Characterization of a Genetically Engineered HUDEP2 Cell Line Harboring a Sickle Cell Disease Mutation As a Potential Research Tool for Preclinical Sickle Cell Disease Drug Discovery

Sickle cell disease (SCD) is a genetic disorder caused by a missense mutation in the beta-globin gene [Glu6Val] resulting in the formation of HbS (sickle hemoglobin). HbS polymerizes under low oxygen tension causing an array of severe pathophysiological complications, including vaso-occlusion, pain and hemolytic anemia. Traditionally, research efforts toward studying anti-sickling effects of various pathways and compounds in vitro have been made using sickle cell patient derived samples. However, such studies are limited based on sample availability and a large degree of phenotypic variability. Thus, a cultured cell system with a sickle mutation mimicking human sickle cell erythroid cells is a potentially useful tool that can be used to interrogate anti-sickling compound action and to study various facets of sickle cell phenotype.

In this study, we generated a genetically engineered sickle mutation (GAG to GTG) in HUDEP2 cell line using CRISPR Cas9 gene editing and homology directed repair mechanism. HUDEP2 cells are immortalized human erythroid progenitor cells, obtained from the Riken Institute and were cultured as described (Kurita et al 2013). CRISPR Cas9 RNP complex with modified guide RNA along with ssODN were electroporated in HUDEP2 cells. Cells were also treated with SCR7, a NHEJ inhibitor to facilitate homology- directed repair events. Cells were sorted for clonal selection, cultured and further screened for homozygous knock-in mutations using digital droplet PCR. The mutation was further confirmed by sanger sequencing.

12 clones consisting of homozygous mutation were identified, with 6 of these clones selected for further characterization. Cells were differentiated for 7 days in culture and hemolysates were analyzed using HPLC. Heterozygote clone showed 32.3% HbS which was comparable to sickle cell trait patient derived blood samples whereas homozygous clones (ssHUDEP2) showed between 71-85% HbS. These results were further confirmed for the presence of HbS using mass spectrometry.

Mutant HUDEP2 cell growth and differentiation were similar to the wild-type HUDEP2 cells. To evaluate sickling in these cells we optimized the differentiation protocol to improve the maturation of these cells by withdrawing SCF and doxycycline gradually.

Hemoglobin polymerization and resulting shape change in sickle HUDEP cells was determined by subjecting mature ssHUDEP2 cells to hypoxia (1% oxygen) for 3 hours. Upon subjecting to hypoxia, ssHUDEP2 cells showed morphological changes indicative of HbS polymerization. These morphological changes were absent in wild type cells under similar conditions of hypoxia. In addition, ssHUDEP2 cells pre-treated with 25µM GBT440, a known anti-sickling compound, for 1 hour prior to hypoxia incubation elicited a reduction in the shape change associated with hypoxia, suggesting inhibition of hemoglobin polymerization and sickling.

These results suggest that genetically-engineered sickle mutant HUDEP cells represent a preclinical model for development of anti-sickling compounds for the potential treatment of Sickle Cell Disease and to explore novel mechanistic pathways.