Γ-globin Expression In Erythroid Cells Research Articles

UM171 Enhances Fitness and Engraftment of Gene Modified Hematopoietic Stem Cells from Sickle Cells Disease Patients

Sickle cell disease (SCD) is one of the most common genetic blood diseases. Autologous hematopoietic stem cell gene therapy using lentiviral vectors is an effective therapeutic strategy for SCD. However, the occurrence of post gene therapy myelodysplastic syndrome and acute myeloid leukemia not related to vector insertion has suggested an underlying genetic risk or acquisition of damage during ex vivo culture or from proliferative stress associated with reconstitution. Here, we aimed to improve the transduction efficiency, stem cell fitness and reconstitution of SCD CD34+ cells transduced with a lentiviral vector (LV) containing BCL11A shmiR currently in clinical trials (Esrick et al. NEJM, 2021, NCT03282656), to minimize culture-induced stress and reduce genomic damage during ex vivo culture. UM171, an HSC self-renewal pyrimidoindole derivative agonist, has been shown to expand HSCs and enhance multilineage blood cell reconstitution in mice. We examined the effect of addition of UM171 during ex vivo transduction on HSCs from SCD patients. Culture of SCD CD34+ HSC in HSC expansion conditions with UM171 increased the proportion of CD34+CD133-CD45RA-CD90+ long term HSCs (LT-HSCs), from 1.1% to 4.1% (N = 3, P < 0.001), and the absolute number of LT-HSCs increased 4-fold (N = 3, P < 0.001). Treatment with UM171 also significantly enhanced the transduction efficiency of BCL11A shmiR containing lentiviral vector (LV-BCL11A) in SCD CD34+ cells from 0.7 vector copies/diploid genome (VCN) to 1.0 VCN (N = 5, P < 0.001), and significantly decreased apoptosis of stem cells from 22.5% to 14.3% (N = 3, P < 0.001), and DNA damage as measured by γ-H2AX from 10.6% to 6.6% (N = 3, P < 0.001). Given these results, we assessed the engraftment capability and clonality of SCD CD34+ transduced in the presence or absence of UM171 in NBSGW mice. UM171 increased the human CD45 engraftment of mice measured in BM at 16 weeks post-transplantation from 32.5% to 69.4% in hCD45+ BM cells (N = 6, P < 0.01) and enhanced the engraftment of transduced in SCD CD34+-derived CD45+ cells from 11.2% to 18.2% in gene-marked BM cells (N = 6, P < 0.01), while maintaining multilineage differentiation in this model. Barcode analysis revealed the engraftment of 500-1800 unique barcodes with a high degree of clonal diversity and a trend towards increased polyclonal engrafting populations in transplanted mice. To assess the effect of UM171 more rigorously on functionally defined HSCs, we performed a competitive transplantation assay using CD34+ cells transduced in the presence vs absence of UM171. Analysis of BM obtained from the mice at 16 weeks post-transplantation showed that cells transduced in the presence of UM171 consistently outcompeted those transduced under control conditions, 62.6% in gene-marked erythroid cells of UM171 treated cells compared with 37.4% of control cells (N = 9, P < 0.001), and UM171 maintains LV-BCL11A induced γ-globin expression in erythroid cells. In summary, a short-term exposure of SCD CD34+ PB cells to UM171 enhances stem cell fitness. Implementing these findings in clinical gene therapy protocols may improve the efficacy and sustainability of gene therapy and generate new opportunities in the field of gene editing.

The CDK-Inhibitor p27 Kip1 Regulates Fetal Hemoglobin Expression

γ-globin upregulation has been shown to be beneficial for individuals with Sickle Cell Disease, leading to improvement in morbidity and mortality rates. Intriguingly, small molecules that modulate the cell cycle have been observed to impact γ-globin expression in erythroid cells, though a causal relationship has not been definitively established. Understanding the connection between cell cycle dynamics/regulation and γ-globin production may shed light into a novel molecular mechanism by which γ-globin expression is regulated, with potential therapeutic implications for Sickle Cell Disease. To evaluate if cell cycle speed correlates with γ-globin expression, we labeled HUDEP-2 cells (which express beta-globin) with carboxyfluorescein diacetate succinimidyl ester (CFSE) at different days of differentiation. With each cell division, CFSE dye intensity is expected to be reduced by half; therefore, faster cycling cells retain less CFSE than slower cycling cells. Forty-eight hours post-CFSE labeling, HUDEP-2 cells were analyzed for γ-globin expression by intracellular flow cytometry at days 2, 4, 6, and 8 of differentiation. Starting at Day 6 of differentiation, the slowest cycling (Top 5% CFSE) HUDEP-2 cells exhibited >2-fold increase in γ-globin expressing cells (F-cells) compared to the fastest cycling (bottom 5% CFSE) cells. Notably, these results were confirmed in erythroid cells differentiated from primary human CD34+ hematopoietic stem and progenitor cells (HSPCs), therefore ruling out the possibility that the above findings were an artifact of the immortalized HUDEP-2 cell line. The correlation between slow cell cycling speed and increased γ-globin expression prompted us to genetically perturb the cell cycle machinery and define the impact of these genetic perturbations on γ-globin expression. Using CRISPR activation (CRISPRa), we increased the expression of 8 Cyclin Dependent Kinase Inhibitors (CDKN2A, CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C and CDKN3) in HUDEP-2 cells. To do so, we first generated a HUDEP-2-MPH cell line that stably expresses the transcriptional activator complex MPH (MS2-P65-HSF1). We then transduced this cell line with a virus that expresses one of 3 sgRNAs targeting each of the 8 CDKIs, a VP64 activation domain fused to catalytically dead Cas9 (dCas9-VP64), and a blasticidin resistance cassette. We found that activation of CDKN1b (P27 Kip1) in particular, resulted in increased F-cell percentage. Indeed, compared to cells transduced with control sgRNAs, HUDEP-2-MPH cells transduced with a CDKN1B-targeting sgRNA (resulting in a 2-fold higher CDKN1B mRNA level; p= 0.006) exhibited an increased proportion of F-cells, from a baseline of ~6% up to ~20% (p= 0.024). Increased CDKN1B expression resulted in a ~14-fold increase in γ-globin mRNA levels (p= 0.026), associated with a mild reduction in beta-globin mRNA level and a reduction in BCL11A mRNA level to ~65% of normal. These results suggest that the increased γ-globin expression resulting from CDKN1B overexpression may result, at least in part, from reduced BCL11A expression (which we are currently validating). To rule out an off-target effect of the CDKN1B targeting sgRNA, we overexpressed CDKN1B in HUDEP-2 cells, using a cDNA expression construct, and found comparable results as observed with CDKN1B CRISPRa. We next overexpressed CDKN1B cDNA in erythroid cells differentiated from CD34+ HSPCs. In early preliminary results, increased CDKN1B expression in the latter cells resulted in increased γ-globin expression, both at the mRNA and protein level, validating the HUDEP-2 data. Additional studies are ongoing to define the role of CDKN1B in the regulation of γ-globin expression, and to define the impact of CDKN1B overexpression on erythroid differentiation. In summary, our preliminary studies have uncovered a link between cell cycle regulation and γ-globin production. These findings may lay the foundation for the development of new therapeutic strategies for Sickle Cell Disease.

Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.