Developing gene therapy to treat blindness caused by Stargardt Disease

Stargardt disease leads to blindness in young people due to loss of the light sensing cells known as photoreceptors that line the retina at the back of the eye. Stargardt disease is caused by the deficiency of a protein known as ABCA4 which stands for "ATP Binding Cassette subtype A4". This protein is very important for recycling the visual pigments necessary for the light-sensitive photoreceptors. When there is a deficiency of ABCA4, the light-sensitive pigments accumulate in the retina which leads to toxic damage and eventual cell death. Stargardt disease usually presents in childhood and there then follows a period of several years of progressive loss of sight and blindness in many cases. There is therefore a window of opportunity of several years after diagnosis in which to intervene with some form of treatment to prevent sight loss.

Gene therapy is a new technique which has been shown to be highly effective at reintroducing defective genes into the retina. Gene therapy was first used in this manner in 2007 in three clinical trials to treat inheriuted blindess caused by defiency of anothe gene known as RPE65. More recently we have started a new gene therapy treatment in Oxford to treat a photoreceptor disease known as choroideraemia and several patients have been treated so far without adverse effects. In these gene therapy trials we are using a small viral particle known as adeno associated virus, or AAV. This viral particle is one of the smallest lifeforms known and it is not associated with any disease in humans. The virus survives by remaining dormant and undetected by the immune system. We have taken advantage of AAV by removing the viral genes and replacing them with the gene that is missing for the particular retinal disease. The ability of the virus to evade the immune system is very helpful because the lack of inflammation means that the virus does not damage the retina when injected into the eye. The AAV particle is however too small to carry many genes that are missing in certain types of retinal degeneration. Unfortunately the ABCA4 gene, which would need to be replaced to cure Stargardt disease, is just too large to fit into AAV.

The purpose of this project is to solve this problem by exploring a new property which has recently been identified in relation to AAV. It has recently been discovered that a large gene can be broken into two segments each of which is carried by an AAV particle and these particles are able to recombine the gene back into full length after infecting nerve cells. In this project we aim to perform a series of experiments whereby we optimise the process of recombining two fragments of the ABCA4 gene in order to deliver its successfully into photoreceptors. We will validate the function of the gene in a genetically engineered mouse, which is also deficient of the same gene and has features similar to Stargardt disease on retinal examination. If we can correct or even improve the deficiency of ABCA4 using our new vector then we would have the ideal background information to support a new clinical trial.

ABCA4 is a protein expressed in photoreceptor cells which is vital for normal photoreceptor function. Mutations in ABCA4 are the commonest cause of monogenic inherited retinal degeneration and usually present in childhood. Although gene therapy with AAV vectors has been shown to be safe and effective at photoreceptor gene replacement, the ABCA4 coding sequence at 6.7 KB is known to be too large to be packaged effectively into standard AAV vectors. We have recently developed an AAV transgene sequence which includes the full length ABCA4 and various regulatory elements to enhance gene expression. We have developed a method of packaging this transgene in two parts into AAV5 and AAV8 capsid serotypes. Through a complex method of intracellular DNA recombination, transduction with these vectors leads to effective expression of ABCA4 protein in human cells in vitro. We wish to take these vectors along a developmental pathway that would result in a Phase 1 clinical trial to treat Stargardt disease. We have a considerable amount of experience with the processes required by GTAC and the MHRA in gaining clinical trial approval for AAV gene
therapy First in Man studies. The method of delivery of the ABCA4 transgene is technically challenging and several steps are now required to optimise vector efficiency and the targeting of photoreceptors. We propose to test several compounds which may improve the efficiency of recombination within photoreceptors and would therefore reduce the total vector dose administered to each patient. We will also confirm functional activity of the ABCA4 protein in the knockout mouse in our laboratory. Whilst lentiviruses provide an alternative way of delivering large genes, these vectors have additional safety concerns for human use, such as the risk of insertional mutagenesis and also an increased risk of immune reactions. AAV vectors in contrast have been shown to be safe and effective for human retinal gene delivery.

Ultimately patients affected by retinal degenerations associated with deficiency of the ABCA4 gene have the most to gain from this research. This will include children with Stargardt disease, which is the commonest recessive inherited macular degeneration affecting the younger age group. It will also be of benefit to those with cone dystrophy which is a rarer variant of ABCA4 deficiency and also some patients with classical retinitis pigmentosa which can be caused by more profound absence of the ABCA4 gene. Beyond this, patients who have retinal degeneration caused by deficiencies of larger genes such as some types of Usher syndrome could also benefit from the technology, although it would need to be redesigned around a different gene. On an even wider basis, any patients with retinal degeneration which would include age-related macular degeneration, the overall commonest cause of blindness in the developed world, would benefit from any technology which aims to develop a single injection gene therapy treatment, as this could be used to deliver any type of gene. The development of retinal gene therapy using AAV has great potential in the future treatment of many currently incurable diseases that lead to blindness. The technical objectives of the proposed research include identifying an optimal means of recombining to viral particles to deliver a large gene and this may also have implications for developing new treatments for other genetic diseases such as muscular dystrophy and cystic fibrosis, which also depend on successful delivery of larger genes.