Background

Background
Technology
Research
Glossary

Background

“Man's conquest of Nature, if the dreams of some scientific planners are realized, means the rule of a few hundreds of men over billions upon billions of men.” … “Each new power won by man is a power over man as well. Each advance leaves him weaker as well as stronger.” … “The final stage is come when Man by eugenics, by pre-natal conditioning, and by an education and propaganda based on a perfect applied psychology, has obtained full control over himself. Human nature will be the last part of Nature to surrender to Man. The battle will then be won.” … “But who, precisely, will have won it?"
        –C. S. Lewis, The Abolition of Man, 1943.

The advent of genetic manipulation techniques has given human beings tremendous power to control the DNA of everything from food to animals, including human beings. Genetic manipulation on human beings may have the potential to combat genetic diseases, but it also has the power to alter the genetic make-up of future generations. As this technology evolves, will prospective parents be able to control the traits of their children? A recent poll by the Genetics and Public Policy Center found that 59% of people approved of genetic engineering to avoid disease (34% disapproved) and 20% approved of genetic engineering to create desirable traits (76% disapproved). And in a Louis Harris poll sponsored by the March of Dimes, 42% of potential parents surveyed said that they would be willing to use genetic engineering on their children to make them smarter, while 43% would be willing to upgrade their children physically. Another survey found that over a third of people surveyed would choose to genetically alter their children to ensure that the children had an appropriate sexual orientation.

There are two distinct types of genetic manipulations: somatic manipulations and germline manipulations. Somatic manipulations are targeted to somatic cells and not intended to affect reproductive cells (also known as germ cells), such as sperm and eggs. However, some somatic manipulations may have germline effects. Somatic gene therapies have been proposed as a means to treat people directly by inserting a functioning gene or replacing a defective gene in specific organs (i.e., inserting genes to correct Alzheimer’s disease or Canavan disease into the brain), without intending to change the genes of the person’s offspring. There have been a significant number of somatic therapy trials both in the United States and abroad without much success.

Germline manipulations, in contrast, would affect future individuals by introducing genetic changes in sperm and eggs at early stages of embryonic development. The current debate regarding genetic manipulation is focused on this technology. With germline manipulation, the effect is to alter the reproductive, or “germline” cells. Germline manipulations create inheritable genetic modifications. The modification is intended to affect the person who develops from the sperm, egg, or embryo, but the change is also passed on to his or her own children and their descendants. Germline interventions are not known to have been intentionally attempted in humans as of 2004.

Those who favor germline changes argue that there is an important distinction between the utilization of inheritable genetic modification to eradicate disease and its utilization to enhance traits, “improve” human beings, and change human nature itself. While the distinction between somatic and germline interventions is clear, the boundary between the use of interventions for the treatment of disease and the use for enhancement is often blurred. Many believe that the problem lies in the fact that the definition of “disease” is constantly changing and has no objective, scientific basis. Should baldness or short statute be considered diseases to be treated by germline manipulations, for example? Some people argue that germline intervention should be used only to correct, treat, or cure debilitating genetic diseases. However, the difficulty lies in classifying what is treatment and what is enhancement. And many experts believe that germline manipulations are too dangerous to attempt in humans at all.

Opposition to germline modification is based on several factors. First, there are safety considerations. Germline interventions may affect gene function in ways that are not immediately apparent, causing devastating problems such as sterility or cancer in the resulting children. Even if the initial child is born healthy his or her offspring might be harmed. In some instances, the damage may not be recognized for a generation or more. Germline introduction of an improperly-regulated normal gene into mice resulted in offspring with unaffected initial development but high tumor incidence during adult life. Furthermore, interactions among genes and their products are highly integrated, have been refined over many generations, and often serve important developmental and physiological purposes. Introducing genetic modifications to such a balanced biological system could cause undesired effects. Presently, inadvertent germline manipulations may have already happened as a result of attempts at somatic gene therapy. Various assisted reproductive technologies, such as artificial insemination and in vitro fertilization have also been used to attempt to enhance offspring.

