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Gene therapy for the mitochondrial genome: Purging mutations, pacifying ailments
Accepted Manuscript Gene therapy for the mitochondrial genome: Purging mutations, pacifying ailments
M. Aravintha Siva, R. Mahalakshmi, Dipita Bhakta-Guha, Gunjan Guha PII: DOI: Reference:
S1567-7249(18)30018-7 doi:10.1016/j.mito.2018.06.002 MITOCH 1292
To appear in:
Mitochondrion
Received date: Revised date: Accepted date:
27 January 2018 24 May 2018 7 June 2018
Please cite this article as: M. Aravintha Siva, R. Mahalakshmi, Dipita Bhakta-Guha, Gunjan Guha , Gene therapy for the mitochondrial genome: Purging mutations, pacifying ailments. Mitoch (2017), doi:10.1016/j.mito.2018.06.002
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ACCEPTED MANUSCRIPT Gene Therapy for the Mitochondrial Genome: Purging Mutations, Pacifying Ailments M. Aravintha Siva, R. Mahalakshmi, Dipita Bhakta-Guha* and Gunjan Guha* Cellular Dyshomeostasis Laboratory (CDHL), School of Chemical and Bio Technology, SASTRA University, Thanjavur 613 401. Tamil Nadu, India. *
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Corresponding authors:
Gunjan Guha
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CDHL, SCBT, SASTRA University, Thanjavur 613 401. Tamil Nadu, India. Email: [email protected]; [email protected] Phone: +91 4362 264101-108 (ext: 3777); +91 4362 304000-010 (ext: 3777).
Dipita Bhakta-Guha
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CDHL, SCBT, SASTRA University, Thanjavur 613 401. Tamil Nadu, India. Email: [email protected]; [email protected] Phone: +91 4362 264101-108 (ext: 3777); +91 4362 304000-010 (ext: 3777).
ACCEPTED MANUSCRIPT Abstract In the recent years, the reported cases of mitochondrial disorders have reached a colossal number. These disorders spawn a sundry of pathological conditions, which lead to pernicious symptoms and even fatality. Due to the unpredictable etiologies, mitochondrial diseases are putatively referred to as “mystondria” (mysterious diseases of mitochondria). Although present-day research has greatly improved our understanding of mitochondrial disorders,
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effective therapeutic interventions are still at the precursory stage. The conundrum becomes further complicated because these pathologies might occur due to either mitochondrial DNA
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(mtDNA) mutations or due to mutations in the nuclear DNA (nDNA), or both. While correcting nDNA mutations by using gene therapy (replacement of defective genes by delivering wild-
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type (WT) ones into the host cell, or silencing a dominant mutant allele that is pathogenic) has emerged as a promising strategy to address some mitochondrial diseases, the complications in
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correcting the defects of mtDNA in order to renovate mitochondrial functions have remained a steep challenge. In this review, we focus specifically on the selective gene therapy strategies
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that have demonstrated prospects in targeting the pathological mutations in the mitochondrial genome, thereby treating mitochondrial ailments.
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Keywords: Mitochondrial diseases; mystondria; mtDNA mutations; gene therapy.
ACCEPTED MANUSCRIPT Abbreviations 3′-UTR
3′-untranslated region
AAV
adeno-associated virus
AAV2/2
AAV serotype 2
AIF apoptosis-inducing factor COX
cytochrome c oxidase
COI cytochrome c oxidase subunit I cytochrome c oxidase subunit II
COIII
cytochrome c oxidase subunit III
COIV
cytochrome c oxidase subunit IV
COVIII
cytochrome c oxidase subunit VIII
CO-X
cytochrome c oxidase subunit X
CPEO
chronic progressive external ophthalmoplegia
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COII
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CRISPR/Cas9 clustered regularly-interspaced short palindromic repeats/CRISPRassociated protein 9 DA dopaminergic displacement loop
DQA
dequalinium chloride
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D-loop
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DF-MITO-porter dual function MITO-porter
DQAsomes DQA-based liposome-like nanovesicles enhanced green fluorescent protein
ETC
electron transport chain
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eGFP
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FLAG epitope tag DYKDDDDK sequence (where D = aspartic acid, Y = tyrosine, K = lysine) familial bilateral striatal necrosis
GFP
green fluorescence protein
HSP KSS LHON
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FBSN
heat shock protein Kearns-Sayre syndrome Leber’s hereditary optic neuropathy
LS Leigh syndrome MELAS
mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
MEMRI
manganese-enhanced magnetic resonance imaging
MEPR
myoclonic epilepsy and psychomotor regression
MERRF
myoclonic epilepsy with ragged-red fibers
MHCM
maternally inherited hypertrophic cardiomyopathy
MICM
maternally inherited cardiomyopathy
MID
maternally inherited deafness
ACCEPTED MANUSCRIPT MIDD
maternally inherited diabetes and deafness
mitoTALEN
mitochondrially targeted transcription activator-like effector nuclease
modRNA synthetic chemically modified mRNA mitochondrial transduction domain
mtDNA
mitochondrial DNA
MTS
mitochondria-targeting sequences
mtZFNs
mitochondrially targeted zinc-finger nucleases
NARP
neurogenic muscle weakness, ataxia and retinitis pigmentosa
NDI1
NADH-quinone oxidoreductase
nDNA
nuclear DNA
ORF
open reading frame
OriH
origin of replication for heavy strand of mtDNA
OriL
origin of replication for light strand of mtDNA
PD Parkinson’s disease
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OX-PHOS oxidative phosphorylation
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MTD
plasmid DNA
PICs
polyion complexes
RGCs
retinal ganglion cells
RNS
reactive nitrogen species
ROS
reactive oxygen species
SOD2
superoxide dismutase 2
TALE
transcription activator-like effector
TALEN
transcription activator-like effector nuclease
TFAM
mitochondrial transcription factor A
TFB1M
mitochondrial transcription factor B1
ZFP
zinc-finger protein
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ΔΨm
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pDNA
mitochondrial membrane potential
ACCEPTED MANUSCRIPT 1. Mitochondria: The dynamics of functions and dysfunctions Mitochondria are eukaryotic cell organelles that arguably evolved from a symbiotic relationship between aerobic microbes and primordial eukaryotic cells (Chan, 2006; Sagan, 1967; Selosse, 2011). They putatively produce energy (ATP) through oxidative phosphorylation (OX-PHOS) by transporting electrons across multiple internal complexes (mitochondrial complexes I-V) of an electron transport chain (ETC) (Das and Guha 2010;
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Kaurola et al., 2016). These double-membrane organelles maintain their redox potential by pumping protons across their inner membrane (Chan, 2006). Mitochondria are involved in
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metabolism of glucose (Krebs cycle) and fatty acids (β-oxidation), and are also associated with calcium homeostasis (Schon and Przedborski, 2011; Palomo and Manfredi, 2015).
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Furthermore, they play a crucial role in apoptosis, which is triggered by the release of cytochrome c and apoptosis-inducing factor (AIF) from the mitochondria into the cytoplasm
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(Frank et al., 2001; Herold et al., 2002; Faridi et al., 2016; Zhao et al., 2016). Mitochondrial redox reactions produce reactive oxygen species (ROS) (such as superoxides, peroxides,
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hydroxides and singlet oxygen) and reactive nitrogen species (RNS) (peroxynitrites and dinitrogen trioxide), which might impair the ETC and cell cycle progression (Das and Guha
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2010; Quijano et al., 2016).
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Human mitochondria contain their own circular, double-stranded genome –– the mitochondrial DNA (mtDNA), which is 16,569 bp in length and constitutes 37 genes (Holt et al., 1989; Van Trappen et al., 2007). It is extremely susceptible to oxidative damage due to its close proximity
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to the mitochondrial ROS generation machinery and the limited availability of DNA repair enzymes (for mtDNA) (Das and Guha, 2010; Lee and Wei, 2007). Moreover, the absence of introns or intergenic sequences and histones in the mtDNA makes it prone to oxidative damage
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(Das and Guha, 2010; DiMauro et al., 2013). The mtDNA encodes for 2 rRNAs, 22 tRNAs and 13 polypeptides –– one subunit of cytochrome bc1 complex (complex III): cytochrome b; two subunits of ATP synthase (complex V): ATP6 and ATP8; three subunits of cytochrome c oxidase (COX; complex IV): COI, COII and COIII; and seven subunits of NADH-ubiquinone oxidoreductase (complex I): ND1, ND2, ND3, ND4, ND4L, ND5 and ND6; while complex II (succinate dehydrogenase), on the other hand, is entirely encoded by the nuclear DNA (nDNA) (Holt et al., 1989; Taanman, 1999; Mishmar et al., 2003; DiMauro et al., 2013). mtDNA also has a non-coding region called the displacement loop (D-loop), containing the origins of replication (OriH and OriL sites) and transcription promoters (Taanman, 1999; Singh, 2013; Kambe and Miyata, 2016). Bioenergetic dyshomeostasis of the mitochondria and consequent
ACCEPTED MANUSCRIPT overproduction of free radicals in the ETC lead to initiation and accumulation of considerable damage to the mtDNA, which can cause a plethora of pathological conditions (Quijano et al., 2016). Such dysfunctional conditions are extensively observed in tissues with higher energy demand like muscles, liver, heart, kidney and brain (Leslie et al., 2016). Since mitochondria are inherited only maternally (Camus et al., 2012; Sato and Sato, 2013), defective mitochondria containing mutant mtDNA are also inherited by the offspring from the mother (Thajeb et al.,
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2006; Tuppen et al., 2010). Mitochondrial diseases constitute a variegated conglomeration of clinical facets and symptoms involving a multitude of diverse tissues and organs, which cause significant morbidity and mortality (Wong, 2007; Moggio et al., 2014; Steele et al., 2017;
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Davis et al., 2018).
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The number of reported cases of mitochondrial disorders has increased in the recent years –– up to one in five thousand children harbor mitochondrial pathologies (Haas et al., 2007). In
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fact, up to four thousand newborns are diagnosed with mitochondrial diseases every year in the United States alone (Tachibana et al., 2013). Similar prevalence of mitochondrial pathologies
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has also been reported from other continents (Nesbitt et al., 2014; Ng and Turnbull 2016; Australian Mitochondrial Disease Foundation 2017). However, mitochondrial diseases are still referred to as “mystondria” (mysterious diseases of mitochondria) due to their unpredictable
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genetic and mipigenetic (mitochondrial epigenetics) etiology (Singh, 2015). Impaired
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respiratory subunits, production of dysfunctional enzymes and leakage of electrons from the ETC (mainly from complexes I and III) cause overproduction of ROS (Tretter and Adam-Vizi 2004; Görlach et al., 2015), which hinder mitochondrial biogenesis, and also lead to lipid
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peroxidation and protein carbonylation, along with significant damage to the mtDNA (Shokolenko et al., 2009; Suzuki et al., 2010; Das and Guha, 2010; Kwiecien et al., 2014; Yao
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et al., 2015; Birch-Machin and Bowman, 2016). Such mitochondrial and cellular dyshomeostases result in myriad pathological conditions. For example, type I and type II diabetes mellitus patients reportedly have defective insulin-producing β cells (in the islets of Langerhans in pancreas) (Lenzen, 2008) due to damaged mtDNA, as well as dysfunctional mitochondrial proteins and lipids (Verdile et al., 2015; Montgomery and Turner 2015). The role of mitochondria has also been established in obesity, where saturated fat in cardiomyocytes was observed to cause mitochondrial dysfunctions, such as decreased rate of OX-PHOS, augmented levels of mitochondrial ROS, and depolarization of the mitochondrial membrane (Joseph et al., 2016).
ACCEPTED MANUSCRIPT The colossal number of mitochondrial disorders has been attributed to a multitude of mutations in the mtDNA (Table 1). It has to be noted that mitochondrial diseases do not only occur due to mtDNA mutations, but also due to nDNA aberrations, since a considerable part of the mitochondrial proteome is encoded by the nucleus (Zhu et al., 2009; Gorman et al., 2015; Viscomi et al., 2015). In this review, however, we focus on the gene therapy strategies targeting
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solely the pathological mutations in the mitochondrial genome.
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2. Pathological phenotypes and mtDNA mutations
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Due to the inherent functional complexity of the mitochondria, it is often onerous to attribute a mitochondrial pathology to a particular mtDNA mutation, as well as to restrict a mutation to one single ailment (Doyle and Chan, 2008; Iyer, 2013). However, few principal types of
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mutations have been frequently reported in the majority of mitochondrial diseases (Yamada et al., 2007) (Fig. 1). For example, an m.11778G>A point mutation in the ND4 gene of complex
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I is observed in Leber’s hereditary optic neuropathy (LHON), a frequently occurring optic neuropathy (1 in 25,000 individuals), which is predominant (with 4-5 times greater probability) in males
(Newman, 1993; Yu-Wai-Man and Chinnery, 1993; Chinnery et al., 2001;
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Chadderton et al., 2013). It is characterized by bilateral acute/subacute visual defects, with
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deleterious effects on retinal ganglion cells (RGCs) (Yu-Wai-Man and Chinnery, 1993; Carelli et al., 2004; Chadderton et al., 2013). Most LHON incidences (~95%) occur via three substitution mutations in the genes encoding for complex I at positions m.3460G>A,
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m.3697G>A and m.4171C>A (ND1), m.11778G>A (ND4) and m.14484T>C (ND6) in the
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mtDNA (Chinnery et al., 2001; Kim et al., 2002; Spruijt et al., 2007). Substitution mutations in mitochondrial ATP6 gene (m.8993T>G; and m.8993T>C in a small percentage of patients) have been attributed to cause neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) (Thorburn and Rahman, 1993; De Meirleir et al., 2004; Kabunga et al., 2015), which is a neurological syndrome with an estimated prevalence rate of 0.81/100,000 individuals (Orphanet, 2017). These mutations lead to the replacement of amino acid leucine by arginine in F1FO-ATPase, thereby altering its function (Thorburn and Rahman, 1993). Similar to NARP, incidences of m.8993T>G and m.8993T>C mutations in the ATP6 gene in mtDNA are also observed in Leigh syndrome (LS), (Thorburn and Rahman, 1993; De Meirleir et al., 2004; Praeter et al., 2014). LS (also known as subacute necrotizing encephalomyelopathy) is a progressive neurodegenerative disorder of the central nervous
ACCEPTED MANUSCRIPT system that primarily affects the spinal cord, basal ganglia, pons, cerebellum and thalamus, as well as causes demyelination of optic nerves (Westarp et al., 1992; Duff et al., 2015; Gerards et al., 2016). Other mtDNA mutations, such as m.4296G>A (tRNAIle) (Martikainen et al., 2013), m.5523T>G and m.5559A>G (tRNATrp) (Mkaouar-Rebai et al., 2009), m.13513G>A (ND5) (Shanske et al., 2008), m.13514A>G (ND5) (Bannwarth et al., 2013), m.9191T>C (ATP6) (Moslemi et al., 2005) and m.9478T>C (COIII) (Mkaouar-Rebai et al., 2011), are also
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reportedly associated with LS (Fig. 1). The ailment has an incidence rate of one in 40,000 live births (Lake et al., 2015). While the maximal onset of this disorder takes place at about 3–12 months of age (with ~50% of the patients dying by the age of 3 years), late onset has also been
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observed in adults (Rahman and Thorburn, 1993). LS is caused by energy deficiency in cells
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due to defective enzymes viz. ETC complex I, complex IV and pyruvate dehydrogenase complex. The symptoms of this disease include hypotonia, ataxia, psychomotor regression,
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dystonia, cranial nerve palsies, spasticity, infantile spasms, sensory neural hearing loss, persistent vomiting, dysphagia and respiratory problems like hyper- and hypoventilation
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(Finsterer, 2008; Sofou et al., 2014; Gerards et al., 2016). The m.8993T>G/C mutation is associated with complex V deficiency too (De Meirleir et al., 2004). In fact, the disease has been associated with the etiologies of NARP and LS (De Vivo,
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1993; National Organization for Rare Disorders, 2016; Rat Genome Database, 2017). Complex
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V deficiency is a very rare metabolic disorder, which is also caused by other mutations in the ATP6 and ATP8 genes (Auré et al., 2013; Kytövuori et al., 2016), such as m.8561C>G (in the overlap region of ATP6 and ATP8) (Kytövuori et al., 2016), m.8969G>A (in ATP6) (Burrage
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et al., 2014) and m.8529G>A (in ATP8) (Jonckheere et al., 2008) (Fig. 1). Myriad mtDNA mutations have been reported in the substantia nigra (a nucleus in midbrain)
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of individuals with Parkinson's disease (PD) (Lin et al., 2012; Coxhead et al., 2016), which is the second most prevalent geriatric neurodegenerative disorder in humans, especially affecting people above 50 years of age (Gibson et al., 2012; Pringsheim et al., 2014). It is characterized by the deterioration of dopaminergic (DA) neurons in the substantia nigra pars compacta and by the accumulation of Lewy bodies (Gibson et al., 2012). PD-associated mutations have been reported in COI, COII, cytochrome b, ND5 and the D-Loop region of the mtDNA. These lead to mitochondrial alterations like decreased complex I activity, excessive ROS production, consequent mtDNA damage and mitophagy, which are associated with the etiology of PD (Winklhofer and Haass, 2010; Moon and Paek, 2015).
