Download Research Reports to April 2015

RP Fighting Blindness Research Reports
May 2014 - April 2015
An omnibus of research reports produced by researchers funded by
RP Fighting Blindness during the period.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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CONTENTS
Page 3
INTRODUCTION
Page 4
STRUCTURAL RESTORATION OF RHODOPSIN ADRP MUTANTS: SUBCELLULAR LOCALISATION AND CO-FACTOR INTERACTIONS
Dr Philip Reeves, University of Essex
Page 5
PATHOLOGY AND TREATMENT OF PROMININ 1 (PROM1)-MEDIATED
RETINITIS PIGMENTOSA
Prof S Ohnuma, Institute of Ophthalmology, London
Page 6
CHARACTERISATION OF SPACEMAKER (SPAM), A PROTEIN ENCODED BY
THE GENE EYS (RP25) IMPLICATED IN AUTOSOMAL RECESSIVE RETINITIS
PIGMENTOSA
Giovanna Alfano & Shomi S Bhattacharya, Institute of Ophthalmology, UCL
Page 7
MODIFICATION OF MUTANT BESTROPHIN-1 TO PREVENT RETINAL
DEGENERATION
Dr F Manson and Dr L Swanton, PhD student: Ms C Uggenti
University of Manchester
Page 8
RP FIGHTING BLINDNESS CENTRE FOR THE DEVELOPMENT OF GENE
THERAPY FOR INHERITED RETINAL DYSTROPHIES
Prof R Ali
Institute of Ophthalmology, University College London
Page 9
MECHANISMS OF PHOTORECEPTOR DEGENERATION IN CHOROIDEREMIA
Prof Clare Futter, Institute of Ophthalmology, University College London
Page 10
PHARMACOLOGICAL THERAPIES FOR RHODOPSIN RETINITIS PIGMENTOSA
Principal investigator: Professor Mike Cheetham, Post-doctoral research associate:
Dr. Dimitra Athanasiou, UCL, Institute of Ophthalmology
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THE DEVELOPMENT OF HUMAN IPSC-DERIVED EX VIVO MODELS OF
RETINAL DEGENERATION. THEIR ANALYSIS IN SPLICING-FACTOR RP
Mr A Webster, N Owen, Institute of Ophthalmology, University College London
Page 13
MAINTAINING EFFECTIVE ANTIOXIDANT CAPACITY IN A DEGENERATING
RETINA: A GENERIC APPROACH TO TREATMENT OF RP
Prof R Ali, Institute of Ophthalmology, University College London
Page 14
RHODOPSIN TRAFFICKING DEFECTS IN RP
Prof M Lako, Newcastle University
Page 15
EXPLOITING THE POWER OF HUMAN INDUCED PLURIPOTENT STEM CELLS
TO GENERATE SYNTHETIC FULLY LAMINATED RETINAE IN VITRO FOR
DISEASE MODELLING, DRUG DISCOVERY AND CELL BASED THERAPIES
Prof M Lako, Newcastle University
Page 17
DEVELOPING NOVEL OPTOGENETIC TOOLS FOR THE TREATMENT OF
RETINAL DYSTROPHIES
Dr J Kapetanovic, University of Manchester
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UK INHERITED RETINAL DYSTROPHY CONSORTIUM RP GENOME PROJECT
Professor Graeme Black - Chief Investigator (Manchester)
Mr Stuart Ingram - acting Project Manager (Manchester)
Professor Alison Hardcastle - Principal Investigator (University College London)
Professor Chris Inglehearn - Principal Investigator (Leeds)
Dr Andrea Nemeth - Principal Investigator (Oxford)
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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INTRODUCTION
(Extracted from the charity’s Annual Report for 2014)
The biggest event in 2014 for RP Fighting Blindness funded research has been the establishment of
the RP Genome Project. This ground-breaking initiative has established a national consortium
including centres in London, Oxford, Manchester and Leeds that has aims including the identification
of new genes that cause retinal dystrophies, the development of genetic testing for clinical use and
developing a database that will allow the rapid identification of patients who might be suitable for
clinical trials. Although this has only very recently got off the ground, there are already tangible
outputs including research papers in which new retinal dystrophy genes are identified. As the
consortium develops, it is expected that it will attract further funding from other sources and be a
major driver for making genetic testing available to everyone in the UK though the NHS.
Funding from RP Fighting Blindness continues to support clinical trials of gene therapy. Genetic
testing that has been facilitated by RP Fighting Blindness has been used to identify patients who are
suitable for clinical trials originating from London and Oxford, and RP Fighting Blindness provides
support to Professor Ali’s group at the Institute of Ophthalmology as they go through the complex
process of setting up new clinical trials.
For the first time, RP Fighting Blindness has funded research in the field of optogenetics. This is an
exciting new approach that aims to restore vision in people with advanced RP. An award has been
made to Dr Jasmina Cehajic-Kapetanovic, from the University of Manchester, that is funding her to
develop optogenetics research in the laboratory of Professor John Flannery at The University of
California, Berkeley. The idea behind optogenetics research is that although the main photoreceptor
cells in the retina, the rods and cones, are irreversibly damaged in advanced RP, other retinal cells
remain viable and these have the potential to be converted into photoreceptors for vision. Whilst this
approach is some way off clinical trials, research into optogenetics complements other more
established strategies aimed at restoring vision such as stem cell based therapies and electronic
retinal implants.
