Gene Therapy (2008) 15, 1017–1023
& 2008 Nature Publishing Group All rights reserved 0969-7128/08 $30.00
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REVIEW
Progress and prospects: gene therapy
for mitochondrial DNA disease
DS Kyriakouli, P Boesch, RW Taylor and RN Lightowlers
Mitochondrial Research Group, Medical School, Newcastle University, Newcastle upon Tyne, UK
Defects of the mitochondrial genome cause a wide variety of
clinical disorders. Except for rare cases where surgery or
transplant is indicated, there is no effective treatment for
patients. Genetic-based therapies are consequently being
considered. On account of the difficulties associated with
mitochondrial (mt) transfection, alternative approaches
whereby mitochondrial genes can be engineered and
introduced into the nucleus (allotopic expression) are being
attempted with some success, at least in cultured cells.
Defects in the activities of multi-subunit complexes
of the oxidative phosphorylation apparatus have been
circumvented by the targeted expression of simple single
subunit enzymes from other species (xenotopic expression).
Although far from the clinic, these approaches show promise.
Similarly, nuclear transfection with genes encoding restriction endonucleases or sequence-specific zinc finger-binding
proteins destined for mitochondria has also proved successful in targeting mtDNA-borne pathogenic mutations. This is
particularly important, as mutated mtDNA is often found in
cells that also contain normal copies of the genome, a
situation termed heteroplasmy. Shifting the levels of heteroplasmy towards the normal mtDNA has become the goal of a
variety of invasive and non-invasive methods, which are also
highlighted in this review.
Gene Therapy (2008) 15, 1017–1023; doi:10.1038/gt.2008.91;
published online 22 May 2008
Keywords: mtDNA; heteroplasmy; mitochondria; mtDNA disease
In brief
Progress
Strategies can be devised for gene therapy of
mitochondrial DNA disorders.
Allotopic and xenotopic expression show great
promise as treatment concepts for mtDNA disease.
The alternative oxidase may prove an attractive
alternative for treating mtDNA disease.
The yeast soluble NADH oxidase can substitute for
defective human complex I.
Allotopic expression and import of nucleic acids are
being considered for rescuing pathogenic mt-tRNA
mutations.
Rescue of mtDNA mutations may be possible
through mitochondrial transfection.
Manipulating the mitochondrial genome may lead to
rescue of an OXPHOS deficiency.
Germline therapy for mitochondrial DNA disease is
being considered.
Introduction
The human mitochondrial genome (mtDNA) is found in
many copies in all nucleated cells (Figure 1). This small
(16 569 bp) genome is housed in the mitochondrial
Correspondence: Professor RN Lightowlers, Mitochondrial
Research Group, Newcastle University, The Medical School,
Framlington Place, Newcastle upon Tyne, Newcastle NE2 4HH, UK.
E-mail: [email protected]
Received 13 February 2008; revised 11 April 2008; accepted 14 April
2008; published online 22 May 2008
Prospects
Generation of numerous mice models heteroplasmic
for pathogenic mtDNA mutations that can routinely
be used to determine efficacy of any therapeutic.
Although such mice do exist, there are currently very
few. It is expected that within 5 years, there will be
many models that can be used.
Determining the feasibility of using genes encoding
single enzyme alternatives to OXPHOS complexes as
possible interventions for mutations that affect
protein-coding genes in mtDNA.
Production of antigenomic drugs that will allow
targeting and removal of pathogenic mtDNA. Current work is promising and it is likely that within 3–5
years a pharmaceutical could be developed for oral or
intravenous delivery that will be able to modulate
levels of heteroplasmy.
matrix and encodes 13 polypeptides, all of which are
integral components of the complexes that couple
oxidative phosphorylation (OXPHOS). In addition to
these polypeptides, this remarkably compact molecule
codes for the two ribosomal RNAs (16S and 12S mtrRNA) and 22 tRNAs that are necessary and sufficient
for intramitochondrial protein synthesis. As all 13
mitochondrially encoded polypeptides are essential
members of the OXPHOS machinery that harnesses
cellular respiration to ATP production, it is not surprising that mutations in this genome can cause a wide
Gene therapy for mitochondrial DNA disease
DS Kyriakouli et al
1018
the field to consider the prospect of gene therapy and
related concepts. The approaches can roughly be divided
into three groups: (1) rescue of a defect by expression of
an engineered gene product from the nucleus (allotopic
or xenotopic expression), (2) import of normal copies or
relevant sections of mtDNA into mitochondria, and (3)
manipulation of the mitochondrial genetic content. We
briefly introduce these concepts below and indicate
where significant progress has been made in the last 2
years.
