Analysis of CircRNAs and CircRNA-Associated Competing Endogenous RNA Networks in β-Thalassemia

The involvement of circRNAs in β-thalassemia and their actions on fetal hemoglobin (HbF) is unclear. Here, the circRNAs in β-thalassemia carriers with high HbF levels were comprehensively analyzed in comparison with healthy individuals. Differential expression of 2183 circRNAs was observed and their correlations with hematological parameters were investigated. Down-regulated has-circRNA-100466 had a strong negative correlation with HbF and HbA2. Bioinformatics was employed to construct a has-circRNA-100466‑associated competing endogenous RNA (ceRNA) network with the determination of hub genes and associated miRNAs. In combination with previous reports, the has-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified. The ceRNA network was verified by qRT-PCR on β-thalassemia samples and RNA immunoprecipitation of K562 cell lysates. Has-circRNA-100466, miR-19b-3p, and SOX6 were present together in anti-argonaute 2 immunoprecipitates, indicating involvement with HbF induction. Furthermore, spearman correlation coefficients revealed their significant correlations with HbF. In conclusion, a novel has-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified, providing insight into HbF induction and suggesting targets β-thalassemia treatment.

Salubrinal induces fetal hemoglobin expression via the stress-signaling pathway in human sickle erythroid progenitors and sickle cell disease mice

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24µM SAL increased F-cells levels 1.4-fold (p=0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p=0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p=0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

LSD1 inhibition enhances robust fetal hemoglobin induction in human β0-thalassemia/HbE erythroid cells

Induction of fetal hemoglobin (HbF) ameliorates the clinical severity of β-thalassemias. Histone methyltransferase LSD1 enzyme removes methyl groups from the activating chromatin mark histone 3 lysine 4 at silenced genes, including the γ-globin genes. LSD1 inhibitor RN-1 induces HbF levels in cultured human erythroid cells. Here, the HbF-inducing activity of RN-1 was investigated in erythroid progenitor cells derived from β0-thalassemia/HbE patients. The significant and reproducible increases in γ-globin transcript and HbF expression upon RN-1 treatment was demonstrated in erythroid cells with divergent HbF baseline levels, the average of HbF induction was 17.7 + 0.8%. RN-1 at low concentration did not affect viability and proliferation of erythroid cells, but decreases in cell number was observed in cells treated with RN-1 at high concentration. Delayed terminal erythroid differentiation was revealed in β0-thalassemia/HbE erythroid cells treated with RN-1 as similar to other compounds that target LSD1 activity. Downregulation of repressors of γ-globin expression; NCOR1 and SOX6, was observed in RN-1 treatment. These findings provide a proof of concept that a LSD1 epigenetic enzymes is a potential therapeutic target for β0-thalassemia/HbE patients.

Combined +58 and +55 BCL11A enhancer Editing Yields Exceptional Efficiency, Specificity and HbF Induction in Human and NHP Preclinical Models

Targeting the BCL11A erythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A +58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2 promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A +58 editing alone, 29.1%±3.9%; BCL11A +55 editing alone, 34.8±5.1%; HBG1/2 promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A +58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed. Together these results suggest that combined BCL11A +58 and +55 erythroid enhancer editing produces highly efficient on-target allelic disruption, erythroid-specific BCL11A downregulation, heightened HbF induction capacity compared to alternative approaches, preserved long-term multilineage engraftment potential by both human xenotransplant and rhesus autotransplant assays, and absence of evident genotoxicity, under clinically relevant SpCas9 RNP electroporation conditions. Disclosures No relevant conflicts of interest to declare.

