Human Reproduction, Vol. 15, (Suppl. 2), pp. 79-85, 2000
In-vitro genetic modification of mitochondrial function
Robert W.Taylor1, Patrick F.Chinnery,
Douglass M.T\irnbull and Robert N.Lightowlers
Department of Neurology, The Medical School, University of Newcastle
upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK
lr
To whom correspondence should be addressed at: Department of Neurology,
The Medical School, University of Newcastle upon Tyne, Framlington Place,
Newcastle upon Tyne, NE2 4HH, UK. E-mail: [email protected]
Defects of mitochondrial (mt) DNA cause
a diverse group of incurable, progressive
diseases that often lead to severe disability
and premature death. Most patients with
pathogenic mtDNA defects have a mixture
of mutant and wild-type mtDNA (heteroplasmy), and the clinical defect is only
expressed when the percentage of mutant
mtDNA exceeds a critical threshold. Since
mtDNA is continually replicating and being
turned over, we have proposed an approach
to the treatment of these disorders that
utilizes sequence-specific antigenomic peptide nucleic acids (PNAs) to hybridize and
specifically inhibit the replication of mutant
mtDNA under physiological conditions. By
allowing the selective propagation of wildtype molecules, it may be possible to correct
the cellular biochemical defect and to prevent the progression of disease. This paper
summarizes the experimental progress in
this area, including the cellular uptake of
PNA molecules and their import into mitochondria both in vitro and in cell culture
by the addition of a nuclear-encoded mitochondrial targeting sequence. The possibilities of extending this strategy to the
treatment of mtDNA deletion disorders are
discussed.
Key words: DNA replication/gene therapy/
© European Society of Human Reproduction and Embryology
mitochondrial DNA/mtDNA heteroplasmy/
peptide nucleic acids
Mitochondrial DNA and disease
Mitochondrial DNA encodes two rRNAs, 22
tRNAs, and some 13 essential polypeptide
subunits of the mitochondrial respiratory
chain, the major cellular function of which is
to generate ATP by oxidative phosphorylation.
As such, defects of mitochondrial DNA are
an important cause of disease in humans.
With >100 pathological mutations described
(Servidei, 1998), they are associated with
a broad spectrum of clinical manifestations
affecting many different tissues (Johns, 1995;
Wallace, 1999); commonly, however, these
disorders present as a progressive neurological
syndrome resulting in severe disability and
premature death (Chinnery and Turnbull,
1997). Pathological mutations can take the
form of large-scale rearrangements or point
mutations within RNA- or protein-encoding
genes. The majority of patients harbour mutant
and wild-type mtDNA within the same cell and
tissue, a phenomenon known as heteroplasmy.
The proportion of mutant to wild-type mtDNA
is the critical determinant of the expression of
the genetic defect, and hence the clinical
79
R.W.Taylor et al.
and to form fully functional muscle fibres is
potentially of therapeutic importance.
Another possible approach has become
evident following recent cell culture studies
in which the mitochondrial inhibitor oligomycin (in conjunction with galactose) has been
used to select for wild-type cells over those
containing the T8993G ATPase subunit 6
mutation. Using cybrid lines containing heteroplasmic levels of this mutation, Manfredi
et al. (1999) have demonstrated that these
conditions are able to induce a significant shift
in the level of mtDNA heteroplasmy in favour
of the wild-type genome, suggesting a basis
for pharmacological intervention in ATPase
gene disorders.
A strategy that we are currently pursuing
makes fundamental use of the heteroplasmic
Approaches to treatment
nature of the majority of pathogenic mtDNA
Notwithstanding problems unfamiliar to con- mutations, and the fact that the mitochondrial
ventional somatic gene therapy approaches, a genome is continuously replicating and thus
number of experimental strategies are cur- being turned over (Gross et al, 1969). Because
rently being pursued towards achieving a mutant mtDNA is functionally recessive, the
gene therapy for mitochondrial diseases specific inhibition of mutant mtDNA replica(Chrzanowska-Lightowlers etal, 1995; Taylor tion by suitable antigenomic agents could
etal, 1997a). These include introducing modi- theoretically correct the imbalance of the ratio
fied genes into mitochondria (Seibel et al, of mutant to wild-type mtDNA that causes
1995; Collombet et al, 1997) and novel the clinical disease. We postulate that if the
approaches that aim to correct the genetic selective binding of an antigenomic agent was
defect in situ (Clark et al, 1997; Shoubridge able to sterically inhibit the mitochondrial
et al, 1997). These authors have shown that DNA polymerase y-initiated replication of the
by inducing focal damage to mature muscle mutant genome, this might allow the selective
that contains high levels of a pathological propagation of wild-type genomes to the extent
tRNA mutation, the satellite cell population that the relative amount of wild-type mtDNA
that proliferates in response to the insult can would be sufficient to restore normal biochemcontain remarkably low levels of the mutant ical function to the cell and to arrest the
mtDNA. As such, subsequent analysis of the progression of disease (Taylor et al, 1997a).
