Professor Dr. Panos Zavos, Ed.S., Ph.D.

TO CLONE OR NOT TO CLONE?

WHOSE CHOICE IS IT ANYWAY?

Professor Emeritus of Reproductive Physiology/Andrology, University of Kentucky Director, Andrology Institute of AmericaAssociate Director, Kentucky Center for Reproductive Medicine & IVF, Lexington, KY, USAExecutive Director, The Zavos Organization

Introduction

Human reproductive cloning today continues to preoccupy the general public and its critics in a very controversial manner. There is also some public hostility directed against it.

The British Medical AssociationThe British Medical Association

“Public hosti l i ty to human reproductive cloning may be

based on an i l logical transient fear of a new technology”

Today, infertility is a disease that reaches epidemic proportions throughout the developing World

Infert i l i ty is a disease

Low sperm count and/or motility

Variety of female factors

Success rate ~ 33% live birth/transfer

Did not overcome severe asthenospermia

In Vitro Ferti l ization (IVF)

If patient has low sperm count or having sperm with no motility

Success rate ~ 32% live birth/transfer

Required presence of sperm in the ejaculate

Intra Cytoplasmic Sperm Injection (ICSI)

No mature sperm present No oocyte (egg) production

Offspring not genetically or biologically related

Sperm and Oocyte Donation

“..we want a child (yesterday, if possible) and a healthy child.”

“..do not want to have another person’s spermor eggs.”

“..what other options do we have?”

“…we want to have a biological child of our own.”

(Received via e-mail from patients)

Quotes from childless patients

1. First, the nucleus of a donor egg is removed.

2. Then a whole somatic cell or the nucleus of a whole somatic cell from a patient is inserted.

3. The result is an egg with the patient's genetic material.

4. The egg is then induced (“jump start”) to divide and become an embryo which grows into several stem cells, all of which are genetically identical to the donor cell.

How Therapeutic Cloning Works

No difference in the type of oocyte (egg) used

Sexual and Asexual Reproduction

Fusion of male and female genetic

material (pronuclei)

Electrofusion of somatic cell that carries ONLY

the male of female genetic material to the

“oocyte”

Sexual and Asexual Reproduction

No difference in the cell division stages after fertilization

Sexual and Asexual Reproduction

Various species have been used as biological models for this effort but extensive research on somatic cell nuclear transfer (SCNT) has been performed using the bovine model.

Cloning in Animals

In our studies we set out to examine the ability of the bovine metaphase oocyte cytoplasm to support mitotic cell cycles under the direction of differentiated somatic cell nuclei of human granulosa cells and fibroblast cells in order to test the efficiency of our SCNT techniques.

Hybrid embryos

Our Studies

Bovine oocytes were randomly treated either for induction of parthenogenesis or for enucleation and SCNT using either human granulosa cells or human fibroblast cells.

Materials and Methods

Bovine oocytes were enucleated by aspiration of the first polar body and the metaphase plate.

Materials and Methods

Human granulosa cells and fibroblast cells were aspirated into a micropipette.

Materials and Methods

One human granulosa cell or fibroblast cell was injected into the perivitelline space of each of the enucleated bovine oocytes.

Injecting the cell

Cell placed subzonally

Materials and Methods

Treated oocytes were evaluated for evidence of cleavage and embryonic development daily.

Embryo quality was assessed using similar grading criteria to those employed in human IVF.

Materials and Methods

Experiment 1 Experiment 2

Granulosa cells

Controls 2 Fibroblast cells

Controls

2

Success rate1

31.2% (15/48)

46.8% (36/77)

24.2 % (15/62)

56.8% (21/37)

CEI 3 66% 42%

1Number of embryos developed from the total number of oocytes;2Parthenogenetic development;3CEI: Cloning Efficiency Index=Embryo success rate/ Parthenogenetic success rate X100

Results

Our results point out that SCNT as applied in the current study fusing human granulosa cells or fibroblast cells can be done.

The technique could be quite sensitive and predictive for similar SCNT attempts in humans for therapeutic or reproductive purposes.

Conclusions

This technology has enabled us to create the first human cloned embryo for reproductive purposes.

Zavos PM: Human reproductive cloning: the t ime is near.

Reproductive BioMedicine Online 6, 397–398, 2003.

First Human Cloned Embryo

Nine human oocytes were enucleated.

Fused with whole human granulosa cells via electrical stimulation and activation.

First Human Cloned Embryo

The resulting cloned embryo reached the 8-10 cell stage and cryopreserved for future molecular analysis.

