O R I AAdvancing eye research

The Ophthalmic Research Institute of Australia

2015 Annual Report

ContentsIntroduction 5

Chairman’s report 6

ORIA Grants 8

Progress reports 12-43

Directors’ Report 48-51

Financial Statements 48-65

Auditors’ Report 66-68

O R I AAdvancing eye research

In 62 years, the ORIA has been in the forefront as a major contributor towards medical eye research in Australia. It has provided over $17million to research all major eye diseases including macular degeneration, glaucoma, low vision, lens and cataract, and diabetic retinopathy.

The ORIA’s contribution benefits many Australians and adds to the knowledge and understanding in finding both cures and treatments for eye diseases generally.

The Annual Report will be presented at the Sixty Third Annual General Meeting to be held in Wellington, New Zealand on Sunday 1 November 2015

Notice of Meeting

THE BOARDProf Stuart Graham, Sydney (Chairman)

Prof Mark Gillies, Sydney (Vice Chairman)

Dr Richard Mills, Adelaide (Honorary Secretary)

Dr W Heriot, Melbourne (Honorary Treasurer)

Dr Fred Chen, Perth

Dr Colin Clement, Sydney

Prof J Crowston, Melbourne

A/Prof Paul Healey, Sydney

Prof David Mackey, Perth

Prof Peter McCluskey, Sydney

Dr John Males, Sydney

Dr Andrea Vincent, New Zealand

Dr Stephanie Watson, Sydney

RESEARCH ADVISORY COMMITTEEFor assessments of funding in 2015

Prof Peter McCluskey, Sydney (Chair)

Dr Richard Mills, Adelaide (Secretary)

Prof Stuart Graham, Sydney (ex officio Chair ORIA)

A/Prof John Foster, Sydney

Dr Kathryn Burdon, Adelaide

Dr Fred Chen, Perth

Dr Samantha Fraser-Bell, Sydney

Dr Alex Hewitt, Melbourne and Hobart

Prof David Mackey, Perth

A/Prof Ian Trounce, Melbourne

Dr Peter Van Wijngaarden, Melbourne

Dr Andrea Vincent, New Zealand

Dr Trevor Sherwin, New Zealand

Dr Stephanie Watson, Sydney

Save Sight Society NZ rep – Dr Graham Wilson

Anne Dunn Snape (as Executive Officer, ORIA)

EXECUTIVE OFFICERMs Anne Dunn Snape, BA (Soc & Pol Phil), (MQ)PostGradC Ethics & Legal Studs.

HONORARY SECRETARY Dr Richard Mills 94-98 Chalmers Street, Surry Hills NSW 2010

HONORARY TREASURER Dr Wilson Heriot 94-98 Chalmers Street, Surry Hills NSW 2010

ACCOUNTANTSMcLean Delmo Bentleys Level 8, 607 Bourke Street, Melbourne Vic 3000

AUDITORS Orr Martin & Waters461 Whitehorse Road, Balwyn Vic 3103

TRUSTEES National Australia Trustees Ltd, Melbourne, Vic

HON SOLICITORSKing and Wood MallesonsAdvance Bank Centre60 Marcus Clarke Street, Canberra 2600

INVESTMENT ADVISORY COMMITTEE Dr Wilson Heriot – Hon Treasurer, ORIA

A/Prof Mark Daniell

Mr Andrew Miller – Senior Adviser, UBS Wealth Management

Mr Andrew Perry – Director-Equities, Lodge Partners Pty Ltd

Mr William Jones – Principal, Goldman Sachs J B Were Limited

Secretary to the Committee, Mr Matthew Timothee – National Australia

Trustees Limited

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Chairman’s Report

The Ophthalmic Research Institute of Australia is the College’s research arm, and aims to: “Advance Eye Research”. The ORIA held its first AGM just over 60 years ago and since that time has distributed literally millions of dollars to provide funding for medical eye research in Australia.

The ORIA’s activities are co-ordinated and managed by up to 16 members of the Board of the ORIA and Executive Officer, Anne Dunn Snape. Using the income from its investments and donor organisations, the ORIA continued to contribute to funding for research projects throughout Australia. During the year the ORIA’s Research Advisory Committee considered 52 applications for project funding from Australian researchers, a significant increase from 19 assessed in 2004. It also assessed 3 New Zealand applications for funding on behalf of the Save Sight Society of New Zealand. The NZ Branch is represented on the committee via its Save Sight Society.

The ORIA’s Research Advisory Committee is composed of leading research scientists and ophthalmologists from Australia and New Zealand. All applications are independently peer reviewed which forms the basis for discussion and recommendation of funding by the Committee. The recommendations of the Committee are put forward to the Board of the ORIA who then indicate what funds are available for the forthcoming calendar year. This year $826,000 has been distributed to fund 18 one year projects throughout Australiaand together with 2014 brings a total of $1.7million supporting research into eye disease”. RANZCO provided funding for three New Investigators and the RANZCO Eye Foundation supported our top rated grant. The New South Wales Branch of

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146ORIA ANNUAL REPORT 2015

RANZCO donated half the funds for the top rated New South Wales project. We are most grateful to all organisations for their continuing support along with previous benefactors whose legacies are acknowledged through the naming of individual grants.

The ORIA also continues its annual support of the Ringland Anderson Chair of Ophthalmology in Victoria.

The ORIA continued funding a New Investigator category in an endeavour to encourage up and coming researchers; five grants were awarded this year.

Significant projects to receive funding from January 2015 were:

ORIA/RANZCO EYE FOUNDATION GRANTProf Ian McAlister, Dr A Shaw & Prof D-Y YuAssessment of tenecteplase (metalyase) as an acute interventional treatment for branch retinal vein occlusion (BRVO).$45,500

ORIA/ESME ANDERSON GRANTDr Alex Hewitt, Prof Andrew Hill, Dr Ruchira Singh & Mr Duncan Crombie Utility of genome editing in correcting inherited retinal disease $50,000

ORIA/RENENSSON BEQUEST GRANTA/Prof Nick Di Girolamo Destiny of limbal epithelial stem cells in the normal cornea $50,000

ORIA/ANSELMI BEQUEST GRANTDr Kathryn Burdon & Dr Shiwani Sharma The Eye Expression Atlas Project $50,000

ORIA/RANZCO NSW BRANCH GRANTA/Prof Robyn Jamieson & A/Prof John Grigg Whole genome sequencing for vitreous and retinal dystrophies $50,000

Details of all other grants awarded can be found on the ORIA website www.oria.org.au and for New Zealand at www.safesightsociety.org.nz.

The ORIA is always mindful of auditing its research funding to assess how well its mission to advance eye research is being achieved. Each year, progress/final reports are provided from researchers funded during the previous year. A financial statement from each project/institution is also secured to ensure funds have been used in the manner previously indicated in the application.

The ORIA continued its presentation of some of the more significant research funded from the 1983 period onwards at its plenary session at RANZCO in Brisbane, Queensland under the chairmanship of Prof.David Mackey. We described the methodology of the research and showed some historical pictures and documents. We chose some contemporary researchers in the same field as the ORIA funded researcher to give a 2-3 minute summary of the individual, 2-3 minute summary of their life’s work and then the remaining time for the ORIA papers. The work of John and Shirley Sarks

was highlighted and their contribution to eye research in Australia was an example of the time required for scientific research to sometimes deliver significant results and a fantastic legacy. Prof. Keryn Williams NHMRC Principal Research Fellow in the Department of Ophthalomology and Associate Dean (Research) within the Faculty of Health Sciences at Flinders University presented the work of her colleague, Prof Doug Coster.

During the year, the Institute has been the recipient of three legacies. Firstly, the estate of Mrs. Betty Marion Roberts contributed $100,000 towards its research. Secondly, the estate of Mrs Gladys Clare Dickson bequeathed around $280,000 to the ORIA. In June, we were thrilled to learn of the formation of the Richard and Ina Humbley Foundation for the sole benefit of the ORIA. The Humbley Foundation has requested that funds be used towards the ORIA’s macular degeneration research projects. The ORIA has a track record of annually supporting research into macular degeneration and has done so over many years. At its inception, the perpetual Foundation is currently funded at around $1.7million. The work of the Foundation will also be to ensure that the ORIA can increase its funding of research into macular degeneration by growing the overall equity. We are very grateful for these very generous legacies.

Prof Stuart Graham Chair, ORIA Anne Dunn SnapeExecutive Officer, ORIA

77

ORIA/RANZCO EYE FOUNDATION GRANT $45,500Prof Ian McAlister, Dr A Shaw & Prof D-Y Yu

Assessment of tenecteplase (metalyase) as an acute interventional treatment for branch retinal vein occlusion (BRVO).

ORIA/ESME ANDERSON GRANT $50,000Dr Alex Hewitt, Prof Andrew Hill, Dr Ruchira Singh & Mr Duncan Crombie

Utility of genome editing in correcting inherited retinal disease

ORIA/RENENSSON BEQUEST GRANT $50,000A/Prof Nick Di Girolamo

Destiny of limbal epithelial stem cells in the normal cornea

ORIA/ANSELMI BEQUEST GRANT $50,000Dr Kathryn Burdon & Dr Shiwani Sharma

The Eye Expression Atlas Project

ORIA/RANZCO NSW BRANCH GRANT $50,000A/Prof Robyn Jamieson & A/Prof John Grigg

Whole genome sequencing for vitreous and retinal dystrophies

ORIA/DAME IDA MANN GRANT $48,500Prof J McAvoy, Dr Y Sugiyama & Prof F Lovicu

A role for planar cell polarity in lens regeneration

ORIA/Mary Tilden Bequest Grant $50,000Dr Vivek Gupta, Dr Stuart Graham & Dr Yuyi You

Developing a new gene therapy approach to protect against glaucoma damage

ORIA GRANT $41,500A/Prof Damien Harkin & Dr Sally Stephenson

Do Ephrin signalling pathways contribute to control of corneal endothelial cell function

ORIA GRANT $49,000Dr Carla Abbott, Dr Penny Allen & A/Prof Erica Fletcher

Effect of electrical stimulation on photoreceptor survival in retinal degeneration

ORIA Project Funding 2015

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

ORIA GRANT $50,000Dr Weiyong Shen

Understanding the role of microglia in retinal health and disease

ORIA GRANT $27,000C/Prof Stephanie Watson, Dr Holly Inglis, Prof Michael Friedlander, Prof Frances Boyle & Dr Yi-Chiao Li

Aromatase inhibitor therapy for breast cancer and dry eyes

ORIA GRANT $49,500Dr Glyn Chidlow

Investigations into retinal pathology in a mouse model of Alzheimer’s disease

ORIA/RANZCO NEW INVESTIGATOR GRANT $49,000Dr Shervi Lie

Defence mechanisms of specialised cells within the eye during parasite infection

ORIA/RANZCO NEW INVESTIGATOR GRANT $16,500Dr Shane Durkin

What is the jelly of the eye made of?

ORIA/RANZCO NEW INVESTIGATOR GRANT $49,500Dr Mojtaba Golzan & Prof Alberto Avolio

Effects of high blood pressure on retinal hemodynamics in glaucoma

ORIA NEW INVESTIGATOR GRANT $50,000Dr Danuta Maria Bukowska

Investigating mechanism of visual scotoma and metamorphopsia due to macular disease using multimodal imaging

ORIA NEW INVESTIGATOR GRANT $50,000Dr Sandy Hung, Dr Raymond Wong, Dr Bryony Nayagam & Prof Ross Hannan

Generation of an immortalised human retinal ganglion cell (RGC) line from pluripotent stem cells

TOTAL $826,000

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Adam Watson

Adrian Fung

Adrian Hunt

Alex Harper

Allison Maree Mckendrick

Andrew Chang Andrew Riley

Andrew Symons

Andrew Thompson

Andrew White

Anne Slavotinek

Annie McCauley

Ashish Agar Bang Bui

Bart Leroy

Binoy Appukuttan

Brendan Vote

Celia Chen

Chi Luu

Christine Younan

Colin R. Green

Colin Willoughby

Colm McAlinden

David Simpson

David Worsley

Deepa Taranath

Dr. Cherie Blenkiron

Dr. Damien Harkin

Duncan Crombie

Elfride de Baere

Eva Fenwick

Fulton Wong Gerald Liew

Glyn Chidlow

Gwyn Rees

Hemal Mehta

Hong Zhang

Ian Constable

Ian McAllister

Ilva Rupenthal

Jan Provis

Jane Khan

Jennifer Craig

Jill Keeffe

John Armitage John Grigg

John McAvoy

John Wood

Justine Smith

Karl Brown

keryn.williams

Kevin Spring

Kimberley Tan

Krisztina Valter-Kocsi

Lisa Keay

Lyndell Lim

Mark Corbett Mark Daniell

Mark Donaldson

Matt Ball

Matthew Simunovic

Michael Coote

Michele Madigan

Mo Dirani

Monica Acosta Nigel Morlet

Paul Leo

Paul Martin Penny McKelvie

Peter Hendicott

Rachel Barnes

Rick Liu

Robert Casson

Robert Finger

Robert Marc

Robyn Guymer

Robyn Jamieson

Rohan Essex

Shervi Lie

Shiwani Sharma

Stewart Lake

Stuart Graham

Stuti Misra

Suriya Foran

Svetlana Cherepanoff

Ted Maddess

Tim Isaacs Tina Lamey

Vicki Chrysostomou

Vivek Gupta

Weiyong Shen Wilson Heriot

The Institute would like to thank our external referees who kindly gave advice which helped with the allocation of the 2015 grants. Their work is invaluable.

RANZCO

RANZCO, New South Wales Branch

The RANZCO Eye Foundation

The Estate of Betty Marion Roberts

The Estate of Gladys Clare Dickson

Richard & Ina Humbley

Thank You

Donations

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

Top right to bottom left: Dr Nicole Van Bergen (page 12); A/Prof Mark Daniell (page 14); Dr Helena Liang, Mr Duncan Crombie, Dr Alice Pébay, A/Prof Alex Hewitt and Dr Raymond Wong (page 12) Photos – courtesy CERA, Dr Angus Turner (Page 30).

Progress Reports

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Progress Reports

ORIA/RANZCO EYE FOUNDATION GRANTDr Raymond Wong. Co-investigators: Dr Alice Pébay, Dr Nicole Van Bergen

Study of Leber’s Hereditary Optic Neuropathy using induced pluripotent stem cells.

The overall aim of this work was to assess the value of using human induced pluripotent stem cells (hiPSCs) to model Leber’s Hereditary Optic Neuropathy (LHON) and correct disease-causing genetic mutations in these cells, using cybrid technology to replace the faulty mitochondria with healthy donor mitochondria.

GENERATION OF LHON-IPSCS AND DIFFERENTIATION INTO RETINAL GANGLION CELLS (RGCS)We have generated and characterised 19 cell lines of iPSCs from LHON patient and controls. Extensive characterisation of these iPSCs confirmed the quality of these iPSCs (Fig. 1). In addition, we have developed an improved protocol for stem cell differentiation to yield an enriched population of RGCs in 45 days (Fig. 2).

