Presented to the U.S. House of Representatives Committee on Energy and Commerce, Subcommittee on Oversight and Investigations, March 28, 2001.

Subject:  Human Cloning

Man has long been interested in nuclear transplantation both as a tool to study developmental biology and as a means for producing genetically identical animals. The basic technique involves the transfer of a nucleus from one cell to another cell which has had its own nucleus removed.  For cloning animals this entails transferring the nucleus of a cell obtained from the individual to be cloned into an unfertilized ovum that has had its chromosomes removed.   If successful, the transferred nucleus is re-programmed so to direct development of a new embryo that is genetically identical to the animal from which the cell was obtained. This embryo can then be transferred into a surrogate mother for gestation to term and birth of a clone.   

In recent years, nuclear transplantation has been employed to clone a number of different animals.  The most acclaimed example is of course the report by Wilmut et al (1997), which was the first to demonstrate cloning of adult mammals was possible.  Nuclei of cultured mammary epithelial cells derived from an adult ewe were transferred into enucleated sheep ova, ultimately resulting in the birth of a cloned lamb (Dolly).  The demonstration that adult cells could be used for cloning mammals sparked enormous new interest in exploring the potential of cloning animals.  As a result, in just the past three years, cloned cattle, sheep, goats, pigs, and mice have been reported (Wilmut et al. 1997; Cibelli et al. 1998; Wakayama et al, 1998; Baguisi et al.  1999; Wells et al. 1999a; 1999b; Polejaeva et al. 2000; Onishi et al. 2000; Hill et al. 2000).

The potential benefits animal cloning will afford mankind are far-reaching, and undoubtedly, many more applications and benefits are yet to be imagined. A current utility includes the production of transgenic animals for use as living bioreactors to produce pharmaceuticals.  Several products produced in milk of transgenic sheep and goats are already in clinical trials (Factor IX, P.P.L., Inc.; anti-thrombin III, Genzyme Inc.; Baguisi et al. 1999; Schnieke et al. 1997), and the estimated market value of pharmaceutical production in the milk of transgenic animals currently exceeds $3 billion per year. A number of other products are targeted for production in milk from transgenic livestock including both nutriceuticals and vaccines. Genetic engineering animals for protein production in milk promises to result in a wide variety of products for human use, many of which will be less expensive and more effective (Stice et al.  1998; Wall, 1996).    Other applications of cloning to produce transgenic animals include the production of livestock that are that are genetically resistant to devastating diseases such as those currently causing major concern throughout the world i.e. Mad Cow Disease and Foot and Mouth disease.  Agricultural applications of animal cloning will result in increased quality and decreased costs for food and fiber (Stice SL et al.  1998; Wall, 1996).  In addition, animal cloning provides for rapid genetic gain in animal breeding programs and could potentially have a great beneficial impact on the conservation, preservation and propagation of endangered species (Wells et al.  1999a).

Anticipated future applications of cloning procedures are nothing short of phenomenal.  These include such things as the production of human embryonic stem cells for tissue transplantation and/or gene therapy and treatments for mitochondrial diseases, just to name a few.  Human cells could potentially be utilized as nuclear donors for transplantation into oocytes, resulting in cell lines that may be useful for human therapy to treat conditions such as Alzheimer’s or Parkinson’s disease (Zawada et al.  1998). 

With animals representing 5 different mammalian species now having been produced by somatic cell nuclear transfer, cloning has been proposed as a tool for assisted reproduction in humans i.e. a means for producing a human baby.  Experiments from our laboratory and others provide strong evidence that the current procedures used for mammalian cloning are not safe and many times result in abnormal development.  This can ultimately lead to death of the cloned offspring and the surrogate mother.  (Campbell et al.  1996; Cibelli et al.  1998; Hill JR et al.  1999; Kato Y et al.  1998; Schnieke et al.  1997; Vignon X et al.  1999; Wakayama et al.  1998; Wells et al.  1997; Wilmut et al.  1997).  Based on these observations and evidence from studies in mice which demonstrate incompatibilities between nucleus and cytoplasm from different strains (Latham, 1999), cloning as an approach to human assisted reproduction is at present both risky and extremely irresponsible.  