SGK1 Inhibition Induces Fetal Hemoglobin in Erythroid Cells

Introduction: Novel and safe therapeutic targets to increase expression of fetal hemoglobin (HbF) have potential to treat b-hemoglobinopathies (Platt, Brambilla et al. 1994, Steinberg 2020), including sickle cell disease (SCD) in which red blood cell (RBC) hemoglobin S resulting from a mutation in the hemoglobin β-globin subunit causes RBC sickling and hemolysis triggering vascular inflammation (Piel, Steinberg et al. 2017, Kato, Piel et al. 2018). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase in the AGK kinase family that controls physiological processes such as cell growth, proliferation, migration, and apoptosis (Hayashi, Tapping et al. 2001, Sang, Kong et al. 2020). SGK1 is regulated by multiple ligands (insulin, cAMP, IGF-1, steroids, IL-2 and TGF-β) and phosphorylation by SGK1 modulates the activity of downstream effectors including ion channels (ENaC), Na-Cl cotransporters (NCC), membrane transporters, cellular enzymes (GSK3B) and transcription factors (FOXO3a, β-catenin, NF-κB and SP1) (Brunet, Park et al. 2001, Snyder, Olson et al. 2002, Loffing, Flores et al. 2006, Bruhn, Pearson et al. 2010, Boccitto and Kalb 2011, Wang, Hu et al. 2017). Previous studies show that SGK1 mediates survival signals in HEK cells by inhibiting FOXO3a through phosphorylation at Ser-315 (Brunet, Park et al. 2001). Recently, metformin was shown to induce HbF in erythroid cells through FOXO3a activation and metformin prevents RBC sickling in SCD (Zhang, Paikari et al. 2018). Thus, we hypothesized that inhibition of SGK1 and subsequent alleviation of SGK1-induced FOXO3a inhibition, may induce expression of erythroid cell HbF. Methods: We studied the ability of SGK1 to inhibit HbF induction in erythroid cells by culturing CD34+ hematopoietic progenitor stem cells from both healthy and SCD blood donors using a 21-day differentiation protocol. After confirming expression of SGK1 in CD34+ cells by Western blot, SGK1 activity was inhibited using the selective and potent SGK1 inhibitor RA04075215A (Halland, Schmidt et al. 2015). SGK1 is activated by phosphorylation at Thr256 and we confirmed target engagement through measurement of Thr256 phosphorylation on Western blots. To decipher the effect of SGK1 inhibition on the SGK1 downstream pathway, we assessed the inhibition of FOXO3a triggered by SGK1 through evaluation of FOXO3a phosphorylation Ser315. In parallel, we quantified HbF gene transcripts by qPCR, determined the level of HbF protein by Western blot, and quantified F-cells by flow cytometry. Finally, to evaluate the effect of SGK1 inhibition on RBC sickling, we performed a cell sickling assay upon completion of erythroid differentiation in culture. Fully differentiated CD34+ cells from SCD blood donors were incubated under in hypoxia (2% O 2) for 4 hours and then abnormal shaped cells were analyzed using the Amnis® ImageStream® flow cytometer. Results: By day 21 of differentiation, HbF protein expression in CD34+ cells increases significantly in RA04075215A-treated cells versus untreated controls. In addition, a combination of SGK1 inhibition and hydroxyurea treatment reveals a potential synergistic induction of HbF. Western blot analysis shows a decrease in phospho-SGK1 phosphorylated at Thr-256 with SGK1 inhibition, confirming target engagement and loss of SGK1 activity. Downstream of SGK1, phospho-FOXO3a phosphorylated at Ser-315 was also decreased significantly following SGK1 inhibition, demonstrating alleviation of FOXO3a inhibition. Finally, in the RBC sickling assay, RA04075215A-treated cells were significantly protected from sickling under hypoxia compared to controls. Conclusion: In summary, this study establishes SGK1 as a potential new therapeutic target in SCD. We demonstrate that SGK1 inhibition induces HbF in CD34+ cells through FOXO3a transcription factor activation and prevents CD34+ cells from sickling. In the future, in vivo studies are necessary to confirm the role of SGK1 in HbF induction and to assess the efficacy of SGK1 inhibition in improving markers of SCD. Disclosures No relevant conflicts of interest to declare.

