Enhancing ocular gene therapy using glycosidic enzymes

Lead Research Organisation: University of Manchester
Department Name: Medical and Human Sciences

Abstract

Gene therapy shows great promise in treating human blindness due to genetic diseases affecting the retina. Healthy genes, contained in modified viruses, are delivered by injection under the retina, a thin later of tissue that converts light into signals that are sent to our brain. However, this approach can potentially damage the retina and cannot deliver the genes across the entire retina. A potentially safer and more effective approach would be to inject the gene-carrying viruses into the vitreous, a gel-like structure in front of the retina. However, to date this approach has not worked very well with only a small number of retinal cells receiving the gene.

My research to date has shown that if we use enzymes in combination with the viruses we achieve much better gene delivery to the retina following injection into the vitreous. The aim of this project is to optimise the intravitreal combination of virus and enzymes to deliver genes right across the retina. I will then see if I can demonstrate recovery of retinal function in a mouse that, because of genetic alterations, has no vision. This may ultimately lead to exciting advancements in treating patients with a variety of retinal diseases.

Technical Summary

The overall aim of this study is to: Investigate strategies to improve retinal transduction efficiency using combinations of intravitreally delivered AAV serotypes and glycosidic enzymes.



Specific objectives of this study are to:

1. Optimize combinations of glycosidic enzymes and AAV serotypes for the transduction of inner and outer retina using an intravitreal approach in a mouse model

2. Assess the safety of these treatment combinations in the mouse using functional studies

3. Confirm our ability to target cells in ganglion cell layer and photoreceptor layer in a therapeutically relevant manner using this technique



Methodology:

AAV containing a cDNA encoding enhanced GFP under the control of a chicken beta-actin promoter will be delivered intravitreally into anaesthetized adult mouse eyes in conjunction with enzymes. Various combinations and doses of glycosidic enzymes including hyaluronan lyase, chondroitin ABC lyase and heparinase III will be co-injected with the AAV2. In addition, I will investigate whether other AAV serotypes (AAV5, AAV8 and AAV9) can achieve retinal transduction following intravitreal injection when used in combination with these enzymes. Two weeks later retina flat mounts will be examined by confocal microscopy to analyze the amount and depth of penetration of the GFP fluorescence (using ImageJ software). Other retinas will be snap frozen, cryostat sectioned and co-stained with antibodies to GFP and to proteins expressed in specific types of retinal neurons to establish which particular cell types are transfected. At two weeks post treatment retinal function will be evaluated with dark- and light- adapted electroretinograms (ERG) and visually evoked potentials (VEPs), which will be compared to control eyes. Next, I will generate triple-knockout (Opn4-/-, Rho-/-, Cnga3-/-) mice lacking rod, cone and melanopsin transduction. I will then be able to use AAV containing cDNA for melanopsin (Opn4), rod opsin (Rho) or the cone cyclic GMP-gated channel A-subunit 3 (Cnga3) to restore phototransduction in melanopsin ganglion cells, rods or cones respectively. The readouts for these experiments will be circadian photo-entrainment (melanopsin photoreceptor activity), pupil reflex activity, ERGs and VEPs. The retinas will also be examined using immunohistochemistry.



Scientific and medical opportunities of the study:

If successful, this work will validate an intravitreal injection as a route for ocular gene therapy. As the current the approach of sub-retinal injection has many practical limitations, my work will meet an immediate clinical need, which is only likely to grow as the range of ocular disorders amenable to gene therapy increases.

