Validation of a sickle cell disease-specific iPSC platform as a preclinical screening tool Free

Sickle cell disease (SCD), caused by a mutation in the β-globin gene, renders hemoglobin prone to polymerization, resulting in anemia, vaso-occlusive crises and a range of downstream complications. Current treatments for SCD primarily aim to reactivate fetal hemoglobin (HbF) expression to offset the defect in adult hemoglobin. While SCD patients all carry the same point mutation in their (adult) β-globin gene, their genetic background to some extent dictates disease severity and response to treatment. Here, we demonstrate the potential of a SCD-specific induced pluripotent stem cell (iPSC) platform to assess HbF induction potential of various therapeutics across the diverse SCD patient population.

Five SCD patient-derived iPSC lines and a healthy control iPSC line were specified towards the erythroid lineage following our previously established erythroid differentiation protocol and cells were dosed with inducers of HbF, mimicking the main therapeutic strategy for SCD. Flow cytometric assessment of HbF induction showed a clear increase in mean fluorescence intensity for cells treated with Hydroxyurea (HU) and LSD1 inhibitor RN-1 compared to control conditions (H2O and DMSO) for healthy control cells as well as some SCD lines, illustrating that predominant expression of fetal globin in iPSC-derived erythroid cells does not present and obstacle for HbF induction screening.

In line with what is seen in the SCD patient population where the response to HU differs between patients, we noted a differential response to HbF-inducing agents across SCD patient backgrounds. Three SCD patient lines failed to show HbF induction whereas healthy control cells and 2 other SCD lines consistently showed robust HbF induction (n=4-10). To verify that this result did not merely reflect insufficient dosing, we increased the HU dose from 50uM (standard dose reported in literature) to 200uM. SCD lines that did not show increased HbF expression upon dosing with 50uM HU also failed to respond to 200uM HU (n=3-6).

Interestingly, we noted absence of HbF induction in SCD line SS100 upon HU treatment corresponding to the clinical representation of the patient that this line is derived from. Despite long-term adherence to HU, HbF levels in this patient show no significant improvement and they experience >2 severe vaso-occlusive crises per year. An advantage of this platform offering an unlimited supply of cells corresponding to a particular genetic background is that it allows for controlled experiments testing several compounds in parallel for their potential to induce HbF expression. We thus set out to explore if HbF inducers other than HU and RN-1 would be more effective at eliciting a response in a SS100 background. Similar to HU and RN-1, pomalidomide and decitabine resulted in increased HbF expression in healthy control iPSC-derived erythroid cells, in agreement with their effect reported in the literature. In SS100 iPSC-derived erythroid cells however, pomalidomide and decitabine were not able to increase HbF expression, in line with what we observed for HU and RN-1 in this background. Treatment with SR-18292 on the other hand did result in HbF induction in this patient background that did not respond to several other known HbF inducers, suggesting that PGC1α activation via SR-18292 could be a more effective therapeutic strategy than HU for this patient.

These findings illustrate the potential of SCD iPSCs to recapitulate treatment responses corresponding to clinical outcomes in SCD patients, showcasing their utility for patient-specific HbF induction screening and associated treatment recommendations. As such, this SCD iPSC platform expands the preclinical toolbox, adding genetic diversity as an important and often overlooked component.