Marilyn A. Mitchell

Myosin gene mutation correlates with anatomical changes in the human lineage

Powerful masticatory muscles are found in most primates, including chimpanzees and gorillas, and were part of a prominent adaptation of Australopithecus and Paranthropus, extinct genera of the family Hominidae. In contrast, masticatory muscles are considerably smaller in both modern and fossil members of Homo. The evolving hominid masticatory apparatus—traceable to a Late Miocene, chimpanzee-like morphology—shifted towards a pattern of gracilization nearly simultaneously with accelerated encephalization in early Homo. Here, we show that the gene encoding the predominant myosin heavy chain (MYH) expressed in these muscles was inactivated by a frameshifting mutation after the lineages leading to humans and chimpanzees diverged. Loss of this protein isoform is associated with marked size reductions in individual muscle fibres and entire masticatory muscles. Using the coding sequence for the myosin rod domains as a molecular clock, we estimate that this mutation appeared approximately 2.4 million years ago, predating the appearance of modern human body size and emigration of Homo from Africa. This represents the first proteomic distinction between humans and chimpanzees that can be correlated with a traceable anatomic imprint in the fossil record.

STABLE RESTORATION OF THE SARCOGLYCAN COMPLEX IN DYSTROPHIC MUSCLE PERFUSED WITH HISTAMINE AND A RECOMBINANT ADENO-ASSOCIATED VIRAL VECTOR

Limb-girdle muscular dystrophies 2C–F represent a family of autosomal recessive diseases caused by defects in sarcoglycan genes. The cardiomyopathic hamster is a naturally occurring model for limb-girdle muscular dystrophy caused by a primary deficiency in δ-sarcoglycan. We show here that acute sarcolemmal disruption occurs in this animal model during forceful muscle contraction. A recombinant adeno-associated virus vector encoding human δ-sarcoglycan conferred efficient and stable genetic reconstitution in the adult cardiomyopathic hamster when injected directly into muscle. A quantitative assay demonstrated that vector-transduced muscle fibers are stably protected from sarcolemmal disruption; there was no associated inflammation or immunologic response to the vector-encoded protein. Efficient gene transduction with rescue of the sarcoglycan complex in muscle fibers of the distal hindlimb was also obtained after infusion of recombinant adeno-associated virus into the femoral artery in conjunction with histamine-induced endothelial permeabilization. This study provides a strong rationale for the development of gene therapy for limb-girdle muscular dystrophy.

Uniform Scale-Independent Gene Transfer to Striated Muscle After Transvenular Extravasation of Vector

Background—The muscular dystrophies exemplify a class of systemic disorders for which widespread protein replacement in situ is essential for treatment of the underlying genetic disorder. Somatic gene therapy will require efficient, scale-independent transport of DNA-containing macromolecular complexes too large to cross the continuous endothelia under physiological conditions. Previous studies in large-animal models have revealed a trade-off between the efficiency of gene transfer and the inherent safety of the required surgical and pharmacological interventions to achieve this. Methods and Results—Rats and dogs underwent limb or hemibody isolation via atraumatic tourniquet placement or myocardial isolation via heterotopic transplantation. Recombinant adenovirus (1013 particles per kilogram) or recombinant adeno-associated virus (1014 genome copies/kg) encoding the lacZ transgene was delivered through pressurized venous infusion without pharmacological mediators. Muscle exhibited almost 100% myofiber transduction in rats and dogs by X-galactosidase staining and significantly higher &bgr;-galactosidase levels compared with nonpressurized delivery. No significant difference was seen in &bgr;-galactosidase levels between 100- or 400-mm Hg groups. The <50-mm Hg group yielded inhomogeneous and significantly lower transgene expression. Conclusions—Uniform scale- and vector-independent skeletal and cardiac myofiber transduction is facilitated by pressurized venous infusion in anatomic domains isolated from the central circulation without pharmacological interference with cardiovascular homeostasis. We provide the first demonstration of uniform gene transfer to muscle fibers of an entire extremity in the dog, providing a firm foundation for further translational studies of efficacy in canine models for human diseases.