Inheritable genetic modification is also commonly criticized as being unnecessary as a response to inheritable disease. Many urge that adoption and assisted reproductive technologies currently offer women and men a variety of reproductive options, as do prenatal selection or the more hotly contested option of preimplantation selection. (This is commonly referred to as preimplantation genetic diagnosis and involves the selection of embryos for desired traits prior to implantation into the womb. The problem with this process is that it creates a number of embryos that are eventually discarded, frozen, or donated for research. Generally, some embryos are rejected and not implanted based on this screening.) With prenatal diagnosis, interested women and men can choose not to transmit specific identifiable genes by selecting against them. If there is no clinical need to perform genetic manipulation, then it follows that the primary reason for such intervention would be solely for human enhancement. Accordingly, it has been argued that the seemingly private personal decisions and “choices” about medical or non-medical program for enhancement would reflect prejudices, socioeconomic and political inequalities, and even current fashion. Some fear that modifications of unborn children undertaken on the basis of existing values and social conditions may later be viewed as disastrous.

Behind safety concerns lies the widely-held conviction that this may be the greatest question ever to confront the human race, since the very boundaries of what is human could be changed by inheritable genetic modifications. These modifications have the potential to be used for enhancing abilities that are thought to be genetically-determined, and may ultimately lead to the selection of desired genetic characteristics in a prospective child, producing a “designer baby.” More broadly, it could lead to the “re-designing” of the human species. The designing of children is occurring subtly as a result of individual choices through an open market. How are we, as a society going to judge such attempts? Should certain genetic manipulations be allowed and others not, as fashions and markets change? Should parents be able to buy height-enhancing genes for their embryos? Brain-enhancing genes? Sexual preference genes? And who will make such decisions? Government, in pushing economic goals? Parents, seeking smart, obedient, hard-working children?

Of course there is no evidence to support genetic determinism—the idea that everything we are is determined by our genes—but there is no doubt that genetic traits have a huge influence in shaping who we are.

Technology

Germline manipulation refers to genetic manipulation of germ cells, fertilized eggs, or the cells of embryonic stem cells (ES cells). These manipulations will be incorporated in all the cells of the resulting offspring, including his or her germ cells. As a result, the altered gene will be passed down to all subsequent generations, resulting in a transgenic animal. Transgenic means that a foreign DNA gene--a transgene--is incorporated into an animal’s genome early in development. The transgene is present in both somatic and germ cells and is expressed in one or more tissues.

Scientists currently employ a number of methods to perform inheritable genetic modifications in animals. The most commonly used method in research and breeding is pronuclear DNA microinjection, either homologous or nonhomologous. Pronuclear DNA microinjection involves the delivery of the genetic material via direct microinjection of extra copies of the desired gene into the pronucleus, which is the nucleus of the egg or sperm after fertilization but before they fuse to form the nucleus of the fertilized egg cell. As a result, the altered genes may then be incorporated into the genetic make-up of the individual and are not only altered in that individual, but are passed on to any offspring.

There are two methods of pronuclear microinjection, nonhomologous recombination and homologous recombination. In nonhomologous recombination, the insertion of DNA using the pronuclear microinjection technique is a nontargeted process, and would present several problems in any application to humans. This technique is characterized by random placement of the extra genes, a lack of control over the number of genes that are incorporated into the egg, rearrangements of genetic material within the egg, and genetic mutations caused by incorrect gene placement.

Animal studies show that nonhomologous recombination is very inefficient. Utilizing this technology, it is not possible to pinpoint the placement of the extra genes in an animal’s DNA. Only a small number of cells actually pick up the genes, and even when the genes are incorporated into the animal’s DNA, they are most often expressed at low levels and in a non-uniform manner. (Gene expression is the process by which a gene's coded information is converted into the structures present and operating within the cell). Specifically, a Science magazine editorial reported that pronuclear microinjection has a success rate of only about 10% in mice. Other reports cite even lower success rates, at less than 3% in rodents and even lower in farm animals.

In addition, this technology may have unpredictable side effects as a result of the extra genes being incorporated into the wrong location, or at incorrect levels, causing problems with over-expression, under-expression, or non-expression of the added genes. Because more than 50 gene copies can be inserted at one site, multiple site insertions are also possible. A gene in the wrong location or expressed at the wrong levels can affect other genes within the cell, altering their expression as well, leading to serious health problems such as developmental anomalies or cancer.