First Human Cloned Embryo

We have produced and transferred the first fresh cloned embryo into the mother and we are awaiting for results

The mother is a 35 year-old woman The embryo was properly

evaluated and transferredat a 4-cell stage

No pregnancy was established

ANNOUNCEMENTThe First Fresh Cloned

Embryo Transfer in Human

Poor cloning response. Poor implantation and pregnancy ratio. Poor health of animals born.

Diff icult ies noted by “Animal Cloners”

Poorly designed experiments.(few animals used with no definite objective)

Poorly executed experiments.(hit and miss type of research)

Poorly approached experiments.(done under non-sterile and uncontrolled environments)

Poorly understood and interpreted.(when animals died, no clear view of their cause of death)

Those diff icult ies are due to:

SOME DONE FOR FAME AND FORTUNE BY THE ANIMAL CLONERS!

Ethics Morality & Hypocrisy

LET US EXAMINE THE FACTS AS THEY APPEAR

WITH THE ANIMAL CLONERS!

Hypocrisy in Action

A number of studies have already demonstrated far higher rates of development, as measured in the proportion of live births to the number of embryos transferred, and in some cases matching or exceeding developmental rates seen in human IVF.

“Animal cloning is ineff icient and is l ikely to remain so for the

foreseeable future” (by Wilmut & Jaenisch, Time, 2001)

Embryos created* 141

Live births 45

Success Rate (%) 32%**

Nuclear-cytoplasmic interaction and development of goat embryos

reconstructed by nuclear transplantation: production of

goats by serial ly cloning embryos(Yong and Yuqiang; Biol. Reprod. 58: 266-269, 1998)

*Re-cloned goat embryos from a previous cloning procedure (serially cloned embryos)

**Similar to Current Human IVF Success Rates

Embryos created 10

Live births 8

Success Rate (%) 80%

Eight calves cloned from somatic cells of a single adult

(Kato et al, Science, 282: 2095-8, 1998)

• “Dolly is not normal. Dolly is overweight.” (Jaenisch, 2001)

*Under oath at the Congressional Hearing on Human Cloning Research, March 28, 2001

Dolly is obese!!!

Dolly has an IQ problem!!!

• “Dolly may have subtle defects like in the brain. Dolly, I believe, is not normal. …. we have no tests to check that.” (Jaenisch, 2001)

Excerpts from the Congressional Hearings on

Human Cloning*

• “Scientists wanting to do human cloning are going to create humans for spare parts and I am against that” (George W. Bush, President of the USA)

Excerpts from President Bush’s talk on Human Cloning

• “You should be ashamed of yourself for wanting to clone a human being. You are going to create human monsters and you will fail.” (Robert Winston, June 5, 2001)

Ironically, he said the exact same things about Robert Edwards 26 years ago about his efforts to create the f irst human being via IVF. Today, Winston embraced IVF and he is known in the UK as Mr. IVF.

That is hypocrit ical! !

Excerpts from the Oxford Union Debate

• “You should be ashamed of yourself for experimenting and killing human embryos.” (Harry Griffin, Roslyn Institute, June 5, 2001)

Today, Mr. Grif f in is given a l icense by the Brit ish Government and HFEA to kil l human embryos and extract stem cells.

That is highly hypocrit ical! ! !

Excerpts from the Oxford Union Debate

In order to avoid “killing human embryos” we have created the Human-Bovine hybrid embryo model to study various phenomena that needed to be evaluated during SCNT.

What has Mr. Griff in done to avoid ki l l ing human embryos?

Human-Bovine Hybrid Embryos Created

• “Animal cloning is ineff icient and is l ikely to remain so for the foreseeable future”

(Ian Wilmut, Roslyn Institute, August 2001)

Excerpts from the National Academy of Sciences

Today the success is tremendous!!

They continue however to misrepresent the facts!

• “Cloning will never be perfected and applied for human or animal purposes. It is impossible to reprogram 35,000 genes present in the human genome and yield a healthy human being.”(Rudolph Jaenisch, MIT Professor, August 2001)

Excerpts from the National Academy of Sciences

Today we are cloning cattle commercially with 83% success rate; astonishing!!

“….in monkeys the removal of the egg nucleus also removes what Schatten called "molecular motors" that are responsible for separating chromosomes during cell division. He explained. "The cells that result after those cell divisions all have the wrong number of chromosomes."

Another “Expert’s” Opinion on Human Cloning

“We cannot do it in monkeys and therefore it cannot be done in humans”

“This is the first concrete genetic data showing that the cloning process could be less complicated in humans than in sheep” Keith Kil l ian, Duke University Medical Center

“…our data show that you don’t necessarily have these problems (with the large offspring syndrome) in humans.” Randy Jirt le, Duke researcher

Humans may be easier to clone than animals!

