SECTION ONE

1.0 INTRODUCTION

Mitochondria are vital organelles that control cell survival and death. They are essential for energy

metabolism and stress management, as well as serving as a central hub for many biological processes

(stenton and prokisch, 2020). Mitochondrial diseases are metabolic disorders that are genetically

determined and are characterized by abnormalities in oxidative phosphorylation (OXPHOS), and are

caused by mutations in genes present in both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA).

These mutations can alter the structural proteins in mitochondria or proteins involved in mitochondrial

function. The OXPHOS system of mitochondria is located in the inner membrane and acts as a final step

in converting nutrients into ATP, the cell's energy currency. It consists of four multi-subunit complexes,

two mobile electron carriers, coenzyme Q10, and cytochrome c. complex i and ii receive electrons from

NADH and FADH2, respectively, and transfer them to coenzyme Q10, which then transports them to

complex iii. From here, electrons are transferred to cytochrome c and to complex iv, where they combine

with molecular oxygen to produce water. Complexes i, iii, and iv also create a proton gradient by moving

protons across the inner membrane, which is then used by complex v to produce ATP. These five

complexes are made up of a total of 92 different proteins. Complex i consists of 44 subunits (7 encoded

by mtDNA and 37 by nDNA), complex ii has 4 subunits, complex iii has 11 subunits (1 mtDNA and 10

nDNA), complex iv has 14 subunits (3 mtDNA and 11 nDNA), and complex v has 19 subunits (2 mtDNA

and 17 nDNA). Mutations in mtDNA or nuclear-encoded mitochondrial genes that disrupt the function of

oxphos can result in a variety of life threatening illnesses. Due to the offloading of electrons at complexes

i and iii, mitochondria are a major source of reactive oxygen species (ros), such as superoxide. The

increased production of ros in mitochondrial diseases can damage proteins, lipids, and DNA, potentially

resulting in additional cell dysfunction and harm.

1.1 BASIC MITOCHONDRIAL BIOLOGY

Organelles called mitochondria composed of an outer and an inner membrane and are used by

cells for a number of purposes. coded proteins transported into mitochondria by a complex

protein transport system (Schmidt et al., 2010) Mitochondria are signaling hubs that integrate the

catabolic and anabolic metabolism and regulate cell growth, differentiation, vitality, and death

(Liu et al.,2020) Mitochondria cannot be synthesized de novo; therefore, they are subjected to

constant quality control and regeneration through budding of mitochondrial-derived ve sicles and

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mitophagy (Quirós et al., 2015) Another important mechanism maintaining stable state of

mitochondria is thousands of copies per cell and it is characterized by high levels of

heterogeneity. It encodes mRNAs for 13 polypeptides of the OXPHOS system, as well as 2

rRNAs and 22 tRNAs for their translation (. D’Souza and Minczuk, 2018) Four out of five

complexes of the ETC and OXPHOS (I, III, IV & V) are composed of subunits, encoded both by

the nuclear and mtDNA, while only one (II) is encoded solely by nuclear genome (Filograna et

al., 2021).

The mitochondrial genome has a higher mutation rate (about 100–1000 fold), than the nuclear

genome (Wallace and chalkia, 2013). The pathological mutations of mitochondria are survived

due to inter-mitochondrial exchange of nutrients and functional complementation taking place

through mitochondrial fusion (Shtolz and Mishmar, 2013). This causes mtDNA

population heterogeneity within a single cell or mitochondria, and as a result mitochondria

become heteroplasmic. There are ~ 1000 mtDNA molecules in a cell, and in case

of mutations, wild-type mtDNA can compensate for the presence of mutant mtDNA up to

threshold levels, which are usually relatively high.

TYPE OF MITOCHONDRIAL DISEASE/DISORDER

Mitochondrial diseases can be group based on mutations affecting the mitochondrial metabolic

pathways into primary and secondary. The former type is due to mutations in the genes, mtDNA

or nDNA, responsible for OXPHOS, or the genes encoding for proteins that interfere with the

OXPHOS pathway (Muraresku et al., 2018). On the other hand, secondary mitochondrial

disorders may involve genetic, non-OXPHOS genes, and environmental factors (Niyazov et al.,

2016). The prevalence of primary mitochondrial diseases was estimated as 1 in every 5000

newborns (Pérez-Albert et al., 2018). In childhood, mitochondrial disorders are not defined by

specific symptoms; instead, a group of genetic, clinical, and biochemical manifestations are used

to diagnose such diseases (Gorman et al., 2016).

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Figured 1: Homoplasmic and Heteroplasmic Mitochondrial DNA(Muraresku et al.,2018).

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A Single cell can have wild type copies of mtDNA (homoplasmy) or a mixture of mutant and

wild-type mtDNA (heteroplasmy)). The proportion of mutant mtDNA copies determines the

penetrance and severity of phenotypic expression, and exceeding a certain limit (threshold)

affects the cell. Abbreviations: mtDNA, mitochondrial DNA.

Neurologic manifestations are among the most severe consequences of mitochondrial diseases

that can result in several conditi ons, such as Leigh syndrome, Epilepsia partialis continua,

occipital stroke-like episodes, axon al sensorimotor neuropathy, myoclonus, and ataxia (Riquin

et al., 2020). In addition, neuropsychological disorders were also recorded, such as impaired

consciousness, hallucinations, reasoning, conf usional sta te, depression, autism and

unsteadiness. In the case of the ophthalmic system, different regions of the eye could be affected,

which are expressed in the form of progressive ptosis and external ophthalmoplegia, visual loss

(retro-chiasmal), pigmentary retinopathy and bilateral optic atrophy ( Lock et al., 2021). In

infants and adolescents, these neuro-ophthalmologic symptoms may be progressive and lead to

serious eye complications with decreased vision efficacy, Hearing loss at both ears (i.e. bilateral)

is usually associated with mitochondrial dysfunctions, particularly in mitochondrial

encephalomyopathy, lactic acidosis and stroke like episodes or MELAS (Chinnery et al., 2000)

In children with bilateral hearing loss, mitochondrial dysfunctions could be the leading cause, or

other neurological disorders can also be involved (Vandana et al., 2016). The heart is another

important organ that can be deteriorate by primary mitochondrial disorder. Lack of functions in

the myocardium cells is characterized by deformed muscle structure and functions (Meyer et al.,

2013). Mitochondrial cardiomyopathy, Wolff-Parkinson-White, and Kearns-Sayre syndromes.

Individuals with mitochondrial cardiomyopathy often display heart hypertrophy and left

ventricular non compaction are some syndrome found in children primary metabolic disorder.

Individuals with MELAS are most likely to develop arrhythmia, including Wolff-Parkinson-

White and ventricul are pre-excitation (Ng and Turnbull, 2016). The disease severity ranges from

asymptomatic to life-threatening tachyarrhythmia and heart failure and could eventually lead to

cardiac death ( Meyer et al., 2013)

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The gastrointestinal tract is also affected by primaryary mitochondrial disorders, in which

numerous manifestations will cause permanent and untreatable disability due to the difficulty of

early diagnosis (Rose et al., 2 017 ). These manifestations include gastroesophageal sphincter

dysfunction, dysphagia., hepatopathy, pancreatitis, intestinal pseudo-obstruction and

gastroparesis. (Moreover, patients diagnosisnosisnosed with Crohn's disease commonly suffer

from mitochondrial neurogastrointestinal encephalopathy ( Patel et al., 2019).

The kidneys are among the organs that are likely to fail due to mitochondrial disorders due to the

high energy consumption in this organ. Even Kidney diseases resulting from mitochondrial

disorders can be categorized into primary and secondary. In the first category, several diseases

have been reported, such as acute or chronic kidney failure, nephrotic syndrome, renal tubular

acidosis, tubulointerstitial nephritis, and nephrocalcinosis. Renal insufficiency in the second

category includes either organ other than the kidneys, for example, the heart and pan creas, or

other diseases such as rhabdomyolysis (Yokoyama et al., 2016). A previous study has reported a

significant relation between mitochondrial dysfunctions, chronic kidney diseases and sarcopenia

(Takemura et al., 2020).

Children with mitochondrial diseases have frequently suffered from several physicals,

neuropsychological disorders, behavior and speech disorders, high morbidity, and recurrent

episodes of anxiety and depression (van de Loo et al., 2020). Due to the impaired mitochondrial

functions, affected children are usually unable to participate in daily activities, which could

aggravate psychiatric disorders ( Riquin et al., 2020). Caregivers and parents might also suffer

from stress that can exacerbate hospitalization and the need for clinical intervention ( Sofou,

2013).

1.4 CLINICAL SIGN OF MITOCHONDRIAL DISORDER.

Mitochondrial and nuclear mutations have lead to about 40 clinical forms of mitochondrial

disorder (MD) with clear molecular-genetical and biochemical disfunction of mitochondria.

More than 30 of these MD have a mutation in nuclear DNA and more than 10 syndromes and

diseases induced by mutations in mtDNA (Chinnery et al., 1993) MD usually affects children,

but the age of MD onset, as well as patient’s lifetime, varies a lot. The most typical clinical

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manifestations of MD are neurological (epilepsy, ataxia, cognitive deficits), sensory (blindness,

hearing loss), and muscular (myopathy) symptoms. However, the clinical picture can vary

greatly and show up as kidney dysfunction, diabetes, liver disease, infertility, arrhythmias, etc.

