Article In Brief
Micro-utrophin gene therapy demonstrated strong proof-of-principle for therapeutic potential in animal models of Duchenne muscular dystrophy. Clinical trials are planned for as early as 2020.
Gene therapy with a highly truncated form of utrophin safely stopped muscle deterioration, providing complete rescue in a mouse model of Duchenne muscular dystrophy (DMD) and widespread correction in a dog model of the disease, investigators reported in the October 7 online issue of Nature Medicine.
What's more, the therapy did not provoke an immune response.
The study demonstrates strong proof-of-principle for the therapeutic potential of this highly engineered and miniaturized dystrophin substitute, the study authors said, and it sets the stage for clinical trials in boys with DMD, which may begin as soon as 2020.
“We believe that treatment with micro-utrophin is likely to be at least as efficacious, and potentially far less immunogenic, than the dystrophin approach,” said the study's lead author Hansell H. Stedman, MD, associate professor of surgery at the Perelman School of Medicine at the University of Pennsylvania.
The challenge for gene therapy of DMD with the dystrophin gene itself has been twofold: The gene is enormous and requires removal of major sections to fit into an adeno-associated virus (AAV) vector. These mini-dystrophin genes have been tested in the clinic with some success, but the expression of such micro-dystrophin in boys with DMD appears to have provoked an immune response, according to a 2010 report in the New England Journal of Medicine. This raised concerns about whether a large-scale introduction of the gene, which would be required for therapeutic benefit, would be safe. In November, the US Food and Drug Administration halted a DMD gene therapy trial by Solid Biosciences using dystrophin via an AAV after one of the study participants experienced blood- and kidney-related adverse events.
“We believe we have the data we need to move to the clinic, and we are exploring a range of possible opportunities to get it there as fast as possible in a responsible way.”
—DR. HANSELL H. STEDMAN
Utrophin is a cytoskeletal protein with significant homology to dystrophin that was discovered in the early days of dystrophin research. It is found at the cell membrane of many tissues and is enriched at the neuromuscular junction. During fetal development, utrophin also localizes to the sarcolemma, but over time is replaced there by dystrophin. Dystrophin and utrophin share similar N-terminal and C-terminal domains, important for tying the proteins to the cytoskeleton and sarcolemma, and both include a long central section composed of multiple repeated units, the number of which differ between the two proteins. Therapeutic strategies to upregulate endogenous utrophin have so far not been successful.
The utrophin gene is smaller than that for dystrophin but is still much too large to fit into the AAV capsid, so Dr. Stedman began with an evolutionary bioinformatic analysis of dystrophin across the animal kingdom “to determine what we could leave out.”
That analysis led to the hypothesis that much of the central portion of the protein might be expendable, despite its presumed role as a “shock absorber” for the physical stresses of contracting muscle, and its role as a platform for neuronal nitric oxide synthase (nNOS), which regulates vascular tone.
“This study served as a test of that hypothesis,” Dr. Stedman said. The final micro-utrophin gene was less than one-third the size of the full-length dystrophin gene.
Study Design, Findings
In a blinded and randomized trial, the team injected AAV vector containing the gene into neonatal mdx mice, the standard mouse model of DMD. After three weeks, they found that treatment completely normalized the degree of myofiber centronucleation, to a level indistinguishable from wild-type mice. This test is “the most sensitive indicator of myoprotection in the mdx mouse,” Dr. Stedman said, “and we found they looked completely cured.”
Serum creatine kinase was also normalized, and expression of micro-utrophin was sustained in both skeletal and cardiac muscle for the four months of follow-up. Treated mice were also indistinguishable from wild-type on tests of strength and activity level.
The golden retriever muscular dystrophy dog is an important large animal model for DMD, but presents several challenges, Dr. Stedman noted. First, the dog increases in weight from birth to adulthood by one hundred-fold, compared to only twenty-fold for mice and humans. Injecting enough vector at birth to provide treatment for a full-size adult dog is not practical; it's costly, making assessments in full-grown dogs problematic. Second, the colonies of research dogs are much less inbred than those of the mdx mouse, and individual dogs display important baseline differences in function, meaning that rigorous, blinded tests of function with statistical power would require many more animals than is possible.