Prof Paul Bishop
Chair of RP Fighting Blindness Medical Advisory Board
April 2015
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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STRUCTURAL RESTORATION OF RHODOPSIN ADRP MUTANTS: SUB-CELLULAR
LOCALISATION AND CO-FACTOR INTERACTIONS
Dr Philip Reeves, University of Essex
Rhodopsin is an abundant light sensitive pigment located in rod cells of the retina. It is responsible for
vision under dim light conditions. Faults in the rhodopsin gene are the most common cause of
Autosomal Dominant Retinitis Pigmentosa (ADRP). These faults lead to the production of incorrectly
folded or unstable rhodopsin proteins that result in cell death of rod photoreceptor cells. The way by
which these faulty rhodopsin proteins trigger death is unclear but we believe there are at least two
separate mechanisms; these are: (1) inability of photoreceptor cells to deal with their build up
defective protein or (2) the capacity of the defective rhodopsin to reach the very sensitive outer region
of photoreceptor cell where they may cause damage. Our main findings this year are described
below.
1) We have examined in exquisite detail the effect of temperature on the stability of the defective
proteins and this has given us greater insight into the type of damage that the faulty receptors incur.
We find that the stability profile of the defective rhodopsins is dramatically different from the normal
rhodopsin. This suggests to us that one side of the rhodopsin protein has a specific function: to hold
rhodopsin together. Rhodopsin has evolved to be extremely stable, a feature that is required for highly
sensitive vision at night. This explains why such minor alterations in the defective rhodopsin proteins
cannot be tolerated, and result in a dramatic deterioration of their function and thermostability. This
might explain why these defective proteins can have such catastrophic consequences on the rod
photoreceptor cell. Furthermore, these defective proteins can evade the cellular surveillance
machinery that usually keeps in check the amount of these defective proteins that are made. We are
now writing a manuscript to describe these important new findings.
2) We are continuing to extract and evaluate our data to help explain how gatekeeper proteins interact
with the defective rhodopsin. This protein surveillance machinery is in place to prevent defective
rhodopsins, such as those described above, from reaching parts of the cell that are particularly fragile
and prone to damage. This work will form the basis of another manuscript.
Publications and Conference Presentations in 2015
Opefi CA, Tranter D, Smith SO, Reeves PJ. Construction of stable mammalian cell lines for inducible
expression of G protein-coupled receptors. Methods Enzymol. 2015; 556:283-305.
Philip J. Reeves. The role of the extracellular domain of rhodopsin in structure, function and disease.
2nd GPCR Targeted Screening Conference, May 7-8, 2015, Berlin, Germany.
Previous publications arising from this grant
1. Opefi CA1, South K, Reynolds CA, Smith SO, Reeves PJ. Retinitis pigmentosa mutants provide
insight into the role of the N-terminal cap in rhodopsin folding, structure, and function. J Biol Chem.
2013 Nov 22;288(47):33912-26. doi: 10.1074/jbc.M113.483032. Epub 2013 Oct 8.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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PATHOLOGY AND TREATMENT OF PROMININ 1 (PROM1)-MEDIATED RETINITIS
PIGMENTOSA
Prof S Ohnuma, Institute of Ophthalmology, London
Retinitis Pigmentosa (RP) and Stargardt’s disease are inherited disorders causing progressive
photoreceptor cell death and affect about one in several thousand people. A number of genes have
been identified, including those encoding transcription factors, splicing regulators, membrane proteins
and proteins involved in visual cycle.
Prominin-1 (Prom1) is one of them. Prom1 encodes a 120 kDa pentaspan transmembrane
glycoprotein, originally identified as a surface antigen of human haematopoietic stem and progenitor
cells. Prom1 is expressed in a number of tissues throughout embryogenesis and adulthood, and has
been implicated in the maintenance of stem cell characteristics and cell proliferation. In addition to
these functions, Prom1 has been shown to be involved in a number of genetic disorders that cause
photoreceptor degeneration, including RP and Stargardt's type 4.
These dystrophies are associated with progressive photoreceptor cell death. There are no effective
therapies for either disorder. The aim of this study was to investigate the mechanism of the retinal
degeneration in Prom1-deficient mouse models.
We constructed Prom1 knockout mice with two distinct genetic backgrounds of C57BL/6 and
C57BL/6xCBA/NSlc, and investigated the photoreceptor degeneration by means of histology and
functional tests. The Prom1-/- knockout mice with both backgrounds developed photoreceptor
degeneration after eye opening, but the CB57/BL6 background mice developed photoreceptor cell
degeneration much faster than the C57BL/6xCBA/NSlc mice, demonstrating genetic background
dependency.
In the cellular level, these observations demonstrate Prom1 is required for the maintenance of both
rod- and cone- photoreceptor cells, and for the correct localization of opsin proteins that act in the
outer segments.
Interestingly, our histological and functional examination showed that the photoreceptor cell
degeneration of Prom1 knockout mice was light dependent. We reared some Prom1-deficient mice
under the dark condition from 8 days to 30 days after the births, and found that the structure of and
the function of the retina were almost kept intact.
During the investigation of the eye-related gene expression, we found that the Prom1 knockout retina
showed strong decrease of expression of the visual cycle components, Rdh12 and Abca4, which
suggests that the visual cycle might be affected in the Prom1-deficient mice. Therefore, for a possible
treatment, we examined the effect of the synthetic retinoid derivative Fenretinide, which reduces the
availability of retinoids and thereby reduces accumulation of A2E in retinal pigment epithelium. As the
result, at 30 days after the births, the Fenretinide injected Prom1-deficient mice had thicker
photoreceptor segments, and increased number of photoreceptor nuclei. In addition, the scores of ERG
(electroretinogram) were improved by the treatment of Fenretinide. These findings improve our
understanding of the mechanism of cell death in Prominin1 related disease and provide evidence that
fenretinide may be worth studying in human disease.