FUNCTIONAL CHARACTERISATION OF STEM CELL-DERIVED RGCSWe carried out electrophysiological analysis to test the functions of the generated RGCs (Fig 3). These stem cell-derived RGCs are capable of firing multiple action potentials in response to membrane depolarisation (Fig. 3A). Voltage-clamp recording showed activation of inward sodium currents followed by outward potassium currents in response to increasing membrane depolarisation (Fig. 3C). In addition, this inward sodium current can be abolished by treatment with the sodium channel blocker tetrodotoxin (TTX), which confirmed the detected current is dependent on sodium channels (Fig. 3D). Together, our results confirm that the stem cell derived-RGCs are functional and display an electrophysiological profile for mature neurons. Our continuing research shows that LHON-RGCs

Figure 1 Representative characterisation of 3 clonal cell lines of iPSCs. Immunocytochemistry showing expression of pluripotent markers Oct4 and TRA-1-60 in all three clonal cell lines of iPSCs (MRU11780c3, MRU11780c4, MRU11780c6). All 3 iPSC lines are capable of differentiating in vitro into cells representative of mesoderm (SMA positive), endoderm (AFP positive) and ectoderm (NESTIN positive). DAPI was used to label the nucleus. Scale bars = 100 µm.

Figure 2 A) Schematic timeline of procedure to generate stem cell-derived RGCs. B) Day 27 Stem cell-derived RGCs. C-G) Characterisation of enriched stem cell-derived RGCs using a panel of known RGC markers C) NFM, D) vGlut1, E) BRN3A, F) HUC/D and G) βIII TUBULIN and vGlut1 (n>3 for all markers). Scale bars = 100 µm.

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

exhibit phenotypes that recapitulate those observed in LHON patient, providing support that disease modelling using iPSC is a feasible approach to study optic neuropathies.

MITOCHONDRIAL GENETIC CORRECTION IN LHON-IPSCSWe have corrected the mitochondrial mutations in LHON patient fibroblasts carrying 14484+4160 mutations using the cybrid technology. Briefly, the defective mitochondria carrying LHON mutations are depleted by R6G treatment, and replaced with mutation-free keratinocyte mitochondria extract by cellular fusion (Fig. 4A). The cybrid fibroblasts are genotyped to confirm genetic correction of LHON mutations (Fig. 4B). Using the genetically-corrected cybrid fibroblasts, we have generated 3 clonal iPSC cell lines. Immunocytochemistry and embryoid body assays confirmed that these are good quality iPSCs. Our results represent the first iPSCs with genetic correction in mitochondrial mutation. Development of this cybrid technology will be crucial to correct mutations in LHON-specific iPSCs before utilisi ng these cells for cell replacement therapy.

This grant has led to further funding from 2 other funding bodies, including:

NHMRC New investigator Project grant (2015-18), ‘Modelling Leber’s Hereditary Optic Neuropathy using human induced pluripotent stem cells’ ($608,813). Chief investigator: Dr Raymond Wong.

Australian Mitochondria Disease Foundation Incubator grant, ‘Genome-wide expression analysis of human induced pluripotent stem cell model of Leber’s Hereditary Optic Neuropathy’ ($25,000), Chief investigator: Dr Raymond Wong, Co-investigators: A/Prof Ian Trounce, Dr Alice Pebay, Dr Alex Hewitt.

RESEARCH OUTPUT

Publications acknowledging the ORIA as a source of funding:1. Hung, S., Pébay, A., Wong, R. (2015)

Generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes. Journal of Visualized Experiments, in press 2015.03.24.

2. Gill, K., Hewitt, A., Davidson, K., Pébay, A., Wong, R. (2014) Methods of retinal ganglion cell differentiation from pluripotent stem cells. Translational Vision Science & Technology, 3(4).

Conference proceeding (Poster):1. Wong, R., Hung, S., Van Bergen, N.,

Jackson, S., Lim, S.Y., Hernandez, D., Gill,

K., Lidgerwood, G., Mackey, D., Liang, H., Kearns, L., Hewitt, A., Trounce, I., Pébay, A. (2015) Using pluripotent stem cells to model optic neuropathies. International Society of Stem Cell Research Annual Meeting, Stockholm, Sweden.

2. Wong, R., Hung, S., Van Bergen, N., Jackson, S., Lim, S.Y., Hernandez, D., Gill, K., Lidgerwood, G., Mackey, D., Liang, H., Kearns, L., Hewitt, A., Trounce, I., Pébay, A. (2015) Using human induced pluripotent stem cells to understand optic neuropathies. Cell Reprogramming Australia, Brisbane.

3. Gill, K., Hung, S., Needham, K., Hewitt, A., Pébay, A., Wong, R. (2015) Differentiation and enrichment of retinal ganglion cells from human embryonic stem cells. International Society of Stem Cell Research Annual Meeting, Stockholm, Sweden

4. Gill, K., Hung, S., Needham, K., Hewitt, A., Pébay, A., Wong, R. (2014) Differentiation and enrichment of retinal ganglion-like cells from human embryonic stem cells. 2014 Biomed link conference, Melbourne.

5. Gill, K., Hung, S., Needham, K., Hewitt, A., Pébay, A., Wong, R. (2014) Differentiation and enrichment of retinal ganglion-like cells from human embryonic stem cells. Australiasian Stem Cell Science and Therapy Meeting, Lorne.

Oral presentations at conferences and academic institutes:1. Wong, R. (2015) Using human induced

pluripotent stem cells to model retinal diseases, academic visit to Advanced Center for Translational and Regenerative Medicine, Karolinska Institutet, Sweden

2. Wong, R. (2014) Modelling retinal diseases by large scale manufacture of human induced pluripotent stem cells. Frontiers in Human Engineering symposium, Melbourne, Australia

3. Wong, R. (2014) Understanding ocular disease using human induced pluripotent stem cells. 4th Annual World Congress of Molecular & Cell Biology, Dalian, China.

4. Wong, R. (2014) Using human induced pluripotent stem cells to model Leber’s Hereditary Optic Neuropathy, Stem Cells Australia Neuro-workshop, Queensland Brain Institute, University of Queensland, Australia

Figure 3 A) Stem cell-derived RGCs are capable of firing multiple action potentials (black trace). B) RGCs with recording microelectrode attached. C) Voltage-clamp recording shows activation of inward sodium currents (arrow) followed by outward potassium currents in response to increasing membrane depolarisation. D) The inward sodium current (arrow; black trace) is abolished in the presence of 1 mM tetrodotoxin (TTX; grey trace), a potent blocker of voltage-gated sodium channels.

Figure 4 A) Schematic diagram of cybrid technology to correct mitochondria mutations. B) Genotyping results confirm correction of mitochondrial mutations in cybrids.

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PROJECT SCOPE AND AIMSThe aim of this project was to develop a ‘web-based’ national registry, to collect high quality data outcomes on the clinical effectiveness and safety of emerging therapies for keratoconus from patients in real life clinical settings.

Despite many therapies, devices and surgical procedures being used to treat keratoconus, few have been evaluated using post-market surveillance. Further, there is no web-based system in place to collect such data nationally. Cross-linking (CXL), a relatively new treatment aimed to prevent keratoconus progression, will be the first therapy to be evaluated via the registry.

This project is unique as the national data collected with our world-leading technology will be used to formulate evidence-based management guidelines, evaluate new interventions and report patient related outcomes. As although randomized clinical trials are the gold standard for treatment evaluation, they cannot be used once therapies are widely available. Using web-based scientific data collection systems to gather all the information from clinical consultations is a simple yet effective way of “collecting all the evidence” from clinical consultations (1).

BACKGROUNDKeratoconus reduces vision by altering the biomechanical properties of the cornea. It affects 50 to 200 per 100,000 of the general population. Severe visual deterioration affects 20% of keratoconics and usually occurs in the second and third decades of life due to irregular astigmatism, corneal scarring or both. As it affects young adults it has

a significant public health impact. If vision loss from keratoconus cannot be corrected by spectacles or contact lenses, corneal grafting maybe needed which confers a life-long risk of graft rejection and weakening of the structural integrity of the eye.

Corneal cross-linking can reduce the risk of progression of keratoconus

Corneal collagen cross-linking (CXL) is a new technique that utilises riboflavin and UVA to strengthen the cornea. Cross-linking can prevent the progression of keratoconus, when measured by key topographic and refractive outcomes, and thus the need for corneal grafting in some patients. The long-terms effects and durability of this treatment are not known. Further there is emerging evidence that the corneal stroma is irreversibly changed (7). Serious complications including corneal oedema, microbial keratitis, corneal melting and perforation along with sterile corneal infiltrates have been reported. However, data is lacking on the complication rates, side-effects and long-term results of this procedure in routine clinical practice (2,3).

Evidence to support current clinical variations in the standardized protocols is lacking

Corneal cross-linking is most commonly carried out according to the ‘Dresden protocol’ which involves epithelial removal, isotonic riboflavin given for half an hour followed by half an hour of UV light at 3 mW (4). To improve patients’ comfort, reduce risk of complications, allow treatment of thin cornea and reduce procedure time, variations in the reported protocols have included leaving the corneal epithelium intact, use of hypo-

ORIA/RANZCO EYE FOUNDATION GRANTChief investigator: Clinical Professor Stephanie WatsonCo-investigators: Associate Professor Mark Daniell, Dr John Males, Dr Yves Kerdraon, Dr Martina Bosch, Dr Daniel Barthelmes

Technology for improving health outcomes in ocular surface and corneal disease – the keratoconus registry

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ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

osmolar riboflavin to swell the cornea and increasing the power of the UVA as well as using adjunct therapies.

There is little evidence in the literature to support such variations in the treatment protocol and the prevalence of variations in the current protocols for cross-linking is not known. However, to ensure the “safety and effectiveness of CXL, strict adherence to standardized CXL procedural protocols is essential”. As many young patients with keratoconus are being treated with protocol variations; research to establish the effects of protocol variations and long-term outcomes of CXL is needed.

KEY PROGRESSStage 1

A draft module design was initially complied after consultation with the Fight Retinal Blindness! Project team and corneal specialists with experience in corneal cross-linking. Factors taken into consideration included ease of use of the registry in the clinical setting, key outcomes measures, key research questions to be answered, and clinically relevant parameters.

Following completion of the module design in October 2014, software development was undertaken by The University of Sydney’s Information and Communications Technology (ICT) Department. This team has extensive experience in programming for the FRB! Project. Programmers from the ICT Department were engaged to develop the scientific web-based data collection system from October 2014, with the first launch of the system in April 2015.

Stage 2

Currently the system is undergoing beta-testing with the chief investigator and co-investigators involved in the project. Data pertaining to the corneal cross-linking procedure and patient related outcomes are now being collected by the web-based system. This data will be used to determine whether clinical outcomes measure correspond to improvements in the patients’ visual function and quality of life. Once user-acceptance has been approved by the chief investigator and co-investigators, the system will be deployed to other corneal clinics nationally.

ORAL PRESENTATIONThe Keratoconus Registry: Technology to audit corneal cross-linking. Fight Corneal Blindness Project. Watson SL. RANZCO Annual meeting 2014

References1. Kelman CW, Pearson S-A, Day RO, Holman

CDJ, Kliewer EV, Henry DA. Evaluating medicines: let’s use all the evidence. Med J Aust. 2007 Mar 5;186(5):249–52.

2. Chan E, Snibson GR. Current status of corneal collagen cross-linking for keratoconus: a review. Clin Exp Optom. 2013 Mar;96(2):155–64.

3. Abdelghaffar W, Hantera M, Elsabagh H. Corneal collagen cross-linking: promises and problems. Br J Ophthalmol. 2010 Nov 22;94(12):1559–60.

4. Spoerl E, Mrochen M, Sliney D, Trokel S, Seiler T. Safety of UVA-Riboflavin Cross-Linking of the Cornea. Cornea. 2007 May;26(4):385–9.

Figure 1 A cornea with keratoconus. Note the conical shape of the cornea, this distorts vision.

Figure 2 Scarring (whitish opacity) of the cornea in keratoconus.

Figure 3 Corneal topography showing a corneal with keratoconus. This map is like a geography map, it shows where the cornea is elevated.

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OVERVIEWGlaucoma is the leading cause of irreversible blindness in the world. In recent years, through genetic association studies in large cohorts of Australian cases with blinding glaucoma, our group identified association of genetic variants at multiple chromosomal regions with increased risk of blinding glaucoma as well as less severe glaucoma. The aim of this project was to investigate the most recently associated genes with glaucoma blindness in the eye for gaining an insight into their functions in the eye and the disease. This understanding would lay the foundation for deciphering the biological relevance of the associated genes in the disease, which would open the possibility of their manupulation to delay glaucoma progression in the future.

BACKGROUNDGlaucoma is a heterogeneous group of optic neuropathies with multifactorial aetiologies all converging at the loss of retinal ganglion cells (RGCs) in the retina and their associated axons at the optic nerve head (ONH), resulting in progressive visual loss. The disease mechanism is complex and poorly understood. Primary open-angle glaucoma (POAG) is the most common form in Caucasian populations including Australia. It is characterised by an open iridocorneal angle and often an elevated intraocular pressure (IOP). However, the IOP is within the normal range in a subset of patients with ‘normal-tension glaucoma’. Onset is most common after age 40 years, but a less common subgroup of juvenile OAG manifest signs earlier. Regardless of the glaucoma subtype, lowering of the IOP with drugs, laser or surgery is the only available treatment. Despite treatment, some

patients still experience progression of the disease albeit slower than without treatment. Thus strategies for early diagnosis, delaying disease progression and alternative treatments are warranted.

Glaucoma has been widely acknowledged as a disease with complex heritability. Pathogenic mutations in the Myocilin and Optineurin genes and copy number variation in the Tank-binding kinase 1 together account for a small proportion of POAG cases. In the recent years, genome-wide association studies (GWAS) in large case-control cohorts have led to identification of several genetic susceptibility factors for POAG. Our group has pioneered the majority of these discoveries. Through GWAS in a large Australian cohort of blinding glaucoma recruited through the Australian & New Zealand Registry of Advanced Glaucoma (ANZRAG), we identified genome-wide significant association of two loci, in and near TMCO1 at 1q24 and CDKN2B-AS1 gene at 9p21 with blinding disease. Variants at these loci are also associated with less severe disease. Association of variants at these loci with the disease has been subsequently also reported in other populations by other groups. More recently, through GWAS in an even larger Australian cohort of blinding glaucoma and meta-analysis in 4 further cohort with varying disease severity from Australia and the United States, we identified genome-wide significant association of three additional loci with POAG: 9q31.1 near ABCA1, 4p16.1 in AFAP1 and 6p24 in GMDS. Association of variants near ABCA1 with POAG was also independently reported in an Asian cohort. The other loci associated with the disease, discovered in other world populations

ORIA/RENENSSON BEQUEST GRANTDr S Sharma and Prof J E Craig

Studying two new genes implicated in glaucoma blindness

Figure 1 AFAP1and actin do not co-localize in trabecular meshwork cells from a normal (NTM-5) or glaucomatous (GTM-3) eye. AFAP1 (green) was labelled using a specific antibody and actin (red) stained with phalloidin. Nucleus stained blue. Separate green and red signal indicates no co-localization of proteins.

