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BACKGROUND: Stem cell products are increasingly entering early stage clinical trials for treating retinal degeneration. The field is learning from experience about comparability of cells proposed for preclinical and clinical use. Without this, preclinical data supporting translation to a clinical study might not adequately reflect the performance of subsequent clinical-grade cells in patients. METHODS: Research-grade human neural progenitor cells (hNPC) and clinical-grade hNPC (termed CNS10-NPC) were injected into the subretinal space of the Royal College of Surgeons (RCS) rat, a rodent model of retinal degeneration such as retinitis pigmentosa. An investigational new drug (IND)-enabling study with CNS10-NPC was performed in the same rodent model. Finally, surgical methodology for subretinal cell delivery in the clinic was optimized in a large animal model with Yucatan minipigs. RESULTS: Both research-grade hNPC and clinical-grade hNPC can survive and provide functional and morphological protection in a dose-dependent fashion in RCS rats and the optimal cell dose was defined and used in IND-enabling studies. Grafted CNS10-NPC migrated from the injection site without differentiation into retinal cell phenotypes. Additionally, CNS10-NPC showed long-term survival, safety and efficacy in a good laboratory practice (GLP) toxicity and tumorigenicity study, with no observed cell overgrowth even at the maximum deliverable dose. Finally, using a large animal model with the Yucatan minipig, which has an eye size comparable to the human, we optimized the surgical methodology for subretinal cell delivery in the clinic. CONCLUSIONS: These extensive studies supported an approved IND and the translation of CNS10-NPC to an ongoing Phase 1/2a clinical trial (NCT04284293) for the treatment of retinitis pigmentosa.

Inactivating mutations in the thyroid hormone (TH) transporter Monocarboxylate transporter 8 (MCT8) cause severe psychomotor retardation in children. Animal models do not reflect the biology of the human disease. Using patient-specific induced pluripotent stem cells (iPSCs), we generated MCT8-deficient neural cells that showed normal TH-dependent neuronal properties and maturation. However, the blood-brain barrier (BBB) controls TH entry into the brain, and reduced TH availability to neural cells could instead underlie the diseased phenotype. To test potential BBB involvement, we generated an iPSC-based BBB model of MCT8 deficiency, and we found that MCT8 was necessary for polarized influx of the active form of TH across the BBB. We also found that a candidate drug did not appreciably cross the mutant BBB. Our results therefore clarify the underlying physiological basis of this disorder, and they suggest that circumventing the diseased BBB to deliver active TH to the brain could be a viable therapeutic strategy.

Abstract Background The ability to specifically label proteins within living cells can provide information about their dynamics and function. To study a membrane protein, we fused a multi-functional reporter protein, HaloTag®, to the extracellular domain of a truncated integrin. Results Using the HaloTag technology, we could study the localization, trafficking and processing of an integrin-HaloTag fusion, which we showed had cellular dynamics consistent with native integrins. By labeling live cells with different fluorescent impermeable and permeable ligands, we showed spatial separation of plasma membrane and internal pools of the integrin-HaloTag fusion, and followed these protein pools over time to study bi-directional trafficking. In addition to combining the HaloTag reporter protein with different fluorophores, we also employed an affinity tag to achieve cell capture. Conclusion The HaloTag technology was used successfully to study expression, trafficking, spatial separation and real-time translocation of an integrin-HaloTag fusion, thereby demonstrating that this technology can be a powerful tool to investigate membrane protein biology in live cells.