CTLA4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle

Adenoviral (Ad) vector-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD), an X-linked recessive, dystrophin-deficient muscle disease. Studies in animal models suggest that successful DMD gene therapy by Ad vector-mediated gene transfer would be precluded by cellular and humoral immune responses induced by vector capsid and transgene proteins. To address the immunity induced by Ad vector-mediated dystrophin gene delivery to dystrophic muscle, we developed high-capacity adenoviral (HC-Ad) vectors expressing mouse dystrophin driven by the muscle creatine kinase promoter (AdmDys) and mCTLA4Ig (AdmCTLA4Ig) individually, or together from one vector (AdmCTLA4Ig/mDys). We found stable expression of dystrophin protein in the tibialis anterior muscles of mdx mice, coinjected with AdmCTLA4Ig and AdmDys, or injected alone with AdmCTLA4Ig/mDys, whereas the expression of dystrophin protein in the control group coinjected with AdmDys and an empty vector decreased by at least 50% between 2 and 8 weeks after administration. Additionally, we observed reductions in Ad vector-induced Th1 and Th2 cytokines, Ad vector-specific cytotoxic T lymphocyte activation and neutralizing anti-Ad antibodies in both experimental groups that received a mCTLA4Ig-expressing vector as compared to the control group. This study demonstrates that the coexpression of mCTLA4Ig and dystrophin in skeletal muscle provided by HC-Ad vector-mediated gene transfer can provide stable expression of dystrophin in immunocompetent, adult mdx mouse muscle and applies a potentially powerful strategy to overcome adaptive immunity induced by Ad vector-mediated dystrophin gene delivery toward the ultimate goal of treatment for DMD.     

Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse - an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD.     

Long-term preservation of cardiac structure and function after adeno-associated virus serotype 9-mediated microdystrophin gene transfer in mdx mice

Dystrophin plays an important role in muscle contraction, linking the intracellular cytoskeleton to the extracellular matrix. Mutations of the dystrophin gene leading to a complete loss of the protein cause Duchenne muscular dystrophy (DMD), frequently associated with severe cardiomyopathy. Early clinical trials in DMD using gene transfer to skeletal muscle are underway, but gene transfer to dystrophic cardiac muscle has not yet been tested in humans. The aim of this study was to develop an optimized protocol for cardiac gene therapy in the mouse model of dystrophin deficiency (mdx), using a cardiac promoter for expression of a microdystrophin (μDys) transgene packaged into an adeno-associated virus serotype 9 vector (AAV9). In this study adult mdx mice were intravenously injected with 1×10(12) genomic particles of AAV9 vectors carrying a cDNA encoding μDys under the control of either a ubiquitously active cytomegalovirus (CMV) promoter or a cardiac-specific CMV-enhanced myosin light chain (MLC0.26) promoter. After 10 months, both AAV9 vectors led to sustained μDys expression in cardiac muscle, but the MLC promoter conferred about 4-fold higher protein levels. AAV9-CMV-MLC0.26-μDys resulted in significant protection of cardiac morphology and function as assessed by histopathology, echocardiography, and left ventricular catheterization. In conclusion, we established an AAV9-mediated gene transfer approach for efficient and specific long-term μDys expression in the hearts of mdx mice, resulting in a sustained therapeutic effect. Thus, this approach might be a basis for further translation into a treatment strategy for DMD-associated cardiomyopathy.     

mdx小鼠骨骼肌组织成肌、成脂、成骨基因的特异性表达

背景：干细胞移植是治疗肌营养不良症的有效方法之一,但移植的干细胞在病理骨骼肌中成肌表达较低。目的：通过比较mdx小鼠和C57小鼠的骨骼肌形态及成肌、成脂、成骨基因表达的差异,探讨mdx小鼠骨骼肌病理改变的可能机制。方法：取mdx小鼠与C57小鼠的骨骼肌组织行冰冻切片,苏木精-伊红染色和Vonkossa染色观察两种小鼠肌肉组织的形态特征;提取mdx小鼠和C57小鼠骨骼肌组织总RNA,real-timePCR检测成肌、成脂、成骨相关基因的表达。结果与结论：mdx小鼠骨骼肌有肌纤维坏死和再生,伴有轻度脂肪、纤维结缔组织增生,Vonkossa染色可见钙结节沉积,而C57小鼠的骨骼肌细胞形态清晰,核位于细胞周边。与C57小鼠比较,mdx小鼠肌肉组织成骨、成脂基因表达有不同程度的上调（P〈0.05）,而成肌基因表达下调（P〈0.05）。dystrophin基因缺失及成肌基因表达下调、成骨和成脂基因上调是造成mdx小鼠肌肉组织变性坏死的原因。     

Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, recessive disease caused by a defect in the dystrophin gene. No effective therapy is available. Dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. However, successful treatment for DMD requires restoration of dystrophin in the affected muscle fibers to at least 20% of the normal level. Current gene transfer methods such as intramuscular injection of viral vector or naked DNA can only transfect a small area of muscle, and therefore is of little clinical utility. We have developed a semisystemic method for gene transfer into skeletal muscle of mdx mice, an animal model for DMD. Naked DNA was injected through the tail artery or vein of mice, in which the aorta and the vena cava were clamped at the location just below the kidneys. The DNA solution was thus forced into the blood vessels of both legs. Luciferase gene expression was detected in all muscle groups in both legs. The effects of injection speed, injection volume, and ischemia time on gene expression were also optimized. LacZ staining was used to check the spread of gene expression in muscle. Although the percentage of transfected fibers was modest (approximately 10%), beta-galactosidase was found in all muscle groups of both legs. Finally, plasmid DNA encoding full-length dystrophin gene was injected into mdx mice and widespread restoration of dystrophin protein was observed in all muscles of both hind limbs. In conclusion, these results demonstrate that the semisystemic delivery of naked DNA is a potential approach towards the long-term goal of gene therapy for DMD.     

Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscular disorder caused by a defect in the DMD gene. AAV vector-mediated micro-dystrophin cDNA transfer is an attractive approach to treatment of DMD. To establish effective gene transfer into skeletal muscle, we examined the transduction efficiency of an AAV vector in skeletal muscles of dystrophin-deficient mdx mice. When an AAV vector encoding the LacZ gene driven by a CMV promoter (AAV-CMVLacZ) was introduced, beta-galactosidase expression markedly decreased in mdx muscle 4 weeks after injection due to immune responses against the transgene product. We also injected AAV-CMVLacZ into skeletal muscles of mini-dystrophin-transgenic mdx mice (CVBA3'), which show ameliorated phenotypes without overt signs of muscle degeneration. AAV vector administration, however, evoked substantial immune responses in CVBA3' muscle. Importantly, AAV vector using muscle-specific MCK promoter also elicited responses in mdx muscle, but at a considerably later period. These results suggested that neo-antigens introduced by AAV vectors could evoke immune reactions in mdx muscle, since increased permeability allowed a leakage of neo-antigens from the dystrophin-deficient sarcolemma of muscle fibers. However, resident antigen-presenting cells, such as myoblasts, myotubes and regenerating immature myofibers, might also play a role in the immune response.     

Autologous transplantation of muscle precursor cells modified with a lentivirus for muscular dystrophy: human cells and primate models.

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. We tested the ability of lentiviral vectors to deliver a transgene into myogenic cells before their transplantation. Enhanced green fluorescent protein (eGFP) transgene was efficiently transferred into cells and eGFP-positive fibers were generated following transplantation. An eGFP-micro-dystrophin transgene under the control of a cytomegalovirus promoter was then transferred with the same viral vector but caused some toxicity to the mono-nucleated cells. We then used instead a muscle creatine kinase promoter. Dystrophin expression was observed in the muscle fibers after the transplantation of such genetically modified cells into mdx and severe combined immunodeficient mice. Micro-dystrophin expression was also observed in monkey muscles a month after allogenic or autologous transplantation of genetically modified myoblasts. Therapeutic exon skipping was induced by infecting myoblasts of a DMD patient, deleted for dystrophin exons 49 and 50, with a lentivirus expressing a U7 small nuclear RNA containing antisense sequences against exon 51. The modification led to correct exon skipping and to the expression of a quasi-dystrophin in vitro and in vivo. These results demonstrate the feasibility of lentiviral-based ex vivo gene therapy for DMD.     

AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype.

