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  • Mid-Term Report Summary - RET-IPSC (Exploiting the power of human induced pluripotent stem cells to generate synthetic retinae in vitro for cell based therapies, drug discovery and disease modelling)

RET-iPSC Report Summary

Project ID: 614620
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Mid-Term Report Summary - RET-IPSC (Exploiting the power of human induced pluripotent stem cells to generate synthetic retinae in vitro for cell based therapies, drug discovery and disease modelling)

Blindness has a significant impact on the quality of a person’s life often resulting in depression, social isolation and premature death. This poses a major burden to society due to lost productivity and earnings as well as the costs of treatment, rehabilitation and education of the visually impaired and provision of visual aids. Recent estimates indicate that the overall number of people with sight loss is 285 million globally, of whom 39 million are blind. Diseases affecting the outer retina including age related macular degeneration (AMD) and inherited retinal dystrophies (HRDs) account for approximately 26% of blindness and the number affected is expected to double by the 2020 due to ageing of the world’s population. Although in some cases, the initial trigger event is the degeneration of retinal pigmented epithelium (RPE, a monolayer of pigmented cells forming part of the blood/retina barrier), the final impact in both AMD and HRDs is the loss of photoreceptors, a specialised type of photosensitive neurons that are capable of photo-transduction. Whilst there are a number of agents (including high dose antioxidants, neuronal survival agents, vascular endothelial growth factor inhibitors etc) that have been shown to slow disease progression, to date there are no treatments to restore lost photoreceptors and visual function, hence there is a pressing need for research into the replacement and/or reactivation of dysfunctional photoreceptors and RPE. The feasibility of this approach is supported by the fact that new photoreceptors need only make a single synaptic connection with the inner retina. The eye is well suited for development of cell transplantation therapies as it is easily accessible and allows local application of cells with reduced risk of systemic effects. To date most of the clinical trials for treatment of eye disease have focused on gene therapy; however for cases that are not amenable to gene therapy or where substantial death of photoreceptors and RPE has already occurred, cell replacement therapies offer an attractive approach. In January 2011, Advanced Cell Technology in USA received FDA clearance to perform a phase I/II multicentre clinical trial to treat dry AMD patients with RPE cells derived from human embryonic stem cells (hESC). In parallel, Japan has embarked on clinical trials of AMD using his pioneering technology of induced pluripotency. Although the long term outcomes of these trials are eagerly awaited, early results from the ACT study have underlined the safety and tolerability of these cells for clinical trials and have set the scene for pioneering new therapies for retinal disease.

Human pluripotent stem cells is the common term used to describe two types of pluripotent stem cells characterised by indefinite self-renewal ability and the capacity to give rise to any cell type in the adult: hESC and human induced pluripotent stem cells (hiPSCs) . hESC are derived from spare in vitro fertilised embryos after parental consent and have been widely used in the last decade as a generic tool to understand maintenance of pluripotency, human embryonic development and congenital disease. Destruction of human embryos for research purposes is surrounded by a number of ethical issues, prohibiting hESC derivation and application in several countries. However the main issue related to their application is the evidence that their differentiated progeny express human leukocyte antigens (HLAs) that will probably result in graft rejection and could be bypassed only by creation of HLA-typed hESC banks, from which a best match can be selected. Human iPSC bypass both of these issues as they are generated by reprogramming somatic cells back to the pluripotent state akin to embryonic stem cells. As such, these cells share all the characteristics of hESC including the ability to proliferate indefinitely and differentiate into many cell types, but also represent a source of autologous stem cells for cell replacement therapies given that they are derived from the patients themselves.

