In Research
Subscribe to this blog
Find out when new entries are posted the instant they are published on Action Duchenne via RSS.
Disclaimer
The Action Duchenne website aims to offer a unique forum for sharing information and ideas in the search for a cure and better medical care for Duchenne and Becker and will at all times attempt to ensure postings are accurate, scientifically peer reviewed and not offensive to individuals. Action Duchenne, however, cannot take responsibility for personal opinions, information and the content of links to other websites. It is essential that you always contact your clinician or GP before taking any course of medication, therapy or supplement.
Viral based Gene Therapy
Previously I discussed Exon-Skipping, in section 2 and recent stem cell research in section 3. This review will deal with aspects of recent viral based gene therapy also with the hope to obtain a cure as envisioned in heading 3.
The object of viral based gene therapy, with respect to DMD, is to attempt to place a corrected dystrophin gene or part of one into all the affected muscle cells and thereby have fully operational muscles instead.
In principle viruses carrying the dystrophin gene would be used to replace the dystrophic gene in the muscle cells.
The papers under discussion.
Generally viruses have had a bad press. In the public view they are associated with some of the nastiest diseases to which humanity has been subjected over the years from smallpox (now fortunately globally eradicated) to HIV-AIDS and including such unpleasant conditions like the common cold, influenza, measles, mumps etc. In addition they are associated with many economically crippling diseases of domestic plants and animals as well as similar diseases of plants and animals in the wild.
That is the bad news, now for the good news. As far as is known viruses have been on this planet since the beginning of life and it is even postulated that the origin of life can be attributed to coincidental conjunction of some primitive viral forms that formed the first self-replicating unit or life form. Regardless of whether this hypothesis is true or not viruses have played a crucial role in the evolution of life by transmitting genes from one creature to another in addition to the better-known method of transmitting genes by sexual reproduction. So the really good news is that viruses are excellent vehicles for putting genes into the genome (the complete genetic material of the living thing) of a host plant, animal or bacterium. It is what they do best. It has been estimated that a high proportion of the genes in the human genome are actually virally derived.
So here we have vehicles that can put genes into the human genome, that are good at entering cells so why have scientists not put the dystrophin gene into a virus and let it infect a patient with DMD, integrate its dystrophin gene into the host genome and the patient is cured? As always there are problems and to understand these we must look at the basic structure of viruses.
Viruses contain nucleic acids, either RNA or DNA, never both, which are surrounded by a protein capsule, which comprises some form of attachment to the cells, which the virus will then infect. These attachment mechanisms are generally very cell-specific. The nucleic acid is introduced into the susceptible cell, where it basically takes over the cell’s functions, subverting them now to the process of making new virus particles. The viruses themselves lack all means of reproducing themselves outside a cell. On some occasions the viruses will cause their nucleic acid to be integrated into the genome, where it may rest for the life of the host or occasionally burst forth and cause symptoms. A well-known example of this is the virus causing chicken pox, which at the end of the infectious state integrates itself into the host and many years or even decades later can burst forth and cause shingles.
Thus to summarise, the ideal virus for our purposes would contain the dystrophin gene, be able to enter muscle cells only, integrate the dystrophin gene into the host genome such that it will start making dystrophin, and not in any other way affect the person.
In order to improve understanding of these papers, I would like to give a brief note on virus classification. The group of viruses to be discussed below are known as adeno-associated virus (AAV). This is a group like the influenza viruses or the measles viruses. Like the influenza viruses, which are subtyped in groups like ‘H1N1; H5N2; N2H4 etc, so the AAV are subtyped into serotypes AAV-1; AAV-2; AAV-3 etc. A modified AAV by recombining it with another gene then becomes a recombinant adeno-associated virus (rAAV), which can belong to any of the subtypes and thus be an rAAV-1; rAAV-2 etc. Now let us discuss the papers.