CD117 Antibody Drug Conjugate-Based Conditioning Allows for Efficient Engraftment of Gene-Modified CD34+ Cells in a Rhesus Gene Therapy Model

Hematopoietic stem cell (HSC) gene therapy is now curative for multiple genetic diseases; however, it is limited by morbidity and mortality from cytotoxic chemotherapy-based conditioning. To overcome these limitations, we developed an antibody drug conjugate (ADC) targeting CD117 (c-Kit) to specifically deplete both HSCs and progenitor cells. In our preliminary study, 0.2 mg/kg CD117-ADC conditioning resulted in >99% bone marrow depletion, detectable engraftment of gene-modified cells (vector copy number per cell (VCN) ~0.01), and minimal toxicities in a rhesus HSC gene therapy model (ASH 2019). In this study, we investigated escalating doses of CD117-ADC to determine the optimum conditioning dose to enable engraftment of gene-modified CD34+ HSCs in rhesus macaques. We evaluated autologous CD34+ cell transplantation with lentiviral gene marking following conditioning using a single injection of CD117-ADC at the 0.3 mg/kg dose for ZL13 and ZJ62, and the 0.4 mg/kg dose for H635 and H96G. The extent of gene marking was compared with myeloablative busulfan conditioning (5.5 mg/kg x 4 days) for 12U018 and 12U020. Mobilized rhesus CD34+ cells (ADC 3.8±1.9x10e7 vs. Busulfan 2.9±0.2x10e7, n.s.) were transduced with a lentiviral vector encoding BCL11A-targeting microRNA-adapted short hairpin RNA (shmiR-BCL11A) co-encoding a truncated human erythropoietin receptor (thEpoR) for stable fetal hemoglobin (HbF) induction (Sci Transl Med. 2021). These cells (in vitro VCN 10.1±3.8 vs. 10.2±7.3, n.s.) were transplanted into autologous animals 6 or 10 days after ADC conditioning (0.3 or 0.4 mg/kg, respectively) or 1 day after busulfan conditioning. Blood counts, gene-marking levels, and HbF induction were evaluated for 0.3-1.2 years post-transplant in ADC conditioning and for 1.5 years in busulfan conditioning. After a reduction of blood counts post-transplantation with ADC or busulfan conditioning, all lineages recovered. Granulocyte (>500/μl, day 6-9 vs. day 8-9), reticulocyte (>50,000/μl, day 10-14 vs. day 11), and platelet (>30,000/μl, day 2-8 vs. no reduction) recoveries were similar for ADC and busulfan conditioning, respectively. Only ADC conditioning resulted in a reduction of platelet counts as well as a novel transient rebound in all major lineages. Two months post-transplant, efficient gene marking (VCN in granulocytes 0.28±0.16 vs. 0.44±0.17, n.s.) was observed in 3 of 4 animals in ADC-conditioning (ZJ62 with 0.3 mg/kg ADC, and H635 and H96G with 0.4 mg/kg ADC). This marking level was similar to busulfan conditioning (Left panel in Figure). Robust and durable HbF induction was also detected by both HbF-positive percentages (F-cell 8.5±1.8% vs. 13.7±5.8%, n.s.) and HPLC-quantitated HbF amounts (8.0±2.9% vs. 11.1±5.2%, n.s.) in these 3 animals, similar to busulfan conditioning (Right panel in Figure). In ZL13 (1 of 2 animals in 0.3 mg/kg ADC), lower gene marking (VCN in granulocytes 0.02) was obtained, along with low HbF induction (F-cell 1.0% and HbF amounts 0.9%), suggesting that 0.3 mg/kg ADC is marginal and 0.4 mg/kg ADC is sufficient for robust engraftment of gene-modified cells. Importantly, CD117-ADC conditioning resulted in minimal toxicities unlike busulfan conditioning. In summary, we demonstrated that a single dose of CD117-ADC allows for efficient engraftment of gene-modified CD34+ HSCs in a rhesus gene therapy model, achieving a similar level as myeloablative busulfan conditioning. Robust HbF induction was also confirmed at the protein levels in this rhesus gene therapy model with ADC conditioning. This targeted approach for safer conditioning could improve the risk benefit profile in HSC gene therapy. Figure 1 Figure 1. Disclosures Latimer: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bhattarai: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Yoder: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Palchaudhuri: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Li: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bertelsen: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Olson: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