In 2011, a pioneering breakthrough highlighted the possibility of generating synthetic retinae from pluripotent stem cells under laboratory conditions, a finding with immense relevance for basic research, in vitro disease modelling, drug discovery and cell replacement therapies. Soon after, my group was able to develop a unique method that resulted in conversion of hESC cells to fully laminated retinae containing all retinal cell types under laboratory conditions. This research is very exciting, however it needs to be developed so human synthetic retinae containing functional photoreceptors can be produced at high efficiency and reproducibly from a large number of patient-specific pluripotent stem cells. This forms the key goal of this ERC funded proposal which is organised to achieve three main objectives: (1) to design robust differentiation protocols that result in efficient differentiation of hiPSC to fully laminated synthetic retinae in vitro, containing sufficient numbers of functional photoreceptors; (2) to construct genetic tools and marked hiPSC lines to enable identification of photoreceptor precursors that are optimal for transplantation and can give rise to new photoreceptors in the degenerate retina, contributing towards restoration of vision and (3) to identify cell surface markers that will enable the enrichment of functional photoreceptor precursors arising from hiPSC differentiation for therapeutic use.

To date we have made great progress in several areas of this project which has resulted in publication of nine directly related manuscripts. In particular, we have optimised each step of differentiation process in a large number of hiPSC and are now able to direct with great efficiency the generation of photoreceptor precursors within a window of 35 days as well as produce laminated retina containing all retinal cell types and responding to electrophysiological stimuli as early as day 100 of differentiation process. We can further enhance the generation of laminated retina by encapsulating the developing optic vesicles within hydrogels. We have identified a number of key extracellular matrix components (ECM) which are present during development of human retina but are missing from our hiPSC derived retinae. Our ongoing work is aiming at adding these to the differentiation process to enhance its efficiency and ability to generate hiPSC derived retinae that are as close as possible to the human retinae. We are in a fortunate position to obtain human embryonic and fetal retina sample (after appropriate consent) from the Human Developmental Biology Resource (HDBR) which we use as gold comparative standard to evaluate the morphology, cellular composition and functionality of hiPSC derived retina. We are using RNA-seq methodology to perform these comparisons and to draft an early atlas of human embryonic and fetal retinal development in the next 12-18 months.
A key question for our work is whether hiPSC derived retinae contain functional photoreceptors that can engraft in demised retina and contribute to restoration of vision. Since these retina develop as three dimensional structures with close interactions between various retinal cells types (akin to development), it is essential to develop strategies for purification of photoreceptor precursors with ease. To date we have generated pluripotent stem cell lines which harbour a fluorescent reporter (GPF) under the control of a gene which is expressed in photoreceptor precursors (CRX). This strategy has enabled us to select a large number of single photoreceptor precursors which we are now comparing to equivalent cells from developing human retina by RNA-seq. Furthermore, we are in the process of constructing hiPSC with multiple reporters under the control of genes involved in determining the eye field, photoreceptor precursors and mature rods or cones. This approach will allow to assess and enhance our differentiation process and to track the emergence of these cellular subpopulations during the differentiation process and engraftment into normal and demised retina. Whole transcriptome analysis at single photoreceptor precursors by RNA-seq are also enabling us to pinpoint important proteins that are expressed at the cell surface. We will validate these proteins in samples of developing and adult human retina. Our intention is to select a subset of these cell surface markers to enrich for photoreceptor precursors prior to transplantation studies.
In parallel to the molecular work, we will start subretinal transplants of hiPSC derived photoreceptor precursors early in 2017. We have purchased the necessary equipment and have arranged staff training visits in our collaborator’s lab (Prof. Marius Ader in Dresden) as well as obtained the necessary permission to work with animal models of retinal disease. Our aim is to test the engraftment capacity of hiPSC derived photoreceptor precursors at different stages of differentiation process to pinpoint the most optimal stage for transplantation. Once this proof of principal work is completed, we intend to apply for funds to develop a pre-clinical project which will ensure all the components of our differentiation protocols and procedures = are compatible with Good Manufacturing Practice Protocols and are amenable to scale up. This is an important and necessary step before moving to Phase 1 clinical trials which we are committed to developing here in Newcastle in collaboration with our Cell Therapy Facility and retinal clinician, Mr. David Steel.

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