MOLECULAR MEDICINE TODAY, JANUARY 1998 Reviews

Towards gene therapy of

mitochondrial disorders
Jean-Marc Collombet and Charles Coutelle

Mitochondrial disorders are characterized by protein deficiencies affecting the structure and function of
mitochondria. The protein deficiencies are caused by mutations either in a nuclear gene or in the
mitochondrial genome. Most current approaches to gene therapy of mitochondrial diseases aim at
expression of the corrective gene sequence by nuclear/cytoplasmic expression. However, the
mitochondrial genome and its autonomous expression system offer the potential of an alternative gene
therapy strategy: the introduction of nuclear gene sequences into the mitochondrial genome and their
expression by the mitochondrial gene expression system. In addition to its potential for gene therapy, the
introduction and expression of an exogenous gene in mitochondria would provide an invaluable tool
towards the understanding of mitochondrial genome expression and its regulation.

MITOCHONDRIA are small organelles (0.5–1 µm) located in the

cytoplasm of all eukaryotic cells (reviewed in Ref. 1). They are re-
sponsible for the generation of ~90% of the total ATP supply of a
cell, the main energy source on which cell life depends. The number
of mitochondria per cell (10–2000 in somatic cells and up to
100 000 in oocytes) is dependent on the energetic requirements of
the respective organ. Metabolically very active organs such as the
liver, the brain and skeletal muscles possess the largest number of
mitochondria and are the organs primarily affected by mitochondrial
pathologies.
Mitochondria contain their own genome, the mitochondrial DNA
(mtDNA). Each mitochondrion has 2–10 copies of mtDNA, which
codes for some of the proteins involved in ATP synthesis as well as
for mitochondrial ribosomal RNA (rRNA) and transfer RNA
(tRNA). The presence of an autonomously replicating and express-
False coloured immunoelectron micrograph of a mitochondrion showing internalization of a

ing genome in mitochondria has been explained by endocytosis of

Jean-Marc Collombet PhD
Research Associate
Charles Coutelle* MD, DSc
Professor of Gene Therapy
plasmid after electroporation. (Modified from Ref. 41.)
prokaryotes into primitive eukaryotes followed by the development
of a symbiotic relationship2. However, mitochondria have lost much
of their autonomy during evolution as a consequence of the decrease
of their genome size2. They are completely dependent on proteins
encoded by the nuclear genome, which are synthesized in the cell
cytoplasm and are transferred into the mitochondria by a specific

Imperial College–School of Medicine,
Section of Molecular Genetics,
Division of Biomedical Sciences,
St Mary’s Hospital,
Norfolk Place,
London UK W2 1PG.
Tel: +44 171 594 3797
Fax: +44 171 706 3272
*e-mail: c.coutelle@ic.ac.uk
import system3.
Mitochondria are composed of two highly specialized structures:
the external and the internal membranes, which divide them into two
compartments – the intermembrane space and the matrix.
The main constituents of the external membrane are channels that
facilitate the transport across the membrane of molecules with a
molecular mass of up to 10 kDa. This membrane also contains en-
zymes that convert lipids into substrates for the metabolic cycles
located in the mitochondrial matrix.
The internal membrane is tightly folded to create cristae that in-
crease the membrane surface. It is impermeable to ions, a property

Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1357 - 4310/98/$19.00 PII: S1357-4310(97)01146-5 31
Reviews MOLECULAR MEDICINE TODAY, JANUARY 1998

that is essential for maintaining an electrochemical

gradient for the functioning of ATPase/ATP synthase,
an enzyme located in the internal membrane and
which produces ATP by oxidative phosphorylation.
Two other types of proteins are integral components of
the internal membrane: specific channels that regulate
substrate transfer across the membrane, and proteins
of the respiratory chain (complexes I, II, III and IV).
The latter reoxidize the two coenzymes NADH + H+
and FADH2, which are located in the matrix and per-
mit electron transfer and proton pumping to create the
electrochemical gradient.
The matrix contains enzymes involved in the oxi-
dation of pyruvate and fatty acids, the tricarboxylic
acid cycle and the urea cycle. It also contains the
mtDNA, mitochondrial polysomes, tRNA and various
enzymes needed for the autonomous replication, tran-
scription and translation of the mtDNA.

