Using zinc finger nuclease technology to generate reporter-labelled human pluripotent stem cells as a tool to optimize photoreceptor transplantation

Pluripotent stem cells are unspecialized cells that can be grown in the laboratory and programmed to become specialized cells of a desired type, such as blood cells, muscle cells etc. Human pluripotent stem cells can be derived in different ways, from very early embryos when they become available as surplus products during in vitro fertilization, or more generally by re-programming easily accessible cells from individuals, such as from a sample of skin cells. This possibility has led to great interest in using stem cells for therapeutic applications to treat disorders caused by loss of cells of a particular type. For example, blindness due to loss of retinal cells could in principle be treated by taking a sample of skin cells from the patient, re-programme the skin cells to make unspecialized pluripotent stem cells and then programme the resulting stem cells to give retinal cell progenitors that can be grafted into the patient's eye to give rise to retinal cells. One of the difficult problems in differentiating human pluripotent stem cells is to track the cells as they change into specialized cells and then to purify the desired specialized cells. This can be done relatively easily for mouse pluripotent stem cells by inserting genes that make reporter molecules tagged with a fluorescent that makes the cells glow under suitable conditions, but the method is very inefficient in human pluripotent stem cells. A new cutting edge technology now offers a potential solution. Zinc finger nucleases are artificially created scissors that can be designed to specifically cut both strands of DNA molecules at just one specific location. These nucleases create a gap in the DNA structure which activates the cell's response for DNA repair. Upon presence of a short DNA stretch which shows similarity to the region containing the excision but also harbouring the reporter gene, it is possible to introduce the reporter gene into the gene of interest in human pluripotent stem cells. This technology is very recent and has only been applied twice in human pluripotent stem cells; however the efficiency has been much higher than other reported methods and as such the potential applications are immense. In this proposal we seek to implement this technology to create labelled human pluripotent stem cells lines that will be used as tools to optimise cell transplantation into the degenerate retina. The retina has a very complex structure consisting of several layers of neurons that are interconnected with each other. The two main cell types that are directly sensitive to light are the rod and cone photoreceptors cells. Our group has shown that it is possible to produce human cells that have the characteristics of cones and rods from human pluripotent stem cells. Despite this progress, we are not able to select these cells amongst other cell types that arise during differentiation process. Normally cell selection is achieved using a technique called fluorescence activated cell sorting (FACS). The different cells of the body have specific proteins on their surface to which antibodies tagged with coloured or fluorescent molecules can bind, allowing us to identify and sort them using FACS, however, there are few such markers that can be used for isolating cones and rods. We intend to introduce a reporter into an important retinal gene that marks their differentiation to cone and rods. The presence of the fluorescent reporter will allow us to use the cell selection strategy mentioned above to purify these cells. We can then ask the question of whether these cells exhibit the properties associated with rods and cones using a variety of in vitro stem cell assays and electrophysiological analysis. If successful, this approach will allow us to prospectively isolate rod and cone cells, define their molecular phenotype and test their ability to restore vision in animal models of retinal disease.

Blindness arising from outer retinal degeneration affects 314 million people worldwide. To date there are no effective treatments that can reverse the primary pathological abnormalities of retinal degeneration. For these reasons there is an emerging need for research into the replacement and/or reactivation of dysfunctional photoreceptors. New photoreceptors only make single synaptic connections to the inner retina, thus retinal repair by cell transplantation is a feasible approach. The only cells that have been shown to integrate successfully into host retina and restore vision are retinal cells that are already committed towards a photoreceptor fate. An equivalent human donor cell would have to be obtained from the second trimester embryo which raises ethical concerns and hence attention has been focused on differentiation of human pluripotent stem cells. Work carried out by our group and others has shown that it is possible to direct human pluripotent stem cell differentiation to cells that express photoreceptor markers in vitro, however their integration into the compromised retina in vivo is poor. This raises the question of whether the transplanted cells have matured to the correct ontogenetic stage that is needed for an efficient integration. To address this question we propose to use zinc finger nucleases to construct labelled human pluripotent stem cell lines in which a reporter gene is introduced into the endogenous loci of a key retinal transcription factor CRX shown to be expressed in postmitotic photoreceptor precursors and vital for their cell fate determination. This approach would enable isolation of photoreceptor precursors from human pluripotent stem cells, testing of their transplantation potential and full characterisation of their transcription profile with the aim of identifying cell surface markers that can be used for their easy isolation prior to transplantation.

Blindness is often assumed to be avoidable or reversible with modern medicine, however many causes of blindness remain untreatable. Severe visual loss has a very significant impact on quality of life leading to social isolation and depression. Perhaps surprisingly the impact of significant visual impairment on an individual's quality of life is similar to that encountered with such devastating diseases as catastrophic stroke and cancer. Two of the most prevalent causes of blindness, namely age related macular degeneration (AMD) and hereditary retinal dystrophies (HRD), share a final common pathway of irreversible visual loss resulting from outer retinal degeneration with photoreceptor loss. AMD in particular is the most common cause of blindness in the western world. It is estimated to currently affect over 400,000 people in the UK and 8 million worldwide with a predicted increasing prevalence as the population ages and developing countries adopt the western lifestyle and diet. Although treatments are evolving for early AMD there is currently no cure, and treatments are largely palliative. Unfortunately clinical experience shows that patients are frequently seen with advanced disease, impaired vision and established outer retinal degeneration. HRD, although less prevalent, have a high health impact often causing profound visual loss from an early age. Until treatments are found that can reverse the primary pathological abnormalities in AMD and HRD, there is a need for research into the reactivation and replacement of non-functioning and lost cells within compromised outer retinal circuitry, whilst, critically, the inner retina is still intact. Transplantation of retinal progenitor cells into the eyes of such patients is a plausible and achievable treatment. Substantial evidence suggests that if such cells can be isolated and differentiated to the optimum stage for integration into the retina, then transplantation could result in the restoration of visual function. Quite apart from the obvious benefit to the patient, the ability to restore sight in these patients would have enormous social and economic benefits following the cessation of their long term palliative care. Indeed AMD is not only a major public health problem that has a devastating effect upon patients; it also has marked adverse financial consequences for the economy. An economic analysis based upon losses to gross domestic product in the USA suggests that AMD alone has an approximate $30 billion annual negative impact. The return on investment is therefore potentially high for the research costs invested in the development of new treatment modalities.