Removing the Immune Response From Muscular Dystrophy Research
Authors: Jeffrey S Chamberlain
Journal: Molecular Therapy (2013)

Immune responses against exogenous cells, vectors, and proteins have emerged as a significant barrier limiting development of therapies for inherited disorders. This is especially true when testing human cells and genes in animal models of disease. In this issue of Molecular Therapy, Vallese et al.1Vallese D Negroni E Duguez S Ferry A Trollet C Aamiri A et al.The Rag2−Il2rb−Dmd− mouse: a novel dystrophic and immunodeficient model to assess innovating therapeutic strategies for muscular dystrophies.Mol Ther. 2013; 21: 1950-1957Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar describe an improved immunodeficient mouse model for Duchenne muscular dystrophy (DMD) that should facilitate the development of therapies. The authors show that these mice enable extensive testing of human stem cells and methods of delivery without immune-mediated rejection of donor cells. The utility of this model is further enhanced by use of a specific mutant allele that eliminates background dystrophin expression in muscle tissues, providing a sensitive platform for monitoring cell engraftment and exogenous gene expression. As such, the model could accelerate development of interventions based on both stem cell transplantation and genetic manipulation. The study focused on DMD, a relatively common human genetic disorder resulting from mutations in the dystrophin gene. DMD is characterized by progressive muscle wasting leading to skeletal muscle loss followed by cardiac and respiratory failure.2Emery AE Muntoni F Duchenne Muscular Dystrophy. Oxford University Press, Oxford, UK2003Google Scholar Numerous therapeutic approaches have been tested, and many have shown early promise in mdx mouse models of the disorder (official nomenclature, Dmdmdx). These include cell transplantation using a variety of different types of stem cells, gene and protein replacement therapies, antisense oligonucleotides to skip mutant exons during pre–messenger RNA splicing, small molecules to bypass missense mutations, and various drugs to enhance muscle mass or reduce inflammation.3Goyenvalle A Seto JT Davies KE Chamberlain J Therapeutic approaches to muscular dystrophy.Hum Molec Genet. 2011; 20: R69-R78Crossref PubMed Scopus (85) Google Scholar Of these approaches, direct gene therapy using adeno-associated viral vectors to replace dystrophin production has shown the most dramatic and widespread effects in animal models.4Konieczny P Swiderski K Chamberlain JS Gene and cell-mediated therapies for muscular dystrophy.Muscle Nerve. 2013; 47: 649-663Crossref PubMed Scopus (63) Google Scholar However, gene therapy is particularly effective before extensive loss of muscle mass, whereas stem cell therapies have potential for both replacing the mutant dystrophin gene and generating new skeletal muscle cells. Developing stem cell therapies presents challenges beyond delivering a new gene or modifying the expression of a mutant allele. Myogenic progenitors from different species do not always behave in similar ways, requiring different methods for their identification, isolation, expansion, and transplantation.5Peault B Rudnicki M Torrente Y Cossu G Tremblay JP Partridge T et al.Stem and progenitor cells in skeletal muscle development, maintenance, and therapy.Mol Ther. 2007; 15: 867-877Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar For example, while the major skeletal muscle stem cell—the satellite cell—can be isolated and propagated in vitro from muscle biopsy samples of various species, human and mouse satellite cells display different cell surface receptors, growth properties, and engraftment potential.6Boldrin L Muntoni F Morgan JE Are human and mouse satellite cells really the same?.J Histochem Cytochem. 