Modular Organization of Phylogenetically Conserved Domains Controlling Developmental Regulation of the Human Skeletal Myosin Heavy Chain Gene Family

The mammalian skeletal myosin heavy chain locus is composed of a six-membered family of tandemly linked genes whose complex regulation plays a central role in striated muscle development and diversification. We have used publicly available genomic DNA sequences to provide a theoretical foundation for an experimental analysis of transcriptional regulation among the six promoters at this locus. After reconstruction of annotated drafts of the human and murine loci from fragmented DNA sequences, phylogenetic footprint analysis of each of the six promoters using standard and Bayesian alignment algorithms revealed unexpected patterns of DNA sequence conservation among orthologous and paralogous gene pairs. The conserved domains within 2.0 kilobases of each transcriptional start site are rich in putative muscle-specific transcription factor binding sites. Experiments based on plasmid transfection in vitroand electroporation in vivo validated several predictions of the bioinformatic analysis, yielding a picture of synergistic interaction between proximal and distal promoter elements in controlling developmental stage-specific gene activation. Of particular interest for future studies of heterologous gene expression is a 650-base pair construct containing modules from the proximal and distal human embryonic myosin heavy chain promoter that drives extraordinarily powerful transcription during muscle differentiation in vitro.

Diaphragm remodeling and compensatory respiratory mechanics in a canine model of Duchenne muscular dystrophy

Ventilatory insufficiency remains the leading cause of death and late stage morbidity in Duchenne muscular dystrophy (DMD). To address critical gaps in our knowledge of the pathobiology of respiratory functional decline, we used an integrative approach to study respiratory mechanics in a translational model of DMD. In studies of individual dogs with the Golden Retriever muscular dystrophy (GRMD) mutation, we found evidence of rapidly progressive loss of ventilatory capacity in association with dramatic morphometric remodeling of the diaphragm. Within the first year of life, the mechanics of breathing at rest, and especially during pharmacological stimulation of respiratory control pathways in the carotid bodies, shift such that the primary role of the diaphragm becomes the passive elastic storage of energy transferred from abdominal wall muscles, thereby permitting the expiratory musculature to share in the generation of inspiratory pressure and flow. In the diaphragm, this physiological shift is associated with the loss of sarcomeres in series (∼ 60%) and an increase in muscle stiffness (∼ 900%) compared with those of the nondystrophic diaphragm, as studied during perfusion ex vivo. In addition to providing much needed endpoint measures for assessing the efficacy of therapeutics, we expect these findings to be a starting point for a more precise understanding of respiratory failure in DMD.

85. AAV9-Utrophin Prevents Myonecrosis in Dystrophic Mice and Dogs without Immunosuppression

The majority of mutations causing Duchenne muscular dystrophy (DMD) are multi-exon, frameshifting deletions, complicating therapy with recombinant dystrophin because of the potential for chronic immune recognition of the “non-self” protein. The paralogous protein utrophin is ubiquitously expressed at levels insufficient to prevent myonecrosis in animal models for DMD, but may confer central immunological tolerance through early developmental expression in the thymus. Here we show for a first time histological evidence for the complete prevention of myonecrosis in dystrophin-deficient striated muscles following systemic administration of an AAV9 vector carrying a 3.5 kb synthetic utrophin transgene (AAVµU). The cDNA was miniaturized by removal of domains least conserved in a comprehensive evolutionary comparison, and further optimized for maximal expression in striated muscle by using the codon bias of mammalian genes encoding contractile proteins. Administration of 1015 AAVµU vector genomes (vg) per kg to neonatal mice prevented centronucleation and saturated global recovery of the sarcoglycan complex, despite a subsequent tenfold increase in striated muscle mass with growth. In neonatal dystrophic dogs, intravenous injection of 1013.5 AAVµU vg/kg without immunosuppression restored sarcoglycan levels and normalized the myofiber size-distribution following a fourfold increase in muscle mass. Interferon-gamma ELISpot assays using utrophin-derived peptides revealed no reactivity in injected dogs, consistent with central immunological tolerance. These findings provide a rationale for high dose, neonatal gene therapy using utrophin as a “self” protein to forestall disability and mortality in DMD, while minimizing the risk of chronic immunotoxicity.