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are, CAV1/CAV2, GAS7 and SIX1/SIX6. These loci are also associated with POAG in the Australian cohort. Additionally, the TMCO1 and ABCA1 loci are associated with IOP, the major risk factor for glaucoma.

Identification of multiple genetic risk factors for POAG has opened up the possibility of advancing the understanding of glaucoma pathogenesis. As a first step in this direction, the aim of this project was molecular characterisation in the human eye of the genes recently associated with POAG in the Australian population, for understanding their functional involvement in the disease.

PROGRESS TO DATEABCA1 (ATP-binding cassette, subfamily A, member1) encodes an integral membrane receptor that is involved in export of excess phospholipid and cholesterol from the cell. It also has role in suppressing inflammation. It is expressed in retinal ganglion cells in monkey eye. However, its expression in the human eye was not known. We determined expression of ABCA1 transcript by RT-PCR (reverse transcription-polymerase expression) and distribution of the encoded protein by immunolabelling in human eye tissues. This gene encodes multiple mRNA transcripts. We found expression of its main transcript in all the analysed tissues, that is, iris, ciliary body, retina, optic nerve head and optic nerve. Expression was also found in cultured trabecular meshwork cells from a normal and a glaucomatous eye. In addition, we found that ciliary body, retina, optic nerve head and the trabecular meshwork cells expressed an alternative transcript resulting from skipping of exon 4 of the gene. These results suggest that

ABCA1 is ubiquitously expressed in the human eye and regulated by a complex mechanism. Consistent with expression of the transcript, the protein was present in the trabecular meshwork, retina and optic nerve head. All the layers of the retina including the RGCs were found to express the protein. In the optic nerve head, the protein distributed in the nerve fibre bundles and in astrocytes in the glial columns. Similar tissue distribution of the protein was observed in a glaucomatous eye. In mouse brain, ABCA1 has been reported to regulate neuroinflammation and neurodegeneration. Whether it plays a similar role in the retina in glaucoma warrants an investigation.

AFAP1 (actin filament associated protein 1) gene encodes a protein that binds to actin filaments and is involved in their cross-linking. Actin cytoskeleton modulation is involved in the regulation of aqueous outflow and IOP; both these are important in glaucoma pathogenesis. AFAP1 encodes two transcripts, one ubiquitously expressed and the other specific to neural cells. Similar to ABCA1, we determined expression of its transcripts and the encoded protein in human eye. We found expression of the ubiquitous transcript in all the eye tissues including iris, ciliary body, lens, retina, optic nerve head and optic nerve, and in cultured trabecular meshwork cells. The retina in addition expresses the neural cell specific transcript. The AFAP1 protein was found to be present in the trabecular meshwork, retina and optic nerve. In the retina, it distributed in the photoreceptor inner segment, some cells in the inner nuclear layer, RGCs and retinal blood vessel wall. In the optic nerve the protein was present in the astrocytes. Similar distribution of AFAP1 was observed in

a glaucomatous eye. Further, because of the pivotal role of actin cytoskeleton in the trabecular meshwork, we determined co-localization of AFAP1 with actin filaments in cultured trabecular meshwork cells derived from a normal and a glaucomatous eye. Our work shows that AFAP1 does not co-localize with actin filaments in these cells (Figure 1). Whether it co-localize with the actin cytoskeleton in the trabecular meshwork tissue and/or in the retina, remains to be investigated.

Furthermore, we analysed expression of the GMDS (GDP-mannose 4,6-dehydratase) gene transcript in human eye tissues. We found that all eye tissues express one of the transcripts encoded by GMDS. Distribution of the encoded protein in the eye is yet to be determined. GMDS is involved in synthesis of a sugar, fucose, required for glycosylation of cell surface proteins. Fucose is required for various biological as well as pathological processes, which may explain its association with glaucoma.

These data provide an insight into expression and distribution of ABCA1 and AFAP1 and expression of GMDS, the genes associated with glaucoma susceptibility, in the eye. This work has laid the foundation for investigating the mechanisms of association of these genes with glaucoma.

Research training achieved: A Doctor of Medicine student pursued a part of this research project and successfully completed his research placement.

Publication arising from this work: Gharahkhani et al, 2014. Common variants near ABCA1, AFAP1 and GMDS confer risk of primary open-angle glaucoma. Nature Genetics 46(10):1120-25.

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The Eye Genetics Research Group is using next-generation sequencing (NGS) to identify novel disease genes and variants causing blinding eye diseases in humans. Model systems are used to determine the underlying pathophysiological mechanisms and for preclinical work to develop new treatment strategies for the vision impairment.

Anterior segment disorders complicated by glaucoma, cause debilitating and progressive vision loss. Disorders of the anterior segment of the eye include primary congenital glaucoma (PCG), abnormalities of iris development, congenital cataracts and corneal abnormalities. Molecular diagnosis and management in anterior segment diseases of the eye is extremely challenging, due to the clinical and genetic heterogeneity in these conditions. Many underlying genetic causes are unknown. Next-generation sequencing (NGS) strategies in novel disease-gene identification benefit from a cost-effective method for biological validation. The zebrafish provides an excellent system for functional studies and examination of development, differentiation and maintenance of the eye due to its rapid and external development. After just five days of zebrafish development, the eye is clearly formed with distinct formation of the lens, anterior segment and lamination across the retina. The genome of the zebrafish is almost completely sequenced and the majority of genes and pathways are conserved with humans. We have applied NGS strategies and use of the zebrafish for functional assessment of candidate disease genes to accelerate progress in understanding of genetic causes of anterior segment abnormality and their functional consequences.

In this project, we undertook morpholino (MO) knockdown experiments in the zebrafish of novel candidate disease genes in anterior segment eye disease, as functional validation of the pathogenicity of the candidate disease genes. Our zebrafish system has proven integral in rapid functional validation of candidate disease genes affecting the anterior segment of the eye and a manuscript has recently been provisionally accepted, reporting this work (1). A second manuscript is in preparation from this work. We also performed NGS studies in several families with abnormalities of the anterior segment of the eye. We used our bioinformatics pipeline to determine predicted pathogenic variants in known and novel candidate disease genes. Candidates in the WNT signalling pathway have been identified and our functional assays have provided validation of these candidates. Due to the success of this project, we have developed a clinical exome sequencing pipeline in collaboration with Associate Professor Bruce Bennetts, Molecular Geneticist, at The Children’s Hospital at Westmead, Sydney. This will allow rapid genetic testing in anterior segment disorders of the eye as a comprehensive clinical service test in Australia. This provides genetic information for patients and families with these conditions, and paves the way for improved treatments and participation in clinical trials.

Publication from this work has recently been provisionally accepted by Human Molecular Genetic (1), and another publication is in preparation for journal submission. Findings from this work have been presented at the Human Genetics Society of Australasia Annual Scientific Meeting, Adelaide, August, 2014 (2), the COMBIO Meeting, Canberra, October

ORIA GRANTAssociate Professor Robyn Jamieson, Associate Professor John Grigg

Applying rapid genetic and model systems in eye disease

Progress Reports

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2014 (3), the American Society of Human Genetics Annual Meeting, San Diego, USA, October 2014 (4 & 5), the Australian and New Zealand Society for Cell and Developmental Biology, NSW group meeting, Sydney, March 2015 (6), and the Association for Research in Vision and Ophthalmology, Annual Meeting, Denver, Colorado, US, May 2015 (7). Support from the ORIA is gratefully acknowledged in publications and presentations of this work.

Publications & Presentations:

1) Greenlees R, Mihelec M, Yousoof S, Speidel D, Wu SK, Rinkwitz S, Prokudin I, Perveen R, Cheng A, Ma A, Nash B, Gillespie R, Loebel DA, Clayton-Smith J, Lloyd IC, Grigg JR, Tam PP, Yap AS, Becker TS, Black GC, Semina E, Jamieson RV. Mutations in SIPA1L3 cause eye defects through disruption of cell polarity and cytoskeleton organization. Human Molecular Genetics, Accepted subject to revision, 27 May 2015. (ORIA support recognised in acknowledgements)

2) Prokudin I, Chen Y, Storen R, Grigg J, Jamieson RV. Mutations in the WNT signalling pathway and eye development. Human Genetics Society of Australasia Annual Scientific Meeting, Adelaide, 4-6 August 2014. (Platform presentation - ORIA support recognised in

acknowledgement slide)

3) Jamieson RV, Greenlees R, Prokudin I, Yousoof S, Loebel D, Becker T, Tam P, Semina E, Davila S. Genomic strategies and functional insights to novel eye disease genes. COMBIO, Canberra. 29 September – 2 October 2014. (Platform presentation - ORIA support recognised in acknowledgement slide)

4) Prokudin I, Kumar V, Davila S, Jamieson RV. WNT signalling and eye development disease genes. American Society of Human Genetics Annual Meeting, San Diego, USA, 18-22 Oct 2014. (Poster presentation - ORIA support recognised in acknowledgements)

5) Greenlees R, Yousoof S, Semina E, Jamieson RV. Rapid Approaches in Functional Validation of Candidate Disease Genes Identified from Structural Rearrangements and Next-Generation Sequencing. American Society of Human Genetics Annual Meeting, San Diego, USA, 18-22 Oct 2014. (Poster presentation - ORIA support recognised in acknowledgements)

6) Greenlees R, Ma A, Grigg JR, Jamieson RV. Functional genomics and identification of a Rap1 signalling role in the lens. Australian and New Zealand Society for Cell and Developmental Biology, NSW group meeting, 16 March 2015. (Platform presentation - ORIA support recognised in acknowledgement slide)

7) Jamieson RV, Ma A, Grigg JR, Greenlees R. Genomic approaches in disease gene identification in congenital cataract: new diagnoses and new insights to Rap1 signalling in the lens. Association for Research in Vision and Ophthalmology, Annual Meeting. Denver, Colorado, US, 3-7 May 2015. (Platform presentation - ORIA support recognised in acknowledgement slide)

Publications enclosed acknowledging ORIA contributions:

1. Prokudin I, Li D, He S, Guo Y, Goodwin L, Wilson M, Rose L, Tian L, Shen Y, Liang J, Keating B, Xu X, Jamieson RV, Hakonarson H. Value of whole exome sequencing for syndromic retinal dystrophy diagnosis in young patients. Clinical and Experimental Ophthalmology. 2015; 43: 132-138. PMID: 25060287

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Our research program is dedicated to improving eye health and restoring vision in patients with a spectrum of diseases that affect the cornea. This ORIA award was directed towards identifying corneal wound-healing factor(s) and devising an improved delivery method for such factors. More specifically the study was designed to target a medical therapy for patients with persistent corneal epithelial defects. To this end we successfully identified an extracellular matrix factor known as vitronectin (VN) that enhanced wound closure in cultured human corneal epithelial cells. This activity was independent of cell proliferation; instead we demonstrated that VN promoted cell migration to cover the defect. Finally we showed that a chemically-induced corneal epithelial wound rapidly re-epithelialised after applying a VN-coated therapeutic contact lens over the native cornea

which acted as a bandage, but more importantly as a ‘slow-release’ device for the VN in an organ culture model. We are now primed to take advantage of these exciting ‘proof-of-concept’ in vitro and ex vivo findings and with future funding we anticipate commencing trials in animal models before developing a clinical therapy for patients.

We are pleased that a publication has arisen out of the ORIA co-funded research.

Full details on these results can be accessed through the reference below.

Chow S, Di Girolamo N. Vitronectin: A migration and wound-healing factor for human corneal epithelial cells. Investigative Ophthalmology & Visual Science 2014; 55:6590-6600.

ORIA GRANTA/Prof Nick Di Girolamo

Factors that heal corneal wounds

Progress Reports

A/Prof Nick Di Girolamo

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

This project has been highly successful in elucidating the genetic causes of primary congenital glaucoma (PCG) in Australia.

Mutations in the TEK gene were originally identified in 3 of 35 PCG patients from the USA by our collaborators (unpublished). We have subsequently screened this gene in 53 Australian patients and identified four mutations including two substitution and two loss of function mutations. When compared to controls, we see a significant enrichment for loss of function mutations in PCG patients (2 mutations in 61,486 publicly available controls from the ExAC database [odds ratio of 1205.57, two-tailed Fisher’s exact p-value of 4.359 x 10-6]). This highly significant enrichment suggests that although rare, this gene is highly likely to be involved in PCG. More importantly, our findings mirror those seen in the USA cohort where a similar mutation spectrum and inheritance pattern were observed. Of note, we see that patients tend to only carry a single TEK mutation as opposed to two mutations seen with other PCG genes such as CYP1B1. Further, analysis of a mouse model shows that mice null or heterozygous for TEK in the eye develop elevated

intraocular pressure and a phenotype reminiscent of human PCG. This work has been submitted for publication.

Stage 2 of this project aimed to identify further novel genes for PCG using exome sequencing. Analysis of the data is still ongoing in conjunction with that of our international collaborators, however, several important findings have been made so far.

The aim was to identify novel genes in families. We sequenced 4 families with multiple PCG cases and analysis is ongoing. A candidate gene has been identified in one of the families and we are currently evaluating its potential role in PCG. We also sequenced a number of unrelated PCG cases. Four show evidence of consanguinity (related parents) which is further assisting the analysis.

Several mutations have been identified in candidate genes. Two patients carried deletion mutations in FOXC1, 1 has a mutation in MYOC, 1 has homozygous mutations in TMEM98 and 1 has a mutation in SLC4A11. FOXC1 mutations are typically associated with anterior segment dysgenesis while MYOC mutations are seen in primary open angle glaucoma including those with juvenile onset. Both genes have now been implicated in PCG by us and others. TMEM98 was identified by our group (funded by ORIA) as a cause of nanophthalmos, a condition which leads to angle closure glaucoma. SLC4A11 is associated with congenital hereditary endothelial dystrophy, as condition also associated with PCG.

One patient in our cohort was misdiagnosed with PCG. This error was noted by the clinical care team at the same time as we received sequencing results which confirmed

the updated clinical diagnosis of X-linked Megalocornea with a mutation in CHRDL1. Although clinical examination identified the error, this highlights how genetic screening can be used to verify or alter clinical diagnoses.