(August, 2001)

“to reconsider the prominence given to repeated claims by certain scientists that they have cloned a human being, including those made by Dr. Panos Zavos last weekend.” (21st January 2004)

We have NEVER claimed to have We have NEVER claimed to have cloned a human being!cloned a human being!

Response to Open Letter to Brit ish News Editors by “leading UK scientists”

“none of those involved have produced a shred of evidence to substantiate their assertions .” (21 st January 2004)

We have published and continue to publish in We have published and continue to publish in peer-reviewed scientific journals!peer-reviewed scientific journals!

It appears that these “leading scientists” do It appears that these “leading scientists” do not do their homework nor READ and get not do their homework nor READ and get

informed before they offer an opinion.informed before they offer an opinion.

Response to Open Letter to Brit ish News Editors by “leading UK scientists”

Zavos PM: Human reproductive cloning: the time is near. Reproductive BioMedicine Online 6, 397–398, 2003.

Illmensee K, Levanduski M, Zavos PM: Development of an interspecies-specific bioassay using the bovine oocyte model to evaluate the potential of SCNT in humans. Journal of Assisted Reproduction and Genetics, 2004 (Accepted, in press, withdrawn due to leading UK scientists pressure).

Zavos PM, Illmensee K: First Embryo Transfer of a Cloned Human Embryo. Middle East Fertility Society Journal (Submitted for publication).

Zavos PM, Illmensee K: Human Reproductive Cloning: The Post Mortem Effort. (Currently in preparation).

Publications

The American Society for Reproductive Medicine, San Antonio, Texas, October 11-15, 2003.

The Austrian Society of Reproductive Medicine and Endocrinology, Bregenz, Austria, October 17-18, 2003.

The Middle East Fertility Society, Beirut, December 10-13, 2003. The 12th World Congress on Human Reproduction, Venice, Italy, March

14-16, 2005 The World DNA and Genome Day, Dalian, China, April 25-30, 2005. The Indian International Conference on Update in Infertility, Bangalore,

India, April 25-May 1, 2005. The American Society for Reproductive Medicine and the Canadian

Fertility and Andrology Society, Montreal, Quebec, Canada, October 15-19, 2005 (Submitted).

Scientif ic Presentations

Is it an act of God?Is it an act of God?

“HFEA grants the f irst therapeutic cloning l icense for

research”(11 August 2004)

Increasing knowledge about the development of embryos

Increasing knowledge about serious disease Enabling any such knowledge to be applied in

developing treatments for serious diseaseAfter destroying the embryo and After destroying the embryo and

its potential for lifeits potential for life

HFEA purposes

Human reproductive cloning is illegal in the UK. As a result of the Human Reproductive Cloning Act (2001) nobody in the UK is allowed to use cell nuclear replacement, or any other technique, to create a child.

But Therapeutic Cloning for Stem Cell But Therapeutic Cloning for Stem Cell Research is allowedResearch is allowed

Therapeutic vs Reproductive Cloning in the UK

Stem cells from Living Human Embryos

Kil l ing & Dismembering Human Embryos

Grow stem cells in culture

Used in treatment of various diseases

Somatic (body) cells Inject into enucleated

oocytes Induce embryo

development Used to create

healthy babies for childless couples and treating inferti l i ty

Therapeutic Cloning & Stem Cell Research vs Reproductive

Cloning

Further elucidation of the molecular mechanisms involved during the processes of embryogenesis

Careful tailoring of subsequently developed culture conditions and manipulation strategies

Appropriate screening methods

The Future of Cloning

will eventually al low inferti le couples to safely have healthy, genetically related children through Somatic Cell Nuclear

Transfer (SCNT) technology

understand this type of work and the seriousness for its development

should be focused on carrying out this project, and

should work with leaders and governments at the International level, to ensure that this technology can be made safe and be disseminated properly

This technology should be developed by scientists and

medical experts that:

Selecting appropriate cell lines for SCNT Proper reprogramming the cell

lines in tissue culture prior to SCNT

Screening the cloned embryosprior to embryo transferinto recipients

Monitoring the ongoingpregnancies from the cloned embryos

We intend to develop this technology by:

Sunday Herald, Glasgow, Scotland, 10/21/01

Vil if ied, r idiculed, accused of pervert ing nature….

But the cloning pioneers are in good company

“Cloning, too, wil l probably come to be

accepted as a reproductive tool if

i t is carefully controlled”

Professor Robert Edwards, 2001

Sunday Herald, Glasgow, Scotland, 10/21/01

The Future of Reproductive Cloning