The mitochondrial genome

Figure 1. The human mitochondrial genome, genetic and transcription map. HSP, promoter for
heavy (H) strand transcription; LSP, promoter for light (L) strand transcription; OH and OL, origins
of replication for H and L strands, respectively; D-loop, displacement loop; 12S and 16S rRNA,
mitochondrial 12S and 16S ribosomal RNAs; ND 1 to ND 6, genes encoding complex I sub-
units; Cyt b, cytochrome b of complex III; COX I to COX III, genes encoding complex IV sub-
units; ATPase 8 and ATPase 6, genes encoding ATPase/ATP synthase; F, V, L, I, M, W, D, K, G,
R, H, S, T, Q, A, N, C, Y, E, P, transfer RNAs using single letter amino-acid abbreviations.
Numbered portions of the genome represent transcripts of H and L strands according to Ref. 4.
Several mammalian mitochondrial genomes have been
sequenced5. They are circular, double-stranded DNA
with a size of 16–17 kb. The origins of replication for
the two strands have different locations on this DNA.
The mitochondrial genome harbours 37 genes. They en-
code the two ribosomal RNAs, 22 transfer RNAs and
13 remaining genes coding for subunits of several com-
plexes of the mitochondrial respiratory chain; 28 of
these genes are located on the H strand of the mtDNA,
among them 12 of the 13 genes that encode protein sub-
units. The remaining genes are located on the L strand.
The presence of genes on both DNA strands implies
that both strands are transcribed (Fig. 1).
The mtDNA is organized in contiguous genes without
introns. Intergenic sequences are rare. The largest one,
the D-loop (displacement loop), which is about 1 kb
long, contains several regions regulating the replication

Box 1. Organ manifestations of mitochondrial disorders

Skeletal muscle Endocrine glands
Progressive external ophthalmoplegia; proximal myopathy; intolerance to Diabetes mellitus; hypothyroidism; hypoparathyroidism; adrenal
exercise; hypotonia; pulmonary insufficiency insufficiency; sexual and growth retardation; sterility

Heart Kidney
Hypertrophic myocardia; disorder of impulse conduction Tubulopathy: glycosuria; aminoaciduria

Central nervous system Digestive apparatus

Cerebellar ataxia; epilepsy; myoclonic convulsions; mental retardation; Liver: hepatic insufficiency
dementia; ischaemia
Pancreas: insufficiency of exocrine glands
Neurosensory pathways
Optic atrophy; retinitis pigmentosa; perceptive deafness Intestine: malabsorption; diarrhoea

Peripheral nervous system Blood

Peripheral neuropathy Pancytopenia; anaemia

32
MOLECULAR MEDICINE TODAY, JANUARY 1998 Reviews

and transcription of the mitochondrial genome6.

