Select Committee on Science and Technology Written Evidence


Memorandum 38

Submission from Dr Elizabeth Allan

EXECUTIVE SUMMARY

A.  ANIMAL/HUMAN HYBRID EMBRYOS

  Regarding proposals for the formation of animal/human hybrid embryos using human nuclear DNA and enucleated animal eggs which would contain mitochondrial DNA: it has been widely stated in the Media that 99.9% of the DNA would be human DNA. However, this is misleading, and misses out several fundamental points:

  1.  Reprogramming of the human genome to become an embryo would be driven by animal reprogramming factors from animal eggs which have evolved to create cows, rabbits etc. The biological and ethical boundaries that would be crossed would therefore be far greater than the combination of animal mitochondrial genes and human nuclear genes in the same cell.

  2.  Mitochondrial DNA from the animal source would be far more than indicated by the Media figure of around 0.1%. Although the number of mitochondrial genes is relatively small, mammalian eggs including cow, rabbit and human eggs contain hundreds of thousands of copies of mitochondrial DNA and mitochondria. In mature human eggs, around half the total DNA is mitochondrial. Using eggs from animals such as cow and rabbit to make hybrid embryos would therefore result in early embryos where a very substantial percentage of the DNA would be animal, and a high percentage of the cell volume would also consist of animal organelles. For example, there were 1,300,000 copies of rabbit mitochondrial DNA at the1-cell stage immediately after nuclear transfer using enucleated rabbit eggs and nuclei from non-human primates.

  3.  Mitochondria also make up a high percentage of the cell volume of certain mature cell types such as heart, liver and muscle cells. The animal component of such cells generated from animal/human hybrid embryos or embryonic stem cells would therefore be very significant.

  4.  Bearing in mind points 1, 2 and 3 above, the Human Fertilisation and Embryology Authority therefore does not have the authority to grant licences to create such embryos, since the HFE Act is confined to "human" embryos. The Government should therefore legislate against the formation of these embryos.

  5.  Mitochondria are of far greater significance than is suggested by the modest number of genes encoded by mitochondrial DNA. Although a primary function relates to energy production, mitochondrial dysfunction is now believed to be a key factor in many neurodegenerative diseases. However, these are the main diseases proposed to be investigated using animal/human hybrid embryos. Attempting to research such diseases or find cures for them using cells which contain animal mitochondria, which are defective in relation to human mitochondrial function, could therefore be a waste of time, and even counter-productive.

  6.  The overwhelming majority of, and possibly all, cloned embryos have a high degree of altered gene expression, which is further complicated by being somewhat random. Use of animal/human hybrid cloned embryos would add a further layer of altered gene expression owing to varying degrees of incompatibility between animal mitochondrial DNA and the human nuclear genome, and the reprogramming of human nuclei by enucleated animal eggs. At best this would severely complicate interpretation of experiments; and would more probably lead to the experiments being simply a study of artifacts rather than providing any useful information relating to the disease under study.

B.  CHIMERAS

  Regarding chimeras, a number of different types of chimera can be made such as tissue-specific, mixed-parent or mixed-sex embryos, by aggregating cells in either animal/human or human/human cell combinations at the embryonic stage.

  Creation of such chimeras should not be permitted. Similarly, creation of chimeras where human brain cells or germ cells could be created in animals, or vice versa, should not be permitted. These types of chimera are fundamentally different, for example, from the grafting of human tissue onto animals.

A.  ANIMAL/HUMAN HYBRID EMBRYOS

  Regarding proposals for the formation of animal/human hybrid embryos using human nuclear DNA and enucleated animal eggs which would contain mitochondrial DNA: it has been widely stated reassuringly in the Media that 99.9% of the DNA would be human DNA. However, this is misleading, and misses out several fundamental points:

1.   Human genome reprogrammed by animal reprogramming factors

  In such embryos, reprogramming of the human genome would be driven by animal reprogramming factors from animal oocytes which have evolved to create cows, rabbits etc. This is fundamentally different, for example, from adding a human gene into the embryo of another species, or even of fusing cytoplasm containing mitochondria from animal cells with human cells.

  Animal/human hybrid embryos created by using enucleated animal eggs and human nuclear DNA are therefore not simply embryos, or even a collection of cells, that are basically human but which have a few additional animal genes. The biological and ethical boundaries that would be crossed in terms of formation of animal/human hybrid embryos would be far greater than simply the combination of some animal mitochondrial genes with human nuclear genes.

