At the recent World Muscle Society meeting, which took place in Portugal, a great deal of new data on exon skipping was presented, both on the status of current human clinical trials and on the efforts of investigators to improve the efficiency of the technique by combining exon-skipping with gene and stem cell delivery. Below are a few highlights from the meeting with the caveat that it was impossible to cover every poster and talk. 


Muscle Stem Cells

Jenny Morgan of UCL Institute of Child Health in London found that irradiating the muscles of a host mouse produced good incorporation of donor satellite cells, although it was clear that too much radiation was also detrimental. Mayana Zatz of the University of Sao Paulo, Brazil, presented data demonstrating that human mesenchymal stem cells, originally isolated from adipose (fat) tissue, were able to fuse with host cells in the mouse without rejection. In the mice, she found human DNA, but no dystrophin—even so, the mdx mice showed some improvement in symptoms.  She showed video of two GRMD dogs who had undergone the same procedure and seemed to improve clinically. The dogs also both lived many years beyond their normal lifespan with the disease, but Zatz cautioned that the dogs are so variable the results can be difficult to interpret. The dogs showed some dystrophin in biopsy samples early on, but not after six months, suggesting that the transplant itself may have conveyed the  benefit if any. 


The Mystery of Ringo

Diane Shelton of UC San Diego described Labrador retrievers that had been discovered to make no detectable dystrophin and yet had no symptoms of weakness. The problem was discovered when an owner brought one of the lab pups (Ringo) to be neutered and regular blood work turned up what appeared to be a liver problem. Upon further investigation, the dog was found to have an elevated CK in the range of 30-40,000 IU/Liter and significantly decreased dystrophin staining. A muscle biopsy also showed “dystrophic pathology,” in other words evidence of inflammation and muscle cell death. The dog otherwise appeared perfectly healthy and seemed to have normal strength. It has since been determined that there are two breeders on the North East coast who have dogs with these characteristics and 12 of Ringo’s male relatives have now been identified. Efforts are underway to understand why the apparent lack of dystrophin in these dogs does not lead to muscle weakness—one hypothesis is that the dogs may have some other change in their genetic background that compensates for the loss of dystrophin. If such a change could be identified, it might lead to a new approach for a human therapy.


Stem Cells Corrected with Exon-skipping

Torrente’s group from the University of Milan described their efforts to remove stem cells from dogs that lack dystrophin (called “GRMD dogs”), correct the cells via exon-skipping, and then reintroduce the cells back into the same dogs. The group isolated a cell line from the muscles of two mildly affected GRMD dogs and three severely affected GRMD dogs. While the cells were being grown in culture, the investigators used a modified virus carrying the coding sequence for antisense oligonucleotides (AONs) that would allow skipping of exons 6-8 to put the dystrophin in the dogs’ muscle stem cells back into frame. The corrected cells of each dog were then reintroduced by intra-arterial injection into the femoral artery. Biopsies later showed dystrophin in 2-7% of fibers in the muscles of the injected leg.  The absolute amount of dystrophin protein ranged from nothing detectable to 6% of normal dystrophin. The dogs showed improvement in functional measurements such as the six minute timed walk and timed swimming and stair climbing exercises. Two affected dogs received their own uncorrected stem cells as controls and both died at age two, while all the dogs who received corrected cells have survived to at least four years. The cautionary note here is that the number of dogs used in these studies is so low that it’s very difficult to verify a functional improvement or even survival time. Based on these results the group is planning a clinical trial in Italy aimed at boys with DMD from age 6-14. In preparation they have been pre-screening donors of potential trial participants for cells.


Permanent Exon-skipping in the dog

A member of Thomas Voit’s group from the Institute of Myology in Paris described the results of injecting a modified virus carrying coding sequences for antisense oligos designed to skip exons 5-10. The vectors were injected intravenously into the foreleg and three months later dystrophin could be found in most of the leg muscles and other muscles in the body. Higher doses of vector produced higher amounts of dystrophin and the group presented evidence for what could be improved muscle strength as well. A human clinical trial to skip exon 53 is planned in boys from 6-15 years in London, Newcastle, Paris and Toulouse. 


