There’s often a fine line between “out-of-the–box” and “out-in-left-field.” I guess when my blog about Duchenne research starts with that statement you know I’m probably going to come down one way or another about something, but it might not be the direction you’re expecting.

This community lives with a lot of hope and hype. Many of you have learned that when you hear something too good to be true that it’s probably not. You know the principles of scientific experimentation, you understand the need for placebos (even if you loath the idea), and you know that progress tends to come in little baby steps and not giant leaps. You may have even found yourself in arguments with people who want to try unproven exotic devices or cell therapies.

But I think it’s also important to remember, at the end of the day, that nothing about the scientific method dictates a priori what answer is right and what is wrong. The true spirit of science is the ability to embrace anything for which there is evidence. Galileo was branded a heretic and imprisoned for supporting what he felt the evidence told him about the planets revolving around the sun and Lister’s theory of germs as a source of infection was mocked for years in surgeries where people left sicker than they arrived. For many years I’ve had a scrap of paper taped to my computer with the quote “I am too much of a skeptic to deny the possibility of anything” (from Thomas Huxley, a contemporary of Charles Darwin).

So all of that being said, I just returned from a visit to the MDA Western Australia meeting in Perth and I want to address some of the elephants in the room surrounding the technique of exon skipping. Just for reference for those of you who are geographically challenged like me, if you get on a plane in LA and fly through five feature films, two meals and a variety of turbulence and finally land in Sydney…you are still five hours from Perth. It’s a lovely city nestled on the West side of Australia along a river and the Indian Ocean and, I understand from the locals, it’s the most isolated state capital in the world. It’s also the home of a thriving group of researchers and clinical investigators focused on Duchenne muscular dystrophy and other muscle disease, including Steve Wilton, Sue Fletcher, Nigel Liang and Miranda Grounds. In addition to the science, I heard some fantastic talks from parents and young men with DMD.

Dr. Steve Wilton, as many of you may know, was one of the pioneers who developed the technique of exon skipping that we are now following so closely as the Prosensa/GSK and AVI trials progress. Most cases of DMD occur when a piece of the gene is deleted and when the number of nucleotides that makes up that missing piece is not a multiple of three--this throws the three letter “words” of the genetic code out of sync or “out-of-frame” and the protein produced from this transcript is just gobbledygook. When, about a third of the time, the missing piece of genetic code is made up of a multiple of three nucleotides you get an “in-frame” deletion, which is usually associated with milder symptoms. Guys with Becker often have in-frame deletions.

As early as 1999 Wilton’s group had published its first evidence that you could “trick” muscle cells into carving out a carefully measured extra piece of the code that is normally left in, called an “exon” (in addition to the already missing piece) during the normal trimming process of the dystrophin RNA, bringing the sequence back into frame so that a protein could still be made. It’s the equivalent of tidying up the rough edges of a cut by removing more material before putting the clean edges back together. Although a Japanese group actually published the first report describing the use of antisense oligonucleotides to alter the reading frame of dystrophin in cultured cells in 1996, Wilton’s group was responsible for much of the early work to perfect the technique in muscle cells. PPMD and MDA were early funders and supporters of the technology during its development.

Unfortunately, the very large dystrophin gene is susceptible to losing pieces of its genetic code at any point along its length, although there are deletion hot spots where such mutations are more common. This means that different exons would need to be carved out or “skipped” to accommodate deletions in different parts of the gene—this is not a one-size-fits-all solution. AVI Biopharma and GSK both have exon 51 skipping strategies in the clinic ( the GSK exon 51 program originated at Prosensa and was funded, in part, by Cure Duchenne) and Prosensa has started a phase I study to look at skipping exon 44. At the meeting, Sue Fletcher, Wilton’s long-time collaborator on this technology, described their efforts to look beyond exon 51.

The area around exon 51 is one of the hot spots for deletions in the dystrophin gene. According to Fletcher and Wilton, approximately 6000 boys worldwide have deletions that could probably be addressed by skipping exon 51—these include deletions of exons 45-50, 47-50, 48-50, 49-50, 50, and 52. Next on deck for AVI is skipping exon 50, a project supported by Charley’s Fund, which could potentially address deletions of exons 51, 51-53, and 51-55 and Prosensa’s PRO044 oligos could address deletions of exons 43, 45, 38-43, 40-43, 42-43 and 45-54 (about 6% of boys with DMD). Clinical results from both exon 51 skipping programs have been promising with some evidence that dystrophin can be produced and potentially some trends (not statistically significant) in improved 6 minute walk tests.

