Regulatory and Ethical Aspects

(Chapter 21)

Gene Therapy

A "perfect" cure of a genetic disease would be to provide the patient's cells with a corrected copy of the faulty gene. The first examples of success with such "gene therapy" now are available. Gene therapy is the correction of a heritable disease by the addition of a functional gene. Technically, the term can be divided into two categories: "germ-line gene therapy" and "somatic cell gene therapy". On ethical grounds, the latter is the only kind of gene therapy being considered in humans and involves the treatment of different cells in the body, but does not allow inheritance of the genetic changes; it is analogous to an organ transplant. For a disease to be a candidate for gene therapy the biochemistry of the disorder must be well understood, a cloned functional gene must be available, and there must be means available to transfer that gene into a tissue where the gene will be beneficial. Current conventional treatment protocols must be nonexistent or inadequate. Somatic gene therapy entails isolation of cells from the patient, in vitro transformation of these cells to introduce the desired gene, and reintroduction of transformed cells into the patient. A virtual requirement for successful gene therapy is a self-propagation of the transformed cells in the patient, as only then the product of the restored gene will continue to be produced and will continue to circulate in the body (remember that all proteins have a limited lifetime). A suitable type of cells for introduction of a functional gene are bone marrow stem cells, which divide and can differentiate to various types of white blood cells (lymphocytes). An early example of gene therapy applied to humans was the case of a young girl, who was born with adenosine deaminase (ADA) deficiency. This deficiency leads to an impairment of production and maintenance of two types of lymphocytes, yielding a defective immune system. A functional ADA gene was introduced in T cells (also a type of lymphocytes), and the transformed T cells were introduced into the patient. She has responded well to the treatment, and apparently is leading a normal life. Subsequent young patients have fared equally well as a result of the gene therapy treatment. Encouraged by this success, gene therapy is now applied (mostly on an experimental basis) to treat cystic fibrosis. In this case, the functional gene is delivered to lung tissue by a viral vector that has been disabled so as to not be infectious.

Other potentially suitable cell types for gene therapy are skin fibroblasts (which grow well in culture), and hepatocytes (liver cells). The problem with both cell types is that they are restricted to a certain part of the body, and do not circulate. Therefore, gene therapy using such cells will be most effective if it is to treat diseases that affect mostly the skin and the liver, respectively, or that are metabolic in nature and affect the level of certain compounds in the bloodstream.

One problem with gene therapy is that one does not have control over where the gene will be inserted into the genome. The location of a gene in the genome is of importance for the degree of expression of the gene and for the regulation of the gene (the so-called "position effect"), and thus the gene regulatory aspects are always uncertain after gene therapy. Another problem is that gene therapy is still experimental and is not without risks. One example was the 1999 death of Jesse Gelsinger, a gene therapy patient who lacked ornithine transcarbamylase activity.

The vector by which the appropriate gene will be introduced into the body is of major importance for the success of gene therapy. The earliest vectors were based on the murine leukemia virus, and carried their genetic passengers into dividing cells. The drawback of this is that most of the body's cells are non-dividing. The next generation made use of adenoviruses, the same kind of viruses that cause the common cold. These viruses had a higher rate of delivery, but the immune system quickly kicked the foreign material out of the body. Lentiviruses, including HIV, promise to incorporate their passenger genes into non-dividing cells, but the use of an attenuated strain that is closely related to deadly siblings causes much concern. Probably the most promising group of viruses in terms of gene therapy are the adeno-associated viruses (AAVs) that cause no known disease in humans; however, they are difficult to produce in mass quantities.

A matter of mostly ethical consequence is the question whether human gene therapy may involve cells that will be transmitted to the next generation (egg or sperm cells). In the case of transformation of bone marrow cells, the patient will be cured, but he or she will still be a carrier of the disease in the sense that progeny will inherit non-transformed cells. This problem would be avoided if generative cells would be transformed as well. However, there is not yet a consensus on whether this would be ethically correct. Even though most people may agree that transformation of generative cells to combat life-threatening diseases should be allowed, it is difficult to decide on where to draw the line.

