Responding to concern within the community regarding the potential for an immune response to exon skipping in the recent Quest Magazine article from the MDA, Annemieke Aartsma (Leiden University Medical Center) formulated the answer for families as follows:

First of all, the exon skipping trials are still ongoing, so unfortunately I cannot give you a definitive answer about a possible immune response to dystrophin after exon skipping. However, I can explain why we think that for exon skipping it is less likely to have an immune response against dystrophin than for a gene therapy.

1. Exon skipping is a genetic therapy - it targets a gene product and does not provide a new gene, as regular gene therapy does. The problem with delivering a new gene for DMD is twofold. First, viral vectors are needed to deliver the new gene. While this viral vector has "good intentions" the body does not know this. What it sees is a virus - a foreign invader that needs to be destroyed. Therefore, using viral vectors, the immune system is on high alert anyway and therefore more likely to also attack the dystrophin. Secondly, because the dystrophin gene is so big, the viral particles do not contain a gene for normal dystrophin, but a much truncated version - which is new to the body (this dystrophin is "artificial" - it contains bits and pieces of the original protein joined together at abnormal places). Therefore, this gene therapy "micro"dystrophin is more likely to induce an immune response than regular dystrophin.

2. Exon skipping aims to skip a single exon (or in rare cases two or three) - the dystrophin that is produced is therefore very similar to normal dystrophin. In fact, many patients spontaneously skip exons at a very low level, resulting in a small number of dystrophin positive "revertant fibers". The number of these fibers is too low to be of functional benefit, but most patients will make a low level of dystrophin - therefore for most patients the dystrophin that is made after exon skipping is not "new" and as such the immune system will not respond to it.

3. Nevertheless during the exon skipping trials that are currently ongoing, they monitor for immune responses to dystrophin. A response to dystrophin has thus far not been found in any of the patients involved in the Prosensa or AVI trials.

So in summary, we cannot exlude an immune response, which is why we carefully check for this - but it is much less likely to occur than with "real" gene therapy.

Hidde Ploegh (Whitehead Institute/MIT) was also asked to comment and provide an explanation

Introduction of any foreign protein into an immunocompetent host always entails the risk of an immune response. In the case of DMD, one should distinguish between cases where no dystrophin is produced at all, and those where a partial or non-functional fragment is produced. To a first approximation, as the target for a potential immune response increases in size, the risk of an immune response to it increases as well. Therefore, gene therapy that results in restoration of dystrophin expression in an individual with a null mutation has the highest likelihood of evoking an immune response, restoration of a complete DMD gene in a patient where the defect is relatively minor would pose a lesser risk.

This immune response is minimally composed of two components: antibodies made by B lymphocytes, and the T cells that provide assistance to B cells in their attempt to make antibodies ("helper T cells"), as well as other T cells that can cause inflammation and tissue damage ("inflammatory T cells" and "killer T cells"). We know that - in principle - antibodies can cause muscle damage, as seen in myasthenia gravis, but in this case the antibodies are directed against a muscle component (acetylcholine receptor) that is accessible on the outside of the cell. This scenario is less likely to occur for dystrophin, which sits inside the muscle fiber. T cells can attack tissues also, the best example perhaps being the kind of rejection reaction that occurs in transplant patients who receive a tissue graft from an unrelated donor. These T cells can "see" foreign proteins that originate inside the cell, or at any other location (surface, nucleus, other; the reasons for this are complicated, but not relevant now).

However, not all tissues are equally good targets for an attack by T cells. Liver transplants are at a lower risk of rejection than, for example, kidneys or maybe even islets of Langerhans (transplanted as a possible cure for type I diabetes). Rejection occurs because our immune system detects the presence of a substance it hasn't seen before (in the case of tissue transplantation: the products of the major histocompatibility complex, which almost always differ between unrelated individuals) and starts a response against this foreign substance. Over time, the target of the immune response is no longer limited to the foreign substance that got the process started in the first place, and the rejection episode itself is a complicated combination of the response carried out by T cells, and the antibodies that can be produced in the course of the immune response. We can suppress these rejection episodes very effectively with immunosuppressive drugs, such that post-operative survival for kidney transplants is > 5 yrs.