AVI and Prosensa have plans to follow up with AONs to skip additional exons, but at this point, it looks like each pair of oligos will be treated as a new drug from a regulatory standpoint and require the full pharm/tox package along with phase I, II and III testing. The AON technology used in this way is very new and regulatory agencies worry about toxicity developing if the AONs bind to inappropriate pieces of the genetic code for other genes, potentially disrupting their function. No exon-skipping trial has yet begun in the US.

Although collectively, the ability to skip these three exons might benefit a fair proportion of boys with DMD, there are actually 77 “skippable exons” according to Fletcher and Wilson. In some cases, skipping one of these other exons to correct a rarer deletion might only be applicable to a handful of boys, or even a single boy. The irony, Wilson says, is that some of these other exons can be skipped with much higher efficiency and potentially better results than exons 50, 44 and 51, but there’s no business model to move them forward. How do you spend $1.4 billion to develop a drug for one boy? And if technology looks really promising and is potentially life-saving, how do you not?

So Wilton’s group has been wrestling with this problem and has proposed a solution that is either “innovative and out-of-the-box” or “out-in-left-field” depending on how you look at it, and, to a certain extent depending on how they choose to go about it: the “N=1 Study.” “N” is the number of replicates you use in a study—in a clinical study “N” usually represents the number of participants.

Wilton is proposing to manufacture and test oligos for the rarer mutations in very small studies of one to a handful of participants. These studies will likely not be powered appropriately to ever get a drug approved based on the results, but one solution would be to maintain a “perpetually open IND” and continue to treat the boys with what would be categorized as an experimental therapy. A major shortcoming of this approach is that without marketing approval, there is no way to get insurance to pay for the drugs, so Wilton is proposing to set up a manufacturing facility that will be run as a nonprofit foundation to supply oligos to the boys for the rest of their lives at no cost. He would donate all of the intellectual property (IP) around the other exons to this foundation and if anyone ever did want to license the IP to market one of these oligos, the proceeds would go back to the foundation to continue to help supply other oligos to boys who need them. Sue Fletcher mentioned that the labs are focusing now on skipping exons 8 (9 comes out with 8 so you skip both), 16, 46, 43, 53, 41 and 74. They would like to tailor a strategy to each boy’s individual mutation.

So, I like some things about this approach. I like the fact Wilton’s group has recognized that you don’t actually have to have marketing approval to make a drug available—you just need to meet the regulatory requirements to open an Investigational New Drug application, or IND. For the less common deletions, this might be the only approach that ever makes exon-skipping feasible. I like the fact that he’s thinking ahead about the need to supply oligos for an entire lifetime, which could stretch on many additional years if the oligos work like we all hope they will (adding the caveat that we don’t yet know how effective AONs will be if they work and this efficacy will very likely vary with different exons and AON pairs).

I have a lot of concerns as well, starting with the fact that efficacy may be difficult or impossible to demonstrate in such a small number of boys—can you justify keeping an IND open if you can’t show efficacy? One potential answer to this question is that if the boys can stay on drug for several years you might be able to measure the effect over time even in a small group of boys. The other potential answer is that maybe some of the oligos might work well enough that you can measure definitive changes with very small numbers. At the very least, you should be able to see if the AONs are triggering dystrophin production even with smaller numbers of boys. If there are only five boys in the world that might benefit from skipping a certain exon and two respond and three don’t, it doesn’t mean the drug fails, it just becomes a two person drug—it’s like the difference between quantum and classical physics. The rules are different when you are dealing with very small numbers.

Next concern—is it really realistic to think that you can raise enough funds to build a manufacturing plant and then supply an expensive oligo for the life time of these boys pro bono? This question is complicated by the chicken-and-egg nature of the issue. If you do a study with a set of AONs and there is a dramatic positive response, ethically you have to be prepared to continue to supply the oligos in perpetuity, so you need a manufacturing source. At the same time, it’s hard to convince people to put up money to build a manufacturing plant before you even know if the approach is viable. What I think is likely to happen in reality is that any oligos that produce a dramatic positive response probably could be licensed to one of the drug companies to develop for marketing and the licensing fees could go back into the kitty to help support the manufacturing facility. It’s the manufacturing of oligos with more subtle results that would need to be supported on the proceeds of any oligos that could be licensed for marketing. That’s if any of the oligos do produce a dramatic response without limiting side-effects (let’s not forget the potential for side effects).
And finally, can Wilton pull it off? He admits that he’s not a “business guy,” at least not by choice. He knows he needs help but sees this n=1 strategy as the only way forward for the less common deletions whether it’s realistic or not.

Personally, I think it’s probably worth knowing if mutation independent approaches like Acceleron’s muscle building compound might not have a greater impact and it’s also worth knowing if AVI, GSK and Prosensa can show any functional results with exons 50, 51 and 44. But as personalized medicine gets more and more personal, the $1.6 billion model of drug development is untenable. Somehow, some way, the costs to develop a drug will have to come down and maybe the perpetual IND with a nonprofit payment system is one way to go. At this point I’d call it “innovative.” The execution will prove whether or not it’s out-in-left-field.