Several features distinguish the mitochondrial genome
from the nuclear genome: (1) mtDNA is maternally in-
herited, being transmitted with the oocyte cytoplasm;
the very low amount of paternal mitochondria con-
tained in spermatozoids, which might even be elimi-
nated in the embryo7, has an insignificant effect on the
mitochondrial content in the offspring. (2) The mito-
chondrial genome is not replicated in synchrony with
cell division. (3) The mammalian mitochondrial gen-
etic code differs from the universal genetic code in four
codons (UGA: Stop → Trp; AUA: Ile → Met; AGA:
Arg → Stop; AGG: Arg → Stop)8. (4) The high fre-
quency of mutations observed in the mitochondrial
genome (10–100 times more than in the nuclear
genome) is probably the result of the lack of histones
and their protective effect, and perhaps the absence of
an effective and accurate DNA repair system9.
However, the latter is still controversial10. (5) In con-
trast to inherited genetic disorders caused by mutations
in the nuclear genome which are present in every cell
of the individual, patients with mitochondrial disease
rarely have only mutant mtDNA (homoplasmy). In
general, wild-type and mutant mtDNA coexist in their
cells (heteroplasmy), and the amount of both types Figure 2. The location of the most common point mutations, deletions or duplications of the
varies between tissues and cells throughout life, owing mitochondrial genome responsible for disorders. Point mutations are indicated by an arrow at
to the random distribution of mitochondria into the the nucleotide position in the mitochondrial genome according to the numbering in Ref. 4.
Numbers in parentheses give the position of less frequent mutations. Names of associated
daughter cells during cell division11. For each tissue, a syndromes in parentheses: LHON, Leber’s hereditary optic neuropathy; NARP, neurogenic
different threshold of mutant mtDNA has to be reached muscle weakness, ataxia and retinitis pigmentosa; MERRF, myoclonic epilepsy and ragged red
before the pathological phenotype caused by the mu- fibre; MELAS, mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes; PEO,
tation becomes manifest12. progressive external ophthalmoplegia; AID, aminoglycoside-induced deafness. 1 and 2 indicate
the region of the common 4997 and 7436 bp deletions, respectively; 3 indicates the region of
deletion for autosomal-dominant multiple deletion syndrome and for most of the spontaneous
Mitochondrial disorders deletions cases.
Traditionally, mitochondrial diseases have been de-
fined as inherited conditions caused by deficiencies of
proteins that function in mitochondria (reviewed in
Refs 13,14). However, more recently, acquired mitochondrial mu- However, mutations or deletions of the mitochondrial genome are
tations have also been recognized as contributing to degenerative also responsible for a number of mitochondrial myopathies caused
diseases15. by respiratory chain deficiencies14.
Patients with mitochondriopathies present with a variety of clinical Three types of mutations in the mitochondrial genome have been
symptoms, and the organ manifestation depends not only on the tissue observed.
expression of mutated genes but also on the energetic requirements of
the respective tissue. The organs usually affected are the heart, the Point mutations
skeletal muscle and the central nervous system, but other organs can Point mutations occur in mitochondrial tRNA and rRNA genes, and
also be involved either alone or in combination (see Box 1). in protein-coding genes (Fig. 2). Mutations in genes encoding tRNA
Mitochondrial diseases can be classified biochemically into three [associated with myoclonic epilepsy and ragged red fibre (MERRF)
categories: (1) deficiencies of enzymes involved in substrate transport and mitochondrial encephalomyopathy with lactic acidosis and
across mitochondrial membranes; (2) deficiencies of enzymes of the stroke-like episodes (MELAS)] and rRNA [associated with amino-
mitochondrial metabolic cycles; and (3) deficiencies of the respiratory glycoside-induced deafness (AID)] are maternally inherited and lead
chain, which mainly cause mitochondrial myopathies. Different mu- to inefficient translation of mitochondrial mRNAs. Mutations in
tations in the nuclear genome, some of which have been identified, protein-coding genes [associated with Leber’s hereditary optic neu-
are responsible for the first two categories of defects13. ropathy (LHON) and neurogenic muscle weakness, ataxia and reti-
Several deficiencies of the respiratory chain have been observed nitis pigmentosa (NARP)] are responsible for amino acid substitu-
(reviewed in Ref. 14). They are (1) single-complex deficiencies found tions in the protein sequence and are also maternally inherited.
in the following decreasing order of frequency: complex I ⬎ IV ⬎ III, Mutations in the ATPase 6 gene are also maternally inherited in
II and V; (2) combined deficiencies (in general complexes I and IV); Leigh syndrome. However, this syndrome has also recently been re-
and (3) more rarely, generalized deficiencies of the respiratory lated to a complex II deficiency of nuclear origin16.
chain. Some of the respiratory chain deficiencies, such as that of
complex II, are the result of mutations of the nuclear genome16. Deletions

