nature publishing group
Aminoglycoside-induced Translational
Read-through in Disease: Overcoming
Nonsense Mutations by Pharmacogenetic
Therapy
LV Zingman1,2, S Park1,2, TM Olson1,2, AE Alekseev1,2 and A Terzic1,2
A third of inherited diseases result from premature
termination codon mutations. Aminoglycosides have
emerged as vanguard pharmacogenetic agents in treating
human genetic disorders due to their unique ability to
suppress gene translation termination induced by nonsense
mutations. In preclinical and pilot clinical studies, this
therapeutic approach shows promise in phenotype correction
by promoting otherwise defective protein synthesis. The
challenge ahead is to maximize efficacy while preventing
interaction with normal protein production and function.
The information encoded in DNA and the proper decoding
into proteins are fundamental to life.1 Change in the genetic
code brings the danger of life-threatening disease. There are
more than 1,800 inherited human diseases caused by
nonsense mutations, i.e., alterations in the genetic code that
prematurely stop the translation of proteins.2,3 Indeed, about
30 percent of heritable disorders result from premature
termination codon (PTC) mutations.2,3 This frequency may
be even higher in certain syndromes or in specific human
populations. Moreover, different classes of mutations,
including frameshift insertions and/or deletions, splice-site
intron inclusions, or simple substitutions of a normal codon
with UAA, UAG, or UGA codons, can all introduce a
premature stop signal, thus arresting protein synthesis.4 Well-
known examples include Duchenne muscular dystrophy, the
most common X-linked fatal genetic disease, where 410–20
percent of patients carry a nonsense mutation resulting in
premature translation termination. Similarly, in cystic
fibrosis, B2–5 percent of patients (60 percent for Ashkenazi
Jews) have nonsense mutations in the cystic fibrosis
transmembrane regulator gene.2,4–6 PTC mutations in
1
Marriott Heart Disease Research Program, Department of Medicine, Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota, USA; 2Department
of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, USA. Correspondence: LV Zingman (zingman.leonid@mayo.edu)
doi:10.1038/sj.clpt.6100012
CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 81 NUMBER 1 | JANUARY 2007
DISCOVERY
tumor-suppressor genes are also common in the develop-
ment and progression of cancer.2
At the molecular level, PTC mutations have major
consequences. Typically, a PTC mutation precipitates rapid
degradation of the mRNA carrying the premature codon, after
a single cycle of translation, through nonsense-mediated
mRNA decay. Activation of this mechanism, while protective
against potentially lethal production of truncated protein by
nullifying deleterious suppression of synthesis and/or function
of vital cell proteins, may not prevent the development of a
disease phenotype due to the absence of the translationally
defective protein itself.2,4,7 In the absence of nonsense-
mediated mRNA decay, truncated polypeptides could be
produced.2,4,7 Assembly of a truncated protein in vivo is,
however, a rare event, and is usually related to a PTC
positioned within the last exon or in a splicing region resulting
in mutation-induced exon skipping. The biological effect of a
mutated gene may be difficult to predict, as it depends on the
extent of protein truncation, stability of the polypeptide
product, and degree of interference with the expression of the
normal allele (e.g., dominant-negative effect).2,7
Two major therapeutic approaches to overcome nonsense
mutations are presently considered. Gene therapy is, in
principle, the treatment of choice due to the potential for
targeted repair, but, to date, several limitations have
precluded clinical success.3,7 Alternatively, pharmacological
approaches can modify gene expression and block nonsense-
mediated mRNA destruction, without correcting the under-
lying gene defect. The rationale supporting such emerging
pharmacogenetic strategies is the premise that even limited
expression of a mutated gene could result in the production
of a partially or fully functional protein, sufficient for
therapeutic benefit.2,4,6,7
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DISCOVERY
AMINOGLYCOSIDES: PROTEIN TRANSLATION-MODIFYING
DRUGS
Aminoglycosides are widely used in clinical practice as
bactericidal antibiotics with established effects on transla-
tional accuracy or efficiency.8 Their utility as antibacterial
agents arises from binding to the highly conserved 16S
ribosomal RNA (rRNA) at the decoding center (Figure 1).