Allotopic and xenotopic expression show
great promise as treatment concepts
for mtDNA disease
Figure 1 The human mitochondrial genome. This small (16 569 bp)
genome is almost completely transcribed from both strands, initiating
from one of two promoters (IH1, IH2) on the Heavy (H-) strand or the
single promoter (IL) on the Light (L-) strand. All of these promoters
and elements involved in replication initiation are found in the
displacement (D-) loop, the only major non-coding region. The
genome encodes 22 mt-tRNA (black diamonds), 2 mt-rRNA genes
(fuscia) and 13 protein-coding genes (olive, ND1-6 encoding
members of NADH:ubiquinone oxido-reductase; blue, Cytb encoding
apocytochrome b of ubiquinol:cytochrome c oxido-reductase; orange,
COI-III encoding members of cytochrome c oxidase: aquamarine,
ATPase 6, 8 encoding two members of Fo-F1 ATP synthase).
Important mutations that are referred to in this article are indicated.
Single letter code is given for each mt-tRNA-encoding gene.
variety of disorders such as mitochondrial encephalopathy lactic acidosis with stroke-like episodes (MELAS),
myoclonic epilepsy with ragged red fibres (MERRF) and
leber’s hereditary optic neuropathy (LHON).1 In addition, due to the ubiquity of mitochondria, the clinical
presentation of mitochondrial DNA disorders can
be multi-system, but often causes profound chronic
progressive defects in muscle and nerve.
Cells are multiploid with respect to mtDNA, with
most somatic cells containing at least 103 copies of the
genome. Consequently, a pathogenic mutation may be
present in just one, many, or all, depending on when the
mutation occurred and how the mutated molecule was
replicated and segregated during development. For most
disorders, the pathogenic mutation is recessive, requiring
a substantial percentage of the genome to carry the defect
before an effect on OXPHOS can be measured. This
‘threshold’ varies between cell types and mutation sites
but is normally between 60 and 95%.
Strategies can be devised for gene
therapy of mitochondrial DNA disorders
There is currently no effective treatment for the majority
of these debilitating diseases. This has led researchers in
Gene Therapy
In 1988, Nagley and colleagues2 were able to show that
the respiratory defect in yeast carrying mutations in the
mitochondrial MTATP8 gene could be rescued if a
normal copy of the gene was engineered and introduced
into the nucleus, resulting in cytosolic translation of the
gene product, ATPase 8. Phenotypic rescue required the
addition of a well-defined mitochondrial targeting and
import sequence at its N-terminal. This ‘allotopically’
expressed mitochondrial gene product could be functionally integrated into the mitochondrial ATP synthase
(complex V) and the deficiency could be rescued. Several
years later, this finding was extended to cultured human
cells. A different mitochondrial gene to encode a member
of the ATP synthase, MTATP6, was also engineered for
expression in the nucleus. Successful targeting of the
ATPase 6 gene product to the mitochondrion was shown
to partially suppress a respiratory-deficient phenotype
caused by a pathogenic mutation in MTATP6. These
experiments were met with optimism as a potential
method for treating patients with mutations in mitochondrial protein-encoding genes such as those with
neurogenic weakness, ataxia with retinitis pigmentosum
(NARP) which are caused by a specific point mutation
(m.8993T4G) in MTATP6. More recent and extensive
analysis by Bokori-Brown and Holt conflict with these
data.3 Similar allotopic experiments were performed to
rescue a cell line carrying the identical mutation in
MTATP6. The authors optimised the import of nuclearencoded ATPase 6 into the human cell lines but after
careful analysis of assembled complexes were unable to
show integration of the imported protein into mature
functional ATP synthase. Restoration of complex V
activity in the original experiments was suggested to
have been due to random clonal variations in ATP
synthesis as a consequence of aneuploidy in the
transfected population. Despite these discrepancies, the
authors remained optimistic about the potential for
allotopic expression as a treatment method.