33
Reviews
Figure 3. Strategies for gene therapy of mitochondrial disorders. (a) For a mi-
tochondrial protein produced by nucleus/cytoplasmic expression, standard
methods of gene transfer to the nucleus are used and the protein (containing
a mitochondrial targeting peptide) is cytoplasmically synthesized before se-
lective import into the mitochondrion. (b) The concept of using mitochondrial
DNA (mtDNA) as a gene therapy vehicle involves the manipulation of mtDNA
to contain the gene of interest constructed according to mitochondrial codon
usage, the transfer of this DNA into mitochondria and the subsequent trans-
fer of these mitochondria into cells. Mitochondrial (mt) gene expression
should then lead to production of the respective protein which should func-
tion in the mitochondrion.
Deletions of mtDNA are found in patients with ocular myopathies,
Kearns–Sayre syndrome, Pearson disease and Wolfram syndrome.
These deletions usually occur in a particular region of the mtDNA
bordered by the beginning of the COX I gene (encoding the complex
I subunits) and the end of the cyt b gene (encoding cytochrome b).
The size of these deletions varies between 1 and 8 kb. More than
120 different deletions have been classified. Two of them are called
‘common’ because they are present in ~ 60% of patients affected by
deletions. The size of these deletions is 4997 and 7436 bp, respec-
tively (Fig. 2). Deletions are, in most cases, sporadic. Only a few
cases of maternal inheritance have been reported. Recessive and
dominant Mendelian inheritance has been shown in progressive ex-
ternal ophthalmoplegia (PEO); patients harbouring multiple deletions
spanning 2.0–10.0 kb and at least two nuclear loci have been related
to their presence17,18. It has been suggested that they encode nuclear
factors that are imported into mitochondria and play an important
role in mitochondrial replication. Mutation or absence of these fac-
tors could cause deletions by faulty mtDNA replication19.
Duplications
34
MOLECULAR MEDICINE TODAY, JANUARY 1998
Glossary
Artificial chromosome – A minimal episomally stable gene delivery
construct containing telomeres, origins of DNA replication and el-
ements for centromere function. It can replicate and segregate
during cell division as an independent entity and can carry large
genomic fragments of the gene of interest into target cells.
Biolistic bombardment – Physical means of gene delivery to cells
by high-speed bombardment (particle gun) of cells with tungsten
microparticles coated with the DNA of interest.
Cytoplast fusion – Cytoplasts are cells enucleated by a combi-
nation of cytochalasin treatment and centrifugation. When cytoplasts
are fused with nucleated cells, they transfer their mitochondria to the
other cell.
Heteroplasmy – Describes cells containing both mutant and wild-
type mitochondrial genome.
Histones – A group of highly positively charged nuclear proteins.
They are the main protein component of the nucleus and play a
major role in DNA packaging to chromatin structures.
Homoplasmy – Describes cells containing only wild-type or mutant
mitochondrial genome.
In organello transcription/translation – Because of the specific
features of mitochondrial gene expression, mitochondrial in vitro
transcription/translation has to be performed with intact isolated
mitochondria.
Leader peptide – A short amino acid sequence located at the N-
terminus of some immature proteins. It has the ability to target pro-
teins to their intracellular site of function such as mitochondria or the
endoplasmic reticulum. The leader peptide is cleaved off in the
mature functional protein.
Mega-mitochondria – Large mitochondria up to about six times the
size of normal mitochondria, generated in vivo by feeding mice with
cuprizone (bis-cyclohexanone oxaldihydrazone).
Non-viral vectors – These avoid the toxicity/immunogenicity and
possible insertion mutagenesis/oncogenesis of viral vectors. Cationic
liposomes with the ability to bind DNA and to transfer it into cells by
endocytosis/phagocytosis are the most widely used.
Viral vectors – Recombinant viruses that are usually rendered
replication-deficient by deletion of essential parts of the wild-type
genome, which are replaced by an expression cassette containing
the therapeutic gene.
Duplications of mtDNA have also been reported. Partial dupli-
cations arise by insertion of a deleted mtDNA molecule into a nor-
mal mtDNA molecule. In tandem duplications, two deleted mtDNA
form a dimer. Duplicated mtDNA of varying size between 23 and
26.5 kb could, in some cases, represent an intermediate state for the
formation of deleted mtDNA molecules20.
Current treatments for mitochondrial disorders
At present, there is no efficient therapy for most mitochondrial dis-
orders. The main approach that has been used to date is the appli-
cation of drug treatment to bypass the metabolic defect or to favour
MOLECULAR MEDICINE TODAY, JANUARY 1998 Reviews