regenerated mature muscle reveals much lower This approach depends on the designing of
levels of mutation, consistent with an increase suitable antigenomic molecules, as an opporin the number of muscle fibres that have tunity already exists for the binding of
normal cytochrome c oxidase (COX) activity sequence-specific agents to mutant mtDNA.
(an enzyme of the mitochondrial respiratory Capitalizing on the extensive displacement of
chain that is in part encoded by the mitochon- the two separate strands of mtDNA that occurs
drial genome). A treatment that stimulates the during the replication of the mitochondrial
proliferation of satellite cells to regenerate genome (Clayton, 1991), it has been possible
phenotype (Lightowlers et al,
1997).
Although this threshold level is dependent
upon both the nature of the mutation and the
tissue affected, the vast majority of pathological mtDNA mutations investigated are
highly recessive and biochemical dysfunction
is only apparent with low levels (<10%)
of wild-type mtDNA (Boulet et al, 1992;
Chomyn et al, 1992; Attardi.^ al, 1995).
Despite an increased awareness of their
clinical importance and considerable progress
towards understanding the relationship
between pathogenic defect and clinical phenotype, effective biochemical and pharmacological treatments are not available (Taylor
et al, 1997a).
80
In-vitro manipulation of mtDNA heteroplasmy
to design an antigenomic molecule that binds
selectively to a mutant mtDNA strand.
Antigenomic PNA therapy for
mtDNA disorders
We designed peptide nucleic acids (PNAs)
complementary to human mtDNA sequences,
and assessed their ability to inhibit mtDNA
replication in an in-vitro assay. PNAs are
polyamide-nucleic acid derivatives in which
the deoxyribose phosphate backbone of DNA
is replaced by an uncharged Ar-(2-aminoethyl)glycine polymer (Nielsen et al, 1991). PNAs
bind complementary DNA and RNA strands
with higher affinities than an equivalent oligodeoxynucleotide (Egholm et al, 1993). Mutation of a single base within a PNA:DNA
duplex can disrupt the thermal stability of the
complex by up to 20°C (0rum et ai, 1993).
This characteristic is particularly important as
it confers upon PNA the ability, at a given
temperature, to selectively discriminate
between two nucleic acid templates that differ
by a single base substitution. Coupled with
enhanced stability in serum and cell extracts
(Demidov et al., 1994), these properties make
PNAs therapeutically attractive for antisense
and antigene strategies to modulate gene
expression (Hanvey et al., 1992; Knudsen and
Nielsen, 1996; Good and Nielsen, 1998).
A PNA was designed to be complementary
to a human mtDNA template containing the
A8344G tRNA1^ point mutation associated
with myoclonus, epilepsy and ragged-red fibre
(MERRF) disease, with the base substitution
in the middle of the sequence. This PNA is
100% complementary to the mutant sequence,
and so would hybridize at physiological temperature. In theory, the mismatched base
present in the wild-type mtDNA sequence
ought to disrupt the stability of the PNA:DNA
binding to such an extent that at physiological
temperature it would not hybridize. In an invitro mtDNA replication run-off assay, this
PNA was shown under physiological conditions to specifically inhibit the replication of
the mutant (G8344) but not wild-type (A8344)
template (Taylor et al., 1997b). Moreover, the
inhibitory effects of this PNA were maintained
in the presence of saturating concentrations of
E.coli single-stranded binding protein (SSB),
a protein analogous to the mitochondrial SSB
that binds single-stranded DNA during replication.
