers MK: Gene therapy for inherited muscle diseases: where genetics meets rehabilitation medicine. Am J Phys Med Rehabil Disclosures: 2014;93(Suppl):S97YS107. Dr. Childers is a paid member of the Scientific Advisory Board for Audentes The development of clinical vectors to correct genetic mutations that cause Therapeutics and holds stock options inherited myopathies and related disorders of skeletal muscle is advancing at an in the company. impressive rate. Adeno-associated virus vectors are attractive for clinical use because Financial disclosure statements have been obtained, and no conflicts of (1) adeno-associated viruses do not cause human disease and (2) these vectors are interest have been reported by the able to persist for years. New vectors are now becoming available as gene therapy authors or by any individuals in control of the content of this article. delivery tools, and recent preclinical experiments have demonstrated the feasibility, safety, and efficacy of gene therapy with adeno-associated virus for long-term cor- rection of muscle pathology and weakness in myotubularin-deficient canine and 0894-9115/14/9311(Suppl)-S97 murine disease models. In this review, recent advances in the application of gene American Journal of Physical Medicine & Rehabilitation therapies to treat inherited muscle disorders are presented, including Duchenne Copyright * 2014 by Lippincott muscular dystrophy and x-linked myotubular myopathy. Potential areas for thera- Williams & Wilkins peutic synergies between rehabilitation medicine and genetics are also discussed.

DOI: 10.1097/PHM.0000000000000138 Key Words: Myopathy, Muscle Disease, Gene Therapy, Canine, Rehabilitation

WHAT IS GENE THERAPY, AND WHEN WILL IT BE USED IN CLINICAL PRACTICE? Gene therapyVthe process of introducing foreign genomic materials into host cells to elicit therapeutic benefitVbecame available for clinical practice on November 2, 2012, when Glybera (alipogene tiparvovec) became the first gene therapy in the Western world to receive market approval for patients with lipo- protein lipase deficiency, a rare genetic disease previously without effective treatment.1,2 Since 1989, gene therapy clinical trials have been undertaken in 31 countries, with more than 1,800 human trials ongoing, completed, or ap- proved worldwide.3 Many of these trials target rare Borphan diseases.[ The Orphan Drug Act of 1983 defined an orphan product as a drug intended to treat a condi- tion affecting fewer than 200,000 persons in the United States or a drug that would not be expected to be profitable within 7 yrs after Food and Drug Administration approval.4 Orphan disease designation allows a sponsor to apply for market pro- tection for the product after approval. A total of 2,700 orphan drug designations and more than 400 approvals associated with these designations have been ap- proved as of 2012.4 Whereas most gene therapy trials have addressed cancer or www.ajpmr.com Gene Therapy for Inherited Muscle Diseases S97

Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. cardiovascular disease, a significant number of and Leber congenital amaurosis. In the first example, gene therapy trials have targeted rare monogenic X-linked severe combined immunodeficiency results (single gene) diseases (Table 1).3 These ground- in recurrent and often fatal infections caused by ge- breaking therapies involve the insertion of DNA netic deficiency in cellular and humoral immunity. sequences that encode functional, therapeutic Long-term follow-up of nine X-linked severe com- genes into patients to replace mutated dysfunc- bined immunodeficiency boys treated in a French tional genes causing disease. In the case of Glybera, adenovirus-mediated gene therapy trial reported a DNA sequence encodes a therapeutic lipoprotein eight survivors after nearly 10 yrs.5 In the second lipase gene packaged within a vector, in this case an example, Leber congenital amaurosis leads to con- adeno-associated virus (AAV). The recombinant genital blindness caused by mutations in a retinal AAV is injected into a patient harboring a mutant gene that causes progressive loss of vision; young disease-causing lipoprotein lipase gene. The injected patients become completely blind by adulthood. AAV is capable of shuttling the replacement DNA Three independent gene therapy trials for Leber sequence from inside the vector to the cells of the congenital amaurosis patients have been initiated, targeted tissue. Once inside, the patient’s own cellu- and follow-up results indicate improvement in vision lar machinery works to transcribe the new replace- for up to 2 yrs with no serious adverse events.6Y8 ment DNA sequence to produce the therapeutic These and other gene therapy successes have been protein to treat the patient’s disease (Fig. 1). Besides offset by serious, and rarely fatal, adverse events that the success of Glybera, other notable examples of lead to early clinical trial Bholds.[ The most famous gene therapy successes are highlighted in clinical case was the death of Jesse Gelsinger in 2000, who trials undertaken in rare genetic childhood diseases was the 19th patient enrolled in a gene therapy trial such as X-linked severe combined immunodeficiency of a deficiency of ornithine transcarbamylase.9 De- spite these early setbacks, the field of gene therapy continues to move forward at an extraordinary pace. TABLE 1 Monogenic disorders reported in gene therapy clinical trials Adrenoleukodystrophy WILL GENE REPLACEMENT THERAPY >-1 antitrypsin deficiency BE USEFUL FOR INHERITED MUSCLE Becker muscular dystrophy DISEASES LIKE MUSCULAR A-thalassaemia Canavan disease DYSTROPHY? Chronic granulomatous disease Most gene therapy clinical trials have targeted Cystic fibrosis genes involved in cancer,3 with fewer trials initiated Duchenne muscular dystrophy Fabry disease in monogenic diseases, such as Duchenne muscular Familial adenomatous polyposis dystrophy (DMD). Muscular dystrophies make up Familial hypercholesterolemia only a small percentage of the monogenic diseases Fanconi anemia Galactosialidosis (Table 1), notably Becker, Duchenne, and limb Gaucher disease girdle muscular dystrophy. Pompe disease, among Gyrate atrophy others listed in Table 1, is not considered a mus- Hemophilia A and B Hurler syndrome cular dystrophy even though skeletal muscles of Hunter syndrome affected patients grow progressively weaker because Huntington chorea of accumulation of abnormal proteins within the Junctional epidermolysis bullosa muscle’s contractile tissue. Therapeutic approaches Late infantile neuronal ceroid lipofuscinosis Leukocyte adherence deficiency at replacing defective genes in monogenic diseases Limb girdle muscular dystrophy of muscle, like DMD, include the use of recombi- a Lipoprotein lipase deficiency nant viral vectors engineered to target specific tis- Mucopolysaccharidosis type VII Ornithine transcarbamylase deficiency sues. In the 1960s, the discovery of naturally occurring Pompe disease AAV isolates led to the clinical application of recom- Purine nucleoside phosphorylase deficiency binant AAV vectors with early successes in clinical Recessive dystrophic epidermolysis bullosa 10 Sickle cell disease trials. AAV vectors are attractive for clinical use be- 2 Severe combined immunodeficiency cause AAVs are not associated with human disease, Tay Sachs disease the virus persists in the infected host for years, and a Wiskott-Aldrich syndrome large Btoolkit[ of AAV vectors is available as clinical aGlybera approved in Europe. gene therapy delivery tools.11 In addition to muscle diseases, AAV vectors have been used in clinical gene

