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.     

Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles.

Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.     

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.     

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.     

AAV-mediated Overexpression of Human α7 Integrin Leads to Histological and Functional Improvement in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by mutations in the DMD gene, with loss of its gene product, dystrophin. Dystrophin helps link integral membrane proteins to the actin cytoskeleton and stabilizes the sarcolemma during muscle activity. We investigated an alternative therapeutic approach to dystrophin replacement by overexpressing human α7 integrin (ITGA7) using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal and cardiac muscle that links the extracellular matrix (ECM) to the actin skeleton. It is modestly upregulated in DMD muscle and has been proposed to be an important modifier of dystrophic symptoms. We delivered rAAV8.MCK.ITGA7 to the lower limb of mdx mice through isolated limb perfusion (ILP) of the femoral artery. We demonstrated ~50% of fibers in the tibialis anterior (TA) and extensor digitorum longus (EDL) overexpressing α7 integrin at the sarcolemma following AAV gene transfer. The increase in ITGA7 in skeletal muscle significantly protected against loss of force following eccentric contraction-induced injury compared with untreated (contralateral) muscles while specific force following tetanic contraction was unchanged. Reversal of additional dystrophic features included reduced Evans blue dye (EBD) uptake and increased muscle fiber diameter. Taken together, this data shows that rAAV8.MCK.ITGA7 gene transfer stabilizes the sarcolemma potentially preserving mdx muscle from further damage. This therapeutic approach demonstrates promise as a viable treatment for DMD with further implications for other forms of muscular dystrophy.     

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.     

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.     

Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations in the dystrophin gene. Transplantation of autologous myogenic cells genetically corrected ex vivo is a possible treatment for this disorder. In order to test the regenerative efficiency of freshly isolated satellite cells, we purified quiescent satellite cells from limb muscles of 8-12-week-old green fluorescent protein-transgenic (GFP-Tg) mice using SM/C-2.6 (a recently developed monoclonal antibody) and flow cytometry. Freshly isolated satellite cells were shown to participate in muscle regeneration more efficiently than satellite cell-derived myoblasts passaged in vitro do, when transplanted into tibialis anterior (TA) muscles of 8-12-week-old cardiotoxin-injected C57BL/6 mice and 5-week-old dystrophin-deficient mdx mice, and analyzed at 4 weeks after injection. Importantly, expansion of freshly isolated satellite cells in vitro without passaging had no detrimental effects on their regenerative capacity. Therefore we directly isolated satellite cells from 5-week-old mdx mice using SM/C-2.6 antibody and cultured them with lentiviral vectors expressing micro-dystrophin CS1. The transduced cells were injected into TA muscles of 5-week-old mdx mice. At 4 weeks after transplantation, the grafted cells efficiently contributed to regeneration of mdx dystrophic muscles and expressed micro-dystrophin at the sarcolemma. These results suggest that there is potential for lentiviral vector-mediated ex vivo gene therapy for DMD.     

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。     

Prevention of the dystrophic phenotype in dystrophin/utrophin-deficient muscle following adenovirus-mediated transfer of a utrophin minigene

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by the lack of a subsarcolemmal protein, dystrophin. We have previously shown that the dystrophin-related protein, utrophin is able to compensate for the lack of dystrophin in the mdx mouse, the mouse model for DMD. Here, we explore whether utrophin delivered to the limb muscle of dystrophin/utrophin-deficient double knockout (dko) neonatal mice can protect the muscle from subsequent dystrophic damage. Utrophin delivery may avoid the potential problems of an immune response associated with the delivery of dystrophin to a previously dystrophin-deficient host. Dko muscle (tibialis anterior) was injected with a first generation recombinant adenovirus containing a utrophin minigene. Up to 95% of the fibres continued expressing the minigene 30 days after injection. Expression of utrophin caused a marked reduction from 80% centrally nucleated fibres (CNFs) in the uninjected dko TA to 12% in the injected dko TA. Within the region of the TA expressing the utrophin minigene, a significant decrease in the prevelance of necrosis was noted. These results demonstrate that the utrophin minigene delivered using an adenoviral vector is able to afford protection to the dystrophin/utrophin-deficient muscle of the dko mouse. Gene Therapy (2000) 7, 201-204.     

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.     

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.     

Immune responses to dystropin: implications for gene therapy of Duchenne muscular dystrophy

Introduction of dystrophin by gene transfer into the dystrophic muscles of Duchenne muscular dystrophy (DMD) patients has the possibility of triggering an immune response as many patients will not have been exposed to some (or all) of the epitopes of dystrophin. This could in turn lead to cytotoxic destruction of transfected muscle fibres. We assessed such concerns in the dystrophin-deficient mdx mouse using plasmid DNA as the gene transfer system. This avoids complications associated with the administration of viral proteins. Gene transfer of cDNAs encoding mouse full-length or a truncated minidystrophin did not evoke either a humoral or cytotoxic immune response. Mdx mice may be tolerant due to the presence of rare 'revertant' dystrophin-positive fibres in their skeletal muscles. In contrast, gene transfer of human full-length or minidystrophin provoked both humoral and cytotoxic responses leading to destruction of the transfected fibres. These experiments demonstrate the potential risk of deleterious effects following gene therapy in DMD patients and lead us to suggest that patients enrolled in gene therapy trials should ideally have small, preferably point, mutations and evidence of 'revertant' dystrophin-positive muscle fibres.     

Muscle Function Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 Exon Skipping

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.     

High-efficiency plasmid gene transfer into dystrophic muscle

The efficiency of plasmid gene transfer in skeletal muscle is significantly enhanced by pretreatment with hyaluronidase and the application of an electrical field to the muscle following the injection of plasmid DNA, a process referred to as electrotransfer. However, the presence of increased levels of connective tissue in muscular dystrophies, such as Duchenne muscular dystrophy (DMD), may affect the efficiency of this process. Here we demonstrate that the efficiency of electrotransfer is not affected by increased levels of connective tissue in the mdx mouse model of DMD and that any damage induced by the electrotransfer process is not exacerbated in the dystrophic phenotype. However, increasing the concentration of hyaluronidase does not improve transfection efficiencies further. Unlike direct injection of plasmid DNA, the efficiency of electrotransfer is not dependent upon the sex and age of mice used. The combined treatment of hyaluronidase and electrotransfer results in highly efficient gene transfer in dystrophic muscle with limited muscle damage.     

Prevention of Dystrophic Pathology in Severely Affected Dystrophin/Utrophin-deficient Mice by Morpholino-oligomer-mediated Exon-skipping

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon-skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of dystrophin expression in the mdx mouse model. Here, we report the efficiency of the PPMO-mediated exon-skipping approach in the utrophin/dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of dystrophin pre-mRNA in dKO mice induce a near-normal level of dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These findings suggest great potential for PPMOs in systemic treatment of the DMD phenotype.