In conclusion, we have provided evidence of light-dependent photoreceptor degeneration in the Prom1deficient mice, and have identified possible downstream signaling targets. Further investigation of the
downstream target genes and interacting proteins will allow a better understanding of the mechanisms
of photoreceptor cell death and more targeted therapeutic approaches in the future.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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CHARACTERISATION OF SPACEMAKER (SPAM), A PROTEIN ENCODED BY THE GENE EYS
(RP25) IMPLICATED IN AUTOSOMAL RECESSIVE RETINITIS PIGMENTOSA
Giovanna Alfano & Shomi S Bhattacharya, Institute of Ophthalmology, University College London
Retinitis pigmentosa (RP) is a group of inherited degenerative eye disease that causes severe vision
impairment due to the progressive degeneration of photoreceptor cells (light-sensitive cells) in the
retina (back of the eye), eventually leading to blindness. RP can be passed through generations with
different modes of inheritance. It is autosomal recessive (arRP) when both parents are unaffected
carriers of the same defective gene and then pass on both defective changes (mutations) from each
parent to the affected child. Mutations at the genetic locus RP25 have been found to cause arRP. The
gene affected is called EYS and it is a major cause of arRP worldwide. EYS encodes a protein termed
SPAM (spacemaker) whose biological role in human is presently unclear. In flies (Drosophila) Spam
has been shown to participate in the organisation of photoreceptors and for this reason the human
protein is thought to maintain the integrity of photoreceptor cells. This study was undertaken to
investigate the role of EYS/SPAM in the human retina. The project focused on a detailed
characterisation of protein localisation in retinoblastoma Y79 cells (retinal cell line) and monkey
retinas whose structure is similar to the human retina. In Y79, SPAM localises to several cellular
compartments (cytoplasm, cell membrane, primary cilium and centrosomes). In monkey retinas SPAM
localises to the connecting cilium of rods and cones and to the cytoplasm of ganglion cells. Moreover,
it is known that from the same gene it is possible to make more than one protein (alternative forms or
isoforms) and isoforms can play different roles in health and disease. As part of this study,
investigation of EYS/SPAM isoforms was performed. Expression studies revealed that previously
uncharacterised EYS isoforms 2 and 3 are present in the retina, testes and Y79 cells. Overexpression
studies demonstrated that both isoforms localise to the cytoplasm of cultured cells.
Our findings suggest that SPAM has a diverse localisation, likely due to the different isoforms, and it
may have multiple function. Interestingly, our results indicate that SPAM may be a novel ciliary
protein. Many genes encoding for ciliary proteins, when defective, have been associated with retinal
dystrophy. It is tempting to speculate that the ciliary function could play a key role in retinal
degeneration in patients, however, further analysis is required to elucidate the pathogenesis of RP
due to mutations in the EYS gene.
Besides, it is known that in flies Spam works in concert with another protein called Prominin, and that
Prominin-1 is implicated in eye diseases in humans. The putative interaction between these two
proteins has been investigated in mammals. Our results suggest that, in humans, SPAM and
Prominin- 1 are likely to follow different functional pathways compared with flies.
Overall, our findings provide useful insights into EYS function in humans and raise new questions for
further investigation. We are hopeful that a better understanding of EYS function should eventually
result in the development of future therapies for patients.
We are very grateful to RP Fighting Blindness for the generous support of our research.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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MODIFICATION OF MUTANT BESTROPHIN-1 TO PREVENT RETINAL DEGENERATION
Dr F Manson and Dr L Swanton, PhD student: Ms C Uggenti, The University of Manchester
Correcting the function of a protein mutated in RP
Bestrophin-1 is a protein that forms a channel for chloride ions in the RPE, the pigmented layer that
supports the function of the rod and cone photoreceptors. Mutations in bestrophin-1 cause a number
of inherited retinal dystrophies, including retinitis pigmentosa (RP), collectively known as
bestrophinopathies. The vast majority of mutations in bestrophin-1 means the protein cannot fold into
its correct shape, which in turn abolishes it function. The aim of our RPFB-funded work was to identify
a way of restoring the function of mutant bestrophin-1 proteins. To do this we tested several different
drugs, known as chemical chaperones, for their ability to help the mutant bestrophin-1 protein regain
its correct shape and function.
To study the effects of chemical chaperones we first developed a cellular model of the RPE. This was
necessary because it is not possible to take biopsies of RPE from patients. The cell models made one
of four different mutant bestrophin-1 proteins, and we compared these mutant cell models to control
cells that made normal bestrophin-1 protein. We grew the cell models in the presence or absence of a
number of different chemical chaperones, or combinations of them, before the cells were analysed to
determine whether the treatment had resulted in increased levels of mutant bestrophin-1 being made,
or whether the treatment had corrected where the protein was located inside the cell. Our research
identified that one chemical chaperone in particular, 4PBA, did both. The next step was to test
whether 4PBA could correct the function of the 4 mutant bestrophin-1 proteins under investigation.
We did this using an extremely fine glass electrode to measure how well the chloride ions moved
through the bestrophin-1 protein channel in individual cells. We have been able to show that the
chloride ion channel function of all 4 of the mutant bestrophin-1 proteins we tested was fully restored
after treatment with 4PBA.