The findings from this project indicate that PCG is not necessarily always a recessive disease and that dominant mutations with reduced penetrance may account for more cases than previously recognised. Further, it seems likely that PCG is a cluster of diseases with overlapping molecular causes. Clinical diagnoses are often unable to separate patients with similar molecular causes of disease such as TMEM98, FOXC1 and MYOC identified in this study. This has implications for diagnosis and genetic counselling in the modern genomics era and supports the idea of exome sequencing as a front line diagnostic tool for congenital diseases.

ORIA/W A QUINLIVAN & GLAUCOMA AUSTRALIA INC GRANTDr Kathryn Burdon and Ms Emmanuelle Souzeau

Identifying genetic causes of Primary Congenital Glaucoma in Australia

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THE INFLUENCE OF RBP3 DYSREGULATION ON A2E PRODUCTION It is known that RBP3 facilitates retinoid transport between photoreceptors and the RPE cells through the retinoid cycle. Accumulation of A2E in the retina is one of the hallmarks of retinoid cycle disruption and is also closely related to retinal degeneration. A2E is an autofluorescent compound, which can accumulate in aged eyes as well as in conditions affecting the visual cycle and RPE function. We examined changes in fundus autofluorescence after selective Müller cell ablation using confocal scanning laser ophthalmoscopy. We detected focal hyperfluorescent dots in mice with induced Müller cell disruption but not in control mice. The autofluorescent signals may derive from A2E accumulation but may also have other causes. We performed HPLC to measure A2E levels in mice after Müller cell disruption compared with controls. We found that down-regulation of RBP3 was accompanied by significant elevation of A2E levels in mice after selective Müller cell disruption. A2E accumulation was observed in transgenic mice 5 weeks after Müller cell disruption. By 25 weeks after Müller cell disruption, the level of A2E in transgenic mice was still significantly increased by ~5.4 fold of that in control mice. These findings suggested that RBP3 dysregulation caused by selective Müller cell disruption leads to dysfunction of the retinoid cycle, which is consistent with a previous report [1].

DYSREGULATION OF RBP3 IN PHOTORECEPTORS TREATED WITH CONDITIONED MEDIUM COLLECTED FROM STRESSED MÜLLER CELLS It is known that RBP3 is synthesized in photoreceptors, secreted into the IPM and rapidly turned over by endocytosis of photoreceptors and the RPE cells [2]. Although there is no direct contact between RBP3 and Müller cells, our further in vitro experiments indicated a regulatory effect of Müller cells on RBP3 expression in photoreceptors. RBP3 expression in mouse photoreceptor-derived 661W cells was significantly decreased following the treatment with conditioned medium collected from LPS-stressed mouse primary Müller cells. Similar results were also obtained in human retinoblastoma Y79 cells treated with conditioned medium collected from LPS-stressed human MIO-M1 Müller cells.

TNFα IS ONE OF THE FACTORS RELEASED FROM STRESSED MÜLLER CELLS THAT DOWN-REGULATES RBP3 EXPRESSION IN PHOTORECEPTORS.Garcia-Ramírez et. al. reported a dose-dependent down-regulation of RBP3 in TNFα treated Y79 human retinoblastoma cells in vitro [3]. We recently reported that TNFα was overexpressed in stressed surviving Müller cells after induced Müller cell ablation, which occurs in patches in the model we have developed [4]. In order to examine whether TNFα can directly cause down-regulation of RBP3 in photoreceptors, control mice were intravitreously injected with recombinant mouse TNFα, with one eye receiving 0.2µg TNFα while the contralateral eye received the same concentration of BSA as a control in each mouse. Western blot analysis

ORIA/RANZCO EYE FOUNDATION GRANTProf Mark Gillies & Dr Ling Zhu

Novel treatment of retinal diseases targeting Retinol Binding Protein 3 dysregulation.

Progress Reports

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

using retinal samples collected 1 week after injection showed that RBP3 in TNFα-treated eyes was decreased to 41.2% of that in control eyes (P<0.05,).

SIMVASTATIN TREATMENT INHIBITED JAK/STAT ACTIVATION, RESTORED RBP3 EXPRESSION AND ATTENUATED PHOTORECEPTOR DEGENERATION AFTER INDUCED MÜLLER CELL DISRUPTION.Induced disruption of Müller cells is the primary event driving the changes observed in our transgenic model. Patches of Müller cells (~40%) die while surviving Müller cells (~60%) become active, or gliotic [5]. Since RBP3 is specifically synthesized by photoreceptors, we believe that there are signals derived from gliotic Müller cells that regulate IRBP expression in photoreceptors. We have showed that TNFα is over-expressed by gliotic Müller cells and regulates IRBP expression in photoreceptors [6]. Precisely how TNFα affects IRBP expression by photoreceptors is unknown. TNFα activates the Jak/STAT signalling pathway, the activation of which might down regulate the expression of CRX proteins, major transcription factors that activate IRBP expression in photoreceptors. Consistent with this, we have also found the Jak/STAT signalling was activated, particularly in photoreceptors, following induced Müller cell disruption [7]. Therefore, we examined the effect of a specific Jak/STAT signalling inhibitor on the regulation of IRBP expression in vivo. We pre-treated the mice with simvastatin, a known Jak/STAT signalling inhibitor, in drinking water 2 weeks before the induction of Müller cell disruption. We found that simvastatin pretreatment in our

transgenic mice significantly inhibited Jak/STAT signalling activation and restored IRBP expression at the protein level (Fig 1A-C). Further staining of fluorescence-conjugated peanut-agglutinin (PNA) labelled cone photoreceptor outer segments (green) showed that this treatment prevented the photoreceptor degeneration that follows induced Müller cell disruption (Fig 1D-G).

Reference1. Jin, M., et al., The role of interphotoreceptor

retinoid-binding protein on the translocation of visual retinoids and function of cone photoreceptors. J Neurosci, 2009. 29(5): p. 1486-95.

2. Cunningham, L.L. and F. Gonzalez-Fernandez, Internalization of interphotoreceptor retinoid-binding protein

by the Xenopus retinal pigment epithelium. J Comp Neurol, 2003. 466(3): p. 331-42.3. Garcia-Ramirez, M., et al.,

Interphotoreceptor retinoid-binding protein (IRBP) is downregulated at early stages of diabetic retinopathy. Diabetologia, 2009. 52(12): p. 2633-41.

4. Shen, W.Y., et al., Effect of glucocorticoids on neuronal and vascular pathology in a transgenic model of selective Müller cell ablation. GLIA, 2014.

5. Shen, W., et al., Involvement of NT3 and P75(NTR) in photoreceptor degeneration following selective Muller cell ablation. J Neuroinflammation, 2013. 10: p. 137.

6. Zhu, L., et al., Dysregulation of Interphotoreceptor Retinoid-Binding Protein (IRBP) after induced Muller Cell disruption. J Neurochem, 2015. (ORIA supported)

7. Coorey, N.J., et al., Differential expression of IL-6/gp130 cytokines, JAK-STAT signalling and neuroprotection after Muller cell ablation in a transgenic mouse model. Invest Ophthalmol Vis Sci, 2015.

Figure 1 Simvastatin treatment inhibited Jak/STAT activation, restored RBP3 expression and attenuated photoreceptor degeneration after induced Müller cell disruption. Control with Vehicle treatment (Ctrl+V); Induced photoreceptor degeneration with Vehicle treatment (MCKO+V); Induced photoreceptor degeneration with simvastatin treatment (MCKO+S). (A) Inhibition of the Jak/STAT signalling (phosphoSTAT3) and restored RBP3 expression were observed in the retina of mice with induced photoreceptor degeneration treated with simvastatin. (B-C) Densitometry analysis of the western blot in (A). n=7, *:p<0.01, †p<0.05. (D-F) fluorescence-conjugated peanut-agglutinin (PNA) labelled cone photoreceptor outer segments (green) in (Ctrl+V, D), (MCKO+V, E) and (MCKO+S, F). (G) Quantitative analysis of PNA staining of cone outer segments in (D-F), n=8/group. *:p<0.01

Left to right - Dr Belinda Yau, Dr Sook Chung, Prof Mark Gillies, Miss Michelle Yam, Dr Ling Zhu, Miss Ying Wang and Dr Weiyong Shen

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AIM OF THE PROJECTOur goal was to engineer a biologic that targets vascular endothelial growth factor-B (VEGF-B), and to assess its efficacy at reducing corneal neovascularisation in an animal model.

BACKGROUNDThe normal human cornea is avascular. Corneal neovascularization is a sight-threatening condition that may follow trauma or infectious keratitis, or may accompany ocular surface disease. Whatever the aetiology, there are serious sequelae. Blood vessels and related secondary changes may obscure the visual axis, reducing vision. The presence of corneal vessels erodes immune privilege and is a strong and independent risk factor for the failure of a subsequent corneal graft in the affected eye. There is thus a clinical need for drugs that can reduce corneal neovascularization prior to transplantation. We were interested in the possibility that biologics, especially recombinant antibody-based molecules or fusion proteins that target members of the VEGF family, might be useful in this context.

VEGF-B is part of a family of peptide growth factors that include VEGF-A, VEGF-B, VEGF-C and VEGF-D, and placental growth factor (PlGF). VEGF-A is involved in both normal and pathological vasculogenesis and angiogenesis, VEGF-B is thought to maintain vessel viability and regulate lipid metabolism in the heart, whereas the C and D isoforms are primarily key regulators of lymphangiogenesis. Recently, Petsoglou and his colleagues demonstrated in a randomised controlled trial that subconjunctival injections of bevacizumab (anti-

VEGF-A) were significantly more effective than placebo at preventing development of new corneal vessels in humans, with an intention-to-treat difference of 126% between test and control groups (Petsoglou et al. Br J Ophthalmol 2013;97:28-32). However, although anti-vascular endothelial growth factor-A (VEGF-A) therapy has shown promise for treating newly-formed corneal vessels in humans, it has little effect on established vessels. VEGF-B, in contrast, is a potent survival factor for vascular endothelial cells. We argued that an anti-VEGF-B antibody might regress established vessels, and be a useful adjunct to bevacizumab.

PROGRESS TO DATEAn anti-VEGF-B antibody fragment in the scFv was first generated by assembly polymerase chain reaction from an existing murine hybridoma by our Flinders Ophthalmology PhD student, Yazad Irani, and its binding to VEGF-B was confirmed by ELISA. The antibody fragment was sent to colleagues Pierre Scotney and Andrew Nash at Bio21 and CSL Ltd in Melbourne, where it was codon-optimised for expression in mammalian cells. Functional binding was assessed by surface plasmon resonance and confirmed in a cell based assay. The scFv was shown to be human and rat reactive. Purified protein was returned to Flinders Ophthalmology, where it was formulated both for topical delivery in eye drop form (1 mg/ml scFv, 1.5% hypromellose, 1% capric acid in normal saline), and in a thermoresponsive gel (5 mg/ml scFv in PluronicF127) for subconjunctival injection. A control scFv with an irrelevant specificity was formulated in an identical manner.

ORIA/G J WILLIAMS GRANTProf KA Williams and Dr S Klebe

Testing new drugs to reduce corneal transplant failure

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The anti-angiogenic activity of the scFv was then assayed in a rat model of corneal neovascularization. Neovascularization was induced in adult outbred Sprague-Dawley male and female rats by superficial cautery of the central cornea, using a silver nitrate applicator. New vessels sprouting from the limbal arcades were apparent at 3-4 days post cautery, and had reached the site of cautery in the central cornea at between 7-10 days. The effect of the scFv on developing vessels was assessed by commencing therapy on the day after cautery. To test the effect on established vessels, treatment was delayed for 14 days after cautery, at which point the vessels had formed an anastomosed network. Treatment involved the application of a 5 µl eye drop containing 5 µg scFv, 5 times each day for 14 consecutive days, supplemented in some cohorts with subconjunctival injections of the scFv on two occasions (50 µg per injection, day 1 and day 8 of treatment). At the conclusion of treatment, corneal vessels were perfused with haematoxylin and the corneal was flat mounted. The percentage of the cornea covered by vessels was determined with NIH ImageJ.

Corneal neovascular area was not significantly different between untreated, control scFv treated or anti-VEGF-B scFv treated animals (growing vessels p=0.46, established vessels p=0.86), after topical therapy alone. However, topical therapy supplemented with subconjunctival injections significantly reduced the area of established corneal vessels (12.9%, SD±3.6, n=21) when compared with untreated (20.5%, SD±7, n=19) or control scFv treated (22.5%, SD±3.2, n=9) rats (p<0.001).

SUMMARYWe engineered a rat and human-reactive anti-VEGF-B antibody fragment (scFv format) and tested its activity on growing and established corneal blood vessels in the rat. Topical scFv supplemented with subconjunctival injection of scFv caused significant regression of established corneal vessels. We suggest that the anti-VEGF-B scFv may be used in conjunction with an anti-VEGF-A agent for the treatment of human corneal neovascularization, prior to corneal transplantation.

Presentations on this work, acknowledging the ORIA

Yazad Irani, Pierre Scotney, Andrew Nash, Sonja Klebe, and Keryn A Williams. 2015. Anti-VEGF-B therapy in a rat model of corneal neovascularization. 32nd Australia and New Zealand Cornea Society Meeting, Perth, March 2015 (oral presentation).

Yazad Irani, Pierre Scotney, Andrew Nash, Sonja Klebe, and Keryn A Williams. 2015. Anti-VEGF-B therapy in a rat model of corneal neovascularization. ARVO Annual Meeting, Denver, Colorado, May 2015 (oral presentation).

Publications arising from ORIA support for projects in the recent past

Colella AD, Chegenii N, Tea MN, Gibbins IL, Williams KA, Chataway TK. Comparison of stain-free gels with traditional immunoblot loading methodology. Anal Biochem 2012;430:108-110.

Appleby SL, Jessup CF, Mortimer LA, Kirk K, Brereton HM, Coster DJ, Tan S, Anson DS, Williams KA. Expression of an anti-CD4 single chain antibody fragment from the donor cornea can prolong corneal allograft survival in inbred rats. Br J Ophthalmol 2013;97:101-105.

Tea MN, Brereton HM, Michael MZ, Williams KA. Stability of small RNA reference gene expression in the rat retina during exposure to cyclic hyperoxia. Mol Vis. 2013;19:501-8.

Appleby SL, Irani Y, Mortimer LA, Brereton HM, Klebe S, Keane MC, Cowan PJ, Williams KA. Co-expression of a scFv antibody fragment and a reporter protein using lentiviral shuttle plasmid containing a self-processing furin-2A sequence. J Immunol Methods 2013; 397:61-65.

Mills RAD, Klebe S, Coster DJ, Williams KA. Comparative outcomes of penetrating and component endothelial cell corneal allografts in outbred sheep. Cell Transplant 2014;23:133-138.