Duchenne muscular dystrophy (DMD) is a lethal disorder of skeletal muscle caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vector-mediated gene therapy is a promising approach to the disease. Although a rod-truncated microdystrophin gene has been proven to ameliorate dystrophic phenotypes, the level of microdystrophin expression required for effective gene therapy by an AAV vector has not been determined yet. Here, we constructed a recombinant AAV type 2 vector, AAV2-MCKDeltaCS1, expressing microdystrophin (DeltaCS1) under the control of a muscle-specific MCK promoter and injected it into TA muscles of 10-day-old and 5-week-old mdx mice. AAV2-MCKDeltaCS1-mediated gene transfer into 5-week-old mdx muscle resulted in extensive and long-term expression of microdystrophin and significantly improved force generation. Interestingly, 10-day-old injected muscle expressed microdystrophin in a limited number of myofibers but showed hypertrophy of microdystrophin-positive muscle fibers and considerable recovery of contractile force. Thus, we concluded that AAV2-MCKDeltaCS1 could be a powerful tool for gene therapy of DMD.     

Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin

Gene therapy for Duchenne muscular dystrophy (DMD) will require sustained expression of therapeutic dystrophins in striated muscles. Lentiviral vectors have a relatively large transgene carrying capacity and can integrate into nondividing cells. We therefore explored the use of lentiviral vectors for transferring genes into mouse skeletal muscle cells. These vectors successfully transferred a minidystrophin expression cassette into mdx muscles, and minidystrophin expression persisted and prevented subsequent muscle fiber degeneration for at least 6 months. However, only low to moderate levels of skeletal muscle transduction could be obtained by intramuscular injection of the highest currently available lentiviral doses. Using cultured cells, the lentiviral vectors effectively transduced proliferating and terminally differentiated muscle cells, indicating that cell cycling is not essential for transduction of myogenic cells. We further showed that lentiviral vectors efficiently transduced both primary myoblasts and multipotent adult progenitor cells (MAPCs) in vitro, and the cells persistently expressed transgenes without any obvious toxicity. When mdx primary myoblasts were genetically modified with minidystrophin vectors and transplanted into mdx skeletal muscles, significant numbers of dystrophin-expressing myofibers formed. Finally, we showed that a short, highly active CK6 regulatory cassette directed muscle-specific activity in the context of the lentiviral vectors. The ability of lentiviral vectors to 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.     

Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA-DNA chimeric oligonucleotides

Point mutations in the dystrophin gene cause dystrophin deficiency and muscular dystrophy in the mdx mouse and a subset of patients with Duchenne muscular dystrophy. As an approach to gene therapy for muscular dystrophies due to point mutations, we have studied the ability of RNA-DNA chimeric oligonucleotides (chimeraplasts) to induce repair of the dystrophin gene in mdx mice. We have previously demonstrated that targeting chimeraplasts can repair the exon 23 point mutation in differentiated myofibers in vivo after intramuscular injection. For long-term benefit to patients with muscular dystrophy, any gene therapy technology must target not only differentiated myofibers but also undifferentiated muscle precursor cells that are involved in ongoing muscle repair. The focus of the current studies was to test whether chimeraplasts could repair the dystrophin mutation in mdx muscle precursor cells. Initial studies were done by transfecting a targeting chimeraplast into mdx myoblasts in vitro. Gene repair was demonstrated at the DNA, RNA, and protein levels in these cells, whereas treatment of the cells with a control chimeraplast resulted in no gene correction. After differentiation of mdx cells that had been treated with a targeting chimeraplast, immunoblot analysis demonstrated full-length dystrophin expression. By quantitative analysis of independent cultures, the amount of dystrophin expressed ranged from 2 to 15% of that expressed in wild-type cells, providing a measure of the efficacy of gene conversion in vitro. To extend the assessment to muscle precursor cells in vivo, we injected targeting and control chimeraplasts into muscles of mdx mice. When muscle precursor cells were subsequently derived from muscles injected with a targeting chimeraplast, we found that gene repair had occurred in these cells as well. These results, taken together, further demonstrate that chimeraplast-mediated gene repair may be effective as an approach to gene therapy for muscular dystrophies due to point mutations.     

Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products

Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer.     

Genetic correction of dystrophin deficiency and skeletal muscle remodeling in adult MDX mouse via transplantation of retroviral producer cells.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD.     