Although animals can be cloned by nuclear transplantation using somatic cells as nucleus donors, the efficiency of the technique is still extremely low.  In cattle where the majority of the work has been completed, problems with early embryonic development do not seem to be a major factor affecting the efficiency of cloning, as development rates to the blastocyst stage in vitro are similar to those of normal embryos produced by in vitro fertilization.  Maternal recognition and the establishment of pregnancy as indicated by pregnancy rates at 35 days of gestation are also similar between normal embryos and those produced by nuclear transplantation.  However, after 35 days of gestation, pregnancy loss is dramatic and very few fetuses survive to term.  Approximately 90% of the pregnancies are lost and abort between days 35 and 90 of gestation (the first trimester).  The most common developmental malformation observed to date is aberrant placentation (Hill et al. 1999; Stice et al. 1996).   Of those calves that do survive, most exhibit placental edema and a reduced number of enlarged placentomes.  These placental abnormalities pose serious health risks not only to the developing fetus and offspring but also to the surrogate mothers carrying the pregnancies.  In several cases involving cattle, both the surrogate mother and the bovine fetuses have died during late gestation due to a variety of complicated health issues related to the abnormal pregnancy.  Moreover, even if the cloned offspring survive to term, many of the resulting calves exhibit developmental abnormalities and die at birth or shortly thereafter, normally a result of cardiopulmonary abnormalities (Cibelli et al. 1998; Garry et al.  1996; Kato et al. 1998; Kruip et al. 1997; Renard et al. 1999; Vignon et al. 1999; Wilson et al. 1995; Hill et al. 1999).  In general, regardless of the species, only 1%-5% of cloned embryos survive to term.

In our laboratory we have utilized nuclear transfer to try and reproduce the genotypes of several different animals, selected for cloning based on their inherent genetic value.  Results we have obtained to date are similar to those reported by other laboratories regardless of the species involved.  The first case involved a Brahman steer named “Chance”, known to be at least 21 years old.  Adult fibroblasts were obtained from a skin biopsy and expanded in culture using standard methods for tissue culture prior to being frozen and stored in liquid nitrogen.  When nuclear transfer was performed using the fibroblast cells derived from Chance, 28% of the fused couplets (53 of 190) developed into blastocysts in culture.  Twenty-six of these were transferred into 11 recipient cows resulting in 6 pregnancies.  Three of these continued to develop through 90 days of gestation but only one survived to term.  “Second Chance” is now over a year old and appears normal and healthy for his age.  However during the first week of life he required intensive monitoring and therapy to treat lung dysmaturity and pulmonary hypertension.  At 7 days of age he was also diagnosed and treated for Type 1 insulin-dependent diabetes, which is extremely rare in cattle.  He also lacked the expression of an important T-cell antigen CD45, indicating his immune system was in some way abnormal (Hill et al, 2000).

The second and third attempts at reproducing desired genotypes by cloning involved two middle-aged cows, one Brangus and one Charolois.  These were selected based on being top performers in the herd.  Fibroblasts were again obtained from skin biopsies.  Development rate to the blastocyst stage following nuclear transfer and embryo culture averaged 16%.  Thirty-seven blastocysts derived from the Charolois cow were transferred into 13 recipients.  Six of these were diagnosed as pregnant at 30 days of gestation but only 4 remained pregnant through 60 days.  One of these pregnancies was subsequently lost.  In two cases the fetus was removed for research purposes.  The final pregnancy was allowed to proceed to term resulting in twin heifers.  However, both calves died between 7-10 days after birth due to complications related to the cloning procedure.  Forty-three blastocysts derived from the Brangus cow were transferred into 14 recipients resulting in 3 pregnancies.  However none of these survived past 90 days of gestation.