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Comment by cheryl cliff on September 28, 2010 at 12:10pm
Maybe by the end of the conference we all will have more concrete ideas as to what is most likely to happen. But because they seem to maintain ambiguity, per what you mentioned as "no one size fits all", I suspect that may be as far as they are willing to go. Please try to remind them Sharon, our sons have a concrete date of expiration in their near futures. Their deaths will happen, therefore these boys deserve and require concrete information from our civil servants - with or without being shamed in front of congress. CYA is typical in business and governments but doesn't take priority over the lives of american children.

I know you will do the best you possibly can, Pat too. Sending cyber hugs to you both.
Comment by Sharon Hesterlee on September 28, 2010 at 10:10am
So, the FDA is giving examples from other situations but maintaining that there's no "one-size-fits-all" solution. Ultimately we will need to have enough information about using AONs for exon skipping to determine if they can all be treated as a class or if they will have individual toxicity profiles that depend on the sequence of the AONs. So, probably at least two AONs approved and on the market before there will be a big enough body of information to determine risks for the others. If they do all seem to behave as a class, there may be an abbreviated path to development, but the FDA won't comment on this situation specifically until we have the data in hand. I know, very frustrating. They are trying to keep everyone safe and prevent FDA officials from being dragged in front of congress for screwing up with very little resources, and they have to balance that with the urgency of getting drugs developed for DMD. Because having no drugs is decidedly not safe either. There are no simple solutions--it's a big complicated thing, but I have been impressed by the fact that the FDA is generally trying to work with the community, at least within the constraints that they are forced to operate. I'll post something longer in the next few days specifically on this meeting.
Comment by cheryl cliff on September 28, 2010 at 9:53am
I sure hope those nice representatives from FDA are explaining how, and on what specifically they can be flexible, regarding multiple pathways to development. Then I have the question as to WILL they do what they say they can do? Stating capablilties and doing them are two different things. My son doesn't have time for the type of political games that can (and I expect will) occur. Please, please convince me I am wrong.
Comment by Sharon Hesterlee on September 28, 2010 at 8:52am
Keith, I think those key questions are what Steve is trying to work out now. He's been talking to potential manufacturers and will have to have some concrete numbes to be able to answer those questions. In the meantime, I'm at the AON meeting in Washington DC and listening to the FDA describe the drug approval process for extremely small populations--they are emphasizing that they can be extremely flexible on the requirements for very small groups. So there might be multiple pathways to development available here...
Comment by Keith Van Houten on September 25, 2010 at 8:54pm
Awesome idea, and there's clearly no shortage of parents willing to try non-FDA approved courses of treatment.

So - when's the non-profit being set up, and how much money does he need to get it going?

In a ballpark sense, what kind of manufacturing cost are we talking about to produce the AON's in a non-profit setting? Without an FDA approval, there's no insurance coverage. Is it $10k a year, or $100k a year?

I think having a treatment available on the marketplace, but financially unattainable would be even worse to deal with emotionally than today's situation of having nothing available at all.
Comment by Sharon Hesterlee on September 23, 2010 at 4:03pm
Laura--sorry, just noticed that you said he needs exon 62 skipped. Where is his mutation?
Comment by Laura Kew on September 23, 2010 at 3:24pm
My son needs skipping of exon 62 which is not even mentioned. I guess this means that he may be the only one (or one of a very small number) with this mutation. I see Sharon mentions that skipping exons 52 - 62 might be more problematic. Any more information on this?
Comment by Sharon Hesterlee on September 23, 2010 at 11:54am
Kelly--here's the results and conclusion section of a paper from Judith van Deutekom and Annemieke Aartsma that suggests what I thought--smaller duplications may be skippable, but larger ones may be more problematic. I (or you) would have to ask Steve specifically about skipping an exon 8 and 9 duplication.

(from: Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy.Annemieke Aartsma-Rus1 , Anneke AM Janson2 , Gert-Jan B van Ommen1 and Judith CT van Deutekom2 . BMC Medical Genetics 2007, 8:43)

For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62), by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons.

The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.
Comment by KellyR on September 23, 2010 at 11:25am
Thank-you, Sharon.
Comment by Sharon Hesterlee on September 23, 2010 at 11:03am
Hi Kelly--I think it might be feasible to skip small duplications but I have specific query about that in to Steve Wilton now--unfortunately he's out of the office for the next two weeks, but I promise I will post when I hear from him. I may try to get in touch with Judith van Deutekom (from Prosensa) in the meantime.

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