This exciting finding is made even more so as 4PBA is already a licenced drug which has been in use
for over 20 years to treat kidney disease. It can be taken by adults and children, and has the
necessary properties that allow it to reach all parts of the body, including the RPE.
In future we would like to test whether 4PBA is equally as effective at restoring mutant bestrophin-1
function in the RPE cells of patients with a bestrophinopathy. To do this we will use special growth
conditions to turn skin cell samples into RPE. If this is successful the next stage will be to test the
efficacy of 4PBA on animals that have a bestrophinopathy before embarking on a human clinical trial.
As 4PBA is already licensed for human use the prospect of a treatment for bestrophinopathies, and
possibly other diseases caused by improperly folded proteins, could be in the not too distant future.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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RP FIGHTING BLINDNESS CENTRE FOR THE DEVELOPMENT OF GENE THERAPY FOR
INHERITED RETINAL DYSTROPHIES
Prof R Ali, Institute of Ophthalmology, University College London
The inherited retinal dystrophies (IRDs) are a large group of sight loss conditions that are caused by
mutations in over 150 genes. They affect around 1/3000 people worldwide with no effective
treatments currently available.
In January 2015, we published in Nature Communications the first highly effective rescue of the rd1
mouse model of retinitis pigmentosa (RP), caused by defects in PDE6B. Numerous attempts have
been made to preserve and restore function to degenerating rod cells in rd1 mice with limited success
until now. Our research has also uncovered a previously unreported second defect in the rd1 mouse
model, in the gene GPR179. This defect prevents proper signalling of visual impulses from reaching
the brain, therefore making any correction of PDE6B with gene therapy ineffectual. Through a process
of selective breeding of different mouse models we have successfully removed the GPR179 defect
from rd1 mice and treated them with PDE6B gene therapy. Treated mice show preservation and
restoration of rod cell function and are still sensitive to light a year after treatment. This study provides
strong evidence to support the use of gene therapy for RP caused by defects in PDE6B.
In May 2015, long term data from our clinical trial of RPE65 gene therapy for Leber Congenital
Amaurosis Type 2 (LCA2) was published in the New England Journal of Medicine (NEJM) and was
presented for the first time at the 2015 Association for Research in Vision and Ophthalmology (ARVO)
conference. This study confirms our preliminary findings, previously published in NEJM in 2008, that
gene therapy can improve night vision and retinal sensitivity in patients. Improvements peaked at six
to twelve months after treatment but showed decline in the following two to three years. Whilst our
latest results provide confirmation of efficacy, the data together with results of a parallel study in dogs,
indicate that the demand for RPE65 in the eye is not fully met with the current generation of gene
therapy vectors. These results are consistent with the published findings of other groups. We have
concluded that earlier intervention using a more potent delivery system that produces higher levels of
RPE65 is likely to provide greater benefit and protection against progressive degeneration in LCA2.
We have now developed a new, more powerful RPE65 gene therapy vector and have applied for and
secured new funding for a second clinical trial of RPE65 gene therapy with this new vector. The
Medical Research Council (MRC) have awarded us £2.97M to complete the necessary pre-clinical
testing and then commence a second phase I/II clinical trial next year.
In January 2015 we launched our new commercial venture, Athena Vision Ltd - an ocular therapeutics
company specialising in novel gene-based therapies for IRDs. Athena has exclusively licenced from
University College London the rights to develop a number of near to clinic therapies for a range of
retinal conditions. We have already raised substantial funding from investors that will enable us to
accelerate the development of promising new therapies.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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MECHANISMS OF PHOTORECEPTOR DEGENERATION IN CHOROIDEREMIA
Prof Clare Futter, Institute of Ophthalmology, UCL
Choroideremia (CHM) is a retinitis pigmentosa (RP)-related disease characterized by progressive
vision loss caused by degeneration of the photoreceptors (PR), the neighbouring retinal pigment
epithelium and the choroid, which provides the blood supply to the retina. CHM is caused by mutation
of Rab Escort Protein-1 (Rep1), a protein involved in the function of Rab GTPases, a protein family
that coordinates membrane transport steps within the cell. The purpose of this study was to use our
CHM models to define how PR die and to identify defects in membrane transport that could lead to
PR death.
Cell death can occur via a number of different mechanisms, one of which is apoptosis. We have found
that the rate of PR death in our CHM mouse models is slow and so have developed assays that
allowed us to quantitate dying cells in the entire retina and conclusively identify the mechanism of PR
cell death in CHM as apoptosis.
