LONDON A controversial technique for Duchenne muscular dystrophy (MD) that involves hundreds of intramuscular injections and can cost $150,000 has come under fresh scrutiny with the publication of new data from a competing group of researchers.
The treatment in question, called myoblast transfer therapy, has been pioneered by the Cell Therapy Research Foundation (CTRF), of Memphis, but a group of scientists in the U.K. and the U.S. said they hope their latest results will spur the foundation into carrying out similar tests to verify the success or failure of the procedure on other patients.
Terry Partridge, head of the muscle cell biology group of the Medical Research Council¿s Clinical Sciences Centre, in London, last year challenged Peter Law, chairman of CTRF, to prove that the technique works. In a letter to the journal Cell Transplantation, he and other researchers outlined an experimental method they believe would prove once and for all whether the technique is helpful to boys with Duchenne muscular dystrophy.
Now, in the November issue of Nature Medicine, Partridge, who works at Imperial College School of Medicine, has published a letter titled ¿Is myoblast transplantation effective?¿ In it, he reports using his suggested method to evaluate the effectiveness of the injection technique on a five-year-old boy who had previously received therapy at CTRF.
Law has rejected Partridge¿s findings, on the grounds that Partridge did not know which of the boy¿s muscles had been injected. He told BioWorld International: ¿We believe that the research conducted by Dr. Partridge and his colleagues is scientifically invalid.¿ Law said that the design of his studies had been approved by the FDA, and that Partridge¿s letter examined only a minute detail of the work.
Muscular dystrophy affects one in every 3,500 boys. It is caused by a defect in the gene coding for the protein dystrophin, which is carried on the X chromosome. In the severe (Duchenne) form, the defective gene fails to produce functional dystrophin, although in the milder Becker¿s form, a partially functional protein is produced.
Dystrophin is a key component of muscle, forming part of the protein complex which anchors the cytoskeleton of the muscle fiber to the extracellular matrix. Without it, the muscle fiber degenerates.
The disease is frequently diagnosed only when an afflicted boy begins walking. In the case of Duchenne muscular dystrophy, the boy may be wheelchair-bound by the age of 10 or 12 and, because the muscles of the heart and chest are eventually affected, death occurs at about the age of 20.
Some attempts to treat muscular dystrophy have therefore focused on delivering a good copy of the dystrophin gene to the wasting muscles. One strategy, offered at CTRF, is called myoblast transfer therapy (MTT). Despite this name, CTRF¿s website (http://www.celltherapy.com) denies that MTT is a ¿treatment,¿ since it is still undergoing clinical trials.
Myoblasts are immature muscle cells capable of fusing with each other to form myotubes, which mature into muscle fibers. CTRF¿s website says: ¿Under appropriate conditions, such as regeneration after injury, myoblasts will fuse with a subject¿s existing myofibers. In MTT, [donor] myoblasts are injected into the subject¿s muscles ... An immunosuppressant is administered following the procedure; however, unlike organ transplants, after a short treatment period, the immunosuppressant is gradually withdrawn. At this point, the subject¿s muscles contain (healthy) genetic information from the donor myoblasts.¿
Law told BioWorld International, ¿The absence of dystrophin is the primary biochemical defect of Duchenne muscular dystrophy and the CTRF has found dystrophin in at least 26 treated muscles, but not in the placebo-injected muscles of the same patients.¿ Some of these results were published in the journal Gene Therapy and Molecular Biology earlier this year, he said.
Preliminary results, adds the website, have shown that the MTT-treated muscles gained strength. The CTRF says that, under a protocol which involves injecting 22 boys with Duchenne muscular dystrophy with 50 billion myoblasts into 82 muscles, results showed ¿significant clinical benefits,¿ including ¿improvements in the ability to balance, stand, walk, manipulate, right oneself and breathe in about 50 percent of the subjects.¿ The foundation says the results also showed ¿improved cell structure, pulmonary function and reduced serum level of leaked muscle enzymes.¿
Donor Dystrophin Test Disputed
However, as Partridge and 13 other researchers outlined in their letter to Cell Transplantation last year, most people working on human myoblast transplantation have found little or no evidence of donor dystrophin following the procedure. The group said the evidence provided by Law, that donor dystrophin is present in muscle fibers in biopsies from patients who have undergone MTT, is inadequate.
Last week, Partridge told BioWorld International that many boys with Duchenne muscular dystrophy ¿have a bit missing from the gene that codes for dystrophin, and the result is a truncated non-functional protein. But, sometimes, the genetic reading apparatus manages to skip over the deleted part and carries on, so that you get a protein which is shorter than usual, but which is missing only a bit in the middle, corresponding to where the boy¿s deletion is.¿ Muscle fibers in which this has occurred are called revertant fibers, and little is known about the process by which dystrophin appears in them.
In their letter to Cell Transplantation, Partridge and his colleagues pointed out that the type of antidystrophin antibody used by Law stains these revertant fibers, making it an inappropriate reagent to demonstrate the presence of donor dystrophin.
To settle the question, the group suggested selecting patients with known deletions from the dystrophin gene who had been treated with MTT. Monoclonal antibodies which bind to the parts of the protein they could not make from their own dystrophin gene could then be used to stain biopsies from these patients. The group wrote: ¿It is generally accepted that fibers stained with these antibodies could only have been derived from the grafted cells, because they contain parts of dystrophin that the patient¿s own cells cannot synthesize.¿
Partridge and his colleagues suggested that an independent study should be organized to carry out these tests, with blind assessment. Major national charities would be prepared to pay the cost of this operation, they said.