alternative metabolic pathways. For ex-

ample, sodium benzoate or sodium
phenylacetate is given to patients with en-
zyme defects located in the urea cycle in
order to develop an alternative route for
the excretion of excess metabolic nitro-
gen21. For mitochondrial myopathies,
electron acceptors such as vitamin C,
menadione, riboflavin or coenzyme Q10
are used to compensate for the deficient
respiratory chain complexes19. This im-
proves the patient’s condition in the short
term but cannot stop disease progression.
In severe cases, organ transplantation has
been attempted: for example, liver
transplantation for ornithine transcar-
bamoylase (OTC) deficiency21 and heart
transplantation for cardiomyopathy22.
Table 1. Viral vector systems used for gene therapy approaches
to mitochondrial diseases
Property Adenovirus
Suitability for in vivo application Very high virus titres (up to 10 10
infectious units ml⫺1)
Transduces a broad variety of
dividing and post-mitotic cells
Capacity Can package ~38 kb of DNA:
capacity for ~8 kb of foreign
DNA
Safety considerations Virus proteins cause immune
and inflammatory reactions:
repeated application limited
Retrovirus
Relatively low virus titre
(107 infectious units ml⫺1)
Virus not very stable
Infects only dividing cells
Small and simple genome:
capacity for ~10 kb of foreign
DNA
Random integration into host
genome