PR are highly specialised cells that have inner segments connected to long outer segments by a
narrow tube called the connecting cilium. The inner segment contains the protein and lipid synthesis
machinery and the outer segment is filled with packed membrane discs containing light sensitive
pigments and is the site where phototransduction, and hence vision is initiated. We have used highresolution electron microscopy to analyse the structure of photoreceptors in CHM models and found
disorganisation and a general shortening of the outer segments. The entire outer segment of the PR
is turned over every 10 days. Although the exquisitely ordered structure of the PR outer segment was
first described more than 50 years ago the mechanisms underlying its biogenesis and daily renewal
remain subject to intense debate. Understanding these mechanisms is critical to establishing how
they breakdown in CHM and other forms of RP where outer segment structure is defective. We have
used electron microscopy, together with 3D modelling, to map the trafficking events involved in
transport of rhodopsin from its site of synthesis in the inner segment to its incorporation into stacked
discs in the outer segment. We then analysed these processes in our CHM models and found that
rhodopsin transport was indistinguishable from healthy controls, indicating that defects in rhodopsin
transport are not the primary cause of apoptosis of PR in CHM. Our study will, however, shed light on
how rhodopsin transport and outer segment disc renewal breaks down in other forms of RP,
characterised by defective disc biogenesis. Furthermore our study will lead to a shift in focus in the
search for compromised membrane traffic pathways within PR in CHM and suggests that secretory
pathways regulating the release of survival factors may lead to PR apoptosis in CHM.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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PHARMACOLOGICAL THERAPIES FOR RHODOPSIN RETINITIS PIGMENTOSA
Principal investigator: Professor Mike Cheetham, Post-doctoral research associate: Dr. Dimitra
Athanasiou, UCL, Institute of Ophthalmology
Background
Retinitis pigmentosa (RP) is a group of hereditary disorders that leads to blindness due to the loss of
specialised cells found in the retina at the back of the eye. These cells are called photoreceptors and
are either responsible for dim light vision (rod cells) or detailed and colour vision (cone cells). Spelling
mistakes in the gene that encodes rhodopsin, the protein responsible for light detection in rod cells,
are the most common cause of dominant RP. These mistakes cause rhodopsin to take up an
abnormal shape, being recognised as faulty and trapped in the wrong part of the cell, with detrimental
effects on rod function and viability. However, photoreceptor cells have evolved several mechanisms
to protect themselves from stress insults such as that caused by faulty rhodopsin. Over the past years
we have identified some of the key members of these mechanisms that are responsible for the
manufacture of normal rhodopsin and the disposal or handling of faulty rhodopsin. We have also
shown that manipulating these systems directly or by using drugs we could alleviate some of the toxic
effects of faulty rhodopsin. The aim of this project is to test different drugs in models of RP to identify
the best candidates that could be used safely in RP patients with the aim of slowing down visual loss
and preserving vision.
Progress
During the first two years of this project we have completed the initial screening of 7 different drugs in
animal models of rhodopsin RP. These drugs were either already approved for clinical use, or are
being developed for other diseases that have common features with rhodopsin RP, and all gave
encouraging results in a cell model of rhodopsin RP. We found that some of these drugs protected
photoreceptor function and survival, some had no effect and others exacerbated RP symptoms. All of
these results are interesting and important, even the negative ones. For example, if there is no effect
upon treatment with a drug that targets a specific cellular pathway that means that this pathway can
be excluded in future studies. Whereas, if a drug exacerbates RP this means that its target might be
important for the retina to function properly or respond to the disease process and provides valuable
information of what to avoid in future therapies. For instance, we have tested a drug commonly used
to control type II diabetes, which had the ability to rescue the production of faulty rhodopsin in cells.
However, it accelerated blindness in a rat model of RP. This finding suggested that therapies for this
type of rhodopsin RP should focus on improving the removal of faulty rhodopsin, instead of rescue, as
the rescue is only transient and the faulty protein is still inherently dangerous. These findings suggest
future studies should focus on removing faulty rhodopsin to improve rhodopsin RP; however, it is also
possible that this drug could be beneficial for some other types of RP. So it is vital that RP patients
with diabetes (which can also cause vision loss and needs to be controlled) do not stop taking their
medication without discussing it with their clinician. Importantly, we have identified three drugs that
protect against vision loss and improve photoreceptor survival in this form of RP and we will
investigate these drugs in more detail in the final year and test their potential clinical application.
Future work
We have selected the three most promising drugs and we will extensively test their long term effect
and efficacy in animal models of RP that have a slower rate of retinal degeneration and their ability to
preserve cone function and survival. In parallel, we will test the efficacy of these drugs in a spectrum
of rod opsin inherited changes that are most common in the UK patients.
Publications
Aguilà M, Bevilacqua D, McCulley C, Schwarz N, Athanasiou D, Kanuga N, Novoselov SS, Lange CA,
Ali RR, Bainbridge JW, Gias C, Coffey PJ, Garriga P, Cheetham ME. (2014) Hsp90 inhibition protects
against inherited retinal degeneration. Hum Mol Genet. 15;23(8):2164-75 PMID: 24301679.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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Presentations
Michael E. Cheetham, Dimitra Athanasiou, Monica Aguila, Kieron South, Chikwado Opefi, Dalila
,
Bevilacqua Francesca Mackenzie, Peter M. Munro, Michael Y. Sherman, and Philip J. Reeves.
Improvement of mutant rhodopsin folding accelerates the rate of retinal degeneration in retinitis
pigmentosa (2015) Presentation EMBO conference: Molecular chaperones: From molecules to cells
and misfolding diseases 8-13 May 2015 Heraklion, Greece Dimitra Athanasiou, Monica Aguila, Kieron
South, Dalila Bevilacqua, Francesca Mackenzie, Peter M. Munro, Michael Y. Sherman, Philip J.
Reeves, Michael E. Cheetham (2015) Improvement of P23H rod opsin traffic after metformin
treatment accelerates the rate of retinal degeneration in P23H retinitis pigmentosa, Paper
presentation number 1701; 04/05/15, ARVO Denver USA 2015.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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THE DEVELOPMENT OF HUMAN IPSC-DERIVED EX VIVO MODELS OF RETINAL
DEGENERATION. THEIR ANALYSIS IN SPLICING-FACTOR RP
Mr A Webster, N Owen, Institute of Ophthalmology, University College London
The retina, the vital photosensitive layer of the eye, degenerates in retinitis pigmentosa due to events
occurring within specialised retinal cells. The disorder in any one person and family is due to a defect
in a single gene. Many genes are known and depending on the specific gene, can cause either
dominant inheritance (in which one copy from one parent is enough to cause the disorder) or
recessive inheritance (in which two copies, one from each parent, is required to cause the disorder). A
significant proportion of the first type, dominant RP, is due to defects in genes that code for proteins
involved in a process called splicing. This is a basic function required by all cells in the body, in fact by
all cells in all organisms. This is the first enigma of this type of RP - why should a defect in a gene that
is required by all cells, only appear to affect the retina? There is a second enigma too. Many people
who inherit defects in such genes, which in relatives would lead to visual loss, in some cause no
problems at all. Hence there is tremendous variability in those gene carriers in the same family, and
the cause for this is only partly understood.
