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.     

Dystrophin delivery in dystrophin-deficient DMDmdx skeletal muscle by isogenic muscle-derived stem cell transplantation

Duchenne's muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMD(mdx) (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology.     

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.     

Amphiphilic block copolymers promote gene delivery in vivo to pathological skeletal muscles

We reported that amphiphilic block copolymers hold promise as nonviral vectors for the delivery of plasmid DNA, ranging from 4.7 to 6.2 kb, to healthy muscle for the production of local or secreted proteins. To evaluate the efficiency of these vectors to deliver large plasmid DNA molecules to pathological muscles, plasmid DNAs of various lengths were complexed with Lutrol or poloxamine 304 and injected intramuscularly into dystrophic muscles. Lutrol-DNA and poloxamine 304-DNA complexes promoted gene transfer into muscles of the naturally occurring mouse model for DMD (mdx) in a dose- and plasmid DNA size-dependent manner. For small plasmid DNAs encoding reporter genes, this improvement over naked DNA was smaller in mdx than in the wild-type control strain. By contrast, Lutrol enabled us to deliver the large plasmid (16.1 kb) encoding the rod-deleted dystrophin in mdx mouse muscle, whereas the same amount of naked DNA did not lead to dystrophin expression, under the same experimental conditions. Lutrol-treated mdx mice showed the production of dystrophin in large numbers of muscle fibers. More importantly, we also found that expressing dystrophin with Lutrol led to restoration of the dystrophin-associated protein complex. Thus, we conclude that block copolymers constitute a novel class of vectors for the delivery of large plasmid DNA not only to healthy muscles but also to pathological muscle tissues.     

Enhanced expression of recombinant dystrophin following intramuscular injection of Epstein-Barr virus (EBV)-based mini-chromosome vectors in mdx mice.

Gene transfer by direct intramuscular injection of naked plasmid DNA has been shown to be a safe, simple but relatively inefficient method for gene delivery in vivo. Eukaryotic plasmid expression vectors incorporating the Epstein-Barr virus (EBV) origin of replication (oriP) and EBNA1 gene have been shown to act as autonomous episomally replicating gene transfer vectors which additionally provide nuclear matrix retention functions. Prolonged expression of a LacZ reporter gene and recombinant human dystrophin was shown using EBV-based plasmid vectors transfected into C2C12 mouse myoblast and myotube cultures. Intramuscular injection of EBV-based dystrophin expression plasmids into nude/mdx mice resulted in significant enhancement in the number of muscle fibres expressing recombinant dystrophin compared with a conventional vector. This effect was observed for over 10 weeks after a single administration. These results indicate the potential advantage of EBV-based expression vectors for focal plasmid-mediated gene augmentation therapy in Duchenne muscular dystrophy (DMD) and a range of other gene therapeutic applications.     

Local and distant transfection of mdx muscle fibers with dystrophin and LacZ genes delivered in vivo by synthetic microspheres.

Patterns of dystrophin and beta-galactosidase expression were examined in mdx mice after i.m. injections of synthetic microspheres (MF-2) loaded with full-length (pHSADy) or mini-dystrophin gene (pSG5dys) cDNA plasmid constructs or with LacZ marker gene (pCMV-LacZ). A single injection of 25 microg pHSADy into quadriceps femoris muscle resulted in 6.8% of dystrophin positive myofibers (DPM) in a given muscle; 8.4% of DPM in glutaeus muscle and 4.3% of DPM in quadriceps femoris muscle of contralateral limb on day 21 after exposure compared with only 0.6% DPM in intact (non-injected) mdx mice. A high proportion of DPM (17.6% and 10.8%, respectively) was registered in both injected and contralateral muscles after mini- gene cDNA administration. MF-2/dystrophin cDNA particles were detected by FISH analysis in about 60-70% of myofiber nuclei in muscles of injected and contralateral limbs 7 days after application. The presence of human dystrophin cDNA and its products in all skeletal muscles and in different internal organs was proven by PCR and RT-PCR analysis. Patches of beta-galactosidase expression were abundant in injected muscle, and frequent in the contralateral and other skeletal muscles as well as in diaphragm, heart and lungs. High levels of dystrophin cDNA expression, and an efficient distant transfection effect with preferential intranuclei inclusion of MF-2 vehicle, are very encouraging for the development of a new constructive strategy in gene therapy trials of DMD.     