  In Section 1.7 and 1.8 of the Department of Health Review of the Human Fertilisation and Embryology Act, the Government has affirmed the principle of the special status of the human embryo. To use animal reprogramming factors to reprogramme the human genome to animal specifications is inconsistent with this principle.

2.   Mitochondrial DNA: High concentration in eggs and early embryos

  Mitochondrial DNA from the animal source would be far more significant than indicated by the Media figure of 0.1%. Although the number of mitochondrial genes is relatively small, mammalian eggs contain hundreds of thousands of copies of mitochondria and therefore of the mitochondrial genome. For examples, studies on mitochondria in human eggs have found averages of 193,000-795,000 copies of mitochondrial DNA (Barritt et al, 2002; Reynier et al, 2001; Steuerwald et al, 2000), with the higher estimates likely to be more accurate (Barritt et al, 2002).

  The two main mammalian species proposed for animal/human hybrid formation by nuclear transfer are cow and rabbit. Cow eggs have been found to contain around 260,000 copies, or 4.5 pg (Michaels et al, 1982). Perhaps surprisingly, there appears to be an even greater number of copies of mitochondrial DNA in rabbit eggs than human or cow eggs (Yang et al, 2004).

  In a direct comparison of nuclear and mitochondrial DNA, Reynier et al (2001) found that around 50% of the DNA in unfertilized human eggs is mitochondrial DNA (around 3.4 pg mitochondrial DNA compared to 3 pg of DNA in the nucleus per haploid genome).

  Since cow eggs were found to contain 4.5 pg of mitochondrial DNA, if an enucleated cow egg and a diploid human nucleus were used to create a cloned animal/human embryo, mitochondrial DNA would probably be around half of total DNA in the earliest stages.

  In an experiment particularly relevant for this issue, Yang et al (2004) used nuclear DNA from non-human primates and enucleated eggs from rabbits to create rabbit—primate cloned embryos. He found that there were 1,300,000 copies of rabbit mitochondrial DNA at the 1-cell stage immediately after nuclear transfer using enucleated rabbit eggs and nuclei from macaque. The percentage of animal mitochondrial DNA to human nuclear DNA would therefore probably be even higher if using rabbit eggs rather than cow eggs.

3.   Mitochondria: High percentage of cell volume in certain mature cell types

  It should be noted that mitochondria in some mature cell types also make up a high percentage of cell volume, and there are also high copy numbers of mitochondrial DNA relative to nuclear DNA, for example in heart, muscle and liver. For example, in human heart muscle, there is an average of almost 7,000 copy numbers of mitochondrial DNA per diploid nuclear genome, and in skeletal muscle, an average of 3650 (Miller et al, 2003).

  At this stage of development, the mitochondria would be composed of animal DNA, and both animal and human gene products. Nevertheless, there would be a significant amount of animal DNA and its gene products in any such cells generated from the hybrid embryos or from embryonic stem cells derived from them.

4.   The HFEA has no authority to grant licences to create animal / human hybrid embryos from enucleated animal eggs and nuclear human DNA

  The Human Fertilisation and Embryology Act clearly applies only to human embryos. The definition of an embryo in the Act is as follows:

  1.— (1)  In this Act, except where otherwise stated—

(a)  embryo means a live human embryo where fertilisation is complete, and

(b)  references to an embryo include an egg in the process of fertilisation, and, for this purpose,     fertilisation is not complete until the appearance of a two cell zygote.

  Therefore, the authority of the Human Fertilisation and Embryology Authority does not extend to granting a licence to create an animal / human hybrid embryo from enucleated animal eggs and nuclear human DNA, since around half of the total DNA in the early embryo would be animal, a substantial percentage of cell volume in the early embryo would also be animal, and the human genome would have been reprogrammed by animal reprogramming factors.

  The Government should therefore legislate against the formation of these embryos.

5.   Mitochondria and neurodegenerative diseases

  Mitochondria are of far greater significance than is suggested by the modest number of genes encoded by mitochondrial DNA. Although a primary function relates to energy production, there is substantial and growing evidence that mitochondrial dysfunction is a key factor in many neurodegenerative diseases including Parkinson's, ALS, Huntington's and Alzheimer's disease (eg Beal, 2005; Lin and Beal, 2006). However, neurodegenerative diseases are among the main diseases proposed to be investigated using animal/human hybrid embryos.