Modulation of Starling forces and muscle fiber maturity permits adenovirus-mediated gene transfer to adult dystrophic (mdx) mice by the intravascular route

Duchenne muscular dystrophy (DMD) and other inherited myopathies lead to progressive destruction of most skeletal muscles in the body, including those responsible for maintaining respiration. DMD is a fatal disorder caused by defects in the dystrophin gene. Recombinant adenovirus vectors (AdV) are considered a promising means for therapeutic delivery of a functional dystrophin gene to DMD muscles. If AdV-mediated dystrophin gene replacement in DMD is to be successful, development of a systemic delivery method for targeting the large number of diseased muscles will be required. In this study we investigated two major factors preventing efficient AdV-mediated gene transfer to skeletal muscles of adult animals after intravascular AdV administration: (1) an inability of AdV particles to breach the endothelial barrier and enter into contact with myofibers, and (2) a relatively nonpermissive myofiber population for AdV infection due at least in part to insufficient levels of the coxsackie/adenovirus attachment receptor (CAR). On the basis of established principles governing the transendothelial flux of macromolecules, we further hypothesized that an alteration in Starling forces (increased hydrostatic and decreased osmotic pressures) within the intravascular compartment would facilitate AdV transendothelial flux via convective transport. In addition, experimental muscle regeneration was employed to increase the prevalence of immature myofibers in which CAR expression is upregulated. Here we report that by employing the above-described strategy, high-level heterologous reporter gene expression was achievable in hindlimb muscles of normal rats as well as dystrophic (mdx) mice (genetic homolog of DMD) after a single intraarterial injection of AdV. Microsphere studies confirmed enhanced transport into muscle of fluorescent tracer particles in the size range of AdV, and there was a high concordance between CAR upregulation and myofiber transduction after intraarterial AdV delivery. Furthermore, in mdx mice examined 10 days after intraarterial AdV delivery, the aforementioned procedures had no adverse effects on the force-generating capacity of targeted muscles. These findings have implications for eventual AdV-mediated gene therapy of generalized skeletal muscle diseases such as DMD using a systemic intraarterial delivery approach.     

Aquaporin 4 mRNA levels in neuromuscular tissues of wild-type and dystrophin-deficient mice

Aquaporin (AQP) 4 is a water-specific channel protein and is abundant in central nervous tissues and skeletal muscles. Recently, the AQP4 molecule has been increasingly highlighted in its pathophysiological role of several neurological diseases, such as stroke, muscular dystrophy and neuromyelitis optica. We therefore measured the levels of AQP4 mRNA and glyceraldehyde-3 phosphate dehydrogenase mRNA (an internal control) in muscle and brain tissues of wild-type mice (C57BL10/ScSn) and age-matched dystrophin-deficient mdx mice (C57BL10/ScSn mdx) by real-time quantitative RT-PCR. The relative AQP4 mRNA level was highest in the spinal cord among the neuromuscular tissues examined in wild-type mice. Among the muscle tissues of wild-type mice, the relative AQP4 mRNA level was higher in extensor digitorum longus (EDL) muscles, and its descending order was EDL, quadriceps femoris, soleus and heart muscles. It is noteworthy that there was no difference in the relative AQP4 mRNA levels in the brain tissues between wild-type mice and age-matched mdx mice. In contrast, the AQP4 mRNA level in the quadriceps femoris muscle was significantly lower in mdx mice than in wild-type mice. The fact that the spinal cord contains the highest AQP4 mRNA may be related to the pathogenesis of neuromyelitis optica, in which AQP4 protein is the target antigen. In addition, the low expression level of AQP4 mRNA in the mdx mouse muscle suggests a functional link between AQP4 and dystrophin in the muscle tissue. We suggest that a similar pathomechanism may underlie the phenotypic consequences of the mdx mouse and Duchenne muscular dystrophy.     