Figure 1 Effect of topical anti-VEGF-B scFv supplemented with subconjunctival injections of scFv on established corneal vessels. Corneal neovascularization was induced in inbred Sprague-Dawley rats by silver nitrate cautery. Treatment commenced 14 days after cautery. At euthanasia, the corneal vessels were perfused with haematoxylin and the percentage of the cornea covered by vessels was quantified. The percentage of cornea covered by vessels was significantly smaller in the anti-VEGF-B scFv treated group. *p<0.001 #p=0.66. The bars represent the mean corneal vessel area in biological replicates, and the error bars represent the standard deviation. n = number of rats in each group.

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BACKGROUNDKeratoconus (KC) is a bilateral, asymmetric and progressive corneal disease, which causes corneal thinning and development of a conical cornea associated with significant visual loss[1]. The hypothesis of this project is that SFRP1 affects proliferation of KC and control central corneal epithelial cells, the response to oxidative stress and possibly cell migration. In the absence of a suitable animal model, the aim of the proposed research was to 1) to set up a cell culture model to examine the human corneal epithelial cell morphology in both control and KC corneas, and 2) to investigate the effect of SFRP1 in both normal and KC human cornea epithelium.

RESEARCH PROGRESS OUTCOMEAim 1: Establishing a tissue culture model

We started to collect human corneal tissues from both control and KC participants from 28th January 2014 (HREC Approval #2013_1041). Up to Dec 2014, we had obtained 15 corneal samples (6 KC buttons and 9 corneas from Lions NSW Eye Bank).

The first samples from each group (three from control groups and two from KC group), were used to optimise an experimental protocol to successfully establish primary corneal explants. We found that primary limbal epithelial cells can be grown for several generations (passaged more than three times, >P3). Only the first three generations, P0-P2, were used for experiments. The central corneal regions of both control and KC corneas (central 8mm region of the cornea) showed less growth potential. Around half of the central region of

control corneas and one third of the KC button could generate cell growth for a few generations (up to P2), the rest of cells did not survive beyond after P0. This is plausible because the central region of cornea is reported to not contain stem cells and have low capability to regenerate compared to the limbal region.

Due to the limited cells available, we modified our experimental design and introduced a new technique, Incucyte Zoom (a live cell imaging system, USyd Bosch Institute) to monitor the cell outgrowth from corneal explants of first generation (P0) cells. We find that the cells from the central region of control corneas grow faster (covering a larger area) compared to KC button (Figure 1 A& B). After removing the epithelial cells from the explants and with the same number of primary epithelial cells seeded to a new culture vessel, control central epithelial cells formed a compact layer compared to the obvious more sparse surviving KC epithelial cells that were loosely adherent (Figure 1C&D). These findings showed that KC corneal epithelial cells having reduced growth potential compared to the control epithelial cells even when cultured independently (without the association with Bowmen’s layer and keratocytes.

ORIA NEW INVESTIGATOR GRANTDr Jingjing You (New Investigator), Prof Gerard Sutton and Dr Lucy DawesSupervisor: Michele C Madigan

Secreted frizzled related protein 1 (SFRP1) and Keratoconus

Figure 1 Cell outgrowth from central region corneal explants observed with Incucyte Zoom. A: Control corneal epithelial cells outgrowth after 1week, B: KC button epithelial cells outgrowth after 1 week, C: primary control corneal epithelial cells harvested from explant

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ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

Aim 2: Effect of SFRP1 in both normal and KC cornea epithelium

The effect of SFRP1 on normal corneal epithelial cells was tested using limbal epithelial cells and control central epithelial cells. These experiments showed that SFRP1 had a dose-dependent pro-proliferation effect on limbal epithelial cells (Figure 2). When compared to other growth factors [for example, hepatocyte growth factor (HGF) and human epidermal growth factor treatment (hEGF)], SFRP1 showed a similar effect on cell proliferation. Primary control central epithelial cells reached 90% confluence at 60 hours (hr) with SFRP1 (10ng/mL), compared 64 hrs with no treatment, and 74 hrs with HGF (10ng/mL) (Figure 3). These findings confirmed our hypothesis that SFRP1 can increase corneal epithelial cell growth. To date, we have not been able to culture sufficient KC epithelial cells to investigate the effects of SFRP1 on KC epithelium. These studies are ongoing.

Aim 3: Examine the HGF and c-Met distribution in KC button

Our findings indicated that corneal epithelial cells do not grow at the same rate as control epithelial cells. As such, we decided to also examine the HGF and its receptor c-Met in KC button to further analyse potential growth pathways that may be affected in KC epithelium. We found that the protein expression of HGF and c-Met localised to a similar location as SFRP1, being overexpressed in the basal epithelial layer in KC compared to control corneas (Figure 4).

In conclusion, adding SFRP1 to our cultured corneal epithelial cells increased cell growth, which could give a new direction for potential approaches to improving epithelial integrity in KC.

Publications generated from the grant:

You JJ, Wen L., Roufas A., Hodge C., Sutton G., Madigan M (2014) Expression of HGF/c-Met in control and keratoconus corneas. Acta Ophthalmologica DOI: 10.1111/j.1755-3768.2014.S023.x (Conference Proceedings)

You J., Wen L., Roufas A., Hodge C., Sutton G., Madigan M. (2015) Expression of HGF and c-Met Proteins in Human Keratoconus Corneas. Eye (currently under review).

You J., Wen L., Hodge C., Sutton G., Madigan M. In vitro effects of SFRP1 on normal and keratoconus corneal epithelium. (In preparation).

Findings from this grant were presented as follows:

2015 Invited Vaegan Seminar, School of Optometry and Vision Science, UNSW, Australia

2014 European Association for Vision and Eye Research, Nice, France

Figure 2 SFRP1 induced corneal epithelial cell proliferation; 5 µg/mL was most effective.

Figure 4 c-Met and HGF are over-expressed in the basal epithelial cells in KC, compared to control epitehlium. Epi: Epithelium, Stro: stroma.IgG immunoglobulin control.

Figure 3 SFRP1 had similar and slightly higher pro-proliferation effect on central primary corneal epithelial cells.

Dr Jingjing You

27

BACKGROUND AND AIMSA large US-based study of over 27,000 runners has shown that regular exercise reduces the risk of glaucoma. Self-reported incident glaucoma rates were halved in those who ran the furthest (>6km per day) or the fastest (>4m/s for 10km race time). This led us to investigate the impact of exercise on optic nerve injury using the aged mouse as a model system. Specifically, we tested whether exercise could protect retinal ganglion cells (RGCs) after an intraocular pressure (IOP) injury. We found that regular exercise significantly improved RGC functional and ameliorated cellular signs of stress in retinal glia, macrophages and neurons after injury. The mechanisms that underlie this protection, however, remain unknown and were the focus of our ORIA-funded project.

Accumulating evidence suggests that the pleiotropic effect of exercise on different organ systems is mediated by myokines, factors that are produced and secreted by exercising skeletal muscle. Skeletal muscle can therefore be considered an endocrine organ, whose secretome has both autocrine and paracrine functions. Members of the interleukin family, IGF, Irisin, BDNF and CNTF are examples of such myokines. The signalling pathways that regulate myokine production and secretion are yet to be fully elucidated but the metabolic sensor, AMP-activated protein kinase (AMPK) appears to play a central role. Exercise is a powerful physiological activator of AMPK in skeletal muscle, which is activated in an intensity-dependent manner and exerts numerous effects on skeletal muscle function, metabolism and signalling. Recent reports show a critical role for AMPK in stimulating the expression,

release and action of myokines from contracting skeletal muscle cells.

This led us to hypothesise that exercise-induced retinal protection may be conferred by AMPK-dependent production of myokines that are secreted by exercising skeletal muscle and act remotely on the retina. The aim of this study therefore is to determine whether AMPK activation in skeletal muscle is a key requirement for exercise-mediated protection of RGCs against IOP injury.

RESULTS TO-DATETo address our project aim, we used a unique mouse strain that has muscle-specific knock-out of both AMPK β1 and β2 subunits (β1β2M-KO). These mice are characterised by dramatic reductions in metabolic responses of muscle to exercise such as contraction-stimulated glucose uptake. If AMPK activation in skeletal muscle mediates retinal protection by exercise, we expected to see no protection in exercised β1β2M-KO mice, whilst protection will be maintained in wild-type (WT) littermates.

In the past year, AMPK β1β2M-KO and WT mice were successfully sourced from the Steinberg laboratory and aged to 12 months (experimental age). Firstly, we assessed AMPK protein expression levels in skeletal muscle and retina of β1β2M-KO and WT animals. Expression of AMPK subunits β2, α1, α2 was absent from the tibialis anterior muscle in β1β2M-KO mice, confirming muscle-specific AMPK deficiency. In contrast, retinal expression of β2, α1, α2 subunits was normal in KO mice, indicating intact retinal AMPK activity.

ORIA/RANZCO EYE FOUNDATION GRANTProf Jonathan Crowston, Dr Vicki Chrysostomou, A/Prof Greg Steinberg

Does AMPK activation in skeletal muscle boost endogenous neuroprotection and protect the optic nerve from injury?

Progress Reports

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

We next compared retinal function between β1β2M-KO and WT mice up to 12 months of age using the electroretinogram (ERG). Figure 1 shows that ERG signals derived from outer (a-wave), mid (b-wave) and inner (pSTR) retinal neurons were similar between genotypes. These results indicate that AMPK deficiency in muscle does not impair normal retinal function in mice at rest. This the first analysis of the retina performed in this strain of mouse.

We next tested if AMPK activation in skeletal muscle is critical for exercise-mediated retinal protection. WT and KO mice were exercised by swimming for 60 min/day, 5 days/week. Although it has been reported that β1β2M-KO mice have reduced tolerance for treadmill running, we did not have any difficulties in exercising these mice by the daily swimming task. Swimming behaviour and ability of β1β2M-KO mice was indistinguishable from WT littermates. After 6 weeks of exercise, animals were subject to unilateral IOP elevation (50 mmHg for 30 min). Using the ERG, RGC function was assessed before (baseline) and 7 days after IOP injury.

In response to pressure-induced injury, RGC function was significantly reduced in non-exercised WT mice, as shown by reductions in pSTR amplitudes to approximately 50% of baseline values (Figure 2). In contrast, exercised WT mice were significantly protected against RGC dysfunction after injury; pSTR amplitudes were preserved at 80%. Interestingly,

exercise conferred identical levels of functional protection in β1β2M-KO mice. These data suggest that that activation of AMPK in skeletal muscle is not required for retinal protection by exercise.

Data obtained from this study were used to support a NHMRC Project Grant application in 2015.

Presentations

Chrysostomou V, Kezic JM, Trounce IA & Crowston JG. How does exercise protect retinal ganglion cells from injury? Optic Nerve Conference, Dec 2014; Obergurgl, Austria

Chrysostomou V, Trounce IA, Fahy E & Crowston, JG. Exercise reverses functional and structural loss after optic nerve injury. Biennial Meeting of the International Society for Eye Research, July 2014; San Francisco, California, USA

Publications

Chrysostomou V, Kezic JM, Trounce IA, Crowston JG (2014). Forced exercise protects the aged optic nerve against intraocular pressure injury. Neurobiol Aging. 35(7):1722-5.

Figure 1 Retinal function, measured by amplitudes of the electroretinogram (ERG), was normal in mice with skeletal muscle AMPK deficiency.

Figure 2 Exercise significantly improved functional recovery of the optic nerve after pressure-induced injury in AMPK β1β2M-KO mice.

Prof Jonathan Crowston – photo courtesy CERA

29

ORIA NEW INVESTIGATOR/REF GEORGE AND CARMEL LEADER GRANTDr Angus Turner & Dr Roderick O’Day

Collaborative care between ophthalmologists and optometrists using telehealth for better eye health care in remote Australia

An audit of 100 cases of teleophthalmology was conducted in 2012 at Lions Outback Vision, LEI, Perth, Western Australia. Medicare Australia provides a telehealth item number for General Practitioners to refer to specialists in 2011. This included incentives and educational promotion to encourage uptake. The study demonstrated limited use by GPs and that the primary care referrers were most commonly optometrists. This was despite a lack of incentivisation and educational promotion to optometrists.

The ORIA grant allowed for the design of a study that included incentives for optometrists and educational resources and booking systems to improve logistics of patient teleophthalmology consultations.

This study was granted exemption from formal ethics approval by the Human Research and Ethics Committee at the University of Western Australia on the basis that it was a clinical audit.

A website was developed with ability to create appointments. Optometrists were educated in techniques for telehealth and CPD /upskilling provided regularly.

Results of the 2014 audit using optometrists as the primary eye health referrers were compared to those in the 2012 audit. A summary of the main findings is included in the table below. An increase in referrals was noted with more non-urgent referrals and clinical caseload more representative of routine outreach clinics. Patients were booked directly for procedures given the quality of the referral information.

This ORIA research has been submitted for publication in a peer-reviewed journal and is pending a decision.

The audit research was used to support a business case (by McKinsey and Co. pro bono) and medico-legal assessment of telehealth (Clayton & Utz pro bono matter) and support from professional bodies (RANZCO and OA) was provided to approach Medicare Australia and suggest modification to the Telehealth item numbers. A positive response was received from the Commonwealth in the May 15 budget with suggested changes being implemented 1st September, 2015.

This is an excellent example of research translation being supported by ORIA.

Progress Reports

Dr Angus Turner

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

Paper abstract:

Roderick O’Day1 MBBS Constance Smith2 BMedSci Josephine Muir3 PhD Angus Turner2,3 MBBS MSc FRANZCO

1 Royal Victorian Eye and Ear Hospital, Melbourne, Australia

2 University of Western Australia, Perth, Australia

3 Lions Eye Institute, Perth, Australia

Corresponding Author: A/Prof Angus Turner, Lions Eye Institute 2 Verdun Street, Nedlands, Western Australia, 6009.Email: angus.turner@gmail.comPh: 08 9381 0777 Fax: 08 9381 0700

Running Title: Tele-ophthalmology in remote Australia

The authors have no competing / conflicts of interest to declare.This study was funded by a combined Ophthalmic Research Institute of Australia New Investigator and RANZCO Eye Foundation George and Carmel Leader Grant.

Abstract:

Background: Telehealth using videoconferencing can provide timely specialist ophthalmic care to rural and remote communities. A multifaceted intervention was designed to increase the use of Lions Outback Vision’s telehealth service (‘the telehealth service’) by optometrists.

Design: Prospective interrupted time series analysis.

Participants: Patients from rural and remote Western Australia referred by optometrists to the telehealth service.

Methods: Two five-month prospective audits of the telehealth service were compared. The first was prior to the implementation of the intervention, which included logistical support, remuneration to optometrists, a more user-friendly referral pathway and awareness raising.

Main Outcome Measures: Number of consultations conducted during the two audit periods and qualitative changes to the telehealth service.

Results: After implementation of the intervention, use of the telehealth service increased 3.5 fold. A greater percentage of referrals were non-urgent (145 [69%] vs 16 [32%], p < 0.001) and less consultations recommended follow up with an ophthalmologist in clinic (42 [20%] vs 17 [28%], p = 0.04). Imaging studies were frequently used to supplement information provided by referrer to specialist during both audit periods.

