Robert Crawford

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.

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.

Health as a meaningful social practice

The pursuit of health has become a highly valued activity in modern and contemporary life, commanding enormous resources and generating an expansive professionalization and commercialization along with attendant goods, services and knowledge. Health has also become a focal, signifying practice. As a ‘key word’, health is constructed in relation to social structures and experience and systematically articulated with other meanings and practices. Although the cogency of health as a practical concept is largely a product of the enormous influence of modern medicine, medical conceptions have never been able to contain the irrepressible proliferation of meanings associated with health. The meaningful - and ideological - practices of health can be illustrated by comparing three periods in American culture: (1) the late 19th and early 20th century; (2) the 1970s and 1980s; and (3) the first years of the 21st century.

Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin

Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult mdx (dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin expression cassettes are able to efficiently transduce muscles of 1-yr-old mdx mice. Single i.m. injections of viral vector restored dystrophin production to 25–30% of mouse limb muscle 1 mo after injection. Furthermore, functional tests of virally transduced muscles revealed almost 40% correction of their high susceptibility to contraction-induced injury. Our results show that functional abnormalities of dystrophic muscle can be corrected by delivery of full-length dystrophin to adult, immunocompetent mdx mice, raising the prospects for gene therapy of muscular dystrophies.

Gallstones play a significant role in Salmonella spp. gallbladder colonization and carriage

Salmonella enterica serovar Typhi can colonize the gallbladder and persist in an asymptomatic carrier state that is frequently associated with the presence of gallstones. We have shown that salmonellae form bile-mediated biofilms on human gallstones and cholesterol-coated surfaces in vitro. Here, we test the hypothesis that biofilms on cholesterol gallbladder stones facilitate typhoid carriage in mice and men. Naturally resistant (Nramp1+/+) mice fed a lithogenic diet developed cholesterol gallstones that supported biofilm formation during persistent serovar Typhimurium infection and, as a result, demonstrated enhanced fecal shedding and enhanced colonization of gallbladder tissue and bile. In typhoid endemic Mexico City, 5% of enrolled cholelithiasis patients carried serovar Typhi, and bacterial biofilms could be visualized on gallstones from these carriers whereas significant biofilms were not detected on gallstones from Escherichia coli infected gallbladders. These findings offer direct evidence that gallstone biofilms occur in humans and mice, which facilitate gallbladder colonization and shedding.

Risk Ritual and the Management of Control and Anxiety in Medical Culture

Medical culture in advanced technological societies is characterized by an abundance of warnings about health hazards, along with an equally abundant flow of prescriptive advice for protecting individuals against them. Knowledge of health hazards also defines living and working environments and has spawned a politics of regulatory control. These features of contemporary health consciousness and action, along with deepening insecurities in the larger body politic, precipitate a spiral of anxiety and control. The spiral disrupts the presumed security derived from medical knowledge and medically informed behaviors and threatens to erode the boundaries of sanctioned action for health protection. In the context of the volatile political economy of health, the spiral, with its excesses of anxiety and demands for unauthorized controls, elicits efforts to contain it. I explore the symbolic dimensions of risk, what I call ‘risk ritual’ - the prevailing form of managing this troublesome contradiction.

Efficient Rescue of Gutted Adenovirus Genomes Allows Rapid Production of Concentrated Stocks Without Negative Selection

Gutted adenoviral (Ad) vectors have a greater cloning capacity and elicit less immune response than conventional Ad vectors. Unfortunately, clinical use of gutted vectors has been slowed by production difficulties, including low yield and a tendency for recombinant virus to emerge. These two problems are related, because expansion of dilute vector stocks requires selective pressure against helper virus. The ability to rescue gutted virus at high titer would lessen the requirement for selective pressure, thereby limiting the advantage afforded to undesirable recombinants. We tested gutted virus rescue from plasmids and from synthetic terminal protein (TP)-DNA complexes by transfection/infection or cotransfection with various forms of helper viral DNA. Optimal rescue required cotransfection of gutted and helper genomes with identical origins of replication. Transfection/infection, which introduces unequal origins, was 30 times less efficient than cotransfection of genomes that had been released from plasmid DNA and bore identical origins. Cotransfection of TP-linked genomes was several times more efficient than that of unlinked genomes, yielding average gutted viral titers above 10(7) transducing units (TU)/ml. In addition, we found that limited expression of Cre recombinase doubled the yield of gutted virus. Using these techniques, gutted viruses can be rescued at titers greater than 3 x 10(7) TU/ml, about 100 times higher than is usually achieved. Finally, we found that high-titer lysates could be serially passaged on Cre-negative cells without loss of titer, further reducing selective pressure. These methods allow large-scale production of gutted virus in three or four serial passages, while minimizing exposure to Cre recombinase.

Fluorophore-labeled myosin-specific antibodies simplify muscle-fiber phenotyping.

Skeletal muscles are frequently analyzed for composition of phenotypically distinct myofibers, as a functional determinant. We describe an improved myofiber phenotyping procedure, involving cryosection co‐incubation with fluorophore‐labeled myosin heavy‐chain (MyHC)‐isoform–specific antibodies. This technique identifies multiple fiber “types” on a single section, thereby reducing reagents and processing, and offers side‐by‐side comparison of samples from multiple species including mice. These advances are valuable for studying the physiological attributes of skeletal muscle in health and disease. Muscle Nerve, 2007