In a twist to this approach, Corral-Debrinski et al.4
have attempted to optimise the allotopic expression of
such highly hydrophobic mitochondrial proteins by
suggesting that targeting of the transcript to the outer
mitochondrial membrane may facilitate co-translational
translocation of the gene product, thus preventing the
accumulation of cytosolic aggregates. Further evidence
was generated by using primary fibroblasts from patients
with mutations in MTND4 (a mitochondrial gene
encoding a member of complex I of the respiratory
Gene therapy for mitochondrial DNA disease
DS Kyriakouli et al
chain) known to cause Leber’s hereditary optic neuropathy, thereby avoiding the complexities of aneuploid
immortalised cell lines.5 Cis-acting elements in the
30 -untranslated regions from transcripts that had previously been shown to localise to mitochondria (SOD2
and COX10) were used in tandem with in-frame
presequence coding regions to improve mitochondrial
import of the allotopically expressed proteins. Transfections resulted in the rescue of the deficiency as evidenced
by the restoration of OXPHOS and growth with galactose
as a sole carbon source, a medium that requires efficient
OXPHOS for cell proliferation. Although impressive,
additional analysis by gel electrophoresis to show
unequivocally that imported proteins are indeed integrated into fully assembled OXPHOS complexes is still
required.
The alternative oxidase may prove
an attractive alternative for treating
mtDNA disease
Allotopic expression is an encouraging strategy, but does
require efficient mitochondrial import and correct
integration of the allotropic protein into an assembled
protein complex. Two entirely separate but equally
exciting prospects have received attention in the last 2
years. Human mitochondria rely on a single cyanidesensitive cytochrome c oxidase (COX) to reduce molecular oxygen. This multi-component complex is the
terminal member of the mitochondrial electron transfer
chain and contains three essential polypeptides encoded
by mtDNA. Consequently, mtDNA mutations often
cause defects in COX activity. Importantly, many other
species encode cyanide-insensitive alternative oxidases
(AOX). A very encouraging report recently showed that a
single subunit AOX from the sea squirt Ciona intestinalis
could be synthesised in the cytosol of cultured human
cells and successfully targeted to the mitochondrial inner
membrane.6 AOX caused no measurable cytotoxicity and
transfectants showed impressive levels of cyanideinsensitive oxidation of mitochondrial metabolites. The
authors also highlighted its important function as an
anti-oxidant, preventing over-reduction of the ubiquinone pool, which is believed to be an important source of
reactive oxygen species. To date, there has been no report
of its use in rescuing a mitochondrial COX defect, but the
potential for its use in treating human COX deficiencies
is clear.
The yeast-soluble NADH oxidase can
substitute for defective complex I
A similar and equally elegant approach has also been
used to by-pass defects in the initial part of the electron
transfer chain. Human complex I, NADH:ubiquinone
oxidoreductase is a profoundly complicated assembly of
approximately 45 proteins, 7 of which are encoded by the
mitochondrial genome. Numerous mitochondrial defects
have been linked with mutations in these mitochondrial
genes. In contrast, the yeast Saccharomyces cerevisiae does
not possess a similar membrane-bound complex, but
instead, uses just a single subunit NADH oxidase,
termed as Ndi1. Back in 2001, Yagi and colleagues7
showed that mitochondrial targeting of Ndi1 in human
cells defective in complex I activity corrected this defect.
In the intervening years, the authors have built on this
observation and showed that in addition to the potential
for Ndi1 activity to treat patients with mtDNA disease
associated with mtDNA-borne MTND mutations, this
enzyme may prove useful for other complex I disorders.
In particular, the authors have focussed on complex I
deficiency in Parkinson’s disease (PD). That the hallucinogen MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes a form of PD has been well described. This
drug selectively inhibits complex I activity in humans,
but has no effect on yeast Ndi1.7 Yagi and colleagues8
have now demonstrated that viral-mediated delivery
and expression of Ndi1 in the substantia nigra protected
against neurodegeneration after feeding rats with MPTP.
These data tentatively support the use of yeast Ndi1
expression to compensate for any complex I deficiency in
patients with PD.