Cellular uptake and targeting of
PNA to mitochondria
Although these in-vitro data clearly indicate
the potential of this approach for providing
antigenomic gene therapy for mtDNA point
mutation disorders, a fundamental prerequisite
is good cellular uptake of these molecules and
accurate targeting and delivery of PNA into
the mitochondrial matrix. Previous reports had
suggested that unmodified antisense PNAs
were poorly imported across cell membranes,
on account of their low phospholipid permeability (Hanvey et al., 1992; Wittung et al,
1995), or were otherwise sequestered away
from the nucleus in cytoplasmic endosomes
(Bonham et al., 1995). Using fluorescence
confocal microscopy to visualize a biotinylated PNA, we have investigated the ability of
several cell lines to import PNA into the
cytoplasm. These molecules were rapidly
taken up by a number of cell lines in a timeand temperature-dependent manner during
which the PNA was localized and concentrated
in the nucleus (Taylor et al., 1997b; Chinnery
et al, 1999). In order to target the PNA to
the correct intracellular location, we have
utilized the protein import machinery to
deliver PNA into mitochondria. Previous
studies have shown that the addition of a
nuclear-encoded N-terminal mitochondrial
leader sequence to a nucleic acid will promote
targeting to mitochondria isolated from yeast
(Vestweber and Schatz, 1989) or rat liver
81
R.W.Taylor et al.
Wild-type mtDNA
4977 bp
1
5
13 bp
13 bp
3"
deletion event
Deleted
mtDNA
51
31
13 bp
Figure 1. Deleted mitochondrial genomes contain just one of two similar sequences found in the intact genome. The 'common
deletion' (AmtDNA4997) is flanked by a perfect 13 bp direct repeat present in the normal mitochondrial genome (Schon et al.,
1989), one copy of which is lost with the missing segment and one copy of which remains in the deleted circle.
Wild-type mtDNA
51
Antigenomic molecule
does not bind
repeat
repeat
31
Antigenomic molecule binds
co-operatively to 31 and 51 ends
Deleted
mtDNA
5'
repeat
Figure 2. Once the deletion has been defined, an antigenomic peptide nucleic acid (PNA) can be constructed that binds cooperatively across one copy of the repeat sequence only when deletion has taken place.
(Seibel et al, 1995). By synthesizing a PNApeptide conjugate that contains the N-terminal
leader sequence of cytochrome c oxidase subunit VIII, we have been able to demonstrate
targeting of antigenomic PNA molecules to
the matrix compartment within isolated rat
liver mitochondria in vitro and cultured human
myoblasts in vivo (Chinnery et al, 1999).
Treatment of mtDNA deletion disorders
Although much attention has focused on the
growing number of pathological mtDNA
82
tRNA abnormalities associated with disease
(Schon et al., 1997), it is large-scale rearrangements that represent the major subset of pathogenic mtDNA mutations, accounting for some
60% of reported mtDNA gene defects
(Chinnery and Turnbull, 1997). Gross deletions of mtDNA have been observed at high
levels in progressive external ophthalmoplegia
(PEO), Kearns-Sayre syndrome (KSS) and
Pearson marrow/pancreas syndrome (Holt
et al, 1988; Moraes et al., 1989; Rotig et al,
1991). In addition, somatic mtDNA deletions
have been shown to accumulate with normal
In-vitro manipulation of mtDNA heteroplasmy
human ageing, particularly in post-mitotic tissues such as brain and skeletal muscle
(Cortopassi and Arnheim, 1990; CorralDebrinski et al., 1992). The level of mutant
mtDNA has been shown to clonally expand
within individual cells to a level past
the threshold required to cause a focal
biochemical abnormality (Brierley et al.,
1998).
Mitochondrial DNA deletions often occur
at the site of perfect repeat sequences of 4 13 nucleotides in length; such repeats that
are remarkably common in the mitochondrial
genome. One of these repeats is always
present in the deleted molecule (Schon et al.,
1989; Mita et al., 1990) and, as such, the
repeat sequence and either the 3' or 5'
flanking sequence will be common to both
the wild-type and the deleted molecule
(Figure 1). This makes the deleted genome
refractory to the antigenomic approach for
treatment in which a potential therapeutic
molecule is designed to selectively hybridize
to the aberrant molecule at physiological
temperatures and salt conditions. Although
there is no unique sequence in deleted
mtDNA that can be targeted to give selective
hybridization, we believe that it is possible
to exploit the close proximity of the 3' and
5' flanking regions in the deleted genome
as a template for selective hybridization
(Figure 2). To achieve this goal, it must be
established that the 5' and 3' complementary
regions of an antigenomic PNA molecule
can act co-operatively to increase the binding
affinity of such a molecule to a deleted
mtDNA template, as compared to either the
3' or 5' regions alone. This would involve
bridging the repeat sequence, and experiments are currently being attempted in
our laboratory to determine binding cooperativity using both thermal melt spectrophotometry and surface plasmon resonance
technology (Jensen et al., 1997).