This center normally facilitates accurate codon–anticodon
pairing. In the presence of an aminoglycoside, the conforma-
tion of rRNA becomes altered, inducing codon misreading
that causes either incorporation of an erroneous amino acid
(mis-incorporation) at a sense codon or failure of recognition
of the stop codon, leading to translational read-through
rather than chain termination (Figure 2).9–11 Although
aminoglycosides target a conserved region of the rRNA
sequence, these agents are highly active against bacterial and
mitochondrial ribosomes, but have limited interaction with
human ribosomes. The specificity is related to the high-
affinity binding of aminoglycosides to prokaryotic rRNA,
which has an adenine at position 1408 of the 16S rRNA
(numbered according to the Escherichia coli sequence). In
contrast, eucaryotic ribosomes have a guanine at the
corresponding position, causing a low affinity towards
aminoglycosides (Figure 2).11 The effects of aminoglycosides
on protein translation in eucaryotic cells have been demon-
strated at concentrations 10 to 15 times higher than the
typical therapeutic antibacterial concentrations. Partial sus-
ceptibility of eucaryotic ribosomes toward aminoglycoside
interaction has been traditionally viewed as the underlying
mechanism of drug toxicity. However, this side effect may
provide the opportunity for the treatment of human genetic
diseases associated with translational defects.
Figure 1 Structure of aminoglycoside bound to rRNA. Paromomycin (red)
binds to helix 44 in 16S rRNA. Adenine in position 1408 is critical for
high-affinity interaction with bacterial ribosome, with other residues
critical for codon–anticodon cognition also indicated. Atomic coordinates
are from 1IBK.
100
AMINOGLYCOSIDES: PHARMACOGENETIC AGENTS
In 1985, Burke and Mogg12 were the first to demonstrate that
the aminoglycoside antibiotics paromomycin and G-418
could partially restore the synthesis of a full-size protein from
a mutant gene with a premature UAG mutation in cultured
mammalian cells. Later, G-418 and gentamicin were shown to
restore the expression of the cystic fibrosis transmembrane
conductance regulator protein in a cell line carrying a
nonsense mutation in cystic fibrosis transmembrane conduc-
tance regulator.13,14 In 1999, the ability of aminoglycosides to
promote protein translation, despite the presence of a PTC
mutation, was first demonstrated in vivo in a model of
Duchenne muscular dystrophy.15 In this way, the concept of
aminoglycoside-induced read-through emerged as a thera-
peutic strategy in human genetic disorders. In the current era,
aminoglycosides have been shown to suppress premature
translation termination at nonsense codons when bound to
the rRNA decoding site interacting with codon–anticodon
pairing in a series of clinically relevant conditions (Table 1).
The efficiency of read-through varies from 1 percent to 25
percent in human cell lines, largely depending on the context
of the stop mutation, with UGA showing greater translational
read-through than UAG, and UAA exhibiting the most
resistance to suppression.16,17 Several studies demonstrate the
critical influence of upstream and downstream sequences on
overall efficiency of translational termination.18,19 In mam-
malian cells, the action of the antibiotic is governed by
nucleotides surrounding the stop mutation. The nucleotide
in the position immediately downstream from the stop
codon has a particularly significant impact on the efficiency
of aminoglycoside-induced read-through, as follows:
C4U4AXG.17
AMINOGLYCOSIDES: TRANSLATION OF PHARMACOGENETIC
AGENTS IN MEDICINE
Production of protein in the presence of a nonsense PTC
mutation resulting from aminoglycoside therapy, even if only
in low amount, may be functionally significant. This is
especially the case in recessive disorders, where protein
expression is essentially absent. In such cases, even 1 percent
of normal protein function may restore a near-normal or
clinically less severe phenotype.2,4,6,7 Accordingly, it is
primarily in recessive disorders that aminoglycosides have
provided the greatest promise in both cell culture experi-
ments and clinical trials. Examples include interventions with
defective genes in cystic fibrosis, Duchenne muscular
dystrophy, cystinosis, mucopolysaccharidosis type I (Hurler
syndrome), X-linked nephrogenic diabetes insipidus, ataxia
–telangiectasia, hemophilia, factor VII deficiency, and in-
fantile neuronal ceroid lipofuscinosis (Table 1).20–29
Recent studies have indicated that read-through induction
may be a promising therapy in autosomal-dominant
disorders as well (Table 1).30–32 This is somewhat surprising
since haploinsufficiency is the common pathogenic mechan-
ism in nonsense mutation-related dominant disorders, i.e.,
B50 percent decrease in the amount of protein produced
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 DISCOVERY

Figure 2 Aminoglycoside interaction with ribosomal protein translation. (a–c) Interaction of paromomycin with 16S rRNA of the 30S ribosome subunit.