1019
Allotopic expression and import
of nucleic acids are being considered for
rescuing pathogenic mt-tRNA mutations
It has been a dramatic few years for reports of nucleic
acid import into mammalian mitochondria, any of which
may eventually prove beneficial for genetic therapies of
mitochondrial disorders. In contrast to many eukaryotes,
mammals encode all tRNAs required for mitochondrial
protein synthesis on the mitochondrial genome (mttRNA). As many disorders of the mitochondrial genome
are caused by mutations in mt-tRNA encoding genes,
there has been great interest in rescuing these mutations
by importing normal copies of the relevant tRNA from
the cytosol.
Tarassov et al.9 showed that a respiratory defect in cell
lines containing mutated mt-tRNALys could be partially
rescued when the yeast homologue was expressed in the
nucleus. These results suggested that a cryptic tRNA
import system remains in human mitochondria. The
exact tRNA import mechanism in human mitochondria
is unclear, although the authors have begun to dissect the
pathway in yeast mitochondria. Building on the theme of
tRNA import as a potential mechanism for treating
mtDNA disorders, Mukherjee et al.10 have exploited the
highly prolific tRNA import process in protists. Initially,
a large tRNA import complex (RIC) was isolated from
the inner mitochondrial membrane of Leishmania tropica
and reconstituted into phospholipid vesicles. The complex comprised 11 major polypeptides, three of which
were mitochondrially encoded and five of the eight
nuclear gene products were also members of the
OXPHOS machinery. Remarkably, bathing cultured human cells in the purified RIC led to receptor-mediated
import of the complex into the cytoplasm and integration
into mitochondria via a mechanism that is currently
unclear.11 Compelling evidence of cytosolic tRNA import
comes from work with a cell line containing mtDNA
with a large-scale deletion that removes various genes
including MTTK, which encodes mt-tRNALys (Figure 1).
Prior to incubation of cells with RIC, there was no
evidence of any mitochondrial protein synthesis, which
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DS Kyriakouli et al
1020
can normally be visualised after selective poisoning of
cytosolic protein synthesis. Following RIC addition,
protein translation was restored for gene products whose
genes were not removed by the deletion. This restoration
is likely to be due to imported cytosolic tRNALys
functioning in mitochondrial translation.
It is remarkable that the large RIC complex can be
transported across the plasma membrane and the outer
mitochondrial membrane and integrated into the inner
membrane. Work with RIC has now been extended to
show that complexes preloaded with antisense RNA
designed against various mt-mRNA can successfully
transport these RNAs to the mitochondrial matrix of
intact cells in culture. This leads not only to selective
impairment of protein synthesis from the mt-mRNA
target, but also degradation of this mt-mRNA.12 This is a
very striking result as it is consistent with an RNAmediated targeting and degradation pathway similar to
that noted in the cytosol. The latter is mediated by the
RISC complex and dicer for which there is no evidence of
mitochondrial localisation. Clearly, this work from the
laboratory of Adhya has quite profound consequences
for the field of mammalian mitochondrial transfection
and would benefit enormously from confirmatory
experiments from independent laboratories.
Rescue of mtDNA mutations
may be possible through mitochondrial
transfection
When a pathogenic nuclear mutation has been identified,
one approach may be to re-introduce normal copies of
the gene that might lead to rescue of the phenotype.
There are major obstacles in using this strategy to
overcome mitochondrial gene mutations. First, transfection is difficult, as there are effectively three membrane
barriers. Second, even assuming that exogenous DNA
can be imported, gene expression will be transient.
Stable integration may be mediated by homologous
recombination. There is evidence that such recombination does occur in some somatic cells at low frequency,
but it appears to be a rare event. Consequently, any
imported DNA must contain the correct cis-acting
elements to promote DNA replication, resolution and
maintenance. To date, these elements are not fully
characterised and even the detailed mechanism of
mtDNA replication itself is controversial. Finally, assuming transfection and maintenance, there are still many
hundreds or thousands of mtDNA molecules that will
outweigh the transfected molecule. How can it be
selected for and maintained in such a background?