After ethical issues would be resolved, a practical consideration would be how one should go about altering germ cells in humans. This does not appear to be simple. In mice three methods have been described to work reasonably well: (1) Manually inject 100-1000 copies of a gene into the pronucleus of a recently fertilized egg. The embryos are then transferred back to a mother mouse, and usually a few % of the offspring will inherit the gene and pass it on to succeeding generations. Of course, even though this works well for mice, its low percentage would not make it suitable for humans. Also, it can be carried out only at the single-cell stage of the fertilized egg, making it useless for gene therapy after birth. (2) Transform embryonal stem cells. These cells are early embryonic cells that can grow in culture. When mixed with non-transformed cells of the growing embryo, they are capable of giving rise to all cell types of an organism, including germ cells. However, it is unpredictable to what cells the embryonic stem cells will give rise in any particular case, and again this requires treatment at the embryo stage. Thus, this also is not particularly applicable to humans. (3) Genes can be introduced into retroviruses, which have a certain probability to integrate into genomes, taking inserted genes with them. But again, this is unpredictable in its frequency and target. Thus, for better or worse, human gene therapy of germ cells still may be a long way off.

The general success of gene therapy experiments, however preliminary and limited in scope, now has led gene therapy more into the mainstream of medical science. The federal government has moved towards relaxing its scrutiny of human gene therapy experiments, so that the approval process of most gene-therapy protocols are much quicker and simpler. Not all gene-therapy protocols need to be subjected to public examination before the Recombinant DNA Advisory Committee (RAC) anymore. Only if new and cutting-edge experiments are proposed, or if important safety or policy issues are concerned, researchers may be asked to appear before the RAC. The reason for the change in regulation mostly is caused by the fact that many of the protocols are rather standard, and there is no need to go through a lengthy approval process for something that has been approved in essentially identical form already. However, several unsuccessful gene therapy treatments in the past year have caused public concern, and a tightening of the protocols and improvement of patient monitoring is being considered.

Further information on gene therapy can be found at links at http://www.ornl.gov/hgmis/medicine/genetherapy.html.

Ethical considerations

Medical applications of biotechnology may have far-reaching ethical consequences. For example, in 1993 an announcement was made that "scientists had cloned human embryos"; three copies of human embryos were created outside the body, and were allowed to develop for six days. A more recent stir was the 2001 announcement by Advanced Cell Technology that cloned human embryos of 4-6 cells had been grown. In essence, human cloning qualitatively is not new, as identical twins are clones. However, the test-tube approach is different in that embryos are selected (which is not the case for identical twins) and can be divided many times, to create a large number of embryos with genetically identical makeup. Since then, developments have been rapid, and there no longer are technological reasons why one could not clone a human. A human embryo reportedly has been cloned in Britain in 2004. There is even a Human Cloning enthusiasts website (http://www.humancloning.org) as well as several websites with lots of information and links (http://www.globalchange.com/clonlink.htm; http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml; http://www.religioustolerance.org/cloning.htm). The federal government has stipulated that federal money cannot be used for cloning research involving human embryos, but this is quite an ineffective measure as now much of the scientific work in the field is carried out by private firms. Several states, including Arizona, also have laws on human cloning (http://www.ncsl.org/programs/health/genetics/rt-shcl.htm). There is no informed consensus yet on cloning, but certainly this issue will need to be raised in the public arena. There still is a long way to go on this. A good web site is that of the Center for Genetics and Society (http://www.genetics-and-society.org), which conducts research on the implications of the new human genetic technologies.

Bioethics also extend into the traditional medical arena. With the development of life-extending tools (including mechanical ventilators, exotic drugs, organ transplants, and artificial nutrition and hydration devices), one must ask the question whether we should keep a person alive just because we can. And who should answer that question? Also, should particular medical products (such as human growth hormone) be available to everyone, or what are the grounds for selection? Or who should have access to anyone's DNA fingerprint? And how ethical is the business standpoint of pharmaceutical companies, who invest a lot of money to develop "luxury" medicine and treatment for the rich in the Western world, but who have invested 100-fold less in new drugs for malaria, cholera, and other lethal maladies of the tropics? These are issues that clearly go beyond what is covered in the classroom, but important to think about and form an informed opinion about.