2010; 58: 941-955Crossref PubMed Scopus (99) Google Scholar Therefore, unique methods for purification, expansion, characterization, and transplantation are required for myogenic stem cells from each species. A more central concern is the ability of a human cell to engraft into mouse muscle, because such xenografts invariably elicit immune responses that lead to donor cell rejection. Most approaches to developing DMD stem cell therapies have relied on working with mouse cells and testing transplants in mdx mice. Promising methods have then typically moved to the canine DMD model. Several successful studies have moved forward into human clinical trials following identification and characterization of appropriate cell types from human muscle biopsy samples.5Peault B Rudnicki M Torrente Y Cossu G Tremblay JP Partridge T et al.Stem and progenitor cells in skeletal muscle development, maintenance, and therapy.Mol Ther. 2007; 15: 867-877Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar,7Gussoni E Blau HM Kunkel LM The fate of individual myoblasts after transplantation into muscles of DMD patients.Nat Med. 1997; 3: 970-977Crossref PubMed Scopus (272) Google Scholar Unfortunately, it is not clear whether the human cells being studied are truly homologous to the murine or canine cells, or whether they are simply similar. Results using murine cells in animal models may not predict results in patients, and it becomes difficult to ascertain the reasons for clinical results that are disappointing compared with mouse studies. One approach to avoiding many of these differences is to focus animal studies on the testing of human stem cells. Testing human cells in mice requires continuous immune suppression. One approach is to use immunodeficient mice, but such mice need to be on a dystrophic background. Several studies have crossed various immune-deficient strains onto one or more of the available mouse DMD models. These studies have relied primarily on mice with dystrophin gene mutations combined with nude mice that lack T cells, or severe combined immunodeficient (scid) and Rag1− or Rag2− mice that lack both T and B cells (Figure 1). However, each of these models expresses natural killer (NK) cells, which can also lead to destruction of xenografts. The value of the new model described by Vallese et al. results primarily from an extensive breeding scheme to generate dystrophin-deficient mice that lack B, T, and NK cells.1Vallese D Negroni E Duguez S Ferry A Trollet C Aamiri A et al.The Rag2−Il2rb−Dmd− mouse: a novel dystrophic and immunodeficient model to assess innovating therapeutic strategies for muscular dystrophies.Mol Ther. 2013; 21: 1950-1957Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar This was accomplished by backcrossing mutations in the genes encoding the β-subunit of the interleukin-2 receptor (Il2rb), which prevents NK cell maturation, and the Rag2 gene (no T or B cells) onto the Dmd(mdxβgeo) mouse, which produces no detectable dystrophin in striated muscles.8Wertz K Fuchtbauer EM Dmd(mdx-beta geo): a new allele for the mouse dystrophin gene.Dev Dyn. 1998; 212: 229-241Crossref PubMed Scopus (51) Google Scholar The result is an mdx mouse strain (Rag2−Il2rb−Dmd−) that is almost fully immunodeficient and thus able to serve as a robust host for human cell transplantation. The choice of mdx strain was important for future research studies. Numerous DMD mouse models (mdx) have been generated, and, although all are genetic phenocopies of the human disorder, each has shown limitations for various types of research.9Ng R Banks GB Hall JK Muir LA Ramos JN Wicki J et al.Animal models of muscular dystrophy.Prog Mol Biol Transl Sci. 2012; 105: 83-111Crossref PubMed Scopus (29) Google Scholar Virtually all strains of mdx mice display various levels of dystrophin expression in a small percentage of myofibers.10Danko I Chapman V Wolff JA The frequency of revertants in mdx mouse genetic models for Duchenne muscular dystrophy.Pediatr Res. 1992; 32: 128-131Crossref PubMed Scopus (123) Google Scholar The mechanism generating these “revertant” myofibers is unknown, but it probably arises from a stable, epigenetic event in myogenic stem cells such that the number of revertant myofibers increases with advancing age.11Yokota T Lu QL Morgan JE Davies KE Fisher R Takeda S et al.Expansion of revertant fibers in dystrophic mdx muscles reflects activity of muscle precursor cells and serves as an index of muscle regeneration.J Cell Sci. 2006; 119: 2679-2687Crossref PubMed Scopus (72) Google Scholar Some mdx mouse strains express far fewer revertant fibers than others, making low levels of exogenous gene expression easier to detect and quantify. For stem cell transplants, which have displayed a low efficiency and which often lead to transient dystrophin expression due to loss of the transplanted cells, gauging the efficiency of engraftment requires distinguishing an increase in dystrophin expression above that attributable to epigenetic reversion. By contrast, the Dmdmdx-βgeo mouse displays