“No one has done that,” Dr. Stedman noted. “All the studies in the golden retriever have been open-label.”
That led Dr. Stedman to focus on histological and biochemical tests as the proof of principle for micro-utrophin therapy in the model. The team randomized five dogs to receive intravenous treatment at four to seven days of age, at two different doses, without immunosuppression. After six weeks, they saw multiple indications of benefit, including wild-type levels of the sarcoglycan complex at the sarcolemma, and a reduction in myonecrosis, with no evidence of immune reaction.
To better mimic the likely clinical scenario of delayed treatment, they injected two other dogs at 7.5 weeks along with short-term prednisone anti-inflammatory treatment. Similar benefit was observed in these animals.
Surprisingly, neither the mouse nor the dog showed any signs that the absence of nNOS scaffolding made necessary by the shortening of the utrophin gene had any clinical effect. The reasons for this are unclear, Dr. Stedman said, but raise the question of whether the importance of nNOS at the sarcolemma is limited to periods of extreme muscle exertion.
Finally, the team tested the treatment in a second dog model, the German short-haired pointer, which, unlike the golden retriever, expresses no dystrophin at all, and so might be expected to have a stronger immune response than the retriever, which retains some expression. Treatment with AAV carrying a micro-dystrophin gene led to a strong T-cell mediated response and lack of long-term gene expression. In contrast, treatment with AAV/micro-utrophin provoked no such response, and continued expression after four weeks.
Because of the limited number of successful therapies for DMD, “The ability of the animal models and ages used in this study to predict success in boys with Duchenne muscular dystrophy is not yet established,” George Dickson, PhD, emeritus professor of molecular cell biology at Royal Holloway University of London told Neurology Today.
Dr. Dickson, who was a co-discoverer of utrophin in 1991, said, “It is not yet clear what the clinical significance of the lack of nNOS scaffolding may be for this therapy, at least in part because the importance of nNOS signaling in DMD is poorly understood.”
“The main advantage of utrophin is that it is non-immunogenic,” commented Michio Hirano, MD, FAAN, professor of neurology and director of the H. Houston Merritt Neuromuscular Research Center at Columbia University Medical Center in New York City.
“I think the specific utrophin construct used here was well thought through,” he added, “and the indications are that it is rather successful. But it is very difficult to extrapolate from the dog to humans” without functional confirmation of the histologic benefit. “Nevertheless, the results are striking and very encouraging.”
In an editorial accompanying the Nature Medicine paper, Kay E. Davies, PhD, of the MDUK Oxford Neuromuscular Center at the University of Oxford in the UK, and Jeffrey S. Chamberlain, PhD, of the neurology, medicine and biochemistry department at the University of Washington School of Medicine, agreed that the findings were promising. They offered an analysis of the differences between using the dystrophin and utrophin genes. They noted that various studies using the truncated dystrophin gene had modified phenotypes into mild forms of muscular dystrophy such as Becker MD. But they also offered cautious optimism about the alternative utrophin.
“Although current gene therapy trials for DMD are showing encouraging results, a rigorous test of dystrophin immunity has not been performed, and the availability of μ-Utro vectors provides an important alternative approach to treating this devastating human genetic disorder,” they wrote.
Dr. Stedman agreed. A clinical trial is likely the only way to confirm whether utrophin can live up to its promise, and such a trial is likely to come soon, he said, perhaps as early as the coming year. “We believe we have the data we need to move to the clinic, and we are exploring a range of possible opportunities to get it there as fast as possible in a responsible way.”
Dr. Stedman is an inventor on several patents assigned to the University of Pennsylvania and subject to the institutional patent policies, which may include royalty distribution in the event of licensure. Dr. Chamberlain is a member of the scientific advisory board for Solid Biosciences. Dr. Davies had no declared conflicts.