Our most recent attempt at cloning a specific animal has involved a deceased Black Angus bull previously shown to be naturally (genetically) resistant to Brucellosis.  Of the oocyte-fibroblast couplets fused and cultured, 44% developed to the blastocyst stage.  Thirty-nine blastocysts were transferred into 20 recipients resulting in 10 pregnancies at 35 days of gestation.  One of these survived to approximately 150 days of gestation and was then lost.  Another single pregnancy survived to term resulting in a healthy bull calf.

Prior to any attempt to use nuclear transplantation/cloning as a means of human assisted reproduction, it is imperative that many additional animal studies evaluating the safety of somatic cell cloning be carried out. These studies should also include efforts to evaluate the safety of applying nuclear transplantation procedures for treatment of human disease or infertility by manipulating oocyte cytoplasm and/or genetically modifying human cells prior to cloning.   Proponents of human cloning as a means of assisted reproduction have pointed out that even with accepted practices of assisted reproduction such as in vitro fertilization, success rates are low and pregnancy losses higher than in natural reproduction.  This is indeed the case, but hardly to the extent seen in cloning where only 1-5% of the procedures performed result in offspring, and a significant number of these either die at birth or require intensive care for several weeks to keep them alive. 

Moreover, the claim that cloned embryos could be screened prior to embryo transfer so to select those that will develop normally is simply not a possibility at this time.  Research conducted in our laboratory and several others now points very strongly to the fact that problems seen in cloned embryos/pregnancies are likely epigenetic effects brought on by the cloning techniques themselves and causing abnormal expression of important developmental genes.  Techniques to evaluate for these abnormalities are simply not yet available and it will likely be years before such diagnostics do become available.  Procedures to determine whether cloned embryos and fetuses appear to have normal and the right number of chromosomes are woefully inadequate as there is no indication to date that abnormal karotypes are a problem i.e. chromosomes in cloned embryos appear normal.  If one wanted to screen for abnormal gene expression, which of the tens of thousands of genes would one screen for? There is no solid data yet to point to one gene/cause for developmental failure.  In addition, given the small size and few cells available, current techniques will not allow any type of adequate analyses of an embryo so to determine in fact that it is normal.  At best, with ultrasound, one could determine that the fetus is dead, which based on animal studies is likely to be the situation in 90% of the cases during the first trimester of pregnancy.

Finally, even the apparently healthy animals that are produced by cloning should be studied and observed for a number of years to evaluate their long-term health status prior to any applications in humans.  Considerable evidence has now been accumulated to suggest that insults occurring during the critical period of embryo and fetal development may have long-term effects on the health of offspring and resulting adults.  Cloned animals produced to date have not yet lived long enough to evaluate this potential risk.  Undoubtedly it would be a devastating case to produce cloned humans only to find out that they all developed serious disease/health problems and/or died during childhood or adolescence or even early in their adult life.  At this point it is simply impossible to eliminate this potential disastrous outcome. 

Ethical Concerns Involving Human Cloning:

I have previously been quoted in the popular press as saying that while there are enormous beneficial applications to cloning animals, “I have never met a human worth cloning”.  Although my wife may take some exception to this statement, I still stand behind it.  In part, this is due to the fact that as human beings, none of us are perfect.  Also, expectations of what a human clone would be or do are many times, exaggerated.  Cloning animals by nuclear transplantation is simply a technology that can be used to produce another individual with the same genetic make up.  What cloning absolutely is not, is a means of resurrection. I think it best we leave this business to God as we have enough problems to deal with just trying to be decent human beings.   It is indeed extremely troubling to me however, that with the successful cloning of animals, many people in society still seem to have no understanding of the difference between “reproduction” and “resurrection”.  A significant number of requests for human cloning involve the utilization of cells from “beloved family members” that are in fact deceased.  Undoubtedly, those requesting such services, whether they would admit it to themselves or not, in some way believe cloning is a form of resurrection, not reproduction.  It is deeply concerning that individuals offering human cloning services could take advantage of highly emotional situations involving the death of a loved one by selling resurrection vs reproduction.

