Jeffrey S. Chamberlain

Systemic delivery of genes to striated muscles using adeno-associated viral vectors

A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant adeno-associated virus pseudotype 6 vectors. The inclusion of vascular endothelium growth factor/vascular permeability factor, to achieve acute permeabilization of the peripheral microvasculature, enhanced tissue transduction at lower vector doses. This technique enabled widespread muscle-specific expression of a functional micro-dystrophin in the skeletal muscles of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy. We propose that these methods may be applicable for systemic delivery of a wide variety of genes to the striated muscles of adult mammals.

X-linked dilated cardiomyopathy. Molecular genetic evidence of linkage to the Duchenne muscular dystrophy (dystrophin) gene at the Xp21 locus.

Background. X‐linked cardiomyopathy (XLCM) is a rapidly progressive primary myocardial disorder presenting in teenage males as congestive heart failure. Manifesting female carriers have later onset (fifth decade) and slower progression. The purpose of this study was to localize the XLCM gene locus in two families using molecular genetic techniques. Methods and Results. Linkage analysis using 60 X‐chromosome‐specific DNA markers was performed in a previously reported large XLCM pedigree and a smaller new pedigree. Two‐point and multipoint linkage was calculated using the LINKAGE computer program package. Deletion analysis included multiplex polymerase chain reaction (PCR). Dystrophin protein was evaluated by Western blotting with N‐terminal and C‐terminal dystrophin antibody. Linkage of XLCM to the centromeric portion of the dystrophin or Duchenne muscular dystrophy (DMD) locus at Xp21 was demonstrated with combined maximum logarithm of the scores of +4.33, &thgr;=0 with probe XJ1.1 (DXS206) using two‐point linkage and +4.81 at XJ1.1 with multipoint linkage analysis. LOD scores calculated using other proximal DMD genomic and cDNA probes and polymerase chain reaction polymorphisms supported linkage. No deletions were observed. Abnormalities of cardiac dystrophin were shown by Western blotting with N‐terminal dystrophin antibody, whereas skeletal muscle dystrophin was normal, suggesting primary involvement of the DMD gene with preferential involvement of cardiac muscle. Conclusions. XLCM is due to an abnormality within the centromeric half of the dystrophin genomic region in heart. This abnormality could be due to 1) a point mutation in the 5′ region of the DMD coding sequence preferentially affecting cardiac function, 2) a cardiac‐specific promoter mutation that alters expression in this tissue, 3) splicing abnormalities, resulting in an abnormal cardiac protein, or 4) deletion mutations undetectable by Southern and multiplex polymerase chain reaction analysis. (Circulation 1993;87:1854‐1865)

Modular flexibility of dystrophin: Implications for gene therapy of Duchenne muscular dystrophy

Attempts to develop gene therapy for Duchenne muscular dystrophy (DMD) have been complicated by the enormous size of the dystrophin gene. We have performed a detailed functional analysis of dystrophin structural domains and show that multiple regions of the protein can be deleted in various combinations to generate highly functional mini- and micro-dystrophins. Studies in transgenic mdx mice, a model for DMD, reveal that a wide variety of functional characteristics of dystrophy are prevented by some of these truncated dystrophins. Muscles expressing the smallest dystrophins are fully protected against damage caused by muscle activity and are not morphologically different from normal muscle. Moreover, injection of adeno-associated viruses carrying micro-dystrophins into dystrophic muscles of immunocompetent mdx mice results in a striking reversal of histopathological features of this disease. These results demonstrate that the dystrophic pathology can be both prevented and reversed by gene therapy using micro-dystrophins.

Identification and Characterization of the Dystrophin Anchoring Site on β-Dystroglycan

Dystrophin, the product of the Duchenne muscular dystrophy gene, is tightly associated with the sarcolemmal membrane to a large glycoprotein complex. One function of the dystrophin-glycoprotein complex is to link the cytoskeleton to the extracellular matrix in skeletal muscle. However, the molecular interactions of dystrophin with the membrane components of the dystrophin-glycoprotein complex are still elusive. Here, we demonstrate and characterize a specific interaction between β-dystroglycan and dystrophin. We show that skeletal muscle and brain dystrophin as well as brain dystrophin isoforms specifically bind to β-dystroglycan. To localize and characterize the dystrophin and β-dystroglycan interaction domains, we reconstituted the interaction in vitro using dystrophin fusion proteins and in vitro translated β-dystroglycan. We demonstrated that the 15 C-terminal amino acids of β-dystroglycan constituted a unique binding site for the second half of the hinge 4 and the cysteine-rich domain of dystrophin (amino acids 3054-3271). This dystrophin binding site is located in a proline-rich environment of β-dystroglycan within amino acids 880-895. The identification of the interaction sites in dystrophin and β-dystroglycan provides further insight into the structure and the molecular organization of the dystrophin-glycoprotein complex at the sarcolemma membrane and will be helpful for studying the pathogenesis of Duchenne muscular dystrophy.

Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy

Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated alpha-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.

rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice.

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector–mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.

Force and power output of fast and slow skeletal muscles from mdx mice 6‐28 months old

1 Differences in the effect of age on structure‐function relationships of limb muscles of mdx (dystrophin null) and control mice have not been resolved. We tested the hypotheses that, compared with limb muscles from age‐matched control mice, limb muscles of 6‐ to 17‐month‐old mdx mice are larger but weaker, with lower normalised force and power, whereas those from 24‐ to 28‐month‐old mdx mice are smaller and weaker. 2 The maximum isometric tetanic force (Po) and power output of limb muscles from 6‐, 17‐, 24‐ and 28‐month‐old mdx and control mice were measured in vitro at 25 °C and normalised with respect to cross‐sectional area and muscle mass, respectively. 3 Body mass at 6 and 28 months was not signifcantly different in mdx and control mice, but that of control mice increased 16 % by 17 months and then declined 32 % by 28 months. The body masses of mdx mice declined linearly with age with a decrease of 25 % by 28 months. From 6 to 28 months of age, the range in the decline in the masses of EDL and soleus muscles of mdx and control mice was from 16 to 28 %. The muscle masses of mdx mice ranged from 9 % to 42 % greater than those of control mice at each of the four ages and, even at 28 months, the masses of EDL and soleus muscles of mdx mice were 17 % and 22 % greater than control values. 4 For mdx mice of all ages, muscle hypertrophy was highly effective in the maintenance of control values for absolute force for both EDL and soleus muscles and for absolute power of soleus muscles. Throughout their lifespan, muscles of mdx mice displayed significant weakness with values for specific Po and normalised power ≈20 % lower than values for control mice at each age. For muscles of both strains, normalised force and power decreased ≈28 % with age, and consequently weakness was more severe in muscles of old mdx than in those of old control mice.

Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma

Duchenne muscular dystrophy (DMD) is the most common, lethal genetic disorder of children. A number of animal models of muscular dystrophy exist, but the most effective model for characterizing the structural and functional properties of dystrophin and therapeutic interventions has been the mdx mouse. Despite the ~20 years of investigations of the mdx mouse, the impact of the disease on the life span of mdx mice and the cause of death remain unresolved. Consequently, a life span study of the mdx mouse was designed that included cohorts of male and female mdx and wild‐type C57BL/10 mice housed under specific pathogen‐free conditions with deaths restricted to natural causes and with examination of the carcasses for pathology. Compared with wild‐type mice, both mdx male and female mice had reduced life spans and displayed a progressively dystrophic muscle histopa‐thology. Surprisingly, old mdx mice were prone to develop muscle tumors that resembled the human form of alveolar rhabdomyosarcoma, a cancer associated with poor prognosis. Rhabdomyosarcomas have not been observed previously in nontransgenic mice. The results substantiate the mdx mouse as an important model system for studies of the pathogenesis of and potential remedies for DMD.–Chamberlain, J. S., Metzger, J., Reyes, M., Townsend, D., Faulkner, J. A. Dystrophin‐deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyo‐sarcoma. FASEB J. 21, 2195–2204 (2007)

Sarcolemma-localized nNOS is required to maintain activity after mild exercise

Many neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response that is disproportionate to activity level. This fatigue is not necessarily correlated with greater central or peripheral fatigue in patients, and some patients experience severe fatigue without any demonstrable somatic disease. Except in myopathies that are due to specific metabolic defects, the mechanism underlying this type of fatigue remains unknown. With no treatment available, this form of inactivity is a major determinant of disability. Here we show, using mouse models, that this exaggerated fatigue response is distinct from a loss in specific force production by muscle, and that sarcolemma-localized signalling by neuronal nitric oxide synthase (nNOS) in skeletal muscle is required to maintain activity after mild exercise. We show that nNOS-null mice do not have muscle pathology and have no loss of muscle-specific force after exercise but do display this exaggerated fatigue response to mild exercise. In mouse models of nNOS mislocalization from the sarcolemma, prolonged inactivity was only relieved by pharmacologically enhancing the cGMP signal that results from muscle nNOS activation during the nitric oxide signalling response to mild exercise. Our findings suggest that the mechanism underlying the exaggerated fatigue response to mild exercise is a lack of contraction-induced signalling from sarcolemma-localized nNOS, which decreases cGMP-mediated vasomodulation in the vessels that supply active muscle after mild exercise. Sarcolemmal nNOS staining was decreased in patient biopsies from a large number of distinct myopathies, suggesting a common mechanism of fatigue. Our results suggest that patients with an exaggerated fatigue response to mild exercise would show clinical improvement in response to treatment strategies aimed at improving exercise-induced signalling.

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.