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Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. FIGURE 1 AAV-mediated gene therapy. A, Genome structure of wild-type AAV, a linear single-stranded DNA. ITR, inverted terminal repeats, act in cis and important for AAV replication, genome packaging, and transcription; Rep, rep gene produces four Rep proteins that act in trans in all phases of AAV life cycle; Cap, capsid gene, produces three viral capsid proteins; Genome structure of recombinant AAV vector, AAV rep and cap genes are replaced by transgene of interest under the control of a promoter of interest. B, AAV vector production, delivery, and assessments of clinical outcome. Cotransfection of AAV pro- duction cells such as HEK293 cells with three plasmids: (1) an AAV ITR-containing plasmid carrying the gene of interest, (2) a plasmid carrying the AAV rep and cap genes, and (3) a plasmid providing the helper genes isolated from adenovirus (Ad). Recombinant AAV vectors will be administrated via in- tramuscular or intravascular or other routes into subjects, up-taken by target cells, and transgene will be expressed and clinical outcomes will be measured as a result of functions of the transgene product. AAV, adeno-associated virus. therapy trials targeted to the liver for the treatment of immune responses to either self or nonself dystro- hemophilia B, to the lung for treatment of cystic fi- phin epitopes. In the study, AAV vectors carrying a brosis, to the brain for treatment of Parkinson, Batten, truncated but functional dystrophin gene under the and Canavan disease, to joints for treatment of rheu- control of a CMV promoter were injected into the matoid arthritis and to the eye for treatment of Leber bicep of six DMD patients. None of the patients congenital amaurosis.10 displayed transgene expression, with four of six In recent years, AAV-mediated gene replace- patients showing detectable T-cell responses against ment has rapidly moved from preclinical studies to the transgene product. Two of the patients had clinical trials because of encouraging results from dystrophin-specific T-cell responses before the treat- animal models. Major challenges surrounding this ment. Taken together, these trials are informative strategy include (1) effective delivery methods to to emphasize the importance of prescreening pa- target muscles throughout the body, including di- tients for preexisting immune responses to both aphragm and cardiac muscle, and (2) host immune AAV capsid proteins and transgene product and also responses to the therapeutic vector.12 The latter to develop strategies to circumvent immune re- challenge was encountered in a double-blinded, sponses, such as using a transient course of im- randomized, controlled phase I trial of limb-girdle munosuppression, shown to be effective in a dog muscular dystrophy,13,14 AAV1-MCK.human-alpha- model of DMD.16,17 Sarcoglycan (SGCA) was injected locally into the extensor digitorum brevis muscle in three patients, and sustained transgene expression was observed in CAN GENES BE REPAIRED? two of three patients after 6 mos. Humoral and Although AAV-mediated gene therapy is regarded cellular immune responses to the AAV capsid pro- as a gene replacement strategy, another exciting de- teins were detected in the patient who failed to show velopment, termed exon skipping, focuses on gene expression. Another phase I trial on DMD from repair. Approximately 70% of all DMD mutations Mendell et al.15 also raised the potential of cellular arecausedbysingleormultipleexondeletions.Such www.ajpmr.com Gene Therapy for Inherited Muscle Diseases S99

Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. deletions disrupt the open reading frame of dystro- È13% of DMD boys with premature termination phin and hence result in a premature truncated codon mutations. Aminoglycoside antibiotics ini- protein. In most cases, selective removal of specific tially demonstrated the capacity to induce ribo- exons can restore the reading frame and produce a somal read-through of premature stop codon27 but partially functional dystrophin protein for clinical not efficient and too toxic to be used for long-term benefit.18Y21 Antisense oligonucleotides (AOs) target- treatment. Ataluren (premature termination codon ing pre-mRNA to modulate splicing have been used 124) was identified to be effective in this matter in to induce exon skipping, and the first human phase mdx mice and subsequently tested in human DMD I trials22,23 focused on skipping exon 51, which, if trials.28Y30 The drug was well tolerated in general successful, would be able to correct È13% of DMD and the dystrophin protein was detected with treated patients with specific deletions within exons 42 to 50. patients showing improvement in the 6-min walking A therapeutic exon skipping AO, 2-O-methyl AO, distance test. However, correlation between the level termed PRO051,23 and AVI-4658, a morpholino con- of dystrophin expression and the 6-min walking dis- jugated AO22 that targets an internal sequence of tance test remains unclear. More trials are currently exon 51, was injected intramuscularly into DMD pa- underway and the same treatment strategy could be tients. AOs used in these trials were well tolerated potentially applied to other muscle diseases, includ- and demonstrated successful exon skipping, and all ing spinal muscular atrophy. Progress in develop- patients demonstrated dystrophin expression to levels ing other approaches targeting repair of the muscle between 3% and 12% and between 22% and 35% membrane due to lack of dystrophin is also under by PRO051 and AVI-4658, respectively. After these development, for example, increasing the level of initial clinical trials of intramuscular administration, the compensatory protein utrophin31,32 and upregu- phase I/II trials using systemic delivery of the two lation of glycosylation of a-dystroglycan to improve drugs were tested for dose, safety, and efficacy.24,25 extracellular matrix attachment.33 Dose-dependent restoration of dystrophin expression was observed after weekly administration of the ex- CAN DEFECTIVE GENES BE perimental compounds through abdominal subcu- COMPLETELY REPLACED? taneous injections in two cohorts. Although no SURPRISING LESSONS LEARNED significant differences were detected in patient’s FROM DOGS walking ability after a 12-wk-long treatment,24 In 2008, a case report of a 5-mo-old Labrador patients treated with weekly phosphorodiamidate Retriever was published in a Canadian veterinary morpholino oligomer-AO (AVI-4658)25 for 48 wks medical journal.34 The dog presented with weak- demonstrated improvement in stabilization of the ness, muscle atrophy, and histopathologic changes muscle and in the 6-min walking distance test.26 A in skeletal muscle consistent with a centronuclear larger confirmatory phase III trial is in the planning myopathy. Because male littermates were similarly for 2014. Clinical trials for skipping other exons are affected, the authors postulated that the disease was also underway or in planning.18 These Bpersonalized X-linked, giving rise to the possibility that the dis- gene therapy medicines[ generate hope for DMD order could be analogous to X-linked myotubular patients, with the caveat that effective therapy myopathy (XLMTM). It was through the tireless and will need to restore dystrophin expression in both extraordinary efforts of Alison Rockett Frase that skeletal and cardiac muscle and that treatment will this study’s research group was able to acquire a need to be persistent. The future challenge for first-degree relative, a dog named BNibs,[ a Labra- clinical development with AOs is that current forms dor retriever coming from a line of dogs with a have a short half-life, and more than 85% of AOs history suspicious for XLMTM. It was later discov- are cleared from the circulation within 24 hrs. This ered that Nibs harbored a canine MTM1 mutation,34 short half-life requires weekly injections to main- the same gene known to cause myotubular myop- tain a therapeutic level. Long-term outcomes of athy in patients (reviewed in BThe Miracle of Nibs[35). these AOs are also unknown. Intramuscular ad- Mrs Frase recalls the story of her odyssey locating ministration of these agents may be limited in and retrieving the founding carrier dog from a farmer treating skeletal muscle, and treating the cardiac in Canada. The following excerpt describes how muscle will need to be addressed for this strategy a determined mother of an affected child can help to be an effective treatment for DMD-associated shape the future of research for a disorder like myo- cardiomyopathy. tubular myopathy: BIn the fall of 2008, a female NonYsense stop codon read-through is another Labrador Retriever was discovered to carry the same method of gene editing and potentially benefits gene as I do for my son’s muscle disorder called