What progress has been made towards overcoming
these barriers? It has been known for several years that
isolated plant mitochondria can spontaneously import a
low level of naked linearised DNA. This observation has
now been extended to mammalian mitochondria, which
also show a low efficiency of natural DNA import.13
Unlike previous anecdotal reports of DNA uptake, data
showed that DNA could be imported and could act as a
template for DNA synthesis. In addition, transcription
was shown to occur only when the template carried a
transcriptional promoter element and the primary
transcript could be successfully processed and matured.
Gene Therapy
This process of natural competence is not at all understood but has recently been used by Weissig and
colleagues to load isolated mitochondria that were
subsequently shown to be engulfed by cells when coincubated.14 Uptake of isolated murine mitochondria had
initially been shown by the authors to restore respiration
on fusion with human A549 lung carcinoma cells devoid
of mtDNA. This internalisation, similar to the method
described over 25 years ago by Clark and Shay may
prove to be a powerful technique for studying mitochondrial gene expression in humans but is unlikely to be
immediately relevant to gene therapy for mtDNA
disease.
Are there any other methods being investigated for
transfecting mitochondria in whole cells? Several years
ago, there was substantial interest in the use of
DQasomes. These nonviral mitochondriotropic liposome
derivatives could be loaded with DNA and had been
shown to co-localise with mitochondria when added to
cultured cells. Unfortunately, these earlier reports have
not been greatly extended in the past 2 years. Another
liposome-based carrier, MITO-Porter has also been
shown to co-localise to mitochondria in intact cells.15
MITO-Porter loaded with GFP was shown in isolated
mitochondria to be delivered to the intermembrane space
and to co-localise with mitochondria in cultured cells.
These data suggest that such liposomal preparations can
fuse with the outer mitochondrial membrane, but will it
prove to be able to access the inner membrane that is
necessary for the cargo to access the mitochondrial
matrix? Another approach to mitochondrial transfection
is protofection. To our knowledge, however, details of
this protein-mediated method for delivering complete
mitochondrial genomes to mitochondria have not appeared as an original article in any peer-reviewed
journal, so it is difficult to assess and to determine its
potential for mediating gene therapy.
Manipulating the mitochondrial genome
may lead to rescue of an OXPHOS
deficiency
Many patients that suffer from mtDNA disease are
heteroplasmic, harbouring two subpopulations, one
normal and one pathogenic. One approach to genetic
therapy for these intractable disorders is to manipulate
the relative amounts of these two subpopulations. This is
particularly attractive, as most heteroplasmic pathogenic
mutations are recessive, such that levels do not have to
be altered greatly before biochemical defects can be
rescued. Which methods are currently being investigated
for manipulating levels of heteroplasmy (Figure 2)?
Exercise training, either by endurance training to
promote mitochondrial biogenesis or by resistance
exercise protocols to promote muscle satellite cell
activation has long been proposed as a means of treating
patients with high levels of heteroplasmic mtDNA
mutations in muscle. Endurance training improves
exercise capacity, inducing physiological adaptations,
which reverse the effects of muscle deconditioning and
lead to a resolution of the underlying mitochondrial
biochemical defect. Nevertheless, concerns have been
raised as to whether endurance training has potentially
Gene therapy for mitochondrial DNA disease
DS Kyriakouli et al
Figure 2 Manipulating mtDNA heteroplasmy. An approach to
treating patients who suffer from an mtDNA disorder where both
pathogenic and wild type mtDNA co-exist (heteroplasmy) is to
manipulate these levels with the aim of increasing the wild type at
the expense of the pathogenic mtDNA, potentially reversing the
biochemical defect. In this figure, heteroplasmic mitochondria
containing both wild type (grey) and pathogenic (black) mtDNA
are schematically represented. Transverse muscle sections illustrate
how high levels of mutated mtDNA in heteroplasmic patients can
result in mosaic muscle fibre staining for cytochrome c oxidase
activity (left panel), a situation that can potentially be reversed by
increasing the level of wild type mtDNA (right panel). Several
different methods to modulate heteroplasmy are currently being
explored. (A) Patients are being subjected to endurance or resistance
training, increasing muscle bulk and promoting growth of new
muscle. (B) Targeting restriction endonucleases to mitochondria has
already been successfully employed in tissue culture to remove
pathogenic mtDNA where the mutation generates a specific
restriction site. This approach has also been used to modulate
levels of heteroplasmy for polymorphic mtDNA in mice. (C) Cell
membrane crossing oligomers (CMCOs) are being optimised to
penetrate cells, target mitochondria and selectively bind to mutated
mtDNA. These molecules are potentially capable of inhibiting
mtDNA replication, although this has yet to be demonstrated. (D)
Zinc-finger binding proteins that show sequence binding specificity
have been targeted to mitochondria. By fusing the ZFB chimaera to
a DNA methylase, it was shown that mtDNA carrying a specific
mutation can be selectively methylated. It is postulated that ZFB
proteins could be fused to DNases, leading to selective cleavage of
mutated mtDNA.
