making muscle wasting history

Article submitted by Dr Karl Bettelheim published on the 18th of July 2009

There has recently appeared an excellent review (1) of current studies on gene therapy for muscular dystrophies. While pointing out in their introduction, that so far, there is no effective treatment for muscular dystrophies, a number of new gene-based therapies are being developed at present. They especially note the use of conventional gene replacement strategies, RNA-based approaches, or cell-based gene therapy. All of these have as their main focus Duchenne muscular dystrophy (DMD). For us this is a most favourable situation.
They point out that especially exon-skipping induced by antisense strategies, and the development of corrective gene therapy using functionally engineered dystrophin genes appear particularly promising. There are several clinical trials currently in progress. Exon-skipping has been shown in animal models, and most recently in clinical trials to be effective and this approach certainly represents a promising therapy for a subset of patients. A photomicrograph is shown of muscle cells from DMD patients after treatment with phosphorodiamidate morpholino oligomers and it clearly shows the presence of dystrophin. The selective removal of exons flanking an out-of-frame DMD mutation by use of exon-skipping can result in an in-frame mRNA transcript which would then be translated into an internally-deleted, Becker muscular dystrophy (BMD)-like but nevertheless functionally active dystrophin protein and thus therapeutic results would be obtained. They refer to the first report of an antisense oligonucleotide (AO) having been used successfully in a single DMD patient as long ago as 2006 (2). They then give an extensive account of the various studies on exon-skipping, which have been and continue to be performed in a number of laboratories around the world.
There are strategies in place for direct gene-delivery-based techniques for both the restoration of the reading frame using antisense-induced techniques as well as for a highly efficient delivery of functional dystrophin mini- and micro-genes to muscle fibers, either directly into the patients’ muscles or into muscle stem cells, which are used to treat the patients.

Additionally, the use of Adeno-associated virus (AAV)-based methods, have been shown to provide efficient systemic gene delivery to skeletal muscle directly in patients. A photomicrograph is shown of muscle cells from DMD patients after treatment with AAV carrying a microdystrophin construct clearly shows the presence of dystrophin. Other viruses such as lentivirus-based methods have shown promise in combining two techniques. Outside the patient the gene modifications are carried out on cells and these cells are then used as infecting cells, which are then introduced into the patients. Of particular interest is also the report of some advances, which use non-viral protocols (3). The technique of nucleofection, (direct infection of the nucleus of the cell) of muscle precursor cells is performed with a special system known as phi31-integrase (it causes the integration of a piece of ‘foreign’ DNA in the form of a plasmid into the human DNA) causes a plasmid carrying the full length human dystrophin gene to be incorporated into the patients’ genes. Such integrases are well known as also being the causes of the transfer of antibiotic resistance between bacteria.
They provide an extensive table, in which are listed all the various muscular dystrophies and the genetic basis of these conditions. DMD heads the list and it still sad to see that with the dystrophin gene having been identified as long ago as 1986, we still do not have a cure. There is also a useful diagram of part of a muscle cell showing where the various mutations have their effects.
In their penultimate section they discuss the development of therapies to try to block the effects of myostatin also known as growth/differentiation factor-8 (GDF-8). It is a member of the TGF-β family of hormones. It is predominantly expressed in skeletal muscle tissue and is secreted into the blood stream where it circulates as one of the many proteins found in serum. It is a negative regulator of muscle mass. They describe the various techniques under study at present to reduce the effects of this factor. The inhibition of myostatin was shown not only to lead to increased muscle mass, but there was also an enhancement of muscle regeneration after injury. It reduced fibrosis and reduced the pathophysiology of the mdx mouse. Unfortunately other studies have shown that in the more severely affected mdx mice, no improvement was observed. Ideally they suggest that this myostatin inhibition strategy will have to be combined with gene replacement or RNA-based strategies to combine repair of the muscle fibres with improvement of muscle physiology.

In their final section labeled ‘Expert Opinion’, they report on the various clinical trials currently in progress and they are enthusiastic that gene based cures for DMD will become available. After pointing out that exon-skipping though promising, only provides a partial solution because it leads to BMD they conclude with the following remark:
“Researchers and therapists in this field are thus looking ahead enthusiastically to the prospect of distributing genes and/or stem cells to the whole body musculature using AAV and lentiviral vector technology to provide realistic therapeutic opportunities for dystrophic patients.”

Acknowledgement: I would like to thank Dr. Dickson, for kindly sending me a copy of the paper, to which I otherwise would not have had access.