IMR-261, a Novel Oral Nrf2 Activator, Induces Fetal Hemoglobin in Human Erythroblasts, Reduces VOCs, and Ameliorates Ineffective Erythropoiesis in Experimental Mouse Models of Sickle Cell Disease and Beta-Thalassemia

Background Sickle cell disease (SCD) is an autosomal recessive disorder where mutated hemoglobin (HbS) polymerizes and can lead to irreversible red blood cell (RBC) sickling and painful vaso-occlusive crisis (VOC). The RBC sickling is amplified by inflammation, resulting in tissue and organ damage. The transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates the expression of antioxidant genes in response to oxidative stress, regulates inflammation, inhibits the NFkB pathway, and induces fetal hemoglobin (HbF), making it an attractive target in SCD and beta-thalassemia. IMR-261 is a novel oral activator of Nrf2 and has been tested in Phase 2 clinical trials (previously as CXA-10). Methods & Results CD14+ human monocytes were exposed to IMR-261 at 3µM and 10µM for 3 hours, to determine via quantitative PCR (qPCR) its ability to induce expression of antioxidant genes. IMR-261 at 10 µM significantly increased (p<0.05) the expression of Nrf2-dependent genes (p<0.05), including HMOX1, HSPA1A, HSP90, GCLM, SOD1 and TXNRD1. Human monocytes were treated with lipopolysaccharide (LPS) to test the ability of IMR-261 to block inflammatory genes with a NFkB target dataset. IMR-261 significantly inhibited (p<0.05) LPS-induced expression of IL-1-beta, TNF-alpha and IL-6 in human monocytes. To test the effects of IMR-261 on HbF induction, human erythroblasts were derived from CD34+ blood marrow progenitor cells sourced from healthy or SCD subjects. IMR-261 induced expression of the gamma-globin gene (4.0-fold change at 3µM and 7.18-fold change at 6 µM). This was accompanied by increased %F-cells (2.8-fold change at 3µM and 3.0-fold change at 6 µM). IMR-261 was also tested in the Townes HbSS mouse model of SCD to assess the potential for HbF induction. Mice were dosed with IMR-261 at 12.5 mg/kg or 37.5 mg/kg BID for 4 weeks (N=4-8/group). After 4 weeks of treatment, IMR-261 at 12.5 mg/kg and 37.5 mg/kg resulted in a significant increase in HbF relative to control, and 37.5 mg/kg resulted in a significant increase in %F-cells relative to control (Table 1, p<0.05). In addition, both doses of IMR-261 led to significant increases in RBC counts and total hemoglobin (Hb) (Table 1, p<0.05). IMR-261 at 37.5 mg/kg also significantly decreased (p<0.05) both reticulocyte counts and spleen cellularity. The ability of IMR-261 to reduce VOCs was assessed in separate Townes HbSS mice after the administration of TNF-alpha (0.5 µg/mice i.p.). IMR-261 was dosed at 37.5 mg/kg BID for 5 days before triggering VOCs. RBCs were stained with Ter-119 antibodies on spleen and liver of mice. Compared to controls, IMR-261 significantly reduced the presence of RBC on occluded vessels. This was coupled with a reduction of P-selectin (3109±97 Mean Fluorescence Units [MFI] in vehicle-treated vs. 1974±379 MFI in IMR-261 group, p<0.05) and L-selectin (375±20 MFI in vehicle-treated vs. 242±60 MFI in IMR-261 group, p<0.05). IMR-261 also reduced select hemolysis biomarkers: bilirubin (11.2±0.3 mg/dL in vehicle-treated vs. 8.4±0.7 mg/dL in IMR-261 group, (p<0.05) and free-heme (325±52 µM in vehicle-treated vs. 203±51 µM in IMR-261 group, p<0.05). A beta-thalassemia experimental model Hbb th1/th1 was tested to evaluate whether IMR-261 could improve ineffective erythropoiesis seen in beta-thalassemia. IMR-261 treatment at 37.5 mg/kg BID significantly increased hemoglobin levels, RBC counts and hematocrit (p<0.05), with significant reductions observed in reticulocytes (p<0.05). flow cytometry analysis (CD71/Ter119) showed that IMR-261 significantly decreased late basophilic and polychromatic erythroblasts (Ery.B) and increased orthochromatic erythroblasts and reticulocytes (Ery.C) cell numbers in the spleen (p<0.05). Conclusions IMR-261 activates Nrf2-dependent antioxidant genes and inhibits NFkB-induced pro-inflammatory genes in human monocytes. In human erythroblasts, IMR-261 significantly increased HbF and %F-cells. In vivo SCD models show that IMR-261 significantly induced HbF and %F-cells, improved hemolytic markers, and decreased VOCs. IMR-261 also increased Hb and improved ineffective erythropoiesis in a beta-thalassemia in-vivo model. Together these data suggest that IMR-261 is a promising, novel, oral therapy that warrants clinical testing in SCD and beta-thalassemia. Figure 1 Figure 1. Disclosures Maciel: Imara Inc.: Research Funding. Carvalho: Imara Inc.: Research Funding. Rignault: Imara Inc.: Research Funding. Pace: Imara Inc.: Consultancy. OCain: Imara Inc.: Current Employment, Current equity holder in publicly-traded company. Ballal: Imara Inc.: Current Employment, Current equity holder in publicly-traded company.

