Stopcodon readthrough approach as a therapeutic approach in genetic diseases
ABSTRACT • Approximately one-third of human genetic diseases are caused by alleles carrying premature termination codons, which lead to the production of truncated proteins that result in loss of function and are often accompanied by low levels of mRNA transcripts. Some chemicals referrred to as stopcodon readthrough drugs (or translational read-through-inducing drugs) including aminoglycosides and non-aminoglycoside small molecules restore full-length functional protein by inducing ‘stopcodon readthrough’ of premature termination codons. During stopcodon readthrough (or translational readthrough), the translational machinery recognizes the stop codon of the premature termination codon as a triplet coding for an amino acid resulting in the translation of a full-length protein from mutant messenger RNA. Considering that >1,800 distinct genetic disorders are caused by premature termination codons, the readthrough of primary premature termination codons has potential for the treatment of a wide range of genetic diseases mainly diseases with neurological symptoms for which enzyme replacement is not an option. Especially it is likely to have an impact on disease pathology for lysosomal storage diseases since even a minor increase in enzyme activity is suffcient to prevent storage. The success of stopcodon readthrough therapy has been shown for many diseases by using in vitro and animal disease models and is currently in clinical trials for treatment of several genetic diseases caused by premature termination codons.
Approximately 30% of human disease causing alleles are premature termination codons (PTCs),
which lead to the production of truncated proteins
(1). PTCs can arise from various types of mutations
in germ or somatic cells including nonsense mutations that change a sense codon to an in-frame premature termination codon (PTC), insertion or deletions that alter the reading frame, and mutations
that lead to mRNA splicing defects (2). Stopcodon
readthrough therapy, also called translational readthrough or nonsense suppression therapy, is
an approach aimed at treating or alleviating the
phenotypic consequences of a wide range of genetic diseases caused by in-frame PTC or nonsense
mutations (3). Some compounds can induce read
through of PTCs by reducing proofreading of codon-anticodon recognition in the ribosome and
result in translation of full-lenght protein. The
induced protein might gain function although it
carries a missense amino acid but it may have a reduced half-life due to the post-translational surveillance system such as endoplasmic reticulum
associated degradation (ERAD) but the recovered
enzymatic activity might allow improvement of
the biochemical phenotype (4). As a general rule
glutamine or tryptophan is inserted at premature
UAG/UAA or UGA codons, respectively (5). The
best characterized of these drugs are the aminoglycosides (6).
Aminoglycosides act as antibiotics in high doses by
inhibiting protein synthesis, where they bind to a
region of the 16S ribosomal RNA in the bacterial
ribosome called the decoding center (7). By binding to the complementary sequences 1404–1412
and 1488–1497, respectively at the decoding center, aminoglycosides displace non-complementary
adenines and locking them into a ‘flipped out’ configuration, which results in reduced discrimination
between cognate and near-cognate tRNA:mRNA
complexes and hence reducing translational fidelity with an end result of nonfunctional truncated
proteins and final cell death (8). Due to the fundamental differences in the nucleotide sequences that is necessary for hydrogen bond formation
(A2408 and G1491 in bacteria, G1408 and A1491
in mammalian cells), the interaction between aminoglycosides and human 18S rRNA is less stable
but sufficient to reduce the proofreading to cause
the insertion of a near-cognate aminoacyl-tRNA
into the ribosomal A site that is subsequently incorporated into the polypeptide chain (8). Studies
in eukaryotic cells have found that aminoglycosides that bind to the eukaryotic ribosome do not
appear to induce significant misreading at sense
codons, but can induce low levels of misreading at
The first demonstration that aminoglycosides
could suppress PTC in a defective gene was carried out in cystic fibrosis (10). Since then stopcodon
readthrough has been reported in cell and animal
models of different disorders including cystic fibrosis (11), Duchenne muscular dystrophy (12), phenylketonuria (13), Rett syndrome (14), ataxia-telangiectasia (15), xeroderma pigmentosum (16),
mucopolysaccharidosis type I-Hurler syndrome
(17,18), Nieman–Pick A/B, mucopolysaccharidosis type IIIB, mucopolysaccharidosis type II (4),
mucopolysaccharidosis VI (19), Usher syndrome
(20), methylmalonic academia (21), proximal spinal muscular atrophy (22), and Stüve-Wiedemann
Syndrome (23). Several pilot clinical trials with
patients carrying nonsense mutations with cystic
fibrosis (24,25) and Duchenne muscular dystrophy
(26,27) have shown the partial restoration of fulllength functional protein to a variable extent with
gentamicin administration. However, the toxicity
of most aminoglycosides in mammals has greatly restricted their potential as readthrough drug
(28). Therefore, efforts have been spent to develop
aminoglycoside derivatives with reduced toxicity
and enhanced activity. NB30, NB54, and NB84
are among these aminoglycoside derivatives with
lower toxicity and exhibiting higher readthrough
PTC therapeutics described an efficient nonaminoglycoside readthrough compound, PTC124 (AtalurenTM), which was developed synthetically by
screening >800,000 chemicals and analogues using a luciferase-based high-throughput screening
(HTS) assay (31,32). A phase-I clinical study in
cystic fibrosis confirmed that PTC124 is generally
well tolerated and appears to have more efficient
readthrough activity than aminoglycosides (32).