Homologous recombination also uses the pronuclear microinjection method for gene delivery, where insertion of the extra genetic material is targeted to a specific location in a chromosome, ensuring that the DNA will be placed in the normal chromosome location. This is currently the preferred technique for germline intervention, involving the replacement of a corrected DNA segment for the defective DNA segment. As a result, the inserted DNA will serve to function in the correct cells, at the correct time, and at the correct level.

ES cells are harvested from an embryo, grown in culture in vitro and are maintained under stringent conditions. The desired gene is replicated and developed into DNA that is analogous to a specific segment of the host DNA. The copied gene is then introduced into the nuclei of the ES cells by means of a vector or by electroporation, which is a process that uses strong, short pulses of electrical current to create pores across the cells’ membranes so the copied DNA can enter the cells. It can then incorporate into the cells’ nuclei, replacing the existing segment of the DNA.

The cells are then screened to determine whether the extra gene properly incorporated into the cells. If the gene has been properly incorporated, the transformed ES cells are then microinjected into embryos. Following the microinjection, the embryos are implanted into the uterus of the foster female. The resulting transgenic animals are tested for the desired gene. Animals identified as having incorporated the transgene may then be bred to produce offspring in which the transgene is present.

Only about one-third of the implanted animal embryos develop into healthy viable offspring. The frequency of correct insertion of the desired gene is no more than 10-20%. One advantage of the homologous recombination technique is that only one copy of the desired gene is incorporated into the animal’s genome and the site of the integration is highly controlled due to the targeting at a specific chromosomal location. However, the process is very time consuming and the desired DNA sequence must already be known. At this time, while allowing greater control than other techniques over the integration of the gene into the animal’s genome, homologous recombination is currently restricted to the mouse.

As an alternative to inserting extra copies of purified DNA by pronuclear microinjection, pronuclear microinjection of artificial chromosomes has also been used to create transgenic mice. Artificial chromosomes are chromosomes produced to contain several extra genes together with their appropriate regulatory sequences. This method enables introduction of relatively large numbers of genes, proper gene expression, and autonomous chromosome-like behavior of these entities during cell division without integration or translocation into the genome. Artificial chromosomes represent a means to alter characteristics that depend on complex gene combinations.

In one recent study utilizing artificial chromosome technology, around 50% of the mouse embryos survived the microinjection, and 50% of the viable fertilized egg cells developed. The copied gene was detected in 44% of the mouse embryos that were analyzed, though the embryos did exhibit varying degrees of gene expression, ranging from 8-67% of the cells expressing the gene. Analysis showed that 7% of the embryos that survived to term expressed the desired gene. The one female mouse that expressed the gene was bred with a normal male mouse and ultimately produced six litters of offspring of which 46% were transgenic for the desired gene.

Researchers at Case Western Reserve University in Cleveland have been working on developing human artificial chromosomes (HACs), which could potentially be used for transgenesis and as germline intervention vectors. In vitro laboratory studies indicate that the HACs do not elicit an immune response within the host cells and have the ability to allow expression in the correct cells.

Another method, commonly used for in vitro fertilization with men who have low sperm counts, is intracytoplasmic sperm injection (ICSI). It involves the injection of sperm directly into the cytoplasm of an unfertilized egg cell using a glass needle pipette. In the use of ICSI for germline intervention, sperm obtained from the male are placed in a detergent or are rapidly frozen and thawed to disrupt the cell membranes. This increases the chance that the desired gene will incorporate into the host DNA. The sperm are then incubated in a solution that contains the desired gene. The treated sperm act as carriers for the desired gene once it is microinjected into the unfertilized egg cell. The resulting embryos are then transplanted into a uterus for gestation, and the subsequent transgenic offspring will pass the incorporated genes to future generations. As with all previous methods, there are concerns about incorrect placement of the gene into the genome.

One advantage of intracytoplasmic sperm injection is that this method yields a higher percentage (above 80%) of offspring expressing the desired gene than those obtained through pronuclear microinjection of DNA or artificial chromosomes. The introduction of transgenes into sperm is also a procedural simplification when compared to the pronuclear microinjection technique using fertilized egg cells. Yet although homologous recombination via pronuclear DNA microinjection is the most successful method for germline intervention in mice, it is not currently available for non-mouse species.