Certain MD symptoms tend to co-occur, making it possible to group them into syndromes like

MIDD (maternally inherited diabetes and deafness) or MELAS (Mitochondrial Encephalopathy,

Lactic Acidosis, and Stroke-like events). Certain combinations of symptoms and indicators are

brought on by gene mutations, and vice versa, the same mutations might result in other

syndromes. Disturbances in the respiratory chain complex I are the primary cause of MD,

accounting for almost one-third of cases.

Certain symptoms of multiple disorders (MD) can cooccur, allowing for the creation of syndrom

es such mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) or mat

ernally inherited diabetes and deafness (MIDD).

Mutations in distinct genes can result in a variety of symptom constellations, and conversely, the

same mutations might induce distinct syndromes. Almost one-

third of MD cases are triggered by disruptions in the respiratory chain complex I.

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Figured 2: Overview of the MD in the clinical and pharmacological contexts. (Kirby et

al.,1999:Fassone and Rahman, 2012:Abramov and Angelova, 2019).

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1.5 OPTION FOR STANDARD TREATMENT OF MITOCHONDRIAL DISORDER.

The treatment of MD remains anecdotal due to a weak body of evidence and scant data from

randomized trials. Furthermore, the majority of recommended remedies are regarded as medical

meals. (Avula et al., 2014). Interventions are mostly focused on vitamin-based and cofactor-

based mitochondrial therapies intended to promote critical enzymatic reactions, reduce oxidative

stress, and scavenge toxic acyl coenzyme A (acyl CoA) molecules, which accumulate in

mitochondrial disease (Parikh et al., 2009). Using cocktails of vitamins and co-factors is more

justified when factors in question are decreased either due to deficiency or defect in their

transport (Khan et al., 2015).Additionally, patients struggling with MD are recommended to diet

and lifestyle changes (Bough et al., 2006). However, only few patients respond to this treatment

since all the methods relying on metabolic gene therapy are poorly effective because of their

non-selectiveness.

SECTION TWO

2.0 GENERAL CONSIDERATION AND TECHNICAL ASPECTS

One appealing and simple therapeutic option for monogenic recessive disorders is gene therapy.

In the clinic today, severe genetic disorders are treated by reintroducing the wild-type form of a

mutant gene or other therapeutic genes with the proper delivery strategy. Even while it's still

difficult to transfer and express an ectopic gene throughout the entire body, modern technology

can target particular cells or tissues to provide a therapeutic impact. Through the development of

novel recombinant DNA technology, the concept of gene therapy was initially envisaged in the

1980s. However, genome sequencing and biotechnology advancement have made gene therapy a

real medical revolution, offering the possibility to cure many otherwise deadly genetic diseases

(Mendell et al., 2021)The first official gene therapy product of the western world was Glybera,

which is to treat lipoprotein lipase deficiency (Melchiorri et al., 2013). Many successful clinical

trials, mainly using viral vectors, have followed, resulting in the more recent commercialization

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of Luxturna to treat inherited retinal dystrophy (Russell et al., 2017) and Zolgensma to treat

pediatric patients with spinal muscular atrophy While the majority of preclinical and clinical

research focuses on specific organs or tissues, like the nervous system or the eye, treating many

MDs would need widespread systemic gene expression, which makes creating an efficient

treatment plan difficult due to the difficulties in getting proteins or genetic material into the

mitochondria. For a variety of hereditary illnesses, distinct gene delivery strategies using viral or

non-viral methods have been developed. Here, we first cover the pertinent technical details in

brief before concentrating on the main treatment approaches for MDs. A variety of viral and non-

viral gene delivery strategies have been developed for different genetic illnesses.

Here, we first address the pertinent technical elements and then move on to discuss the main treatment str

ategies for Mitochondrial disorder (Mendell et al., 2017).

2.1 Gene Therapy for Mitochondrial Diseases Caused by Mutations in Nuclear Genes

Gene therapy is the easiest "precision medicine" approach for many doctors, although there are a

number of factors that must be considered for its successful use. Most MDs

are multisystem syndromes, meaning they affect multiple organs; therefore, a significant

improvement in the patient's condition should, at least in theory, require widespread, if not

ubiquitous, gene expression of the vector carrying the therapeutic transgene. However, it has

been shown that single-organ gene therapy can successfully improve the phenotype of MDs in at

least two different conditions: tissue-specific clinical presentation of mitochondrial

dysfunction (e.g, LHON, liver-specific mtDNA depletion syndrome due to MPV17

mutations) and systemic accumulation of toxic compounds (e.g, ethylmalonic

encephalopathy, multiorgan involvement caused by mitochondrial neurogastrointestinal

encephalomyopathy). Among other things, the high risk of an immune response against the

delivery vector or therapeutic cargo requires practical consideration of how to achieve such a

broad expression pattern. First, high vector doses are required to achieve long-term and

widespread systemic effects, which involve issues of toxicity and immunogenicity. Large sets of

vectors must be produced in high throughput, which greatly increases production costs and

ultimately processing costs. Second, a virus particle with the correct tropism must

be selected based on the target tissue. In most cases, systemic transport to multiple tissues is

required. Third, because mitochondrial diseases usually affect the central nervous

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system, the virus particle must be able to cross the BBB. Here, we review the preclinical and

clinical applications of gene therapy to restore the function of mutated genes that cause MD.

2.2. Mitochondrial Myopathy (MM) and Cardiomyopathy

As a paradigmatic example of mitochondrial myopathy, we will discuss gene therapy approaches

available for ANT1. ANT1 encodes the adenine nucleotide translocator, an integral inner

mitochondrial membrane (IMM) protein operating as electrogenic pumps that export ATP in

exchange for cytosolic ADP (Kaukonen et al., 2000). Mutations in ANT1 lead to MM associated

with ragged red muscle fibers and PEO triggered by the paralysis of the extraocular eye muscles

(Kaukonen et al., 2000). One of the earliest preclinical gene therapy studies for mitochondrial

myopathy (MM) was performed with an ssAAV2 vector carrying murine Ant1 cDNA

administered intramuscularly (1 × 109 i.u.) to an Ant1 knockout (KO) mouse model. Ant1

KO mice showed severe exercise intolerance and metabolic acidosis. Histological and

ultrastructural analysis showed the presence of torn red muscle fibers with

mitochondrial deficient respiration, while cardiac examination showed cardiac

hypertrophy and mitochondrial proliferation. AAV2-Ant1 transduction resulted in long-term

stable expression in muscle precursor cells and differentiated muscle fibers. The transgenic

ANT1 protein was targeted to the IMM and formed a functional ADP/ATP carrier, resulting in a

5–30% increase in ANT1 protein, a 25–45% increase in ATP production, and a reversion of the

histopathological changes associated with the MM (Flierl et al., 2005).

2.3 Metabolic Disorder Caused by the Accumulation of Toxic Compounds

We discuss available gene therapy approaches for ethylmalonic encephalopathy caused

by ETHE1 mutations as an emblematic example of mitochondrial encephalopathy caused by

toxic sulfide accumulation. ETHE1 is a nuclear gene encoding sulfur dioxygenase (SDO),

which is involved in a mitochondrial pathway that converts sulfide to harmless sulfate. Recessive

mutations in ETHE1 lead to ethylmalonic encephalopathy (EE), a fatal mitochondrial disease

characterized by the accumulation of hydrogen sulfide (H2S) and ethylmalonic acid (EMA)( Di

Meo et al., 2015). At higher concentrations, H2S acts as a toxic compound that inhibits several

enzymes, such as cytochrome c oxidase (COX) ( Di Meo et al., 2011). and short-chain acyl-CoA

dehydrogenase (Tiranti et al., 2009). leading to the progressive accumulation of necrotic and

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hemorrhagic brain lesions (Yang et al., 2004). chronic hemorrhagic diarrhea, vascular petechiae

purpura and orthostatic acrocyanosis. The Ethe1 KO mouse shows growth retardation from

postnatal day 15 and reduced motor activity and premature death at the fifth to sixth postnatal

week. In addition, it partially reproduces the biochemical changes of the

patients, with clearly low COX activity in muscle, brain and jejunum, but normal activity in the

liver. By expressing the ETHE1 gene lacking circulating H2S clearance in a filtering organ such

as the liver, H2S levels can be lowered, thus acting as a detoxification therapy. A single systemic

injection of 4 × 1013 viral genome (vg)/kg ssAAV2/8 vector expressing human hETHE1 cDNA

under the liver-specific thyroxine-binding globulin (TBG) promoter into three-week-old early

symptomatic Ethe1 KO mice resulted in clear recovery. of the phenotype and a strong

extension of mouse lifespan. This remarkable clinical result was associated with the partial or

complete correction of the disease’s main metabolic and biochemical indexes, including the

EMA and thiosulfate levels in plasma and the COX activity in tissues (Di Meo et al., 2012).

Iiving-donor orthotopic liver transplantation also resulted in an effective option to treat EE since

the transplanted organ substituted the deficient ETHE1 enzyme and cleared the circulating toxic

H2S ( Olivieri et al., 2021). These results demonstrated the efficacy and safety of AAV2/8-

mediated liver gene therapy for EE and related conditions caused by the accumulation of toxic

compounds in body fluids and tissues.

2.4 Mitochondrial DNA depletion syndrome

Mitochondrial DNA depletion syndrome DNA (MDS) is an autosomal

recessive condition, characterized by variable organelles and a reduced number of copies

of mtDNA and enzyme activity of the mitochondrial respiratory chain in damaged tissues.