Technology moves on, and a recent innovation allows the creation of stem cells from human skin and
the transformation of these into retinal-type cells (retinal pigment epithelial cells) for investigation and
potential treatment in the laboratory. As part of this project we intend to use this technique to compare
cells from specific patients with those from controls. One set of measurements we are working on is
based on determining an accurate tally of all the genes that are expressed in the cells. For this we
use another relatively new technology, 'RNA-Seq' in which many millions of molecules made by the
cells' genes (called messenger RNAs) have their exact sequence determined. Given the underlying
problem in this type of RP is the processing of genes (splicing) we would expect there to be
abnormalities in these measurements compared to controls. Exactly how we measure such an
abnormality, quantify it and correct for its inherent variation, we need to determine. Presently the
project is using blood samples from patients and controls; we are applying computer algorithms to
best compare the levels of correctly spliced genes, the proportion of incorrectly spliced genes, to
allow us to apply this to other cells from the patients. Something in a person's DNA also determines
whether they will be affected or not, and counting the RNA molecules in the same region of the
chromosome in which the splicing gene is situation, might allow us understand why this is.
Importantly, we wish to find out whether these genes have a second moonlighting function, in the
retina which might explain why they cause RP and to this end we are testing various antibodies to
best determine the gene products' precise location in the cells as well as tissue from Macaque retina
(very similar structure to our own).
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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MAINTAINING EFFECTIVE ANTIOXIDANT CAPACITY IN A DEGENERATING RETINA: A GENERIC
APPROACH TO TREATMENT OF RP
Prof R Ali, Institute of Ophthalmology, UCL
Oxygen is essential for the vast majority of life and plays an important role in the generation of energy required
for different cellular processes. Mitochondria as powerhouses of the cell are the major places for this energy
generation. However, byproducts of these energy generating processes are reactive oxygen species (ROS) that
have long been implicated in different conditions, including neurodegenerative diseases like Alzheimer’s and
Parkinson’s, as well as ageing.
The retina is a tissue with high oxygen supply and demand. In retinitis pigmentosa (RP), retinal degeneration
results primarily in the loss of rods cells but some central cones remain. These surviving cones experience
considerable stress and damage from ROS, a process known as oxidative stress, which can lead to further cell
death and loss of remaining central vision
Through this project we are seeking to explore two lines of enquiry. First, is an exploration of the emerging notion
that ROS are not only toxic but can also be signaling molecules that can provide valuable information of the
health of cells and tissues. To this end we will use mitochondrially-targeted molecules to measure the levels of
ROS in the mitochondria of normal mouse retina at different ages. These levels will be used, together with other
readouts of oxidative stress such as mitochondrial DNA damage and protein and lipid oxidation as baseline
values against which we can compare the amount of ROS RP mouse models as a measure of disease
progression. We are currently investigating levels of the ROS, hydrogen peroxide, in fast and slow models of
retinal degeneration.
Through our second line of enquiry we intend to investigate whether antioxidants and defence enzymes which
cells naturally employ to deal with damaging ROS have potential as therapeutic agents to protect and preserve
vision. Currently we are investigating whether supplementation of retinal cells with a mitochondrially targeted
antioxidant (MitoQ) can lead to the prevention of cone loss. We have so far determined that delivery of MitoQ
directly into the eye may not be ideal as the compound is cleared too fast from the retina for effective long term
treatment. We are currently exploring other forms of delivery include supplementation of drinking water as well as
injection into the body.
In the future we would like to test the effect of antioxidant supplementation on cone survival through the use of
MitoQ, as well as via delivery of defence enzymes such as peroxiredoxin 3 (Prx3), which detoxifies hydrogen
peroxide and thioredoxin 2 (Trx2), which recycles used Prx3. For this we will be using mouse models of RP with
different rates of rod cell degeneration to get an indication on whether there is a point at which patients with
advanced RP may benefit from this approach to maintain functional cones after loss of rod photoreceptor cells.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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RHODOPSIN TRAFFICKING DEFECTS IN RP
Dr R Megaw, University of Edinburgh
Mutations in the RPGR gene are responsible for 20 per cent of all retinitis pigmentosa and cause a
particularly severe form of the disease. At present the exact function of RPGR is unclear and as a
result it has no treatment. My work focuses on trying to understand RPGR’s role in the retina. To do
this I have taken advantage of the Nobel prize-winning discoveries of Shinya Yamanaka and John
Gurdon in stem cell biology.
Stem cells, by definition, are cells which can self-renew and can differentiate into any type of cell in
the body. It is possible to continually maintain stem cells in a dish in a laboratory and, when required,
differentiate them into any cell type. Importantly, we now have the ability to generate stem cells from
adult skin. As a result, these ‘induced’ stem cells can be generated from those who carry diseasecausing mutations. I have made ‘induced’ stem cells from the skin of several very generous donors
who have mutations in the RPGR gene. Their unaffected relatives have also provided skin samples in
order to generate ‘control’ stem cells which don’t carry the mutation. With these induced stem cells I
can then make use of the seminal work of Yoshiki Sasai in order to model RPGR disease ‘in a dish.’