a In the absence of antibiotic, the conserved 16S rRNA (yellow strands) nucleotides A1492 and A1493 are stacked in the interior of helix 44. b Binding
of mRNA codon and cognate tRNA causes conformational change of A1492, A1493, and G530 to accommodate energetically favorable interactions of
ribosome components, tRNA and mRNA. c Paromomycin binding to the interior of helix44 induces a local conformational change in nucleotides A1492
and A1493, flipping them out. These changes facilitate binding of near-cognate tRNA. Black lines represent H-bonds. Crystal atomic coordinates are
from 1J5E, IBK, and 1IBM. (d–f) Aminoglycoside induced read-through of the premature E375X stop codon in Kv1.5. d Normally, tRNA carrying glutamic
acid (E) matches the mRNA codon to process Kv1.5 polypeptide elongation. Matching of the mRNA codon to the proper tRNA anticodon results in
conformational alignment of A1492 and A1493 in the ribosomal decoding center (red dashes) and polypeptide chain elongation. e E375X (UAA codon in
mRNA) mutation prevents codon–anticodon pairing and excludes the possibility of the A1492 and A1493 alignment in the ribosomal decoding center
(red dashes) terminating protein translation. f Aminoglycoside binding to 16S rRNA induces conformational alignment in the ribosomal decoding center
despite codon/anticodon mismatch. In the presence of aminoglycosides, the UAA codon may be paired with CUU or GUU tRNA anticodon promoting
polypeptide chain elongation with glutamate or glutamine.

due to the heterozygous loss of one allele causes clinical
symptoms. Just as a mutated protein can have a dominant-
negative effect on the protein produced by the normal allele,
even a small increase in full-length functional protein
resulting from aminoglycoside therapy may improve out-
come.30–32 A case in point is Hailey–Hailey disease, caused by
a nonsense mutation in the Ca2 þ -ATPase2C1 transporter
gene (ATP2C1),32 and familial atrial fibrillation related to a
PTC mutation in KCNA5 encoding the voltage-sensitive
potassium channel Kv1.5.31 In both cases, aminoglycosides
have been suggested as corrective therapy.30,31
A limitation to aminoglycoside use as pharmacogenetic
agents is necessary to reach concentrations sufficient to
interfere with protein synthesis at human ribosomes,
increasing risk of significant toxicity. Therefore, clinical
success to date has been observed with local aminoglycoside
delivery, particularly treatment of cystic fibrosis, the most
studied disease in the context of pharmacogenetic-based
therapy. Systemic delivery of currently available aminoglyco-
sides at clinically approved doses has failed, as in the case of
McArdle disease and Duchenne muscular dystrophy.33,34
Thus, developing effective drug delivery strategies that would
minimize side effects and optimize safely achievable ther-
apeutic levels is warranted. For example, percutaneous drug-
eluted patch atrial implantation could be considered for
patients with PTC mutations causing atrial arrhythmias.
Furthermore, after treatment with aminoglycosides, en-
hanced production of functional protein from defective
genes can be boosted through simultaneous application of
protein-specific promoters.35 Also, recent discoveries in the
structure of ribosomes and their interactions with aminogly-
cosides provide a platform on which to engineer new
generations of pharmacogenetic agents with tissue predilec-
tion and molecular specificity, and limit or prevent undesired
interactions with normal protein synthesis and function.9–11
CONCLUSION
Currently, aminoglycosides are the only clinically available
drug family known to consistently affect gene translation in
eucaryotes. This property has provided the foundation for
preclinical and clinical testing of aminoglycosides to rescue
protein production arrested by nonsense mutations. Ami-
noglycosides have shown initial promise in diverse genetic
disorders due to PTC mutations, particularly when applied
locally to secure sufficient therapeutic levels while avoiding
systemic toxicity. The challenge in the emerging field of
pharmacogenetic therapy will be to define the optimal
strategy that would prevent interference with the normal

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DISCOVERY

Table 1 Genetic diseases and aminoglycoside-based pharmocogenetic therapy

Affected
Disease gene Model Treatment outcome Reference
22
Ataxia–telangiectasia ATM In vitro cDNA-coupled Full-length functional protein
transcription
Cell culture
2,4,6,7,13,14,20,26
Cystic fibrosis CFTR Cell culture Full-length functional protein
Transgenic mice Amelioration of phenotype
Patients Amelioration of clinical
symptoms
23
Cystinosis CTNS Cell culture Full-length functional protein
2,7,15,17,24,34
Duchenne muscular dystrophy DMD Cell culture Variable
Transgenic mice
Patients
28
Factor VII deficiency F7 Cell culture Full-length functional protein
31
Familial atrial fibrillation KCNA5 Cell culture Full-length functional protein
30
Hailey–Hailey disease ATP2C1 Cell culture Full-length protein synthesis
27
Hemophilia F8 and F9 Patients Full-length functional protein
25
Infantile neuronal ceroid lipofuscinosis TPP1 Cell culture full-length functional protein
33
McArdle disease PYGM Patients No significant benefit
16,21
Mucopolysaccharidosis Type 1 (Hurler IDUA In vitro cDNA-coupled Full-length functional protein
syndrome) transcription
Cell culture
29
X-linked nephrogenic diabetes insipidus AVPR2 Cell culture Full-length functional protein
Transgenic mice Amelioration of phenotype

protein synthesis process. In this regard, development of new
drugs based on deciphering the intimate structural and
functional determinants of aminoglycoside interaction with
ribosomes, as well as the establishment of new modalities for
targeted drug delivery are priorities. Indeed, a better under-
standing of the benefits afforded by combinatorial use of
aminoglycosides with gene-of-interest-specific promoters,
enhanced local application, as well as determining the most
favorable schedule for long-term therapy will be critical for
the future of this promising strategy for gene code repair in
otherwise untreatable genetic diseases.