ACCEPTED MANUSCRIPT An m.8344A>G mutation in the gene encoding tRNALys is the most common cause of myoclonic epilepsy with ragged-red fibers (MERRF) syndrome (Shoffner et al., 1990; Jacobs, 2003), a disease that commonly affects muscles and the central nervous system, and is characterized by the presence of myoclonus, myopathy and spasticity. It has been estimated to affect one in every 5,000 individuals worldwide (US National Library of Medicine, Genetics Home Reference, 2017). MERRF can also occur due to point mutations in the mtDNA tRNAPhe gene at nucleotide position 616 and is observed in skeletal muscles (Zsurka et al., 2010).
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Mutations (Fig. 1) in the encoding region of tRNALeu (m.3243A>G and m.3271T>C) and tRNAVal (m.1642G>A) have been reported in mitochondrial encephalomyopathy, lactic
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acidosis, and stroke-like episodes (MELAS) (Goto et al., 1990), and maternally inherited
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cardiomyopathy (MICM) (Silvestri et al., 1994; Santorelli et al., 1996, 1999; Dipchand et al., 2001). MELAS is also associated with mutations in the COIII (m.9957T>C) and ND5
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(m.12770A>G, m.13045A>C, m.13513G>A and m.13514A>G) genes (Tuppen et al., 2010; Hashimoto et al., 2015).
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mtDNA deletions have been encountered in the chronic progressive external ophthalmoplegia (CPEO) (Van Goethem et al., 2003; Sundaram et al., 2011) and Kearns-Sayre syndrome (KSS) (Shanske et al., 1990). A number of other mutations are also associated with a host of other
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mitochondrial disorders (Fig. 1), such as familial bilateral striatal necrosis (FBSN)
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(Thyagarajan et al., 1995), maternally inherited diabetes and deafness (MIDD) (Mangiafico et al., 2004), myoclonic epilepsy and psychomotor regression (MEPR) (Shtilbans et al., 1999), maternally inherited hypertrophic cardiomyopathy (MHCM) (Thambisetty and Newman,
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2004; Tuppen et al., 2010; Hashimoto et al., 2015), and maternally inherited deafness (MID)
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(Yang et al., 2011; Zheng et al., 2012; Hoptasz et al., 2014).
3. Gene therapy: Confronting mitochondrial pathologies Although contemporary research has considerably enhanced our understanding of the genetic and biochemical etiology of mitochondrial disorders, therapeutic interventions for the same have been preliminary at best. Nevertheless, with recent advances in technology, gene therapy has emerged as a potential strategy to address mitochondrial diseases, either by manipulating the nuclear genome or by targeting the mitochondrial paraphernalia (Weissig and Torchilin, 2001). Gene therapy is the replacement of a defective gene by delivering a corrective allele into the host cell, or silencing a dominant mutant allele that is pathogenic to the host (Kaufmann
ACCEPTED MANUSCRIPT et al., 2013; Choong, Baba, and Mochizuki 2015). Since a large number of mitochondrial disorders are rooted to mutations in the mtDNA (Fig. 1), an intervention can be achieved by corrective gene therapy against the defective/missing facets of the mitochondrial genome. This can be materialized by delivery of functional therapeutic genes as substitutes for the endogenous counterparts (Yamada et al., 2007; Kyriakouli et al., 2008; Heller, Brockhoff, and Goepferich 2012). For most mitochondrial gene therapies, one of the paramount criteria is the
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construction of suitable vectors to accommodate the genes of interest (Yasuzaki et al., 2015). Among a plethora of vectors, adenoviruses have been most commonly used (accounting for 23.3% of the trials) because of their high transduction efficiency (leading to high gene
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expression levels) and larger DNA load carrying capacity in comparison to other viruses (Ginn
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et al., 2013). Adeno-associated virus (AAV) has also been extensively used due to its broad spectrum of hosts (Gao et al., 2002; Wang et al., 2016). Herpes simplex, pox and vaccinia
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viruses have also been successfully used in gene therapy (Ginn et al., 2013). Mitochondrial gene therapy has also been accomplished by administration of the naked plasmid DNA (pDNA) in vivo resulting in production of mRNA in the mitochondria (Yasuzaki et al., 2015).
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Variegated modules of mitochondrial gene therapy have demonstrated positive results in a host of neuromuscular and metabolic disorders related to mitochondrial dyshomeostasis. However,
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although diverse in their characteristics, all applied strategies broadly follow one of the three major approaches: (1) allotopic expression (of mtDNA genes in the nucleus); (2) selective
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inhibition of mutant mtDNA; (3) delivery of WT mtDNA into the mitochondria.
3.1. Allotopic expression-mediated gene therapy
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Endosymbiotic gene transfer from the mitochondria to the nucleus is a ubiquitous ongoing evolutionary process that occurs at a substantial frequency (Timmis et al., 2004). This phenomenon is emulated for allotopic expression-mediated gene therapy for defective mtDNA genes for the integration of the corresponding corrective mitochondrial genes into the nucleus (Guy et al., 2002; Manfredi et al., 2002; Shokolenko et al., 2010; Cwerman-Thibault et al., 2015). Transport of the subsequently expressed protein into the mitochondria is facilitated by a mitochondria-targeting sequence (MTS; which helps in transporting a protein of interest into the mitochondria) (Shokolenko et al., 2010). nDNA-encoded proteins that are destined to be localized in the mitochondria contain an MTS at their N-terminal ends, which is recognized by mitochondrial protein import channels, thereby allowing protein import (Chin et al., 2018). Moreover, specific 3’- untranslated regions (3′-UTRs) are used to locate mRNAs on the
ACCEPTED MANUSCRIPT mitochondrial surface and help in co-translational import of these nDNA-encoded mitochondrial proteins (Kaltimbacher et al., 2006; Bonnet et al., 2007a). However, it has been recently reported that it is the choice of MTS that determines the efficiency of delivery of allotopically expressed proteins to the mitochondria, rather than the selected 3’-UTR (Chin et al., 2018). This method was first tested for its efficacy in mitochondrial ATP8 gene-deficient models of Saccharomyces cerevisiae, where the incorporation of the WT mtDNA gene in the
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nucleus resulted in successful restoration of function (Nagley et al., 1988). This strategy of integrating mtDNA genes in the nDNA has been applied to correct defects arising from several
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mtDNA mutations (Fig. 2A).
As a key strategy for treating LHON, allotopic expression-mediated gene therapy has been
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accomplished in vitro in cybrid cells (derived from homoplasmic osteosarcoma) harboring m.11778G>A mutation in the ND4 gene (Guy et al., 2002). Through this method, the WT ND4
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gene was integrated into the nucleus by recombinant AAV (rAAV) vector. The plasmid was constructed by fusing the ND4 gene with an MTS (from the P1 isoform of human ATP synthase
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subunit c), and a FLAG epitope tag. This sequence was then integrated with green fluorescence protein (GFP) via an internal ribosomal entry site (IRES) sequence, a chicken β-actin promoter and a cytomegalovirus enhancer. The final construct was cloned into pTR-UF12 plasmid vector
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and packaged to generate rAAV particles. The cybrid cells were then stably transfected with
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3×107 rAAV particles. The expression of the fused protein was driven by the chicken β-actin promoter and the enhancer. The transfected cells subsequently showed GFP and FLAG expression in the cytoplasm and mitochondria respectively. The FLAG localization to the
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mitochondria implied successful transport of the ND4 gene, which was further confirmed by the observed three-fold increase in ATP synthesis in the cybrid cells (Guy et al., 2002).
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In an in vivo LHON model (rats), the disease was developed using allotopic expression of mutant human ND4 gene (bearing m.11778G>A mutation) in the RGCs, thereby creating mitochondrial heteroplasmy (Ellouze et al., 2008), which is a condition where a single cell can harbor mitochondria with both WT and mutant genomes (Das and Guha, 2010; Wallace and Chalkia, 2013; Picard et al., 2014; Wernick et al., 2016). The WT ND4 gene was incorporated with pAAV-IRES-GFP and transformed pDNA was injected into the vitreous body of the eye through electroporation. The plasmid was constructed by fusion of CO-X (which is a nuclear gene) and ND4 into the pAAV-IRES-GFP vector. CO-X encodes for both MTS (present at the amino termini of pre-proteins) and the sequence for MTS cleavage (Omura, 1998; Ellouze et al., 2008). MTS helps to import ND4 proteins from cytoplasm into the mitochondria, while the
ACCEPTED MANUSCRIPT MTS cleavage sequence functions to generate the mature ND4 once the ND4-MTS complex is inside the mitochondria (Ellouze et al., 2008). GFP gene was used for transgene selection. The ND4-CO-X gene was integrated into the nuclear genome of RGC and the ND4 subunit protein was synthesized in the cytoplasm. With the help of MTS, ND4 was imported into the mitochondria, thus replacing the defective ND4, thereby restoring normal bioenergetics (Ellouze et al., 2008).
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In another approach, trans-kingdom mitochondrial gene therapy was accomplished by intraocular delivery of NDI1 (NADH-quinone oxidoreductase; a nuclear gene from S.
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cerevisiae that encodes for single subunit complex I) in mice. NDI1 gene contains endogenous MTS, which assists in efficient transport of the protein into the mitochondria (which is more
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effective than allotopic expression with introduced MTS) (Chadderton et al., 2013). AAV2 plasmid containing NDI1 (AAV2-NDI1) was constructed and delivered intravitreally into
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rotenone-treated (induces RGC loss and inhibits complex I) mice (Chadderton et al., 2013). Optokinetic assessment of the treated eye showed significant reduction in RGC death and optic
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nerve defects. Also, manganese-enhanced magnetic resonance imaging (MEMRI) exhibited improved functions of optic nerve by active transport of Mn+2 into the cells through voltagegated calcium channels (Chadderton et al., 2013; Marella et al., 2010). Observations up until
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six months post-treatment showed no remarkable amount of anti-NDI1 antibody in the sera
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(Marella et al., 2011), proving the potency of NDI1 for the replacement of defective complex I proteins. An added advantage of this approach is that it is not restricted to a particular mutation
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(Chadderton et al., 2013).
In an ongoing (2014–2019) clinical trial (NCT02161380) (Guy, 2018) at the Bascom Palmer Eye Institute (Miller School of Medicine, University of Miami, USA), AAV2-mediated gene
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therapy containing ND4 (AAV(scAAV)2(Y444,500,730F)-P1ND4v2) was done intravitreally in five patients with LHON (m.11778G>A mutation) (Feuer et al., 2016). The selfcomplementary genome improved the transgene expression, and the AAV2 capsid promoted efficient tissue-specific uptake (Koilkonda et al., 2010; Feuer et al., 2016). The patients were treated with differential doses of transformed vector genomes. The initial results of this clinical trial showed an improved vision in all the patients with none of them suffering from any severe side-effects. However, it has to be noted that the sampling size was very low and that the study is still recruiting patients (Feuer et al., 2016; Wan et al., 2016; Yang et al., 2016). Another set of recent prospective clinical trials (NCT02064569, NCT02652767, NCT02652780, NCT03406104 and NCT03293524) are testing the efficacy and safety of GS010 (rAAV2/2-
ACCEPTED MANUSCRIPT ND4) in patients affected by ND4 mutations, the results of which are currently unavailable (Newman, 2018; Subramanian, 2018; Vignal, 2018; Yu-Wai-Man, 2018a, 2018b). As discussed earlier (section 2), the m.8993T>G substitution mutation in the mitochondrial ATP6 gene is extremely common in NARP, LS and complex V deficiency (De Meirleir et al., 2004). To develop an allotopic expression-mediated gene therapy strategy to correct this mutation, skin biopsies from an 8-month old child with NARP syndrome (m.8993T>G
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mutation) were used for establishing fibroblast culture as the experimental model (Bonnet et al., 2007a). For effective localization of ATP6 mRNA into the mitochondria, the MTS sequence
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of superoxide dismutase 2 (SOD2) gene and its 3′-UTR were combined with the ATP6 gene (Bonnet et al., 2007a, 2007b). The transfection plasmid was constructed by inserting ATP6
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gene (with a FLAG epitope at the C terminus of the ATP6 ORF) into pCMV-Tag 4A vector, along with the MTS of the SOD2 gene and either the 3′-UTR of SOD2 or the 3′-UTR of SV40,
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thereby giving rise to the two following final constructs: SOD2MTSATP6-3′UTRSOD2 and SOD2MTSATP6-3′UTRSV40 (Kaltimbacher et al., 2006). It was observed that when both MTS
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and the 3′-UTR sequence were present (i.e., SOD2MTSATP6-3′UTRSOD2), ATP6 mRNA sorting to the mitochondrial surface was augmented, leading to 1.8 times higher ATP6 protein expression, than that with the MTS alone (i.e., SOD2MTSATP6-3′UTRSV40). SOD2-ATP6 co-
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localization was observed by indirect immunofluorescence using antibodies against FLAG or
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ATP synthase (-subunit). Complex V activity was evidently restored using this gene therapy approach, which was also verified by the increased production of ATP (Bonnet et al., 2007a).