Conclusions: Optometrists used the telehealth service more frequently after the implementation of an intervention that addressed the barriers to its use. This has a number of potential benefits to rural and remote eye health service provision in Australia.

KEYWORDS: telehealth / telemedicine, ophthalmology, optometry

Table Comparison of findings between two 5 month telehealth audits. *Significant difference

2012 2014

Total 60 211

Non-urgent 32% 69%*

Required clinic follow-up

28% 20%*

Booked for procedure

0 26%*

No 1 Diagnosis Uveitis (22%) Cataract (27%)

OCT used 12% 21%

31

A new mouse and retinal ganglion cell model of mitochondrial optic neuropathy

HYPOTHESIS A new xenomitochondrial mouse will provide a transformative model of in vivo and in vitro retinal ganglion cell pathology secondary to mitochondrial impairment

Xenomitochondrial mice: Most inbred strains of laboratory mice have identical mtDNA sequences, indicating a single maternal ancestor so they cannot be used in studies that model human mtDNA variation. The lack of homologous recombination in mtDNA precludes the use of conventional genetic engineering approaches to alter this organelle genome. To circumvent these limitations, we have produced a xenomitochondrial mouse line harbouring an interspecific mtDNA that introduces 155 amino acid polymorphisms in the respiratory chain protein subunits, with a preponderance of changes in complex I genes, and a partial complex I defect. We have established that the optic nerve of the animals is more susceptible to acute pressure stress, while age-related loss of retinal ganglion cells was not accelerated compared with control C57Bl/6 mice.

Here we aimed to produce a second xenomouse with further diverged mtDNA, resulting in a greater degree of oxidative phosphorylation (OXPHOS) impairment and consequent retinal ganglion cell pathology.

AIMS1. Create new mouse embryonic

stem cell ‘xenocybrid’ line and a xenomitochondrial mouse.

2. Optimize differentiation protocols for retinal ganglion cell production in vitro from the xenocybrid and control ES cells.

EXPERIMENTAL APPROACHAim 1: Xenomitochondrial mouse ES cells will be produced by introduction of mitochondria from cultured cells of the mouse species Mus pahari into female ES cells. These cells will then be used by the Monash Gene Targeting Facility to produce a mouse line by blastocyst injection.

Rationale: We have previously shown that this species is further divergent than our first xenocybrid, such that a greater OXPHOS impairment will result.

PROGRESS We succeeded in producing the new xenomitochondrial ES cell cybrid, as planned. These cybrid ES cells contained mitochondria from the donor mouse species Mus Pahari. Blastocyst injections proceeded in 3 iterative attempts (over 40 rounds of injections) during mid-2014 with these cells, however chimeric offspring (indicative of the cybrid ES cells populating significant levels in offspring) were not obtained. At the same time our initial differentiation attempts with the same cells led to limited differentiation, with some embryo-body formation but with poor neuronal yields.

We then chose to re-examine the mitochondrial oxidative phosphorylation (OXPHOS) capacities

ORIA/ESME ANDERSON GRANTA/Prof Ian Trounce & Dr Matthew McKenzie

A new pre-clinical model of mitochondrial optic neuropathy

Progress Reports

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

Figure 1 Mitochondrial oxygen consumption was measured in Mm, Ms, Mc, Md and Mp using the high-resolution Oxygraph-2k respirometer (Oroboros Instruments, Innsbruck Austria) and 2x106 cells/chamber. Sequential measures were made of the endogenous respiration rate (with endogenous substrates), ADP-stimulated complex-I respiration (CxI; glutamate + malate), dual ADP-stimulated complex-I+II respiration (CxI+II; glutamate + malate + succinate) and the uncoupled maximal respiration (UC, after CCCP). The Ms and Mt xenocybrids showed no impairments compared with the Mm control, whereas the Mp cybrid showed a severe complex I-linked defect and a milder complex II-linked defect. The Mc cybrid exhibited a milder complex I and II-linked defect, suggesting this to be the most promising construct to use for xenomouse production.

Figure 2 Gels showing resolved restriction fragments of a PCR product from putative cybrid clones. The left gel shows homoplasmic cybrids containing the Mus Pahari mtDNA, while the right gel shows cybrid clones with the Mus caroli mtDNA. Asterisks indicate the homoplasmic cybrid ES clones; at right on each gel the uncut PCR fragments are shown along with donor cell DNA controls cut with the respective restriction enzyme to show the expected restriction fragments.

A/Prof Ian Trounce – photo courtesy CERA

of our set of L-cell xenocybrids, suspecting that the OXPHOS defects in the Mus Pahari (Mp) cybrids may be too great to allow normal differentiation. This analysis has provided interesting results, showing the pahari construct to have significantly greater OXPHOS defects than our earlier work had suggested. But another construct investigated, with L-cells harbouring mtDNA from another mouse species called Mus caroli (Mc) showed milder defects (Figure 1).

We then proceeded to produce ES cell cybrids containing the Mc mtDNA. These are shown in figure 2 along with the first produced Mp ES cell cybrids.

We have now progressed to blastocyst injections using the second, Mus caroli cybrid construct. These injections began early in 2015 and continue. While the funds for injections were expended on the first construct, the relationship built with the Monash Gene Targeting facility has allowed ongoing injections ‘pro-bono’ due to their interest in the challenge of the project. We therefore hope to obtain the goal of chimeric animals in the second half of 2015.

The 0.4 FTE research assistant effort (and additional postdoctoral fellow efforts) was largely taken up in 2014 by the additional construct production and OXPHOS investigations after the lack of success with the first cybrid. However with reagents in hand we continue to develop the retinal ganglion cell differentiation of the new cybrid ES cells. We therefore request a no-cost extension to end 2015, after which a follow-up report will be sent to ORIA.

CONCLUSIONWhile challenging, we believe this project has progressed significantly toward the goal of a new mouse model of mitochondrial optic neuropathy. A report is being prepared for publication on the OXPHOS characterization of the cybrids and an additional report should result from the in vitro ES cell differentiation work to be completed. The ultimate success will be the generation of the new mouse line, which remains uncertain. If successful this would form the basis for a major new grant application to Australian and US Federal agencies.

33

DEFOCUS VS. OCCLUSION.Most compensation to ±10D defocussing lenses in chick occurs within the first 24hours, with compensation almost complete at 48 hours. Comparatively, myopia increases steadily across the first week of occlusion (see below). Thus, although initial siRNA and carrier trials were conducted with lenses, we are now using occlusion only as this effectively increases the time-window available for knock-down to affect growth.

CARRIERSLipofectamine. Lipofectamine (liposome carrier not designed for in vivo delivery) enhanced myopic growth in response to negative lenses by approximately 4 diopters across 96hours rearing. Lipofectamine delivers to the endosomal pathway, resulting in exposure to endosomally located TLRs 3/7/8 (Forsbach et al., 2012). Given that induction of immune and inflammatory pathways has been shown to promote myopic growth (Wan, Lin, & Hsu, 2014), it is not surprising that Lipofectamine has this effect.

Invivofectamine. Following Lipofectamine ,we trialled delivery with another liposomal carrier, Invivofectamine, that has shown recent success for intravitreal injection delivery to retina (Rutar, Natoli, & Provis, 2012). In contrast to Lipofectamine, Invivofectamine appears to slightly inhibit myopic growth relative to PBS (Figure 2, below).

ORIA/RANZCO EYE FOUNDATION GRANTDr Alex Hewitt, Prof Sheila Crewther, Dr Helena Lang & Prof David Crewther

Validation of therapeutic targets for myopia siRNA Summary

Figure 1 Chick refraction following 0-96 hours occlusion (left) and 24-72 hours ±defocus (right)

Figure 2 Mean difference in refractive state (experimental-fellow eye) following no injection, PBS, or Invivofectamine injections and 96h occlusion (a). Cy-3 tagged siRNA (red) and DAPI stain (blue) in the retina 1 hour (b) and 3 hours (c) following intravitreal injection of Cy-3-tagged negative control siRNA conjugated with Invivofectamine carrier.

A

B C

Progress Reports

A/Prof Alex Hewitt – photo courtesy CERA

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

A primary concern in using positively charged liposomal carriers for delivery was aggregation with negatively charged glycosaminoglycans in the vitreous impeding diffusion to the retina (Peeters et al., 2005; Pitkanen, Ruponen, Nieminen, & Urtti, 2003). Pilot results (Figure 2 above) with Invivofectamine and Cy-3-tagged negative control siRNA indicate that delivery to the nerve-fibre layer occurs within 1-3 hours of intravitreal injection, suggesting that aggregation in the vitreous is not problematic. In contrast, following injection with Cy-3-tagged siRNA only (without carrier), no fluorescence was visible at any time-point (although it is unclear whether this reflects faster degradation of the fluorophore or a failure to reach the retina).

SIRNAInitial siRNA trials without carrier demonstrated refractive change (for some agents) at low concentrations (300nM) with some signs of inflammation when dissecting tissue. After purchasing Invivofectamine we ran a dose response with BMP2 across similarly low concentrations (100-900nM) with no change in ocular growth. These concentrations are ~15times lower than those used in published studies for intravitreal siRNA delivery with Invivofectamine (e.g. Rutar et al., 2012). We repeated across a concentration range closer to that used in previous studies (5uM-40uM).

With some attrition, we used most of the BMP2 available conducting the dose response biometrics. If qPCR demonstrates that knock-down was effective, some molecular data from

earlier time-points will be needed to demonstrate that the knock-down occurred early enough to have time to affect growth (and build an argument re the importance of the protein). To ensure that we have enough siRNA to assess multiple time-points in future, subsequent biometrics have been conducted at a single high dose of 30uM (this allows enough siRNA to collect 2 time-points for molecular analysis).

IMMEDIATE PLANS- Run qPCR/ELISA on tissue collected

from 30uM concentration BMP2/3/4 following 48 and 96h occlusion.

- If knock-down is effective run another batch of chicks to boost numbers for biometrics stats before moving on to other targets.

Figure 3 Mean difference in refractive state (experimental-fellow eye) following no injection, injection of carrier only (PBS or Invivofectamine), BMP2 and CHRNG siRNA without carrier and BMP with Invivofectamine carrier and 72hrs occlusion.

Figure 4 Mean difference in refractive state (experimental-fellow eye) following 30uM injections of Invivofectamine, BMP3 or BMP4 and 48 or 96 hours occlusion

35

In accordance with a resolution of the directors, the directors submit herewith the financial statements of The Ophthalmic Research Institute of Australia for the year ended on 30 June 2015 and report as follows:

1. DIRECTORSThe names of the Directors of the company in office at the date of this report are:

Professor Stuart L Graham, Sydney (Chairman)

Professor Mark Gillies, Sydney (Vice Chairman)

Dr Richard Mills, Adelaide (Honorary Secretary)

Dr Wilson Heriot, Melbourne (Honorary Treasurer)

Dr Fred Chen, Perth

Dr Colin Clement, Sydney

Professor Jonathon Crowston, Melbourne

A/Prof Paul Healey, Sydney

Professor David Mackey, Perth

Professor Peter J McCluskey, Sydney

Dr John Males, Sydney

Dr Andrea Vincent, New Zealand

Dr Stephanie Watson, Sydney

2. INFORMATION ON DIRECTORS

The names, qualifications and period membership commenced and position held are as follows:

Dr Fred Chen MB BS (Hons), PhD (London), FRANZCO, CSA (Cert) 2011

Dr Colin Clement BSc (Hons), MB BS PhD, FRANZCO 2011

Professor J Crowston BSc, MB BS, FRCOphth, FRANZCO, PhD 2008

Professor Mark Gillies MB BS, PhD, FRANZCO Vice Chairman 2004

Professor Stuart L Graham MB BS, MS, FRANZCO, FRACS Chairman 2001

A/Prof Paul Healey MB BS (Hons), B (Med) Sc, MMed PhD, FRANZCO 2011

Dr Wilson Heriot MB BS, FRANZCO, FRACS Honorary Treasurer 2009

Professor David Mackey MB BS, MD, FRANZCO, FRACS 2005

Professor Peter J McCluskey MB BS, FRANZCO, FRACS 1984

Dr John Males MB BS, M Med, FRANZCO 2009

Dr Richard Mills MB BS, FRCS, FRACS, FRANZCO, PhD Honorary Secretary 2003

Dr Andrea Vincent MBChB, FRANZCO 2008

Dr Stephanie Watson BSc, MB BS, FRANZCO, PhD 2006

No shares are held by Directors.

Directors’ Report

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

3. MEETINGS OF DIRECTORS

During the financial year three meetings of directors were held. Attendances were:

Board Members Number Eligible to Attend Number Attended

Dr Fred Chen, Perth 3 3

Dr Colin Clement, Sydney 3 1

Professor J Crowston, Melbourne 3 2

Professor Mark Gillies, Sydney 3 2

Professor Stuart L Graham, Sydney 3 3

A/Prof Paul Healey, Sydney 3 3

Dr Wilson Heriot, Melbourne 3 3

Professor David Mackey, Perth 3 2

Professor Peter J McCluskey, Sydney 3 3

Dr John Males, Sydney 3 3

Dr Richard Mills, Adelaide 3 3

Dr Andrea Vincent, New Zealand 3 3

Dr Stephanie Watson, Sydney 3 3

Dr Andrea Vincent, New Zealand 3 3

Dr Stephanie Watson, Sydney 3 3

4. INDEMNIFYING OFFICER OR AUDITORThe company has not during or since the financial year in respect of any person who is or has been an officer or auditor of the company or a related body corporate indemnified or made any relevant agreement for indemnifying against a liability incurred as an officer including costs and expenses in successfully defending legal proceedings or paid or agreed to pay a premium in respect of a contract of insurance against a liability incurred as an officer for the costs or expenses to defend legal proceedings.

5. PRINCIPAL ACTIVITIESThe principal activity of the company in the course of the financial period was to provide funds for ophthalmic research. There has been no significant change in the nature of this activity during that period.

6. SHORT-TERM AND LONG-TERM OBJECTIVESThe company’s short-term objectives are to:- continue to fund research into all types of eye diseases annually in Australia- continue to be in the forefront of advancing eye research in Australia - continue to support the presentation of research and the publication of the results of research for vision scientists and

ophthalmologists for the benefit of all Australians.- continue to support new scientists by providing a percentage of its annual funding to support this category

The company’s long-term objectives are to:- increase the funds available for the provision of research funding in order to achieve its mission statement of advancing

eye research in Australia. - ensure that the funding it provides leads to researchers gaining a track record to enable them to secure larger grants

towards bigger and successful projects.