Exon-skipping: progress in humans

Mathew Wood of the University of Oxford discussed the current status of exon skipping, listing as limitations: the variability from person-to-person and from dose-to-dose; the need to target the AONS more specifically to the muscle; and the inability to reach cardiac muscle with current methods. He went on to describe his own work in which he conjugated “CPPs” or “Cell-Penetrating-Peptides” to the AONS to help them get into muscle more efficiently. One such construct, PiPe5-PMO was able to get into both skeletal and cardiac muscle. His group is aiming to use this or a similar compound in a clinical trial within 2-3 years.


Prosensa, 96 week follow-up data on open label

Natalie Goemens of the University Hospital Leuvens reported on this open label follow-on to the original Prosensa phase I/IIa study using AONs injected under the skin to skip exon 51. The boys first participated in a dosing study in which different groups of boys received different doses of AONs and then they were put on a “drug holiday” that lasted from 6-15 months before being enrolled in the open label extension study at 6mg/kg. After 72 weeks in the open label study the boys were moved from a once a week injection to a pattern of 8 weeks on, and then 4 weeks off drug.  There are 12 subjects in this open label extension whose ages now range from 7.9-16.3 years of age. When the group started the trial, one boy was not walking, several were walking well and some were in decline. At the end of 96 weeks, two of the boys who were in the group that was declining have lost ambulation. Overall, the six minute walk test shows improvement over baseline (for boys still walking) but the numbers in this study are very low and there is no placebo. They also found evidence for some protein in the urine that investigators felt bears watching, although it goes away when the boys are off drug. In addition, Prosensa has PRO44 in a dose escalation study similar in design to the phase I/II for PRO 51, except that they are doing pre-, post- and intermediate biopsies. PRO45 and PRO53 are due to start next year and PRO52 and PRO53 are in the preclinical stages. Dr. Goemans discussed overall concerns about exon skipping:

  • The functionality and stability of the shorter versions of dystrophin produced by this technique is not known—it’s likely that some forms of dystrophin that can produced by skipping exons will be non-functional
  • It’s not clear how effective the treatment will be
  • Effects of chronic dosing are not known
  • No current ability to treat the heart (standard AONs don’t get into heart muscle)
  • Each set of AONs must be approved as a separate drug
  • Endpoints in a degenerative disease in a pediatric population mean different stages produce different slopes;  there’s a lack of measures and scales that are relevant across the entire spectrum of the disease
  • There is a need for larger placebo-controlled studies
  • There is a need better upper limb assessments
  • Finally, everyone needs to answer the question “What are we aiming for?  It’s an open question, but “we are aiming for improvement”


Skipping Duplications

Kevin Flanigan of the Research Institute at Nationwide Children’s Hospital in Ohio reported on efforts in his lab to develop lines of cells derived from boys with duplications. So far he has developed 11 human cell lines with different representative duplications that have been “immortalized” so that they will continue to divide and can be maintained in the lab indefinitely.  He described preliminary results with a duplication of exons 8 and 9 and was able to skip them as a single unit. He also described results with a cell line containing an exon 2 duplication (duplications are found in 7% of patients and the most common duplication is in exon 2). The first question to ask is whether you can get a functional protein if exon 2 is skipped and there seems to be evidence that the loss of this exon actually leads to no muscle weakness. Using AONs in cultured cells, the group was able to skip an exon 2 duplication in about 40% of the dystrophin made by those cells. He also reported that his group has recently made a mouse model with an exon 2 duplication that could be used for additional studies on correcting duplications.