long-term deleterious effects, given that the mutated
mtDNA load in some patients was noted to
increase following training. To assess the safety of
endurance training, Taivassalo et al.16 studied the
effects of 14 weeks of exercise in a group of patients
with single, large-scale mtDNA deletions. Markers
of physiological improvement (oxygen utilisation,
skeletal muscle oxygen extraction, peak capacity
for work and submaximal exercise tolerance) were
evident, although genetic analysis did not reveal any
change in the level of deleted mtDNA following exercise
training. Further training for an additional 14 weeks
maintained these benefits, whereas detraining resulted in
a loss of the physiological adaptations. Follow-up studies
to assess whether long-term training might induce
changes in mtDNA mutation load are currently in
progress.
Some patients with sporadic mtDNA mutations
harbour high levels of mutated mtDNA in mature
muscle fibres, but low or undetectable levels in muscle
satellite cells. The stimulation of muscle regeneration,
and concomitant incorporation of satellite cell mtDNA
into mature muscle (gene shifting), is therefore an
attractive approach to manipulate mtDNA heteroplasmy
levels in vivo. Indeed, anaesthetic injection, traumatic
biopsy injury and resistance exercise (both eccentric and
concentric) training protocols have all been shown to be
of benefit in inducing muscle regeneration in individual
patients. Ongoing studies in patients with single, largescale mtDNA deletions suggest that 12 weeks of
resistance training induces an increase in satellite cell
number and muscle regeneration, leading to improvements in strength and oxidative capacity as reflected by a
decrease in the percentage of muscle fibres deficient in
mitochondrial COX activity.17
An elegant approach to removing pathogenic mtDNA
is by targeting restriction endonucleases to mitochondria
of heteroplasmic cells. First pioneered by Carlos Moraes
in rodent and Masashi Tanaka in human cell lines,
specific mtDNA subpopulations in a heteroplasmic
background can be degraded by endonucleases expressed from introduced genes engineered to generate
enzymes with mitochondrial targeting sequences. Heteroplasmy was shown to shift substantially towards the
genome lacking the restriction site. This was potentially
of direct relevance to disease, as the heteroplasmic
m.8993T4C mutation was shown to be markedly
reduced on targeting of the restriction site by endonuclease SmaI. It could be envisaged that the engineered
gene encoding the targeted endonuclease could be
delivered to patients, but the approach would appear
to be limited to the very few human pathogenic mtDNA
mutations that result in a restriction site unique to
the pathogenic mtDNA. Moraes and colleagues
have, however, shown that the generated restriction
site need not be unique.18 Heteroplasmic mice carrying
two distinct genomes were subjected to adenoviralmediated delivery of a gene encoding the targeted
endonuclease ScaI. The two genomes each carried
multiple ScaI sites, five in one genome and three in the
second genome. Heteroplasmy was shown to shift in the
liver and skeletal muscle, although high-level expression
did lead to mtDNA depletion. These data, however, are
promising and suggest that if expression levels are
controlled it may be possible to treat patients who have
pathogenic mtDNA mutations that create additional
restriction sites.