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Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. myotubular myopathy (MTM). To date, this was the canine breeding colony to study the effects of the first MTM large animal ever discovered by researchers disease in dogs. Initial data revealed that affected anywhere in the worldI. Nibs was a beautiful Lab- males display a phenotype directly analogous to rador Retriever that was instrumental in giving us human XLMTM: progressive and severe muscular puppies that carry the myotubular myopathy (MTM1) weakness45 leading to the inability to walk, weak gene that affects our son Joshua. I am so grateful to ventilatory muscles leading to respiratory impair- Nibs for the initial 12 puppy litterI. Our second ment,46 and early death. Based upon the early ex- litter of MTM pups has been bornI.Knowledge perience with gene replacement in the Mtm1 gained from these animals may one day lead to knockout mouse,44 a similar gene replacement treatments not only for MTM, but other neuromus- strategy was initiated in the XLMTM dog in 2011. cular diseases. It will be a miracle for our son Josh and For eventual gene therapy of XLMTM patients, thousands of children like him if our goals are the goal was to use a predictive large animal model achieved.[ Joshua Frase died on December 24, 2010, (the XLMTM dog) to refine the delivery system, to less than 2 yrs after this was written. His legacy, the assess critical safety parameters such as the potential Joshua Frase Foundation, set into motion research host immune response to vector and transgene, and that will hopefully develop the first effective treat- to optimize efficacy measurements. In collaboration ment for this devastating disorder. with the French nonprofit institute Ge´ne´thon,47 XLMTM is an orphan disease, affecting 1 of 50,000 cohorts of Mtm1 knockout mice were first tested for live male births worldwide,36 with only supportive, response to systemic Mtm1 gene replacement via tail palliative care available for patients.37 This inherited vein injection. Results indicated that a single sys- muscle disease results from loss-of-function muta- temic treatment with AAV-Mtm1 sufficed for long- tions in the Myotubularin 1 gene (MTM1)38 that en- term (at least 1 yr) survival and essentially complete codes the founder of a family of 3-phosphoinositide amelioration of symptoms of mice with myotubularin- phosphatases acting on the second messengers phos- deficient muscles.48 Using the same AAV vectors phatidylinositol 3-monophosphate and phosphatidy- produced by Ge´ne´thon scientists and tested in mice, linositol 3,5-bisphosphate.39,40 Although myotubularin the authors’ collaborative research group confirmed is expressed ubiquitously, loss of this enzyme pro- that local gene replacement therapy, delivered in- foundly affects skeletal muscles causing hypotrophic tramuscularly into the hind limb of young XLMTM myofibers and structural abnormalities, with associated dogs, reversed pathologic changes in myotubularin- weakness.41 No effective therapy exists for XLMTM. deficient skeletal muscles. Remarkably, the treated Management of the disease generally consists of me- muscles also showed nearly normal strength at 6 wks chanical ventilation, gastrostomy feeding tubes, antibi- after injection, compared with very weak muscles otics (for respiratory infections), orthotics to prevent (only 20% of normal strength) in saline-injected skeletal limb contractures, and surgical treatment to contralateral limbs. In subsequent experiments, in- alleviate severe spinal deformities. Despite aggressive travascular administration of AAV8-MTM1 at the medical care, the average life expectancy is only about same dose used in mice was well tolerated in dogs, 2 yrs, and most who survive beyond this age require rescued the skeletal muscle pathology and respi- mechanical ventilation. ratory function, and prolonged life for over 1 yr Animal models of the disease currently exist (Fig. 2). Together, these initial studies demon- in zebrafish, mice, and notably, in dogs.41Y43 The strated the feasibility, safety, and efficacy of gene murine phenotype resembles human XLMTM, with therapy with AAV for long-term correction of muscle similar pathology and early mortality. In a mouse pathology and weakness observed in myotubularin- knockout model of XLMTM, local delivery of the deficient mouse and dog models and paved the way wild-type myotubularin gene (MTM1) via an AAV for clinical trials aimed at correcting this devastating vector reversed characteristic pathologic features disease in patients. and rescued the function of injected limb muscles.44 Buj-Bello et al.44 were the first to report gene therapy success in the Mtm1 knockout mouse in REHABILITATION AND GENE THERAPY 2008. That same year, the Joshua Frase Foundation As highlighted in the discussion above, re- provided this research group access to a female search on the role of gene therapy in the treatment Labrador retriever harboring an MTM1 gene mu- of muscular disorders has undergone impressive tation that was later proven by Beggs et al.43 to growth over the course of the past decade, with a cause a canine version of the human disease. From considerable portion of this research being focused this single founding female, the group established a on inherited myopathies such as DMD and XLMTM. www.ajpmr.com Gene Therapy for Inherited Muscle Diseases S101

Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. FIGURE 2 The first XLMTM dog treated with intravascular gene replacement therapy (AAV8-MTM1) given at 9 wks of age. Untreated XLMTM dogs do not survive beyond 6 mos of age and succumb to severe muscle weakness and respiratory insufficiency. Shown here is an affected dog 1 yr after infusion with AAV8- MTM1. Systemic treatment resulted in amelioration of the severe muscle pathology and weakness observed in untreated dogs. AAV, adeno-associated virus; MTM, myotubular myopathy.

