Removing the immune response from muscular dystrophy research.

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.1 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.2 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.3 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.4 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.5 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.6 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.5,7 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. Figure 1 Immunomodulated mouse strains and the immune cells they lack. NK, natural killer; NOD, nonobese diabetic; SCID, severe combined immunodeficient. 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.1 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.8 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.9 Virtually all strains of mdx mice display various levels of dystrophin expression in a small percentage of myofibers.10 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.11 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.1 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.12 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.13 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.14 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.4 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.15 Consequently, the new immunodeficient mdx mouse models are likely to become an invaluable resource for developing DMD therapeutics.1,13