Immune response to adenovirus-delivered antigens upregulates utrophin and results in mitigation of muscle pathology in mdx mice

The upregulation of endogenous utrophin in skeletal muscle may lead to a new approach to the treatment of Duchenne muscular dystrophy (DMD). We found that injection of an E1, E3-deleted adenovirus vector expressing beta-galactosidase (beta-Gal) or green fluorescent protein (GFP) into the skeletal muscle of neonatal dystrophin-deficient mdx mice alleviated dystrophic pathology. In the adenovirus-infected muscles, an evaluation of sarcolemma stability showed low permeability and immunohistochemistry revealed utrophin upregulation at the extrasynaptic sarcolemma of mature muscle fibers. This utrophin upregulation was concomitant with endomysial cellular infiltration from a host immune reaction. There was no evidence of active muscle regeneration. In normal C57BL/10 mice, utrophin was also upregulated in adenovirus-injected skeletal muscles, where upregulated utrophin often coexisted with dystrophin. FK506 and anti-CD4 antibody administration decreased utrophin expression in adenovirus-injected mdx muscles and prevented the dystrophic phenotype from being mitigated, suggesting that an immune reaction is involved in utrophin upregulation. This is the first report demonstrating the improvement of the dystrophic phenotype as a result of the acquired overexpression of endogenous utrophin. Our findings provide an important clue to understanding the mechanism of utrophin expression and the development of an effective treatment for DMD.     

Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice

Adeno-associated virus (AAV)-mediated microdystrophin gene therapy holds great promise for treating Duchenne muscular dystrophy (DMD). Previous studies have revealed excellent skeletal muscle protection. Cardiac muscle is also compromised in DMD patients. Here we show that a single intravenous injection of AAV serotype-9 (AAV-9) microdystrophin vector efficiently transduced the entire heart in neonatal mdx mice, a dystrophin-deficient mouse DMD model. Furthermore, microdystrophin therapy normalized the heart rate, PR interval, and QT interval. The cardiomyopathy index was also significantly improved in treated mdx mice. Our study demonstrates for the first time that AAV microdystrophin gene therapy can ameliorate the electrocardiographic abnormalities in a mouse model for DMD.     

Sustained improvement of muscle function one year after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector

Dystrophin gene transfer using helper-dependent adenoviral vectors (HDAd) deleted of all viral genes is a promising option to treat muscles in Duchenne muscular dystrophy (DMD). Previously, we reported high-level dystrophin expression and functional correction of dystrophin-deficient (mdx) mouse muscle 60 days after gene transfer with an HDAd encoding two full-length murine dystrophin cDNAs (referred to as HDCBDysM). In the present study, we tested the long-term efficacy of HDCBDysM by examining muscle contractility parameters and the stability of dystrophin expression 1 year after injection into neonatal mdx muscles. At this point, HDCBDysM-treated muscles averaged 52% dystrophin-positive fibers. Treated muscles also displayed significantly greater isometric force production as well as greater resistance to the force deficits and damage caused by eccentric contractions. The level of protection against eccentric contraction-induced force deficits correlated with the percentage of dystrophin-positive fibers. Furthermore, HDCBDysM treatment restored the dystrophin-glycoprotein complex (DGC) to the sarcolemma and improved other aspects of mdx muscle histopathology examined (central nucleation, muscle hypertrophy, and mononuclear [phagocytic] cell infiltration). These improvements occurred despite the induction of a humoral response against murine dystrophin. Our results indicate that major therapeutic benefits of HDCBDysM are maintained for a long period of the animals' lifespan and suggest that HDCBDys holds promise for treating DMD by gene therapy.     

Dystrophin expression in host muscle following transplantation of muscle precursor cells modified with the phiC31 integrase

Duchenne muscular dystrophy (DMD) is the most severe muscular dystrophy. It is caused by the absence of dystrophin in muscle fibers. The autologous transplantation of genetically corrected muscle precursor cells (MPCs) is a possible cure for DMD. A non-viral method of genetic modification was tested in this study. The co-transfection (nucleofection) of a phiC31 integrase and a transgene expressing plasmid in MPCs led to an increased stable expression in vitro. The stable expression of a small transgene (eGFP) in muscle fibers was initially demonstrated following the transplantation of the genetically modified cells. The stable expression of a truncated version of dystrophin as well as the full-length dystrophin fused with eGFP was then demonstrated in MPCs obtained from an mdx mice. The transplantation of these cells led not only to the expression of these fusion proteins in muscle fibers but also to the reconstitution of the dystrophin complex. Human MPCs were also genetically modified with a plasmid coding for the full-length human dystrophin gene fused with eGFP and transplanted in severe combined immuno deficient mice leading to the expression of eGFP dystrophin in muscle fibers. This work indicates that cell transplantation after correction of MPCs with phiC31 integrase is a possible approach to treat DMD.