PTC124 was initially shown to suppress nonsense
mutations associated with Duchenne muscular
dystrophy (DMD) and cystic fibrosis in mouse models (2).
The success of suppression therapy to provide a
therapeutic benefit in various individuals depends
on many factors. One particularly important factor is the threshold of correction for a particular
disorder that varies upon the function of the factor
and the tissues where the protein is expressed. For
example, for some disorders that result from an enzyme deficiency such as mucopolysaccharidosis
type I-Hurler (MPS I-H), as little as 1% of wildtype enzymatic activity can significantly alleviate
the disease phenotype (33).
FACTORS AFFECTING THE RESPONSE TO
Several factors were reported to affect the efficiency
of stopcodon readthrough treatment including the
identity of the PTC, the sequence context around
the PTC and nonsense mediated decay (NMD).
UGA stop codon exhibit the highest readthrough
efficiency, followed by UAG and, to a lesser extent,
UAA (5,34). Regarding the effect of sequence context, the fourth position of the tetranucleotide also
plays a role in determining the efficiency of readthrough; however, its effect depends on largely to
the PTC itself (4,5). For example, the UGA C was
shown to exhibit a three-to sixfold higher level of
readthrough than the other UGA (N) signals. The
relative order of susceptibility to readthrough as a
function of the fourth base was C>A, G>U. However, readthrough of the UAG C and UAA C signals
was not significantly higher than the readthrough
observed at the other UAG (N) and UAA (N) signals, respectively (5).
Gentamicin treatment of C2C12 mouse myoblast
cells transfected with PTC-bearing dual luciferase vector resulted in ~8% readthrough for UGA
C while little measurable increase in readthrough
was observed for UAA A (mdx) premature stop
codon for Duchenne’s muscular dystrophy (DMD)
and Becker’s muscular dystrophy (BMD). Gentamicin-induced readthrough levels for eight PTCs identified in DMD and BMD patients varied between
approximately 1 and 10% and roughly paralleled
as a function of the fourth base (UGA>UAG>UAA;
+4 C>U>G≥A) (35).
Chemical composition of aminoglycosides is another factor affecting the response to readthrough.
When compared in human cells expressing reporter constructs, gentamicin and paromomycin were most effective at inducing readthrough while
tobramycin and neomycin showed lower readthrough efficiency than gentamicin, amikacin and
One factor leading to low readthrough efficiency is the low amount of PTC-bearing transcripts
caused by nonsense mediated decay (NMD) which
degrades mutated mRNAs (36). NMD is a surveillance system detecting and commiting PTC-bearing
transcripts to rapid decay to prevent the synthesis
of unstable proteins that might be deleterious for
the cell (37). NMD is an evolutionary conserved
mechanism to be implicated in surveillance and
regulation of gene expression in all eukaryotes
(38). NMD downregulates not only PTCs but also
one-third of alternatively spliced mRNAs, certain
selenoprotein mRNAs, some mRNAs that have
upstream open reading frames, and some mRNAs
that contain an intron within the 3´ untranslated
region (39,40).The NMD process could be relevant
in terms of the phenotypic presentation of human
diseases caused by nonsense mutations. In some
cases, the lack of a mutant protein due to NMD
could result in a milder phenotype since the deleterious effect of the aberrant protein is partially
abolished. In other cases, the NMD could eliminate
a partially active mutant protein and produce a
more severe phenotype (41,42). PTCs in mammalian systems are targeted for NMD when located
more than 50-54 nucleotides upstream the last
exon-exon junction whereas PTCs located downstream of this boundary are not. Recognition of
PTC- mRNAs and their targeting for degradation
requires a set of conserved NMD effectors, which
include the Up-frame shift (UPF) proteins UPF1,
UPF2 and UPF3B and some exon junction complex
(EJC) proteins (2). The EJC complex was shown to
constitute a binding platform for the NMD effectors UPF2 and UPF3 (up-frameshift) (37). When
the ribosome reaches a PTC, interaction of the
release factors eRF1 and eRF3 with downstream
EJCs bridged by the UPF proteins triggers the phosphorylation of UPF1 and subsequent degradation of the mRNA (for detailed information about
NMD, see the reviews (37,43,44,45).
