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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Therapy. Author manuscript; available in PMC 2010 June 10.
Published in final edited form as:
Therapy. 2010 May 1; 7(3): 287–290.
doi:  10.2217/thy.10.14
PMCID: PMC2883255
NIHMSID: NIHMS207284

Could gene therapy be the future for muscular dystrophy?

Muscular dystrophy (MD) represents a group of hereditary disorders characterized by muscle weakness and wasting. The degree of disability depends upon the type of MD, and severe forms can lead to loss of ambulation, respiratory problems and death. Some genes linked to MD have been known to encode structural proteins in the muscle membrane-associated complex [1,2]. For example, the most common types of MD, Duchenne and Becker MD, are caused by mutations in the dystrophin gene that lead to either absence (Duchenne) or reduction (Becker) of the protein product [3,4]. Although the genetic causes for many types of MD have been identified, there is currently no cure for MD. Patients typically rely on physical therapy and oral corticosteroids for symptom management [5].

Recent research progress has offered great hope for cell and gene therapies. Since many forms of MD are monogenic disorders, gene replacement is an attractive approach. A wide array of strategies have been developed to deliver dystrophin or other affected genes to muscles, many of which have been highly promising in preclinical studies. In particular, the first report of viral vector-mediated gene delivery for MD recently demonstrated successful transgene expression in human muscle for up to 3 months [6].

Recombinant adeno-associated virus (AAV) has emerged as the most promising gene delivery vector owing to its high tropism for skeletal muscle and low immunogenicity. AAV is a replication-defective parvovirus that can be produced relatively efficiently. Although most adults have been infected with AAV, no pathogenicity has been associated with this virus, making it an ideal vector for gene transfer.

Despite the small packaging capacity of the AAV vector (<5 kb), dystrophin, the largest gene in the human genome (2600 kb) [4], has been modified into ‘mini’ and ‘micro’ forms and packaged [7,8]. These smaller versions of dystrophin retain their binding domains with actin in the cytoskeleton and dystroglycan at the cell membrane, but lack most of the central rod domain. The shortened dystrophin is thought to preserve critical regions for signaling and structural support. It is unclear whether these small forms of dystrophin could provide complete rescue in humans, but preclinical studies in the mdx mouse model of Duchenne MD have shown that mini- and micro-dystrophins can reverse the dystrophic phenotype [8,9].

One challenge for clinical translation is to deliver the vector to multiple muscle groups and potentially the affected cardiac muscle. Several serotypes of AAV (i.e., AAV6, AAV8 and AAV9) have shown considerable preclinical success in targeting muscle after vascular delivery in the mdx mouse and dystrophic canine [1012]. Importantly, AAV9 appears to be particularly efficient in transducing cardiac tissue, which is of high interest considering the prevalence of cardiomyopathy in MD [13,14].

It seems likely that regional vascular delivery will be the first step toward systemic vector delivery. Isolating the circulation for as little as 10 min through balloon catheters or uniquely placed tourniquets on the extremity can target one to two critical muscle groups. This approach has recently been demonstrated to safely and efficiently transduce the limb muscle of the rhesus macaque, which has high anatomical similarity to humans [15]. Although AAV has not yet been administered systemically in humans, regional vascular delivery for muscle transduction is on the close horizon. Safety in regional vascular delivery will open the door for systemic approaches.

Another critical issue in gene therapy is evasion of the adaptive and innate immune systems. The primary concern is the role of the adaptive immune system in prohibiting delivery of the virus to the targeted cells. Since humans are a natural host for AAV, much of the population possess binding antibodies to AAV [16,17]. It is still unclear whether the majority of humans have a level of circulating neutralizing antibodies high enough to significantly affect viral gene delivery. However, by prescreening patients for neutralizing viral antibodies or using rare viral serotypes to deliver genes, this issue can potentially be circumvented.

A more unexpected observation has been the cytotoxic T-cell response to the AAV capsid peptides following vector administration. In a 2006 clinical trial for hemophilia, AAV expressing factor IX was delivered to the liver, but only resulted in transient gene expression [18]. The reduction in gene expression was attributed to T cells forming a cytotoxic response to AAV capsid peptides presented on the surface of transduced liver cells. This observation prompted discussions regarding the potential use of immunosuppressants in patients at least until after capsid peptides are fully cleared from transduced cells [16,19]. Interestingly, this damaging cytotoxic T-cell response has not been seen in all clinical trials where AAV has been administered. A recent clinical trial for limb-girdle MD type 2D successfully delivered the missing α-sarcoglycan protein to muscle and showed continued gene expression after 3 months [6]. Out of three subjects, only one displayed a minimal cytotoxic T-cell response to AAV capsid peptides, which did not preclude gene expression. Differences between these two trials include the AAV serotype, route of administration and the targeted tissue. Additional studies will be required to determine whether immunosuppression is needed to avoid a T-cell-mediated response.

