1. Investigating Ataluren for the treatment of genetic
disorders
Matthew Guo, Alex Mackenzie
Children’s Hospital of Eastern Ontario - University of Ottawa Department Biochemistry, Microbiology and Immunology
Abstract
Cowden syndrome is a rare autosomal dominant
inherited disorder characterized by multiple
tumor-like growths called hamartomas and an
increased risk of certain forms of cancer. Cowden
syndrome is associated with mutations in PTEN, a
tumor suppressing gene. A Nonsense mutation in
the PTEN gene has been observed in some
patients which causes the protein to be
prematurely terminated and therefore producing
a non-functioning protein. A mutation in the PTEN
gene can also contribute to the development of
other diseases that will not be discussed on this
poster.
Ataluren, formerly known as PTC124, is a small-
molecular agent designed by PTC Therapeutics. It
is an orally delivered investigational drug that has
the potential to overcome the effects of nonsense
mutation on the affected gene. It is hypothesized
that Ataluren interacts with the ribosomes which
enable the ribosomes to read through premature
nonsense stop codon on mRNA, thereby
producing a functional protein. If successful, this
drug represents a unique mutation-specific
approach to treating Cowden syndrome that is
caused by nonsense mutation on the PTEN gene
and also a broad range of other inherited
diseases.
Background and Hypothesis
FIGURE 1. Comparison of normal translation. (A),
premature termination of translation (B), and
treatment with Ataluren inducing translation of the
functional protein (C).
Objectives
1. Optimize the protocol for growing lymphoblasts in
culture
2. Demonstrate that the PTEN protein concentration
is lower in patient’s lymphoblasts when compared
to normal lymphoblasts
3. Test the efficiency of Ataluren at repairing
truncated proteins and its degree of effectiveness
on the patient’s lymphoblasts
Results
• It is hypothesized that cells treated with Ataluren
will produce a higher concentration of the
functional PTEN protein
Conclusions
• Ataluren does not to induce proper translation of the
PTEN protein, as the concentration of functional protein
in patient cell lines does not increase with dosage.
• There is a small increase of PTEN production in normal
human lymphoblast cells when treated with Ataluren,
and a small decrease of PTEN production in patient’s
lymphoblast cells.
Future Studies
Ataluren does not seem to have any effect on the mutated PTEN gene in
the GM10080 lymphoblasts. However, all non-sense mutations are
different and therefore it is impossible to determine the usefulness of a
drug from one trial alone. The different nucleotide triplets associated
with stop codons and the location of the premature stop codons may all
alter the effectiveness of Ataluren. Therefore, multiple cell lines with
different mutations will need to be tested to further understand the
functionalities of this drug. Once a trial has produced successful results,
the optimal dosage will need to be optimized for future experiments.
Acknowledgements
Thank you to the Mackenzie lab for all their help with this project and
to the UROP community for providing me with the opportunity to
further my scientific research experiences.
FIGURE 3. Bands were normalized to GAPDH using ImageJ and relative protein levels were calculated and graphed using Excel. A) The
relative PTEN levels in NHL(T). B) The relative PTEN levels in GM10080. C) The relative PTEN levels in NHL(A). D) All PTEN levels were
normalized to protein levels in cells treated with 0uM Ataluren.
Materials and Methods
Cell Culturing:
• Patient’s lymphoblasts were obtained from
Corielle Institute (GM10080) and normal
lymphoblasts were isolated from the peripheral
blood of two members in Mackenzie Lab
• Lymphoblasts were grown in Gibco® RPMI 1640
medium with 10% serum and 10ug/mL Penicillin-
Streptomycin Solution and was kept at 37°C in an
incubator
• Medium was changed twice every week to
replenish the nutrients
Drug treatment:
• Ataluren was added to the patient’s lymphoblasts
(GM10080) and normal lymphoblasts (NHL(T) and
NHL(A)) in concentrations of 0uM, 5uM, 10uM and
15uM
• The lymphoblasts were allowed to grow for
24hours
Protein extraction and quantification:
• Lymphoblasts were harvested and added to
centrifuge tubes along with 1mL RIPA buffer for
cell lysis and protein extraction
• Proteins were purified and Bradford protein
bioassay was conducted to determine total
concentration of protein
Protein visualization and analysis:
• 40ng of total protein was loaded along with
loading dye and RIPA buffer onto wells for SDS-
PAGE
• Proteins were then transferred onto a
nitrocellulose membrane using wet-transfer
method
• Western blot was developed on film using ECL
method
• ImageJ was used to determine the concentration
of PTEN protein for each treatment
PTENGAPDH
0uM 5uM 10uM 15uM 0uM 5uM 10uM 15uM 0uM 5uM 10uM 15uM
GM10080 NHL(T) NHL(A)
FIGURE 2. Western blot of GM10080, NHL(T) and
NHL(A) cells treated with Ataluren (0uM, 5uM, 15uM
and 15uM) for 24 hours.
0.74
0.76
0.78
0.8
0.82
0.84
0.86
0.88
0.9
0.92
0.94
0uM 5uM 10uM 15uM
RelativePTEN
24 Hour Ataluren Treatment
PTEN - NHL(A)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0uM 5uM 10uM 15uM
RelativePTEN
24 Hour Ataluren Treatment
PTEN - NHL(T)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0uM 5uM 10uM 15uM
RelativePTEN
24 Hour Ataluren Treatment
PTEN - GM10080
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0uM 5uM 10uM 15uM
NormalizedPTEN
24 Hour Ataluren Treatment
PTEN - Lymphoblasts
NHL(A)
NHL(T)
GM10080
A B
C D
References
1. Peltz, Stuart. et al (2013) Ataluren as an agent for
Therapeutic Nonsense Suppression. Annu. Rev. Med.
2013. 64:407–25
2. Ellen M. Welch et al. (2007). PTC124 targets genetic
disorders caused by nonsense mutations. Nature,
447:87-91.