Gene therapy for mitochondrial
disorders
Gene therapy can provide strategies for a
causal treatment of such pathologies.
This approach can be defined as the de-
livery of the functional homologue of a
defective gene to a somatic cell in order
to correct a deficient cellular function. Ideally, this should lead to
persistent expression of the therapeutic gene at physiological levels
in the relevant cells after a single application. Obtaining this goal by
homologous recombination in vivo is currently not feasible for tech-
nical reasons. However, several alternative gene therapy strategies
have been developed that are also applicable to some mitochondrial
pathologies (Fig. 3).
Gene delivery to the cell nucleus
To correct a deficiency caused by a mutation in a nuclear gene the ob-
vious strategy is to complement the defective gene with a gene therapy
vector expressing the correct gene after reaching the cell nucleus. In
this case, the transgene is transcribed in the nucleus and the transcript
transported to the cytosol for translation. The resulting normal protein
is selectively imported into the mitochondria by the natural import
mechanism based on the presence of a leader peptide (about 20 amino
acid residues located N-terminal to the sequence of the active protein),
which is later cleaved off inside the mitochondrial matrix3.
This approach follows the ‘classical’ gene therapy strategies de-
veloped for nuclear encoded genes, and the choice of the vector sys-
tem carrying the therapeutic gene defines the efficiency of transfec-
tion, the level and duration of expression of the therapeutic gene and
possible side effects of the system. Recombinant viral vectors (see
Table 1) and non-viral delivery systems can be used for this ap-
proach. Non-viral vectors are expected to be less immunogenic and
safer than viral vectors. However, they are also still much less effi-
cient for gene delivery and are currently mainly used for the transfer
of plasmids which are not maintained in dividing cells and thus ne-
cessitate repeated applications. It is therefore no surprise that non-
viral vectors have not been tried in gene therapy approaches for
mitochondrial diseases.
Because of the low number of known nuclear genes encoding mi-
tochondrial proteins and the absence of animal models for mito-
No integration into host genome No toxic effects on host cells
No insertion mutagenesis but only Potential for long-term
transient expression expression, but also potentially
mutagenic/oncogenic
chondrial diseases, only a few gene therapy experiments have been
attempted to correct mitochondrial pathologies. However, two mouse
models for OTC deficiency, sparse-fur (Spf) and Spf–ash (where ash
stands for abnormality of skin and hair) mice23,24, have provided the
basis for gene therapy approaches to this disease using adenoviral
and retroviral vectors, demonstrating the proof of principle of gene
therapy for chromosomally inherited mitochondrial diseases.
Intravenous injection of a first-generation recombinant adenovirus
vector containing the rat OTC cDNA into neonatal Spf–ash mice pro-
duced varying increases in hepatic OTC activity in some animals.
The virally transferred OTC gene was still expressed in one mouse
15 months after viral administration, revealed by a hepatic OTC
activity of 50% of normal levels and normalization of the sparse-fur
phenotype that is characteristic of the OTC-deficient mice25.
Using a second-generation adenovirus–OTC construct, Ye et al.26
achieved correction of the metabolic defect in adult Spf mice for up
to 2 months. A partial biochemical correction of OTC deficiency was
also obtained in primary hepatocytes from Spf and Spf–ash mice
after transduction with a retroviral vector containing the human OTC
cDNA (Ref. 27).
Deficiencies of branched-chain keto acid dehydrogenase
(BCKDH) and ornithine aminotransferase (OAT) have also been ap-
proached by nuclear expression strategies. Mueller et al.28 obtained
the total correction of BCKDH deficiency in cultured fibroblasts
after infecting these cells with a retrovirus expressing gene se-
quences encoding the E2 subunit of the BCKDH multi-enzyme com-
plex; 75% of the wild-type activity was still found in cultured fibrob-
lasts at least 7 weeks after transduction. Sullivan et al.29 have
overexpressed OAT to more than 150 times the wild-type activity in
primary cultures of human retinal pigment epithelium using a first-
generation adenovirus vector.
These ‘conventional’ approaches to gene therapy could also be
used to correct mitochondrial diseases caused by a single mutation