Law responded to their letter in the same issue of Cell Transplantation, but ignored their suggested strategy. Last week, he told BioWorld International that, in his study, the FDA ¿insisted on a randomized, double-blind controlled study design to offset the presence of revertant fibers.¿ He also claimed that the type of study suggested by Partridge and his colleagues could produce false-negative results. He said that the parts of donor dystrophin which the patient¿s own cells cannot synthesize ¿may exist transiently in treated patients and escape detection.¿
This year, however, Partridge himself had the opportunity to carry out one such evaluation of a patient treated at CTRF. ¿The child¿s father had raised $140,000 from his local community to send him to the CTRF,¿ Partridge said. ¿Afterwards, the father became more and more convinced that it had done no good. He withdrew the child from the trial and had him biopsied.¿ Although the biopsies were obtained in the U.S., they were brought back to the U.K. because the only two groups which make the required panel of antibodies are based here.
Biopsies May Not Invalidate Method
Partridge and his colleagues Qi Long Lu, also of the Muscle Cell Biology Group, and Glen Morris, of North West Wales Institute, in Wrexham, U.K., and Eric Hoffman of the University of Pittsburgh School of Medicine, report their method and results in their letter to Nature Medicine. They took a series of neighboring sections from the biopsies so that each section showed part of the same muscle fibers and stained them with a panel of antibodies which bind to different parts of the dystrophin molecule, starting at one end and finishing at the other. They found that the antibodies specific for the first part of the molecule stained, as did those specific for later parts of the molecule, but a section in the middle did not stain. The biopsies had been taken from both the boys¿ bicep muscles, and the results were the same for both biopsies.
¿The antibodies did not stain the part where the boy had a deletion,¿ Partridge said. ¿We actually found out where his deletion was after we had done the staining and it was quite a relief that the two coincided. We then decided to publish this work as an object lesson in being careful when you assess these things.¿
The letter to Nature Medicine said: ¿Failure of myoblast transplantation in a single instance does not prove that the procedure as a whole is ineffective, but these results do caution against interpretation of data from a single antibody stain.¿ Partridge and his colleagues urged families contemplating myoblast transplantation to have this type of analysis, and invited any patients already participating in CTRF¿s trials to seek similar verification of treatment they have received.
Asked whether CTRF accepted that these data prove that, in this patient, there was no survival of donor myoblasts, Law told BioWorld International: ¿[We] are concerned that the peer review board at Nature Medicine allowed the publication of a case report based on incomplete information. The FDA-permitted clinical trials conducted at CTRF were randomized double-blind trials. How could Dr. Partridge be certain whether the patient received myoblasts in the upper or lower body? Had the patient received myoblasts in the lower body, then of course all of the muscle biopsies in the upper body would show no dystrophin. We do not know which muscle was biopsied. It is possible that the physician took the muscle biopsy from a site that was not injected. It is not possible to determine whether or not the results are valid.¿
If You Look Carefully, It Is A Negative Result¿
Law also said: ¿Although they were not able to find any dystrophin, this does not mean that there was no survival of donated myoblasts.¿ He added that CTRF had tested five other parameters by which the success of MTT could be judged, and that all showed statistically significant improvement.
In response, Partridge said the patient¿s family had told him that the boy had received injections in the upper body, and had also sent him background information from CTRF that had been supplied to them detailing the conduct of the trial on their son. He agrees that he did not have access to the unblinded protocol concerning this patient, but says that at least one of the biopsies would have been from a treated muscle.
Turning to the question of whether dystrophin was present, Partridge said: ¿We do not comment on a lack of dystrophin. We comment on the fact that there is dystrophin present, and that by the criteria of the CTRF, they would regard it as a positive result, whereas in fact if you look carefully, it is a negative result. That is the entire point of the letter.¿
Sarah Yates, Director of Scientific Affairs at the Muscular Dystrophy Group of Great Britain and Northern Ireland, in Oxford, U.K., said the phenomenon of revertant fibers is ¿so well-recognized that one would expect someone performing a clinical study to control for it. If a researcher does not control for it, then we are dependent on someone else doing it. We are very grateful that this work is being performed to examine exactly what is going on.¿
The group receives calls from families every week, from parents asking if they should send their sons to CTRF. Yates said: ¿We don¿t recommend this at the present time. We tell them that, in our view, Peter Law has not proven that he has an efficient therapeutic regime. We would love for him to back up many of the statements that he has made with data that other scientists find convincing, but at the moment we don¿t think he has done that.¿
Partridge added: ¿Many patients¿ families who have problems raising money to put their children into the CTRF¿s trials get upset because national muscular dystrophy charities will not support them in their efforts. If Law¿s work could be verified, this would change the attitudes of the charities and benefit both the CTRF and the patients. It is hard to see why they do not cooperate enthusiastically.¿
Law said CTRF would not agree to conduct the type of study suggested in Cell Transplantation. ¿The foundation¿s main goal is to develop a treatment for muscular dystrophy,¿ he said. ¿We will not be distracted ... with not-meaningful collaboration [sic] suggested by our critics.¿ Law suggested that Partridge conduct myoblast transfer on his own patients and subsequently test them with his own method, rather than testing patients who had been treated at CTRF, without having access to the unblinded protocols.
He added that, following a meeting between CTRF and FDA representatives Oct. 13, at which the foundation¿s recent results were reviewed, the FDA ¿encouraged us to move forward with a larger pivotal, Phase III clinical study which, if successful, would serve as the basis for a request for marketing approval of MTT as a treatment for muscular dystrophy.¿
CTRF¿s website currently displays a letter from the FDA, dated Oct. 16, indicating the agency has designated the technique as a fast-track development program. Partridge said he is ¿distressed¿ by this news, and added that he hoped the FDA would take the latest evidence, published in Nature Medicine, into account when reviewing the CTRF¿s work.