PTC readthrough compounds may increase the
stability of mutant RNA by limiting NMD. Infact, several papers have reported that gentamicin and other readthrough agents inhibit NMD
and increase the amount of PTC-containing RNAs
(4,46,47). In an effort to determine the correlation
between the recovery residual enzymatic activity
and mRNA expression in response to gentamicin
treatment, mRNA expression levels of about 20-
40% that of controls for SMPD1 (Nieman-Pick
A/B disease) and NAGLU (MPS III disease) genes
except IDS gene (MPS II disease) was observed
(4). Although these levels did not reach those obtained after treatment with cycloheximide, which
is a general translation inhibitor used to assay for
the occurrence of NMD, suggesting that gentamicin readthrough was not totally efficient and some
mRNA was still being degraded. Interestingly, the
gentamicin treated culture of MPS II (Hunter) disease presented mRNA expression levels similar to
controls, which is explained by the location of the
relavant PTC in IDS gene where it is located in the
last exon, so that the resulting mRNA might elude
the NMD surveillance mechanism resulting in normal mRNA levels (4). In the same study, gentamicin treatment of two different MPS III patients,
one with p.W168X and p.R234C and another with
p.W168X and Q566X resulted in approximately
20% and 40% increase in NAGLU gene expression,
respectively. Although one of the alleles of MPS
III patient (p.Q566X) is located in the last exon of
NAGLU gene, mRNA expression in fibroblasts was
low (20% of control values), which was explained
by the presence of p.W168X mutation that did not
elude the NMD surveillance (4).
In a study investigating the readthrough effects
of gentamicin, G148 (geneticin) and five non-aminoglycoside compounds (PTC124, RTC13, RTC14,
BZ6 and BZ16) on fibroblasts from one patient with MPS IIIB (Sanfilippo B) harbouring p.W168X-
/p.Q566X, and one with MPS IIIC (Sanfilippo C)
harbouring p.R384X/c.1542+dupA mutations, it
was found that although no recovery was detected for relevant enzyme activities, mRNA recovery
was observed in both cases, nearly a two-fold increase for Sanfilippo B fibroblasts with G418 and
around 1.5 fold increase for Sanfilippo C cells with
RTC14 and PTC124 (48).
Strategies have been developed to inhibit NMD
and hence increase the expression of PTC containing mRNAs by using small molecules. Through a
high-throughput screening, amlexanox was found
to inhibit to increase the amount of PTC-bearing mRNAs in cell lines from patients suffering
from nonsense-mutation mediated lung cancer,
Duchenne muscular dystrophy (DMD) or cystic
fibrosis (CF) (49). In addition to acting as NMD
inhibitor, amlexanox leads to the readthrough of
mutated mRNA and results in the synthesis of
full-lenght protein (49). Amlexanox was found to
be as potent as G418 and PTC124 and more effective than combinations of them at higher concentrations due to the combined function of amlexanox in both NMD and readthrough processes (49).
Premature termination codons (PTCs) or nonsense mutations account for about 11% of all described gene defects that cause inherited diseases. Stopcodon readthrough, also called nonsense suppression, hold promise as a therapeutic strategy for the treatment of a broad range of genetic diseases caused by nonsense mutations. An advantage of stopcodon readthrough therapy is that it can be applied to any disease provided that the molecular cause is a primary nonsense mutation in which the PTC results directly from a point mutation in the DNA. In the case of neurological lysosomal storage diseases, additional advantage is the potential penetrance through the blood brain barrier. The fact that a slight recovery of protein levels could be enough to alleviate disease phenotype, and potential penetrance of readthrough drugs through the blood brain barrier makes the stopcodon readthrough therapy an ideal treatment method mainly in diseases with neurological symptoms that are caused by nonsense mutations and for which enzyme replacement is not an option. As the resulting readthrough proteins contain a different amino acid that might cause misfolded protein, combined application of readthough drugs with pharmacological chaperones or proteostasis regulators should be considered.
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