[转贴]局部基因治疗对肌肉生长和修复的作用

Localized gene therapy promises muscle growth and repair
Oren Bassik

The New York Times Magazine article in question, a lengthy article on the abuse of medical technology in sports, describes the potential application of research conducted at the University of Pennsylvania. This research, some of which has already been published, is attempting to use gene therapy to promote muscle growth, by increasing the production of a growth-promoting factor by muscle cells.

Gene therapy is a promising new technique which provides the only real cure for many diseases. As covered in class, “genetic diseases” is a term covering both chromosomal diseases, such as Down's syndrome and triple-X syndrome, as well as diseases which are the expression of a certain set of genes inherited normally, from here on referred to as “hereditary diseases” or “inheritable diseases”. Gene therapy is not useful in combating chromosomal disorders, as will be made clear below. Rather, gene therapy is useful when modifying or adding a specific gene is enough to cure or alleviate a disease.

In recent years, many specific genes have been identified which either cause a specific disease or cause a higher probability of developing a disease – examples include genes for hemophilia and cystic fibrosis in the former case, and breast cancer in the latter case. Traditionally, therapies for these diseases have only been able to attack symptoms caused by the defective gene. For instance, in the case of hemophilia, the current therapy consists of regular injections of clotting factor – a blood protein essential to clotting – and a lifestyle which minimizes the possibility of bleeding. Gene therapy aims to cure the disease at its source, replacing or augmenting the old defective gene with a new gene.

For a gene therapy to be successful, a disease must meet a number of requirements. First, the gene which is responsible for a given disease must be isolated, as well as the “normal type” for this gene. The best candidates for gene therapy are diseases which are determined only by a single site on the DNA; many diseases are caused by the lack of a single enzyme or other protein (for instance, phenylketonuria, sickle-cell anemia, and hemophilia) and these have been the first diseases for which gene therapies have been researched.

The next requirement for a successful gene therapy is a vector to transmit the gene. So far, scientists have been experimenting with vectors which are carefully modified viruses, engineered to do no damage but to inject their genome into human DNA. Viruses are the obvious choice, because they have already in nature evolved the capacity to inject their DNA into a cell and to use that cell to reproduce. The choice of virus is very important, as different viruses have evolved to infect rather specific target cells. For instance, retroviruses are used to target immune cells; adenoviruses are used to target cells in the respiratory and digestive system; and various herpes simplex viruses have been used because they target neurons. Another vector which has been experimented with involves the creation of a lipid sphere, with an aqueous core containing DNA. Another non-viral method researches are investigating adds a 47th chromosome to the target cell, leaving all other DNA unchanged. These methods have the advantage of being able to transmit large amounts of DNA (viruses are generally quite limited by size) and of avoiding the immune response normally associated with viruses. Researchers have had difficulty, however, transmitting such large molecules into the nuclei of cells.

Another requirement for successful gene therapy is a very targeted administration of the vector. Many hereditary diseases are only problems in specific cells – cystic fibrosis, for instance, requires that a gene be transmitted to lung tissue. Hemophilia therapies have generally focused on the liver, where clotting factor is normally produced, but there has also been experimentation with muscle tissue. This need for specific targeting already affects the choice of virus, but also affects the method in which this virus is administered. Therapies are either in vivo, where the gene transmission occurs inside the body, or ex vivo, where selected tissues are removed from the body, modified by the viral vector, and then reinserted into the body. For an in vivo gene therapy, vector is usually injected at or near the target cells or into the bloodstream and the virus is left to its own devices. Ex vivo therapies allow for much more control and choice of vector; they also permit techniques such as electroporation, where an electric current is used to open up cell walls and allow DNA to enter into cells, to be used. In general, for very specific tissues, an ex vivo approach is often easier to get working (in rats and dogs, at least) and safer. However, an in vivo approach is generally more desirable, because a treatment can consists of a simple injection, as opposed to laborious surgical techniques.

The specific research in question is targeted at a set of diseases which does not entirely fit the above specifications. In fact, the research in question aims to develop a therapy for a whole group of diseases, with mostly unrelated causes: muscular dystrophy, “sarcopenia” (a name for the decay of muscle in old age), as well as almost any disease in which the body does not produce enough muscle. This is possible because they are not, in this case, attacking these diseases at their source – the sources are varied, and not generally genetic at all (old age, for example.) However, a therapy is possible in this case because by stimulating the production of a single protein, it is possible to stimulate the growth of muscle.
Specifically, the protein used in this study is known as muscle insulinlike growth factor-1, or IGF-1. IGF-1 is a protein which promotes muscle growth, speeds up reparation, and delays decay. mIGF-1, is different from other IGF-1 proteins because it is normally only produced in skeletal (voluntary) muscle, and also normally stays there. Previous attempts at therapy have used less specific forms of IGF-1; these have promoted muscle growth throughout the body and specifically, have promoted growth of cardiac muscle in the heart. Mice upon whom this has been tried die of an abnormal enlargement of the heart. The switch to this specific protein was essential for the research to show results.