You may want to look at the web site of the Council of Responsible Genetics, which can be found at http://www.gene-watch.org. This Council may seem a little overcautious, but many of the issues brought up are very valid regardless the position one takes on them.

Stem cells

Although mouse embryonic stem (ES) cells have been isolated more than 20 years ago and have successfully been used in creating transgenic mice, it was not until 1998 when Dr. James Thomson from the University of Wisconsin, Madison succeeded in isolating the first human ES cell line (http://www.news.wisc.edu/packages/stemcells/). Embryonic stem cells are cells derived from the inner mass of the blastocyst (4-5 day old human embryo) that are capable of self-reproduction for long periods of time and that can yield specialized cells that constitute various tissues and organs. A nice illustration of the procedure can be found at: (http://www.news.wisc.edu/packages/stemcells/illustration.html).

Because of the pluripotent nature of stem cells, researchers and patients alike put great hopes into the use of ES cells for treating diseases such as Parkinson's, Alzheimer's, diabetes and repairing damaged hearts and spinal cords. As often is the case with exciting new technologies, ES cells are surrounded by a cloud of political, ethical and other issues that make them very controversial.

Stem cell research funding is an example of how changes in the political climate and public opinion affects scientific research almost overnight. After initial backing by the Clinton administration in 1999-2000 to allow the use of NIH funds for ES research using already existing embryos frozen in fertility clinic labs, in August 2001 President Bush announced the decision that human embryos no longer be used as a ES cell source; only currently existing ES cell lines could be used for research approved by NIH. The text of the Bush speech can be found at http://www.washingtonpost.com/wp-srv/onpolitics/transcripts/bushtext_080901.htm. However, there is serious concern on whether existing ES cell lines are sufficiently viable to support the myriad of potential applications with great medical benefit. (http://www.sabr.us/medical.htm), and currently there is pressure from the scientific community in the US to increase the number of ES cell lines (http://www.aaas.org/spp/cstc/briefs/stemcells/index.shtml).

An excellent source of information about ES and other types of stem cells can be found on http://stemcells.nih.gov. The AAAS 1999 report on ethical and policy issues is available at http://www.aaas.org/spp/sfrl/projects/stem/main.htm.

Besides ES cells, other cell lines are being developed for potential therapeutic applications. These are embryonic germ cells, derived from human fetal tissue and adult stem cells derived from tissues of adult humans. These cells, however, do not share the same proprieties as ES cells (e.g., they have a more limited repertoire of differentiation) and further research is necessary to establish their usefulness in practical applications. (more about embryonic germ cells and adult stem cells in http://stemcells.nih.gov/info/scireport)

The "early" (1977-1983) applications of genetic engineering all involved expression of human genes (such as the insulin, growth hormone and interferon genes) in bacteria that were kept in the laboratory. The level of expression was up to a million protein molecules per bacterial cell, which means that about a mg of protein could be extracted from 100 ml of cell culture. This was a marked improvement over the traditional method of extraction of relatively rare enzymes from tissue. For many purposes, it suffices to grow genetically engineered organisms in the laboratory, and to obtain the desired product from them. However, laboratory containment of production-scale numbers of transgenic plants and animals is impractical, and such genetically altered species will need to be released. Therefore, it is important to consider the requirements that need to be met for a genetically modified organism to be released in the environment.

The first genetically engineered organism that was approved for release in the US in the early nineties was a bacterium, Pseudomonas syringii, which, in contrast to most of its naturally occurring kin, does not make a protein that acts as a nucleation site for ice formation during light freezing conditions. Thus, if the genetically engineered bacteria rather than the naturally occurring ones are sitting on (and in) a plant leaf, ice formation (leading to rupture of cell walls and to plant damage) will not easily occur. Natural Pseudomonas syringii strains with properties identical to the genetically modified one do occur, so one can argue that no new bacterial characteristics are added to the ecosystem.