no revertant myofibers and provides a clean background with which to quantify cell engraftment. Vallese et al. demonstrated the utility of their new model in several ways.1Vallese D Negroni E Duguez S Ferry A Trollet C Aamiri A et al.The Rag2−Il2rb−Dmd− mouse: a novel dystrophic and immunodeficient model to assess innovating therapeutic strategies for muscular dystrophies.Mol Ther. 2013; 21: 1950-1957Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar A basic characterization of the dystrophic phenotype in the mice showed that general features of the disorder were similar to those in immunocompetent mice. The newer model displayed a somewhat delayed period of intensive myofiber necrosis and regeneration, probably reflecting a role for immune cells in modulating the dystrophic process.12Spencer MJ Montecino-Rodriguez E Dorshkind K Tidball JG Helper (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficient muscle.Clin Immunol. 2001; 98: 235-243Crossref PubMed Scopus (208) Google Scholar Primary human myoblasts were also transplanted into muscles of the Rag2−Il2rb−Dmd− mice. Dystrophin-expressing myofibers were found at least 4 weeks after transplantation, and numerous human-derived satellite cells were also found in the transplanted muscles. The dystrophin-positive myofibers also expressed members of the dystrophin–glycoprotein complex, indicating functional association of the exogenous dystrophin with the sarcolemma. It should be noted that a similar immunodeficient mdx mouse model was developed nearly simultaneously by Arpke et al.13Arpke RW Darabi R Mader TL Zhang Y Toyama A Lonetree CL et al.A new immuno- dystrophin-deficient model, the NSG-mdx4Cv mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation.Stem Cells. 2013; (e-pub ahead of print 19 April 2013)PubMed Google Scholar The availability of immunodeficient mdx mouse models will enable future studies to be focused on obtaining more efficient cell engraftment without concern for issues related to immune rejection or background dystrophin expression. A critical first step will be to explore the longevity of dystrophin expression in transplanted muscles, which will probably depend on the efficiency of engraftment. Preventing ongoing myofiber necrosis in dystrophic muscles requires approximately 20% of the normal levels of dystrophin expression within individual myofibers as well as a critical number of dystrophin-positive myofibers.14Chamberlain JS Dystrophin levels required for correction of Duchenne muscular dystrophy.Basic Appl Myol. 1997; 7: 251-255Google Scholar Current transplant methods have largely failed to achieve these levels of expression. Also, longer time frames will be important to assess the role of nonimmune mechanisms in supporting engraftment and persistence of cells transplanted into muscle. Methods to improve engraftment can now focus on the source or type of stem cell (e.g., human myoblasts, pericytes, CD133+ cells, or myogenic progenitors derived from embryonic or induced pluripotent stem cells), variables in culturing and expanding the donor cells, and optimizing transplantation via intramuscular or intravascular delivery protocols.4Konieczny P Swiderski K Chamberlain JS Gene and cell-mediated therapies for muscular dystrophy.Muscle Nerve. 2013; 47: 649-663Crossref PubMed Scopus (63) Google Scholar The immunodeficient models may also prove useful in testing alternative and novel therapies such as gene transfer and exon skipping, by revealing the extent that immune mechanisms might limit restoration of dystrophin expression.15Mendell JR Campbell K Rodino-Klapac L Sahenk Z Shilling C Lewis S et al.Dystrophin immunity in Duchenne's muscular dystrophy.N Engl J Med. 2010; 363: 1429-1437Crossref PubMed Scopus (453) Google Scholar Consequently, the new immunodeficient mdx mouse models are likely to become an invaluable resource for developing DMD therapeutics.1Vallese D Negroni E Duguez S Ferry A Trollet C Aamiri A et al.The Rag2−Il2rb−Dmd− mouse: a novel dystrophic and immunodeficient model to assess innovating therapeutic strategies for muscular dystrophies.Mol Ther. 2013; 21: 1950-1957Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar,13Arpke RW Darabi R Mader TL Zhang Y Toyama A Lonetree CL et al.A new immuno- dystrophin-deficient model, the NSG-mdx4Cv mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation.Stem Cells. 2013; (e-pub ahead of print 19 April 2013)PubMed Google Scholar