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Another similar strategy was developed using a B6(B6SJLF1) (mice) model (Dunn and Pinkert, 2012). First, WT ATP6 genes were cloned in frame into the pEF/myc/mito plasmid, containing the promoter for human EF-1α, the MTS from the human COVIII gene (nuclear), and an in-
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frame 3’ myc epitope tag. This pEF/myc/mito/ATP6 plasmid was used to generate transgenic mice. Mitochondrial localization of allotopically expressed corrective ATP6 protein was confirmed by electron microscopy (Dunn and Pinkert, 2012). In a recent study on mtDNA complex V-deficient patient cells (null for the ATP8 protein, with significantly less ATP6 expression) harboring the m.8529G>A mutation (in the mitochondrial ATP8 gene) (Boominathan et al., 2016), researchers allotopically expressed ATP6 and ATP8 genes in the nucleus to correct the deficiency using a pCMV6 vector. Corrective DNA sequences for ATP6 and ATP8 were incorporated into the vector between the MluI and XhoI sites. A CO-X or ATP5G1- or ATP5G2-based MTS was also inserted into the pCMV6 at the 5′ end (between sites AsiSI and MluI) of the genes. Myc and FLAG or V5 epitope were also
ACCEPTED MANUSCRIPT introduced immediately downstream of the codon-corrected genes. This technique rescued expression of both the proteins and recovered their functions in the cell significantly (Boominathan et al., 2016). A strategy for allotopic expression of mitochondrial proteins without altering the nuclear genome uses synthetic chemically modified mRNA (modRNA) encoding for the corrective protein (Fig. 2B), whose uridine and cytosine have been substituted respectively by
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pseudouridine and 5-methyl-cytosine (Chin et al., 2018). These modifications allow the modRNA to avoid the Toll-like receptors of innate immune system (that would have otherwise
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degraded them) in vivo while facilitating efficient translation of the protein of interest (Karikó et al., 2005). A report published in 2018 has demonstrated allotopic expression of
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mitochondrial proteins by using modRNAs. In this study, the researchers constructed modRNAs for allotopic expression of MTS-ATP6 fusion protein in ATP6-mutant cybrids
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(harboring the m.8993T>G mutation), which consequently led to improved mitochondrial
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functions and cell survival (Chin et al., 2018).
3.2. Gene therapy by selective inhibition of mutant mtDNA
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Mitochondrial heteroplasmy is implicated in various mitochondrial disorders including
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mitochondrial myopathy, MERRF, and MELAS (Ye et al., 2014). In such a state, a certain threshold of mutant mtDNA gene(s) will be required to elicit the corresponding disease condition(s) (Taylor et al., 1997; Schon et al., 2012). A prudent strategy to alleviate such
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diseases is to selectively inhibit mutant mtDNA (Taylor et al., 1997; Russell and Turnbull, 2014), thereby mitigating the negative effects of heteroplasmy. This approach has shown
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promise in treating mitochondrial ailments that arise due to mutations in both protein- and RNA-coding genes (Tanaka et al., 2002; Hashimoto et al., 2015). In section 3.1, we have discussed how alloptopic expression-mediated gene therapy has found success in correcting the m.8993T>G mutation, which is involved with NARP, LS and complex V deficiency (De Meirleir et al., 2004) (as discussed in section 2). Gene therapy by selective inhibition of mutant mtDNA has also been used as a potent strategy to inhibit the effects of this substitution mutation in the mtDNA. One approach to reach this goal is to use mitochondriadirected restriction endonucleases specific for unique mutations in the mutant mtDNA (Fig. 3A). For this, a group of researchers (Tanaka et al., 2002) at the Gifu International Institute of Biotechnology in Japan transiently expressed the restriction endonuclease SmaI (fused with
ACCEPTED MANUSCRIPT MTS) in cells harboring the m.8993T>G mutation in a heteroplasmic state. SmaI selectively cleaved mutant mtDNA at the guanine residue (CCCGGG) of m.8993, while not affecting the thymine residue (CCCTGG) of m.8993 in the WT mtDNA. The same strategy was also applied by using EcoRI (another restriction endonuclease) instead of SmaI. The plasmid for transfection was constructed by fusing the cDNA encoding pCOIV (pre-sequence of cytochrome c oxidase subunit IV from S. cerevisiae) with pMACSKKII (mammalian expression vector) and either EcoRI or SmaI. The resultant gene therapy vectors were
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pMACSKKII-pCOIV-EcoRI and pMACSKKII-pCOIV-SmaI (Tanaka et al., 2002). pCOIV guided the delivery of restriction enzymes selectively into the mitochondria, hence avoiding
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cleavage of the nDNA. Interestingly, while SmaI required five transfection and selection cycles
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to successfully eliminate the mutant mtDNA, EcoRI needed only one to two cycles (probably due to differential numbers of recognition sites for the restriction enzymes) (Tanaka et al.,
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2002). Another restriction endonuclease, XmaI, has also been used to correct the m.8993T>G mutation by selectively targeting mutant mtDNA (Alexeyev et al., 2008).
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The research group of Prof. Carlos T. Moraes at the Miller School of Medicine (University of Miami, USA) has done some comprehensive work in using restriction endonucleases to obliterate mutant mtDNA. In an in vitro study (Srivastava and Moraes, 2001), they expressed
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mitochondria-targeted PstI in murine cells harboring heteroplasmy of mtDNA (the mutant
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containing two PstI-specific sites). In another study (Bayona-Bafaluy et al., 2005), performed both in vivo (brain and muscle of heteroplasmic mice) and in vitro (heteroplasmic mice hepatocytes), they transfected ApaLI fused with MTS of COVIII to selectively ablate the
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mutant mtDNA containing ApaLI-specific sites. The group later demonstrated that systemic delivery of viral vectors containing MTS-fused ApaLI to specific organs accomplished in
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treating mtDNA-associated disorders (Bacman et al., 2010, 2012). In yet another study, the same group has demonstrated application of mitochondria-targeted ScaI restriction endonuclease to scythe mutant mtDNA, thereby mitigating heteroplasmy-associated dyshomeostasis (Bacman et al., 2007). Although the restriction endonuclease approach has shown significant success, it is unfortunately limited to only a small number of mtDNA mutations with unique restriction sites, which are recognized by these enzymes (Choo and Klug, 1994). To avoid this disadvantage, mtDNA-binding zinc-finger nucleases (mtZFNs) have been engineered, which have preset selectivity for specific mtDNA sequence(s) (Kim et al., 1996; Isalan et al., 2001; Papworth et al., 2006; Minczuk et al., 2006; Gammage et al., 2016). mtZFNs are chimeric enzymes
ACCEPTED MANUSCRIPT containing a Cys2His2 zinc-finger protein (ZFP), which is conjugated with an MTS and the catalytic domain of restriction endonucleases (such as FokI), along with nuclear export signal (NES) peptides. This ensures their localization exclusively in the mitochondria (Minczuk et al., 2006; Gammage et al., 2016). This strategy demonstrated that mtZFNs can specifically localize to the mitochondria when combined with MTS, and can selectively cleave mutant mtDNA (Fig. 3B). The approach was successfully used in reducing the levels of mutant
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mtDNA harboring the m.8993T>G mutation (Gammage et al., 2016), which is associated with NARP, LS and complex V deficiency (De Meirleir et al., 2004). The mtZFN-based gene therapy also restored ATP homeostatsis and ΔΨm on selective removal of the mutant mtDNA
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(Gammage et al., 2016).
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Apart from restriction endonucleases and mtZFNs, there is another potent gene therapy strategy for selective elimination of mutant mtDNA in a heteroplasmic situation. This involves
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designing mitochondrially targeted transcription activator-like effector (TALE) nucleases (mitoTALENs), which are novel nucleases (containing FokI catalytic domains) that can
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selectively target and cleave mutant mtDNA (Hashimoto et al., 2015; Gammage et al., 2016; Gómez-Tatay et al., 2017) (Fig. 3C). TALENs work as heterodimers, which need two TALEN monomers to bind closely-spaced DNA sequences, allowing their FokI nuclease domains to
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dimerize and cleave the flanked sequence of the bound DNA (Bacman et al., 2013). In addition
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to the work done in restriction endonuclease-based gene therapy to obliterate mutant mtDNA, Prof. Moraes and his group have also worked on designing mitoTALENs, which demonstrated significant decline in the number of mutant mtDNA molecules in patient–derived cells
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(Bacman et al., 2013). This strategy was further applied by the group in reducing mtDNA with m.8344A>G (tRNALys) and m.13513G>A (ND5) mutations (in cybrids), which are respectively
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associated with MERRF and MELAS (Jacobs, 2003; Hashimoto et al., 2015). The researchers constructed plasmids coding for mitoTALEN monomers. They removed any nuclear localization signal from the construct, while incorporating (i) an SOD2- or COVIIIA/Su9derived MTS, (ii) a FLAG or hemagglutinin immuno-tag, (iii) a 3′-UTR, (iv) a fluorescent marker (eGFP or mCherry), and (v) a recoded picornaviral 2A-like sequence (T2A) between the mitoTALEN and the fluorescent marker. MitoTALENs showed significant mitigation in the levels of the mutant mtDNA (harboring m.8344A>G or m.13513G>A), along with considerable recovery in respiratory capacity and OX-PHOS efficiency (Hashimoto et al., 2015).
ACCEPTED MANUSCRIPT 3.3. Gene therapy via delivery of WT mtDNA Targeting WT mtDNA (containing the corrective genes) directly into the mitochondrion has emerged as a potent strategy to rectify pathogenic mtDNA mutations (Vaidya et al., 2009; Yu et al., 2012). Carrier-based delivery systems, such as mitochondrial transduction domain (MTD) conjugated to mitochondrial transcription factor A (MTD-TFAM), MITO-porters and DQAsomes, have shown great promise in this mitochondriotropic approach (Niazi et al., 2013).
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However, the paramount challenge still remains in understanding the mechanism of nucleic acid import into the mitochondria (Weber-Lotfi et al., 2015). Nevertheless, several mtDNA
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mutations, such as m.8993T>G and m.11778G>A, have been successfully tackled by
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employing this strategy (Iyer et al., 2012).
In an in vivo study (Yu et al., 2015), transgenic mice were generated as a disease model for
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LHON by introducing mutant human ND4 (mutND4) gene (m.11778G>A) into the mice mtDNA through rAAV (containing self-complementary sc-HSP-mutND4-FLAG+mCherry).
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The AAV vector was modified by appending a COVIII presequence that facilitated its localization directly to the mitochondria, rather than the nucleus. This strategy caused the mutant ND4 gene to assemble in the mitochondrial complex I, thereby causing visual loss in
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the mice. The condition was found to be reversed when the animals were treated with
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mitochondria-targeted self-complementary AAV serotype 2 (scAAV2) containing the WT human ND4 (scAAV2-HSP-ND4) (Yu et al., 2015). The m.11778G>A mutation has also been successfully rectified in SH-SY5Y cells using the
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protein transduction (protofection) technique (Iyer et al., 2009, 2012). In this approach, mtDNA (corrective, WT) cargo is delivered into the mitochondria using engineered protein
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vectors, such as recombinant TFAM conjugated with MTD (combination of an N-terminal protein transduction domain and the MTS of SOD2) (Khan and Bennett, 2004; Iyer et al., 2009; Keeney et al., 2009; Thomas et al., 2011; Iyer et al., 2012; Iyer, 2013). In the MTD-TFAM conjugate, TFAM carries the WT mtDNA cargo, the protein transduction domain facilitates conveyance through the cell membrane, and the MTS ensures mitochondriotropic targeting and uptake (Fig. 4A). This has reportedly corrected the mtDNA mutation and significantly enhanced mitochondrial function (Iyer et al., 2009). This strategy has also shown success in correcting the m.8993T>G mutation in the ATP6 gene, which is involved with the etiologies of NARP, LS and complex V deficiency (De Meirleir et al., 2004). MTD-TFAM conjugated with WT mtDNA improved mitochondrial functions of fibroblast-derived cybrid cells with 95% of mitochondrial genomes bearing the m.8993T>G mutation (Iyer et al., 2012).
ACCEPTED MANUSCRIPT Furthermore, in an in vitro model for PD (cybrids generated by fusing SH-SY5Y ρ0 cells with platelets obtained from PD patients) with reduced complex I activity, MTD-TFAM-mediated delivery of corrective mtDNA into the mitochondria showed increased mitochondrial gene expression with augmented mitochondrial functions (Keeney et al., 2009). Yet another evolving strategy for delivery of WT mtDNA into mitochondria (harboring mutant genomes) is by use of MITO-porters, which are liposome-based nanocarriers that can deliver
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nucleic acids, proteins or drugs selectively to the mitochondria via a membrane-fusion mechanism (Niazi et al., 2013; Yamada and Harashima, 2015; Ishikawa et al., 2018). These
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MITO-porters can be coated with two membrane-fusogenic outer envelopes to produce the dual function MITO-porters (DF-MITO-porters) (Yamada, 2014). After internalization into
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cells, the outer envelope of the DF-MITO-porters fuses with the endosomal membranes (the MITO-porter thus escaping from the endosome sans the outer envelope), while the inner one
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fuses with the mitochondrial membranes, thereby delivering the cargo exclusively into the mitochondria (Yamada et al., 2011; Niazi et al., 2013; Yamada, 2014; Yamada and Harashima,
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2015) (Fig. 4B). This approach has accomplished in delivering a variety of macromolecules into the mitochondria (Yamada et al., 2008; Yamada and Harashima, 2012; Yamada, 2014; Yamada and Harashima, 2015; Ishikawa et al., 2018). A recent study has reported successful
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delivery of WT ND4 gene into the mitochondria in both in vitro (HeLa) and in vivo (C57BL/6
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mice) systems (Ishikawa et al., 2018). The researchers designed a construct containing human ND4 gene (pCMV-mtLuc (CGG) [ND4]) and transfected it into the mitochondria by using a MITO-porter (Ishikawa et al., 2018; Yamada et al., 2017) that is conjugated with a DNA-
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binding a cationic amphipathic peptide called KALA, which causes membrane destabilization and facilitates DNA transfection (Wyman et al., 1997). These results make it prudent to explore
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further in the precincts of the MITO-porter systems to develop novel gene therapy strategies for delivery of WT mtDNA into the mitochondria. Another significant approach for delivery of nucleic acids into the mitochondria involves the application of dequalinium chloride (DQA) as a targeted mitochondrial vesicular delivery system. DQA forms cationic liposome-like nanovesicles (DQAsomes) at concentrations above the critical vesicular values (Weissig et al., 2000; Weissig, 2015). These mitochondriotropic nanocarriers directly transfer exogenous DNA, peptides, oligonucleotides, nucleic acids, pDNA and low molecular weight drugs into the mitochondria (Weissig, 2015) (Fig. 4C). Exogenous DNA along with an MTS can be combined with the DQAsome to form a DQAsome-DNA-MTS complex, which might then be injected into the host cell (D’Souza et
ACCEPTED MANUSCRIPT al., 2005; Lyrawati et al., 2011). Its contact with the mitochondrial membrane results in cleaving of the conjugate from the DQAsome and its selective accumulation on mitochondria. This selectivity is ascribed to the difference in the mitochondrial membrane potential (ΔΨm) and charge gradient across the mitochondrial matrix. The splitting is followed by its entry into the matrix with the help of protein import machinery (Weissig et al., 2000; Weissig and D’Souza, 2004). Hence, DQAsome-mediated transfection of functional mtDNA genes into the
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mitochondria is an effective technique of gene therapy.