Successful compensation for dystrophin deficiency by a helper-dependent adenovirus expressing full-length utrophin.

Helper-dependent adenovirus vector (AdV)-mediated full-length dystrophin expression leads to significant mitigation of the dystrophic phenotype of the mdx mouse. However, dystrophin, as a neoantigen, elicits antibody formation. As an alternative approach, we evaluated gene transfer of full-length murine utrophin, a functional homologue of dystrophin that is normally present only at the neuromuscular junction. A single injection in the tibialis anterior (TA) muscle of the helper-dependent adenovirus vector encoding utrophin provided very good transduction, with 58% of fibers demonstrating sarcolemmal utrophin expression in the neonates, and 35% utrophin-positive (Utr(+)) fibers in adults. The presence of utrophin prevented extensive necrosis in the neonates, halted further necrosis in the adults, and led to restoration of sarcolemmal expression of dystrophin-associated proteins up to 1 year after injection. Marked physiological improvement was observed in both neonates and adults. Neither increased humoral responses nor cellular immune responses were evident. However, there was a time-related decline of the initial high utrophin expression. Although viral DNA persisted in animals that were injected in the neonatal stage, viral DNA levels decreased in muscles of adult mice. These results demonstrate that although utrophin gene transfer leads to amelioration of the dystrophic phenotype, the effects are not sustained upon loss of utrophin expression.     

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.     

Matching host muscle and donor myoblasts for myosin heavy chain improves myoblast transfer therapy

Intensive efforts have been made to develop an effective therapy for Duchenne muscular dystrophy (DMD). Although myoblast transplantation has been found capable of transiently delivering dystrophin and improving the strength of the injected dystrophic muscle, this approach has been hindered by the immune rejection problems as well as the poor survival and limited spread of the injected cells. In the present study, we have investigated whether the careful selection of donor myoblasts and host muscle for the myosin heavy chain expression (MyHCs) plays a role in the success of myoblast transfer. Highly purified normal myoblasts derived from the m. soleus and m. gastrocnemius white of normal mice were transplanted into the m. soleus (containing 70% of type I fibers) and gastrocnemius white (100% of type II fibers) of dystrophin deficient mdx mice. At several time-points after injection (10, 20 and 30 days), the number of dystrophin-positive fibers was monitored and compared among the different groups. A significantly higher number and better persistence of dystrophin-positive myofibers were observed when the injected muscle and donor myoblasts expressed a similar MyHC in comparison with myoblast transfer between host muscle and donor myoblasts that were not matched for MyHC. These results suggest that careful matching between the injected myoblasts and injected muscle for the MyHC expression can improve the efficiency of myoblast-mediated gene transfer to skeletal muscle. Gene Therapy (2000) 7, 428-437.     

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.     

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.     

Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy

Our previous studies have demonstrated that the intraarterial delivery of naked plasmid DNA leads to high levels of foreign gene expression throughout the muscles of the targeted limb. Although the procedure was first developed in rats and then extended to nonhuman primates, the present study has successfully implemented the procedure in normal mice and the mdx mouse model for Duchenne muscular dystrophy. After intraarterial delivery of plasmid DNA expressing the normal, full-length mouse dystrophin from either the cytomegalovirus promoter or a muscle-specific human desmin gene control region, mdx mouse muscle stably expressed dystrophin in 1-5% of the myofibers of the injected hind limb for at least 6 months. This expression generated an antibody response but no apparent cellular response.     

Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers.

