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Agresti et al. J Transl Genet Genom 2018;2:9 I http://dx.doi.org/10.20517/jtgg.2018.05 Page 7 of 11

mostly be found in all tissues, the existence of heteroplasmy can cause mutated mtDNA to be variably
distributed in tissues that leads to a changeable disease-triggering threshold level .
[36]

Similar to MERRF, MELAS is also a multisystem disease, with an origination usually in childhood,
that is characterized by stroke-like episodes, dementia, seizures, lactic acidosis, ragged red fibers (visible
during muscle biopsy), vomiting and headaches [37,38] . The most prominent pathogenic variant (present in
approximately 80% patients) is MT-TL1 (encodes for tRNALeu(UUA/UUG)), which can be observed by an
A-to-G nucleotide switch at position 3243 (m.3243A>G) [Figure 1] . Other common pathogenic variants, such
[37]
as MT-ND5, are associated with MELAS . Confirming a patient positive for MELAS focuses on correctly
[37]
diagnosing phenotypic characteristics associated with the disease, as well as employing genetic testing for
further validation (extraction of mtDNA from leukocytes acts as a reliable source) . Like MERRF, the
[37]
existence of mtDNA heteroplasmy can cause mutated mtDNA to be variously distributed in tissues, which
leads to a mutable disease-triggering threshold level . Studies have shown the A3243G mutation affects
[37]
mitochondrial protein synthesis, as well as the respiratory chain, when the threshold of approximately 85%
mutant mtDNA is reached (as determined by Kobayashi et al. ); even though mutant mtDNA levels vary
[39]
among individuals, as well as organs and tissues of a single individual . On average, individuals with
[40]
MELAS do not have a favorable prognosis, as the 34.5 ± 19 years is the average age of death .
[38]
MERRF and MELAS - current advancements in gene therapy
The current state of care for MERRF and MELAS patients focuses on a combination of genetic counseling,
surgery, and palliative treatment. Potential reasons why a pharmacological cure does not currently exist (for
either disease) may be attributed to the location of mtDNA within the mitochondrial matrix, followed by an
inability to correct the disease-causing point mutation. It seems plausible that developing a gold-standard
capable of overcoming these hurdles, and cure a mitochondrial disease, is likely difficult at best. The field
of gene therapy may offer hopeful and potentially realistic possibilities that may lie beyond routine patient
symptom management. It is an emerging field that seeks to treat or prevent disease using viral and/or nonviral
modalities by inducing correction (of a particular cellular gene) via an exogenous payload (i.e., nucleic acids).
While gene therapy may theoretically sound like a promising prospective, downsides such as mistargeting of
the exogenous payload, transient correction, inciting an immune response, and cellular apoptosis, do exist.
While these existing downsides have a direct relationship with nuclear targeting, integrating mitochondrial
biology brings forth an additional obstacle a gene therapy agent would have to be capable of overcoming.

For these reasons, previous gene therapy research efforts, with a focus on correcting MERRF and
MELAS diseases, have been conservatively progressing for the past few decades. Gene therapy research
efforts have been focused on three main approaches: nucleic acid delivery, peptide-mediated therapy, and
cleavage of pathogenic mutations by mitochondrial-targeted transcription activator-like effector nucleases
(mitoTALENS) [Table 3] [41-56] .

The goal of nucleic acid therapy is to deliver exogenous material to mitochondria harboring pathogenic
mutations to restore function. Mahata et al. [41,42] demonstrated the RNA import complex from Leishmania
can induce tRNALys import into mitoplasts isolated from a transmitochondrial cybrid MERRF cell line and
in MERRF cybrid cells. Following the import of tRNALys, Mahata et al. [41,42] observed restored mitochondrial
translation to near-wild-type levels and suppression of mutant polypeptide production. Likewise,
Kolesnikova et al. demonstrated that mitochondrial functions were partially restored due to the import of
[43]
yeast tRNALys into homoplasmic transmitochondrial cybrid MERRF lines and primary human fibroblasts
that were 70% ± 5% heteroplasmic for MERRF. Following this research, Karicheva et al. transfected cybrid
[44]
cells that were 90% ± 5% heteroplasmic for MELAS with tRNALeu(UUR) transcripts. Authors observed
an improvement in mitochondrial translation, higher levels of mtDNA encoded respiratory subunits, and
increased respiration activity .
[44]