Debunking the "junk": Unraveling the role of lncRNA–miRNA–mRNA networks in fetal hemoglobin regulation

Fetal hemoglobin (HbF) induction is considered to be a promising therapeutic strategy to ameliorate the clinical severity of β-hemoglobin disorders, and has gained a significant amount of attention in recent times. Despite the enormous efforts towards the pharmacological intervention of HbF reactivation, progress has been stymied due to limited understanding of γ-globin gene regulation. In this study, we intended to investigate the implications of lncRNA-associated competing endogenous RNA (ceRNA) interactions in HbF regulation. Probe repurposing strategies for extraction of lncRNA signatures and subsequent in silico analysis on publicly available datasets (GSE13284, GSE71935 and GSE7874) enabled us to identify 46 differentially expressed lncRNAs (DElncRNAs). Further, an optimum set of 11 lncRNAs that could distinguish between high HbF and normal conditions were predicted from these DElncRNAs using supervised machine learning and a stepwise selection model. The candidate lncRNAs were then linked with differentially expressed miRNAs and mRNAs to identify lncRNA-miRNA-mRNA ceRNA networks. The network revealed that 2 lncRNAs (UCA1 and ZEB1-AS1) and 4 miRNAs (hsa-miR-19b-3p,hsa-miR-3646,hsa-miR-937 and hsa-miR-548j) sequentially mediate cross-talk among different signaling pathways which provide novel insights into the lncRNA-mediated regulatory mechanisms, and thus lay the foundation of future studies to identify lncRNA-mediated therapeutic targets for HbF reactivation.