In addition to the generation of AAV capsid immunogenesis, there may also be an immune response mounted to the AAV-expressed therapeutic protein. In the case of Duchenne dystrophy, the dystrophin peptides expressed by gene transfer could be seen as a foreign antigen. Scientists had anticipated that the revertant fibers found in many patients would be protective. These revertant fibers result from spontaneous second-site mutations that skip exons and restore the open reading frame, producing dystrophin. It has been the hope that dystrophin expression on revertant fibers would generate tolerance (i.e., recognition as ‘self ’ and not ‘foreign’). Instead, recent findings in a Duchenne MD gene therapy clinical trial have revealed that novel dystrophin epitopes on revertants are immunogenic rather than protective, threatening newly expressed microdystrophin. An advantage of translational therapy is that these immunogenic epitopes on revertant fibers can be assessed prior to gene transfer. Their presence favors alternate approaches, including gene delivery of utrophin, an endogenous homolog of dystrophin [20].

While AAV has been the most promising gene therapy vector for MD, other considerations influence its use depending on the target tissue and specific goals to be achieved. AAV vectors infect both dividing and nondividing cells and express the inserted transgene DNA as an episome [21]. However, the episomal transgene will eventually be diluted out in dividing cells, making AAV less desirable for rapidly dividing cell populations. Overall, it is advantageous that AAV does not integrate into the host DNA since integrating retroviral vectors can present a significant risk for cancerous transformation related to insertional mutagenesis [22,23]. Fortunately for MD gene therapy, integration does not appear to be a required goal because muscle fibers are terminally differentiated, nondividing cells. Concerns have been raised that in cases of Duchenne MD where there are prolific cycles of muscle fiber degeneration and regeneration, the AAV episome could be in jeopardy. Nevertheless, it is worth emphasizing that robust and stable AAV gene expression has been a reproducible finding in mdx mouse studies for well over a year [24], even in the face of pronounced muscle regeneration and degeneration. Should this issue become a concern for human gene therapy, it might be necessary to reconsider viral vectors that integrate into the genome, such as lentivirus. A lentiviral vector carrying microdystrophin has been demonstrated to infect muscle stem cells and muscle fibers after intramuscular injection in the mdx mouse [25]. The transduced muscle stem cells were able to differentiate into new muscle fibers expressing microdystrophin. Although lentivirus is less efficient than AAV in transducing muscle, a scenario could be envisioned where AAV is utilized to transduce existing muscle fibers while muscle stem cells are infected by lentivirus either in vivo or ex vivo.

The genetic cause of some forms of MD, such as facioscapulohumeral MD, is still unclear. One gene replacement strategy for such a muscle wasting disease is to develop approaches to increase muscle mass and strength. Disruption of the myostatin signaling pathway has been shown to significantly enlarge muscle mass [26]. Myostatin is a member of the TGF-β superfamily and is a negative regulator of muscle growth [27]. A wide variety of both pharmacological and gene therapy approaches have been developed to inhibit myostatin [2830]. We recently demonstrated that AAV delivery of a myostatin antagonist protein, follistatin, increases muscle mass and strength in the mdx mouse and in the cynomolgus macaque [24,31]. Significantly, AAV expressing follistatin proved to be safe and effective in the nonhuman primate, setting the stage for clinical translation for treating muscle diseases.

Adeno-associated virus-mediated gene therapy is presently at the forefront of promising treatments for MD. Certainly, the development of gene therapy for these diseases has not been without challenges. Translational scientists have used creativity and persistence to develop novel approaches, such as truncated dystrophin isoforms and optimized viral vectors for gene delivery. This is further exemplified in the efforts to achieve success in the human gene transfer trial for limb-girdle MD type 2D [6]. Collectively, these achievements provide the impetus to move forward, optimizing regional and systemic delivery approaches creating the potential to change the quality of life for patients with these devastating muscle disorders.

Acknowledgements

The authors would like to thank all members of the Kaspar and Mendell laboratories.

Biographies

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Footnotes

Financial & competing interests disclosure

The Kaspar and Mendell Laboratories are funded by the NIH, Muscular Dystrophy Association, the Myositis Association and Jesse’s Journey. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Contributor Information

Amanda M Haidet, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA and The Ohio State University, Columbus, OH, USA.

Jerry R Mendell, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA and The Ohio State University, Columbus, OH, USA.

Brian K Kaspar, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA and College of Medicine & Integrated and Biomedical Science Graduate Program, The Ohio State University, Columbus, OH, USA, Tel.: +1 614 722 5085, Fax: +1 614 355 5247, gro.snerdlihcediwnoitan@rapsaK.nairb.

Bibliography

1. Davies KE, Nowak KJ. Molecular mechanisms of muscular dystrophies: old and new players. Nat. Rev. Mol. Cell Biol. 2006;7(10):762–773. [PubMed]
2. Michele DE, Campbell KP. Dystrophin–glycoprotein complex: post-translational processing and dystroglycan function. J. Biol. Chem. 2003;278(18):15457–15460. [PubMed]
3. Hoffman EP, Brown RH, Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987;51(6):919–928. [PubMed]
4. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell. 1987;50(3):509–517. [PubMed]
5. Mendell JR, Moxley RT, Griggs RC, et al. Randomized, double-blind six-month trial of prednisone in Duchenne’s muscular dystrophy. N. Engl. J. Med. 1989;320(24):1592–1597. [PubMed]
6. Mendell JR, Rodino-Klapac LR, Rosales-Quintero X, et al. Limb-girdle muscular dystrophy type 2D gene therapy restores α-sarcoglycan and associated proteins. Ann. Neurol. 2009;66(3):290–297. [PubMed]
7. Phelps SF, Hauser MA, Cole NM, et al. Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice. Hum. Mol. Genet. 1995;4(8):1251–1258. [PubMed]
8. Harper SQ, Hauser MA, DelloRusso C, et al. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat. Med. 2002;8(3):253–261. [PubMed]
9. Wang B, Li J, Xiao X. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. Proc. Natl Acad. Sci. USA. 2000;97(25):13714–13719. [PubMed]
10. Rodino-Klapac LR, Janssen PM, Montgomery CL, et al. A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy. J. Transl. Med. 2007;5:45. [PMC free article] [PubMed]
11. Gregorevic P, Allen JM, Minami E, et al. rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice. Nat. Med. 2006;12(7):787–789. [PubMed]
12. Yue Y, Ghosh A, Long C, et al. A single intravenous injection of adeno-associated virus serotype-9 leads to whole body skeletal muscle transduction in dogs. Mol. Ther. 2008;16(12):1944–1952. [PMC free article] [PubMed]
13. Inagaki K, Fuess S, Storm TA, et al. Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol. Ther. 2006;14(1):45–53. [PMC free article] [PubMed]
14. Pacak CA, Mah CS, Thattaliyath BD, et al. Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo. Circ. Res. 2006;99(4):e3–e9. [PubMed]
15. Rodino-Klapac LR, Montgomery CL, Bremer WG, et al. Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery. Mol. Ther. 2009;18(1):109–117. [PMC free article] [PubMed]
16. Jiang H, Couto LB, Patarroyo-White S, et al. Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy. Blood. 2006;108(10):3321–3328. [PubMed]
17. Scallan CD, Jiang H, Liu T, et al. Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice. Blood. 2006;107(5):1810–1817. [PubMed]
18. Manno CS, Arruda VR, Pierce GF, et al. Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat. Med. 2006;12(3):342–347. [PubMed]
19. Wang Z, Kuhr CS, Allen JM, et al. Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression. Mol. Ther. 2007;15(6):1160–1166. [PubMed]
20. Tinsley J, Deconinck N, Fisher R, et al. Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat. Med. 1998;4(12):1441–1444. [PubMed]
21. Duan D, Sharma P, Yang J, et al. Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue. J. Virol. 1998;72(11):8568–8577. [PMC free article] [PubMed]
22. Schnepp BC, Clark KR, Klemanski DL, Pacak CA, Johnson PR. Genetic fate of recombinant adeno-associated virus vector genomes in muscle. J. Virol. 2003;77(6):3495–3504. [PMC free article] [PubMed]
23. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302(5644):415–419. [PubMed]
24. Haidet AM, Rizo L, Handy C, et al. Long-term enhancement of skeletal muscle mass and strength by single gene administration of myostatin inhibitors. Proc. Natl Acad. Sci. USA. 2008;105(11):4318–4322. [PubMed]
25. Kimura E, Li S, Gregorevic P, Fall BM, Chamberlain JS. Dystrophin delivery to muscles of mdx mice using lentiviral vectors leads to myogenic progenitor targeting and stable gene expression. Mol. Ther. 2009;18(1):206–213. [PubMed]
26. Lee SJ, McPherron AC. Regulation of myostatin activity and muscle growth. Proc. Natl Acad. Sci. USA. 2001;98(16):9306–9311. [PubMed]
27. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member. Nature. 1997;387(6628):83–90. [PubMed]
28. Wagner KR, McPherron AC, Winik N, Lee SJ. Loss of myostatin attenuates severity of muscular dystrophy in mdx mice. Ann. Neurol. 2002;52(6):832–836. [PubMed]
29. Lee SJ, Reed LA, Davies MV, et al. Regulation of muscle growth by multiple ligands signaling through activin type II receptors. Proc. Natl Acad. Sci. USA. 2005;102(50):18117–18122. [PubMed]
30. Bogdanovich S, Krag TO, Barton ER, et al. Functional improvement of dystrophic muscle by myostatin blockade. Nature. 2002;420(6914):418–421. [PubMed]
31. Kota J, Handy CR, Haidet AM, et al. Follistatin gene delivery enhances muscle growth and strength in nonhuman primates. Sci. Transl. Med. 2009;1(6) 6ra15. [PMC free article] [PubMed]