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
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