Genomic Characterization of Sickle Cell Mouse Models for Therapeutic Genome Editing Applications

Sickle cell disease (SCD) is caused by a mutation of the β-globin gene (HBB), resulting in abnormal hemoglobin molecules that polymerize when deoxygenated, forming “sickle” shaped red blood cells (RBCs). Sickle RBCs lead to anemia, multi-organ damage and pain crises, beginning the first year of life. The onset of symptoms coincides with the developmental switch of β-like globin gene expression from fetal stage γ-globin to adult stage β-globin, resulting in a shift from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2β2). Some individuals harbor rare genetic variants in the extended β-globin gene cluster that cause constitutively elevated postnatal HbF, a benign condition known as hereditary persistence of fetal hemoglobin (HPFH) which alleviates symptoms of co-inherited SCD. Previously, we showed that CRISPR-Cas9-mediated genome editing can recreate a naturally occurring HPFH variant in the γ-globin (HBG1 and HBG2) promoters. Disruption of a TGACC nucleotide motif within this region by Cas9-mediated non-homologous end joining in human erythroid cells or their progenitors caused induction of HbF by interfering with recruitment of the transcriptional repressor, BCL11A. This strategy results in potent HbF induction in human cells and is a promising therapeutic strategy. However, the efficiency of genome editing and the level of HbF induction required to arrest or reverse the pathologies of SCD are unknown. In this work, we investigated the utility of humanized mouse models for SCD to answer this question. We further characterized the genomic configurations of two models: Berkeley mice, which harbor multiple tandem copies of three separate transgenes encoding human α-globin, sickle β-globin (βS) and a segment of the locus control region (LCR) a powerful enhancer that drives high-level erythroid-specific expression of linked genes; and Townes mice, in which the endogenous α-globin gene is replaced by the homologous human gene and the endogenous β-globin gene is replaced by human γ-globin (γA) and βS-globin genes. Genome editing of human γ-globin promoter in the Berkeley mouse induced a massive DNA damage response and cell death caused by the accumulation of multiple double-stranded DNA breaks (DSB) within the highly repetitive human transgene. In contrast, it was possible to achieve high-level editing of the single copy human γ-globin gene in the Townes model. However, induction of HbF was approximately 10-fold less that what occurred after generating the same edits in human cells, possibly because the mouse model lacks essential non-coding DNA regulatory sequences. Together, these limitations rule out the Berkeley mouse for DSB-inducing gene-editing purposes and the Townes mouse for HbF induction by regulatory element targeting. Despite these limitations, we determined the Townes model to be a good candidate for a base editing strategy to directly alter the SCD mutation. This work sought to edit the sickle T to a G, resulting in the Hb G-Makassar variant suspected to be benign and non-sickling. Recipient mice transplanted with successfully edited (55-60%) Townes HbSS Lin- cells show marked improvement in blood count values and splenomegaly. This Hb G-Makassar strategy allows for a better understanding of the levels of hematopoietic stem cell editing required to correct the SCD phenotype.

Hematopoietic stem cell transplantation and gene therapy are the sole treatments for sickle cell disease and other hemoglobinopathies

Linus Pauling and colleagues originally discovered sickle cell disease (SCD) as a molecular genetic abnormality in 1949, and after more than five decades of study, the FDA authorized hydroxyurea as the first HbF-inducing drug for the treatment of VOC in SCD patients in 1998. L-glutamine, crizanlizumab, and voxelotor were authorized by the FDA for the treatment of SCD in adults 20 years ago. There are several FDA-approved medications for treating SCDs in the United States, but the HU is the only EMA-approved medication in Europe.Hematopoietic stem cell transplantation (HSCT) and gene therapy are the sole treatments for SCD and other hemoglobinopathies. Furthermore, miRNAs contribute to the development of novel therapeutics by delineating the molecular mechanisms and signaling networks involved in HbF induction. The transcription factors BCL11A, MYB, KLF-3, and SP1 regulate miRNAs, which have a variety of consequences on target expression. HbF induction is now an important aspect of minimizing hospitalizations and improving survival. A comprehensive perspective on miRNA regulation mechanisms and comprehensive study on miRNAs as a possible SCD treatment are relevant, based on the rising number of detailed miRNA functional investigations. As a biomarker, it might be used to quantify VOCs and discriminate between acute and chronic pain. This is the first study we've seen that suggests miRNAs may be used to treat SCD.