  Attempting to research such diseases, for example using hybrid ntES cells cloned from a patient but which contain animal mitochondria, which are deficient in relation to human mitochondrial function, could therefore be counter-productive if the disease has a mitochondrial component.

  Adding human mitochondria seems unlikely to be the answer since, for example, mitochondria from primate donor cells in rabbit/primate cloned embryos do not appear to replicate during early embryogenesis, and then decrease at the blastocyst stage (Yang et al, 2004).

  There is considerable interaction between nuclear DNA and mitochondrial DNA in relation to mitochondrial biogenesis and function. As pointed out by Barrientos et al (1998) and Kenyon and Moraes (1997), the tight interdependence of a large set of gene products coded by both mitochondrial and nuclear genomes has led to the close co-evolution of these two genomes in a species-specific manner, resulting in species-specific compatibility between the nuclear- and mitochondrial-encoded factors.

  Although little is yet known about how effectively animal mitochondrial DNA would combine with human nuclear DNA in terms of efficiency of mitochondrial biogenesis and function, the research so far carried out indicates very clearly that they would be largely, or even completely incompatible.

  For example, in studies of oxidative phosphorylation following the fusion of enucleated primate cells with human cells lacking mitochondrial DNA, it was found that mitochondria from species biologically very similar to Man such as New World Monkeys, Old World Monkeys, orangutans and lemurs, could not functionally replace human mitochondria in cells. Mitochondria from primates more similar to Man (gorilla, chimpanzee and pigmy chimp) were partially successful in restoring oxidative phosphorylation, but even then, oxygen consumption was 20-34% lower than that of the human parental cell line (Kenyon and Moraes, 1997), and subsequent studies by the same group showed that there was a considerable deficiency in the function of mitochondrial complex I in these cells (Barrientos et al, 1998).

  Regarding the partial incompatibility of gorilla and chimpanzee mitochondria with human nuclei owing to complex I deficiency, it should be noted that many human mitochondrial diseases result from complex I deficiencies. For example, a decrease or disturbance of activity of complex I of the mitochondrial respiratory chain has been strongly implicated in at least some types of Parkinson's Disease, and inhibitors of complex I reproduce the clinical, neurochemical and neuropathological features of Parkinson's Disease (Lin and Beal, 2006; Ebadi et al 2001; Betarbet et al 2000).

  Since it has been found that a reduction in only 25% of activity of complex I causes a major change in oxygen consumption and ATP synthesis, severely impairing energy metabolism (Ebadi et al, 2001), the level of decrease in complex I activity associated with gorilla and chimpanzee mitochondrial/human nucleus cybrids could readily lead to pathological consequences.

  Even although cells generated using interspecies SCNT would not be used clinically, but solely for research, the high degree of incompatibility between nuclear and mitochondrial genomes could generate artifactual results. The involvement of mitochondria in diseases would further complicate results, for example if a risk factor for the disease being examined was mitochondrially-encoded, or even related indirectly to mitochondria. There would therefore also be dangers in extrapolating results for clinical use.

6.  Altered gene expression in cloned hybrid embryos

  The overwhelming majority of, and possibly all, cloned embryos are abnormal, and have a high degree of altered gene expression which is further complicated by being somewhat random. (Reik et al, 2001; Dean et al, 2001; Ohgane et al, 2001; Han et al, 2003; Humpherys et al, 2001; Bourc'his et al, 2001; Rideout et al, 2001; Fairburn et al, 2002; Slimane-Bureau and King, 2002 Boiani et al, 2002; Kang et al, 2001, 2002).

  The use of animal/human hybrid cloned embryos would add a further layer of altered gene expression owing to varying degrees of incompatibility between animal mitochondrial DNA and the human nuclear genome, and the reprogramming of human nuclei by enucleated animal eggs.

  Regarding the use of enucleated animal eggs for somatic cell nuclear transfer for research into diseases, there are already so many profound genetic and epigenetic flaws in cloned embryos, that to use embryos created by interspecies nuclear transfer would be liable to amount to a study of artefacts, rather than a study of the disease in question.

B.  CHIMERAS

  Regarding chimeras, a number of different types of chimera can be made such as tissue-specific, mixed-parent or mixed-sex embryos, by aggregating cells in either animal/human or human/human cell combinations at the embryonic stage (for example, Ryan and Townes, 2001; Alikani and Willadsen, 2002).