35
Reviews
The outstanding questions
Short term
• Can the size and amount of exogenous DNA transferred into
mitochondria be increased?
• Can exogenous gene sequences be correctly expressed in
mitochondria?
Medium term
• Is it possible to introduce mitochondria containing exogen-
ous DNA sequences into cells in vitro and in vivo and thereby
correct mitochondriopathies?
Long term
•release
Can mitochondria be used as vector systems to produce and
proteins into the cytoplasm?
in a protein-coding gene of the mitochondrial genome, such as
LHON or NARP. In such cases, the difference in the genetic code
between nucleus and mitochondrion has to be taken into account for
the construction of the corrective gene sequence. With the great ad-
vances in oligonucleotide synthesis and PCR technology, this prob-
lem has now been solved in principle30. In addition, a sequence en-
coding a specific leader peptide, necessary for the import of the
therapeutic protein into mitochondria, would have to be added to the
5' region of the therapeutic gene.
Nagley et al.31 have demonstrated the potential of this approach
for correction of mtDNA defects in mutant yeast with a deletion
of the ATPase 8 gene. After expression in the yeast nucleus of a
mitochondrial ATPase 8 gene modified according to the above-
mentioned conditions, a functional ATPase 8 subunit was synthe-
sized in the cytosol and imported into mitochondria, correcting the
ATPase defect in this organism.
Gene delivery to the mitochondrion
Because the mammalian mitochondrial genome is essentially a
multi-copy episome present in all cell types, we and others have ad-
dressed the question of whether this genome could be manipulated
in order to correct a mitochondrial mutation or even express a for-
eign gene inside the mitochondrion. As mitochondria possess their
own machinery for replication, transcription and translation, an ex-
ogenous gene inserted into the mtDNA could be stably propagated
and expressed without integration into the nuclear genome.
Furthermore, if mitochondria could be exploited for this purpose,
perhaps they could also serve as a non-pathogenic, non-immuno-
logic delivery system. However, despite the small size of the mito-
chondrial genome, progress in this area is lagging far behind the
advances in nuclear gene transfer.
Probably the most important challenge at present is the develop-
ment of procedures for the introduction of DNA into mitochondria.
Ideally this should be achieved with intact cells or even tissue.
However, the mitochondrial double membrane has made this a chal-
lenging task, even with the isolated organelle. As outlined above,
these membranes present strict physical and functional barriers,
which are traversed naturally only by selective active transport pro-
cesses. If other means of entry are employed, the internal membrane
has to be crossed without loss of the coupling state that is essential
for mitochondrial function.
36
MOLECULAR MEDICINE TODAY, JANUARY 1998
Natural import mechanisms for nucleic acids are only known for
a few RNA molecules. tRNALys has been shown in vivo and in vitro
to be selectively imported into yeast mitochondria; this transport re-
quires the presence of at least two cytoplasmic proteins in the in
vitro system32. A similar mechanism in mammalian cells is unknown
but a nuclear encoded 135-nucleotide RNA (MRP) is thought to be
required for mammalian mitochondrial RNA processing33 and
would, therefore, have to be imported into the mitochondria.
However, this is still controversial34,35 and, because the mechanisms
of such import processes are still unknown, their use for the import
of foreign nucleic acids is currently not feasible.
Various techniques for the introduction of foreign DNA into mi-
tochondria have been tested. Early attempts to transfer liposome-
encapsulated fluorescence-labelled phage DNA into the ‘mega’-
mitochondria of mouse liver cells by injection into the tail vein36
have not been continued, probably because of the low efficiency of
this procedure.
Biolistic bombardment
Several investigators have successfully introduced foreign DNA
into mitochondria and chloroplasts, respectively, of yeast,
Chlamydomonas and plants by biolistic bombardment of whole cells
with DNA-coated tungsten particles of ~0.5–1 ␮m (reviewed in
Ref. 37). Although not without problems, this has led to restoration
of function in these organelles, but it has not been successful in
mammalian cells or isolated mammalian mitochondria.
Hijacking the protein import pathway
Co-transport of DNA with nuclear coded proteins, which are naturally
imported into the mitochondria, or the use of their mitochondrial-tar-
geting signal peptides, is a more physiological approach to this prob-
lem. Oligonucleotides (24 nucleotides) can be transferred in vitro into
isolated yeast mitochondria as conjugates with a fusion protein con-
taining a mitochondrial presequence (derived from the yeast cy-
tochrome oxidase subunit IV precursor) and a modified nuclear en-
coded enzyme (mouse dihydrofolate reductase)38. In an extension
of this approach, Seibel et al.39 have introduced a 320 bp
sequence of foreign DNA covalently linked to an oligopeptide N-ter-
minal leader sequence of OTC into rat mitochondria; 37% of this con-