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4. Epilogue: The promises and pitfalls of mitochondrial gene therapy Gene therapy is currently the ‘closest to cure’ approach in the treatment of mitochondrial ailments (Doyle and Chan, 2008). Nevertheless, its strategies have their own share of
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impediments, such as concerns regarding the safety of different modes of therapies, stability of the introduced gene(s) over generations, actual in vivo efficacy and the extent to which it can
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be applied in practice (Kagawa and Hayashi, 1997; Doyle and Chan, 2008). The complexity in the genotype-phenotype relationship and the process of stochastic segregation of mitochondria make it difficult to diagnose the specific disorder, thereby rendering further complications to
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design therapeutic approaches using gene therapy (Kagawa and Hayashi, 1997; Doyle and
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Chan, 2008). Moreover, although about 67% of contemporary clinical trials use viral vectors, their immunogenicity and carcinogenicity pose a major challenge (Kamimura et al., 2015). To circumvent these plausible dire outcomes, application of liposome- or nanoparticle-based
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delivery systems are gaining significance. However, limited cargo-carrying capacity, low and transient gene expressions of these non-viral delivery systems, plague their ubiquitous use (Yu-
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Wai-Man, 2016). Also, for carrying out prospective clinical trials, extensive heterogeneity of mtDNA mutations in patients make it a challenging exercise to perform appropriate sampling of patient groups with significant homogeneity with respect to their genetic disorders, disease progression, biochemical facets and mtDNA mutation load (Kanabus et al., 2014; Rahman, 2015). Individual gene therapy strategies have also been marred with several problems. For example, allotopic expression-mediated gene therapy strategy, although proficient, often gets limited by the need of effective mitochondrial import and accurate incorporation of the allotopically expressed protein (Kyriakouli et al., 2008). Moreover, it has been recently shown that constructs used to deliver allotopically expressed proteins into the mitochondria often result in
ACCEPTED MANUSCRIPT mitochondrial fragmentation (Chin et al., 2018). The hurdles in transition of present gene therapy approaches from laboratory to clinic are, however, not limited to allotopic expressionmediated gene therapy only. For instance, in gene therapy via RE-mediated selective ablation of mutant mtDNA, a major issue is that only a handful of mutations harbor specific restriction sites for known REs (Nightingale et al., 2016). In the mtZFN approach (which moderately overcomes the above-mentioned problem by using ZFPs to bind to a specific DNA sequence),
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occurrence of cytotoxicity has been detrimental in molding this approach into a safe clinical intervention tactic (Nightingale et al., 2016). Also, the RE FokI, which is commonly used in this strategy, is known to dimerize, which in turn triggers off-target binding and subsequent
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cleavage (Ramalingam et al., 2011). In case of mitoTALENs, a pertinent problem lies in the
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fact that, owing to their large size and requirement of two monomers, it is often difficult to pack the large cargo into a suitable vector (Hashimoto et al., 2015). In case of carrier-based
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systems such as MITO-porters, selective delivery of the cargo into the mitochondria has been reported to be difficult, particularly during delivery of hydrophobic molecules or macromolecules (Yamada et al., 2011). Also, it has to be noted that most of these strategies
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have been predominantly tested in cell-based systems and/or animal models. Their individual efficacy in the clinical scenario still needs to be ascertained.
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Despite the enormous promise that gene therapy offers, it is indispensible to explore further for
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novel approaches to invoke safer and more efficient strategies to combat mitochondrial conundrums. Although more than five hundred mtDNA mutations are reportedly associated with mitochondrial diseases (Ye et al., 2014), the availability of gene therapy is restricted to
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only a handful of mutations, as discussed in section 3. For instance, although substitution mutations in the mitochondrial genome, such as m.4296G>A (tRNAIle) (Martikainen et al.,
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2013), m.5523T>G and m.5559A>G (tRNATrp) (Mkaouar-Rebai et al., 2009), m.9185T>C and m.9191T>C (ATP6) (Moslemi et al., 2005), m.9478T>C (COIII) (Mkaouar-Rebai et al., 2011), m.13513G>A and m.13514A>G (ND5) (Bannwarth et al., 2013) are associated with LS, gene therapy exists only for the commonly reported m.8993T>G (ATP6) mutation (Iyer et al., 2012). MELAS, which is caused by m.3243A>G (tRNALeu), m.1642G>A (tRNAVal), m.9957T>C (COIII), m.12770A>G (ND5), m.13045A>C (ND5), m.13514A>G (ND5), and m.3271T>C (tRNALeu) mutations (Tuppen et al., 2010), has so far only received gene therapy for the m.13513G>A (ND5) mutation (Hashimoto et al., 2015). In the case of LHON, gene therapy for mutations like m.3697G>A (ND1) (Spruijt et al., 2007), m.14484T>C (ND6), m.3460G>A (ND1) (Chinnery et al., 2001), and m.4171C>A (ND1) (Kim et al., 2002) is still speculative at best. Mutations implicated in complex V deficiency, such as m.8561C>G (in the overlap region
ACCEPTED MANUSCRIPT of ATP6 and ATP8) (Kytövuori et al., 2016) and m.8969G>A (in ATP6) (Burrage et al., 2014), are yet to have an efficient mode of gene therapy. In spite of the diverse hurdles, mitochondrial gene therapy has evolved exponentially in the recent years owing to the emergence of myriad novel approaches. For example, in polyion complexes (PICs), where methylated carrier molecules (such as poly-L-histidine) containing DNA for the effective transgene expression were used, higher gene expressions were reported
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(Asayama et al., 2015). Another emerging technique involves the use of DQAsomes for gene therapy, where augmented in vivo efficacy has been observed, which might be due to the fact
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that DQAsomes are not disintegrated by the plasmalemmal anionic lipids (i.e., they do not release the DNA during endocytosis) (Zupančič et al., 2014; Weissig, 2015). Furthermore, they
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strongly bind to the exogenous DNA and protect them from nuclease digestion (Zupančič et al., 2014; Weissig, 2015). Yet another advancement in tackling mitochondrial diseases has
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been observed in germline therapy, where the mutant mtDNA is replaced by normal mtDNA in the zygote (Kyriakouli et al., 2008). Contemporary breakthroughs in CRISPR/cas9
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technology have also shown significant promise in germline therapy of mitochondrial pathologies. This strategy overcomes the ethical concerns associated with embryonic gene therapy by avoiding the necessity of a WT mtDNA donor. The CRISPR/Cas9 technique is
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easily programmable and facilitates targeting of single gene mutations in mtDNA (Fogleman
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et al., 2016). Recent studies have further utilized a specially engineered Cas9 called mitoCas9, which have localization specificity for the mitochondrial matrix, thereby averting off-target delivery (Gómez-Tatay et al., 2017). In another approach, a 2015 study by Mitalipov and co-
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authors at Oregon Health and Science University (USA) used genetically rectified pluripotent stem cells in patients harboring mtDNA mutations (Ma et al., 2015). Among other strategies,
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the hydrodynamic injection approach might show potential in mitochondria-targeted gene therapy, where a momentary increase of hydrostatic pressure in the target tissue due to hydrodynamic injection facilitates easy passage of a corrective DNA into the mitochondria (Nagata et al., 2014). Using this approach, mitochondrial anomalies in skeletal muscles of rats have been targeted by hydrodynamically injecting pcDNA3.1(+)-luc plasmid into the limb veins, which resulted in specific delivery of the corrective gene into the mitochondria with minimum and reversible damage to the muscle cells (Yasuzaki et al., 2013, 2015). Therefore, this strategy might be adopted to treat neuromuscular mitochondrial disorders, such as MERRF and MELAS. Although a few vagaries plague the current strategies, the need of the hour is to plunge deeper
ACCEPTED MANUSCRIPT into the available approaches, and to improvise them further in order to bolster their efficacies against myriad mitochondrial diseases. The ‘powerhouse’ of the cell is an indispensable facet of human health, which necessitates further research. The staggering task of curbing mitochondrial pathologies via gene therapy might then be translated from bench to bedside.
Conflict of interests
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The authors declare that there is no conflict of interest.
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Yu-Wai-Man, P., 2016. Genetic manipulation for inherited neurodegenerative diseases: myth or reality? Br. J. Ophthalmol. 100, 1322–1331. https://doi.org/10.1136/bjophthalmol2015-308329 Yu-Wai-Man, P., Chinnery, P.F., 1993. Leber Hereditary Optic Neuropathy, in: Pagon, R.A., Adam, M.P., Ardinger, H.H., Wallace, S.E., Amemiya, A., Bean, L.J., Bird, T.D., Ledbetter, N., Mefford, H.C., Smith, R.J., Stephens, K. (Eds.), GeneReviews(®). University of Washington, Seattle, Seattle (WA). Zhao, L., Wen, Q., Yang, G., Huang, Z., Shen, T., Li, H., Ren, D., 2016. Apoptosis induction of dehydrobruceine B on two kinds of human lung cancer cell lines through mitochondrial-dependent pathway. Phytomedicine 23, 114–122. https://doi.org/10.1016/j.phymed.2015.12.019 Zheng, J., Ji, Y., Guan, M.-X., 2012. Mitochondrial tRNA mutations associated with deafness. Mitochondrion 12, 406–413. https://doi.org/10.1016/j.mito.2012.04.001 Zhu, X., Peng, X., Guan, M.-X., Yan, Q., 2009. Pathogenic mutations of nuclear genes associated with mitochondrial disorders. Acta Biochim. Biophys. Sin. 41, 179–187.
ACCEPTED MANUSCRIPT Zsurka, G., Hampel, K.G., Nelson, I., Jardel, C., Mirandola, S.R., Sassen, R., Kornblum, C., Marcorelles, P., Lavoué, S., Lombès, A., Kunz, W.S., 2010. Severe epilepsy as the major symptom of new mutations in the mitochondrial tRNA(Phe) gene. Neurology 74, 507–512. https://doi.org/10.1212/WNL.0b013e3181cef7ab
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Zupančič, Š., Kocbek, P., Zariwala, M.G., Renshaw, D., Gul, M.O., Elsaid, Z., Taylor, K.M.G., Somavarapu, S., 2014. Design and development of novel mitochondrial targeted nanocarriers, DQAsomes for curcumin inhalation. Mol. Pharm. 11, 2334–2345. https://doi.org/10.1021/mp500003q
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Fig. 1. mtDNA loci map for mitochondrial pathologies. Respective mutations are denoted in parentheses. CPEO: chronic progressive external ophthalmoplegia; FBSN: familial bilateral striatal necrosis; KSS: Kearns-Sayre syndrome; LHON: Leber’s hereditary optic neuropathy; LS: Leigh syndrome; MEPR: myoclonic epilepsy and psychomotor regression; MELAS: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MERRF: myoclonic epilepsy with ragged-red fibers; MHCM: maternally inherited hypertrophic cardiomyopathy; MICM: maternally inherited cardiomyopathy; MID: maternally inherited deafness; MIDD: maternally inherited diabetes and deafness, NARP: neurogenic muscle weakness, ataxia, and retinitis pigmentosa.
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Fig. 2. Allotopic expression-mediated gene therapy. (A) Stable transfection of corrective gene in the nDNA: A recombinant DNA construct comprising of a promoter, a mitochondriatargeting sequence (MTS), the gene of interest (GOI) (encoding the corrective protein), a tag and a 3’-untranslated region (3’-UTR) is stably transfected into the nuclear DNA using a suitable vector. Post transcription, the mRNA is exported into the cytoplasm where it gets translated to yield the protein of interest (POI) fused with MTS, which directs the POI specifically into the mitochondria, thereby subsequently restoring mitochondrial functions. (B) Chemically modified mRNA (modRNA)-mediated therapy: A modRNA (containing the open reading frames of an MTS, a GOI, a tag and a 3’-UTR) is inserted into the cytoplasm. The modRNA, being an mRNA, is translated by the ribosome machinery of the cell to form a fused protein (MTS-POI). The translated MTS-conjugated POI enters specifically into the mitochondria, thereby functionally restoring them.
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Fig. 3. Gene therapy by selective inhibition of mutant mtDNA. (A) Restriction endonucleases (REs): A construct, which contains a suitable promoter, the gene for a mitochondria-targeting sequence (MTS) and a specific RE-encoding gene, is transiently transfected into the nucleus. On translation, the MTS-conjugated RE localizes to mitochondria and cuts (obliterates) the mutant (MT) mtDNA. (B) mtDNA-binding zinc-finger nucleases (mtZFNs): The construct contains a promoter, an MTS, a tag, a nuclear export signal (NES) and genes for a specific zinc-finger protein (ZFP) and a RE. It is transiently transfected into the nucleus. The translated MTS-ZFP-RE fusion protein localizes to the mitochondria, where the ZFP recognizes specific sequence in the MT mtDNA, while the fused RE subsequently cuts the mtDNA at the recognized site. (C) Mitochondrially targeted trancription activator-like effector (TALE) nucleases (mitoTALENs): Two monomeric constructs, each containing a promoter, an MTS, a tag, and genes encoding for TALE (left or right) and FokI nuclease, are transiently transfected into the nucleus. On translation, the fused proteins are delivered to the mitochondria by the MTS. Here, TALE (left) and TALE (right) from the corresponding fused proteins bind to specific sequences of the MT mtDNA, thereby flanking a specific site. The two FokI nuclease proteins dimerize and cut the flanked site, thereby destroying the MT mtDNA.
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Fig. 4. Gene therapy by delivery of wild type (WT) mtDNA. (A) Mitochondrial transcription factor A (TFAM): This carrier-based delivery system comprises of a mitchondrial transduction domain (MTD), which is a combination of a mitochondria-targeting sequence (MTS) and a protein transduction domain (PTD), conjugated with TFAM (MTDTFAM). While TFAM carries the WT mtDNA, PTD facilitates transport of the MTD-TFAM conjugate across the cell membrane. This is then followed by MTS-mediated delivery of the WT gene into the mitochondria harboring the mutant (MT) mtDNA. (B) Dual function MITOporters (DF-MITO-porters): These liposome-based nanocarriers deliver WT mtDNA to the mitochondria with the MT mtDNA. The DF-MITO-porters are two membrane-fusogenic systems comprising of outer and inner envelopes capable of fusing specifically with the endosomal and mitochondrial membranes respectively. DF-MITO-porters containing the WT mtDNA are endocytosed within the cell and are captured in the endosome. The outer envelope fuses with the endosomal membrane, subsequently triggering the escape of the WT mtDNA still packed within the inner envelope. Selective fusion of the inner envelope with the mitochondrial membrane releases the WT mtDNA inside the mitochondria. (C) DQAsomes: Liposome-like nanovesicles made of dequalinium chloride (DQA) carry WT mtDNA tagged with MTS (DQAsome-DNA-MTS complex) inside the cell. The MTS delivers the DQAsome to the mitochondria. On contact with the mitochondrial membrane, the DQA-coated membrane of the nanovesicle disintegrates and releases the WT mtDNA on to the mitochondria from where it is taken into the matrix via the protein import machinery.
ACCEPTED MANUSCRIPT Table 1: Major mitochondrial diseases arising from mutations in the mtDNA
# Mitochondrial disease
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tRNAAla ATP6, ATP8 ATP6 COII, COIII
ND1, ND4, ND6 tRNALeu, tRNAVal rRNA12s, tRNAThr, Auditory defects tRNALeu, tRNASer tRNALeu, Diabetes and hearing loss tRNALys, tRNAGlu tRNALeu, Neuromuscular disorder of the cardiac tRNAIle, tissue tRNALys, ND5
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Chronic progressive external Visual defects due to neurological ophthalmoplegia (CPEO) degeneration Metabolic disorder due defective ETC Complex V deficiency complex V Familial bilateral striatal necrosis Striatonigral degeneration in brain (FBSN) Neuromuscular disorder causing Kearns-Sayre syndrome (KSS) retinopathy, cardiomyopathy, deafness, ataxia and other defects Leber’s hereditary optic Visual defects due to mutant retinal neuropathy (LHON) ganglion cells (RGCs) Maternally inherited Neuromuscular disorder of the cardiac cardiomyopathy (MICM) tissue
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Maternally inherited deafness (MID)
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Maternally inherited diabetes and deafness (MIDD)
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Maternally inherited 9 hypertrophic cardiomyopathy (MHCM) Mitochondrial 1 encephalomyopathy, lactic 0 acidosis, and stroke-like episodes (MELAS) 1 Myoclonic epilepsy and 1 psychomotor regression (MEPR) 1 Myoclonic epilepsy with ragged2 red fibers (MERRF) Neurogenic muscle weakness, 1 ataxia, and retinitis pigmentosa 3 (NARP)
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2
Characteristics
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1
Mutant mtDNA gene(s)
Neuromuscular disorder
tRNAAsp
Neuromuscular disorder
tRNALys, tRNAPhe
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Neurodegenerative disorder
tRNALeu, tRNAVal, ND5, COIII
Neurodegenerative disorder arising from ATP6 defective F1FO-ATPase activity
COI, COII, Neuronal defects in the substantia nigra of cytochrome b, brain ND5, D-Loop Subacute necrotizing Neurodegenerative disorder due to defects tRNAIle, 1 encephalomyelopathy / Leigh in ETC complex I, complex IV and tRNATrp, ATP6, 5 syndrome (LS) pyruvate dehydrogenase complex ND5, COIII 1 Parkinson's disease (PD) 4
ACCEPTED MANUSCRIPT Highlights
Variegated mutations in the nuclear and/or mitochondrial DNA (mtDNA) give rise to a plethora of mitochondrial diseases.
