Developing rhodopsin gene therapy to treat dominant retinitis pigmentosa

Lead Research Organisation: University of Oxford
Department Name: Clinical Neurosciences


Affecting approximately 1 in 4000 of the population, retinitis pigmentosa (RP) is the most common genetic cause of irreversible sight loss within the developed world. Individuals with RP are born with normally functioning eyes but go on to develop progressive loss of vision in the form of night-blindness, loss of peripheral vision (resulting in 'tunnel vision') and in more severe cases, reduced central clarity and/or sight. This is a highly variable condition that progresses at different rates within different individuals: some develop functionally apparent visual impairment in childhood whilst for others, this may not become manifest until later in life. This variability reflects the large number of genetic mutations ('mistakes' within the genetic code) that can result in the clinical syndrome of RP. By far the most common set of gene mutations to cause RP are those which interfere with the protein rhodopsin. This protein plays a central role within rod photorecepor cells, the functional units or 'pixels' of the retina (the light sensitive layer at the back of the eye). These cells convert light into a meaningful message to convey to the brain thereby mediating the sense of sight. Mutations in the rhodopsin gene in 'dominant' RP (which is the most frequently occurring form) lead to the production of a defective rhodopsin protein within all rod cells of both eyes of the individual. The presence of this abnormal protein causes a degeneration of the retina and gradual sight loss.

The aim of this PhD is to develop a new treatment strategy for dominant RP. The suggested approach involves inserting additional healthy rhodopsin genes into the defective rods. This gene encodes the normally functioning rhodopsin protein. It is anticipated that through this gene transfer, rod cells will produce a greater quantity of healthy protein which should overcome the deleterious effects of the mutant rhodopsin, thus preserving rods and maintaining vision. Delivery of the gene to the retina will be performed with a specially engineered virus (AAV) containing the healthy rhodopsin gene. The virus, which has no capacity to replicate and in itself cause disease, will be delivered to rod cells through an injection underneath the retina. This strategy has previously proved successful in restoring vision in a number of mouse models of retinal degenerative disease and in early human clinical trials.

In my PhD project, I will aim to develop a new AAV vector to optimise the delivery of healthy rhodopsin in mouse models of human dominant RP. This is important as an efficient viral vector will allow fewer viral particles to be delivered (thus minimising any potential side-effects), whilst allowing for the high levels of healthy rhodopsin that are likely to be required to rescue the degenerating retina in dominant RP. Having developed the optimised AAV, I will first test its effect in normal mice to establish the safe dose range, and in mice lacking rhodopsin to demonstrate functional efficacy of the inserted gene. I then plan to test the new virus at its optimised dose in human retinal tissue (from consenting patients who have undergone retinal surgery for unrelated reasons), and in a mouse model of dominant RP to assess the success of this strategy in preserving retinal structure and function. It is my hope that this work will form the basis for a clinical trial in human subjects with dominant RP and that I can be active in overseeing this transition in the years following my PhD. The ultimate goal of this project is thus to develop a genetic technology which may slow or even halt the progression of this hitherto untreatable and debilitating disease. It has the potential to impart a significant improvement in quality of life and ability to work for many thousands of individuals with RP across the world.

Technical Summary

Retinitis pigmentosa (RP) is the most common genetic cause of irreversible sight loss in the developed world with the dominant form being the most frequent. Around 25% of cases of dominant RP are caused by mutations in the rhodopsin gene. As a single gene disorder involving a small protein, gene therapy using an adeno-associated virus (AAV) vector (which has been used successfully in a number of mouse models of retinal degenerations and Phase 1 human clinical trials) may represent a viable treatment option for RP. Recent evidence has shown that gene augmentation may be of benefit in treating dominant conditions- the assumption being that augmentation of the wild-type allele may increase the ratio of wild-type to mutant gene at the mRNA level, leading to reduced mutant protein. Rescue of dominantly inherited RP by over-expression of the wild-type gene (but without direct suppression of the mutant gene) has been demonstrated in the P23H-rhodopsin mouse, in which one allele contains a human rhodopsin variant that causes RP (Mao et al., 2011).

The purpose of this proposed PhD (DPhil) project is to develop an efficient AAV vector that expresses the human rhodopsin gene to a sufficient level that might be applicable for a follow-on clinical trial in patients. The first phase will involve the generation of an AAV rhodopsin vector by assessing the optimal AAV capsid combinations and DNA cloning sequence to produce highly efficient rhodopsin expression. This will be achieved through the use of self-complementary double stranded DNA. In the second phase, I will assess the effect of this vector in wild type and rhodopsin knock-out mice to ascertain the optimal dosage. The final third phase will consist of an analysis of gene silencing activity of the optimized vector in the P23H or P347S rhodopsin mouse model of dominant RP and in human retinas in vitro. It is expected that all data generated from this project would be submitted for future Phase I clinical trial approval.

Planned Impact

1. Quality of life of patients currently afflicted with incurable forms of blindness.
2. Cost to the NHS of patients currently afflicted with incurable forms of blindness.
3. Cost to the economy of patients currently afflicted with incurable forms of blindness.