Sasai discovered that if you can successfully push stem cells towards an eye field fate then these
cells have the inherent ability to organize themselves and form 3-dimensional eye-like structures all
by themselves. These cultures can mature over time and result in a structure containing all the main
cells seen in a human eye which are organized in the correct manner. Importantly the eye-like
structures contain photoreceptors; the cells in the retina which degenerate in retinitis pigmentosa.
This provides an exciting new way to model disease and I am able to pattern my ‘induced’ stem in
such a way in a dish in my lab. Thus, I can make these eye-like structures both with RPGR mutations
and without and compare the two in an attempt to discover RPGR’s true function in photoreceptors.
My research has uncovered a key abnormality in photoreceptors when RPGR is mutated. I have also
been able to determine a mechanism by which this happens. With these discoveries we now have a
better understanding of what RPGR’s role is in the human photoreceptor and what goes wrong in
disease. It is hoped continuing research with this ‘disease in a dish’ model can begin to identify
possible therapies which would correct the abnormalities seen in my photoreceptor cultures. I believe
this use of ‘induced’ stem cell technology will lead, eventually, to the development of treatments for
this condition. None of this work would have been possible without the generous financial support of
RPFB.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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EXPLOITING THE POWER OF HUMAN INDUCED PLURIPOTENT STEM CELLS TO GENERATE
SYNTHETIC FULLY LAMINATED RETINAE IN VITRO FOR DISEASE MODELLING, DRUG
DISCOVERY AND CELL BASED THERAPIES
Prof M Lako, Newcastle University
Background
Loss of vision due to degeneration of retinal cells characterises many genetic disorders including
Retinitis Pigmentosa (RP). One of the problems with studying RP is that the retinal tissue affected is
difficult to obtain and hence working out what is going wrong can be complex. Furthermore laboratory
animals often do not have the same pattern and mechanisms of disease. Stem cells are cells which
can keep on dividing for ever and have the ability to become any type of cell in the body. Certain
types of retinal cells have been grown from human stem cells and are being evaluated for possible
transplantation. However recent developments in stem cell research have shown that not only can
individual retinal cells be produced but that it may be possible to grow a fully formed retina in the
laboratory from patient-specific stem cells which could then be studied to help us understand human
retinal disease and its treatment, or used for transplantation.
Aims
In this project we focus on two aims: (1) developing robust methods for generating retina in the lab
from a large number of person-specific stem cells and (2) optimising the environment in which stem
cells are cultured so that the lab-made retina is as similar as possible to the retinal tissue found in the
back of our eyes.
Results
Previously, we reported that the derivation of retina from stem cells can be enhanced by addition of a
growth factor known as ‘IGF-1’ (Mellough et al. 2015). These findings were derived from work
performed in only one cell type. In the last year, we have performed experiments in 7 person-specific
stem cell lines and we have observed variations in their ability to give rise to neural tissue from which
retina is derived. We have undertaken further work to understand why this variations arises (Zhu et al.
2015) and, on this basis, we have selected three person-specific stem cell lines which show the
greatest ability to differentiate into neural and retinal tissue. Work performed with these three personspecific stem cell lines has shown that IGF-1 confers an enhanced ability to differentiate these stem
cells into retinal tissue. Furthermore, we have tested three differentiation methods across these
person-specific stem cell lines and have selected the best method that results in an enhanced ability
to give rise to retinal tissue and which can easily be automated and scaled up for clinical applications.
Different cell types found in the adult retina are surrounded by a complex matrix of different proteins
and sugars which provide a supportive meshwork which the cells can stick to, known as the
extracellular matrix (‘ECM’). It is thought that hyaluronic acid (HA), a sugar molecule that is abundant
in this matrix may be important for retinal development. We have been investigating whether the
growth of stem cells in HA gels enhances retinal formation and maturation. Our results to date have
shown that retina can be grown in HA gels and interestingly the gels enhance the formation of retinal
pigmented epithelium (RPE), which sits behind the retina and is important for the maturation and
maintenance of the photoreceptors (the light-sensing cells). Ongoing work is looking at whether the
photoreceptors are more mature when grown in the HA gels, compared with normal culture
conditions. The ECM of the retina does not only contain HA, it also contains many other molecules. A
greater understanding of the composition of the retina’s ECM by looking at it during development and
in the mature retina will allow us to replicate the ECM more accurately to further enhance retinal
formation from person-specific stem cells. We are currently characterising the distribution of different
important molecules known to be present in the retina, including hyaluronan and proteoglycan link
protein 1 (‘HAPLN1’), versican, interphotoreceptor matrix proteoglycans 1 and 2 (IPM 1 and 2). It is
important to understand whether the cells themselves produce and distribute these proteins correctly
in the retina generated in the lab, or whether it is necessary to add these proteins to the stem cell
cultures. We are therefore currently assessing the ECM of lab-generated retina at different time points
and comparing it with the developing human retina.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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Publications
Mellough CB, Collin J, Khazim M, White K, Sernagor E, Steel DH and Lako M. IGF-1 signalling plays
an important role in the formation of three dimensional laminated neural retina and other ocular
structures from human embryonic stem cells. Stem Cells. 2015 Apr 1. doi: 10.1002/stem.2023.