Limited gene therapy options exist for mtDNA mutation-associated disorders in comparison to conditions arising from mutations in the nuclear DNA. Contemporary gene therapy strategies for correcting mtDNA mutations include
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allotopic expression of corrective proteins, site-specific ablation of mtDNA and carrier-
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There is a need for further exploration into novel strategies for improved efficacy and
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safety in gene therapy against mtDNA mutation-associated ailments.
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based delivery of wild-type mtDNA.
M. Aravintha Siva, R. Mahalakshmi, Dipita Bhakta-Guha, Gunjan Guha PII: DOI: Reference:
S1567-7249(18)30018-7 doi:10.1016/j.mito.2018.06.002 MITOCH 1292
To appear in:
Mitochondrion
Received date: Revised date: Accepted date:
27 January 2018 24 May 2018 7 June 2018
Please cite this article as: M. Aravintha Siva, R. Mahalakshmi, Dipita Bhakta-Guha, Gunjan Guha , Gene therapy for the mitochondrial genome: Purging mutations, pacifying ailments. Mitoch (2017), doi:10.1016/j.mito.2018.06.002
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ACCEPTED MANUSCRIPT Gene Therapy for the Mitochondrial Genome: Purging Mutations, Pacifying Ailments M. Aravintha Siva, R. Mahalakshmi, Dipita Bhakta-Guha* and Gunjan Guha* Cellular Dyshomeostasis Laboratory (CDHL), School of Chemical and Bio Technology, SASTRA University, Thanjavur 613 401. Tamil Nadu, India. *
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Corresponding authors:
Gunjan Guha
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CDHL, SCBT, SASTRA University, Thanjavur 613 401. Tamil Nadu, India. Email: [email protected]; [email protected] Phone: +91 4362 264101-108 (ext: 3777); +91 4362 304000-010 (ext: 3777).
Dipita Bhakta-Guha
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CDHL, SCBT, SASTRA University, Thanjavur 613 401. Tamil Nadu, India. Email: [email protected]; [email protected] Phone: +91 4362 264101-108 (ext: 3777); +91 4362 304000-010 (ext: 3777).
ACCEPTED MANUSCRIPT Abstract In the recent years, the reported cases of mitochondrial disorders have reached a colossal number. These disorders spawn a sundry of pathological conditions, which lead to pernicious symptoms and even fatality. Due to the unpredictable etiologies, mitochondrial diseases are putatively referred to as “mystondria” (mysterious diseases of mitochondria). Although present-day research has greatly improved our understanding of mitochondrial disorders,
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effective therapeutic interventions are still at the precursory stage. The conundrum becomes further complicated because these pathologies might occur due to either mitochondrial DNA
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(mtDNA) mutations or due to mutations in the nuclear DNA (nDNA), or both. While correcting nDNA mutations by using gene therapy (replacement of defective genes by delivering wild-
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type (WT) ones into the host cell, or silencing a dominant mutant allele that is pathogenic) has emerged as a promising strategy to address some mitochondrial diseases, the complications in
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correcting the defects of mtDNA in order to renovate mitochondrial functions have remained a steep challenge. In this review, we focus specifically on the selective gene therapy strategies
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that have demonstrated prospects in targeting the pathological mutations in the mitochondrial genome, thereby treating mitochondrial ailments.
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Keywords: Mitochondrial diseases; mystondria; mtDNA mutations; gene therapy.
ACCEPTED MANUSCRIPT Abbreviations 3′-UTR
3′-untranslated region
AAV
adeno-associated virus
AAV2/2
AAV serotype 2
AIF apoptosis-inducing factor COX
cytochrome c oxidase
COI cytochrome c oxidase subunit I cytochrome c oxidase subunit II
COIII
cytochrome c oxidase subunit III
COIV
cytochrome c oxidase subunit IV
COVIII
cytochrome c oxidase subunit VIII
CO-X
cytochrome c oxidase subunit X
CPEO
chronic progressive external ophthalmoplegia
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COII
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CRISPR/Cas9 clustered regularly-interspaced short palindromic repeats/CRISPRassociated protein 9 DA dopaminergic displacement loop
DQA
dequalinium chloride
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D-loop
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DF-MITO-porter dual function MITO-porter
DQAsomes DQA-based liposome-like nanovesicles enhanced green fluorescent protein
ETC
electron transport chain
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eGFP
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FLAG epitope tag DYKDDDDK sequence (where D = aspartic acid, Y = tyrosine, K = lysine) familial bilateral striatal necrosis
GFP
green fluorescence protein
HSP KSS LHON
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FBSN
heat shock protein Kearns-Sayre syndrome Leber’s hereditary optic neuropathy
LS Leigh syndrome MELAS
mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
MEMRI
manganese-enhanced magnetic resonance imaging
MEPR
myoclonic epilepsy and psychomotor regression
MERRF
myoclonic epilepsy with ragged-red fibers
MHCM
maternally inherited hypertrophic cardiomyopathy
MICM
maternally inherited cardiomyopathy
MID
maternally inherited deafness
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maternally inherited diabetes and deafness
mitoTALEN
mitochondrially targeted transcription activator-like effector nuclease
modRNA synthetic chemically modified mRNA mitochondrial transduction domain
mtDNA
mitochondrial DNA
MTS
mitochondria-targeting sequences
mtZFNs
mitochondrially targeted zinc-finger nucleases
NARP
neurogenic muscle weakness, ataxia and retinitis pigmentosa
NDI1
NADH-quinone oxidoreductase
nDNA
nuclear DNA
ORF
open reading frame
OriH
origin of replication for heavy strand of mtDNA
OriL
origin of replication for light strand of mtDNA
PD Parkinson’s disease
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OX-PHOS oxidative phosphorylation
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MTD
plasmid DNA
PICs
polyion complexes
RGCs
retinal ganglion cells
RNS
reactive nitrogen species
ROS
reactive oxygen species
SOD2
superoxide dismutase 2
TALE
transcription activator-like effector
TALEN
transcription activator-like effector nuclease
TFAM
mitochondrial transcription factor A
TFB1M
mitochondrial transcription factor B1
ZFP
zinc-finger protein
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ΔΨm
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pDNA
mitochondrial membrane potential
ACCEPTED MANUSCRIPT 1. Mitochondria: The dynamics of functions and dysfunctions Mitochondria are eukaryotic cell organelles that arguably evolved from a symbiotic relationship between aerobic microbes and primordial eukaryotic cells (Chan, 2006; Sagan, 1967; Selosse, 2011). They putatively produce energy (ATP) through oxidative phosphorylation (OX-PHOS) by transporting electrons across multiple internal complexes (mitochondrial complexes I-V) of an electron transport chain (ETC) (Das and Guha 2010;
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Kaurola et al., 2016). These double-membrane organelles maintain their redox potential by pumping protons across their inner membrane (Chan, 2006). Mitochondria are involved in
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metabolism of glucose (Krebs cycle) and fatty acids (β-oxidation), and are also associated with calcium homeostasis (Schon and Przedborski, 2011; Palomo and Manfredi, 2015).
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Furthermore, they play a crucial role in apoptosis, which is triggered by the release of cytochrome c and apoptosis-inducing factor (AIF) from the mitochondria into the cytoplasm
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(Frank et al., 2001; Herold et al., 2002; Faridi et al., 2016; Zhao et al., 2016). Mitochondrial redox reactions produce reactive oxygen species (ROS) (such as superoxides, peroxides,
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hydroxides and singlet oxygen) and reactive nitrogen species (RNS) (peroxynitrites and dinitrogen trioxide), which might impair the ETC and cell cycle progression (Das and Guha
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2010; Quijano et al., 2016).
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Human mitochondria contain their own circular, double-stranded genome –– the mitochondrial DNA (mtDNA), which is 16,569 bp in length and constitutes 37 genes (Holt et al., 1989; Van Trappen et al., 2007). It is extremely susceptible to oxidative damage due to its close proximity
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to the mitochondrial ROS generation machinery and the limited availability of DNA repair enzymes (for mtDNA) (Das and Guha, 2010; Lee and Wei, 2007). Moreover, the absence of introns or intergenic sequences and histones in the mtDNA makes it prone to oxidative damage
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(Das and Guha, 2010; DiMauro et al., 2013). The mtDNA encodes for 2 rRNAs, 22 tRNAs and 13 polypeptides –– one subunit of cytochrome bc1 complex (complex III): cytochrome b; two subunits of ATP synthase (complex V): ATP6 and ATP8; three subunits of cytochrome c oxidase (COX; complex IV): COI, COII and COIII; and seven subunits of NADH-ubiquinone oxidoreductase (complex I): ND1, ND2, ND3, ND4, ND4L, ND5 and ND6; while complex II (succinate dehydrogenase), on the other hand, is entirely encoded by the nuclear DNA (nDNA) (Holt et al., 1989; Taanman, 1999; Mishmar et al., 2003; DiMauro et al., 2013). mtDNA also has a non-coding region called the displacement loop (D-loop), containing the origins of replication (OriH and OriL sites) and transcription promoters (Taanman, 1999; Singh, 2013; Kambe and Miyata, 2016). Bioenergetic dyshomeostasis of the mitochondria and consequent
ACCEPTED MANUSCRIPT overproduction of free radicals in the ETC lead to initiation and accumulation of considerable damage to the mtDNA, which can cause a plethora of pathological conditions (Quijano et al., 2016). Such dysfunctional conditions are extensively observed in tissues with higher energy demand like muscles, liver, heart, kidney and brain (Leslie et al., 2016). Since mitochondria are inherited only maternally (Camus et al., 2012; Sato and Sato, 2013), defective mitochondria containing mutant mtDNA are also inherited by the offspring from the mother (Thajeb et al.,
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2006; Tuppen et al., 2010). Mitochondrial diseases constitute a variegated conglomeration of clinical facets and symptoms involving a multitude of diverse tissues and organs, which cause significant morbidity and mortality (Wong, 2007; Moggio et al., 2014; Steele et al., 2017;
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Davis et al., 2018).
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The number of reported cases of mitochondrial disorders has increased in the recent years –– up to one in five thousand children harbor mitochondrial pathologies (Haas et al., 2007). In
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fact, up to four thousand newborns are diagnosed with mitochondrial diseases every year in the United States alone (Tachibana et al., 2013). Similar prevalence of mitochondrial pathologies
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has also been reported from other continents (Nesbitt et al., 2014; Ng and Turnbull 2016; Australian Mitochondrial Disease Foundation 2017). However, mitochondrial diseases are still referred to as “mystondria” (mysterious diseases of mitochondria) due to their unpredictable
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genetic and mipigenetic (mitochondrial epigenetics) etiology (Singh, 2015). Impaired
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respiratory subunits, production of dysfunctional enzymes and leakage of electrons from the ETC (mainly from complexes I and III) cause overproduction of ROS (Tretter and Adam-Vizi 2004; Görlach et al., 2015), which hinder mitochondrial biogenesis, and also lead to lipid
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peroxidation and protein carbonylation, along with significant damage to the mtDNA (Shokolenko et al., 2009; Suzuki et al., 2010; Das and Guha, 2010; Kwiecien et al., 2014; Yao
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et al., 2015; Birch-Machin and Bowman, 2016). Such mitochondrial and cellular dyshomeostases result in myriad pathological conditions. For example, type I and type II diabetes mellitus patients reportedly have defective insulin-producing β cells (in the islets of Langerhans in pancreas) (Lenzen, 2008) due to damaged mtDNA, as well as dysfunctional mitochondrial proteins and lipids (Verdile et al., 2015; Montgomery and Turner 2015). The role of mitochondria has also been established in obesity, where saturated fat in cardiomyocytes was observed to cause mitochondrial dysfunctions, such as decreased rate of OX-PHOS, augmented levels of mitochondrial ROS, and depolarization of the mitochondrial membrane (Joseph et al., 2016).
ACCEPTED MANUSCRIPT The colossal number of mitochondrial disorders has been attributed to a multitude of mutations in the mtDNA (Table 1). It has to be noted that mitochondrial diseases do not only occur due to mtDNA mutations, but also due to nDNA aberrations, since a considerable part of the mitochondrial proteome is encoded by the nucleus (Zhu et al., 2009; Gorman et al., 2015; Viscomi et al., 2015). In this review, however, we focus on the gene therapy strategies targeting
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solely the pathological mutations in the mitochondrial genome.