Benefit to patients
The proposed technology is directly relevant to preserving the sight of patients who are gradually going blind from photoreceptor loss caused by retinitis pigmentosa (RP). Affecting approximately 1 in 4000 individuals, RP is the most common genetic cause of sight loss in the developed world and at present there is no treatment available. The disease process commonly begins early in life and thus has a dramatic impact on quality of life and ability to work for many individuals with the disease. It is highly likely that patients with RP will benefit directly through the development of a clinical trial at the end of this project. Professor Robert MacLaren has a strong track record of developing laboratory research into new treatments for patients who are blind with incurable retinal disease.

Benefit to the NHS
The cost of blindness is significant and is largely borne by the government in the UK. The cost of a guide dog is approximately £50,000 for 12 years and the cost of an electronic retinal implant is in the region of £100,000 based on the Second Sight device. A gene therapy which halts or slows down photoreceptor loss may not only be more effective in the long run but it is possible that it may become more affordable if the technology is more widely adopted.

Benefit to the economy
The United Kingdom already occupies a leading international position in biotechnology research. Successful results in NHS funded clinical trials led by Professor Robert MacLaren have been reported around the world and have further highlighted the important role of UK research in developing new treatments for blindness. The high-profile research in Oxford further underlies the reputation the UK holds as a ctre of excellence for biomedical
Description Graduate Research Award, Merton College, University of Oxford
Amount £1,500 (GBP)
Organisation University of Oxford 
Department Merton College
Sector Academic/University
Country United Kingdom
Start 01/2018 
End 01/2018
Title nrl.GFP.RHO-/- Mouse Line 
Description As part of my research I have generated a transgenic mouse line nrl.GFP.RHO-/- which expresses GFP in rods which themselves degenerate due to homozygous knockout of the rhodopsin gene. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This transgenic mouse model is anticipated to allow the retinal degeneration associated with knock out of the rhodopsin gene to be quantified in vivo using the scanning laser ophthalmoscope and should represent a good model to compare efficacy of various rhodopsin adeno-associated viral vectors. 
Title Mirtron-mediated gene therapy 
Description Development of mirtron-based gene therapy for the treatment of dominant diseases. Two patent applications have been filed on behalf of the University of Oxford by Oxford University Innovations to protect the intellectual property associated with this technology. One relates to the use of artificial mirtrons in the 5' untranslated region of an adeno-associated virus-based construct, whilst the second applies to the specific application for the treatment of autosomal dominant retinitis pigmentosa caused by mutations in the rhodopsin gene. 
Type Therapeutic Intervention - Cellular and gene therapies
Current Stage Of Development Refinement. Non-clinical
Year Development Stage Completed 2018
Development Status Under active development/distribution
Impact Still under development in the laboratory. 
Description Cafe Scientifique (Basingstoke, UK) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Cafe Scientifique events generally talk the form of an invited speaker talking at regional meetings on scientific concepts aimed at the general public. i was invited to deliver one such talk on retinal gene therapy at the Basingstoke branch. It consisted of a 45 minute lecture followed by a 20 minute question and answer session. Around 50 people attended the event and the talk was also filmed for wider online circulation. The audience were highly engaged and posed some insightful questions. The talk received a 5 star rating on the Basingstoke Cafe Scientifique website with one attendee commenting "Excellent, thank you!".
Year(s) Of Engagement Activity 2017
Description Invited Lecture at Therapeutics Manchester Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited to give a lecture at the Therapeutics Manchester Conference organised by the Association of Optometrists on 24th June 2018. This meeting was for optometrists interested in prescribing/therapeutics. Lecture title: "New and Emerging Treatments for Inherited Diseases of the Retina". Around 70 participants.
Year(s) Of Engagement Activity 2018
Description Micro.X Conference Panel Member 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Invited to the Oxford University medical School's Microsurgical Conference ("Micro.X") to sit on a panel discussing aspects of clinical academia. Also acted as poster judge and demonstrated at workshops throughout the meeting. Feedback from the organisers was very positive:

"Dear Harry,

Thank you for speaking on the panel, judging the posters and running the techniques in ophthalmology workshop at Micro.X2017.

Several of the attendees have mentioned how much they enjoyed both your panel discussion and workshop, and I am sorry to have been unable to see either of them in full. The opportunity to learn skills used in ophthalmology was highly praised by attendees, and much of that was down to your enthusiasm in teaching them. It was wonderful that you could stay for the afternoon, and I hope you had a very enjoyable weekend.

We hope to run Micro.X next year, making it both bigger and better. We would greatly appreciate your feedback about the event."
Year(s) Of Engagement Activity 2017
Description RP Fighting Blindness Seminar (Oxford, UK) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact Invited to deliver a talk on Gene Therapy for Inherited Retinal Diseases during a seminar organised for visitors from the charity RP (Retinitis Pigmentosa): Fighting Blindness. The talk was very well received and stimulated much discussion from the audience.
Year(s) Of Engagement Activity 2016
Description Regional Teaching for Ophthalmology Registrars 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Invited to deliver a talk at a regional teaching seminar for Ophthalmology trainees in the Oxford Deanery on my research. The talk was well received.
Year(s) Of Engagement Activity 2017
Description School Visit (Repton, UK) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Surgery & Science for Saving Sight': A 40 minute invited lecture delivered to the Medical Society of Repton School, Derbyshire. The talk covered careers in Ophthalmology as well as aspects of my own research and that of the MacLaren group as a whole with regards to the development of gene therapy treatments for inherited diseases of the retina. Feedback from students and teachers at the school was universally very positive.
Year(s) Of Engagement Activity 2017