One of the viruses, which has rapidly gained popularity in gene therapy is the adeno-associated virus (AAV) and it is discussed in a recent review (1). As long ago as 1982 it has been considered for such a role because of its lack of pathogenicity (ability to cause illness), wide range of infectivity, and ability to establish long-term transgene expression, i.e. to continue to follow the revised genetic instructions intrpduced by the virus for a long time). In addition it needs a helper virus to complete a productive life cycle, i.e. it needs an adenovirus as a colleague to perform its role. Over 2,000 PubMed references on AAV have been published during the last ten years and great advances in the knowledge of the virus, have been achieved. These significantly improve the construction of AAV vectors (transmitting agents) and give a greater understanding on their use. The recent discovery of the existence of previously unknown AAV serotypes (variants) suggest, that there may be one preferred serotype for the cells in nearly every organ or tissue to target. The authors therefore conclude that AAV-based vectors have been successful in overcoming the main challenges of gene therapy. These include transgene maintenance, safety and host immune response (possible rejection of the therapy). AAV thus have been shown to have a high level of safety combined with clinical efficacy (i.e. they work well) and versatility in terms of potential applications. AAV are the vector of choice for a wide range of gene therapy approaches. In their review they report that recombinant AAV-2 (AAV serotype 2) vectors have been tested in pre-clinical studies for a variety of diseases such as haemophilia, 1anti-trypsin deficiency, cystic fibrosis, Duchenne Muscular Dystrophy, rheumatoid arthritis and others.
In the second study to be reviewed (2), the authors note that there remains a problem with the large-scale production for clinical use of recombinant adeno-associated virus (rAAV), i.e. the virus re-engineered to contain the desirable gene to be transferred. This remains one of the major challenges for continued development of pre-clinical and clinical studies, and for its potential commercialisation. In this study they examined the baculovirus expression vectors (BEVS) and insect cells as a potential method to produce rAAV economically on a large scale. The conditions for maintenance and storage of baculovirus stocks have been described previously and thus the supply of sufficient stable baculovirus stocks, to be used to produce rAAV at large scale, in a reproducible manner have been achieved, i.e. he good news of this study is that the problem of large scale production of rAAV has been solved.
In their recent study (3) Ghosh and Duan note that in the presence of a helper virus, AAV is reproduced but without a helper virus, a latent infection cycle is established i.e. the virus is dormant awaiting the bright conditions and AAV is propagated as an integrated provirus. In human cells, it has been shown that AAV-2 integrates in a specific point of chromosome 19, not the X-chromosome, which is where the dystrophin gene is situated. They note that most of the work has used AAV-2, but investigators have begun to appreciate the newly-identified AAV serotypes as gene therapy vehicles over the past few years, some display unique transduction patterns (i.e, ways of getting inside the cells and integrating into the chromosome) in different tissues. Some serotypes, especially AAV-6, -8 and -9, can achieve efficient whole body gene transfer, thus getting to parts of the body that other viruses cannot get to. Therefore these viruses can potentially put an correct version of a protein-coding gene where it will be able to produce the missing protein – hopefully dystrophin.
However, unfortunately, AAV cannot be used for a large therapeutic gene such as the dystrophin gene or the cystic fibrosis transmembrane conductance regulator (CFTR) gene (the gene responsible for cystic fibrosis) as it is too big to fit into the virus. In order to deal with this problem of size, efforts to delete the less important regions in a therapeutic gene have been made. The production of a minimized gene that can fit into a single AAV capsid is being considered. The problem is that such mini-genes may well be less functional than the full-length gene. Thus alternative strategies are being considered to deliver a large gene with an AAV vector. It has been demonstrated that AAV genomes undergo intermolecular recombination and with appropriate genetic manipulation it may be possible to double the AAV packaging capacity with two vectors and several distinctive dual vector approaches have being developed and these are discussed in this paper. In principle this means that the dystrophin gene would be split between two viruses and rejoined when they the target. This is still in the developmental stage.
In conclusion, it seems clear that AAV is the virus of choice for effective gene therapy. The large-scale production of rAAV is in the process of development and its ability to carry and therefore transmit the large dystrophin gene is being successfully tackled. We can but hope that the remaining problems will be solved soon so that our boys will be able to benefit from this treatment. Though the use of viruses to transmit a beneficial gene can be seen as a ‘Brave New World’ approach, we should not forget that it has actually gone on in nature for millennia.
References
1. Coura, R.D. & Nardi, N.B. (2007) The state of the art of adeno-associated virus-based vectors in gene therapy - art. no. 99. Virology Journal. 4: 99.
2. Negrete, A., Yang, L.C., Mendez, A.F., Levy, J.R. & Kotin, R.M. (2007) Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system. Journal of Gene Medicine. 9(11):938-948.
3. Ghosh, A. & Duan, D. (2007) Expanding adeno-associated viral vector capacity: A tale of two vectors. Biotechnology and Genetic Engineering Reviews. 24:165-178.
Karl A. Bettelheim
26.2.2008