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2. Pathological phenotypes and mtDNA mutations
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Due to the inherent functional complexity of the mitochondria, it is often onerous to attribute a mitochondrial pathology to a particular mtDNA mutation, as well as to restrict a mutation to one single ailment (Doyle and Chan, 2008; Iyer, 2013). However, few principal types of
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mutations have been frequently reported in the majority of mitochondrial diseases (Yamada et al., 2007) (Fig. 1). For example, an m.11778G>A point mutation in the ND4 gene of complex
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I is observed in Leber’s hereditary optic neuropathy (LHON), a frequently occurring optic neuropathy (1 in 25,000 individuals), which is predominant (with 4-5 times greater probability) in males
(Newman, 1993; Yu-Wai-Man and Chinnery, 1993; Chinnery et al., 2001;
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Chadderton et al., 2013). It is characterized by bilateral acute/subacute visual defects, with
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deleterious effects on retinal ganglion cells (RGCs) (Yu-Wai-Man and Chinnery, 1993; Carelli et al., 2004; Chadderton et al., 2013). Most LHON incidences (~95%) occur via three substitution mutations in the genes encoding for complex I at positions m.3460G>A,
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m.3697G>A and m.4171C>A (ND1), m.11778G>A (ND4) and m.14484T>C (ND6) in the
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mtDNA (Chinnery et al., 2001; Kim et al., 2002; Spruijt et al., 2007). Substitution mutations in mitochondrial ATP6 gene (m.8993T>G; and m.8993T>C in a small percentage of patients) have been attributed to cause neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) (Thorburn and Rahman, 1993; De Meirleir et al., 2004; Kabunga et al., 2015), which is a neurological syndrome with an estimated prevalence rate of 0.81/100,000 individuals (Orphanet, 2017). These mutations lead to the replacement of amino acid leucine by arginine in F1FO-ATPase, thereby altering its function (Thorburn and Rahman, 1993). Similar to NARP, incidences of m.8993T>G and m.8993T>C mutations in the ATP6 gene in mtDNA are also observed in Leigh syndrome (LS), (Thorburn and Rahman, 1993; De Meirleir et al., 2004; Praeter et al., 2014). LS (also known as subacute necrotizing encephalomyelopathy) is a progressive neurodegenerative disorder of the central nervous
ACCEPTED MANUSCRIPT system that primarily affects the spinal cord, basal ganglia, pons, cerebellum and thalamus, as well as causes demyelination of optic nerves (Westarp et al., 1992; Duff et al., 2015; Gerards et al., 2016). Other mtDNA mutations, such as m.4296G>A (tRNAIle) (Martikainen et al., 2013), m.5523T>G and m.5559A>G (tRNATrp) (Mkaouar-Rebai et al., 2009), m.13513G>A (ND5) (Shanske et al., 2008), m.13514A>G (ND5) (Bannwarth et al., 2013), m.9191T>C (ATP6) (Moslemi et al., 2005) and m.9478T>C (COIII) (Mkaouar-Rebai et al., 2011), are also
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reportedly associated with LS (Fig. 1). The ailment has an incidence rate of one in 40,000 live births (Lake et al., 2015). While the maximal onset of this disorder takes place at about 3–12 months of age (with ~50% of the patients dying by the age of 3 years), late onset has also been
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observed in adults (Rahman and Thorburn, 1993). LS is caused by energy deficiency in cells
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due to defective enzymes viz. ETC complex I, complex IV and pyruvate dehydrogenase complex. The symptoms of this disease include hypotonia, ataxia, psychomotor regression,
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dystonia, cranial nerve palsies, spasticity, infantile spasms, sensory neural hearing loss, persistent vomiting, dysphagia and respiratory problems like hyper- and hypoventilation
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(Finsterer, 2008; Sofou et al., 2014; Gerards et al., 2016). The m.8993T>G/C mutation is associated with complex V deficiency too (De Meirleir et al., 2004). In fact, the disease has been associated with the etiologies of NARP and LS (De Vivo,
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1993; National Organization for Rare Disorders, 2016; Rat Genome Database, 2017). Complex
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V deficiency is a very rare metabolic disorder, which is also caused by other mutations in the ATP6 and ATP8 genes (Auré et al., 2013; Kytövuori et al., 2016), such as m.8561C>G (in the overlap region of ATP6 and ATP8) (Kytövuori et al., 2016), m.8969G>A (in ATP6) (Burrage
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et al., 2014) and m.8529G>A (in ATP8) (Jonckheere et al., 2008) (Fig. 1). Myriad mtDNA mutations have been reported in the substantia nigra (a nucleus in midbrain)
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of individuals with Parkinson's disease (PD) (Lin et al., 2012; Coxhead et al., 2016), which is the second most prevalent geriatric neurodegenerative disorder in humans, especially affecting people above 50 years of age (Gibson et al., 2012; Pringsheim et al., 2014). It is characterized by the deterioration of dopaminergic (DA) neurons in the substantia nigra pars compacta and by the accumulation of Lewy bodies (Gibson et al., 2012). PD-associated mutations have been reported in COI, COII, cytochrome b, ND5 and the D-Loop region of the mtDNA. These lead to mitochondrial alterations like decreased complex I activity, excessive ROS production, consequent mtDNA damage and mitophagy, which are associated with the etiology of PD (Winklhofer and Haass, 2010; Moon and Paek, 2015).
ACCEPTED MANUSCRIPT An m.8344A>G mutation in the gene encoding tRNALys is the most common cause of myoclonic epilepsy with ragged-red fibers (MERRF) syndrome (Shoffner et al., 1990; Jacobs, 2003), a disease that commonly affects muscles and the central nervous system, and is characterized by the presence of myoclonus, myopathy and spasticity. It has been estimated to affect one in every 5,000 individuals worldwide (US National Library of Medicine, Genetics Home Reference, 2017). MERRF can also occur due to point mutations in the mtDNA tRNAPhe gene at nucleotide position 616 and is observed in skeletal muscles (Zsurka et al., 2010).
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Mutations (Fig. 1) in the encoding region of tRNALeu (m.3243A>G and m.3271T>C) and tRNAVal (m.1642G>A) have been reported in mitochondrial encephalomyopathy, lactic
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acidosis, and stroke-like episodes (MELAS) (Goto et al., 1990), and maternally inherited
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cardiomyopathy (MICM) (Silvestri et al., 1994; Santorelli et al., 1996, 1999; Dipchand et al., 2001). MELAS is also associated with mutations in the COIII (m.9957T>C) and ND5
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(m.12770A>G, m.13045A>C, m.13513G>A and m.13514A>G) genes (Tuppen et al., 2010; Hashimoto et al., 2015).
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mtDNA deletions have been encountered in the chronic progressive external ophthalmoplegia (CPEO) (Van Goethem et al., 2003; Sundaram et al., 2011) and Kearns-Sayre syndrome (KSS) (Shanske et al., 1990). A number of other mutations are also associated with a host of other
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mitochondrial disorders (Fig. 1), such as familial bilateral striatal necrosis (FBSN)
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(Thyagarajan et al., 1995), maternally inherited diabetes and deafness (MIDD) (Mangiafico et al., 2004), myoclonic epilepsy and psychomotor regression (MEPR) (Shtilbans et al., 1999), maternally inherited hypertrophic cardiomyopathy (MHCM) (Thambisetty and Newman,
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2004; Tuppen et al., 2010; Hashimoto et al., 2015), and maternally inherited deafness (MID)
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(Yang et al., 2011; Zheng et al., 2012; Hoptasz et al., 2014).
3. Gene therapy: Confronting mitochondrial pathologies Although contemporary research has considerably enhanced our understanding of the genetic and biochemical etiology of mitochondrial disorders, therapeutic interventions for the same have been preliminary at best. Nevertheless, with recent advances in technology, gene therapy has emerged as a potential strategy to address mitochondrial diseases, either by manipulating the nuclear genome or by targeting the mitochondrial paraphernalia (Weissig and Torchilin, 2001). Gene therapy is the replacement of a defective gene by delivering a corrective allele into the host cell, or silencing a dominant mutant allele that is pathogenic to the host (Kaufmann
ACCEPTED MANUSCRIPT et al., 2013; Choong, Baba, and Mochizuki 2015). Since a large number of mitochondrial disorders are rooted to mutations in the mtDNA (Fig. 1), an intervention can be achieved by corrective gene therapy against the defective/missing facets of the mitochondrial genome. This can be materialized by delivery of functional therapeutic genes as substitutes for the endogenous counterparts (Yamada et al., 2007; Kyriakouli et al., 2008; Heller, Brockhoff, and Goepferich 2012). For most mitochondrial gene therapies, one of the paramount criteria is the
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construction of suitable vectors to accommodate the genes of interest (Yasuzaki et al., 2015). Among a plethora of vectors, adenoviruses have been most commonly used (accounting for 23.3% of the trials) because of their high transduction efficiency (leading to high gene
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expression levels) and larger DNA load carrying capacity in comparison to other viruses (Ginn
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et al., 2013). Adeno-associated virus (AAV) has also been extensively used due to its broad spectrum of hosts (Gao et al., 2002; Wang et al., 2016). Herpes simplex, pox and vaccinia
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viruses have also been successfully used in gene therapy (Ginn et al., 2013). Mitochondrial gene therapy has also been accomplished by administration of the naked plasmid DNA (pDNA) in vivo resulting in production of mRNA in the mitochondria (Yasuzaki et al., 2015).
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Variegated modules of mitochondrial gene therapy have demonstrated positive results in a host of neuromuscular and metabolic disorders related to mitochondrial dyshomeostasis. However,
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although diverse in their characteristics, all applied strategies broadly follow one of the three major approaches: (1) allotopic expression (of mtDNA genes in the nucleus); (2) selective
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inhibition of mutant mtDNA; (3) delivery of WT mtDNA into the mitochondria.
3.1. Allotopic expression-mediated gene therapy
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Endosymbiotic gene transfer from the mitochondria to the nucleus is a ubiquitous ongoing evolutionary process that occurs at a substantial frequency (Timmis et al., 2004). This phenomenon is emulated for allotopic expression-mediated gene therapy for defective mtDNA genes for the integration of the corresponding corrective mitochondrial genes into the nucleus (Guy et al., 2002; Manfredi et al., 2002; Shokolenko et al., 2010; Cwerman-Thibault et al., 2015). Transport of the subsequently expressed protein into the mitochondria is facilitated by a mitochondria-targeting sequence (MTS; which helps in transporting a protein of interest into the mitochondria) (Shokolenko et al., 2010). nDNA-encoded proteins that are destined to be localized in the mitochondria contain an MTS at their N-terminal ends, which is recognized by mitochondrial protein import channels, thereby allowing protein import (Chin et al., 2018). Moreover, specific 3’- untranslated regions (3′-UTRs) are used to locate mRNAs on the
ACCEPTED MANUSCRIPT mitochondrial surface and help in co-translational import of these nDNA-encoded mitochondrial proteins (Kaltimbacher et al., 2006; Bonnet et al., 2007a). However, it has been recently reported that it is the choice of MTS that determines the efficiency of delivery of allotopically expressed proteins to the mitochondria, rather than the selected 3’-UTR (Chin et al., 2018). This method was first tested for its efficacy in mitochondrial ATP8 gene-deficient models of Saccharomyces cerevisiae, where the incorporation of the WT mtDNA gene in the
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nucleus resulted in successful restoration of function (Nagley et al., 1988). This strategy of integrating mtDNA genes in the nDNA has been applied to correct defects arising from several
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mtDNA mutations (Fig. 2A).
As a key strategy for treating LHON, allotopic expression-mediated gene therapy has been
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accomplished in vitro in cybrid cells (derived from homoplasmic osteosarcoma) harboring m.11778G>A mutation in the ND4 gene (Guy et al., 2002). Through this method, the WT ND4
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gene was integrated into the nucleus by recombinant AAV (rAAV) vector. The plasmid was constructed by fusing the ND4 gene with an MTS (from the P1 isoform of human ATP synthase
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subunit c), and a FLAG epitope tag. This sequence was then integrated with green fluorescence protein (GFP) via an internal ribosomal entry site (IRES) sequence, a chicken β-actin promoter and a cytomegalovirus enhancer. The final construct was cloned into pTR-UF12 plasmid vector
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and packaged to generate rAAV particles. The cybrid cells were then stably transfected with
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3×107 rAAV particles. The expression of the fused protein was driven by the chicken β-actin promoter and the enhancer. The transfected cells subsequently showed GFP and FLAG expression in the cytoplasm and mitochondria respectively. The FLAG localization to the
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mitochondria implied successful transport of the ND4 gene, which was further confirmed by the observed three-fold increase in ATP synthesis in the cybrid cells (Guy et al., 2002).
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In an in vivo LHON model (rats), the disease was developed using allotopic expression of mutant human ND4 gene (bearing m.11778G>A mutation) in the RGCs, thereby creating mitochondrial heteroplasmy (Ellouze et al., 2008), which is a condition where a single cell can harbor mitochondria with both WT and mutant genomes (Das and Guha, 2010; Wallace and Chalkia, 2013; Picard et al., 2014; Wernick et al., 2016). The WT ND4 gene was incorporated with pAAV-IRES-GFP and transformed pDNA was injected into the vitreous body of the eye through electroporation. The plasmid was constructed by fusion of CO-X (which is a nuclear gene) and ND4 into the pAAV-IRES-GFP vector. CO-X encodes for both MTS (present at the amino termini of pre-proteins) and the sequence for MTS cleavage (Omura, 1998; Ellouze et al., 2008). MTS helps to import ND4 proteins from cytoplasm into the mitochondria, while the
ACCEPTED MANUSCRIPT MTS cleavage sequence functions to generate the mature ND4 once the ND4-MTS complex is inside the mitochondria (Ellouze et al., 2008). GFP gene was used for transgene selection. The ND4-CO-X gene was integrated into the nuclear genome of RGC and the ND4 subunit protein was synthesized in the cytoplasm. With the help of MTS, ND4 was imported into the mitochondria, thus replacing the defective ND4, thereby restoring normal bioenergetics (Ellouze et al., 2008).
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In another approach, trans-kingdom mitochondrial gene therapy was accomplished by intraocular delivery of NDI1 (NADH-quinone oxidoreductase; a nuclear gene from S.
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cerevisiae that encodes for single subunit complex I) in mice. NDI1 gene contains endogenous MTS, which assists in efficient transport of the protein into the mitochondria (which is more
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effective than allotopic expression with introduced MTS) (Chadderton et al., 2013). AAV2 plasmid containing NDI1 (AAV2-NDI1) was constructed and delivered intravitreally into
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rotenone-treated (induces RGC loss and inhibits complex I) mice (Chadderton et al., 2013). Optokinetic assessment of the treated eye showed significant reduction in RGC death and optic
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nerve defects. Also, manganese-enhanced magnetic resonance imaging (MEMRI) exhibited improved functions of optic nerve by active transport of Mn+2 into the cells through voltagegated calcium channels (Chadderton et al., 2013; Marella et al., 2010). Observations up until
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six months post-treatment showed no remarkable amount of anti-NDI1 antibody in the sera
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(Marella et al., 2011), proving the potency of NDI1 for the replacement of defective complex I proteins. An added advantage of this approach is that it is not restricted to a particular mutation
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(Chadderton et al., 2013).
In an ongoing (2014–2019) clinical trial (NCT02161380) (Guy, 2018) at the Bascom Palmer Eye Institute (Miller School of Medicine, University of Miami, USA), AAV2-mediated gene
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therapy containing ND4 (AAV(scAAV)2(Y444,500,730F)-P1ND4v2) was done intravitreally in five patients with LHON (m.11778G>A mutation) (Feuer et al., 2016). The selfcomplementary genome improved the transgene expression, and the AAV2 capsid promoted efficient tissue-specific uptake (Koilkonda et al., 2010; Feuer et al., 2016). The patients were treated with differential doses of transformed vector genomes. The initial results of this clinical trial showed an improved vision in all the patients with none of them suffering from any severe side-effects. However, it has to be noted that the sampling size was very low and that the study is still recruiting patients (Feuer et al., 2016; Wan et al., 2016; Yang et al., 2016). Another set of recent prospective clinical trials (NCT02064569, NCT02652767, NCT02652780, NCT03406104 and NCT03293524) are testing the efficacy and safety of GS010 (rAAV2/2-
ACCEPTED MANUSCRIPT ND4) in patients affected by ND4 mutations, the results of which are currently unavailable (Newman, 2018; Subramanian, 2018; Vignal, 2018; Yu-Wai-Man, 2018a, 2018b). As discussed earlier (section 2), the m.8993T>G substitution mutation in the mitochondrial ATP6 gene is extremely common in NARP, LS and complex V deficiency (De Meirleir et al., 2004). To develop an allotopic expression-mediated gene therapy strategy to correct this mutation, skin biopsies from an 8-month old child with NARP syndrome (m.8993T>G
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mutation) were used for establishing fibroblast culture as the experimental model (Bonnet et al., 2007a). For effective localization of ATP6 mRNA into the mitochondria, the MTS sequence
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of superoxide dismutase 2 (SOD2) gene and its 3′-UTR were combined with the ATP6 gene (Bonnet et al., 2007a, 2007b). The transfection plasmid was constructed by inserting ATP6
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gene (with a FLAG epitope at the C terminus of the ATP6 ORF) into pCMV-Tag 4A vector, along with the MTS of the SOD2 gene and either the 3′-UTR of SOD2 or the 3′-UTR of SV40,
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thereby giving rise to the two following final constructs: SOD2MTSATP6-3′UTRSOD2 and SOD2MTSATP6-3′UTRSV40 (Kaltimbacher et al., 2006). It was observed that when both MTS
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and the 3′-UTR sequence were present (i.e., SOD2MTSATP6-3′UTRSOD2), ATP6 mRNA sorting to the mitochondrial surface was augmented, leading to 1.8 times higher ATP6 protein expression, than that with the MTS alone (i.e., SOD2MTSATP6-3′UTRSV40). SOD2-ATP6 co-
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localization was observed by indirect immunofluorescence using antibodies against FLAG or
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ATP synthase (-subunit). Complex V activity was evidently restored using this gene therapy approach, which was also verified by the increased production of ATP (Bonnet et al., 2007a).