The depletion of mtDNA in humans has been associated with mutations in nine nuclear genes,

six of which (TYMP, TK2; DGUOK; SUCLA2, SUCLG1, and RRM2B) are involved in the

homeostasis of the mitochondrial nucleotide pool ( addition, mtDNA depletion can be caused by

mutations in POLG, encoding the catalytic subunit of mitochondrial polymerase γ; PEO1,

encoding a mitochondrial T4-phage-like helicase (twinkle) (Ronchi et al., 2011). and MPV17,

encoding an inner mitochondrial membrane protein of unknown function ( Spinazzola et al.,

2006 ).Several gene therapy approaches have been explored to restore the physiological activity

of TYMP, TK2, and MPV17 (Bottani et al., 2014).

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  TYMP

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by autosomal

recessive mutations in the nuclear gene TYMP, which encodes the cytosolic enzyme thymidine

phosphorylase (TP). The lack of TP activity leads to the systemic accumulation of TP substrates,

the nucleosides deoxythymidine (dT) and deoxyuridine (dU), which are precursors of the

deoxyribonucleoside triphosphates (dNTPs) used for DNA synthesis (Di Meo et al., 2005). One

of the first strategies developed for restoring TP and reverting dT and dU overload to normal

levels was the lentiviral-mediated hematopoietic ex vivo gene therapy (Torres-Torronteras et al.,

2011).

Immunoselective hematopoietic lineage-negative (Lin) cells from DKO mice were transduced

with a lentiviral vector containing the TYMP cDNA (p305-TP), containing the human

phosphoglycerate kinase (hPGK) promoter and containing the EGFP coding sequence as a

marker. gene Transduced cells were infused into partially myeloablated syngeneic dKO mice.

One month after transplantation, gene expression levels in the peripheral blood (PB) of TP-

transplanted mice ranged from 2.0 to 10.5%, and the average copy number per transduced cell

ranged from 0.5 to 1.5 In the peripheral blood of transplanted mice, TP activity was observed

high level and accompanying decrease in nucleoside concentration. However, a long-term

follow-up revealed a reduced survival rate of treated mice due to the transplantation procedure,

which included total body irradiation of recipient animals before progenitor cell infusion.

 TK2

TK2 is a nuclear gene that encodes a deoxyribonucleoside kinase with mitochondrial

localization and is involved in pyrimidine salvage pathways. TK2 phosphorylates both

deoxycytidine (dC) and dT to produce deoxycytidine monophosphate (dCMP) and

deoxythymidine monophosphate (dTMP), which are then phosphorylated to dCTP and dTTP,

which are essential for mtDNA replication and maintenance. Recessive mutations in the human

TK2 gene cause a lethal early-onset myopathic mitochondrial

depletion/multiple deletion syndrome (Wang et al., 2003)..

 MPV17

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MPV17 is a nuclear gene encoding for a mitochondrial inner membrane protein involved in

mitochondrial deoxynucleotide homeostasis and the maintenance of mtDNA (Wong et al.,

2007).Mutations in human MPV17 cause a hepatocerebral form of MDS hallmarked by early-

onset severe hypoglycemic crises followed by liver cirrhosis and failure leading to premature

death (Wong et al., 2007).Patients who survive liver failure develop progressive peripheral

neuropathy and cerebellar degeneration (Spinazzola et al., 2008).

Although the molecular and biochemical features in the liver of Mpv17 KO mice closely

resemble human alterations, this model does not develop hepatic dysfunction and

neuropathological abnormalities (Viscomi C et al., 2009). However, when Mpv17 KO mice

were fed a high-fat ketogenic diet (CD), they rapidly developed liver cirrhosis and liver failure.

2014. , Bottani and coworkers launched an ssAAV2/8-based preclinical gene therapy protocol

for MPV17-MDS. typical viral vector. hMPV17 cDNA under the control of the liver-specific

TBG promoter was administered systemically by retroorbital injection at a dose of 4 x 1012

vg/kg to 2-month-old Mpv17 KO and control mice. Analysis of a blood sample before AAV

treatment showed elevated alanine aminotransferase and aspartate aminotransferase enzymes,

two markers of hepatocyte leakage. Those signs fully normalized three weeks after AAV

injection. Moreover, this treatment effectively prevented KD-induced liver degeneration and

restored mtDNA depletion (Bottani et al., 2014)

 Leigh Syndrome

Leigh syndrome (LS) is a neurometabolic MD that affects 1 in 36,000 newborns and causes

lactic acidosis and symmetric lesions in the CNS, leading to intellectual disability and muscle

weakness, with a peak of mortality before three years of age. Mutations in more than 75 genes of

nuclear or mtDNA that commonly affect CI and CIV of the mitochondrial respiratory chain can

cause LS (Schubert and Vilarinho, 2020) The following chapters will discuss the gene therapy

approaches recently exploited in KO mouse models for Ndufs4 and Surf1.

 NDUFS4

NDUFS4 is a non-enzymatic, 18 kDa nuclear-encoded CI subunit. Mutations in

the NDUFS4 gene are a frequent cause of LS (Budde et al., 2003) Common symptoms include

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psychomotor arrest or regression, hypotonia, dystonia, ataxia, ophthalmoplegia, lethargy, apneic

spells, and respiratory failure with elevated lactate levels in the blood and cerebrospinal fluid

(Ortigoza-Escobar et al., 2003) .

The constitutive Ndufs4 KO mouse model appeared healthy at birth, but starting from 40 days of

age, it develops progressive encephalopathy, growth retardation, ataxia, hypotonia, and lethargy,

ultimately leading to death at ~7 weeks. Notably, CI activity was reduced, recapitulating the

pathophysiology observed in LS patients (van de Wal et al., 2022)

During the last decade, various research groups have used this model to devise a gene therapy

approach for ls. Using an ssaav2/9 vector, di meo and colleagues delivered the human wild-type

ndufs4 cdna under the control of the cytomegalovirus (cmv) promoter. At postnatal day 21, the

vector was administered (2 1012 vg/mouse) iv by retroorbital injection. The hndufs4 protein was

found to have high amounts in the skeletal muscle, heart, and liver, but no significant increase in

protein content was detected in the brain. Although hndufs4 was able to fully restore the ci

assembly in ndufs4 ko liver mitochondria and restore the ci spectrophotometric activity in the

viscera, no significant improvement in the clinical phenotype was observed. Similar results were

obtained in newborn mice that were injected systematically through the temporal vein. In

newborn mice injected intracerebroventricularly (icv) with a higher dose (3 1011 vg/mouse),

there was a modest increase in body weight and a significant improvement in motor

coordination, without extending their lifespan. Importantly, an imaging study of injected brains

revealed that the bulk of the transduced cells belong to the glia.

Finally, double administration of AAV2/9-hNDUFS4 by both IV and ICV administration caused

a remarkable improvement in the clinical symptoms, such as an amelioration of motor

coordination, a gain of body weight, and a highly significant prolongation of the lifespan (82

days) compared with untreated KO mic (Di Meo et al., 2017).

 SURF1

Surf1 is a nuclear gene that encodes for a mitochondrial inner membrane protein that is involved

in the assembly of civ. Surf1 mutations result in a defect in cox assembly and a severe cox

deficit, which is one of the main causes of ls (tiranti et al., 1999), but the constitutive surf1 ko

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murine model exhibits mild cox deficiency and a slight increase in blood lactate, but it fails to

recapitulate human clinical signs while displaying a remarkable increase in longevity and

improved memory (dell et al., One-month-old ko mice were given an intrathecal injection (8

1011 vg/mouse) of an scaav9 vector carrying hsurf1 cdna under the control of the hybrid chicken

—actin/cmv enhancer (cag) promoter. The treatment effectively increased surf1 mrna expression

in many important organs, including the brain and spinal cord, resulting in a partial recovery of

cox activity in all tissues, as well as the abnormal lactate acidosis caused by intense exercise

(ling et al., 2021).

 Friedrich Ataxia

Friedreich ataxia (frda) is a rare neurodegenerative disease that is characterised by

spinocerebellar and sensory ataxia and is associated with hypertrophic cardiomyopathy and

diabetes (pandolfo and friedreich, 2009). Frda is mainly caused by a homozygous n(gaa)

expansion within the first intron of the frataxin gene (fxn), an essential mitochondrial protein

involved in the biosynthesis of iron This triplet expansion leads to heterochromatinization of the

locus, with a consequent decrease in fxn transcription. A frataxin deficiency leads to impaired fe-

s biogenesis, loss of fe-s enzymes, mitochondrial dysfunction, iron metabolism dysregulation,

and ultimately cell dysfunction and death (campuzano et al., 1996). Several disease models have

been developed in recent years, but most of them failed in recapitulating the biological aspects of

frda. For a detailed description of the characteristics of the main fxn mouse models, please refer

to this paper (ocana et al., 2021). Nevertheless, these models enabled the development of gene

therapy strategies. By retro-orbital injection, the 7-week-old conditional mouse model (mck-cre-

fxnl3/l) was treated with an ssaavrh10-cag-hfxn-ha vector. Whether administered before or after

the onset of the disease, the treatment was effective at preventing or reversing cardiomyopathy

and mitochondrial dysfunction (piguet et al., 2018). In a subsequent study, the same group

developed a pvalb-cre conditional ko model that treats sensory axonopathy and cerebellar ataxia.