726. Prevention of Muscular Dystrophy by an AAV Vector Encoding a Nonimmunogenic Protein Based on the Evolution of Utrophin and Dystrophin

The majority of mutations causing Duchenne muscular dystrophy (DMD) are multi-exon, frameshifting deletions in the dystrophin gene. Expression of recombinant dystrophin in DMD therefore risks chronic immune recognition of the “non-self” protein. Early developmental expression of the paralogous protein utrophin in the thymus may confer central immunological tolerance to its peptide sequence. Here we address previously unanswered questions about the therapeutic efficacy and immunogenicity of “µutrophins” encoded by synthetic minigenes suitable for use in AAV. First we used a comparative phylogenomic approach to address a deceptively simple question with critical implications for this field: “which came first-dystrophin or the sarcomere”. This analysis provided evidence that the long rod-like domain of utrophin and dystrophin was “coopted” from a higher molecular weight protein in dynein-propelled animals lacking striated muscle. When considered in light of recent crystalographic studies, these results suggest that the full functionality of native dystrophin may be conferred by recombinant mini-utrophins with internally deleted rod domains that preserve inter-repeat folding and hence maximal tensile strength. We maximized recombinant µUtrophin (µU) expression from synthetic open reading frames by using the codon bias of striated myosin. Our blinded studies of mdx mice injected with a µU transgene are the first to show COMPLETE recovery of peripheral myofiber nucleation following a single systemic injection of an AAV vector. Sarcoglycan expression, as measured by western blot in the µU-treated mice, are restored to control levels throughout growth to skeletal maturity. To investigate the correlation between our histological findings and functionality, we validated a novel open field cage and attached running wheel system. Our blinded data show remarkable functional rescue in µU-treated mdx mice using this non-invasive test with relevance to the clinical assessment of disease progression in DMD. These data also strongly correlate with those derived from in vivo force grip and ex vivo force transducer measurements. Remarkably, a majority of mdx mice show complete normalization of serum creatine kinase (CK), a first for single-dose treatment by any clinically translatable modality. In dystrophic puppies, intravenous injection of a 30-fold lower relative dose of AAV9U fully restored sarcoglycan levels and normalized the myofiber size-distribution following a threefold increase in muscle mass. Interferon-gamma ELISpot assays using utrophin-derived peptides revealed no reactivity in injected dogs, consistent with central immunological tolerance. These findings suggest a rationale for neonatal gene therapy using utrophin as an internally deleted “self” protein in DMD to minimize the risk of chronic immunotoxicity. We outline a rigorous translational approach using escalating vector doses in GSHPMD dogs harboring a newly defined 5 megabase deletion encompassing the dystrophin ORF.

42. Uniform Scale-Independent Gene Transfer to Striated Muscle after Transvenular Extravasation of Vector

Top of pageAbstract The muscular dystrophies exemplify a class of systemic disorders for which widespread protein replacement in situ is essential for full complementation of the underlying genetic disorder. As a direct approach to this clinical challenge, somatic gene transfer will require efficient, scale-independent transport of DNA-containing macromolecular complexes too large to cross the continuous endothelia under physiological conditions. Previous studies in large animal models have revealed a trade-off between the efficiency of gene transfer and the inherent safety of the required surgical and pharmacological interventions. We tested the hypothesis that rapid, mechanical distention of the post-capillary venular endothelium by afferent infusion from a distal site would safely facilitate macromolecular transport from the vascular space to the striated muscle interstitium. We show that pressurized infusion through a large-bore catheters in either peripheral, superficial veins or the coronary sinus results in uniform, scale-and vector-independent transduction of myofibers in anatomic domains isolated from the remainder of the circulation. This approach is rapid, minimally invasive as applied to the isolated limb, and avoids pharmacological interference with cardiovascular homeostasis. We provide the first demonstration of uniform gene transfer to virtually 100% of the muscle fibers of an entire extremity in the dog, providing a firm foundation for studies of efficacy in canine models for human diseases. Additional data from a combination of angiographic, tracer dye, and marker gene studies suggests that this approach can be modified to meet the requirements for cardiac-specific or systemic gene delivery as appropriate in a variety of inherited and acquired diseases including hemophilia, muscular dystrophy, and cardiomyopathy. Figure 1 |[ndash]| |[beta]|-galactosidase levels of rat limb, rat cardiac and dog limb muscles after no treatment, vector delivery without afferent transvenular retrograde extravasation (ATVRX) and vector delivery with ATVRX. Figure 2 |[ndash]| LacZ expression after rat quadriceps (left), rat heart (middle), and dog vastus medialis (right) stained with x-galactosidase.