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Another similar strategy was developed using a B6(B6SJLF1) (mice) model (Dunn and Pinkert, 2012). First, WT ATP6 genes were cloned in frame into the pEF/myc/mito plasmid, containing the promoter for human EF-1α, the MTS from the human COVIII gene (nuclear), and an in-
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frame 3’ myc epitope tag. This pEF/myc/mito/ATP6 plasmid was used to generate transgenic mice. Mitochondrial localization of allotopically expressed corrective ATP6 protein was confirmed by electron microscopy (Dunn and Pinkert, 2012). In a recent study on mtDNA complex V-deficient patient cells (null for the ATP8 protein, with significantly less ATP6 expression) harboring the m.8529G>A mutation (in the mitochondrial ATP8 gene) (Boominathan et al., 2016), researchers allotopically expressed ATP6 and ATP8 genes in the nucleus to correct the deficiency using a pCMV6 vector. Corrective DNA sequences for ATP6 and ATP8 were incorporated into the vector between the MluI and XhoI sites. A CO-X or ATP5G1- or ATP5G2-based MTS was also inserted into the pCMV6 at the 5′ end (between sites AsiSI and MluI) of the genes. Myc and FLAG or V5 epitope were also
ACCEPTED MANUSCRIPT introduced immediately downstream of the codon-corrected genes. This technique rescued expression of both the proteins and recovered their functions in the cell significantly (Boominathan et al., 2016). A strategy for allotopic expression of mitochondrial proteins without altering the nuclear genome uses synthetic chemically modified mRNA (modRNA) encoding for the corrective protein (Fig. 2B), whose uridine and cytosine have been substituted respectively by
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pseudouridine and 5-methyl-cytosine (Chin et al., 2018). These modifications allow the modRNA to avoid the Toll-like receptors of innate immune system (that would have otherwise
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degraded them) in vivo while facilitating efficient translation of the protein of interest (Karikó et al., 2005). A report published in 2018 has demonstrated allotopic expression of
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mitochondrial proteins by using modRNAs. In this study, the researchers constructed modRNAs for allotopic expression of MTS-ATP6 fusion protein in ATP6-mutant cybrids
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(harboring the m.8993T>G mutation), which consequently led to improved mitochondrial
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functions and cell survival (Chin et al., 2018).
3.2. Gene therapy by selective inhibition of mutant mtDNA
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Mitochondrial heteroplasmy is implicated in various mitochondrial disorders including
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mitochondrial myopathy, MERRF, and MELAS (Ye et al., 2014). In such a state, a certain threshold of mutant mtDNA gene(s) will be required to elicit the corresponding disease condition(s) (Taylor et al., 1997; Schon et al., 2012). A prudent strategy to alleviate such
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diseases is to selectively inhibit mutant mtDNA (Taylor et al., 1997; Russell and Turnbull, 2014), thereby mitigating the negative effects of heteroplasmy. This approach has shown
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promise in treating mitochondrial ailments that arise due to mutations in both protein- and RNA-coding genes (Tanaka et al., 2002; Hashimoto et al., 2015). In section 3.1, we have discussed how alloptopic expression-mediated gene therapy has found success in correcting the m.8993T>G mutation, which is involved with NARP, LS and complex V deficiency (De Meirleir et al., 2004) (as discussed in section 2). Gene therapy by selective inhibition of mutant mtDNA has also been used as a potent strategy to inhibit the effects of this substitution mutation in the mtDNA. One approach to reach this goal is to use mitochondriadirected restriction endonucleases specific for unique mutations in the mutant mtDNA (Fig. 3A). For this, a group of researchers (Tanaka et al., 2002) at the Gifu International Institute of Biotechnology in Japan transiently expressed the restriction endonuclease SmaI (fused with
ACCEPTED MANUSCRIPT MTS) in cells harboring the m.8993T>G mutation in a heteroplasmic state. SmaI selectively cleaved mutant mtDNA at the guanine residue (CCCGGG) of m.8993, while not affecting the thymine residue (CCCTGG) of m.8993 in the WT mtDNA. The same strategy was also applied by using EcoRI (another restriction endonuclease) instead of SmaI. The plasmid for transfection was constructed by fusing the cDNA encoding pCOIV (pre-sequence of cytochrome c oxidase subunit IV from S. cerevisiae) with pMACSKKII (mammalian expression vector) and either EcoRI or SmaI. The resultant gene therapy vectors were
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pMACSKKII-pCOIV-EcoRI and pMACSKKII-pCOIV-SmaI (Tanaka et al., 2002). pCOIV guided the delivery of restriction enzymes selectively into the mitochondria, hence avoiding
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cleavage of the nDNA. Interestingly, while SmaI required five transfection and selection cycles
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to successfully eliminate the mutant mtDNA, EcoRI needed only one to two cycles (probably due to differential numbers of recognition sites for the restriction enzymes) (Tanaka et al.,
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2002). Another restriction endonuclease, XmaI, has also been used to correct the m.8993T>G mutation by selectively targeting mutant mtDNA (Alexeyev et al., 2008).
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The research group of Prof. Carlos T. Moraes at the Miller School of Medicine (University of Miami, USA) has done some comprehensive work in using restriction endonucleases to obliterate mutant mtDNA. In an in vitro study (Srivastava and Moraes, 2001), they expressed
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mitochondria-targeted PstI in murine cells harboring heteroplasmy of mtDNA (the mutant
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containing two PstI-specific sites). In another study (Bayona-Bafaluy et al., 2005), performed both in vivo (brain and muscle of heteroplasmic mice) and in vitro (heteroplasmic mice hepatocytes), they transfected ApaLI fused with MTS of COVIII to selectively ablate the
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mutant mtDNA containing ApaLI-specific sites. The group later demonstrated that systemic delivery of viral vectors containing MTS-fused ApaLI to specific organs accomplished in
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treating mtDNA-associated disorders (Bacman et al., 2010, 2012). In yet another study, the same group has demonstrated application of mitochondria-targeted ScaI restriction endonuclease to scythe mutant mtDNA, thereby mitigating heteroplasmy-associated dyshomeostasis (Bacman et al., 2007). Although the restriction endonuclease approach has shown significant success, it is unfortunately limited to only a small number of mtDNA mutations with unique restriction sites, which are recognized by these enzymes (Choo and Klug, 1994). To avoid this disadvantage, mtDNA-binding zinc-finger nucleases (mtZFNs) have been engineered, which have preset selectivity for specific mtDNA sequence(s) (Kim et al., 1996; Isalan et al., 2001; Papworth et al., 2006; Minczuk et al., 2006; Gammage et al., 2016). mtZFNs are chimeric enzymes
ACCEPTED MANUSCRIPT containing a Cys2His2 zinc-finger protein (ZFP), which is conjugated with an MTS and the catalytic domain of restriction endonucleases (such as FokI), along with nuclear export signal (NES) peptides. This ensures their localization exclusively in the mitochondria (Minczuk et al., 2006; Gammage et al., 2016). This strategy demonstrated that mtZFNs can specifically localize to the mitochondria when combined with MTS, and can selectively cleave mutant mtDNA (Fig. 3B). The approach was successfully used in reducing the levels of mutant
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mtDNA harboring the m.8993T>G mutation (Gammage et al., 2016), which is associated with NARP, LS and complex V deficiency (De Meirleir et al., 2004). The mtZFN-based gene therapy also restored ATP homeostatsis and ΔΨm on selective removal of the mutant mtDNA
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(Gammage et al., 2016).
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Apart from restriction endonucleases and mtZFNs, there is another potent gene therapy strategy for selective elimination of mutant mtDNA in a heteroplasmic situation. This involves
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designing mitochondrially targeted transcription activator-like effector (TALE) nucleases (mitoTALENs), which are novel nucleases (containing FokI catalytic domains) that can
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selectively target and cleave mutant mtDNA (Hashimoto et al., 2015; Gammage et al., 2016; Gómez-Tatay et al., 2017) (Fig. 3C). TALENs work as heterodimers, which need two TALEN monomers to bind closely-spaced DNA sequences, allowing their FokI nuclease domains to
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dimerize and cleave the flanked sequence of the bound DNA (Bacman et al., 2013). In addition
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to the work done in restriction endonuclease-based gene therapy to obliterate mutant mtDNA, Prof. Moraes and his group have also worked on designing mitoTALENs, which demonstrated significant decline in the number of mutant mtDNA molecules in patient–derived cells
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(Bacman et al., 2013). This strategy was further applied by the group in reducing mtDNA with m.8344A>G (tRNALys) and m.13513G>A (ND5) mutations (in cybrids), which are respectively
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associated with MERRF and MELAS (Jacobs, 2003; Hashimoto et al., 2015). The researchers constructed plasmids coding for mitoTALEN monomers. They removed any nuclear localization signal from the construct, while incorporating (i) an SOD2- or COVIIIA/Su9derived MTS, (ii) a FLAG or hemagglutinin immuno-tag, (iii) a 3′-UTR, (iv) a fluorescent marker (eGFP or mCherry), and (v) a recoded picornaviral 2A-like sequence (T2A) between the mitoTALEN and the fluorescent marker. MitoTALENs showed significant mitigation in the levels of the mutant mtDNA (harboring m.8344A>G or m.13513G>A), along with considerable recovery in respiratory capacity and OX-PHOS efficiency (Hashimoto et al., 2015).
ACCEPTED MANUSCRIPT 3.3. Gene therapy via delivery of WT mtDNA Targeting WT mtDNA (containing the corrective genes) directly into the mitochondrion has emerged as a potent strategy to rectify pathogenic mtDNA mutations (Vaidya et al., 2009; Yu et al., 2012). Carrier-based delivery systems, such as mitochondrial transduction domain (MTD) conjugated to mitochondrial transcription factor A (MTD-TFAM), MITO-porters and DQAsomes, have shown great promise in this mitochondriotropic approach (Niazi et al., 2013).
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However, the paramount challenge still remains in understanding the mechanism of nucleic acid import into the mitochondria (Weber-Lotfi et al., 2015). Nevertheless, several mtDNA
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mutations, such as m.8993T>G and m.11778G>A, have been successfully tackled by
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employing this strategy (Iyer et al., 2012).
In an in vivo study (Yu et al., 2015), transgenic mice were generated as a disease model for
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LHON by introducing mutant human ND4 (mutND4) gene (m.11778G>A) into the mice mtDNA through rAAV (containing self-complementary sc-HSP-mutND4-FLAG+mCherry).
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The AAV vector was modified by appending a COVIII presequence that facilitated its localization directly to the mitochondria, rather than the nucleus. This strategy caused the mutant ND4 gene to assemble in the mitochondrial complex I, thereby causing visual loss in
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the mice. The condition was found to be reversed when the animals were treated with
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mitochondria-targeted self-complementary AAV serotype 2 (scAAV2) containing the WT human ND4 (scAAV2-HSP-ND4) (Yu et al., 2015). The m.11778G>A mutation has also been successfully rectified in SH-SY5Y cells using the
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protein transduction (protofection) technique (Iyer et al., 2009, 2012). In this approach, mtDNA (corrective, WT) cargo is delivered into the mitochondria using engineered protein
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vectors, such as recombinant TFAM conjugated with MTD (combination of an N-terminal protein transduction domain and the MTS of SOD2) (Khan and Bennett, 2004; Iyer et al., 2009; Keeney et al., 2009; Thomas et al., 2011; Iyer et al., 2012; Iyer, 2013). In the MTD-TFAM conjugate, TFAM carries the WT mtDNA cargo, the protein transduction domain facilitates conveyance through the cell membrane, and the MTS ensures mitochondriotropic targeting and uptake (Fig. 4A). This has reportedly corrected the mtDNA mutation and significantly enhanced mitochondrial function (Iyer et al., 2009). This strategy has also shown success in correcting the m.8993T>G mutation in the ATP6 gene, which is involved with the etiologies of NARP, LS and complex V deficiency (De Meirleir et al., 2004). MTD-TFAM conjugated with WT mtDNA improved mitochondrial functions of fibroblast-derived cybrid cells with 95% of mitochondrial genomes bearing the m.8993T>G mutation (Iyer et al., 2012).
ACCEPTED MANUSCRIPT Furthermore, in an in vitro model for PD (cybrids generated by fusing SH-SY5Y ρ0 cells with platelets obtained from PD patients) with reduced complex I activity, MTD-TFAM-mediated delivery of corrective mtDNA into the mitochondria showed increased mitochondrial gene expression with augmented mitochondrial functions (Keeney et al., 2009). Yet another evolving strategy for delivery of WT mtDNA into mitochondria (harboring mutant genomes) is by use of MITO-porters, which are liposome-based nanocarriers that can deliver
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nucleic acids, proteins or drugs selectively to the mitochondria via a membrane-fusion mechanism (Niazi et al., 2013; Yamada and Harashima, 2015; Ishikawa et al., 2018). These
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MITO-porters can be coated with two membrane-fusogenic outer envelopes to produce the dual function MITO-porters (DF-MITO-porters) (Yamada, 2014). After internalization into
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cells, the outer envelope of the DF-MITO-porters fuses with the endosomal membranes (the MITO-porter thus escaping from the endosome sans the outer envelope), while the inner one
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fuses with the mitochondrial membranes, thereby delivering the cargo exclusively into the mitochondria (Yamada et al., 2011; Niazi et al., 2013; Yamada, 2014; Yamada and Harashima,
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2015) (Fig. 4B). This approach has accomplished in delivering a variety of macromolecules into the mitochondria (Yamada et al., 2008; Yamada and Harashima, 2012; Yamada, 2014; Yamada and Harashima, 2015; Ishikawa et al., 2018). A recent study has reported successful
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delivery of WT ND4 gene into the mitochondria in both in vitro (HeLa) and in vivo (C57BL/6
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mice) systems (Ishikawa et al., 2018). The researchers designed a construct containing human ND4 gene (pCMV-mtLuc (CGG) [ND4]) and transfected it into the mitochondria by using a MITO-porter (Ishikawa et al., 2018; Yamada et al., 2017) that is conjugated with a DNA-
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binding a cationic amphipathic peptide called KALA, which causes membrane destabilization and facilitates DNA transfection (Wyman et al., 1997). These results make it prudent to explore
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further in the precincts of the MITO-porter systems to develop novel gene therapy strategies for delivery of WT mtDNA into the mitochondria. Another significant approach for delivery of nucleic acids into the mitochondria involves the application of dequalinium chloride (DQA) as a targeted mitochondrial vesicular delivery system. DQA forms cationic liposome-like nanovesicles (DQAsomes) at concentrations above the critical vesicular values (Weissig et al., 2000; Weissig, 2015). These mitochondriotropic nanocarriers directly transfer exogenous DNA, peptides, oligonucleotides, nucleic acids, pDNA and low molecular weight drugs into the mitochondria (Weissig, 2015) (Fig. 4C). Exogenous DNA along with an MTS can be combined with the DQAsome to form a DQAsome-DNA-MTS complex, which might then be injected into the host cell (D’Souza et
ACCEPTED MANUSCRIPT al., 2005; Lyrawati et al., 2011). Its contact with the mitochondrial membrane results in cleaving of the conjugate from the DQAsome and its selective accumulation on mitochondria. This selectivity is ascribed to the difference in the mitochondrial membrane potential (ΔΨm) and charge gradient across the mitochondrial matrix. The splitting is followed by its entry into the matrix with the help of protein import machinery (Weissig et al., 2000; Weissig and D’Souza, 2004). Hence, DQAsome-mediated transfection of functional mtDNA genes into the
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mitochondria is an effective technique of gene therapy.