Zhu L, Gomez A, Saretzki G, Jin S, Anyfantis G, Chinnery P, Lako M and Armstrong L. CHCHD2: a
new OCT4 and SOX2 target and a putative marker for assessing the differentiation potential of human
induced pluripotent stem cells to neuroectodermal lineages (under revision, May 2015).
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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DEVELOPING NOVEL OPTOGENETIC TOOLS FOR THE TREATMENT OF RETINAL
DYSTROPHIES
Dr J Kapetanovic, University of Manchester
Retinal degenerations that affect 1:2500 people worldwide result in photoreceptor loss and represent
major causes of blindness. There are few experimental treatments and none reverse the loss of
vision. Following the loss of photoreceptor function, the surviving non-photoreceptor cells of the retina
often remain structurally intact and potentially capable of function. A very promising treatment strategy
called optogenetics involves converting remaining non-photoreceptor retinal cells into directly light
sensitive photoreceptors.
The technique used for converting cells into photoreceptors is a form of gene therapy whereby
suitable photosensitive proteins are introduced into the non-photoreceptor cells using a gene delivery
system. Current gene therapy clinical trials utilize a subretinal delivery route which allow for limited
gene expression at the site of injection and is technically challenging. We have made a major
advance that allows the widespread delivery of genes to retinal cells following intravitreal injection of a
viral-based gene delivery system in combination with certain enzymes.
Another important factor to consider when developing optogenetic therapy is which cells to target.
Untargetted approach can target any remaining retinal cells, whereas selective targeting is specific to
a particular cell type. A potential concern with untargeted approach is that it might lead to incoherent
visual signal. This research, undertaken at the University of California, Berkeley, USA, focuses on
targetting the gene expression to a particular cell type, the bipolar cells. The bipolar cells are the
highest order surviving retinal cells and have the most potential to preserve retinal computation and
lead to restoration of useful vision. So far, conventional vectors lead to very poor gene expression in
bipolar cells.
We have developed novel vectors that can specifically target bipolar cells in a mouse model of retinal
degeneration via clinically relevant intravitreal injection. We are currently investigating the efficacy of
these novel vectors in terms of the extent of gene expression in the treated retina. Future work will
focus on detailed functional analysis and characterization of the photosensitive proteins expressed in
the bipolar cells via these optimised vectors.
RP Fighting Blindness Research Reports 2014/2015 (Charity No. 1153851)
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UK INHERITED RETINAL DYSTROPHY CONSORTIUM
RP GENOME PROJECT
Professor Graeme Black - Chief Investigator (Manchester)
Mr Stuart Ingram - acting Project Manager (Manchester)
Professor Alison Hardcastle - Principal Investigator (University College London)
Professor Chris Inglehearn - Principal Investigator (Leeds)
Dr Andrea Nemeth - Principal Investigator (Oxford)
Inherited retinal dystrophies are a group of over 200 genetic conditions affecting around 1 in 2500 of
the population. Advances in technology (Next Generation sequencing, or NGS) and a sharp fall in the
cost of this technology mean that we can now generate huge amounts of genetic data in trying to find
the cause of these conditions. However the amount of data is so large, that we believe to use it
effectively now requires collaborative working across a number of sites.
The RP Genome project is an ambitious project operating at four leading research centres in
ophthalmology and genetics. We aim to discover new genes causing inherited retinal dystrophy,
improve the number of diagnoses made, and develop data sharing and databases so that these
improvements can be shared with the medical community at large in the UK and beyond. We also
want to improve access to genetic testing in areas where it may not be currently available.
Since the start of the project on 01/11/2014 we have laid the groundwork for this collaborative
approach to research. This has meant a significant amount of time appointing staff, writing protocols
and ethics applications, as well as creating secure databases for storing the enormous volumes of
data that genomic analyse produce. We have agreed the types of patients to be included in the first
round of studies and are currently in the process of testing databases, data sharing systems and the
NGS “pipeline” to ensure we can all work to the same quality standards. We have recruited our first
participants and have already published a number of papers, two of which are now in press.
The UK IRDC members continue to make contributions to other groups working in the field including
Genomics England, the UK Eye Genetic Group and the European Retinal Dystrophy Consortium, and
are actively ensuring that momentum is maintained in developing genomic testing for patients with
inherited retinal disorders. Our ultimate aim is that the research conducted through this grant leads to
further collaboration and study of the condition, with further improvements in diagnosis, patient
management and access to testing.
References
Biallelic Mutations in the Autophagy Regulator DRAM2 Cause Retinal Dystrophy with Early Macular
Involvement. The American Journal of Human Genetics, in press.
El-Asrag ME, Sergouniotis PI, McKibbin M, Plagnol V, Sheridan E, Waseem N, Abdelhamed Z,
McKeefry D, Van Schil K, Poulter JA, UK Inherited Retinal Disease Consortium, Johnson CA, Carr IM,
Leroy BP, De Baere E, Inglehearn CF, Webster AR, Toomes C, Ali M. (2015)
http://dx.doi.org/10.1016/j.ajhg.2015.04.006
Pinpointing clinical diagnosis through whole exome sequencing to direct patient care: a case of
Senior-Loken syndrome
Jamie M Ellingford, Panagiotis I Sergouniotis, Rachel Lennon, Sanjeev Bhaskar, Simon G Williams,
Kate A Hillman, James O’Sullivan, Georgina Hall, Simon C Ramsden, I Christopher Lloyd, Adrian S
Woolf, Graeme C M Black Lancet. In press
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