In addition, -globin RNA levels were similar in T87Q-globin vector-transduced cells and non-transduced mock control (Figure?S2)

In addition, -globin RNA levels were similar in T87Q-globin vector-transduced cells and non-transduced mock control (Figure?S2). that lentiviral addition of T87Q-globin strongly reduced endogenous -/S-globin expression, resulting in an anti-sickling effect. Our findings should be helpful to understand the anti-sickling effects of therapeutic genes in SCD gene therapy. SCD models regarding their molecular mechanism, efficacy, and safety before testing in animal models and subsequent clinical trials to enhance the success rate for early participants in those trials. In terms of speed and cost, cell culture models have a great advantage over human primary cells for drug candidate screening and molecular mechanism evaluation. To the best of our knowledge, there is no publicly available SCD cell line for research. Here, we introduced the SCD mutation into a previously generated immortalized erythroid progenitor cell line (HUDEP-2)16 using the CRISPR-Cas9 approach, allowing us to evaluate the anti-sickling activity of T87Q-globin, as well as its potential mechanism of action using RNA sequencing (RNA-seq) in this cell line. Results Sickle HUDEP-2 (sHUDEP-2) Cells Produce the S-Globin Protein BMS-582949 To introduce the SCD mutation into the adult -globin gene in HUDEP-2 cells, we used MSH2 the CRISPR-Cas9 approach. The electroporated bulk HUDEP-2 cell population was differentiated in order to determine whether there was any detectable HbS production. While wild-type HUDEP-2 cells mostly expressed adult Hb (HbA), edited cells (bulk) produced HbS and HbA (Figure?1A). To derive an SCD cell line clone, we cloned single cells from the bulk population and performed PCR-based genotyping to determine the editing status of the clones. The results revealed that the total editing ratio was 67% (49 out of 73 clones) and biallelic editing was 22% (16 out of 73 clones) with the homozygous SCD mutation (Figure?1B). To confirm HbS protein expression, homozygous gene-edited clones were differentiated and subjected to Hb electrophoresis. All homozygous gene-edited clones produced HbS protein BMS-582949 expression (Figure?1C), indicating gene conversion was realized at the protein level. Because the seventh clone (hereafter referred to as sHUDEP-2) produced significant HbS protein amount without fetal globin (HbF) expression, it was selected for further characterization, anti-sickling, and RNA-seq experiments. Open in a separate window Figure?1 Sickle HUDEP-2 (sHUDEP-2) Cells Produce Sickle Hemoglobin (HbS) (A) Hemoglobin (Hb) electrophoresis of differentiated cells derived from wild-type HUDEP-2 and cells electroporated with ribonucleoprotein complex and donor BMS-582949 template containing the sickle cell disease (SCD) mutation (Edited). (B) qRT-PCR analysis of single-cell cloned electroporated cells. (C) Hb electrophoresis for single-cell cloned sHUDEP-2 cells. (D and E) Cell BMS-582949 number (D) and cell surface marker (GPA, CD71, and CD36) expression change (E) during red blood cell (RBC) differentiation of sHUDEP-2 cells (n?= 3). (F) Giemsa-wright staining of sHUDEP-2 cells at day 10 of differentiation. sHUDEP-2 cell numbers increased over the course of a 14-day differentiation period (Figure?1D), similar to the parental HUDEP-2 cell line as reported previously.16 There was a 13-fold increase in cell number at day 10 and a 24-fold increase at day 14 of differentiation. sHUDEP-2 cells were evaluated for erythrocyte marker (CD36, CD71, and glycophorin A [GPA]) expressions throughout differentiation. Most of the cells were already positive for CD36 (82.3%? 2.8%), CD71 (68.0%? 2.8%), and GPA (69.0%? 2.9%) at day 0 (Figure?1E), similar to wild-type HUDEP-2 cells.16 Although there was a slight reduction in CD71 and CD36 expression during the early phase of differentiation, expression increased after 5?days. Moreover, GPA levels reached 99.2%? 2.9% at day 10 of differentiation. Because GPA is a terminal marker for erythrocyte differentiation, we used 10?days for the differentiation experiments. Whereas GPA positivity was almost 100% at day 10 of differentiation, enucleation efficiency was only 6.5%? 2.9% as determined by flow cytometric analysis of cells stained with Hoechst 23322 dye (Figure?1F), which is similar?to the enucleation efficiency of wild-type HUDEP-2 cells (8.4%C10%).13,17.