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4. Epilogue: The promises and pitfalls of mitochondrial gene therapy Gene therapy is currently the ‘closest to cure’ approach in the treatment of mitochondrial ailments (Doyle and Chan, 2008). Nevertheless, its strategies have their own share of
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impediments, such as concerns regarding the safety of different modes of therapies, stability of the introduced gene(s) over generations, actual in vivo efficacy and the extent to which it can
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be applied in practice (Kagawa and Hayashi, 1997; Doyle and Chan, 2008). The complexity in the genotype-phenotype relationship and the process of stochastic segregation of mitochondria make it difficult to diagnose the specific disorder, thereby rendering further complications to
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design therapeutic approaches using gene therapy (Kagawa and Hayashi, 1997; Doyle and
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Chan, 2008). Moreover, although about 67% of contemporary clinical trials use viral vectors, their immunogenicity and carcinogenicity pose a major challenge (Kamimura et al., 2015). To circumvent these plausible dire outcomes, application of liposome- or nanoparticle-based
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delivery systems are gaining significance. However, limited cargo-carrying capacity, low and transient gene expressions of these non-viral delivery systems, plague their ubiquitous use (Yu-
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Wai-Man, 2016). Also, for carrying out prospective clinical trials, extensive heterogeneity of mtDNA mutations in patients make it a challenging exercise to perform appropriate sampling of patient groups with significant homogeneity with respect to their genetic disorders, disease progression, biochemical facets and mtDNA mutation load (Kanabus et al., 2014; Rahman, 2015). Individual gene therapy strategies have also been marred with several problems. For example, allotopic expression-mediated gene therapy strategy, although proficient, often gets limited by the need of effective mitochondrial import and accurate incorporation of the allotopically expressed protein (Kyriakouli et al., 2008). Moreover, it has been recently shown that constructs used to deliver allotopically expressed proteins into the mitochondria often result in
ACCEPTED MANUSCRIPT mitochondrial fragmentation (Chin et al., 2018). The hurdles in transition of present gene therapy approaches from laboratory to clinic are, however, not limited to allotopic expressionmediated gene therapy only. For instance, in gene therapy via RE-mediated selective ablation of mutant mtDNA, a major issue is that only a handful of mutations harbor specific restriction sites for known REs (Nightingale et al., 2016). In the mtZFN approach (which moderately overcomes the above-mentioned problem by using ZFPs to bind to a specific DNA sequence),
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occurrence of cytotoxicity has been detrimental in molding this approach into a safe clinical intervention tactic (Nightingale et al., 2016). Also, the RE FokI, which is commonly used in this strategy, is known to dimerize, which in turn triggers off-target binding and subsequent
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cleavage (Ramalingam et al., 2011). In case of mitoTALENs, a pertinent problem lies in the
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fact that, owing to their large size and requirement of two monomers, it is often difficult to pack the large cargo into a suitable vector (Hashimoto et al., 2015). In case of carrier-based
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systems such as MITO-porters, selective delivery of the cargo into the mitochondria has been reported to be difficult, particularly during delivery of hydrophobic molecules or macromolecules (Yamada et al., 2011). Also, it has to be noted that most of these strategies
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have been predominantly tested in cell-based systems and/or animal models. Their individual efficacy in the clinical scenario still needs to be ascertained.
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Despite the enormous promise that gene therapy offers, it is indispensible to explore further for
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novel approaches to invoke safer and more efficient strategies to combat mitochondrial conundrums. Although more than five hundred mtDNA mutations are reportedly associated with mitochondrial diseases (Ye et al., 2014), the availability of gene therapy is restricted to
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only a handful of mutations, as discussed in section 3. For instance, although substitution mutations in the mitochondrial genome, such as m.4296G>A (tRNAIle) (Martikainen et al.,
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2013), m.5523T>G and m.5559A>G (tRNATrp) (Mkaouar-Rebai et al., 2009), m.9185T>C and m.9191T>C (ATP6) (Moslemi et al., 2005), m.9478T>C (COIII) (Mkaouar-Rebai et al., 2011), m.13513G>A and m.13514A>G (ND5) (Bannwarth et al., 2013) are associated with LS, gene therapy exists only for the commonly reported m.8993T>G (ATP6) mutation (Iyer et al., 2012). MELAS, which is caused by m.3243A>G (tRNALeu), m.1642G>A (tRNAVal), m.9957T>C (COIII), m.12770A>G (ND5), m.13045A>C (ND5), m.13514A>G (ND5), and m.3271T>C (tRNALeu) mutations (Tuppen et al., 2010), has so far only received gene therapy for the m.13513G>A (ND5) mutation (Hashimoto et al., 2015). In the case of LHON, gene therapy for mutations like m.3697G>A (ND1) (Spruijt et al., 2007), m.14484T>C (ND6), m.3460G>A (ND1) (Chinnery et al., 2001), and m.4171C>A (ND1) (Kim et al., 2002) is still speculative at best. Mutations implicated in complex V deficiency, such as m.8561C>G (in the overlap region
ACCEPTED MANUSCRIPT of ATP6 and ATP8) (Kytövuori et al., 2016) and m.8969G>A (in ATP6) (Burrage et al., 2014), are yet to have an efficient mode of gene therapy. In spite of the diverse hurdles, mitochondrial gene therapy has evolved exponentially in the recent years owing to the emergence of myriad novel approaches. For example, in polyion complexes (PICs), where methylated carrier molecules (such as poly-L-histidine) containing DNA for the effective transgene expression were used, higher gene expressions were reported
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(Asayama et al., 2015). Another emerging technique involves the use of DQAsomes for gene therapy, where augmented in vivo efficacy has been observed, which might be due to the fact
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that DQAsomes are not disintegrated by the plasmalemmal anionic lipids (i.e., they do not release the DNA during endocytosis) (Zupančič et al., 2014; Weissig, 2015). Furthermore, they
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strongly bind to the exogenous DNA and protect them from nuclease digestion (Zupančič et al., 2014; Weissig, 2015). Yet another advancement in tackling mitochondrial diseases has
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been observed in germline therapy, where the mutant mtDNA is replaced by normal mtDNA in the zygote (Kyriakouli et al., 2008). Contemporary breakthroughs in CRISPR/cas9
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technology have also shown significant promise in germline therapy of mitochondrial pathologies. This strategy overcomes the ethical concerns associated with embryonic gene therapy by avoiding the necessity of a WT mtDNA donor. The CRISPR/Cas9 technique is
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easily programmable and facilitates targeting of single gene mutations in mtDNA (Fogleman
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et al., 2016). Recent studies have further utilized a specially engineered Cas9 called mitoCas9, which have localization specificity for the mitochondrial matrix, thereby averting off-target delivery (Gómez-Tatay et al., 2017). In another approach, a 2015 study by Mitalipov and co-
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authors at Oregon Health and Science University (USA) used genetically rectified pluripotent stem cells in patients harboring mtDNA mutations (Ma et al., 2015). Among other strategies,
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the hydrodynamic injection approach might show potential in mitochondria-targeted gene therapy, where a momentary increase of hydrostatic pressure in the target tissue due to hydrodynamic injection facilitates easy passage of a corrective DNA into the mitochondria (Nagata et al., 2014). Using this approach, mitochondrial anomalies in skeletal muscles of rats have been targeted by hydrodynamically injecting pcDNA3.1(+)-luc plasmid into the limb veins, which resulted in specific delivery of the corrective gene into the mitochondria with minimum and reversible damage to the muscle cells (Yasuzaki et al., 2013, 2015). Therefore, this strategy might be adopted to treat neuromuscular mitochondrial disorders, such as MERRF and MELAS. Although a few vagaries plague the current strategies, the need of the hour is to plunge deeper
ACCEPTED MANUSCRIPT into the available approaches, and to improvise them further in order to bolster their efficacies against myriad mitochondrial diseases. The ‘powerhouse’ of the cell is an indispensable facet of human health, which necessitates further research. The staggering task of curbing mitochondrial pathologies via gene therapy might then be translated from bench to bedside.
Conflict of interests
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The authors declare that there is no conflict of interest.
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Fig. 1. mtDNA loci map for mitochondrial pathologies. Respective mutations are denoted in parentheses. CPEO: chronic progressive external ophthalmoplegia; FBSN: familial bilateral striatal necrosis; KSS: Kearns-Sayre syndrome; LHON: Leber’s hereditary optic neuropathy; LS: Leigh syndrome; MEPR: myoclonic epilepsy and psychomotor regression; MELAS: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MERRF: myoclonic epilepsy with ragged-red fibers; MHCM: maternally inherited hypertrophic cardiomyopathy; MICM: maternally inherited cardiomyopathy; MID: maternally inherited deafness; MIDD: maternally inherited diabetes and deafness, NARP: neurogenic muscle weakness, ataxia, and retinitis pigmentosa.
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Fig. 2. Allotopic expression-mediated gene therapy. (A) Stable transfection of corrective gene in the nDNA: A recombinant DNA construct comprising of a promoter, a mitochondriatargeting sequence (MTS), the gene of interest (GOI) (encoding the corrective protein), a tag and a 3’-untranslated region (3’-UTR) is stably transfected into the nuclear DNA using a suitable vector. Post transcription, the mRNA is exported into the cytoplasm where it gets translated to yield the protein of interest (POI) fused with MTS, which directs the POI specifically into the mitochondria, thereby subsequently restoring mitochondrial functions. (B) Chemically modified mRNA (modRNA)-mediated therapy: A modRNA (containing the open reading frames of an MTS, a GOI, a tag and a 3’-UTR) is inserted into the cytoplasm. The modRNA, being an mRNA, is translated by the ribosome machinery of the cell to form a fused protein (MTS-POI). The translated MTS-conjugated POI enters specifically into the mitochondria, thereby functionally restoring them.
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Fig. 3. Gene therapy by selective inhibition of mutant mtDNA. (A) Restriction endonucleases (REs): A construct, which contains a suitable promoter, the gene for a mitochondria-targeting sequence (MTS) and a specific RE-encoding gene, is transiently transfected into the nucleus. On translation, the MTS-conjugated RE localizes to mitochondria and cuts (obliterates) the mutant (MT) mtDNA. (B) mtDNA-binding zinc-finger nucleases (mtZFNs): The construct contains a promoter, an MTS, a tag, a nuclear export signal (NES) and genes for a specific zinc-finger protein (ZFP) and a RE. It is transiently transfected into the nucleus. The translated MTS-ZFP-RE fusion protein localizes to the mitochondria, where the ZFP recognizes specific sequence in the MT mtDNA, while the fused RE subsequently cuts the mtDNA at the recognized site. (C) Mitochondrially targeted trancription activator-like effector (TALE) nucleases (mitoTALENs): Two monomeric constructs, each containing a promoter, an MTS, a tag, and genes encoding for TALE (left or right) and FokI nuclease, are transiently transfected into the nucleus. On translation, the fused proteins are delivered to the mitochondria by the MTS. Here, TALE (left) and TALE (right) from the corresponding fused proteins bind to specific sequences of the MT mtDNA, thereby flanking a specific site. The two FokI nuclease proteins dimerize and cut the flanked site, thereby destroying the MT mtDNA.
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Fig. 4. Gene therapy by delivery of wild type (WT) mtDNA. (A) Mitochondrial transcription factor A (TFAM): This carrier-based delivery system comprises of a mitchondrial transduction domain (MTD), which is a combination of a mitochondria-targeting sequence (MTS) and a protein transduction domain (PTD), conjugated with TFAM (MTDTFAM). While TFAM carries the WT mtDNA, PTD facilitates transport of the MTD-TFAM conjugate across the cell membrane. This is then followed by MTS-mediated delivery of the WT gene into the mitochondria harboring the mutant (MT) mtDNA. (B) Dual function MITOporters (DF-MITO-porters): These liposome-based nanocarriers deliver WT mtDNA to the mitochondria with the MT mtDNA. The DF-MITO-porters are two membrane-fusogenic systems comprising of outer and inner envelopes capable of fusing specifically with the endosomal and mitochondrial membranes respectively. DF-MITO-porters containing the WT mtDNA are endocytosed within the cell and are captured in the endosome. The outer envelope fuses with the endosomal membrane, subsequently triggering the escape of the WT mtDNA still packed within the inner envelope. Selective fusion of the inner envelope with the mitochondrial membrane releases the WT mtDNA inside the mitochondria. (C) DQAsomes: Liposome-like nanovesicles made of dequalinium chloride (DQA) carry WT mtDNA tagged with MTS (DQAsome-DNA-MTS complex) inside the cell. The MTS delivers the DQAsome to the mitochondria. On contact with the mitochondrial membrane, the DQA-coated membrane of the nanovesicle disintegrates and releases the WT mtDNA on to the mitochondria from where it is taken into the matrix via the protein import machinery.
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# Mitochondrial disease
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tRNAAla ATP6, ATP8 ATP6 COII, COIII
ND1, ND4, ND6 tRNALeu, tRNAVal rRNA12s, tRNAThr, Auditory defects tRNALeu, tRNASer tRNALeu, Diabetes and hearing loss tRNALys, tRNAGlu tRNALeu, Neuromuscular disorder of the cardiac tRNAIle, tissue tRNALys, ND5
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Chronic progressive external Visual defects due to neurological ophthalmoplegia (CPEO) degeneration Metabolic disorder due defective ETC Complex V deficiency complex V Familial bilateral striatal necrosis Striatonigral degeneration in brain (FBSN) Neuromuscular disorder causing Kearns-Sayre syndrome (KSS) retinopathy, cardiomyopathy, deafness, ataxia and other defects Leber’s hereditary optic Visual defects due to mutant retinal neuropathy (LHON) ganglion cells (RGCs) Maternally inherited Neuromuscular disorder of the cardiac cardiomyopathy (MICM) tissue
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Maternally inherited deafness (MID)
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Maternally inherited diabetes and deafness (MIDD)
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Maternally inherited 9 hypertrophic cardiomyopathy (MHCM) Mitochondrial 1 encephalomyopathy, lactic 0 acidosis, and stroke-like episodes (MELAS) 1 Myoclonic epilepsy and 1 psychomotor regression (MEPR) 1 Myoclonic epilepsy with ragged2 red fibers (MERRF) Neurogenic muscle weakness, 1 ataxia, and retinitis pigmentosa 3 (NARP)
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Mutant mtDNA gene(s)
Neuromuscular disorder
tRNAAsp
Neuromuscular disorder
tRNALys, tRNAPhe
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Neurodegenerative disorder
tRNALeu, tRNAVal, ND5, COIII
Neurodegenerative disorder arising from ATP6 defective F1FO-ATPase activity
COI, COII, Neuronal defects in the substantia nigra of cytochrome b, brain ND5, D-Loop Subacute necrotizing Neurodegenerative disorder due to defects tRNAIle, 1 encephalomyelopathy / Leigh in ETC complex I, complex IV and tRNATrp, ATP6, 5 syndrome (LS) pyruvate dehydrogenase complex ND5, COIII 1 Parkinson's disease (PD) 4
ACCEPTED MANUSCRIPT Highlights
Variegated mutations in the nuclear and/or mitochondrial DNA (mtDNA) give rise to a plethora of mitochondrial diseases.
Limited gene therapy options exist for mtDNA mutation-associated disorders in comparison to conditions arising from mutations in the nuclear DNA. Contemporary gene therapy strategies for correcting mtDNA mutations include
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allotopic expression of corrective proteins, site-specific ablation of mtDNA and carrier-
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There is a need for further exploration into novel strategies for improved efficacy and
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safety in gene therapy against mtDNA mutation-associated ailments.
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based delivery of wild-type mtDNA.