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.     

Nerve growth factor improves the muscle regeneration capacity of muscle stem cells in dystrophic muscle

Researchers have attempted to use gene- and cell-based therapies to restore dystrophin and alleviate the muscle weakness that results from Duchenne muscular dystrophy (DMD). Our research group has isolated populations of muscle-derived stem cells (MDSCs) from the postnatal skeletal muscle of mice. In comparison with satellite cells, MDSCs display an improved transplantation capacity in dystrophic mdx muscle that we attribute to their ability to undergo long-term proliferation, self-renewal, and multipotent differentiation, including differentiation toward endothelial and neuronal lineages. Here we tested whether the use of nerve growth factor (NGF) improves the transplantation efficiency of MDSCs. We used two methods of in vitro NGF stimulation: retroviral transduction of MDSCs with a CL-NGF vector and direct stimulation of MDSCs with NGF protein. Neither method of NGF treatment changed the marker profile or proliferation behavior of the MDSCs, but direct stimulation with NGF protein significantly reduced the in vitro differentiation ability of the cells. NGF stimulation also significantly enhanced the engraftment efficiency of MDSCs transplanted within the dystrophic muscle of mdx mice, resulting in the regeneration of numerous dystrophin-positive muscle fibers. These findings highlight the importance of NGF as a modulatory molecule, the study of which will broaden our understanding of its biologic role in the regeneration and repair of skeletal muscle by musclederived cells.     

Effect of injecting primary myoblasts versus putative muscle-derived stem cells on mass and force generation in mdx mice

It is well established that the injection of normal myoblasts or of muscle-derived stem cells (MDSCs) into the muscle of dystrophin-deficient mdx mice results in the incorporation of a number of donor myoblasts into the host muscle. However, the effect of the injected exogenous cells on mdx muscle mass and functional capacity has not been evaluated. This study evaluates the mass and functional capacity of the extensor digitorum longus (EDL) muscles of adult, male mdx mice that received intramuscular injections of primary myoblasts or of MDSCs (isolated by a preplating technique; Qu, Z., Balkir, L., van Deutekom, J.C., Robbins, P.D., Pruchnic, R., and Huard, J., J. Cell Biol. 1998;142:1257-1267) derived from normal mice. Evaluations were made 9 weeks after cell transplantation. Uninjected mdx EDL muscles have a mass 50% greater than that of age-matched C57BL/10J (normal) EDL muscles. Injections of either primary myoblasts or MDSCs have no effect on the mass of mdx EDL muscles. EDL muscles of mdx mice generate 43% more absolute twitch tension and 43% less specific tetanic tension then do EDL muscles of C57BL/10J mice. However, the absolute tetanic and specific twitch tension of mdx and C57BL/10J EDL muscles are similar. Injection of either primary myoblasts or MDSCs has no effect on the absolute or specific twitch and tetanic tensions of mdx muscle. Approximately 25% of the myofibers in mdx EDL muscles that received primary myoblasts react positively with antibody to dystrophin. There is no significant difference in the number of dystrophin-positive myofibers when MDSCs are injected. Regardless of the source of donor cells, dystrophin is limited to short distances (60-900 microm) along the length of the myofibers. This may, in part, explain the failure of cellular therapy to alter the contractile properties of murine dystrophic muscle.     

Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells

Cell transplantation holds promise as a potential treatment for cardiac dysfunction. Our group has isolated populations of murine skeletal muscle-derived stem cells (MDSCs) that exhibit stem cell-like properties. Here, we investigated the fate of MDSCs after transplantation into the hearts of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy (DMD). Transplanted MDSCs generated large grafts consisting primarily of numerous dystrophin-positive myocytes and, to a lesser degree, dystrophin-negative non-myocytes that expressed an endothelial phenotype. Most of the dystrophin-positive myocytes expressed a skeletal muscle phenotype and did not express a cardiac phenotype. However, some donor myocytes, located at the graft-host myocardium border, were observed to express cardiac-specific markers. More than half of these donor cells that exhibited a cardiac phenotype still maintained a skeletal muscle phenotype, demonstrating a hybrid state. Sex-mismatched donors and hosts revealed that many donor-derived cells that acquired a cardiac phenotype did so through fusion with host cardiomyocytes. Connexin43 gap junctions were not expressed by donor-derived myocytes in the graft. Scar tissue formation in the border region may inhibit the fusion and gap junction connections between donor and host cells. This study demonstrates that MDSC transplantation warrants further investigation as a potential therapy for cardiac dysfunction in DMD.     

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.     

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.     

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.     

Effect of VEGF on the Regenerative Capacity of Muscle Stem Cells in Dystrophic Skeletal Muscle

We have isolated a population of muscle-derived stem cells (MDSCs) that, when compared with myoblasts, display an improved regeneration capacity, exhibit better cell survival, and improve myogenesis and angiogenesis. In addition, we and others have observed that the origin of the MDSCs may reside within the blood vessel walls (endothelial cells and pericytes). Here, we investigated the role of vascular endothelial growth factor (VEGF)–mediated angiogenesis in MDSC transplantation–based skeletal muscle regeneration in mdx mice (an animal model of muscular dystrophy). We studied MDSC and MDSC transduced to overexpress VEGF; no differences were observed in vitro in terms of phenotype or myogenic differentiation. However, after in vivo transplantation, we observe an increase in angiogenesis and endogenous muscle regeneration as well as a reduction in muscle fibrosis in muscles transplanted with VEGF-expressing cells when compared to control cells. In contrast, we observe a significant decrease in vascularization and an increase in fibrosis in the muscles transplanted with MDSCs expressing soluble forms-like tyrosine kinase 1 (sFlt1) (VEGF-specific antagonist) when compared to control MDSCs. Our results indicate that VEGF-expressing cells do not increase the number of dystrophin-positive fibers in the injected mdx muscle, when compared to the control MDSCs. Together the results suggest that the transplantation of VEGF-expressing MDSCs improved skeletal muscle repair through modulation of angiogenesis, regeneration and fibrosis in the injected mdx skeletal muscle.     