ACKNOWLEDGMENTS
LVZ is the recipient of the Kogod Program on Aging Career Development
Award. This work was supported by grants from the National Institutes of
Health, Marriott Heart Disease Research Program, Marriott Foundation,
Ted Nash Long Life Foundation, and Ralph Wilson Medical Research
Foundation.
CONFLICT OF INTEREST
The authors declared no conflict of interest.
& 2007 American Society for Clinical Pharmacology and Therapeutics
1. Nirenberg, M. The genetic code: Nobel lecture, http://nobelprize.org/
nobel_prizes/medicine/laureates/1968/press.html (1968).
2. Kellermayer, R. Translational readthrough induction of pathogenic
nonsense mutations. Eur. J. Med. Genet. (2006).
3. Kusik, V. Mendelian Inheritance in Man: A Catalog of Human Genes and
Genetic Disorders, (John Hopkins University Press: Baltimore, 1998).
4. Byers, P.H. Killing the messenger: new insights into nonsense-mediated
mRNA decay. J. Clin. Invest. 109, 3–6 (2002).
5. Roberts, R.G., Gardner, R.J. & Bobrow, M. Searching for the 1 in
2,400,000: a review of dystrophin gene point mutations. Hum. Mutat. 4,
1–11 (1994).
6. Lukacs, G.L. & Durie, P.R. Pharmacologic approaches to correcting
the basic defect in cystic fibrosis. N. Engl. J. Med. 349, 1401–1404
(2003).
7. Holbrook, J.A., Neu-Yilik, G., Hentze, M.W. & Kulozik, A.E.
Nonsense-mediated decay approaches the clinic. Nat. Genet. 36,
801–808 (2004).
8. Luzzatto, L., Apirion, D. & Schlessinger, D. Mechanism of action of
streptomycin in E. coli: interruption of the ribosome cycle at the
initiation of protein synthesis. Proc. Natl. Acad. Sci. USA 60, 873–880
(1968).
9. Yoshizawa, S., Fourmy, D. & Puglisi, J.D. Structural origins of gentamicin
antibiotic action. EMBO J. 17, 6437–6448 (1998).
10. Ogle, J.M. et al. Recognition of cognate transfer RNA by the 30S
ribosomal subunit. Science 292, 897–902 (2001).
11. Recht, M.I., Douthwaite, S. & Puglisi, J.D. Basis for prokaryotic specificity
of action of aminoglycoside antibiotics. EMBO J. 18, 3133–3138 (1999).
12. Burke, J.F. & Mogg, A.E. Suppression of a nonsense mutation in
mammalian cells in vivo by the aminoglycoside antibiotics G-418 and
paromomycin. Nucleic Acids Res. 13, 6265–6272 (1985).
13. Howard, M., Frizzell, R.A. & Bedwell, D.M. Aminoglycoside antibiotics
restore CFTR function by overcoming premature stop mutations. Nat.
Med. 2, 467–469 (1996).
14. Bedwell, D.M. et al. Suppression of a CFTR premature stop mutation in a
bronchial epithelial cell line. Nat. Med. 3, 1280–1284 (1997).
15. Barton-Davis, E.R., Cordier, L., Shoturma, D.I., Leland, S.E. & Sweeney, H.L.
Aminoglycoside antibiotics restore dystrophin function to skeletal
muscles of mdx mice. J. Clin. Invest. 104, 375–381 (1999).