In 3.5-week-old early symptomatic pvalb ko mice, an individual iv injection of ssaav9-cag-fxn-

ha at a 5 1013 vg/kg dose was administered. All behavioral studies revealed a significant

improvement in motor coordination in comparison to untreated mice. Moreover, iv

administration of ssaav9-cag-fxn-ha at a dose of 5 1013 vg/kg alongside intracerebral

administration of ssaavrh.10-cag-fxn-ha (1 1010 vg/kg) was performed post-symptomatically at

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7.5 weeks of age to assess the therapeutic value of the intervention at a later post-symptomatic

stage. Within the first few days after treatment, the treatment reversed fine peripheral

coordination and neurological functions (piguet et al., 2018). Despite these promising results, it

is important to bear in mind that the dose of vector administered following the dual injections

was very high and may not be appropriate in a clinical setting. According to a recent report, fxn

cardiac overexpression was safe up to nine times the physiological value, with significant cardiac

toxicity over twenty times. The toxicity seems to be caused by mitochondria respiratory chain

and ultrastructure dysfunction, which leads to cardiomyocyte subcellular disorganization, cell

death, and fibrosis (Belbellaa et al., 2020).

SECTION THREE

3.0 Mitochondrial disease etiology and pathophysiology

37 genes (16569 Base pair in a circular pattern) that encode 13 proteins (seven subunits of

respiratory chain complex I, three subunits of complex IV, two subunits of complex V, and one

subunit of complex III), 22 tRNAs, and two rRNAs make up the human mitochondria double-

strand DNA, or mtDNA. While the amount of mitochondria in each cell might vary, each

mitochondrion contains several copies of mtDNA (two to ten copies). The inheritance of mtDNA

is maternally transmitted, in contrast to the nDNA, which has a biparental transmission pattern

(An et al., 2020). Additionally, there are 1,500 identified genes presented in the nDNA, which

are essential for the mitochondrial structure and function (Li et al., 2020).

The mtDNA "utation rate" Is considered to be higher (more than ten times) than the nDNA. This

could be due to the inadequate shielding of the histone complex and the high sensitivity of

mtDNA to oxidative stress when using less effective mtDNA repair techniques, such as the base

excision repair technique (Alexeyev et al., 2013). In addition, the DNA polymerase (DNA

polymerase ) activity in the mitochondria has a low fidelity rate, which can increase the mutation

during mitochondrial dna replication (song et al., 2005). As shown in fig. ci, the mutation in the

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mitochondria can affect the whole mtDNA in the cell (homoplasmy) or partially (heteroplasmy).

Both events, therefore, influence the severity of mitochondrial disorders (Moggio et al., 2014). In

addition to the primary mitochondrial function, i.e. ATP, mitochondrial function is a branch of

mitochondrial function. The mitochondria serve other functions in calcium metabolism, innate

metabolism, cell death, stem cell regeneration, autophagy, inflammation, and senescence (smith

et al., 2012). Mutation at the mtDNA or nDNA levels can result in a variety of pathogenesis

effects.

The nuclear genome Is essential for mitochondrial function, with more than 1,000 genes

involved. The mutations of greater than 280 genes have been reported to cause mitochondrial

disorders, where the defect may be inherited as autosomal dominant (8 genes), recessive (262

genes) or X- linked pattern (14 genes) (Stenton and Prokisch, 2020). Historically, the first nDNA

mutation (autosomal recessive) was associated with human mitochondrial disease at the SDHA

gene, which is involved in encoding a structural subunit of complex II, (Bourgeron et al., 1995).

On the other hand, the first X-linked recessive mutations identified in 2007 at the NDUFA1 gene

were p.Gly8Arg and p.Arg37Se (Fernandez-Moreira et al., 2007). The prevalence of nDNA

mutation triggering mitochondrial diseases is estimated to be 1 in 35,000 (Gorman et al., 2016).

Leigh syndrome is a common neurodegenerative mitochondrial disorder with a 1 in 40,000,

which can be caused by approximately 90 gene mutations, including the mtDNA and nDNA

genomes (Rahman et al., 2017). An example of this is the mutation in the SURF1 nDNA gene,

which is linked with Cytochrome c oxidase deficiency (Wedatilake et al., 2013).

The prevalence of mtDNA mutation causing mitochondrial disease is estimated to be 1 in 5,000

(Schon et al., 2021). The most common large-scale mtDNA deletion resulted from the omission

of 4977 bp located between the locus 8470 and 13447, which involved the encoding of 15 genes

from ATPase8 gene to ND5 gene (Yusoff et al., 2019). In contrast, point mutation can involve an

insertion, deletion or substitution, affecting either the respiratory chain coding genes or the RNA

coding genes (mt-rRNA and mt-tRNA). Furthermore, m.3243A > G mutation is one of the most

common mutations in the tRNAs genes, with a heterogeneous effect on the muscles and the

nervous system, causing mitochondrial encephalomyopathy, lactic acidosis and stroke-like

episodes (MELAS) (Pickett et al., 2018).

17
3.1 Clinical features of mitochondrial diseases

The childhood-onset of mitochondrial diseases have mainly resulted from recessive nDNA or

mutations in mtDNA that exist at high levels of mtDNA heteroplasmy (Taylor and Turnbull,

2005). As mitochondrial diseases have diverse phenotypes and usually cause multi-organs

dysfunction, the clinical features and diagnosis are relatively complicated (Alston et al., 2017).

Moreover, the tenuous link between the observed clinical phenotype and the genotype in

mitochondrial disease patients complicates the accurate diagnosis (Stenton and Prokisch, 2020).

The pediatric-onset of mitochondrial disorders have several clinical features that are regularly

observed, such as fatigue, vomiting, failure to thrive, encephalopathy, seizures, hypotonia,

exercise intolerance and dysautonomia, (Kanungo et al., 2018). Nevertheless, the bulk of these

physical abnormalities are not related to mitochondrial disorders, and a confirmation analysis is

required (e.g., a blood test). (analysis of molecular genetic screening tests.) The congenital

disabilities in children with mitochondrial dysfunctions are frequently associated with different

complications, as in the case of renal disease and proximal tubulopathy that occurs due to the

mutations in RMND1 (O’Toole, 2014), RRM2B genes, respectively.

18
Figure 3: clinical features of mitochondrial diseases. (Kanungo et al.,2018).

19
In addition to previously mentioned clinical features of mitochondrial diseases, hypertrophic

cardiomyopathy is frequently observed in children with mitochondrial disorders, and it is

associated with MTO1 or AARS2 mutations (Euro et al., 2015). Moreover, excessive hair

growth (hypertrichosis) and dysmorphic disorder are common clinical manifestations in children

with mitochondrial diseases that are caused by mutations in SURF1 (Baertling et al., 2013) and

FBXL4 (Ballout et al., 2019), respectively. The modifications in SUCLA2, SUCLG1, MT-

RNR1 (m.1555A > G), MT-TL1 (m.3243A > G), RMND1 and RRM2B in children can lead to

sensorineural hearing loss (Rahman, 2020).

20
21
Figure 4: childhood -onset Mitochondrial diseases(Rahman,2020).

3.2 diagnostic evaluation of mitochondrial diseases

various diagnostic tests, such as blood, urine, molecular genetics, and tissue biopsy tests, can be

used to diagnose mitochondrial disorders. To establish the diagnosis of the mitochondrial

disease, several common biomarkers can be used, including specific enzymes, anaerobic glucose

metabolism, and substances or metabolic intermediates (e.g., alanine, lactate, creatine kinase,

pyruvate, deoxyuridine, thymidine, acylcarnitines, and organic acids). To detect mitochondrial

abnormalities in their early stages, it is necessary to perform metabolic screening tests in urine

and blood samples. Different diagnostic biochemical screening tests may also be used, such as a

complete blood count, urine organic acid analysis, urine amino acid analysis, hormone testing,

hemoglobin a1c, comprehensive chemistry panel, blood lactate and pyruvate, creatine kinase,

ammonia and carnitine, acylcarnitine, and lipoprotein profiles (Muraresku et al., 2018). If

necessary, additional biochemical screening tests can be performed to determine the

mitochondrial disorder.

One of the most valuable biomarkers in the diagnosis of mitochondrial disease is the metabolic

fingerprints of OXPHOS deficiency (Finsterer and Zarrouk, 2018). For instance, it has been

demonstrated that the fibroblast growth factor 21 (FGF21) level is a potential biomarker for

muscle-manifesting mitochondrial disease. The elevated growth-differentiation factor 15

(GDF15) has been detected in blood samples collected from mitochondrial dysfunction patients

and is considered another potential biomarker. The measurement of abnormal quantities of

organic acids in urine may be used as a diagnostic tool to detect several mitochondrial disorders

in children (Yatsuga et al., 2015), such as methylmalonic aciduria (caused by mutations in

SUCLA2 and SUCLG1) (Landsverk et al., 2014), and 3-methylglutaconic aciduria (caused by

mutations in TAZ, TMEM70 and SERAC1).

Molecular genetic testing is a vital diagnostic tool that helps identify the molecular etiology of

mitochondrial dysfunction, resulting in improved therapeutic outcomes. This is a critical test to

identify de novo dominant mutations in the affected individuals and improve the interpretation

22
accuracy of variant pathogenicity. The most effective method for identifying primary

mitochondrial disorders may be the next-generation sequencing (ngs) of mtDNA and its

abundance in diseased tissues. Biotin and thiamine responsive basal ganglia disease (slc19a3)

and riboflavin transport deficits (slc52a2 or slc52a3) have been found to have phenotypic overlap

with mitochondrial dysfunctions (Ferreira et al., 2017).