Around the same time, field tests were done with genetically modified plants. In 1992, transgenic tomato that expresses the coat protein from a plant virus, the tobacco mosaic virus, was tested. Plants that express this coat protein (obtained by transforming tomato protoplasts with the virus coat protein gene, and regenerating the protoplast to an entire plant) are less sensitive to the tomato mosaic virus and the tobacco mosaic virus. The insertion and expression of the coat protein gene in tomato does not result in any loss of productivity of the plant, whereas virus infection of non-resistant plants can decrease yields by 20% or more. Thus, the insertion of the gene for the tobacco mosaic virus coat protein into tomato leads to an increased resistance against two viruses without affecting the yield of the crop. The reason why viral coat protein expression in the plant leads to increased resistance of the plant against the "real" virus is not yet understood.

As will become clear in the next few paragraphs, the rate of release of genetically engineered organisms during the past decade has been mindboggling. However, first we need to consider what may be the effects when one introduces genetically engineered organisms into the environment. In the first place, is there any risk to the environment (for example, an upsetting of the ecological equilibrium in the area)? Secondly, does the introduction of the organism make sense in the long term (for example, is it likely that weeds or insects will rapidly become resistant to the herbicide or pesticide upon extensive application)? Thirdly, do we know sufficiently about the nature of the genetically engineered organism to be released to be sure that there will be no side effects (like a higher sensitivity to certain diseases)? Fourthly, is the introduction of the genetically engineered organisms the best way to go, or is it used to temporarily fix other problems? In the case of insect-resistance, most genetic modifications of plants involve the introduction of an appropriate Bt toxin. For introduction of herbicide resistance, there are several possibilities: either (1) introduction of a gene whose product metabolizes the herbicide, (2) introduction of a highly expressed copy of the gene for the receptor protein (thus "catching away" all herbicide molecules, and still have enough receptor protein without herbicide to function with), or (3) introduction of a gene that contains a mutation in the receptor protein so that the receptor protein has a decreased affinity for the herbicide without affecting the functional activity of the protein.

In the United States, herbicide-tolerant soybeans became available to farmers for the first time in 1996. This has proven to be quite a success story. Within two years, over 4 million acres (40 percent of the U.S. soybean acreage) had been planted with herbicide-tolerant soybeans -- making soybeans the number one bioengineered crop in the United States. Why have U.S. farmers taken to the herbicide-resistant soybeans and similar crops? It is because they have found that using these bioengineered seeds reduces the need to plow their fields to control weeds; decreases the amount of chemical herbicide they need to use; produces higher crop yields; and can deliver a cleaner and higher quality harvest. Total herbicide sales have not increased, indicating that environmental impacts are limited. Pesticide tolerance has been introduced primarily by means of introducing the gene for Bt toxin into plants. Corn provided with genetic protection from the European corn borer by insertion of the appropriate Bt gene was approved in the United States in August 1995. Fields of corn with Bt protection, on average, have 7-11 percent increase in yield per acre in comparison to more conventional corn, and the amount of sprayed pesticide has decreased, thus decreasing the environmental impact of agriculture.

Companies want to safeguard their investments in generating new and "better" varieties of agricultural crops. An interesting development over the past few years is the appearance of "terminator technology" designed to prevent propagation of genetically engineered plants. This was developed to prevent farmers from using home-grown seeds of genetically engineered plants, and to force the farmers to buy seeds from the company who developed them. The principles of this technology are explained thoroughly in http://filebox.vt.edu/cals/cses/chagedor/terminator.html, and terminator technology has been introduced briefly (barstar/barnase) in an earlier chapter of this on-line syllabus. The ethics of this approach clearly are multi-faceted: on one hand it is clear that the company needs to recapture the investment they made in developing the genetically engineered plants, but on the other hand poor farmers may not be able to afford going back to the company and buying seeds every year.

