Developing rhodopsin gene therapy to treat dominant retinitis pigmentosa

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.

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.

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 centre of excellence for biomedical
research.