Studies on assisted reproductive technologies have shown that the ICSI procedure increases the risks of chromosomal and other abnormalities in the resulting children. In a recent study, a research team concluded that infants conceived with the use of intracytoplasmic sperm injection or in vitro fertilization have twice as high a risk of a major birth defect as infants conceived naturally. Specifically, 8.6% of infants conceived with ICSI, 9% of infants conceived with in vitro fertilization, and 4.2% of naturally conceived infants were diagnosed with a major birth defect by one year of age. Compared to infants naturally conceived, infants conceived with assisted reproductive technology had more musculoskeletal, gastrointestinal, and chromosomal defects.

Genetic modifications may also be introduced into organisms using two types of viral vectors; retroviral vectors and adenoviral vectors. In both methods, the viral vector is initially modified to prevent it from replicating or causing disease in the target cells of the host embryo. The desired genes are incorporated into the viral genome, and then the virus is used to infect early stage embryos. While often used in animal research, this method has not been proposed for human use due to a number of reasons. First, many of the embryos do not uniformly carry the copied gene. Only some of the cells integrate the copied gene; many of the cells do not incorporate the new genetic material due to the delay in integration. Second, there is a low rate of germline transmission associated with retroviral gene transfer, where subsequent generations do not express the gene. Third, the size of the transgene is limited.

Retroviral vectors are used in a technique where genetic information, transferred as a ribonucleic acid molecule (RNA) rather than DNA, is used to infect egg cells or preimplanted embryos. Retroviruses are single-stranded RNA viruses that contain an enzyme called reverse transcriptase, which enables the production of viral DNA from cellular material of the viral RNA. The retroviral vectors can then be injected into the egg cells. One specific type of retroviruses, lentiviruses, has the ability to infect both dividing and undividing cells. The lentiviral vector is injected into the space surrounding the single-cell embryo, between the egg itself and the cell membrane. The advantages of using lentiviral vectors are that they are more efficient and cost-effective, as well as less evasive than pronuclear microinjection.

Adenoviral vectors are based on a family of viruses that normally cause benign respiratory tract infections in humans. The main advantages of adenoviral vectors in germline interventions are the efficiency of transduction and the high level of gene expression. One disadvantage is that adenoviral vectors do not integrate into the animal’s genome when replicating, causing the high gene expression to be short-lived. Since the expression is short-lived, any therapy that is based on adenoviral gene transfer would require repeated application of the vector.

The use of adenoviral vectors in gene therapy attempts has been shown to have considerable risks. Somatic gene therapy trials at the Institute for Human Gene Therapy at the University of Pennsylvania a number of years ago utilized a form of adenoviral vectors to transport new genes into patients. One of the patients enrolled in the trial, Jesse Gelsinger, who suffered from a rare liver disorder, underwent an injection of the adenoviral vector. Four days later he died of multiple organ failure due to a severe immune system response. Initial reports indicated that an undetected genetic condition or viral infection may have triggered the deadly immune response to the adenovirus. However, subsequent research by other groups on adenoviruses has found that the dose amount Gelsinger received may very well have set off a forceful defense mechanism within the body, targeting the invading pathogens and leading to multiple organ failure.

Recent Research Findings and Risks

Although germline intervention has not been attempted in humans, germline studies have been fairly common in animals, dating back to as early as 1976 when the first gene-altered mouse was created. Since then, genetic researchers have developed a variety of transgenic animals, such as the “geep” expressing both goat and sheep genes. “Obese” mice have also been common germline creations, where human genes that produce the human growth hormone are spliced into the animal, creating larger than average mice. In addition, germline interventions have been used to study the intervention technologies themselves, utilizing genes housing the glowing protein GFP found in jellyfish to detect whether the resulting offspring carry the specific gene. (GFP glows under a special light and allows researchers to study the presence or absence of the GFP in the tissues and organs of the resulting offspring.)

However, findings in animal studies give pause to whether the technology would be safe and effective in humans. There are problems with respect to low efficiency and low rate of integration into the genome. In one GFP study that tested the ability of sperm and altered DNA to transfer into mice egg cells, only 17-21% of live offspring carried the gene with respect to observable GFP in the skin, indicating that while the gene inserted, it was not correctly expressed in the animals.