Use of genetically modified agricultural crops is widespread globally. http://www.isaaa.org/kc/CBTNews/press_release/briefs32/figures/Biotech_map_acreage.jpg provides an overview of the number of acres with "biotech crops" in the various countries. Globally, over 200 million acres now has genetically modified crops. After the USA, the country with the most genetically engineered agricultural crops is Argentina, with 99% of the soybean crop there being of transgenic origin. In 2004, farmers planted biotechnology-derived seed on 56% of global soybean acres, 14% of global corn acres, and 28% of global cotton acres (http://www.isaaa.org/kc/CBTNews/press_release/briefs32/figures/adoption_rates.jpg). Most biotechnology-derived crops most were tolerant of specific herbicides, and some were tolerant to specific insects, or both insect and herbicide tolerant (http://www.isaaa.org/kc/CBTNews/press_release/briefs32/figures/dominant_crops.jpg). Thus far, there is no convincing evidence for negative effects of these transgenic crops on humans or the environment. In 1999, preliminary reports were published indicating negative effects of plants producing the Bt toxin on caterpillars of the monarch and other butterflies, but subsequent field research demonstrated that the Bt level and toxicity in the plants were insufficient to negatively impact butterflies (http://www.nature.com/nsu/010913/010913-12.html).

Release of transgenic crops and animals in some respects is similar to the release of a "new" organism into the environment. The effects may be difficult to predict: there are success stories (many agricultural crops are not native to where they are grown), but (often unintentional) introduction of new species into an ecosystem in some cases has led to huge disasters. For example, a fungus introduced into America from Asia killed almost all of North America's chestnut trees. Another fungus has eliminated most Dutch elm trees from the Eastern US. The myxomatosis virus introduced in Australia (by Australian scientists) almost completely annihilated that continent's rabbit population, where this species had become a major pest within a century after its introduction in Australia. However, it should be mentioned that the rabbit strikes back, and has become essentially resistant to the virus; another virus has now been "accidentally" introduced in Australia. More than half of the insect pests in the US today come from abroad. Similarly, starlings, house sparrows and gypsy moths are all introduced animals that America could have lived without. By the same token, most of the USA's major crops, including soybean, wheat and also rice, are not indigenous to America. Such arguments are valuable reminders of biotechnology's potential to do great good or great harm, and thus one should examine very carefully the potential effects of the release of genetically engineered organisms into the environment. On the other hand, it should be realized that in many cases hybrids with properties close to those of certain genetically altered organisms could be selected by repeated crosses, as has been done virtually throughout history. Thus, it makes no sense to over-react when encountering an organism that is genetically engineered; factors such as the nature of the introduced gene (and its product) need to be evaluated first before any judgment on its potential environmental danger or on its merits can be made.

One particular issue pertaining to the safety of release of genetically modified organisms is whether desired characteristics in crops can confer adaptive advantages to weedy species. Spreading of plant genetic material is very easy. For example, bees are important pollen vectors over a range of distances and farm-to-farm spread of plants such as canola (rapeseed) that have closely related wild relatives. Pollen can also travel for miles in the wind. If crossing with wild plants and native species, herbicide or pesticide resistant weeds might result. Indeed, in Canada canola-related weeds have been found that have become resistant to three types of herbicides. It is not only related plants that may receive the transgene. There is also a report of gene transfer from genetically engineered rapeseed to bacteria and fungi in the gut of honey bees. Therefore, it is clear that one cannot guarantee the absence of spreading of transgenes that have been introduced. The question that then needs to be addressed in the evaluation of the risks of release of a genetically modified organism is whether such a spread would pose unacceptable environmental risks.

Apart from a rational analysis of potential risks, public opinion is also an important factor in the fate and success of transgenic plants and animals. After a decade of research, development, testing, and approvals, Calgene's Flavr Savr^® genetically engineered tomato hit the market place in the nineties. This tomato is slow-ripening due to decreased ethylene production, and thus it can be picked later and still be sold in good shape in the grocery store. This was the first genetically engineered food product to be marketed. However, there was a concern about the public perception of the product, and the Flavr Savr^® tomato no longer is marketed commercially. Another example of where public perception had a major effect was the case of Starlink corn: In 2000, in tacos traces of a Bt corn variety (DNA fingerprinting in action!) were found that had not yet been approved for human consumption (pending the outcome of allergen tests) but had been approved for use as animal feed. A public outcry followed. Even though Starlink corn was approved for public consumption soon thereafter and no proven cases of allergies due to Starlink have been found, this was poor public relations for the biotechnology industry. Currently, no corn varieties will be approved for animal feed if they have not been approved for human consumption as well.