Conclusions
The unrelenting growth in the field of
diagnostic mitochondrial medicine over the
past 10 years impels us to consider possible
options for treatment. The absence of specific
biochemical and pharmacological therapies
reinforces the need to develop realistic gene
therapies. In the case of mitochondrial
disease, it is the important balance between
the level of wild-type versus mutant mitochondrial genomes that determines clinical
phenotype. As such, a subtle manipulation
of mtDNA heteroplasmy has the potential
to redress the imbalance and so to reverse
the biochemical and clinical abnormality. In
view of the in vitro and cell uptake data
available, the sequence-specific inhibition of
mutant mDNA replication by antigenomic
PNAs promises to achieve important goals
to this end. We look forward to the next
10 years.
Acknowledgements
We thank the Muscular Dystrophy Campaign and
The Wellcome Trust for their continued support of
this work.
References
Attardi,G., Yoneda, M. and Chomyn, A. (1995)
Complementation and segregation behavior of
disease causing mitochondrial DNA mutations in
cellular model systems. Biochim. Biophys Acta,
1271, 241-248.
Bonham, M.A., Brown, S., Boyd, A.L. et al. (1995)
An assessment of the antisense properties of RNase
H-competent and steric-blocking oligomers. Nucleic
Acids Res., 23, 1197-1203.
Boulet, L., Karparti, G. and Shoubridge, E.A. (1992)
Distribution and threshold expression of the tRNALys
mutation in skeletal muscle of patients with
myoclonus epilepsy and ragged-red fibers (MERRF).
Am. J. Hum. Gen., 51, 1187-1200.
Brierley, E.J., Johnson, M.A., Lightowlers, R.N. et al.
(1998) Role of mitochondrial DNA mutations in
human aging: implications for the central nervous
system and muscle. Ann. Neuroi, 43, 217-223.
83
R.W.Taylor et al.
Chinnery, P.F. and Turnbull, D.M. (1997) Clinical
features, investigation, and management of patients
with defects of mitochondrial DNA. J. Neurol.
Neurosurg. Psychiatry, 63, 559-563.
Chinnery, RE, Taylor, R.W., Diekert, K. et al.
(1999) Peptide nucleic acid delivery into human
mitochondria. Gene Ther. 6, 1919-1928.
Chomyn, A., Martinuzzi, A., Yoneda, M. et al. (1992)
MELAS mutation in mtDNA binding site for
transcription termination factor causes defects in
protein synthesis and in respiration but no changes in
levels of upstream or downstream mature transcripts.
Proc. Natl Acad. Sci. USA, 89, 4221^225.
Chrzanowska-Lightowlers, Z.M.A., Lightowlers, R.N.
and Turnbull, D.M. (1995) Gene therapy for
mitochondrial DNA defects: is it possible? Gene
Ther, 2, 311-316.
Clark, K.M., Bindoff, L.A., Lightowlers, R.N. et al.
(1997) Reversal of a mitochondrial DNA defect in
human skeletal muscle. Nature Genet., 16, 222-224.
Clayton, D.A. (1991) Nuclear gadgets in mitochondrial
DNA replication and transcription. Trends Biochem.
Sci, 16, 107-111.
Collombet, J.-M., Wheeler, V.C., Vogel, F. et al.
(1997) Introduction of plasmid DNA into isolated
mitochondria by electroporation. A novel approach
toward gene correction for mitochondrial disorders.
J. Biol. Chem., 272, 5342-5347.
Corral-Debrinski, M., Horton, T., Lott., M.T. et al.
(1992) Mitochondrial DNA deletions in human
brain: regional variability and increase with advanced
age. Nature Genet., 2, 324-329.
Cortopassi, G.A., and Arnheim, N. (1990) Detection
of a specific mitochondrial DNA deletion in tissues
of older humans. Nucleic Acids Res., 18, 6927-6933.
Demidov, V.V., Potaman, V.N., Frank-Kamenetskii,
M.D. et al. (1994) Stability of peptide nucleic acids
in human serum and cellular extracts. Biochem.
Pharmacol., 48, 1309-1313.
Egholm, M., Buchardt, O., Christensen, L. et al. (1993)
PNA hybridizes to complementary oligonucleotides
obeying the Watson-Crick hydrogen-bonding rules.
Nature, 365, 566-568.
Good, L. and Nielsen, P.E. (1998) Antisense inhibition
of gene expression in bacteria by PNA targeted to
mRNA. Nature Biotechnol, 16, 355-358.
Gross, N.J., Getz, G.S. and Rabinowitz, M. (1969)
Apparent turnover of mitochondrial deoxyribonucleic
acid and mitochondrial phospholipids in the tissues
of the rat. J. Biol. Chem., 244, 1552-1562.