Another GFP study conducted on monkeys, an animal that more accurately serves to generate data applicable to humans, resulted in only one live birth out of forty implanted embryos actually expressing the gene. One monkey (2.5% of implanted embryos), ANDi (the name is backwards for “inverted DNA’), was born with the altered GFP gene present in all tissues, although no fluorescence was observed. This suggests that the foreign genes are functioning poorly or not at all, and illustrates that the technique is not yet refined enough to be useful. The researchers must wait until ANDi develops through puberty to see whether germline transmission was accomplished by checking for the presence of transgenic sperm.

In addition to the low initial gene implantation and expression rates evinced in the GFP studies, there are also problems that manifest later in the life of the animals following the event of successful gene integration. Genes expressed in wrong tissues or in the developmental stage may have deleterious effects on the proper functioning of the cell, tissues, or organ, causing problems such as developmental complications, sterility, and cancer.

Perhaps the most illustrative of these problems is the much publicized study involving “Doogie Howser” mice. (These mice were aptly named “Doogie Howser” mice, after the brainy television character in the early 1990s who was practicing medicine in his early teens). Researchers at Princeton created mice with enhanced memory by giving them an extra gene associated with long-term memory. The transgenic mice exhibited superior ability in learning and memory tasks such as novel-object recognition, contextual and cued fear conditioning, and emotional learning. But despite the fact that these mice had increased cognitive and mental abilities, a subsequent study showed that mice with increased memory are more susceptible than normal mice to long-term pain.

Scientists at the Washington University School of Medicine found that altering genes to enhance memory (as seen in the “Doogie Howser” study) had broad effects on behavior, including increased responses to tissue injury and inflammation due to the altered gene’s role in pain perception. The Washington University researchers discovered that the transgenic mice with enhanced memory also exhibited an enhancement of persistent and chronic pain, suggesting that while the altered gene enhanced the learning and memory abilities of the transgenic mice, it also exacerbated behavioral responses to pain.

In another study that aimed to increase the muscle mass of cattle by the transfer of a gene known for increasing muscle mass, only one calf (0.2% of the initial implanted embryos) was born alive. The calf exhibited initial muscle increase, but muscle degeneration rapidly followed and, unable to stand, the calf had to be killed.

Other studies that have attempted to determine the effect of providing animals with extra copies of normally-present genes also shed light on potential problems with germline intervention. Where a gene is discovered to control a certain human characteristic and parents seek to have extra copies (or corrected copies) of that gene inserted into their child, the additional gene may not actually enhance but disrupt normal functioning. It has been shown that the over-expression of a single gene may drastically affect the way the body operates. While one human gene may control one specific aspect of an individual’s genetic makeup, such as increased intelligence or memory, it is not clear what havoc an enhancement of this gene may wreak on the body in other functioning.

For example, one study gave mice an extra copy of a gene that had been implicated in tumor expression. The offspring with the transgene appeared unaffected at birth, but developed cancer at 40 times the rate of the normal, unmodified mice. However, the extra gene did not seem to disturb the otherwise normal development of the mice. In another study, researchers investigated the deleterious biological effects of altering a tumor suppressor gene, which typically acts to destroy potentially cancerous cells, causing symptoms of old age and hastening of death in mice. When researchers deleted a section of the gene in some mice, none of the 35 genetically altered mice in the study developed life-threatening tumors, although some did acquire small tumors. In contrast, of the 56 mice with two intact copies of the gene, 45% developed deadly tumors. The age-related conditions observed were shrinking in size, thinning skin, slowly healing wounds, and organ shrinking. These results indicate that the ability to obstruct the cell division leading to cancer may interfere with the ability to replenish essential cells.

The insertion of foreign DNA into inaccurate sites in an animal’s chromosomes can also cause extensive disruption of normal development. Because the technique of pronuclear microinjection cannot accurately target where the gene is inserted, often the gene is placed in the wrong location, leading to devastating results. As an illustration of what can happen as a result of such misplacement, researchers disrupted a normal gene by insertion of foreign DNA via pronuclear microinjection, creating a genetic mutation on one of the chromosomes. As a result, mouse embryos treated with inserted DNA lacked eyes, inner ear canals, and abnormalities in tissues that control smell. A similar study led to mice that had limb, brain, and craniofacial malformations, along with displacement of the heart.

Glossary