This is the analogy I would make with gene therapy in a DMD patient:

It is as if you are carrying out a transplantation, with the only difference between donor and recipient being the presence of a functional copy of the dystrophin gene. A DMD patient with no dystrophin at all, and with a perfectly good immune system, will recognize this protein as foreign and start an immune response against it. The ability to do so is genetically determined: some individuals will be predisposed to make a strong response, some a weaker response, and yet others none at all. The main genetic factors that determine this trait will be the genes that encode the major histocompatibility proteins (the very same proteins that start a graft rejection in a transplant setting). Because dystrophin is so large, the likelihood of such a response would be considered pretty high. If you were born with a partial, non-functioning copy of the dystrophin gene, the immune response would in all likelihood be directed against the missing part only, and your immune system would have received instructions, in the course of its development, to ignore all those pieces of dystrophin that were made at some point during development. Even if dystrophin was present initially in a partially functioning form, and would then be lost progressively with advancing age, your immune system would probably remember what it had seen in early childhood, and not respond against those portions of the dystrophin protein it had already seen. However, this memory declines with time, and it cannot be predicted what and how much would be remembered. These properties are intrinsic to the immune system, and there is very little we can do about it, except by immunosuppression, or - in theory - desensitization, just like is done for allergic reactions to pollen, etc. However, the situation with allergies is easier to deal with for reasons I'd be happy to explain, but that aren't really relevant for this discussion now. Remember that to avoid loss of their graft, many kidney transplant patients remain on immunosuppression for prolonged periods of time, sometimes for life, and tolerate this reasonably well. (Risk: infections, treatable with antibiotics).

In the case of gene therapy, the use of viral vectors for delivery poses a potential additional risk. Anything that causes tissue damage (injection, transplantation) also causes varying degrees of tissue damage and inflammation, and these help get an immune response started. Some of the viral delivery vectors may trigger other components of the immune system (the components of what is called the "innate immune system") and triggering that kind of response makes it much easier for the T cells and the B cells to get started. Tissue damage of any kind (injection, transplantation) results in leakage of proteins that normally sit inside cells into the bloodstream, and this, too can get an immune response started that would not be seen normally. Once immunoglobulins made by the B cells bind to dystrophin released from damaged muscle cells, the complexes that are formed as a consequence can also help cause disease. These antibodies can be removed from the bloodstream by plasmapheresis, a process that would probably have to be repeated with some frequency to keep these antibodies below acceptable levels. The more severe the dystrophin defect, the greater likelihood of antibody production: this is a matter of "target size".

In summary, an immune response to any protein produced by gene therapy is a highly likely outcome, but the severity and type of response are not easy to predict. Even if a response occurs, it is likely to be manageable by procedures very similar to those in wide use to manage transplant patients. A different, far more complex scenario applies if a patient with DMD made (partial or non functional) protein early in life, and then saw most of this disappear over time. The patient's immune system is educated by this early exposure, may remember this exposure entirely or in part, and in the course of gene therapy, the response might be more like that seen in certain types of autoimmune disease (MS, type I diabetes). We still don't know why it is possible to manage kidney transplant patients with immunosuppressants such as cyclosporin, and we cannot really use these drugs to effectively treat, say, MS. For MS we use interferons, but we don't use them for type I diabetes or arthritis. Much will therefore depend on the type of immunity that would be triggered in the case of gene therapy for DMD, and this, in turn, would dictate the treatment and intervention (or even preventive treatment) options available.

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Comment by Karen Stahler on April 22, 2010 at 4:55pm
How do we find out if my grandson has a stop codon or a nonsense mutation? We only know that it is a deletion on chromosome 51.
Comment by Julie Gilmore on April 20, 2010 at 7:57pm

Exon skipping as a treatment for stop codons will only work if the reading frame is maintained. In some cases this may require 2 or more exons to be skipped. (which for some exons is proving to be more challenging than single exon skipping)
Comment by Jonathan on April 20, 2010 at 7:44pm
Exon skipping will work for nonsense mutations aswell. Basically the skip removes the exon where the stop codon is and so the dystrophin is made.
My question is about the revertant fibers. In patients with nonsense mutations, do they also produce revertant fibres? and hence reduce the risk of an immune response?
Comment by Tulika on April 20, 2010 at 4:48pm
Great piece of information. Thanks a ton Pat, for making this so easy to understand.

However a doubt on the future clinicla trials. Before we start measuring the clinical benefits of the exon skipping drug, should there not be a measure point (specially when exon skipping is tried in older patients) to see if the body is not atacking the dystrophine being produced.

Only when the older patients have cleared this gate should the data from them be used for evaluating the success of exon skipping.
Comment by Dalila Rodriguez on April 20, 2010 at 3:49pm
My sons have a nonsense mutation. They have a stop codon. Could this exxon skipping be a possibility for them if it were to become a treatment? Does anyone know. Their codon position is 664, that is what their genetic tests show.

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