Developing an embryonic stem cell-based cell product for age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) remains a major cause of blindness with no current cure. With the increasing ageing population in the developed world, a high number of AMD patients is expected. AMD can be distinguished between dry or wet AMD. 10-15% of patients present wet AMD which can be treated with anti-VEGF injections. In contrast, dry AMD management has just recently advanced towards a new drug to treat and to halt the progression of the disease by targeting the complement system. However, we are still far from cure of AMD and more efforts are needed towards development of stable and long-term treatment. With the lack of conventional drug options, the field has moved into the investigation of stem cell-derived therapies. In the case of AMD, the cells that degenerate are retinal pigment epithelium (RPE) cells. For a decade, the lab has worked towards the development of a xeno-free, chemically defined, and scalable protocol to generate stem cell-derived RPE. This is achieved whereas translation into the clinic remains. Within my PhD studies, I have performed the following studies:

Firstly, by using single-cell RNA sequencing (scRNAseq) we explored the different cell types that emerged during in vitro differentiation towards hESC-RPE at various time points. The transcriptomic profiling showed a diversity of cells recapitulating the early embryonic development in the first weeks of gestation, showing genetic expression of neural crest, placodal and mesenchyme. The differentiation protocol has a replating step in the middle of the protocol, that induced a rapid move of the cell’s profile towards a pure RPE population which by the end of the protocol matured further. At the replating step, cells could be driven to other lineages with the selection of NCAM1 positive population showing the potential of this retinal progenitor’s marker. Thus generated mature, pure, and functional hPSC-RPE were transplanted into rabbit’s eyes, a largeeyed animal model. After 28 days, transplanted cells were retrieved and scRNAseq was performed revealing that those cells were able to mature even further in vivo.

Secondly, cryopreservation offers a highly important step in the manufacturing process of a cell product. Cryostorage offers flexibility between the manufacturing location, the testing of the product, and the patient availability. It also confers the ability to produce larger batches, reducing the costs of manufacturing. After the discovery of the incapability of our hPSC-RPE to cryopreserve, we introduced and optimized an extra replating step, regarding time and densities, to be able to successfully cryopreserve our drug product. This replating step didn’t impact the identity of the hPSC-RPE, since after preservation it had characteristic RPE traits and preserved functionality. Transcriptionally, the replated hPSC-RPE presented a cycling profile and a shrinker maturation profile compared to the terminally mature hPSC-RPE obtained in the original protocol. A noninvasive tool is presented to track the optimal replating time window before cryopreservation based on brightfield images based on the cobblestone morphology of the cells.

Lastly, we addressed translation towards the clinic. We proceeded to good manufacturing practice (GMP) adaptation of our protocol that introduced slight differences to improve cost-effectiveness, scale up the manufacture, and introduce large-batch instruments into our process. We successfully completed one engineering and one GMP batch in the Cell Therapy Center of Karolinska University Hospital and cryopreserved them. Most have been used in the extensive release testing, stability program, and preclinical testing presented here. Conventional release testing was complemented with newly developed and validated assays including a more sensitive in vitro assay for lingering pluripotent stem cells or transformed cells. Dose precision and in-use stability studies were performed to mimic the surgical setting for suspension injections. Route of administration feasibility was tested in nude rats, evaluating subretinal injection through transscleral or transvitreal route, revealing that the latter minimized the risk of procedure-related complications. The 28-days toxicology studies in nude rats showed no human cells outside the eyes. No unexpected pathological outgrowth or clinical symptoms were observed in the nude rats dosed with the drug product. Efficacy studies in Royal College of Surgeons (RCS) rats for 90 days after injections show reduced degeneration of the neuroretina of the rats. Optokinetic response and electroretinography showed higher light sensitivity in dosed animals than in vehicles, showing functionality of the implant. Further application to the Swedish Medical Agency is planned for late this year.

Taken together, the studies presented in this thesis represent a step forward toward the first stem cell-based therapy to treat AMD in Sweden. I) An unbiased characterization of our hPSC-RPE differentiation protocol is presented showing all cellular composition through the different stages by scRNAseq. II) An optimization step to produce large batches and cryostore the hPSC-RPE cells for flexible manufacturing and delivery of cells. III) Our drug product, CellThRPE1, has been extensively tested and is ready for regulatory review prior to entering the clinical phase.