Hanvey, J.C., Peffer, N.J., Bisi, J.E. et al. (1992)
Antisense and antigene properties of peptide nucleic
acids. Science, 258, 1481-1485.
Holt, I.J., Harding, A.E. and Morgan-Hughes, J.A.
(1988) Deletions of muscle mitochondrial DNA in
84
patients with mitochondrial myopathies. Nature, 331,
717-719.
Jensen, K.K., 0rum, H., Nielsen, P.E. et al. (1997)
Kinetics for hybridization of peptide nucleic acids
(PNA) with DNA and RNA studied with the
BIAcore technique. Biochemistry, 36, 5072-5077.
Johns, D.R. (1995) Mitochondrial DNA and disease.
N. Engl. J. Med., 333, 638-644.
Knudsen, H. and Nielsen, P.E. (1996) Antisense
properties of duplex- and triplex-forming PNAs.
Nucleic Acids Res., 24, 494-500.
Lightowlers, R.N., Chinnery, P.E, Turnbull, D.M. et al.
(1997) Mammalian mitochondrial genetics: heredity,
heteroplasmy and disease. Trends Genet., 13, 450455.
Manfredi, G., Gupta, N., Vazquez-Memije, M.E. et al.
(1999) Oligomycin induces a decrease in the cellular
content of a pathogenic mutation in the human
mitochondrial ATPase 6 gene. J. Biol. Chem., 274,
9386-9391.
Mita, S., Rizzuto, R., Moraes, C.T. et al. (1990)
Recombination via flanking direct repeats is a major
cause of large-scale deletions of human mitochondrial
DNA. Nucleic Acids Res., 18, 561-567.
Moraes, C.T., DiMauro, S., Zeviani, M. et al. (1989)
Mitochondrial DNA deletions in progressive external
ophthalmoplegia and Kearns-Sayre syndrome.
N. Engl. J. Med., 320, 1293-1299.
Nielsen, P.E., Egholm, M., Berg, R.H. et al. (1991)
Sequence-specific recognition of DNA by strand
displacement with a thymine-substituted polyamide.
Science, 254, 1497-1500.
0rum, H., Nielsen, P.E., Egholm, M. et al. (1993)
Single base pair mutation analysis by PNA directed
PCR clamping. Nucleic Acids Res., 21, 5332-5336.
Rotig, A., Cormier, V., Koll, F. et al. (1991) Sitespecific deletions of the mitochondrial genome in
the Pearson marrow-pancreas syndrome. Genomics,
10, 502-504.
Schon, E.A., Rizzuto, R., Moraes, C.T. et al. (1989)
A direct repeat is a hotspot for large-scale deletion of
human mitochondrial DNA. Science, 244, 346-349.
Schon, E.A., Bonilla, E. and DiMauro, S. (1997)
Mitochondrial DNA mutations and pathogenesis.
J. Bioenerg. Biomembr., 29, 131-149.
Seibel, P., Trappe, J., Villani, G. et al. (1995)
Transfection of mitochondria: strategy towards a
gene therapy of mitochondrial DNA diseases. Nucleic
Acids Res., 23, 10-17.
Servidei, S. (1998) Mitochondrial encephalomyopathies: gene mutation. Neuromuscul. Disord., 8,
8-11.
Shoubridge, E,A, Johns, T. and Karpati, G. (1997)
Complete restoration of a wild-type mtDNA genotype
in regenerating muscle fibres in a patient with a
In-vitro manipulation of mtDNA heteroplasmy
tRNA point mutation and mitochondrial encephalomyopathy. Hum. Mol. Genet, 13, 2239-2242.
Taylor, R.W., Chinnery, P.F., Clark, K. et al. (1997a)
Treatment of mitochondrial disease. J. Bioenerg.
Biomemb., 29, 195-205.
Taylor, R.W., Chinnery, P.F., Turnbull, D.M. et al.
(1997b) Selective inhibition of mutant human
mitochondrial DNA replication in vitro by peptide
nucleic acids. Nature Genet., 15, 212-215.
Vestweber, D. and Schatz, G. (1989) DNA-protein
conjugates can enter mitochondria via the protein
import pathway. Nature, 338, 170-172.
Wallace, D.C. (1999) Mitochondrial diseases in man
and mouse. Science, 283, 1482-1488.
Wittung, P., Kajanus, J., Edwards, K. et al. (1995)
Phospholipid membrane permeability of peptide
nucleic acid. FEBS Lett., 375, 27-29.
85