With time and education, society will eventually understand the difference between resurrection and reproduction.  I will also predict that given the current state of various assisted reproduction techniques that are already being utilized by humans and readily accepted as ethical, such as in vitro fertilization and intracytoplasmic sperm injection, cloning by nuclear transplantation will eventually also be thought of as simply another form of assisted reproduction, and individuals employing techniques of nuclear transplantation will not be accused of “playing God”.  In short, I predict that humans will someday be cloned.  When this happens, the sky will not fall and the world will not come to an end.  Scenarios such as that seen in “The Boys from Brazil” and armies of clones will remain in the movies.  The number of human babies that would ever be produced by cloning will be infinitesimally small compared to children born by natural reproduction, and will hardly be noticed.  The person (s) that come into this world by way of cloning will be new and unique individuals.  Moreover, I have confidence and a personal faith in God that they will be blessed with a unique spirit and soul.  To think otherwise is to suspect that God hasn’t blessed the thousands of babies already born by other forms of assisted reproduction with a soul, and neither the tens-of-thousands of genetically identical twins that live in this world.  This begs the question, what is it that really makes human cloning so (as it is often referred to) repugnant?  Is it the word “clone” itself and/or the horrendous stories that have been written, or movies that have been made that always depict cloning as a terrible thing leading to a terrible outcome?  Would it be impossible to write a story about human cloning that had a happy ending, or is it just the fact that it wouldn’t sell and therefore no profit would be gained?  Surely it is not the fact that a clone would have a genetically identical copy, either still alive or deceased?  How would this be that much different than an identical twin?

Consider the following scenario.  A skin cell from a human male is inserted into an enucleated human ovum (nuclear transplantation) so to create a cloned human embryo.  However, instead of transplanting this embryo into a surrogate mother, the embryo is placed into culture and treated in such a way that it develops into embryonic stem cells.  Given the enormous and promising success that has been achieved in recent years involving the production of human embryonic stem cells, it is easily conceivable that in the not to distant future, these stem cells could then be directed in culture to undergo gameteogenesis and develop into cell types that represent gametes (sperm and eggs) containing a haploid number of chromosomes (half of that in a normal somatic cell), and the genes will have been rearranged, as occurs during normal gamete development.  Once this has occurred, two of the gamete cells could be selected and using nuclear transfer a second time, placed into another enucleated ovum resulting in a normal embryo that could then be transferred into a surrogate mother for development to term.  While this scenario may be difficult for some to follow, here’s the punch line.  It is entirely conceivable that a single cell originally derived from a single male, with the aid of technology, could be used to produce a new human baby.  This new human being would not at all be a clone, because of the natural process of gene rearrangement that occurs during gamete development, and in fact, could turn out to me a girl! 

If cloning a human being is unethical, would this procedure also be unethical even though the new baby would not be a clone at all but simply derived from an elaborate assisted reproductive technology?  Given the state of currently accepted practices for treating human infertility, I doubt it, but with one caveat.  It would certainly be considered highly unethical and completely irresponsible if 90% of the pregnancies resulted in abortions, the surrogate mother was put in serious health risk, and a significant portion of the offspring that resulted were developmentally abnormal and many died.

            So we are back to square one. Is nuclear transfer to produce a human clone a reasonable thing to consider attempting at this time?  In my opinion absolutely no!  Ethical issues and moral issues aside, at present, cloning is just too risky, many times resulting in serious health problems and/or death the developing fetus, surrogate mother, and resulting offspring.

References:

Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW, Echelard Y, 1999. Production of goats by somatic cell nuclear transfer.  Nat Biotechnol 17:456-461.