In addition to biochemical screening analysis and molecular genetic testing, tissue analysis is

another important test to diagnose mitochondrial diseases, particularly in symptomatic tissues

that have mtDNA mutations or mitochondrial dysfunctions (Dimmock and Lawlor, 2017). The

mitochondrial enzymes and functions can be evaluated through a skin biopsy (Newell et al.,

2019). This tissue screening might include the measurement of integrated mitochondrial

OXPHOS and the analysis of enzymatic activity of the electron transport chain (ETC) complex

(Germain et al., 2019). Biopsy from skeletal muscles can be performed to understand the degree

of mitochondrial dysfunctions or confirm the dysfunction as an alternative approach to a failed

blood genetic analysis (Ahmed et al., 2018). Further clinical diagnostic testing may be conducted

on high energy demanding tissues, i.e. kidneys or muscle, such as ETC complexes I-IV enzyme

activity analyses (Parikh et al., 2015).

3.3 Epigenetic alterations in mitochondrial diseases

The significant differences between the mtDNA and the nDNA are that there are several copies

of mtDNA that lack histone. Epigenetic variation is a change that may occur in the genetic

expression, which is not heritable (Sharma et al., 2019). Several mtDNA variabilities are also

inheritable (D’Aquila et al., 2015).

The primary epigenetic regulation of the mitochondria can occur In the mtDNA methylation,

non-coding RNAs (ncRNA) modification, and posttranslational modification (Sharma et al.,

2019). DNA methylation is the process in which a methyl group from S-adenosyl-methionine

binds to the DNA nucleotide, mainly adenine (A) and cytosine (C), by the support of DNA

methyltransferases (DNMTs) (Maresca et al., 2015).

The communication between mitochondria and the nucleus plays a critical role in cellular

homeostasis. The nucleus controls the mitochondrion’s gene expression and post-translation

23
process; however, the nuclear gene expression and protein activity are mediated by the

mitochondria through signal transport from mitochondria to cytosol. This crosstalk is controlled

by several signals, such as microRNA (miRNA), a subclass of ncRNA (Cavalcante et al., 2020).

The transcription of miRNA, which is non-coding RNA, occurs in the nucleus as primary

miRNA to be transformed to precursor miRNA by Drosha and then to its mature form by Dicer

at the cytoplasm. The primary function of miRNA is to inhibit the translation of mRNA via

destabilizing its binding to the 3′ untranslated region of mRNA (Chipman and Pasquinelli, 2019).

miRNA can play a vital role in mitochondrial function. For instance, several miRNAs such as

miR-138, miR-1291, miR-150, and miR-199a-3p can cause a change in the regulation of the

expression of some glycolytic enzymes’ glucose transporters. This explains the role of miRNA in

controlling glucose uptake by the mitochondrion (Srinivasan and Das, 2015).

Additionally, the production of reactive oxygen species (ROS) from the mitochondria could

trigger the expression of hypoxia-inducible factor 1 (HIF-1) (Weinberg et al., 2015). The ncRNA

has a vital role in regulating gene expression and controlling the mitochondria. For example,

miRNA from the nucleus might control the mitochondrial gene expression, which depends on the

adenosine triphosphate (ATP) produced by the mitochondria (Duarte et al., 2014)

3.4 current and potential therapies for mitochondrial diseases.

There is no cure or an FDA (i.e. Due to the different genes and phenotypes involved with the

development of such disorders, there is currently no FDA-approved treatment for mitochondrial

diseases. In the last decade, however, only few symptomatic therapies have been validated as

palliative therapies by clinical trials. A mitochondrial cocktail, i.e., a microbial cocktail. A blend

of vitamins, cofactors, minerals, and antioxidants can reduce pain, delay disease progression, and

eliminate mitochondrial toxins.

This symptom-based management aims to enhance mitochondrial function by supporting the

electron transport chain and treating mitochondrial dysfunction’s consequences (Parikh et al.,

20019).

3.5 Nutritional supplement and exercise

24
Poor diet and extreme malnutrition lead to secondary mitochondrial dysfunctions, while

overeating increases ROS formation and generates toxic metabolites (Wortmann et al., 2009).

Therefore, specific dietary restrictions have been shown to ameliorate mitochondrial health in

patients with mitochondrial disorders; thus, evaluating individuals’ nutritional necessity and

deficiencies are significantly needed. A High-carbohydrate diet has been reported to increase

oxidative stress, which can be metabolically challenging for those with impaired oxidative

phosphorylation (El-Hattab et al., 2012). On the other hand, the ketogenic diet (high-fat diet) has

been beneficial for patients with pyruvate dehydrogenase deficiency but not helpful in the case of

pyruvate carboxylase deficiency and treating fatty acid oxidation disorders (Bough et al., 2006).

Mitochondrial diseases affecting the respiratory system can be treated with substances that

enhance electron transport and substrate supply while bypassing its components. Several

phenotypes of mitochondrial disorders resulting from the biosynthetic defects of Coenzyme Q10

(CoQ10) were treated with CoQ10 supplementation and found to decrease the elevated levels of

lactate in post-exercise and increase oxygen consumption (Di Giovanni et al., 2001). Other food

supplements, including vitamins and amino acids, may be used as redox agents and intracellular

buffering for ATP. Other symptoms relevant to mitochondrial diseases, including stroke-like

episodes, myopathy, diabetes, and lactic acidosis associated with nitric oxide (NO) depletion,

could be treated with NO natural precursors, for example, arginine and citrulline, to restore the

production of NO (El-Hattab et al., 2012).

Physical therapy and aerobic fitness have been shown to improve mitochondrial health and

reduce mitochondrial dysfunction. Patients with mitochondrial disorders are exercise-intolerant

due to low maximal oxygen uptake, but a gradual endurance exercise could help overcome such

difficulty and enhance mitochondria enzymic activity (Parikh et al., 2009).

In addition, physical therapy promotes mitochondrial biogenesis, which is induced by the high

expression of the master transcription regulator pgc-1. Accumulating evidence was demonstrated

that exercise elevates mitochondrial ROS, which triggers the organelle biogenesis pathway by

the high expression of the master transcription regulator PGC-1α leading to increased

mitochondrial quantity and quality (Kang and Li Ji, 2012). In addition, physical training can

25
improve OXPHOS, respiratory capacity and electron flow, reducing ROS production ( Memme

et al., 2021).

3.6 pharmacological agents

Ccoq analogs such as idebenone, mitoquinone, and short-chain coq10 with improved

pharmaceutical and pharmacological properties were developed to enhance mitochondria's

electron transport chain and were tested clinically for their therapeutic value. These natural and

synthetic quinones demonstrated potential antioxidant activities against toxic metabolites from

the defected mitochondria and accumulated ROS (Suárez et al., 2021). For example, a study by

(Klopstock et al., 2011) showed remarkable success in treating the visual acuity of a large group

of patients using idebenone. Many applications in clinical trials, such as Leber’s Hereditary

Optic Neuropathy (LHON; NLM, 2013), Parkinson’s disease (NLM, 2018), and MELAS

syndrome (NLM, 2016), have assured the safety of idebenone and its efficacy, even at higher

doses. It has passed phase III evaluation (Suárez et al., 2021). Other coq analogues with a diverse

side chain had unique biological functions and enhanced pharmacological properties, such as

bioavailability, mitochondrial accumulation, and antioxidant activity. Coq analogues with shorter

isoprenoid side chains have a higher antioxidant value, according to reduce ros-induced toxicity

and accumulation of ros from the defected mitochondria, antioxidants such as lipoic acid and n-

acetyl-cysteine were used.

3.7 Gene editing technology

Other than the controversial Mitochondrial Replacement Therapy (MRT) utilized to prevent the

inherited mitochondrial DNA (mtDNA) mutations (Klopstock et al., 2016),genome-editing

therapy can be considered the ultimate treatment strategy to which mitochondrial diseases

patients hold their hopes out. Despite being the prominent approach for gene editing, the

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology falls

behind in mitochondrial diseases due to the encountered difficulties in delivering the Cas9

nuclease and a guide RNA (gRNA) along with a homologous repair template coincidentally to

mitochondria for editing to take place (Gammage et al., 2018a). Alternatively, restriction

endonucleases have been used as a novel tool for gene editing, owing to their ability to form

26
linear fragments of mtDNA after selective cleavage of mutant mtdna while leaving the wild type

intact. Elimination of the mtDNA fragments pursues rapidly by the exonucleolytic activity of

enzymatic machinery, including the mitochondrial polymerase gamma (POLG) (Nissanka et al.,

2018), the mitochondrial replicative DNA helicase Twinkle (TWNK), and the mitochondrial

genome maintenance exonuclease 1(MGME1) (Peeva et al., 2018), leading to an increased

population of the wild type mtDNA. However, the use of grnas is a problem that prevents the

production of such restriction endonucleases. This inability has steered the work toward

developing programmable nucleases that can induce specific elimination of the mutant mtDNA.

