Engineering the Germline

All 100 trillion cells in each adult human trace back to a single diploid cell, the fertilized egg. Nearly all of the cell divisions are mitotic, faithfully repli cating the zygotic genome into armies of derivative cells. Most of these are somatic cells that constitute heart, skin, nerves, liver, muscle, blood, and the body's many other parts. However, a significant event occurs early in embryogenesis when a few thousand amoebalike cells begin to form the primordial gonadal tissues of the developing embryo. It is from this small band of dividing cells (founders of the germline), that all of the individual's eggs or sperm eventually will arise, and it is only these germline gametic cells whose DNA has a chance to survive the inevitable death of the individual (i.e., by contributing to zygotes of the next generation).

Whatever hopes or ethical concerns may attend the practice of human somatic gene therapy, they are likely to become magnified in deliberations on germline engineering. In germline engineering, the genetic alterations can affect not only the person receiving the GM procedure, but also his or her descendants. Germline DNA is potentially immortal. Each piece of human DNA in existence today traces its history through an unbroken chain of ancestry extending back across thousands and thousands of generations, each link in the chain forged by gametic cells. Looking ahead, all people in the future will derive their DNA from the germlines of those of us alive today. Thus, any tinkering with the human germline is not to be initiated cavalierly.

Yet, in the first half of the twentieth century, various eugenics movements in effect did just that by encouraging reproduction by people with supposedly desirable genetic features (e.g., fair skin, square jaws, or favored character traits) and discouraging reproduction by those deemed undesirable. A nadir for eugenic efforts came in Nazi Germany, going so far as to promote racial extermination purportedly to improve humanity's gene pool. Now, in the twenty-first century, a new brand of eugenics has become imaginable: direct alteration of the human germline via recombinant DNA techniques. However, the misplaced goals and horrible actions of last century's eugenics crusades should give serious pause to any modern-day genetic engineer who might feel an urge to direct our species toward some genetic ideal or imagined utopia.

On the other hand, would not we wish, if possible, to rid our children and grandchildren of the fear of horrendous genetic disorders such as cystic fibrosis or Huntington's disease, much as we have labored without regret to eradicate external agents of human disease such as the smallpox virus and plague bacterium? Hopefully, this is much closer to the kind of genetic engineering possibilities that societies will address from ethical as well as practical standpoints in any new eugenics movements.

Moral issues aside for the moment, what kinds of technical methods from the field of recombinant DNA might be available to engineer genes in the human germline? In principle, three direct genetic approaches can be envisioned: alter zygotes, alter germline cells within the body, or alter germline cells in vitro before returning them to the human population. These are discussed in turn.

Because the germline, as well as the somatic cells, originate from a single diploid cell via mitotic divisions, any genetic modification of the zygote normally will be passed intact to the germline. In chapter 5, various methods were described by which zygotes from barnyard animals are engineered to carry particular transgenes. For example, a foreign piece of DNA can be microin-jected into the egg directly, or it can be ferried in by a GM viral vector. Comparable approaches can be envisioned for humans. However, the animal studies also indicate that much can go wrong. Only small fractions of tampered zygotes typically incorporate the desired transgene successfully, relatively few of those survive in utero, and relatively few of those survivors develop into fully healthy animals. Likewise, genetic modifications in humans may carry unexpected health consequences even in the best of outcomes.

The second plausible approach is to deliver foreign DNA to the germline within the patient, for example, by using a disabled viral vector that targets the appropriate cells. A likely outcome of this method is genetic mosaicism in which some germ cells carry the transgene and others do not. All else being equal, the earlier and more effectively the foreign DNA is delivered during an individual's development, the higher the fraction of transgenic gametes he or she will produce. A potential drawback of this approach is that each experiment is conducted within a live individual, who therefore could bear the health burden of any procedural failure or unintended action of the transgene.

The third approach is to remove gametes or other germline cells from the patient, insert a specific transgene into them in a test tube, and screen the resulting cells in vitro for viability and presence of foreign DNA. The transformed cells then might be returned to the individual's reproductive tract, or, more likely, used immediately to produce a new embryo (e.g., via artificial insemination or by IVF). The technical feasibility of such germline manipulation recently was addressed in another primate, a rhesus monkey named ANDi (from "inserted DNA" spelled backward). True to his name, ANDi carries a foreign gene for a green fluorescent protein (GFP) delivered to the unfertilized egg from which he arose. Scientists isolated 224 mature oocytes from adult rhesus females, exposed them to GM retroviruses housing the GFP gene, artificially fertilized the manipulated egg cells with rhesus sperm, and implanted the fertilized eggs into the monkeys' reproductive tracts. Three baby monkeys eventually were born, but ANDi alone proved to carry the foreign DNA.

A human female produces only about 400 mature egg cells during her lifetime, releasing them one by one during her monthly ovulatory cycle. By contrast, males produce sperm in abundance. A typical ejaculate contains about 600 million spermatozoa, and a man's lifetime production may exceed 10 trillion cells, roughly 1000 sperm for each and every heartbeat. Genetic engineers are exploring ways to capitalize upon this ready abundance of male sex cells.

In experimental rodents, scientists isolated germline stem cells from healthy males and transplanted them into the testes of infertile males, thereby restoring fertility in the latter. Similar testis-cell transplantations have been initiated in clinical trials on humans, where the intent is to recover fertility in cancer patients that have undergone irradiation or chemotherapy. An extension of this approach, already attempted in mice but not in humans, is to correct additional genetic defects during the transplantation procedure. GM viral vectors, for example, have been used to introduce transgenes to isolated germ cells. By implanting germline cells with specific gain-of-function or loss-of-function genes, it may be possible to correct serious genetic disorders or otherwise improve the health of any resulting progeny.

Notwithstanding recent progress in understanding some of the basic genetic techniques that will be required for any contemplated efforts in human germline engineering, the field has not yet moved far. Thus, it remains for now within the realm of hyperbole, both positive and negative, as people ponder what good and bad might emerge in any new-age eugenics.

BD HY HO BO

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