Hi,
In my kid’s case, it appears that 2 exons , 45 and 51 need to get skipped ( His molecular diagnostic report says ‘ Out of frame deletion of exon 46 to 50’).
I just wanted to understand if there are any work going on which can help in skipping 2 exons (45 and 51). Whether even this is possible also or no.

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Yes, multiple exon skipping is being worked on. The DMD Genetic Group says:
For a small number of deletions, skipping a single exon is not sufficient to restore the open reading frame, e.g. a deletion of exon 46-50. The total number of deleted nucleotides is 695 (not divisible by 3) and neither a deletion of 45-50 (871 nucleotides) or a deletion of 46-51 (928 nucleotides) is divisible by 3 and thus exon 45 skipping or exon 51 skipping will not restore the open reading frame. However, a deletion of exon 45-51 involves 1106 nucleotides, which is divisible by 3. Therefore, to restore the open reading frame for this patient both exon 45 and exon 51 have to be skipped. This can be achieved by using a combination of AONs targeting exon 45 and AONs targeting exon 51. We have indeed confirmed the applicability of double exon skipping in cultured cells from a patient with a deletion of exon 46-50.
We have a similar problem; Alex has a small mutation on exon 44 that creates a stop codon, so exon 44 must be skipped to get past that stop codon, which would then still give him an out of frame dystrophin; to correct that out of frame dystrophin, he would need to skip exon 43 or 44. Based on an email exchange I have had with Dr. Annemieke Aartsma-Rus at Leiden University who is one of the investigators working on Prosensa's exon skipping trials, it is my understanding that the simultaneous administration of the compound to skip exon 44 and either 43 or 45 is predicted to cause Alex to produce an in-frame dystrophin. I don't know if there is any special magic to the fact that the two exons Alex needs to skip are adjacent. I respectfully suggest you email Dr. Aartsma-Rus a copy of your son's genetic test and put the question to her. She is amazingly responsive. Her email address is on her website. http://www.humgen.nl/lab-aartsma-rus/
Hi Ratkim,

My son has the same deletion: 46 - 50. From my discussions with our consultant at St. Thomas' Hospital in London, and the Genetics Dept. at Guys Hospital, a double skip of Exons 45 and 51 will be required.

At the moment the "proof of principle" work on a single exon skip (of Exon 51) looks to be going to clinical trial to determine its effectiveness shortly. I hope that if that has a positive outcome they will try double skipping.

The understanding which I have is that suitable "patches" exist for most of the exons - however it's necessary to first have the safety data from the exon 51 trial to support work on skipping other exons, including simultaneous double-skipping.

Obviously we all hope this is achieved as speedily as possible !
Thanks John.Thouh SINGLE exon skipping(with PMO and PPMO) is getting worked out and getting made more effective, we all have to pray for multi exon skipping also. Not sure whether just a cocktail of 45+51 exon skipping molecule will work for us or something more required.
Keep in touch.
Thanks Paul. I will contact Dr Aartsma-Rus. Guess she is out of office till 20th NOV.Praying that just a cocktail of 2 SINGLE exon skip molecules should work for our kids.
Thanks Lisa.Article quoted by you is very encouraging. Praying that Cocktail of 45+51 SINGLE exon skipping molecules works out speedily for humans also and all our kids get well soon.
Hi,
I was talking with some of the members of group 'MDEX Consortium'. Many of these members are in London/Oxford in UK.
One thought is that for exon 46 to 50 deletion, both exon 45 and 51 need to get skipped. Though a cocktail of 45 exon skip+51 exon skip can work, not everyone is very sure the effectiveness of this. They are trying to create single oligonucleotides so as to skip 45 and 51 together.
Unfornately I gather they are still short of fund for working on this full time.
Not sure how much is needed. Not sure what exactly can be done here to speed up the process.
Any comments/suggestion is welcome.
Hi Ratkim,

That's interesting - I didn't realise they were looking at a single oligonucleotide to remove both Exons.

I can see that two separate oligonucleotides would possibly generate several RNA species [I guess four, these being (1) an RNA with both exons removed which is what we want, (2) a version with just exon 45 removed, (3) a version with just 51 removed, (4) and the current RNA with both 45 and 51 still present]. Clearly this wouldn't be very efficient.

I guess with a single oligonucleotide you would get just two species, these being an RNA with both exons removed, and the current RNA with both exons still present. Clearly this is potentially better, although what the ratio of these two RNA species might be in the cell will depend on how well the oligonucleotide is at binding to the RNA and removing the exons.

Can you find out how much funding they need to progress the single oligonucleotide double-skip approach ? If funding could be made available - as genuine extra funding - could they do the work without prejudicing the other ongoing DMD work ? Like any other parent I would like to see progress which will help my child - but I don't want to see this happen at the expense of others; I have a strongly held belief that no DMD child should be left behind.
Hi John,
I agree with you that all our DMD kids needs equal treatment.We all have to work on solutions which can benefit everyone at same time.
I had tried finding out the cost here for double exon skipping.
What I understood was it would be best to have one or two dedicated people just working on this problem alone for 1-2 years. One person would develop the oligonucleotides and the other would test them in animals.

The cost of one person per year is about £60K British Pounds, so therefore the cost of two people for two years is about £240K.

Any suggestions/inputs are welcome here to contribute our little bit towards DMD treatment.
Her lab certainly believes that it does. http://www.humgen.nl/lab-aartsma-rus/

Click through to "State of the Art" and read the first couple of paragraphs.
Thanks Paul. I am also praying for this to work. I think the little dobt is about how much effective will it be and that's why plan to create single oligonucleotides as theoratically that may be more effective.We have to plan/pray for each treatment.
Hi Raktim,

Do you know how advanced the work on a single oligonucleotide to skip Exons 45 and 51 is ? Other questions which come to mind are:

- does this approach use a the same chemistry as the single exon skipping oligos ? If so it might allow more rapid progress through to human trials.

- does the MDEX consortium see this approach as providing a significant advantage over use of single oligo's in combination ?

- is there a commercial partner who might be willing to take such a product forward to the market ?

- if they have funding for part-time work on this at the moment, how much do they currently have - what is the gap to fill to provide two full time staff ?

If the answers to the above gave grounds for optimism I would be happy consider contributing towards the funding (and I guess there will be others in the same position). I would need to be satisfied first that the approach had merit, that there were reasonable grounds to believe an oligo could get to market (i.e. someone would actually manufacture it), and within a timescale of say 4 years or so ? I also think it's important that knowledge generated from this approach (successful or otherwise) is available to others so the whole field of Exon skipping benefits.

If the answers to these questions were reasonably positive I would be happy to go and speak with your contacts directly - Oxford is only a couple of hours drive for me and I pass it regularly when away on work ?

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