Gene replacement therapy in such disorders, how- mechanisms that are induced and/or enhanced by ever, represents only a fraction of the spectrum of rehabilitation therapies continues to grow.55 muscular disorders addressed in rehabilitation med- Increasingly demonstrated in rehabilitation icine that might benefit from application of a gene medicine literature is research on how mechanical therapy approach. Equally significant are inquiries stimuli such as stretch or compression affect in- into the genetic regulation of age-related muscle tracellular signaling and gene expression (i.e., atrophy, muscle breakdown in hypercatabolic states Bmechanotransduction[).56Y58 Studies on up- (as seen with malignancy or infection), as well as regulation of growth factors in response to tensile recovery from muscle injuries associated with me- and/or compressive loading have been undertaken chanical trauma, ischemia, or denervation. in tendon (showing up-regulation of insulin-like Investigation of potential gene therapy strate- growth factor-1),59,60 as well as muscle (showing gies to enhance muscle regeneration and/or coun- up-regulation of mechano growth factor).57,61 The teract the effects of muscle atrophy and degeneration mechanisms underlying mechanotransduction are is underway to elucidate (and experimentally ma- still being elucidated and seem to involve not only nipulate) molecular events at the transcriptional, chemical signaling pathways but also direct physical translational, and posttranslational stages. Examples coupling via cytoskeletal elements between mem- include research on the bioavailability of growth brane structures and nuclear structures. Less un- factors such as transforming growth factor 1Y3,49,50 derstood are the effects of mechanical stimulus on insulin-like growth factor-1,51,52 and growth- gene therapy delivery and efficacy. Previous studies differentiation factors such as GDF8/myostatin53,54 have shown that cyclical mechanical stretch en- that play a role in the proliferation and differentiation hances plasma-mediated gene transfer to skeletal of muscle stem cells (reviewed in this supplement by muscle cells,62 but to the authors’ knowledge, the Jasuja and LeBrasseur). The ability to therapeutically effects of mechanical stretch on AAV gene transduc- regulate the expression of these factors has the po- tion have not been reported in skeletal muscle. This tential to prevent muscle loss and enhance muscle study’s research group is investigating the effects of recovery and thus is aligned with key concerns in the passive stretch in cultured skeletal muscle on the area of rehabilitation medicine. To date, however, uptake and expression of various AAV serotypes, a much of the research in these areas has remained process thought to occur via clathrin-dependent within the molecular and cellular biology domains, endocytosis.63 A series of follow-up studies will with limited dissemination to the rehabilitation re- examine the effect of exercise on gene therapy up- search community. Translational research on the take/efficacy in vivo. Rehabilitation research is rehabilitation applications of these therapies remains needed using similar paradigms along with mea- in the nascent stages, although advocacy for advance- sures of motor recovery in human subjects under- ment of research to define the cellular and molecular going gene transfer.