37

7. STRATEGIESTo achieve its stated objectives, the company has adopted the following strategies:- The company is partnered with the RANZCO Eye Foundation who are now primarily responsible for raising additional

funding towards the ORIA’s research projects and to raise awareness generally of eye health within Australia.- The company’s Investment Advisory Committee monitors and works towards successfully managing the company’s

invested funds, the profits from which are used annually for research funding.- The company connects with other vision related organisations in Australia to support funding of projects for specific

diseases.- The company strives to attract, support and retain quality staff who are committed to the work of the organization.- The company conducts audits on its previously funded research to ensure the funding it provides is meeting its

objectives.- The company’s Board and Research Advisory Committee is made up of leading vision scientists and ophthalmologists

within Australia.

8. OPERATING RESULTS(1) OPERATING REVENUERevenue is mainly derived from investing in shares and interest bearing securities.

2015 2014 Increase %

Net dividend, interest and trust distribution income

$ 675,334 $ 608,266 $ 67,068 11.03

Less Expenses 43,887 42,387

631,447 565,879

(2) OPERATING SURPLUSThe surplus of the company before other comprehensive income for the year ended 30 June 2015 was $1,049,334 (2014 $680,926). This amount is comprised of the following:

2015 2014

Trust Fund $ 1,049,602 $ 669,214

Administration (268) 11,712

1,049,334 680,926

Other comprehensive income before grants and Director of Research allocation amounted to a deficit of $265,886 (2014: surplus of $929,832) and included a profit on re arrangement of investments of $184,105 (2014: profit of $396,780) and valuation loss on available-for-sale financial assets of $449,991 (2014: gain of $533,052).

9. REVIEW OF OPERATIONS The surplus for the year was $1,049,334 compared to $680,926 in 2014. Distributions from legacies and donations increased to $417,630 from $122,719 in 2014. This increase is due to two large bequests, $100,000 from Betty Marion Roberts and $272,000 from The Estate of the late Gladys Clare Dickson during the 2015 financial year. The administrative operations of the institute for the year resulted in a deficit of $268 compared with a surplus of $11,712 in 2014.

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

10. DIVIDENDSThe company’s Articles of Association preclude the payment of dividends to any of its members.

11. STATE OF AFFAIRSThere has been no significant change in the state of affairs of the company occurring during the year.

12. LIKELY DEVELOPMENTSAt the date of this report, there are no known unusual developments that will affect the results of the company’s operations in subsequent financial years.

13. SHARE OPTIONSNo share options were issued during the year.

14. DIRECTORS’ BENEFITSWith the exception of the grants made or allocated to Dr Stephanie Watson, Professor Richard Mills, Professor Jonathan Crowston, Professor Mark Gillies, Professor Stuart Graham and Dr John Males, no director of the company has since the end of the previous financial year, received or become entitled to receive a benefit not disclosed in the accounts as directors’ emoluments by reason of a contract made by the company or a related corporation with the directors, or with a firm in which he or she has a substantial financial interest.

15. AUDITOR’S INDEPENDENCE DECLARATIONA copy of the auditor’s independence declaration as required under the Australian Charities and Not-for-profits Commission Act 2012 is set out at page 27.

For and on behalf of the Board.

Prof Stuart Graham Dr Wilson Heriot Director Director

Sydney

Signed in accordance with a resolution of directors,

this 12 day of September 2015

39

STATEMENT OF FINANCIAL POSITIONAS AT 30 JUNE 2015

Note2015

$2014

$

Current Assets

Cash and Cash Equivalents 3 1,820,990 1,649,880

Receivables 4 189,670 139,089

Investments 5 9,083,682 9,277,610

11,094,342 11,066,579

Non-Current Assets

Plant & Equipment 6 2,274 3,087

Total Assets 11,096,616 11,069,666

Current Liabilities

Payables 7 662,882 620,256

Provisions 8 21,565 19,689

684,447 639,945

Net Assets 10,412,169 10,429,721

Equity

General Fund 13 (a) - -

Capital Funds

Research Fund 9 1,261,288 1,013,442

Settled Funds 10 472,556 472,556

Financial Assets Reserve 11 1,109,933 1,559,924

Capitalised Profit on Re-arrangement of Investments & Capital Distributions

12 7,152,163 6,968,058

9,995,940 10,013,980

Retained Income- Available for grants 13 (b) 416,229 415,741

Total Equity 10,412,169 10,429,721

The accompanying Notes form part of these financial statements.

Financial Statements

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

TRUST FUND STATEMENT OF COMPREHENSIVE INCOMEFOR THE YEAR ENDED 30 JUNE 2015

Note2015

$2014

$

Income

Dividends received from:

Other Corporations 539,980 470,567

Total Dividends 539,980 470,567

Interest received from:

Other Entities 67,186 75,283

Trust distributions received from:

Other Entities 68,168 62,416

675,334 608,266

Legacies - Anselmi Estate 39,786 51,399

- Ivy May Stephenson 5,844 4,363

- Mary Tilden Bequest - 66,457

- Betty Marion Roberts Bequest 100,000 -

- The Estate of the late Gladys Clare Dickson Bequest 272,000 -

Other Donations and Legacies Received - 500

Sundry Income 525 616

Total Income for the Year 1,093,489 731,601

Expenses

AOVS Meeting Contribution - 20,000

Commission Paid 43,887 42,387

43,887 62,387

Surplus For The Year 1,049,602 669,214

Other Comprehensive Income

Valuation Gains/(Losses) on available-for-sale financial assets (449,991) 533,052

Profit/(Loss) on Re-arrangement of Investments 184,105 396,780

Total other comprehensive income (265,886) 929,832

Surplus for the year before allocation 783,716 1,599,046

Grants Allocated/made during the year 14 441,000 396,365

Allocation to Director of Research - Victoria 15 210,000 184,000

651,000 580,365

Total Comprehensive Income/(Loss) 132,716 1,018,681

Profit Attributable to Members of the Entity 398,602 88,849

Total Other Comprehensive Income Attributable to Members of the Entity (265,886) 929,832

The accompanying Notes form part of these financial statements.41

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.

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

ADMINISTRATION STATEMENT OF COMPREHENSIVE INCOMEFOR THE YEAR ENDED 30 JUNE 2015

Note2015

$2014

$

Income

Membership Fees - RANZCO 142,150 140,000

Total Income 142,150 140,000

Expenses

Accountancy Fees 20,217 21,471

Auditors' Remuneration 16 4,950 4,950

Bank Charges 113 144

Depreciation 813 1,197

General Expenses 9,698 6,429

IT & Webpage Expenses 119 -

Insurance 2,858 2,802

Printing & Stationery 5,251 4,989

Staff Salaries 39,000 43,600

Superannuation Contribution 8,330 7,776

Salary Sacrificed Benefits 30,420 21,200

Provision Employee Benefits 1,876 -

Meeting and Travelling Expenses 18,773 13,730

Total Expenses 142,418 128,288

Surplus/(Deficit) For The Year 13 (a) (268) 11,712

Other Comprehensive Income - -

Total Comprehensive Income (268) 11,712

The accompanying Notes form part of these financial statements.

43

STATEMENT OF CASH FLOWSFOR THE YEAR ENDED 30 JUNE 2015

Note2015

$2014

$

Cash Flows From Operating Activities

Receipts

Dividends Received 489,203 471,819

Interest Received 67,186 75,283

Trust Distributions 68,168 62,416

Legacies 417,630 99,543

Other Revenue 720 142

RANZCO - Reimbursement of membership fees 142,150 140,000

Contributions from Ranzco Eye Foundation 60,000 302,435

Contributions from Ranzco NSW Branch 25,000 -

Contributions from Ranzco 150,000 -

Contribution from Glaucoma Australia Inc - 25,000

Donations Received - 66,957

Payments

Commissions (43,887) (42,387)

Research Grants Paid (808,400) (817,700)

Payments to Director of Research - Victoria (184,000) (175,000)

Other Grants & Contributions - (20,000)

Other (140,703) (123,866)

Net Cash (Used in)/Provided by Operating Activities 17 243,067 64,642

Cash Flows From Investing Activities

Proceeds from Re-arrangement of Investments 1,148,002 1,768,742

Payments for Investments (1,219,959) (1,277,277)

Net Cash Used in Investing Activities (71,957) 491,465

Net Increase in Cash and Cash Equivalents 171,110 556,107

Cash and Cash Equivalents at 1 July 2013 1,649,880 1,093,773

Cash and cash equivalents at 30 June 2014 3 1,820,990 1,649,880

The accompanying Notes form part of these financial statements.

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

1 STATEMENT OF ACCOUNTING POLICIESThe financial statements are for the Ophthalmic Research Institute of Australia, incorporated and domiciled in Australia. The Ophthalmic Research Institute of Australia is a company limited by guarantee.

(A) BASIS OF PREPARATIONThe financial statements are general purpose financial statements that have been prepared in accordance with Australian Accounting Standards (including Australian Accounting Interpretations) and the Australian Charities and Not-for-profits Commission Act 2012.

The accounting policies set out below have been consistently applied to all years presented, unless otherwise stated. The financial report has been prepared on an accruals basis and is based on historical costs and does not take into account changing money values or, except where stated, current valuations of non current assets. Cost is based on the fair values of the consideration given in exchange for assets.

The following is a summary of the significant accounting policies adopted by the company in the preparation of the financial report.

(B) INCOME TAX

The company is an approved research institute and is exempt from income tax.

(C) TRANSFERS TO CAPITAL FUNDS

(i) Capital profits and losses on disposal of investments & capital distributions.

Realised capital profits and losses on disposal of investments are brought to account in the trust fund as profit/(loss) on rearrangement of investments, however, these amounts are transferred to capital funds and do not form part of retained income available for grants.

Capital Distributions and special dividends together with associated imputation credits recognised in the statement of comprehensive income are also transferred to the capital fund and do not form part of retained income available for grants.

(ii) General Research Capital FundTen percent of the net surplus of the General Fund including imputation credits are transferred to the General Research Capital Fund this financial year.

(iii) Allocation of Income to Each Fund

During the year ended 30 June 1993, the investments of the company were separated into the D.W. Research Fund and the General Fund in the ratio of 72% and 28% respectively. As the flow of investment and donation income to and from the two funds does not occur in the same proportion, the ratio of the D.W. Research Fund and the General Fund is no longer at 72% and 28%.

Notes to and forming part of the Financial Statements

45

Income from the General Fund which comprises of all funds except the D.W. Research Fund, is allocated as follows:

Research Fund 10.0%

Esme Anderson 51.4%

G.J.Williams 8.9%

B. Mitchell 8.9%

Dame Ida Mann 12.5%

R. & L. Lowe Research 8.3%

If and when further donations are received by specific fund(s) the allocation of future income will be distributed to each fund in accordance with its revised proportion to the General Fund.

Fifty percent of the income derived from the D.W. Research Fund and its investments is allocated to the Director of Research Victoria.

(D) CASH AND CASH EQUIVALENTS

For the purpose of the statement of cash flows, cash and cash equivalents include cash on hand and at call deposits with banks.

(E) INVESTMENTS

Investments are carried at fair value. Changes in fair value will be held in an equity reserve until the asset is disposed, at which time the changes in fair value will be brought to account through the statement of comprehensive income.

(F) REVENUE

Interest and dividends are recognised when received. Grants, donations and distributions income are recognised when received.

(G) GOODS AND SERVICES TAX (GST)

All revenue, expenses and assets are recognised net of the amount of goods and services tax (GST), except where the amount of GST incurred is not recoverable from the Australian Tax Office. In these circumstances the GST is recognised as part of the cost of acquisition of the asset or as part of an item of the expense. Receivables and payables in the statement of financial position are shown inclusive of GST.

(H) FINANCIAL INSTRUMENTS

Recognition and Initial Measurement

Financial instruments, incorporating financial assets and financial liabilities, are recognised when the entity becomes a party to the contractual provisions of the instrument.

Financial instruments are initially measured at fair value plus transactions costs where the instrument is not classified as at fair value through profit or loss. Financial instruments are classified and measured as set out below.

Classification and Subsequent Measurement

(i) Loans and receivablesLoans and receivables are non-derivative financial assets with fixed or determinable payments that are not quoted in an active market and are subsequently measured at amortised cost using the effective interest rate method.

(ii) Held-to-maturity investmentsHeld-to-maturity investments are non-derivative financial assets that have fixed maturities and fixed or determinable payments, and it is the entity’s intention to hold these investments to maturity. They are subsequently measured at amortised cost using the effective interest rate method.

(iii) Available-for-sale financial assetsAvailable-for-sale financial assets are non-derivative financial assets that are either designated as such or that are not classified in any of the other categories. They comprise investments in the equity of other entities where there is neither a fixed maturity nor fixed or determinable payments.

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

(iv) Financial liabilitiesNon-derivative financial liabilities (excluding financial guarantees) are subsequently measured at amortised cost using the effective interest rate method.

Fair value

Fair value is determined based on current bid prices for all quoted investments. Valuation techniques are applied to determine the fair value for all unlisted securities, including recent arm’s length transactions, reference to similar instruments and option pricing models.

Impairment

At each reporting date, the entity assesses whether there is objective evidence that a financial instrument has been impaired. In the case of available-for-sale financial instruments, a prolonged decline in the value of the instrument is considered to determine whether an impairment has arisen. Impairment losses are recognised in the statement of comprehensive income.

(I) IMPAIRMENT OF ASSETS

At each reporting date, the entity reviews the carrying values of its assets to determine whether there is any indication that those assets have been impaired. If such an indication exists, the recoverable amount of the asset, being the higher of the asset’s fair value less costs to sell and value in use, is compared to the asset’s carrying value. Any excess of the asset’s carrying value over its recoverable amount is expensed to the statement of comprehensive income.

Where it is not possible to estimate the recoverable amount of an individual asset, the entity estimates the recoverable amount of the cash-generating unit to which the asset belongs.

2 MEMBERS’ GUARANTEEIf the company is wound up the Memorandum of Association states that each member is required to contribute a maximum of $ 2.00 each towards meeting any outstanding obligations of the company.