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.     

Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation

Duchenne muscular dystrophy (DMD) is a severe progressive muscle-wasting disorder caused by mutations in the dystrophin gene. Studies have shown that bone marrow cells transplanted into lethally irradiated mdx mice, the mouse model of DMD, can become part of skeletal muscle myofibers. Whether human marrow cells also have this ability is unknown. Here we report the analysis of muscle biopsies from a DMD patient (DMD-BMT1) who received bone marrow transplantation at age 1 year for X-linked severe combined immune deficiency and who was diagnosed with DMD at age 12 years. Analysis of muscle biopsies from DMD-BMT1 revealed the presence of donor nuclei within a small number of muscle myofibers (0.5-0.9%). The majority of the myofibers produce a truncated, in-frame isoform of dystrophin lacking exons 44 and 45 (not wild-type). The presence of bone marrow-derived donor nuclei in the muscle of this patient documents the ability of exogenous human bone marrow cells to fuse into skeletal muscle and persist up to 13 years after transplantation.     

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.     

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.     

Functional Overloading of Dystrophic Mice Enhances Muscle-Derived Stem Cell Contribution to Muscle Contractile Capacity

Ambrosio F, Ferrari RJ, Fitzgerald GK, Carvell G, Boninger ML, Huard J. Functional overloading of dystrophic mice enhances muscle-derived stem cell contribution to muscle contractile capacity. Arch Phys Med Rehabil

Objectives

To evaluate the effect of functional overloading on the transplantation of muscle derived stem cells (MDSCs) into dystrophic muscle and the ability of transplanted cells to increase dystrophic muscle's ability to resist overloading-induced weakness.

Design

Cross-sectional.

Setting

Laboratory.

Animals

Male mice (N=10) with a dystrophin gene mutation.

Interventions

MDSCs were intramuscularly transplanted into the extensor digitorum longus muscle (EDL). Functional overloading of the EDL was performed by surgical ablation of the EDL's synergist.

Main Outcome Measures

The total number of dystrophin-positive fibers/cross-section (as a measure of stem cell engraftment), the average number of CD31+ cells (as a measure of capillarity), and in vitro EDL contractile strength. Independent t tests were used to investigate the effect of overloading on engraftment, capillarity, and strength. Paired t tests were used to investigate the effect of MDSC engraftment on strength and capillarity.

Results

MDSC transplantation protects dystrophic muscles against overloading-induced weakness (specific twitch force: control 4.5N/cm²±2.3; MDSC treated 7.9N/cm²±1.4) (P=.02). This improved force production following overloading is concomitant with an increased regeneration by transplanted MDSCs (MDSC: 26.6±20.2 dystrophin-positive fibers/cross-section; overloading + MDSC: 170.6±130.9 dystrophin-positive fibers/cross-section [P=.03]). Overloading-induced increases in skeletal muscle capillarity is significantly correlated with increased MDSC engraftment (R²=.80, P=.01).

Conclusions

These findings suggest that the functional contribution of transplanted MDSCs may rely on activity-dependent mechanisms, possibly mediated by skeletal muscle vascularity. Rehabilitation modalities may play an important role in the development of stem cell transplantation strategies for the treatment of muscular dystrophy.     

Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder associated with dystrophin deficiency that results in chronic inflammation and severe skeletal muscle degeneration. In DMD mouse models and patients, we find that IkappaB kinase/NF-kappaB (IKK/NF-kappaB) signaling is persistently elevated in immune cells and regenerative muscle fibers. Ablation of 1 allele of the p65 subunit of NF-kappaB was sufficient to improve pathology in mdx mice, a model of DMD. In addition, conditional deletion of IKKbeta in mdx mice elucidated that NF-kappaB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells. Furthermore, specific pharmacological inhibition of IKK resulted in improved pathology and muscle function in mdx mice. Collectively, these results underscore the critical role of NF-kappaB in the progression of muscular dystrophy and suggest the IKK/NF-kappaB signaling pathway as a potential therapeutic target for DMD.     

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.     

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.     

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.     

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.     

Physiological Characterization of Muscle Strength With Variable Levels of Dystrophin Restoration in mdx Mice Following Local Antisense Therapy

Antisense-induced exon skipping can restore the open reading frame, and thus correct the dystrophin deficiency that causes Duchenne muscular dystrophy (DMD), a lethal muscle wasting condition. Successful proof-of-principle in preclinical models has led to human clinical trials. However, it is still not known what percentage of dystrophin-positive fibers and what level of expression is necessary for functional improvement. This study directly address these key questions in the mdx mouse model of DMD. To achieve a significant variation in dystrophin expression, we locally administered into tibialis anterior muscles various doses of a phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 from the mRNA of murine dystrophin. We found a highly significant correlation between the number of dystrophin-positive fibers and resistance to contraction-induced injury, with a minimum of 20% of dystrophin-positive fibers required for meaningful improvement. Furthermore, our results also indicate that a relatively low level of dystrophin expression in muscle fibers may have significant clinical benefits. In contrast, improvements in muscle force were not correlated with either the number of positive fibers or total dystrophin levels, which highlight the need to conduct appropriate functional assessments in preclinical testing using the mdx mouse.