These nucleases include the mitochondrial-targeted- zinc finger nucleases (mtZFN) delivered by

an adeno-associated virus and transcription activator-like effector nucleases (mitoTALENs)

(Bacman et al., 2018), both corrected the mtDNA heteroplasmy through inducing a specific

elimination of the mutant mtDNA in a mouse model. Nonetheless, these programmable

nucleases cannot cause particular nucleotide changes in mtDNA nor be applied to homoplasmic

mtDNA mutations due to the potential harmful destruction of all mtDNA copies (Stewart and

Chinnery, 2015).

A safer gene therapy reliant on base editing was recently developed by Mok et al. (Mok et al.,

2020). The interbacterial cytidine deaminase toxin DddA was effectively modified to divide into

two non-toxic parts that could be activated when they came into contact with mtDNA. These

base editors are made up of a single guide RNA (sgRNA) to aid in the single-nucleotide

conversion process via the deamination reaction and a catalytically inactive Cas9 protein

conjugated to a bacterial deaminase. With great target specificity, DddA-base editors can

catalyze the programmable transformation of C•G-to-T•A, allowing for precise editing of human

mtDNA without causing double-strand cleavage. When combined, these intriguing instruments

demonstrate preclinical proof of a substantial change in mtDNA heteroplasmy.However, their

translation into clinics is still pending, owing to the lack of efficient delivery of nucleic acids to

mitochondria (Gammage et al., 2018a).

27
 SECTION FOUR

4.0 Conclusion

Mitochondrial diseases affecting newborns and the elderly can be distinguished by their clinical,

biochemical, and genetic complexities. Recent advances in medicine have improved our

knowledge of mitochondrial disorders’ characteristics, diagnosis, prognosis, management, and

prevention, as well as the mechanisms responsible for the development of the complex clinical

phenotypes associated with such disorders. Several essential mitochondrial genes have been

identified, and they could be used to enhance mitochondrial function. Although no specific

medication has been developed to target the mitochondrion, palliative therapy is often

recommended. More effective cures can be found by using more specific medical techniques that

target individual genetic variations. In treating mitochondrial disease, new therapeutic strategies

and the increasing number of clinical trials could have a promising future.

28
 Reference

Abramov, A. Y., & Angelova, P. R. (2019). Cellular mechanisms of complex I-associated

pathology. Biochemical Society Transactions, 47, 1963–1969.

An, P., Wei, L.-L., Zhao, S., Sverdlov, D. Y., Vaid, K. A., Miyamoto, M., Kuramitsu, K., Lai,

M., & Popov, Y. V. (2020). Hepatocyte mitochondria-derived danger signals directly activate

hepatic stellate cells and drive progression of liver fibrosis. Nature Communications, 11(1).

Avula, S., Parikh, S., Demarest, S., Kurz, J., & Gropman, A. (2014). Treatment of mitochondrial

disorders. Current Treatment Options in Neurology, 16, 292.

Ahmed, S. T., Craven, L., Russell, O. M., Turnbull, D. M., & Vincent, A. E. (2018). Diagnosis

and treatment of mitochondrial myopathies. Neurotherapeutics : the Journal of the American

Society for Experimental NeuroTherapeutics, 15(4), 943–953.

Alexeyev, M., Shokolenko, I., Wilson, G., et al. (2013). The maintenance of mitochondrial DNA

integrity – critical analysis and update. Cold Spring Harbor Perspectives in Biology, 5.

Alston, C. L., Rocha, M. C., Lax, N. Z., et al. (2017). The genetics and pathology of

mitochondrial disease. The Journal of Pathology, 241, 236–250.

29
Bacman, S. R., Kauppila, J. H. K., Pereira, C. V., Nissanka, N., Miranda, M., Pinto, M.,

Williams, S. L., Larsson, N.-G., Stewart, J. B., & Moraes, C. T. (2018). MitoTALEN reduces

mutant mtDNA load and restores tRNA(Ala) levels in a mouse model of heteroplasmic mtDNA

mutation. Nature Medicine, 24(11), 1696–1700.

Belbellaa, B., Reutenauer, L., Messaddeq, N., Monassier, L., & Puccio, H. (2020). High levels of

frataxin overexpression lead to mitochondrial and cardiac toxicity in mouse models. Molecular

Therapy – Methods & Clinical Development, 19, 120–138.

Bottani, E., Giordano, C., Civiletto, G., Di Meo, I., Auricchio, A., Ciusani, E., Marchet, S.,

Lamperti, C., d’Amati, G., Viscomi, C., et al. (2014). AAV-mediated liver-specific MPV17

expression restores mtDNA levels and prevents diet-induced liver failure. Molecular Therapy,

22, 10–17.

Bough, K. J., Wetherington, J., Hassel, B., Pare, J. F., Gawryluk, J. W., Greene, J. G., Shaw, R.,

Smith, Y., Geiger, J. D., & Dingledine, R. J. (2006). Mitochondrial biogenesis in the

anticonvulsant mechanism of the ketogenic diet. Annals of Neurology, 60(2), 223–235.

Budde, S. M. S., van den Heuvel, L. P. W. J., Smeets, R. J. P., Skladal, D., Mayr, J. A., Boelen,

C., Petruzzella, V., Papa, S., & Smeitink, J. A. M. (2003). Clinical heterogeneity in patients with

mutations in the NDUFS4 gene of mitochondrial complex I. Journal of Inherited Metabolic

Disease, 26, 813–815.

Bourgeron, T., Rustin, P., Chretien, D., Birch-Machin, M., Bourgeois, M., Viegas-Péquignot, E.,

Munnich, A., & Rötig, A. (1995). Mutation of a nuclear succinate dehydrogenase gene results in

mitochondrial respiratory chain deficiency. Nature Genetics, 11(2), 144–149.

Cavalcante, G. C., Magalhães, L., Ribeiro-dos-Santos, Â., & Vidal, A. F. (2020). Mitochondrial

epigenetics: Non-coding RNAs as a novel layer of complexity. International Journal of

Molecular Sciences, 21(5), 1838.

Chipman, L. B., & Pasquinelli, A. E. (2019). miRNA targeting: Growing beyond the seed.

Trends in Genetics, 35, 215–222.

30
Campuzano, V., Montermini, L., Moltò, M. D., Pianese, L., Cossée, M., Cavalcanti, F., Monros,

E., Rodius, F., Duclos, F., Monticelli, A., et al. (1996). Friedreich’s ataxia: Autosomal recessive

disease caused by an intronic GAA triplet repeat expansion. Science, 271, 1423–1427.

D’Souza, A. R., & Minczuk, M. (2018). Mitochondrial transcription and translation: Overview.

Essays in Biochemistry, 62, 309–320.

Di Mauro, S., & Schon, E. A. (2003). Mitochondrial respiratory-chain diseases. New England

Journal of Medicine, 348, 2656.

Di Giovanni, S., Mirabella, M., Spinazzola, A., Crociani, P., Silvestri, G., Broccolini, A., Tonali,

P., Di Mauro, S., & Servidei, S. (2001). Coenzyme Q10 reverses pathological phenotype and

reduces apoptosis in familial CoQ10 deficiency. Neurology, 57(3), 515–518.Dell’Agnello, C.,

Leo, S., Agostino, A., Szabadkai, G., Tiveron, C., Zulian, A., Prelle, A., Roubertoux, P., Rizzuto,

R., & Zeviani, M. (2007). Increased longevity and refractoriness to Ca2+-dependent

neurodegeneration in Surf1 knockout mice. Human Molecular Genetics, 16, 431–444.

Duarte, F. V., Palmeira, C. M., & Rolo, A. P. (2014). The role of microRNAs in mitochondria:

Small players acting wide. Genes, 5, 865–886.

Dimmock, D. P., & Lawlor, M. W. (2017). Presentation and diagnostic evaluation of

mitochondrial disease. Pediatric Clinics of North America, 64(1), 161–171.

D’Aquila, P., Bellizzi, D., & Passarino, G. (2015). Mitochondria in health, aging and diseases:

The epigenetic perspective. Biogerontology, 16(5), 569–585.

El-Hattab, A. W., Hsu, J. W., Emrick, L. T., Wong, L.-J., Craigen, W. J., Jahoor, F., & Scaglia,

F. (2012). Restoration of impaired nitric oxide production in MELAS syndrome with citrulline

and arginine supplementation. Molecular Genetics and Metabolism, 105(4), 607–614.

Fassone, E., & Rahman, S. (2012). Complex I deficiency: Clinical features, biochemistry and

molecular genetics. Journal of Medical Genetics, 49, 578–590.

31
Filograna, R., Mennuni, M., Alsina, D., & Larsson, N. (2021). Mitochondrial DNA copy number

in human disease: The more the better? FEBS Letters, 595, 976–1002.

Fernandez-Moreira, D., Ugalde, C., Smeets, R., Rodenburg, R. J. T., Lopez-Laso, E., Ruiz-Falco,

M. L., Briones, P., Martin, M. A., Smeitink, J. A. M., & Arenas, J. (2007). X-linked NDUFA1

gene mutations associated with mitochondrial encephalomyopathy. Annals of Neurology, 61(1),

73–83.

Finsterer, J., & Zarrouk-Mahjoub, S. (2018). Biomarkers for detecting mitochondrial disorders.

Journal of Clinical Medicine, 7(2), 16.

Flierl, A., Chen, Y., Coskun, P. E., Samulski, R. J., & Wallace, D. C. (2005). Adeno-associated

virus-mediated gene transfer of the heart/muscle adenine nucleotide translocator (ANT) in

mouse. Gene Therapy, 12, 570–578.