3 CASH AND CASH EQUIVALENTS

2015 $

2014 $

General Account 1,078,100 1,067,207

Donations Account 429,313 112,284

D.W. Research Fund Account 313,577 470,389

1,820,990 1,649,880

4 RECEIVABLES

Sundry Debtors 189,670 139,089

189,670 139,089

5 INVESTMENTS

Shares in Listed Corporations & Other Securities 9,083,682 9,277,610

Total Available-for-sale Financial Assets 9,083,682 9,277,610

Total Investments 9,083,682 9,277,610

47

6 PLANT AND EQUIPMENT

2015 $

2014 $

Office Equipment - at cost 9,040 9,040

Less: Accumulated Depreciation (6,766) (5,953)

2,274 3,087

Reconciliation

Reconciliation of the carrying amount of plant and equipment at the beginning and end of the current and previous financial year:

Carrying amount at beginning of year 3,087 4,284

Additions - -

Less: Depreciation expense (813) (1,197)

Carrying amount at end of year 2,274 3,087

7 PAYABLES

Creditors and Accruals 27,356 24,119

Grants Payable 408,900 502,800

Director of Research - Victoria (refer note 15) 184,000 175,000

620,256 701,919

8 PROVISIONS

Employee Benefits 21,565 19,689

9 RESEARCH CAPITAL FUND

General

Balance 1 July 2014 692,087 660,702

Allocation to Capital:

- 10% Surplus & Imputation Credits & Other Legacies 25,846 31,385

- Capitalised Bequests 372,000 -

Transfer from Capital:

-Amount transferred to Income to match RANZCO donation (150,000) -

Balance 30 June 2015 939,933 692,087

Anselmi Estate

Balance 1 July 2014 290,979 290,979

Allocation during year - -

Transfer during year - -

Balance 30 June 2015 290,979 290,979

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

Ivy May Stephenson Estate

Balance 1 July 2014 30,376 30,376

Allocation during the year - -

Transfer during year - -

Balance 30 June 2015 30,376 30,376

Total 1,261,288 1,013,442

10 SETTLED FUNDS

2015 $

2014 $

D.W. Research Funds 200,000 200,000

Esme Anderson 124,326 124,326

G.J. Williams 25,500 25,500

B. Mitchell 26,023 26,023

Dame Ida Mann (Est. 31/03/84) 56,707 56,707

Ronald & Lois Lowe 40,000 40,000

472,556 472,556

11 FINANCIAL ASSETS RESERVE

Balance 1 July 2014 1,559,924 1,026,872

Revaluation increment/(decrement) (449,991) 533,052

Balance 30 June 2015 1,109,933 1,559,924

Financial assets reserve records unrealised gains on revaluation of financial assets to fair value.

12 CAPITALISED PROFIT ON RE-ARRANGEMENT OF INVESTMENTS, CAPITAL DISTRIBUTIONS & TRANSFERS

Balance 30/6/14

$

Allocation of Realised Profit/(Loss) on

Rearrangement of Investments &

Capital Distributions & Transfers

$

Balance 30/6/15

$

Research Fund

General 134,537 4,338 138,875

Anselmi Estate 48,189 1,554 49,743

Ivy May Stephenson 124 4 128

D.W. Research Funds 5,143,218 125,148 5,268,366

Esme Anderson 947,844 30,304 978,148

G.J. Williams 162,831 5,247 168,078

B. Mitchell 160,895 5,247 166,142

Dame Ida Mann 227,220 7,370 234,590

Ronald & Lois Lowe 143,200 4,893 148,093

6,968,058 184,105 7,152,163

49

13 ACCUMULATED FUNDS

Note2015

$2014

$

(a) Administration

Accumluated Deficits - 1 July 2014 - -

Total Comprehensive Income (268) 11,712

Total available for appropriation (268) 11,712

Aggregate of amounts transferred from Administration 13 (b) 268 (11,712)

Accumulated Deficits - 30 June 2015 - -

(b) Trust Fund

Retained income - 1 July 2014 415,741 346,565

Total Comprehensive Income 398,602 88,849

Total available for appropriation 814,343 435,414

Aggregate of amounts transferred to General/Capital Funds

Administration 13 (a) (268) 11,712

Research Trust (397,846) (31,385)

Retained income - 30 June 2015 416,229 415,741

14 GRANTS ALLOCATED / MADE DURING THE YEAR

2015 $

2014 $

Dr Raymond Wong, Dr Alice Pebay & Dr Nicole Van Bergen - 50,000.00

*Dr Stephanie Watson, A/Prof Mark Daniell, Dr Daniel Barthelmes, Dr Martina Bosch, *Dr John Males & Dr Yves Kerdraon

- 50,000.00

Dr Kathryn Burdon & Ms Emmanuelle Souzeau - 50,000.00

A/Prof Ian Trounce & Dr Matthew McKenzie - 49,000.00

Prof K Williams, Dr D Klebe & *Prof R Mills - 45,500.00

*Prof J Crowston, Dr Vicki Chrysostomou & A/Prof Greg Steinberg - 49,500.00

Dr Angus Turner & Dr Roderick O'Day - 36,200.00

Prof Justine Smith - 49,900.00

Dr Shiwani Sharma & Prof Jamie Craig - 50,000.00

*Prof Mark Gillies & Dr Ling Zhu - 50,000.00

Dr Alex Hewitt, Prof Sheila Crewther, Dr Helena Liang & Prof David Crewther

- 50,000.00

Prof Robert Casson - 44,000.00

Dr Jingjing You, Prof Gerard Sutton & Dr Lucy Dawes - 50,000.00

A/Prof Robyn Jamieson & A/Prof John Grigg - 50,000.00

A/Prof Nick Di Girolamo - 49,700.00

Prof Ian McAlister, Dr A Shaw & Prof D-Y Yu 45,500.00 -

Dr Alex Hewitt, Prof Andrew Hill, Dr Ruchira Singh & Mr Duncan Crombie

50,000.00 -

A/Prof Nick Di Girolamo 50,000.00 -

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

Dr Kathryn Burdon & Dr Shiwani Sharma 50,000.00 -

A/Prof Robyn Jamieson & A/Prof John Grigg 50,000.00 -

Prof J McAvoy, Dr Y Sugiyama & Prof F Lovicu 48,500.00 -

Dr Vivek Gupta, *Dr Stuart Graham & Dr Yuyi You 50,000.00 -

A/Prof Damien Harkin & Dr Sally Stephenson 41,500.00 -

Dr Carla Abbott, Dr Penny Allen & A/Prof Erica Fletcher 49,000.00 -

Dr Weiyong Shen 50,000.00 -

*Prof Mark Gillies & Dr Ling Zhu 50,000.00 -

*C/Prof Stephanie Watson, Dr Holly Inglis, Prof Michael Friedlander, Prof Frances Boyle & Dr Yi-Chiao Li

27,000.00 -

Dr Glyn Chidlow 49,500.00 -

Dr Shervi Lie 49,000.00 -

Dr Shane Durkin 16,500.00 -

Dr Mojtaba Golzan & Prof Alberto Avolio 49,500.00 -

Dr Danuta Maria Bukowska 50,000.00 -

Dr Sandy Hung, Dr Raymond Wong, Dr Bryony Nayagam & Prof Ross Hannan

50,000.00 -

826,000 723,800

Deduct Contribution from:

The Ophthalmic Research Institute of Australia 150,000 -

Glaucoma Foundation Australia Inc - 25,000

RANZCO Eye Foundation 60,000 302,435

RANZCO NSW Branch 25,000 -

RANZCO 150,000 -

385,000 327,435

441,000 396,365

* Grant received by director

15 FUNDS ALLOCATED TO DIRECTOR OF OPHTHALMIC RESEARCH - VICTORIA

Balance as at 1 July 2014 184,000 175,000

Interest for the year 1,626 996

Allocation for year 210,000 184,000

395,626 359,996

Payment made to Director of Research 185,626 175,996

Balance as at 30 June 2015 210,000 184,000

16 AUDITORS REMUNERATION

Financial Statements - Audit Service 4,950 4,950

Other services - -

4,950 4,950

51

17 RECONCILIATION OF NET CASH PROVIDED BY OPERATING ACTIVITIES TO RESULTS FOR YEAR

Net Surplus/(Deficit)

- Trust Fund 132,716 1,018,681

- Administration (268) 11,712

132,448 1,030,393

Depreciation 813 1,197

Provision for Employee Benefits 1,876 -

Transfer from Capital to Contribute Towards Grants (150,000) -

(Increase)/Decrease in Receivables (50,582) 44,559

Increase/(Decrease) in Creditors and Accrued Expenses (974) 3,225

Increase/(Decrease) in Grants Payable 17,600 (93,900)

Increase/(Decrease) in allocation to Director of Research - Victoria 26,000 9,000

Valuation (Gains)/Losses on available-for-sale financial assets 449,991 (533,052)

(Profit)/Loss on Rearrangement of Investments (184,105) (396,780)

Net Cash Provided by /(used in) Operating Activities 243,067 64,642

18 DISCLOSURES ON DIRECTORS AND OTHER KEY MANAGEMENT PERSONNELDIRECTORSThe following directors received grants during the year. These are detailed at note 14.

Professor Stuart Graham

Dr Stephanie Watson

Professor Richard Mills

Professor Jonathan Crowston

Professor Mark Gillies

Dr John Males

The names of the directors who have held office during the financial year are:

Professor Stuart L Graham, Sydney (Chairman)

Professor Mark Gillies, Sydney (Vice Chairman)

Dr Richard Mills, Adelaide (Honorary Secretary)

Dr Wilson Heriot, Melbourne (Honorary Treasurer)

Dr Fred Chen, Perth

Dr Colin Clement, Sydney

Professor J Crowston, Melbourne

Dr Paul Healey, Sydney

Professor David Mackey, Perth

Professor Peter J McCluskey, Sydney

Dr John Males, Sydney

Dr Andrea Vincent, New Zealand

Dr Stephanie Watson, Sydney

KEY MANAGEMENT PERSONNELOther Key Management Personnel include Executive Officer, Anne Dunn Snape.

Key management personnel are those persons having authority and responsibility for planning, directing and controlling

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

the activities of the entity, directly or indirectly, including any director (whether executive or otherwise) of that entity. Control is the power to govern the financial and operating policies of an entity so as to obtain benefits from its activities.

KEY MANAGEMENT PERSONNEL COMPENSATIONKey Management Personnel has been taken to comprise the directors and one member of the executive management responsible for the day to day financial and operational management of the entity.

2015 $

2014 $

(a) Short-term employee benefits 69,420 64,800

(b) Post-employment benefits 8,330 7,776

(c) Other long-term benefits - -

(d) Termination benefits - -

(e) Share-based payment - -

77,750 72,576

19 FINANCIAL INSTRUMENTS

(A) FINANCIAL RISK MANAGEMENT POLICIESThe entity’s financial instruments consist mainly of deposits with banks, local money market instruments, short-term investments, accounts receivable and payable.

The entity does not have any derivative instruments at 30 June 2015.

(i) Treasury Risk Management An investment committee consisting of Board members of the entity meet on a regular basis to analyse financial risk

exposure and to evaluate treasury management strategies in the context of the most recent economic conditions and forecasts.

The committee’s overall risk management strategy seeks to assist the entity in meeting its financial targets, whilst minimising potential adverse effects on financial performance.

Risk management policies are approved and reviewed by the Board on a regular basis. These include credit risk policies and future cash flow requirements.

(ii) Financial Exposures and Management Risk The main risks the entity is exposed to through its financial instruments are interest rate risk, liquidity risk and credit risk.

Interest rate risk Interest rate risk is managed with a mixture of fixed and floating rates on investments.

Foreign currency risk The entity is not exposed to fluctuations in foreign currencies.

Liquidity risk The entity manages liquidity risk by monitoring forecast cash flows.

Credit risk The maximum exposure to credit risk, excluding the value of any collateral or other security, at balance date to recognised financial assets, is the carrying amount, net of any provisions for impairment of those assets, as disclosed in the statement of financial position and notes to the financial statements.

The entity does not have any material credit risk exposure to any single receivable or group of receivables under financial instruments entered into by the entity.

Price risk The group is not exposed to any material commodity price risk.

53

(B) F

INA

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2015

%

2014

%

2015

$20

14 $

2014

$20

14 $

2015

$20

14 $

2015

$20

14 $

2015

$20

14 $

Fina

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Cas

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Equ

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2.00

2.50

1,82

0,99

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649,

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N/A

N/A

--

--

--

--

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--

--

--

--

189,

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139,

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1

89,6

70

139,

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s 1,

820,

990

1,64

9,88

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352

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416,

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665,

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620,

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665,

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620,

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Net

Fin

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820,

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1,64

9,88

0-

--

-

8,60

7,47

0

8,79

6,44

3

10,4

28,4

60

10,4

46,3

23

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

(C) NET FAIR VALUESThe net fair values of listed investments have been valued at the quoted market bid price at balance date. For other assets and other liabilities the net fair value approximates their carrying value. No financial assets and financial liabilities are readily traded on organised markets in standardised form other than listed investments.

The aggregate net fair values and carrying amounts of financial assets and financial liabilities are disclosed in the statement of financial position and in the notes to and forming part of the financial statements.

(D) SENSITIVITY ANALYSIS

Interest Rate RiskThe entity has performed a sensitivity analysis relating to its exposure to interest rate risk at balance date. This sensitivity analysis demonstrates the effect on the current year results and equity which could result from a change in this risk.

Interest Rate Sensitivity Analysis:At 30 June 2015, the effect on profit and equity as a result of changes in the interest rate, with all other variables remaining constant, would be as follows:

2015Carrying Amount

Interest Rate Risk

$ -1% Profit +1% Profit -1% Equity +1% Equity

Financial Assets

Cash and Cash Equivalents 1,820,990 (18,210) 18,210 (18,210) 18,210

2014Carrying Amount

Interest Rate Risk

$ -1% Profit +1% Profit -1% Equity +1% Equity

Financial Assets

Cash and Cash Equivalents 1,649,880 (16,499) 16,499 (16,499) 16,499

20 FAIR VALUE MEASUREMENTS

Financial assets and financial liabilities measured at fair value in the statement of financial position are grouped into three Levels of a fair value hierarchy. The three Levels are defined based on the observability of significant inputs to the measurement, as follows:

Level 1: quoted prices (unadjusted) in active markets for identical assets or liabilities;

Level 2: inputs other than quoted prices included within Level 1 that are observable for the asset or liability, either directly or indirectly;

Level 3: unobservable inputs for the asset or liability.

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The following table shows the Levels within the hierarchy of financial assets and liabilities measured at fair value on a recurring basis at 30 June 2014 and 30 June 2013:

NoteLevel 1

$Level 2

$Level 3

$Total

$

30 June 2015

Assets

Listed securities 5 9,083,682 - - 9,083,682

Net fair value 9,083,682 - - 9,083,682

30 June 2014

Assets

Listed securities 5 9,277,610 - - 9,277,610

Net fair value 9,277,610 - - 9,277,610

There were no transfers between Level 1 and Level 2 for assets measured at fair value during 2015 or 2014.

Listed SecuritiesFair values have been determined by reference to their quoted bid prices at the reporting date.

ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

DIRECTORS’ DECLARATION

The directors of the company declare that:

1. the financial statements and notes as set out on pages 5 - 23:

(a) comply with Accounting Standards and Australian Charities and Not-for-profits Commission Act 2012; and

(b) give a true and fair view of the financial position as at 30 June 2015 and performance for the year ended on that date of the company.

2. In the directors’ opinion there are reasonable grounds to believe that the company will be able to pay its debts as and when they become due and payable.

The declaration is made in accordance with a resolution of the board of Directors.

On behalf of the Board.

Prof Stuart Graham Dr Wilson Heriot Director Director

Sydney, this 12 day of September 2015

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ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

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ORIA ANNUAL REPORT 2015 ABN 37 008 393 146

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