Guidelines have been prepared by both national and international institutions for screening and characterization procedures of recombinant DNA organisms. The most well-known are the 1976 NIH (National Institute of Health) guidelines for research involving recombinant DNA molecules, formulated after a meeting of scientists at Asilomar. These guidelines were very strict, since at that time there was not much experience with the potential hazards of biotechnology, and the guidelines were (rightfully) targeted at a "worst-case scenario". After a few years it became clear that many of the risks were initially overestimated, and the guidelines relaxed over the years. Today some 90% of the experiments involving recombinant DNA are exempt from the guidelines. However, now new regulatory concerns have emerged: in some cases the approval for various aspects of release of genetically engineered organisms or the production of drugs etc. by transgenic organisms is spread out over several federal agencies (Food and Drug Administration, the Environmental Protection Agency, and the US Department of Agriculture), so that product approval is a long and tedious process. Although obviously the streamlining of the approval process must not result in a loss of thoroughness and quality of this process, it is obvious that one single agency can do a better and cleaner job than several agencies that often work in parallel (or antiparallel) with respect to each other. In addition, the workload of some agencies has increased dramatically over the last few years (now biotechnology is coming of age), while the staffing of the agencies has not kept pace.

In an attempt to streamline the approval process for field testing of genetically engineered plants, the USDA no longer requires submission and approval of a description of the method for conducting the field tests before the tests actually take place. Now for routine tests, one only needs to notify APHIS (Animal and Plant Health Inspection Service, a branch of the USDA) as long as the field tests follow basic guidelines. Waiting for approval is not necessary in these cases. To qualify for this "express-lane treatment", the researcher must certify that (1) the transgenic plant is one of the following species: corn, cotton, potato, tomato, soybean, or tobacco; (2) the transferred gene is stable; (3) the process will not produce disease in the transgenic plant; (4) the process introduces no infectious material to the plant; (5) the process poses no significant risk of creating new plant viruses; and (6) the transgenic plant does not contain any functionally intact genes from human or animal pathogens. This new notification procedure reduces approval time, cuts cost, encourages biotechnology innovations, and focuses USDA resources on the areas of greatest complexity. In another move to limit unnecessary paperwork, USDA allows specific transgenic plants to be removed from regulation after adequate field testing has been completed.

Whatever one's view on the appropriateness of current rules on approval of genetically modified plants and animals in the environment, there is no doubt that such organisms are hard to avoid in the food that you eat or the clothing you wear. Suitable web sites with additional information on government and regulatory resources are http://www.nbiap.vt.edu/, http://usbiotechreg.nbii.gov/ and http://agnic.umd.edu/.

Release of genetically engineered microbes

Thus far, release of genetically engineered microorganisms has not been looked upon favorably by scientists and regulatory agencies. Microbes cannot be easily tracked in the environment, and it is felt that risks associated with release of genetically altered microbes are too large. Instead, naturally occurring organisms may be fished out of their natural habitat, enriched in the laboratory, and released at a different location. This may be done, for example, in the case of in situ bioremediation.

When discussing the risks associated with biotechnology and the release of genetically engineered organisms, one has to keep in mind that there are risks involved with almost anything. We ride our bicycles or drive a car, use energy from a nearby nuclear power plant, live in a flood plain, get a suntan, or maybe we even smoke, and still may be worried more about genetically engineered foods than about any of the daily hazards we have chosen to take for granted. Our choices may be irrational because we may make decisions based on emotions rather than facts. Some of the factors triggering our emotions may have to do with whether the risks are voluntary (more acceptable) or involuntary (less acceptable), whether we are in control or not, whether the risks are familiar or not, and whether the risk is natural or man-made. However, this may be pretty misleading. For those of you who like(d) cabbage, mushrooms, and peanut butter: did you know that at least for bacteria the carcinogenic potential of these presumably healthy items is very high? Often the media are keen on just reporting "facts" that will draw the attention of the reader, but there is not necessarily a critical comparison with other items.

Risks and benefits need to be put together and compared. Are we prepared to take certain risks to enjoy particular benefits? Depends on the risks, and depends on the benefits, you may answer. The same may be true with genetically engineered materials. What are we comfortable accepting as risk, and what benefits do we expect from it? This may be an important question to consider.... it clearly may have a different answer for any of us individually, but we must think about it rationally.

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