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Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. TABLE 2 Transcription factors in skeletal muscle potentially responsive to rehabilitation strategies

Factor Description Myo D Transcriptional regulatory protein, induced during muscle regeneration65 Myogenic factor 5 (Myf5) Transcriptional regulatory protein induced during muscle regeneration66Y68 Mechano growth factor (MGF) A splice variant of insulin-like growth factor-1 (IGF-1) that leads to muscle hypertrophy via activation of satellite cells69 Myogenic regulatory factor 4 (Mrf4) Myogenic regulatory factor induced during muscle regeneration70Y72 Myogenin Myogenic regulatory factor induced during muscle regeneration73Y75 Transforming growth factor beta (TGF-beta) Considered a key signaling molecule in cardiac hypertrophy and failure, among others76Y78 IGF-1 Implicated in the control of skeletal muscle growth, differentiation, survival, and regeneration, and considered a promising therapeutic agent in staving off the advance of muscle weakness79Y83

In other emerging areas of research on gene to determine whether voluntary exercise enhances therapy for treatment of muscular disorders, the the effects of gene therapy delivered via Schwann interplay of genetic factors and cellular factors is cells overexpressing FGF-2. These authors found central (e.g., up-regulating expression of growth that rats treated with FGF-2 gene therapy plus ex- factors and the availability of responsive precursor ercise showed enhanced regeneration of myelinated cells that retain the ability to differentiate into axons in comparison with sedentary animals, and functionally mature excitable tissues such as nerve furthermore, that mRNA levels of regeneration as- and muscle). Indeed, in some cases, the means for sociated proteins in lumbar spinal cord were sig- delivery of gene therapy relies directly upon trans- nificantly higher in exercised vs. sedentary animals. plant of cells that have been transfected with the Gene therapies and cellular therapies are each im- genetic elements of interest. The importance of this portant tools in the emerging era of biologic ther- synergistic interplay is underscored in a 2008 study apeutics. Although cellular and gene therapies each by Haastert et al.64 in rats with sciatic nerve injury have independent potential as treatments to combat

FIGURE 3 Areas for future research at the interface of rehabilitation medicine and genetic medicine. www.ajpmr.com Gene Therapy for Inherited Muscle Diseases S103

Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. muscular dystrophy, congenital myopathies, and therapeutic exercise in the rehabilitation of in- muscle damage, the potential for synergistic ther- herited muscle disorders exists and, if so, whether a apies is also an important aspect of future treatment minimal effective exercise Bdose[ (vs. toxic Bdose[) strategies. Moreover, as demonstrated by the work can be defined. Similarly for gene therapy, dose- of Hasstert et al. above, further potentiation of response curves need to be established and exam- cellular and gene therapies may be effected by the ined with an eye toward minimizing both potential strategic use of exercise interventions, thus posi- costs and adverse effects associated with the delivery tioning rehabilitation researchers and clinicians to of large inocula. An intriguing possibility, built on offer significant contributions in the development the principles of mechanotransduction discussed of these important therapeutic advances (Table 2). earlier, is that by targeting gene therapy delivery to elements overexpressed in muscle after mechani- AREAS FOR FUTURE RESEARCH: cal loading, an activity-dependent up-regulation of WHERE REHABILITATION therapeutic gene products may be facilitated. Re- MEETS GENETICS habilitation researchers and clinicians thus stand to As exploration continues on how rehabilitation offer important insights on how gene therapies medicine and genetic medicine complement each may be potentiated by the strategic manipulation other, one can begin to define potential therapeutic of exercise timing and intensity. This could have synergies by combining rehabilitation and genetic implications, for example, in myopathic patients approaches. Areas for further research that emerge weaning from chronic ventilator support, where at the intersection of these two clinical/research gene replacement therapy might be coupled with domains are presented in Figures 3 and 4. In this a pulmonary rehabilitation/exercise program. On- review, advances in the application of gene thera- going dialogue about the interplay of biologic and pies to treat inherited muscle disorders such as behavioral interventions will be essential in moving DMD and XLMTM have already been discussed. gene therapy for muscle disorders forward in the Specific inquires that should be addressed in the coming decade and will constitute a central focus future include whether a dose-response effect for in the evolving field of regenerative rehabilitation.

FIGURE 4 Conceptual framework for areas of overlap between rehabilitation and genetic medicine. MGF, mechano growth factor; TGF, transforming growth factor; IGF, insulin-like growth factor.

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