A concept that received wide coverage several years
ago was the possibility of antigenomic therapy. This
required the design and use of a drug that could be
imported into mitochondria, bind selectively to the
mutated mtDNA and inhibit it from replicating, allowing
the normal copy to propagate with time, and thus rescue
the defective OXPHOS phenotype. But what could this
antigenomic molecule be? At first, there was great
interest in the use of mitochondrially targeted peptide
nucleic acids (PNAs). These molecules still retained the
ability to Watson:Crick base pair and showed remarkable
binding selectivity even at physiological salt concentrations and temperatures. Initial experiments were encouraging but eventually it was determined that the
targeted PNAs although co-localised to mitochondria in
whole cells did not get across the inner membrane, and
so could not access mtDNA. Modified molecules termed
1021
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DS Kyriakouli et al
1022
as ‘cell membrane crossing oligomers’ (CMCO) have
now been designed, with greater polarity than traditional
PNAs. It has now proved possible to target and import
CMCO:PNA hybrid molecules into mitochondria in
whole cells. Although these new molecules have great
promise as potential drugs for treating heteroplasmic
patients, many important experiments need to be
performed to determine whether they can inhibit
mtDNA replication in whole cells and be delivered to
defective mitochondria in animals.
The possibility of selective targeting of mtDNA
has also been explored by Minczuk et al.19 These
authors have attempted to use the short zinc-finger
peptides that show sequence-specific DNA binding
capabilities.
The
authors
generated
chimaeric
ZFPs linked to a DNA methylase carrying an additional
mitochondrial presequence. Following import, chimaeras were shown to bind mtDNA and to leave a
signature methylation pattern at various sites on the
mitochondrial genome, a genome that is not normally
methylated. In addition, by using the NARP m.8993T4G
cell line, popular for many of these studies, the
authors showed methylation patterns that differed
from the wild-type mtDNA control. By linking these
domains to a nuclease it may be possible to target a
mutated subpopulation in a heteroplasmic patient. This
is an exciting and impressive achievement, but its
long-term usage will be limited by the requirement for
transfection and successful expression in defective cells
and tissues.
Germline therapy for mitochondrial DNA
disease is being considered
Patients with mtDNA disease often wish to have
children. However, the knowledge that mtDNA disease
is a genetic disorder and can be transmitted down the
maternal line can preclude this decision. Would it be
possible to generate a zygote from the parents’ gametes
using standard in vitro fertilisation techniques and then
replace the defective mitochondrial genetic complement?
A single-cell zygote containing normal copies of mtDNA
would need to be enucleated to produce a cytoplast. An
egg from the patient would then be fertilised in vitro with
the sperm from the partner. Finally, an embryo would
have to be reconstructed from pronuclei carefully
removed from the patients fertilised oocyte and fused
to the cytoplast.20 Studies in mice have indeed suggested
that it may be possible to prevent transmission of
mutated mtDNA by pronuclear transfer. Following
reconstruction of such a murine embryo, fused zygotes
were transferred to the oviducts of pseudopregnant
females and offspring were shown to be normal.21 Could
such an approach be possible in humans? Following the
decision in 2005 by the UK Human Fertilisation and
Embryology Authority to approve research into the
feasibility of this approach in humans, experiments have
been initiated using abnormally fertilised embryos.
Although results of these studies have yet to be
published, this approach may bring hope to patients
with mtDNA disorders who wish to have children and
for whom oocyte donation is either not possible or not
desired.
Gene Therapy
Future prospects for genetic therapy of mtDNA
disease
Considering the recent output, it is clear that scientists are
now claiming a variety of methods for transfecting
mitochondria. Although impressive, it is difficult to see
how this work will be of direct relevance to treatment
unless the transfected material can be autonomously
maintained. Allotopic expression and the use of simple
single-subunit enzymes that can potentially replace the
activity of OXPHOS complexes are both areas of great
potential. The drawback is that they will require the
delivery of foreign genes to affected cells, which is a similar
problem to many conventional gene therapy approaches.
Manipulating heteroplasmy remains a particularly attractive idea, especially if the intervention is non-invasive or is
mediated by a pharmaceutical agent. One area that has
been resurrected recently and shows great promise is the
use of CMCO-based molecules that can enter cells and
mitochondria naturally and initial experiments suggest
these molecules can influence heteroplasmy and mitochondrial gene expression. Before any of these therapeutic
approaches can be used in clinical trials, it will be necessary
to show their efficacy in animal models. Unfortunately, it
has proven difficult to establish mice that are heteroplasmic
for pathogenic mutations. To move forward, it is important
that more models become available.
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