16. Keeling, K.M. & Bedwell, D.M. Clinically relevant aminoglycosides can
suppress disease-associated premature stop mutations in the IDUA and
P53 cDNAs in a mammalian translation system. J. Mol. Med. 80, 367–376
(2002).

102 VOLUME 81 NUMBER 1 | JANUARY 2007 | www.nature.com/cpt


17. Howard, M.T. et al. Sequence specificity of aminoglycoside-induced stop
condon readthrough: potential implications for treatment of Duchenne
muscular dystrophy. Ann. Neurol. 48, 164–169 (2000).
18. Bonetti, B., Fu, L., Moon, J. & Bedwell, D.M. The efficiency of translation
termination is determined by a synergistic interplay between upstream
and downstream sequences in Saccharomyces cerevisiae. J. Mol. Biol.
251, 334–345 (1995).
19. Namy, O., Hatin, I. & Rousset, J.P. Impact of the six nucleotides
downstream of the stop codon on translation termination. EMBO Rep. 2,
787–793 (2001).
20. Clancy, J.P. et al. Evidence that systemic gentamicin suppresses
premature stop mutations in patients with cystic fibrosis. Am. J. Respir.
Crit. Care Med. 163, 1683–1692 (2001).
21. Hein, L.K. et al. Alpha-L-iduronidase premature stop codons and
potential read-through in mucopolysaccharidosis type I patients. J. Mol.
Biol. 338, 453–462 (2004).
22. Lai, C.H. et al. Correction of ATM gene function by aminoglycoside-
induced read-through of premature termination codons. Proc. Natl.
Acad. Sci. USA 101, 15676–15681 (2004).
23. Helip-Wooley, A., Park, M.A., Lemons, R.M. & Thoene, J.G. Expression of
CTNS alleles: subcellular localization and aminoglycoside correction in
vitro. Mol. Genet. Metab. 75, 128–133 (2002).
24. Politano, L. et al. Gentamicin administration in Duchenne patients with
premature stop codon. Preliminary results. Acta Myol. 22, 15–21 (2003).
25. Sleat, D.E., Sohar, I., Gin, R.M. & Lobel, P. Aminoglycoside-mediated
suppression of nonsense mutations in late infantile neuronal ceroid
lipofuscinosis. Eur. J. Paediatr. Neurol. 5, 57–62 (2001).
CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 81 NUMBER 1 | JANUARY 2007
DISCOVERY
26. Wilschanski, M. et al. Gentamicin-induced correction of CFTR function in
patients with cystic fibrosis and CFTR stop mutations. N. Engl. J. Med.
349, 1433–1441 (2003).
27. James, P.D. et al. Aminoglycoside suppression of nonsense mutations in
severe hemophilia. Blood 106, 3043–3048 (2005).
28. Pinotti, M. et al. Intracellular readthrough of nonsense mutations by
aminoglycosides in coagulation factor VII. J. Thromb. Haemost. 4,
1308–1314 (2006).
29. Sangkuhl, K. et al. Aminoglycoside-mediated rescue of a disease-causing
nonsense mutation in the V2 vasopressin receptor gene in vitro and in
vivo. Hum. Mol. Genet. 13, 893–903 (2004).
30. Kellermayer, R., Szigeti, R., Keeling, K.M., Bedekovics, T. & Bedwell, D.M.
Aminoglycosides as potential pharmacogenetic agents in the treatment
of Hailey–Hailey disease. J. Invest. Dermatol. 126, 229–231 (2006).
31. Olson, T.M. et al. Kv1.5 channelopathy due to KCNA5 loss-of-function
mutation causes human atrial fibrillation. Hum. Mol. Genet. 15,
2185–2191 (2006).
32. Porgpermdee, S. et al. Expression of SPCA1 (Hailey–Hailey disease gene
product) in acantholytic dermatoses. J. Dermatol. Sci. 40, 137–140
(2005).
33. Schroers, A. et al. Gentamicin treatment in McArdle disease: failure to
correct myophosphorylase deficiency. Neurology 66, 285–286 (2006).
34. Wagner, K.R. et al. Gentamicin treatment of Duchenne and Becker
muscular dystrophy due to nonsense mutations. Ann. Neurol. 49,
706–711 (2001).
35. Xi, B., Guan, F. & Lawrence, D.S. Enhanced production of functional
proteins from defective genes. J. Am. Chem. Soc. 126, 5660–5661 (2004).
103