Ferreira, C. R., Whitehead, M. T., & Leon, E. (2017). Biotin-thiamine responsive basal ganglia

disease: Identification of a pyruvate peak on brain spectroscopy, novel mutation in SLC19A3,

and calculation of prevalence based on allele frequencies from aggregated next-generation

sequencing data. American Journal of Medical Genetics. Part A, 173(6), 1502–1513.

Gammage, P. A., Moraes, C. T., & Minczuk, M. (2018). Mitochondrial genome engineering: The

revolution may not be CRISPR-ized. Trends in Genetics, 34(2), 101–110.

Gorman, G. S., Chinnery, P. F., DiMauro, S., Hirano, M., Koga, Y., McFarland, R.,

Suomalainen, A., Thorburn, D. R., Zeviani, M., & Turnbull, D. M. (2016). Mitochondrial

diseases. Nature Reviews. Disease Primers, 2(1).

Germain, N., Dessein, A.-F., Vienne, J.-C., Dobbelaere, D., Mention, K., Joncquel, M., …

Marchetti, P. (2019). First-line screening of OXPHOS deficiencies using microscale oxygraphy

in human skin fibroblasts: A preliminary study. International Journal of Medical Sciences, 16(7),

931–938.

Kang, C., & Li, Ji, L. (2012). Role of PGC-1α signaling in skeletal muscle health and disease.

Annals of the New York Academy of Sciences, 1271(1), 110–117.

Mitochondrial disorders. Annals of Translational Medicine, 6, 475.

Klopstock, T., Yu-Wai-Man, P., Dimitriadis, K., et al. (2011). A randomized placebo-controlled

trial of idebenone in Leber’s hereditary optic neuropathy. Brain, 134, 2677–2686.

Klopstock, T., Klopstock, B., & Prokisch, H. (2016). Mitochondrial replacement approaches:

Challenges for clinical implementation. Genome Medicine, 8, 126.

Kirby, D. M., Crawford, M., Cleary, M. A., Dahl, H. -H. M., Dennett, X., & Thorburn, D. R.

(1999). Respiratory chain complex I deficiency: An underdiagnosed energy generation disorder.

Neurology, 52, 1255.

Khan, N. A., Govindaraj, P., Meena, A. K., & Thangaraj, K. (2015). Mitochondrial disorders:

Challenges in diagnosis & treatment. Indian Journal of Medical Research, 141, 13.

Kaukonen, J., Juselius, J. K., Tiranti, V., Kyttälä, A., Zeviani, M., Comi, G. P., … Suomalainen,

A. (2000). Role of adenine nucleotide translocator 1 in MtDNA maintenance. Science, 289, 782–

785.

Landsverk, M. L., Zhang, V. W., Wong, L. -J., & Andersson, H. C. (2014). A SUCLG1 mutation

in a patient with mitochondrial DNA depletion and congenital anomalies. Molecular Genetics

and Metabolism Reports, 1, 451–454.

Ling, Q., Rioux, M., Hu, Y., & Lee, M. (2021). Adeno-associated viral vector serotype 9-based

gene replacement therapy for SURF1-related Leigh syndrome. Molecular Therapy – Methods &

Clinical Development, 23, 158–168.

Liu, Y. J., McIntyre, R. L., Janssens, G. E., & Houtkooper, R. H. (2020). Mitochondrial fission

and fusion: A dynamic role in aging and potential target for age-related disease. Mechanisms of

Ageing and Development, 186, 111212.

Maresca, A., Zaffagnini, M., Caporali, L., et al. (2015). DNA methyltransferase 1 mutations and

mitochondrial pathology: Is mtDNA methylated? Frontiers in Genetics, 6.

33
Mendell, J. R., Al-Zaidy, S. A., Rodino-Klapac, L. R., Goodspeed, K., Gray, S. J., Kay, C. N., …

Sahenk, Z. (2021). Current clinical applications of in vivo gene therapy with AAVs. Molecular

Therapy, 29, 464–488.

Memme, J. M., Erlich, A. T., Phukan, G., & Hood, D. A. (2021). Exercise and mitochondrial

health. The Journal of Physiology, 599(3), 803–817.

Mok, B. Y., de Moraes, M. H., Zeng, J., Bosch, D. E., Kotrys, A. V., Raguram, A., … Liu, D. R.

(2020). A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.

Nature, 583 (7817), 631–637.

Melchiorri, D., Pani, L., Gasparini, P., Cossu, G., Ancans, J., Borg, J. J., … & Hudson, I. (2013).

Regulatory Evaluation of Glybera in Europe—Two Committees, One Mission. Nat. Rev. Drug

Discov., 12, 719.

Mendell, J. R., Al-Zaidy, S., Shell, R., Arnold, W. D., Rodino-Klapac, L. R., Prior, T. W., … &

Berry, K. (2017). Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. *N.

Engl. J. Med., 377, 1713–1722.

Muraresku, C. C., McCormick, E. M., & Falk, M. J. (2018). Mitochondrial Disease: Advances in

clinical diagnosis, management, therapeutic development, and preventative strategies. *Current

Genetic Med. Reports, 6(2), 62–72.

Moggio, M., Colombo, I., Peverelli, L., et al. (2014). Mitochondrial disease heterogeneity: a

prognostic challenge. Acta Myologica : Myopathies Cardiomyopathies : Off. J. Mediterranean

Soc. Myol., 33, 86–93.

Newell, C., Khan, A., Sinasac, D., Shoffner, J., Friederich, M. W., Van Hove, J. L. K., … &

Sosova, I. (2019). Hybrid gel electrophoresis using skin fibroblasts to aid in diagnosing

mitochondrial disease. Neurology. Genetics, 5(3), e336.

Nissanka, N., Bacman, S. R., Plastini, M. J., & Moraes, C. T. (2018). The mitochondrial DNA

polymerase gamma degrades linear DNA fragments precluding the formation of deletions. Nat.

Commun., 9(1).
34
Ocana-Santero, G., Díaz-Nido, J., & Herranz-Martín, S. (2021). Future Prospects of Gene

Therapy for Friedreich’s Ataxia. Int. J. Mol. Sci., 22, 1815.

Olivieri, G., Martinelli, D., Longo, D., Grimaldi, C., Liccardo, D., Di Meo, I., … & Dionisi-Vici,

C. (2021). Ethylmalonic Encephalopathy and Liver Transplantation: Long-Term Outcome of the

First Treated Patient. Orphanet J. Rare Dis., 16, 229.

Ortigoza-Escobar, J. D., Oyarzabal, A., Montero, R., Artuch, R., Jou, C., Jiménez, C., … &

López-Gallardo, E. (2016). Ndufs4 Related Leigh Syndrome: A Case Report and Review of the

Literature. Mitochondrion, 28, 73–78.

O’Toole, J. F. (2014). Renal manifestations of genetic mitochondrial disease. *Int. J. Nephrol.

Renovascular Dis., 7, 57–67.

Parikh, S., Saneto, R., Falk, M. J., Anselm, I., Cohen, B. H., Haas, R., … & Medicine Society

TM. (2009). A modern approach to the treatment of mitochondrial disease. Curr Treat Options

Neurol., 11 414–430.

Parikh, S., Goldstein, A., Koenig, M. K., Scaglia, F., Enns, G. M., Saneto, R., … & DiMauro, S.

(2015). Diagnosis and management of mitochondrial disease: a consensus statement from the

Mitochondrial Medicine Society. Genetics Med. : Off. J. Am. College Medical Genet., 17(9),

689–701.

Pickett, S. J., Grady, J. P., Ng, Y. S., et al. (2018). Phenotypic heterogeneity in m.3243A>G

mitochondrial disease: The role of nuclear factors. Ann. Clin. Transl. Neurol., 5, 333–345.

Peeva, V., Blei, D., Trombly, G., Corsi, S., Szukszto, M. J., Rebelo-Guiomar, P., … & Kunz, W.

S. (2018). Linear mitochondrial DNA is rapidly degraded by components of the replication

machinery. Nat. Commun., 9(1).

Parés, M., Fornaguera, C., Vila-Julià, F., Oh, S., Fan, S. H. Y., Tam, Y. K., … & Borrós, S.

(2021). Preclinical Assessment of a Gene-Editing Approach in a Mouse Model of Mitochondrial

Neurogastrointestinal Encephalomyopathy. Hum. Gene Ther., 32, 1210–1223.

35
Pandolfo, M. (2009). Friedreich Ataxia: The Clinical Picture. J. Neurol., 256, 3–8.

Piguet, F., de Montigny, C., Vaucamps, N., Reutenauer, L., Eisenmann, A., & Puccio, H. (2018).

Rapid and Complete Reversal of Sensory Ataxia by Gene Therapy in a Novel Model of

Friedreich Ataxia. Mol. Ther., 26, 1940–1952.

Quirós, P. M., Langer, T., & López-Otín, C. (2015). New roles for mitochondrial proteases in

health, ageing and disease. Nat Rev Mol Cell Biol., 16, 345–359.

Rahman, J., Noronha, A., Thiele, I., & Rahman, S. (2017). Leigh map: A novel computational

diagnostic resource for mitochondrial disease. Ann. Neurol., 81(1), 9–16.

Russell, S., Bennett, J., Wellman, J. A., Chung, D. C., Yu, Z.-F., Tillman, A., … & High, K. A.

(2017). Efficacy and Safety of Voretigene Neparvovec (AAV2-HRPE65v2) in Patients with

RPE65-Mediated Inherited Retinal Dystrophy: A Randomised, Controlled, Open-Label, Phase 3

Trial. Lancet, 390, 849–860.

