Therapy in the future

Until recently the systematic delivery of a gene to major muscle groups was considered very difficult and was a huge stumbling block regarding gene therapy for all major muscle groups such as the heart and skeletal muscles. The failure and incapability of systematic gene delivery to the muscles of adult organisms has been a hurdle in gene therapy. The difficulty is in achieving transgene expression to all diseased muscle groups rather than only one group.

Duchenne muscular dystrophy is one of the most serious conditions affecting muscle groups; caused by absence of dystrophin in muscle fibres, it results in muscle damage, loss of muscle mass, and respiratory and heart failure. The success of addressing this disease has been minimal with no effective treatment. Gene therapy is a possible treatment strategy which can be used to treat a number of diseases. There are several approaches to using this tharapy such as, creating a swap between an abnormal gene and a normal gene through homologous recombination, or by altering gene regulation “turning it off”.

Other approches include using reverse mutation for gene repair or replacing the non-fucntional gene by insertation of a gene into the non-specific location of the genome, with the latter being the most common. Further research indicates usage of viruses as a potential “transport system” to deliver a new gene into a human cell and have it change the genetic expression of that cell. Paul Gregorevic1 and fellow researchers have managed to demonstrate a method of systematic delivery of genes to all muscle groups compared to the limited success of several plasmid based therapy predecessors.

There were developments in plasmid based technologies2, however this technology faces limitations involving the distribution of the plasmid following an intramuscular injection which didn’t result in widespread distribution. This form of technology also resulted in extra chromosomal plasmid loss. Intramuscular injection had been the most common experimental method but it was shown to be impractical and the results were nonviable. The other method used was local blood perfusion. Both approaches were unable to give gene expression in the whole body.

Other methods included in vivo gene therapy approaches such as lentiviral delivery 3; however this is less effective than delivering viral vectors through circulation, and there is also a safety issues since HIV is used as a retroviral vector. Non-viral methods involving phiC314 integrase and a transgene expressing plasmid have also been used in in vitro gene therapy but have still not resulted with a whole body expression. P. Gregorevic1 and collaborators used an animal based model concentrating on Duchenne Muscular Dystrophy in mice.

In the past the more traditional gene transfer systems could not simultaneously infiltrate the skeletal muscles and the heart, these methods showed a very inefficient and limited potential. A recent discovery of methods involving recombinant adeno-associated viral vectors1,5,8,9,10 has led to a significant breakthrough and the success of this method has been echoed by other experiments. Recombinant adeno-associated viral vectors also known as rAAV’s are very versatile gene delivery agents commonly used in gene therapy as well as functional genomic studies.

They are important in the efficient transfer of genes to mammalian cells. rAAV’s are particularly useful due to their long term expression, they appear at high levels in the cell of interest even after a single application, rAAV’s are also non pathogenic as well as non-inflammatory. The adeno associated viral vector rAAV6 was used in this experiment in combination with vascular endothelium growth factor (VEGF). P. Gregorevic and fellow researchers tried to demonstrate the capabilities of rAAV6 and VEGF regarding transduction. First AAV6 expressed with B-galacosidase was injected on its own via the tail veins of the mice.

B-galactosidase was used in order to observe the degree of transduction in the muscular tissues. The result obtained from this intravenous administration of rAAV6 vector wasn’t decisive and had barely noticeable activity when compared to the untreated mice. However the introduction of VEGF resulted in a dramatic increase in transduction. It was also shown to give rise to higher B-galactosidase expression in skeletal muscle cells throughout the mouse body. The results from the experiment indicated a systemic delivery and a single application of the recombinant vector was enough for this wide expression.

In order to ensure the expression took place in skeletal muscle groups and not in non-muscle cells expression cassettes1, 5, were used with regulating elements such as CK6. The results involving VEGF showed a powerful 100 fold increase. The muscle groups were studied individually so accurate transduction was seen at a cellular level. From these results, greater transduction was expressed when rAAV6 and VEGF were used in combination with each other rather than the rAAV6 on its own. Nevertheless, to conclude that the experiment was a total success and conclude the effects of VEGF were useful is not yet possible.

Although P. Gregorevic1 et al show whole body transduction, there are still doubts whether this method will work on a human subject. The research going into gene therapy is mainly acquired to treat human diseases not mice, therefore until human studies can be completed successfully those conclusions can not be drawn. There is a matter of safety and feasibility in doing those studies. Despite using expression cassettes, it is impossible to exclude genome vector expression in non-muscle cells such as the lungs, even with the use of specific promoters. This can be seen from the results of the experiment1.

The results using rAAV6 are successful but, it fails to answer questions such as how it compares to other genome vectors such as rAAV9, rAVV8 and the commonly used rAAV2. There are other studies which show that rAAV96 and rAAV87 are actually better performers than rAAV6 regarding transduction in certain muscle groups. Other studies suggest AAV68,9,10 as being a very strong rAAV, but there is also some research indicating higher transduction by rAAV69,10 when compared vectors such as rAAV2. The cellular receptor of rAAV6 has shown to be inconclusive11 compared to other vectors such as rAAV2.

It is difficult to analyse how well VEGF works in collaboration with rAAV’s other than rAAV6 because higher dosages of these rAAV’s can transverse the endothelium without using VEGF. Usage of papaverine and histamine5, 12 which can induce vascular permeability similar to VEGF changes the results, however in this study a less effective adenovirus was used. Due to the small size of the rAAV particles, they only carry small amounts of DNA. This can be further developed using RNA interference, which gives us hope of further development in this method regarding gene therapy in the future.

This study shows a promising development in duchenne muscular dystrophy. Instead of impractical intramuscular injections there is now a chance of whole body transgenic expression by a single intravenous administration. There is need of more evidence supporting the effect of VEGF. It is a short lived non-toxic growth factor and needs more study to show its worth. With studies showing impressive results by just using rAAV61,8 ,9,10 as well as other vectors it is showing great promise. One thing is for sure, gene therapy for degenerative disease can be realised13.

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1. If my child was diagnosed with a rare inherited disease, it would be quite unfortunate. However, I wouldn’t lose hope and try to do whatever I can to soothe the situation. In the modern era, there are an eclectic …

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