Publications

10 25 50
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Milosavljevic N (2016) Chemogenetic Activation of ipRGCs Drives Changes in Dark-Adapted (Scotopic) Electroretinogram. in Investigative ophthalmology & visual science

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Cehajic-Kapetanovic J (2018) Efficacy and Safety of Glycosidic Enzymes for Improved Gene Delivery to the Retina following Intravitreal Injection in Mice. in Molecular therapy. Methods & clinical development

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Cehajic-Kapetanovic J (2015) Restoration of Vision with Ectopic Expression of Human Rod Opsin. in Current biology : CB

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Cehajic-Kapetanovic J (2018) Efficacy and Safety of Glycosidic Enzymes for Improved Gene Delivery to the Retina following Intravitreal Injection in Mice. in Molecular therapy. Methods & clinical development

 
Description Centenary Award
Amount £21,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 10/2015
 
Description Clinical Research Training Fellowship
Amount £225,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2010 
End 10/2014
 
Description Confidence in Concept
Amount £75,000 (GBP)
Organisation Medical Research Council (MRC) 
Department MRC Confidence in Concept Scheme
Sector Academic/University
Country United Kingdom
Start 07/2014 
End 07/2015
 
Description Keeler Scholarship
Amount £15,000 (GBP)
Organisation Royal College of Ophthalmologists 
Sector Learned Society
Country United Kingdom
Start 10/2014 
End 10/2015
 
Description Research Grant
Amount £10,000 (GBP)
Organisation University of Manchester 
Sector Academic/University
Country United Kingdom
Start 01/2012 
End 10/2014
 
Description Research Grant
Amount £68,000 (GBP)
Organisation Retinitis Pigmentosa Fighting Blindness 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 10/2015
 
Description Developing gene therapy via vitreous 
Organisation University of California, Berkeley
Department Helen Wills Neuroscience Institute
Country United States 
Sector Academic/University 
PI Contribution Discussion stage
Collaborator Contribution Discussion stage
Impact Discussion stage
Start Year 2012
 
Description Optogenetic therapy 
Organisation University of California, Berkeley
Department Department of Molecular & Cell Biology
Country United States 
Sector Academic/University 
PI Contribution Currently working at University of California Berkeley, CA on an optogenetic project with Professor John Flannery
Collaborator Contribution Currently working at University of California Berkeley, CA on an optogenetic project with Professor John Flannery
Impact Dickinson Trust Award to help with the costs of the proposed visit Retinitis Pigmentosa Fighting Blindness - scholarship award to cover salary costs for this project at Berkeley MRC Centenery Award to help with the consumable costs of the project at Berkeley
Start Year 2012
 
Description Optogenetics 
Organisation University of California
Country United States 
Sector Academic/University 
PI Contribution Working together in a partnership to develop novel optogenetic tools for the treatment of inherited retinal degenerations
Collaborator Contribution Hosted a year of post-doc work / experience to develop these tools at University of California, Berkeley.
Impact Presentations of findings of the research at an International Retina Conference in Dublin, 2015.
Start Year 2014
 
Title Method for Treatment of Retinal Disease 
Description Optogenetic method for treatment of Retinal Degenerations 
IP Reference WO2015128624 
Protection Patent application published
Year Protection Granted 2015
Licensed Yes
Impact IP licencing in progress with an industrial partner, Acucela to develop optogenetic therapy for the treatment of retinal degenerations in humans.
 
Description Manchester Museum A level Study Day 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Schools
Results and Impact Organised and took part in "Engaging with Experts" series - A level study day: Discovering Diabetes- Diabetic Complications Dragons Den and Biomarker Discovery and 'Meet the Scientist' Q & A activity, Oct 2012 and March 2013 and November 2013
30-60 students attending each day

Excellent feedback and demand for future same events
Year(s) Of Engagement Activity 2012,2013
 
Description Manchester Museum A level study day 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Schools
Results and Impact 50 A level students (year 1 and 2) attended a study day/workshop called "Discovering Diabetes" which was organized by the members of our laboratory (including myself). This was in order to engage students in scientific research, teach them about patho-physiology and complications of a particular disease relevant for their studies and make them aware of the ongoing cutting edge research in our laboratory. In addition this was a great opportunity for students to find out more about careers in science, medicine and research.

Extremely successful event - as identified from the feedback from students and organizers. Schools have asked for more alike events. We have now organized a similar event to take place on March 23, 2013 and are looking into different funding opportunities to make this into a regular bi-annual event.
Year(s) Of Engagement Activity 2012