Ronchi, D., Fassone, E., Bordoni, A., Sciacco, M., Lucchini, V., Di Fonzo, A., … & Comi, G. P.

(2011). Two Novel Mutations in PEO1 (Twinkle) Gene Associated with Chronic External

Ophthalmoplegia. J. Neurol. Sci., 308, 173–176.

Schon, K. R., Horvath, R., Wei, W., … & Taylor, R. W. (2021). Use of whole genome

sequencing to determine genetic basis of suspected mitochondrial disorders: cohort study. BMJ,

375 , e066288.

Stenton, S. L., & Prokisch, H. (2020). Genetics of mitochondrial diseases: identifying mutations

to help diagnosis. EBioMedicine, 56, 102784.

Skulachev, V. P. (1999). Mitochondrial physiology and pathology; concepts of programmed

death of organelles, cells and organisms. Mol Aspects Med., 20, 139–184.

Schmidt, O., Pfanner, N., & Meisinger, C. (2010). Mitochondrial protein import: from

proteomics to functional mechanisms. Nat Rev Mol Cell Biol., 11, 655–667.

36
Shtolz, N., & Mishmar, D. (2019). The mitochondrial genome–on selective constraints and

signatures at the organism, cell, and single mitochondrion levels. Front Ecol Evol., 7, 342.

Schon, E. A., & Gilkerson, R. W. (2010). Functional complementation of mitochondrial DNAs:

Mobilizing mitochondrial genetics against dysfunction. Biochim Biophys Acta., 1800, 245–249.

Spinazzola, A., Viscomi, C., Fernandez-Vizarra, E., Carrara, F., D’Adamo, P., Calvo, S., … &

Zeviani, M. (2006). MPV17 Encodes an Inner Mitochondrial Membrane Protein and Is Mutated

in Infantile Hepatic Mitochondrial DNA Depletion. Nat. Genet., 38 , 570–575.

Spinazzola, A., Santer, R., Akman, O. H., Tsiakas, K., Schaefer, H., Ding, X., … & Zeviani, M.

(2008). Hepatocerebral Form of Mitochondrial DNA Depletion Syndrome: Novel MPV17

Mutations. Arch. Neurol., 65, 1108–1113.

Schubert Baldo, M., & Vilarinho, L. (2020). Molecular Basis of Leigh Syndrome: A Current

Look. Orphanet J. Rare Dis., 15, 31.

Song, S., Pursell, Z. F., Copeland, W. C., Longley, M. J., Kunkel, T. A., & Mathews, C. K.

(2005). DNA precursor asymmetries in mammalian tissue mitochondria and possible

contribution to mutagenesis through reduced replication fidelity. PNAS, 102(14), 4990–4995.

Sharma, N., Pasala, M. S., & Prakash, A. (2019). Mitochondrial DNA: Epigenetics and

environment. Environ. Mol. Mutagen., 60(8), 668–682.

Smith, R. A. J., Hartley, R. C., Cochemé, H. M., & Murphy, M. P. (2012). Mitochondrial

pharmacology. Trends Pharmacol. Sci., 33(6), 341–352., J. B., & Chinnery, P. F. (2015). The

dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease. Nat.

Rev. Genet., 16(9), 530–542.

Stenton, S. L., & Prokisch, H. (2020). Genetics of mitochondrial diseases: Identifying mutations

to help diagnosis. EBioMedicine, 56, 102784.

Suárez-Rivero, J. M., Pastor-Maldonado, C. J., Povea-Cabello, S., Álvarez-Córdoba, M.,

Villalón-García, I., Munuera-Cabeza, M., … & Sánchez-Alcázar, J. A. (2021). Coenzyme Q(10)

37
Analogues: Benefits and Challenges for Therapeutics. Antioxidants (Basel, Switzerland), 10(2),

236.

Srinivasan, H., & Das, S. (2015). Mitochondrial miRNA (MitomiR): a new player in

cardiovascular health. Can. J. Physiol. Pharmacol., 93(10), 855–861.

Schmidt, O., Pfanner, N., & Meisinger, C. (2010). Mitochondrial protein import: from

proteomics to functional mechanisms. Nat Rev Mol Cell Biol., 11 , 655–667.

Spinazzola, A., Viscomi, C., Fernandez-Vizarra, E., Carrara, F., D’Adamo, P., Calvo, S., … &

Zeviani, M. (2006). MPV17 Encodes an Inner Mitochondrial Membrane Protein and Is Mutated

in Infantile Hepatic Mitochondrial DNA Depletion. Nat. Genet., 38, 570–575.

Smith, R. A. J., Hartley, R. C., Cochemé, H. M., & Murphy, M. P. (2012). Mitochondrial

pharmacology. Trends in Pharmacological Sciences, 33(6), 341–352.

Stenton, S. L., & Prokisch, H. (2020). Genetics of mitochondrial diseases: Identifying mutations

to help diagnosis. EBioMedicine, 56, 102784.

Sharma, N., Pasala, M. S., & Prakash, A. (2019). Mitochondrial DNA: Epigenetics and

environment. Environmental and Molecular Mutagenesis, 60(8), 668–682.

Taylor, R. W., & Turnbull, D. M. (2005). Mitochondrial DNA mutations in human disease.

Nature Reviews Genetics, 6(5), 389–402.

Tinker, R. J., Lim, A. Z., Stephanotis, R. J., & McFarland, R. (2021). Current and emerging

clinical treatment in mitochondrial disease. Molecular Diagnosis & Therapy, 25 ,181–206.

Tiranti, V., Viscomi, C., Hildebrandt, T., Di Meo, I., Mineri, R., Tiveron, C., … & Zeviani, M.

(2009). Loss of ETHE1, a mitochondrial dioxygenase, causes fatal sulfide toxicity in

ethylmalonic encephalopathy. Nature Medicine, 15 , 200–205.

38
Tiranti, V., Jaksch, M., Hofmann, S., Galimberti, C., Hoertnagel, K., Lulli, L., … & Comi, G.-P.

(1999). Loss-of-function mutations of SURF-1 are specifically associated with Leigh syndrome

with cytochrome c oxidase deficiency. Annals of Neurology, 46, 161–166.

Torres-Torronteras, J., Gómez, A., Eixarch, H., Palenzuela, L., Pizzorno, G., Hirano, M., … &

Martí, R. (2011). Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell

culture and a murine model of MNGIE. Gene Therapy, 18 . 795–806.

Van de Wal, M. A. E., Adjobo-Hermans, M. J. W., Keijer, J., Schirris, T. J. J., Homberg, J. R.,

Wieckowski, M. R., … & Quintana, A. (2022). Ndufs4 knockout mouse models of Leigh

syndrome: Pathophysiology and intervention. Brain, 145 , 45–63.

Viscomi, C., Bottani, E., & Zeviani, M. (2015). Emerging concepts in the therapy of

mitochondrial disease. Biochimica et Biophysica Acta, 1847, 544–57.

Viscomi, C., Spinazzola, A., Maggioni, M., Fernandez-Vizarra, E., Massa, V., Pagano, C., … &

Zeviani, M. (2009). Early-onset liver mtDNA depletion and late-onset proteinuric nephropathy in

Mpv17 knockout mice. Human Molecular Genetics, 18, 12–26.

Wang, L., Saada, A., & Eriksson, S. (2003). Kinetic properties of mutant human thymidine

kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy. Journal of Biological

Chemistry, 278, 6963–6968.

Wallace, D. C., & Chalkia, D. (2013). Mitochondrial DNA genetics and the heteroplasmy

conundrum in evolution and disease. Cold Spring Harbor Perspectives in Biology, 5, a021220–

a021220.

Wedatilake, Y., Brown, R. M., McFarland, R., Yaplito-Lee, J., Morris, A. A. M., Champion, M.,

… & Rahman, S. (2013). SURF1 deficiency: A multi-centre natural history study. Orphanet

Journal of Rare Diseases, 8(1).

Weinberg, S., Sena, L., & Chandel, N. (2015). Mitochondria in the regulation of innate and

adaptive immunity. Immunity, 42(3), 406–417.

39
Wortmann, S. B., Zweers-van Essen, H., Rodenburg, R. J. T., van den Heuvel, L. P., de Vries,

M. C., Rasmussen-Conrad, E., … & Morava, E. (2009). Mitochondrial energy production

correlates with the age-related BMI. Pediatric Research, 65(1), 103–108.

Wong, L.-J. C., Brunetti-Pierri, N., Zhang, Q., Yazigi, N., Bove, K. E., Dahms, B. B., … &

Schmitt, E. S. (2007). Mutations in the MPV17 gene are responsible for rapidly progressive liver

failure in infancy. Hepatology, 46 , 1218–1227.

Yang, G., Sun, X., & Wang, R. (2004). Hydrogen sulfide-induced apoptosis of human aorta

smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3. The

FASEB Journal, 18, 1782–1784.

Yatsuga, S., Fujita, Y., Ishii, A., Fukumoto, Y., Arahata, H., Kakuma, T., … & Koga, Y. (2015).

Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders. Annals of

Neurology, 78(5), 814–823.

Yusoff, A. A. M., Abdullah, W. S. W., Khair, S., et al. (2019). A comprehensive overview of

mitochondrial DNA 4977-bp deletion in cancer studies. Oncology Reviews, 13, 409.

40