Campbell KH, McWhir J, Ritchie WA, Wilmut I, 1996. Sheep cloned by nuclear transfer from a cultured cell line.  Nature 380:64-66

Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon A, Robl J. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 1998; 280:1256-1258.

Garry FB, Adams R, McCann JP, Odde KG. 1996. Postnatal characteristics of calves produced by nuclear transfer cloning.  Theriogenology 45:141-152.

Hill JR, Roussel AJ, Cibelli JB, Edwards JF, Hooper RN, Miller MW, Thompson JA, Looney CR, Westhusin ME, Robl JM, Stice SL. 1999. Clinical and pathologic features of cloned transgenic calves and fetuses (13 Cases).  Theriogenology 51:1451-1465.

Hill JR, Winger QA, Long CR, Looney CR, Thompson JA, Westhusin ME. 2000. Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells. Biol Reprod 62:1135-1140.

Kato Y, Tetsuya T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, Yasue H, Tsunoda Y.1998. Eight calves cloned from somatic cells of a single adult.  Science 282:2095-2098.

Kruip TAM, Daas JHG den, Den Daas JHG. 1997. In vitro produced and cloned embryos: effects on pregnancy, parturition and offspring.  Theriogenology 47: 43- 52.

Latham KE, 1999. Epigenetic modification and imprinting of the mammalian genome during development.  Curr.Top.Dev.Biol. 43:1-49:1-49.

Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry ACF. Pig Cloning by Microinjection of fetal fibroblast nuclei. 2000 Science 289:1188-1190. 

Polejaeva IA, Chen S, Vaught T, Page R, Mullins J, Ball S, Dal Y, Boone J, Walker S, Ayatres D, Colman A, Campbell K. 2000. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86-90. 

Renard JP, Chastant S, Chesne P, Richard C, Marchal J, Cordonnier N, Chavette P, Vignon X. 1999. Lymphoid hypoplasia and somatic cloning.  The Lancet 353:1489-1491.

Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A, Campbell KH. 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts.  Science 278:2130-2133.

Stice SL, Strelchenko NS, Keefer CL, Matthews L. 1996. Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer.  Biol Reprod 54:100-110.

Stice SL, Robl JM, Ponce de Leon FA, Jerry J, Golueke PJ, Cibelli JB, Kane JJ. 1998. Cloning: new breakthroughs leading to commercial opportunities.  Theriogenology 49:129-138.

Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R. 1998. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394:369-374. 

Wall RJ. 1996. Transgenic livestock - progress and prospects for the future.  Theriogenology 45:57-68.

Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813.

Wells DN, Misica PM, Day TA, Tervit HR, 1997. Production of cloned lambs from an established embryonic cell line: a comparison between in vivo- and in vitro-matured cytoplasts.  Biol Reprod 57:385-393

Wells DN, Misica PM, Forsyth JT, Berg MC, Lange JM, Tervit HR, Vivanco WH. 1999a. The use of adult somatic cell nuclear transfer to preserve the last surviving cow of the Enderby Island cattle breed.  Theriogenology 51:217.

Wells DN, Misica PM, Tervit HR. 1999b. Production of Cloned Calves Following Nuclear Transfer with Cultured Adult Mural Granulosa Cells.  Biol Reprod 60:996-1005.

Wilson JM, Williams JD, Bondioli KR, Looney CR, Westhusin ME, McCalla DF. 1995. Comparison of birth weight and growth characteristics of bovine calves produced by nuclear transfer (cloning), embryo transfer and natural mating.  Animal Reproduction Science 38:73-83.

Zawada WM, Cibelli JB, Choi PK, Clarkson ED, Golueke PJ, Witta SE, Bell KP, Kane J, Ponce de Leon FA, Jerry DJ, Robl JM, Freed CR, Stice SL. 1998. Somatic cell cloned transgenic bovine neurons for transplantation in parkinsonian rats. Nat Med 4:569-574.