Gene Therapy

Kevin Flanigan stood in for Jerry Mendell of the Research Institute at Nationwide Children’s Hospital in Ohio to talk about current strategies for gene therapy in DMD, BMD and Limb-girdle muscular dystrophy. A publication last year in the New England Journal of Medicine on the first gene therapy trial for DMD in the US made waves when the investigators reported that some boys seem to have pre-existing immunity to the dystrophin protein due to the presence of rare “revertant fibers” that undergo a kind of natural exon-skipping to make a protein. Scientists have long hoped that the presence of these revertant fibers would allow the boys’ immune systems to accept newly introduced dystrophin. What Mendell’s group actually found was the opposite—it seems that the dystrophin in these revertant fibers was recognized by the immune system.  The investigators found that immune cells called (killer) T cells recognized the same region of the dystrophin protein as that present in the revertant fibers.  What isn’t clear even now is whether or not the presence of these T cells means that the boys’ bodies would recognize as foreign and destroy any new dystrophin introduced as a therapy. To follow up on this work, Mendell’s group examined the biopsies of 63 boys with DMD and found that 47% had revertant fibers. They then looked for the presence of T cells that recognize dystrophin and found that the boys on steroids were less likely to have these cells, suggesting that the T cell response, if it’s a problem, might be modified by steroids or other immunosuppressants. He then described preliminary results from a phase I/II clinical trial to deliver alpha-sarcoglycan by injection to a foot muscle of participants with limb-girdle muscular dystrophy. They investigators reported no pre-existing antibodies to alpha-sarcoglycan and only one case of antibodies developing post-injection. 5 of 6 of the participants showed sustained alpha-sarcoglycan gene expression on biopsy—the patient who did not show alpha-sarcoglycan was the one with antibodies. A trial is being planned now to deliver the same vector through the bloodstream to affect all of the muscles of a single limb—participants with pre-existing antibodies to alpha-sarcoglycan will be excluded from that study. Flanigan also reported good progress on developing reliable outcome measures to correlate knee extensor strength with the six and two-minute timed walk tests. This is in preparation for starting the PPMD-funded gene therapy trial to deliver follistatin to the quad muscles of participants with Becker muscular dystrophy and inclusion body myositis.


Blog by Sharon Hesterlee, Ph.D.
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Comment by Sharon Hesterlee on November 9, 2011 at 6:50am

Hi Ofelia--I don't know the answers to the specific questions about individual boys in the Prosensa extension study but I understand why you are asking.  They did report that overall the boys in the study did better than expected on functional tests so even if a couple stopped walking they may have done so later than would be anticipated.  It may also matter how the boys were and at what stage in the disease process they started the drug.  She didn't get into details like amount of dystrophin in biopsies.


As for the concern about the functionality of the dystrophin produced by exon-skipping, they think that skipping exon 51 should produce a fairly functional protein.  I think she was just stating a general concern, which is a very important point, that any time you create a protein that is missing pieces it may not work as well as the full-length protein.  All of these shortened dystrophins produced from skipping different exons will likely have varying degrees of functionality.  The dystrophin protein is pretty unique in that you can make these shorter dystrophins and still get them to work to some degree.  Smaller proteins and enzymes, in particular, often won't tolerate those kinds of changes at all.  Dystrophin is a big structural protein--the analogy I would make is that it's like a steel girder that can still prop up a building even if it's a bit shorter than intended.  An enzyme would be more like a car engine that just won't run if you remove a piece.  But that being said, there are still parts of the dystrophin protein that are critical for function and if you remove them via exon-skipping the steel girder won't work. 


Comment by Ofelia Marin on November 8, 2011 at 10:18pm

I just wonder why she makes the assumption that some will not be functional. Does she have any data in animals or humans to prove that? Is that just an assumption, guess? It is just strange to state this if they do not have evidence. We all know that the progression is different from BMD patient to patient, I just thought that she had some data proving that claim that some shorter dystrophin will not be functional. I would also like to know what % positive fibers was seen in the 2 boys who stopped walking, was it low, was it due to the fact that the disease was so advanced to begin with (not much muscle mass left), was it due to something else, where they overweight? For example, I do not expect to see any improvement in 6-mwt in an obese boy regardless if they now have some % dystrophin for a number of weeks. They should have more info than the fact that they stopped walking. What about the boys not on decline at trial start, have any of them declined?

Comment by Sharon Hesterlee on November 8, 2011 at 4:50pm

Hi Ofelia: 

I think she just meant that some of the exons that you can skip to put the dystrophin gene back into frame may not actually lead to a functional dystrophin.  When you have an in-frame deletion you create a dystrophin protein that is missing some pieces and is "shorter" that a  normal dystrophin. We've been guessing, based on what you see with similar in-frame mutations in Becker what the outcome will be, but we don't always have BMD examples for every skipped exon.  I don't have additional details beyond what I reported on the Exon 51 study.  It was clear that a couple of the boys had stopped walking though.



Comment by Ofelia Marin on November 8, 2011 at 11:01am

Hi Sharon, what exactly does this mean? "The functionality and stability of the shorter versions of dystrophin produced by this technique is not known—it’s likely that some forms of dystrophin that can produced by skipping exons will be non-functional". What are the "shorter" versions of dystrophin?

Do you have the details about skipping 51 extension, how many were in decline out of 12?

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