Optimisation of human ESC-derived photoreceptor cell differentiation

Retinal degenerations, in which the light-sensing photoreceptor cells of the eye are lost, are a major cause of untreatable blindness in the UK. Inherited sight loss affects 1 in 3,000 of the population, and age-related macular degeneration (AMD) affects 1 in 10 people over 60 yrs. Currently there are no treatments that can restore sight and thus there is a need to develop new therapeutic approaches. As the majority of the neural connections remain following retinal disease, new photoreceptors need only make short, single connections to the remaining retinal cells to contribute to vision. Therefore, retinal repair by the transplantation of new photoreceptors to the diseased retina represents one of the most feasible types of CNS repair.
We have previously made important breakthroughs regarding cell transplantation therapy for retinal repair. In landmark studies, we have demonstrated that photoreceptor cells can be transplanted into an adult retina and restore vision in animal models of retinal degeneration, provided the photoreceptor cells are at the correct stage of development. It is not practical to develop treatments based on obtaining photoreceptor cells from human fetuses and we therefore need to generate photoreceptor cells in the laboratory from human embryonic stem cells. Following the discovery by other groups that embryonic stem cells can spontaneously self-organise and develop into a retina if placed in the right conditions in a three-dimensional culture system, we have shown that we can produce photoreceptors that are suitable for transplantation from mouse embryonic stem cells grown in a dish. In this project we propose to develop a methods to generate, purify and store human photoreceptor precursors that are suitable for transplantation from clinical-grade human embryonic stem cells in order to determine the best cell lines and protocols for clinical application. This project will provide essential underpinning for the development of future clinical trials and the translation of our stem cell therapy programme into the clinic.

Retinal degenerations with loss of photoreceptors are the leading cause of blindness in Europe. As there are no treatments that restore lost photoreceptors and vision, there is a clear need for new therapeutic approaches. In 2006 we showed that photoreceptor can be transplanted into the adult retina and recently we have been able to provide the first definitive evidence of restoration of rod-mediated vision, following transplantation of rods into a model of stationary night blindness. Importantly, we have also recently demonstrated that following transplantation, mESC-derived rod photoreceptors are able to integrate, mature & connect with the adult host retinal circuitry. Thus our research to date has established principles of effective transplantation using donor and mESC-derived photoreceptors. Whilst we demonstrated that retinal repair by photoreceptor transplantation is feasible, for clinical application we need to develop protocols for generating & transplanting human photoreceptors from a renewable source. In this project we aim to develop a defined and xeno-free differentiation system to generate and store photoreceptors from clinical grade hESC lines that will support the development of a clinical trial in retinal dystrophy patients. We will
(i) develop xeno-free protocols for hESC lines
(ii) grow EUCTD-grade hESC lines in the optimal conditions established in (i)
(iii) modify our hESC differentiation protocols to be compatible with clinical application
(iv) characterize the development of xeno-free EUCTD-grade hESC-derived photoreceptors & utilise viral vectors and CD markers to purify photoreceptor precursors for transplantation
(v) transplant photoreceptor precursors generated from EUCTD-grade hESC lines; (vi) develop cryopreservation protocols for photoreceptor & determine their survival after thawing
(vi) determine which EUCTD-grade hESC lines are the most effective for generating photoreceptor that can be transplanted following cryopreservation

Hereditary retinal disease and age related macular degeneration (AMD) leading to photoreceptor loss are the leading causes of untreatable blindness in the UK. Inherited retinal degenerations affect 1 in 3000 of the population whilst AMD affects 1 in 10 people over the age of 60 and its prevalence is increasing. AMD is responsible for over 50% of blind registrations in the UK in patients over 65 years of age. At present, there are no available treatments that offer anything more than a temporary slowing of disease and even then to only a minority of patients. The increasing socio-economic burden of AMD provides an urgent imperative for developing new therapies.
Stem cell therapy for the treatment of blindness generates considerable excitement within the international research community, in government and amongst the wider public. We have recently discovered that transplantation of immature photoreceptors improves rod vision in mouse models of retinal degeneration. These studies provide the first proof-of-concept for effective transplantation of neurons and provide the basis for embryonic stem cell (ESC)-derived photoreceptor transplantation. Importantly, we have also demonstrated the generation of ESC-derived rod photoreceptors that following transplantation into the adult retina, are able to integrate and connect with the host retinal circuitry. These discoveries provide a strong basis for the translation of ESC-derived photoreceptor cell therapy for retinal disease. The proposed project aims to build on our discoveries and to develop human ESC-derived photoreceptor replacement therapy for the treatment of retinal diseases and the restoration of vision. By the end of this 4 year project we aim to have developed GMP-compatible defined, and xeno-free differentiation system to generate and store photoreceptor precursors derived from clinical grade human ES cell lines that will support the subsequent development of a clinical trial in patients with retinal dystrophy. The development of GMP-compliant protocols should be patentable and we will seek to file IP. This should then enable us to attract industrial partners to develop the technology further.