I3 - Pharmacology/Toxicology Studies and Analytics/Assay Development
237: Potency Assay Enabling Both Ex Vivo and In Vivo Genome Editing Therapeutics for Sickle Cell Disease
Type: Oral Abstract Session
Presentation Details
Session Title: Pharmacology Toxicology Studies and Analytics Assay Development
Sickle cell disease (SCD) stems from a point mutation of the two β subunits in adult hemoglobin (HbA) that leads to nucleated polymerization of sickle hemoglobin (HbS) under hypoxia. A critical unmet need for using gene editing-based genomics therapies to cure sickle cell disease is the ability to characterize red blood cell (RBC) quality to assess the potency, regardless of the editing modality. The standard DNA sequencing methods can assess the safety of the product, and conventional hematologic assays such as high-performance liquid chromatography (HPLC) can measure the abundance of various hemoglobin variants in a sample. There is sparse knowledge of the in vitro functional characterization of RBCs. Still, these could assess the biophysical and rheological properties of RBCs derived from genome-edited SCD HSPCs at the single-cell level.
We delivered CRISPR Cas9 RNPs targeting the BCL11a enhancer locus (or controls) to commercially obtained healthy donor peripheral blood mobilized (CD34+) HSPCs to enhance fetal hemoglobin expression. Edited HSPCs were differentiated into erythroid cells using a 21-day 3-phase cytokine cocktail system (including IL-3, SCF, and erythropoietin). Differentiation, proliferation, and enucleation were compared between control and gene-edited samples with flow cytometry. Adhesion and deformability of enucleated RBCs were studied using the SCD-Biochip Assay, a suite of microfluidic devices capable of functionally characterizing several biochemical and biomechanical phenotypes of RBCs namely: adhesion, deformability, and sickling under hypoxic and normoxic conditions [1, 2, 3].
Editing efficiency was estimated as high as ~60% via electroporation. The mean cell amplification of CD34+ cells reached a 695-fold expansion by Day 16. Differentiation of the immature erythroblasts into mature RBCs continued from day 16 to 21, marked by the progressive loss of CD71 expression; ~50% of CD235a+ cells were enucleated. We observed more microfluidic occlusion index (a surrogate for deformability) with glutaraldehyde crosslinked RBCs compared to untreated controls. We found that RBC occlusion and adhesion (to fibronectin) are significantly greater under hypoxia than under normoxia for HbSS RBCs.
Here, we present a matrix-type potency assay to address the critical need for a universal potency assay for editing-based SCD treatments. The assay will be anchored by a suite of novel microfluidic biochip devices that can functionally assess the health of individual RBCs in a clinically relevant human capillary model in unprecedented sensitivity for precision diagnostics. We demonstrate the feasibility of adopting the SCD-Biochip platform to evaluate pertinent biomechanical functions of RBCs derived from CRISPR-edited HSPCs. The application of these functional assays could result in a robust and accurate precision diagnostic that addresses the critical need for a universal potency assay for editing-based SCD treatments.
References:
[1] Goreke, U., Iram, S., Singh, G., Domínguez-Medina, S., Man, Y., Bode, A., ... & Gurkan, U. A. (2023). Catch bonds in sickle cell disease: shear-enhanced adhesion of red blood cells to laminin. Biophysical Journal.
[2] Man, Y., Wu, D. H., An, R., Wei, P., Monchamp, K., Goreke, U., ... & Gurkan, U. A. (2023). Microfluidic concurrent assessment of red blood cell adhesion and microcapillary occlusion: potential hemorheological biomarkers in sickle cell disease. Sensors & Diagnostics, 2(2), 457-467.
[3] Sekyonda, Z., Goreke, U., An, R., Oshabaheebwa, S., Man, Y., & Gurkan, U. A. (2022). Sickling Dynamics Differ Among the Different Sickle Cell Disease Genotypes. Blood, 140(Supplement 1), 5401-5402.
Plain Language Summary
It is difficult to answer how much gene-editing drug is enough to cure a disease. It depends on the patient and the strength of the drug. We could know the answer by measuring the desired cellular function following gene editing. Sickle cell disease is a genetic disease that affects red blood cells. Authors present an assay that puts edited red blood cells to ‘a stress test’. The assay imitates the capillaries outside the body to measure multiple functional properties of red blood cells. The authors can successfully edit and grow red blood cells in the lab. They also show that the devices are sensitive to the effects of disease stressors. This is an innovative approach that takes into account individual differences in patients. The assay can be universally used for diseases that affect red blood cells.
Utku Goreke1, Dipti H. Kamath1, Yaw O. N. Ansong-Ansongton1, Nathan M. Perez1, Umut Gurkan2, Petros Giannikopoulos1, David N. Nguyen1
1Innovative Genomics Institute, University of California Berkeley, Berkeley, CA,2Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, OH"
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