The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

Transgenic Copies of mtDNA: Techniques and Hurdles
means that cells whose mtDNA is perfectly intact, which means nearly all cells, will
nevertheless have nuclear (transgenic) copies of it. Thus, there will be a copy number problem
after all, since these mitochondrially healthy cells will be expressing twice as much of the
mtDNA-encoded proteins as of the solely nuclear-coded ones. A factor of two may not
sound like much of a burden, but the assembly of enzyme complexes is a very subtle process
which is still poorly understood, so there may be very deleterious effects. Indeed, this may
be the reason for the failure, a few years ago, of an in vitro attempt to make a working
nuclear-coded ATPase subunit 6. 7 This is discussed further in Section 15.8.
If this does turn out to be a barrier, however, there are various way that we might get
around it. One is to disable some of the nuclear-coded genes that are responsible for
transcribing and/or translating the mitochondrial copies. This would leave the inserted copies
as the only functional ones, so the correct stoichiometry would be restored. Also, it would
then not matter whether the mitochondrial copies were mutant or not; they would already
be functionless.
However, this approach has the disadvantage that the disruption of mitochondrial
transcription or translation must absolutely be effected only in cells whose inserted mt-coded
genes are all working. It is quite possible that gene therapy will (at least at first) be unable to
incorporate the engineered DNA into every single cell for which it is intended. If copies of
the mitochondrially-coded genes reach, say, 90% of cells, and independently the DNA that
disrupts mitochondrial transcription or translation also reaches 90%, then there will on
average be 9% of cells which are no longer able to use their mtDNA but which also have no
functioning replacement. Since under 1% of cells exhibit OXPHOS failure naturally, this
would be worse than doing nothing at all! It would thus be necessary to arrange a very tight
linkage between the two treatments, so as to allow mtDNA still to be used in cells where the
replacement DNA was ineffective. This may be more easily said than done. A second problem
is that all known techniques for disrupting a particular nuclear gene have an error rate, so
that they occasionally disrupt miscellaneous other genes too. Such disruption might easily
kill the cell, or even induce a tumour.
15.5. Interference by Mutant mt-Coded Proteins
An alternative approach to the stoichiometry problem would be to incorporate into
the inserted DNA a mechanism that stopped it from being transcribed until such time as
the cell got into OXPHOS difficulties. This would have the same effect: until and unless SOS
destroys the cell’s OXPHOS capacity, only the mitochondrially-coded copies are used, and
after that only the transgenic copies are used (since the mutant copies are useless). It should
also be quite easy to arrange, since there will be some fairly unsubtle intracellular signals
(such as the up-regulation of the PMOR) that can be used to trigger the switching-on of the
inserted genes. The switch itself can be constructed by numerous methods that are already
standard molecular biological tools, such as FLP recombination. 8 It may be necessary to
make the switch reversible, since cells may transiently get into a state where, for example,
the PMOR is briefly asked to work very hard but not because of mtDNA failure; if so, then
the detection mechanism would also need to distinguish between OXPHOS recovery due to
the action of the transgenic DNA and recovery that would have occurred anyway, since in
the former case the switch should clearly not be reversed when OXPHOS recovers.
The main shortcoming of this approach to the stoichiometry problem comes from the
mutant mtDNA. Some mutations (e.g. in tRNAs) will simply cause no mt-coded protein to
be constructed; cells taken over by mutations of that sort should respond properly to the
mechanism just outlined. But other mutations may only change the amino acid sequence a
little: enough to stop the protein from working, but not enough to stop it from being
incorporated into an enzyme complex. It will thus compete with the correct copies being
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