  Mixed-sex embryos could be created by using a combination of cells from male and female embryos; mixed-parent embryos could be created by aggregating embryonic cells from several different embryos, resulting in various combinations that had several "parents," some of which could be from an animal source. Tissue-specific chimeras could be made for example by genetic engineering of embryonic stem cells to block development of specific types of cells, and aggregation with embryonic cells from other species.

  Creation of such chimeras should not be permitted. Similarly, creation of chimeras where human brain cells or germ cells could be created in animals, or vice versa, should not be permitted. These types of chimera are fundamentally different, for example, from the grafting of human tissue onto animals.

January 2007

REFERENCES  Alikani, M and Willadsen, S M (2002). RBM Online 5:56-58. "Human blastocysts from aggregated mononucleated cells of two or more non-viable zygote-derived embryos."

  Barrientos, A et al (1998). J Biol. Chem 273: 14210-14217. "Human Xenomitochondrial Cybrids."

  Barritt J A et al (2002). Reproductive BioMedicine Online 4: 243-247. "Quantification of Human Ooplasmic Mitochondria."

  Beal, M F (2005). Ann Neurol 58: 495-505. "Mitochondria Take Center Stage in Aging and Neurodegeneration."

  Betarbet, R et al (2000). Nature Neuroscience 3: 1301-1306. "Chronic Systemic Pesticide Exposure Reproduces Features of Parkinson's Disease."

  Boiani, M et al (2002). Genes and Dev. 16: 1209-1219. "Oct4 Distribution and Level in Mouse Clones: Consequences for Pluripotency."

  Bourc'his, D et al (2001). Curr. Biol. 11: 1542-1546. "Delayed and Incomplete Reprogramming Patterns in Bovine Cloned Embryos."

  Dean, W et al (2001). Proc. Natl. Acad. Sci. USA 98: 13734-13738. "Conservation of Methylation Reprogramming: Aberrant Reprogramming in Cloned Embryos."

  Ebadi, M et al (2001). Biological Signals and Receptors 10: 224-253. "Ubiquinone (Coenzyme Q10) and Mitochondria in Oxidative Stress of Parkinson's Disease."

  Fairburn, H R et al (2002). Curr. Biol. 12: R68-R70. "Epigenetic reprogramming: how now, cloned cow? "

  Han, Y M et al, (2003). Theriogenoloy 59: 33-44. "Nuclear Reprogramming of Cloned Embryos Produced in vitro."

  Humpherys, D et al (2001). Sci. 293: 95-97. "Epigenetic Instability in ES Cells and Cloned Mice."

  Kang, Y K et al (2001). Nat Genet. 28: 173-177. "Aberrant methylation of donor genome in cloned bovine embryos."

  Kang, Y K et al (2002). EMBO J. 21: 1092-1100. "Limited Demethylation Leaves Mosaic-Type Methylation States in Cloned Bovine Pre-Implantation Embryos."

  Kenyon, L and Moraes, C T (1997). Proc Natl Acad Sci 94: 9131-9135. "Expanding the Functional Human Mitochondrial DNA Database by the Establishment of Primate Xenomitochondrial Cybrids."

  Lin, M T and Beal, M F (2006). Nature 443: 787-795. "Mitochondrial Dysfunction and Oxidative Stress in Neurodegenerative Diseases."

  Michaels, G S et al (1982). Dev. Biol. 94: 246-251. "Mitochondrial DNA copy number in bovine oocytes and somatic cells."

  Miller, F J et al (2003). Nuc. Acids Res. 31:... "Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age."

  Ohgane, J et al (2001). Genesis 30: 45-50. "DNA Methylation Variation in Cloned Mice."

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  Reynier, P et al (2001). Mol Hum Reprod 7: 425-429. "Mitochondrial DNA Content Affects the Fertilizability of Human Oocytes."

  Rideout III, W M et al 2001. Sci 293: 1093-1098. "Nuclear Cloning and Epigenetic Reprogramming of the Genome."

  Ryan, T and Townes, T (2001). Patent Application Number WO0069268. World Intellectual Property Organisation. "Production of Human Cells, Tissues and Organs in Animals."

  Slimane-Bureau, W C and King, W A (2002). Cloning and Stem Cells 4: 319-329. "Chromosomal Abnormalities: A Potential Quality Issue for Cloned Cattle Embryos."

  Steuerwald N et al (2000). Zygote 8: 209-215. "Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR."

  Yang, C-X (2004). Reproduction 127: 201-205. "Quantitative analysis of mitochondrial DNAs in macaque embryos reprogrammed by rabbit oocytes."





 
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