Antisense oligonucleotides (AOs) with 2-O-methyl modifications can circumvent dystrophin mutations via exon skipping and, it is hoped, can become drugs for treatment of Duchenne muscular dystrophy (DMD). However, AO-based approaches are hindered by a lack of effective carriers to facilitate delivery of AOs to myonuclei. We examined whether copolymers composed of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) can enhance AO transfection in skeletal muscle of mdx mice. Single intramuscular injections of AO complexed with low Mw PEI2000(PEG550) copolymers into TA muscles of mdx mice resulted in widespread distribution of dystrophin-positive fibers at 3 weeks after injection, with no apparent cytotoxicity. Overall, injections of these low Mw polyplexes, which formed 250-nm aggregate particles, resulted in about sixfold more dystrophin-positive fibers than AO alone. Western analysis confirmed the dystrophin expression in these muscles. Surprisingly, injections of AO complexed with high Mw PEI25000(PEG5000) copolymers, which formed smaller nonaggregated particles, produced about threefold fewer dystrophin-positive fibers than injections of the low Mw polyplexes. We conclude that low Mw PEI2000(PEG550) copolymers function as high-capacity, nontoxic AO carriers suitable for in vivo transfection of skeletal muscle and are promising compounds for potential use in molecular therapy of DMD.     

Full-length dystrophin gene transfer to the mdx mouse in utero

In utero gene therapy for genetic diseases, such as muscular dystrophies, offers potential advantages over postnatal treatment including vector delivery at the earliest point in the disease and treatment prior to full maturation of the immune system. This study examines in utero gene delivery of full-length murine dystrophin to the murine mdx model for Duchenne muscular dystrophy using a high-capacity adenoviral vector. We examined dystrophin expression, spread of vector, morphology and specific force production of the tibialis anterior muscle 9 weeks after intramuscular in utero injection. Recombinant dystrophin was expressed in the hindlimb muscles, with the majority of animals having expression in two muscles of the injected hindlimb. The dystrophin-glycoprotein complex was restored in those muscle fibers expressing recombinant dystrophin. Analysis of the percentage of dystrophin-expressing muscle fibers with centrally placed nuclei revealed effective protection from cycles of degeneration and regeneration normally seen in muscle fibers lacking dystrophin. However, due to low levels of muscle gene transfer, further advances in the efficiency of adenoviral vector-mediated gene delivery would be required for clinical applications of in utero gene therapy for primary myopathies such as Duchenne muscular dystrophy.     

骨髓间充质干细胞移植治疗Duchenne肌营养不良型mdx小鼠后的肌电图和dystrophin蛋白表达改变

目的 研究骨髓间充质干细胞（MSCs）移植治疗Duchenne肌营养不良（DMD）模型鼠mdx鼠后肌电图改变及dystrophin蛋白表达变化。方法 分离培养正常小鼠MSCs局部肌肉注射移植入DMD模型鼠mdx鼠，数周后观察肌电图改变及dystrophin蛋白表达变化。结果 MSCs移植后mdx鼠肌电图明显改善，dystrophin蛋白阳性表达肌纤维增加明显。结论 MSCs局部骨骼肌肌肉内细胞移植治疗mdx鼠有一定效果。     

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.     

Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles.

Intramuscular injection of plasmid is a potential alternative to viral vectors for the transfer of therapeutic genes into skeletal muscle fibers. The low efficiency of plasmid-based gene transfer can be enhanced by electroporation (EP) coupled with the intramuscular application of hyaluronidase. We have investigated several factors that can influence the efficiency of plasmid-based gene transfer. These factors include electrical parameters of EP, optimal use of hyaluronidase, age and strain of the host, and plasmid size. Muscles of very young and mature normal, mdx, and immunodeficient mice were injected with plasmids expressing beta-galactosidase, microdystrophin, full-length dystrophin, or full-length utrophin. Transfection efficiency, muscle fiber damage, and duration of transgene expression were analyzed. The best transfection level with the least collateral damage was attained at 175-200 V/cm. Pretreatment with hyaluronidase markedly increased transfection, which was also influenced by the plasmid size and the strain and the age of the mice. Even in immunodeficient mice, there was a significant late decline in transgene expression and plasmid DNA copies, although both still remained relatively high after 1 year. Thus, properly optimized EP-assisted plasmid-based gene transfer is a feasible, efficient, and safe method of gene replacement therapy for dystrophin deficiency of muscle but readministration may be necessary.