jugate was completely translocated into the matrix. Effective cleavage
of the presequence from the exogenous DNA by the mitochondrial
processing peptidase (MPP)3 after translocation might be essential to
allow its efficient replication and transcription, and the linear confor-
mation of the DNA in this construct could also pose a problem for its
replication and expression in the mitochondria. The authors speculate
that this approach could be used in combination with liposome-medi-
ated transfer of the DNA–protein conjugate into cells. This would,
however, substantially decrease the efficiency of the whole procedure.
Taylor et al.40 have recently suggested the use of this method for
the introduction of antisense protein nucleic acid oligomers into
mitochondria to selectively inhibit replication of defective mitochon-
drial genomes in mitochondrial diseases caused by a heteroplasmic
mtDNA defect.
Electroporation
An electroporation approach to introduce exogenous plasmid DNA
of 7.2 kb into the matrix compartment of isolated mitochondria has
recently been developed in our laboratory. Fully functional DNA
can be introduced into intact mitochondria (as shown by enzymatic
MOLECULAR MEDICINE TODAY, JANUARY 1998
assays of marker enzymes, measurement of the mitochondrial
coupling state and electron microscopy)41. We have also recently
inserted a DNA sequence, coding for human OTC according to mito-
chondrial codon usage, into the mitochondrial genome between two
contiguous tRNAs, giving it the potential to be processed correctly
from the primary mitochondrial transcript42. As with the above-
described procedure for introduction of DNA into mitochondria by
peptide targeting, the only criterion by which to judge the future
potential of this method for gene therapy is function. We are there-
fore currently working on introducing this construct into isolated
mitochondria and testing its expression in an in organello transcrip-
tion–translation system. If successful expression of the
introduced DNA sequence can be shown, the next step would be
the introduction of the manipulated mitochondria into cells.
Introduction of ‘therapeutic’ mitochondria into cells
It should be possible to microinject mitochondria43 containing this
modified genome into OTC-deficient hepatocytes and oocytes from
spf or spf–ash mice23,24, providing the first model system for in vitro
and in vivo correction of mitochondrial disease by manipulation of
the mitochondrial genome.
Another means for the introduction of in vitro manipulated mito-
chondria into cells could be endocytosis44. Such a procedure could
perhaps be enhanced by coating the mitochondria with basic peptide
sequences45 carrying a targeting ligand. We have used such a con-
struct for receptor-mediated gene delivery and have demonstrated
that an integrin-targeting ligand allows the effective intracellular up-
take of a bacteriophage into mammalian cells46.
More recently, Kagawa and Hayashi47 have shown the correction
of cells with a model of mitochondrial deficiency by transfer of nor-
mal mitochondria through cytoplast fusion in vitro. They suggest
that it might be possible to use this technique as a therapeutic strat-
egy in vivo by infusing cytoplasts containing normal mitochondria.
These cytoplasts could be generated from the patient’s heteroplas-
mic cells by in vitro selection for normal mitochondria followed by
treatment with cytochalasin. The cytoplasts should fuse with the af-
fected cells, complementing them with normal mitochondria to a
level above the threshold needed to prevent disease manifestation.
Concluding remarks
The expression of a foreign DNA sequence in isolated mammalian
mitochondria would open the way to new experimental approaches
in the understanding and manipulation of mitochondrial genome ex-
pression and its regulation, which could lead to strategies for a gen-
etic correction of mitochondrial disease. Achieving this goal and/or
the efficient introduction of whole mitochondria into somatic target
cells in vivo would be the final requirement for application to gene
therapy. There is still a very long way to go. However, the introduc-
tion of the whole manipulated organelle is, in principle, similar to
the problem of in vivo delivery of artificial chromosome constructs.
If solved, again in analogy to the artificial chromosome system, it
should be possible to begin to address the multitude of problems that
will have to be solved for the use of mitochondria and their gene ex-
pression system, not only for the correction of mitochondrial disease
but perhaps also as a general stable and non-integrating vector for
therapeutic gene delivery.
Acknowledgements. This work is supported by the Muller-Bequest, the UK Medical
Research Council and the Association Française contre les Myopathies (AFM). The
Reviews
authors thank Patrick Mazoyer (Wellcome Department of Cognitive Neurology, London,
UK) for his helpful contribution to the drawing of the figures.
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04 Anderson, S. et al. (1981) Sequence and organisation of the human mitochon-
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05 Bibb, M.J. et al. (1981) Sequence and gene organization of mouse mitochondr-
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