Another crucial feature of the design of this new research was the choice of vector. For this study, an “adeno-associated virus” was used. An AAV has a number of important features. First, it causes no known disease in human beings. Other gene therapy trials have used viruses which have been dangerous, and in one case complications arising from the viral infection turned out to be fatal. AAV is known (in some areas) to infect a fifth of the human populations with no known consequences. Second, recent research has engineered AAV’s which are very efficient at targeting specific tissues. One variant, which targets the liver very efficiently, is being used in a trial to get the liver to produce blood clotting factor 9, providing a cure for type B hemophilia. Another variant, used in this study, readily infects skeletal muscle cells, which are actually the virus’ preferred tissue target.

One disadvantage of using this strain of AAV’s as a vector is that its entire genome consists of less than 5000 bases. This limits its usefulness in general as a gene therapy solution for human hereditary diseases, because most human genes are longer than this. The gene for mIGF-1, however, fit.

Finally, this research has used old surgical methods in new ways to further improve the targeting of the gene therapy, increasing both the efficiency of transmission and the amount of virus needed to be synthesized. Specifically, by putting specific parts of the body on a bypass, and passing vector infected blood through this bypass, it is possible to apply gene therapy to only a specific set of muscle. This is also helpful because it allows a steady stream of viral blood to be passed over muscle, as opposed to a one-time dose provided by an injection.

The therapy being pioneered at the University of Pennsylvania is very promising. Research on rats and mice has shown greatly increased muscle, even in dormant mice, and has shown that this increased muscle mass stays on even on old age. Results on longevity are as of yet statistically inconclusive, but also seem promising. There have been no immune system reactions, and problems with cardiac muscle have been solved as well.

If all goes well, this gene therapy will provide a cure for a number of muscular dystrophies, which until now have been untreatable, as well as a cure (and even a preventative measure) for the natural decay of muscle that begins in old age. At the same time, however, there is a potential for abuse. It is the stated opinion of the lead researcher in this project, Dr. Sweeney, that “… but on the world stage, in a world where countries in the past have shown that they want their athletes to win no matter what, … , one can imagine that with enough money you could put together a program to genetically engineer your athletes and do it in such a way, which is what one is really concerned about that it would be totally undetectable unless you were to remove tissue from that athlete.” Ignoring the ethical issue of cheating in athletic events – which is not insignificant – the possibility of less democratic countries trying such gene therapy on athletes without any safety testing or even regard for consequences is alarming. The well-known East German practice of forcing steroids on to their swim team without their knowledge destroyed the lives of a number of athletes. Gene therapy, which has proved fatal in some cases and which the FDA has not approved a single instance of, has yet to be determined to be safe and is particularly worrisome because it involves changes to the genome. Thus, on final estimation, the ethical concerns raised by the article are real, but due to the still limited nature of the research they are at least a decade or so away from being “immediate” concerns.

References:

Newspaper Article:
Sokolove, Michael. 2004. “The Lab Animal”, New York Times Magazine. Jan 18, 2004.
Also available at:
http://www.nytimes.com/2004/01/18/magazine/18SPORTS.html

Journal Article:
A.Musarò, K. McCullagh, A. Paul, L.Houghton, G. Dobrowolny, M. Molinaro, E. R. Barton, H. L Sweeney, N. Rosenthal. 2001. Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nature Genetics: vol. 27 no. 2, p. 195 – 200.
Also Available at:
http://www.nature.com/cgi-taf/DynaPage.taf?file=/ng/journal/v27/n2/full/ng0201_195.html

Musaro, A. & Rosenthal, N. Maturation of the myogenic program is induced by post-mitotic expression of IGF-1. Mol. Cell. Biol. 19, 3115-3124 (1999).

President’s Council on Bioethics, transcript of Friday, September 13, 2002, entitled
“Session 7: Enhancement 5: Genetic Enhancement of Muscle”:
http://www.bioethics.gov/transcripts/sep02/session7.html

Muscular Dystrophy Association. “Protein Turns Average Mouse into Mighty Mouse”. April 2001:
http://www.mdausa.org/publications/Quest/q82resup.cfm#protein

Human Genome Project Information. “Gene Therapy”. December 2003:
http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml