HbF Levels Research Articles

Early Results from a Phase 1/2 Study of Aru-1801 Gene Therapy for Sickle Cell Disease (SCD): Manufacturing Process Enhancements Improve Efficacy of a Modified Gamma Globin Lentivirus Vector and Reduced Intensity Conditioning Transplant

Introduction: ARU-1801 is a gene therapy consisting of autologous CD34+ hematopoietic stem cells and progenitors (HSCPs) transduced with a lentiviral vector (LV) encoding a modified γ-globinG16D gene. Preclinical studies in SCD mice have shown that g-globinG16D binds α-globin with higher affinity; hence, the g-globinG16D LV produces 1.5-2x more HbF/vector copy number (VCN) than a g-globin LV. Preliminary studies also show greater reduction in reticulocytes in SCD mice expressing HbFG16D compared to those expressing the same level of HbF, suggesting that HbFG16D may have a more potent anti-sickling effect than HbF. We hypothesized a high potency anti-sickling globin would allow ARU-1801 to be effective with reduced intensity conditioning (RIC). RIC would result in lower toxicities and resource utilization compared to myeloablative approaches, allowing access of gene therapy to a broader group of SCD patients. We previously reported early data from patient 1 (P1) and 2 (P2) in the ongoing Phase 1/2 study (NCT02186418), who were treated with drug product (DP) from the initial ARU-1801 manufacturing process (Process I). We now present the long-term data on these patients and early data from P3, the first patient treated with our new manufacturing process (Process II). Methods: Adults with severe SCD, as defined by recurrent vaso-occlusive events (VOE) and acute chest syndrome deemed eligible were enrolled. Manufacturing process improvements in Process II included optimized timing of HSCP collection after plerixafor mobilization, LV production and improved HSCP transduction. Prior to DP infusion, all patients received a single dose of IV melphalan (140 mg/m2 BSA) and were weaned off transfusions 3-6 months after DP infusion. Patients were monitored for safety, engraftment, VCN, anti-sickling Hb (ASG) expression, and hematological and clinical manifestations of SCD. Levels of ASG (including HbFG16D) are presented as fractions of endogenous Hb. Results: As of 28 July 2020, data from 3 patients treated with ARU-1801 are available. P1 (34yr old) has HbSβ0- and P2 (24yr old) has HbSβ+ thalassemia (2-3% HbA). Both have 30 months (mo) post-transplant (PT) follow up. P3 (19yr old) has HbSS genotype with 6 mo PT follow up. ARU-1801 demonstrated a favorable safety profile with no treatment-related adverse events to date. Time to neutrophil engraftment (ANC ≥500) was 9, 7, and 7 days PT, and time to platelet recovery (Plt >50,000) was 12, 7, and 6 days PT, in P1, P2, and P3, respectively. Figure 1 shows HSPC dose, conditioning exposure and gene transfer; Figure 2 shows ASG over time. Using Process I, P1 has shown stable expression of 20% HbFG16D, 31% ASG and 31%à64% F-cells over 2.5 years, despite a low DP VCN of 0.2 and low HSPC dose of 1.4 x106 cell/kg. P2 received a higher cell dose of 7.1 x106 cell/kg with a DP VCN of 0.47 but had below target melphalan exposure, likely due to rapid clearance from hyperfiltration (GFR= 200 mL/min/1.73m2). Despite lower engraftment and HbFG16D level, P2 maintains stable total ASG of 22% at 30 mo due to a compensatory increase in HbF. Using Process II, P3 received DP of 6.8 x106 cells/kg with a VCN of 1.0, and demonstrated an engrafted VCN of 0.74, 71% F-cells and 91% F-reticulocytes at 6 mo. As P3 is being weaned off transfusions, HbFG16D is progressively rising, showing the selective advantage to HbFG16D-containing RBCs. P1 and P2 have maintained improvements in VOEs, no VOE in P3 so far (data will be presented). Conclusion: We show that engraftment of ARU-1801 and amelioration of disease is possible with RIC using IV melphalan, with persistent stable ASG expression and meaningful improvement in VOEs in P1 and P2. P1 shows stable HbFG16D and high ASG despite low, albeit stable VCN. P2 had lower HSCP engraftment, which we hypothesize was due to below target melphalan exposure. Nevertheless, significant clinical benefit was observed in P2 due to stable ASG of 22% at mo 30. It is likely that the presence of this amount of HbFG16D has provided enough ASG to prevent sickling/ineffective erythropoiesis, resulting in the preferential survival of HbF+HbFG16D-expressing RBC. Process II DP in P3 resulted in 2-4X higher engraftment of transduced HSCPs at 6 mo. Additional process enhancements are under development for future treated patients. ARU-1801, administered with RIC, holds significant promise for achieving durable responses with a favorable safety profile in patients with severe SCD. Disclosures Asnani: Aruvant Sciences: Research Funding; Avicanna Ltd.: Research Funding. Lutzko:Aruvant Sciences: Patents & Royalties: pre-clinical vector development. Lo:Aruvant Sciences: Current Employment. Little:Aruvant Sciences: Current Employment. McIntosh:Aruvant: Current Employment, Current equity holder in private company. Malik:Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties. OffLabel Disclosure: Plerixafor - stem cell mobiliziation Melphalan - chemotherapy conditioning pre autologous transplant with ARU-1801

Novel Genetic Loci That Influence Fetal Hemoglobin Expression in Children with Sickle Cell Anemia

Introduction: Elevated levels of fetal hemoglobin (HbF) are known to ameliorate both the morbidity and mortality of sickle cell anemia (SCA). Sustained post-natal HbF expression is heritable and regulated by multiple quantitative trait loci. Previous genomic studies have identified three major gene loci (BCL11A, HBS1L-MYB, and HBG2) that account for ~40% of HbF variation in SCA, but additional genetic modifiers remain to be discovered. We performed a genome wide association study (GWAS) using DNA collected from multiple cohorts of children with SCA, to identify novel genes and variants involved in HbF expression. Methods: We analyzed genomic DNA from 1009 children with SCA and pre-treatment steady-state HbF levels who enrolled in prospective research trials from the United States (HUSTLE, SWiTCH, TWiTCH), the Caribbean (SACRED) or sub-Saharan Africa (REACH, NOHARM). Whole blood DNA was first genotyped using the H3Africa SNP array (Illumina) that identifies over 2.2 million single nucleotide variants (SNVs) across the genome. Most samples also underwent whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform analysis, which identifies coding variants in all known exons. Square root transformed HbF values were the continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, with both age and sex as co-variates. The GWAS approach included 3 distinct steps. First, we performed two independent GWAS discovery steps using distinct African populations; these were designated Discovery I (N=211) and Discovery II (223). Second, only SNVs that were significant (p<0.05) in both datasets were then selected for two independent replication steps; these were designated either African-American (N=157) or African (N=269). Third, the SNVs that were significant in both dual discovery and at least one of the replication cohorts were then verified using an additional Caribbean cohort (N=149) with TaqMan techniques for genotyping specific variants. Through this multistep process, we searched for genomic loci with consistent HbF associations across multiple cohorts. Results: From the combined SNP and WES dataset, 8 BCL11A variants passed genome wide significance (p<10-8) in the discovery analysis, and 1,048 additional variants were identified with nominal HbF association (p<0.001). We found that 173 of these novel variants had sustained association in at least one of the replication cohorts (p<0.05). We selected 20 variants with the strongest and most consistent associations with HbF from the discovery and replication analyses for further verification (Table 1). Expected HbF associations with BCL11A (rs1427407) and HBS1L-MYB (rs4895441) were identified. Among other 18 novel candidate variants, the rs77737207 variant (allele frequency ~0.10) near the RUNX1T1 locus was strongly associated with lower HbF levels, while coding variant rs2279587 (allele frequency ~0.03) in the ITGA1 gene approached statistical significance (p<0.08) in the final verification cohort and was associated with higher levels of HbF. Conclusions: Our large GWAS of HbF with diverse global cohorts of children with SCA from Africa, the United States, and the Caribbean validated the strong associations of HbF with common genetic variants near the BCL11A and HBS1L-MYB gene loci. We also identified two novel gene loci, ITGA1 and RUNX1T1, that have statistical associations with HbF expression. The RUNX1T1 gene is a broad transcriptional corepressor known to impact myeloid differentiation in hematopoiesis, while ITGA1 encodes the integrin alpha subunit of a cell-surface receptor involved in cell-cell adhesion and inflammation. Both of these genes represent novel loci that may be involved in the regulation of HbF expression in children with SCA and should be investigated further using cellular and animal models. Disclosures No relevant conflicts of interest to declare.

Catalytic Activity of Heme-Regulated eIF2 Alpha Kinase (HRI) Regulates Fetal Hemoglobin

Sickle cell disease (SCD) is a hereditary disorder occurring due to a mutation in the β- globin gene resulting in hemoglobin polymerization and sickling of red blood cells that drives an array of severe pathophysiologies. SCD patients with hereditary persistence of fetal hemoglobin mutations show amelioration of disease symptoms. HRI is a heme sensing eIF2α kinase belonging to the integrated stress response pathway, primarily regulating the hemoglobin synthesis in RBCs. Under low levels of heme, HRI undergoes auto-phosphorylation and subsequently phosphorylates its substrate eIF2α. This impedes the global protein translation and selectively activates ATF4 translation thereby initiating a transcriptional stress response. HRI protein deletion has been shown to induce fetal hemoglobin (HbF) in vitro by reducing BCL11A levels (Grevet et al., 2018). It has also been shown that ATF4 binds to enhancer region of BCL11A promoting its expression, revealing a direct regulation of HRI from eIF2α to ATF4 to BCL11A to γ-globin (Huang P, 2020). As it is well-established that protein kinases can exert dual kinase and scaffold functions, we explored whether the regulation of HRI on HbF is driven by its catalytic or non-catalytic activity. We generated HRI kinase dead (KD) mutant HUDEP-2 cells, with a mutation (K196R) in HRI ATP binding site that is predicted to result in loss of its auto-phosphorylation (Bauer B. N., 2001). HRI KD mutant HUDEP-2 was created using CRISPR Cas9 and homologous recombination approach, followed by clonal selection that yielded two homozygous clones, referred as KD clones. To further compare the scaffold and kinase function of HRI, we created HRI knockout (KO) in HUDEP-2 cell line using CRISPR Cas9 approach. We isolated clonal populations with 3 clones each with heterozygous and homozygous editing. Western analysis of KD clones showed that HRI protein levels remain unchanged. About 50% and a 100% loss in HRI protein level was observed in heterozygous and homozygous KO clones respectively. To confirm the loss in kinase activity of HRI, we differentiated KD clones for 7 days in vitro and subjected them to iron depleted conditions. As expected, HRI WT cells under iron depleted conditions showed robust activation in the p-eIF2α levels indicative of increase in HRI activity. KD clones lacked increase in p-eIF2α levels under these conditions confirming the loss of kinase activity of HRI in KD clones. HRI KD and KO clones were characterized to evaluate the HbF regulation by differentiating in vitro for 7-8 days. KD clones showed 4-5-fold induction in HbF levels measured by flow cytometry. Heterozygous and homozygous KO clones showed an equally strong activation in HbF levels. KD and KO clones showed downregulation in BCL11A and ATF4 protein levels. These results indicate that HRI regulates HbF as a function of its catalytic activity. We further employed RNA-seq to study the global transcriptomics in HRI WT and KD mutant clones at day 0 and day 8, under steady state and iron depleted conditions. RNA-seq confirmed the strong activation of HBG1/2 levels and robust silencing of BCL11A levels in KD clones at day 8. A weaker induction in HBG1/2 levels and minor reduction on BCL11A levels were observed at day 0. These results indicate that HRI may play a role in HbF silencing in a stage specific manner. HRI WT cells under iron depleted condition showed activation of ATF4 pathway, indicative of activation of HRI stress pathway. As expected, HRI KD population showed no regulation of ATF4 pathway under iron depleted conditions. Interestingly, we did not observe any changes in the BCL11A gene expression in WT cells under iron depleted conditions. Further evaluation needs to be carried out in order to understand the regulation of BCL11A by ATF4 under low iron mediated stress. Fewer gene changes were observed in KD populations under iron depleted conditions indicative of HRI playing a vital role as a heme sensor. Overall, our results provide evidence to support that HRI regulates HbF though its catalytic activity and demonstrates its role as heme sensor in HUDEP-2 cells. Disclosures Gupta: Sanofi: Current Employment. Hara:Sanofi: Current Employment. Lessard:Sanofi: Current Employment. Sturtevant:Sanofi: Current Employment. Krishnamoorthy:Sanofi: Current Employment. Demers:Sanofi: Current Employment. Hicks:Sanofi: Current Employment.

Editing the LRF Repressor Binding Site in the γ-Globin Promoters Induces Therapeutically Relevant Fetal Hemoglobin Levels for the Treatment of β-Hemoglobinopathies

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult β-globin chain. In β-thalassemia, the reduced (β+) or absent (β0) production of adult β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent red blood cell (RBC) sickling, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 disrupt the binding sites (BS) for transcriptional repressors (e.g., BCL11A and LRF). Recently, we demonstrated that CRISPR/Cas9-mediated disruption of the LRF BS in the HBG promoters via non-homologous end joining and microhomology-mediated end joining (MMEJ) repair mechanisms mimics the effect of HPFH mutations by impairing the LRF binding and re-activating the γ-globin expression (Weber, Frati et al., Science Advances, 2020). Efficient editing of the LRF BS (≥ 3 γ-globin promoters in >70% of SCD hematopoietic stem/progenitor cells (HSPCs)) resulted in a robust HbF reactivation and a concomitant reduction in βS-globin levels recapitulating the phenotype of asymptomatic SCD-HPFH patients. RBCs derived from edited HSPCs displayed HbF levels sufficient to correct the SCD cell phenotype. Similarly, LRF BS targeting in β0-thalassemic cells results in HbF reactivation potentially correcting the α/β-like globin imbalance. Xenotransplantation of human HSPCs edited using several gRNAs targeting the LRF BS showed a robust engraftment of edited cells that were capable to differentiate into multiple lineages. HBG editing in engrafted cells ranged from 26% to 76% with a decrease (-33%) of editing events compared to the input HSPCs, partially due to a reduced occurrence of MMEJ-mediated events in hematopoietic stem cells (HSCs). Erythroid progenitors (BFU-E) obtained ex vivo from engrafted human cells, showed a relevant γ-globin expression (~40% of the total β-like chain) despite of the reduction in the number of edited promoters per BFU-E after transplantation. Moreover, mature RBCs ex vivo differentiated from edited human cells ensure therapeutically relevant HbF levels. Sequencing of top-scoring off-targets identified by GUIDE-seq showed a relatively high off-target activity within an intergenic site devoid of known regulatory elements both in vitro and in vivo in primary human cells treated with one of the gRNAs targeting the LRF BS. Although the occurrence of this off-target event in repopulating cells suggests that it has no detrimental effect on HSC engraftment and multilineage differentiation, we tested high fidelity Cas9 variants to reduce off-target activity in primary HSPCs. Finally, we used CAST-Seq assay to evaluate the potential chromosomal rearrangements in edited primary human cells in vitro and in vivo. To minimize the potential genotoxicity associated to Cas9 nuclease-mediated double-strand breaks (DSBs) in the genome, we explored the base editing system to introduce C>T point mutations in the LRF BS without generating DSBs. The absence of the canonical SpyCas9 NGG PAM close to the LRF BS, prompted us to use known and novel base editors containing non-NGG Cas9 variants that allowed the editing of up to 6 out of 8 cytosines of the LRF BS in erythroid cell lines and in primary HSPCs from SCD patients. These C>T conversions include not only known HPFH mutations but also mutations that can further impair LRF binding. In the majority of cases, we detected no insertions or deletions in base-edited samples, as compared to nuclease-edited samples. The 4.9-kb deletion that can be generated upon cleavage of the two identical HBG promoters by the Cas9 nuclease was barely detected in base-edited samples. Importantly, disruption of the LRF BS by non-NGG enzymes led to HbF de-repression, without affecting erythroid differentiation. This work identifies the LRF BS as an effective and safe therapeutic target for the treatment of β-hemoglobinopathies. Disclosures Casini: Alia Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Multiplex CRISPR/Cas9 Genome Editing Targeting the BCL11A/HBG Axis Maximizes Fetal Hemoglobin Reinduction but Generates Chromosomal Translocations Which Persist In Vivo

Sickle cell disease and β-thalassemia are among the most common monogenic disorders globally, causing significant morbidity and early mortality. The only curative option available is allogeneic hematopoietic stem cell transplantation, which is limited by a lack of matched donors and risks, including graft versus host disease and secondary malignancy. Retroviral gene transfer is being explored in clinical trials, but an alternative approach is more targeted CRISPR/Cas9 genome editing, to recapitulate naturally occurring hereditary persistence of fetal hemoglobin (HPFH) which ameliorates disease. HbF reinduction is achieved by disrupting either transcription factor binding sites within the HBG promoter, or an erythroid enhancer sequence within the HbF repressor, BCL11A. Results from preclinical studies have suggested that HbF levels may remain suboptimal when each locus is targeted individually. We thus investigated the feasibility of a dual editing approach, targeting both loci simultaneously or sequentially, to maximize HbF production. G-CSF-mobilized human peripheral blood hematopoietic stem and progenitor cells (HSPCs) underwent CRISPR/Cas9 genome editing at either HBG promoter, BCL11A erythroid enhancer, or both loci targeted either simultaneously or sequentially. Immunodeficient mice were transplanted with edited cells. HSPCs cultured in differentiation media demonstrated comparable levels of editing at each locus in the single edited arms (by TIDE).The dual editing approach did not impair editing efficiency at each site when conducted sequentially and only slightly reduced efficiency in the simultaneously-edited reactions. HbF reinduction was also greatest in sequentially double-edited reactions, with HbF/HbA ratio as high as 3.9 times that seen in mock reactions by flow cytometry and 4.1 times by HPLC. In single-edited arms, HbF reinduction was slightly greater with HBG promotor than BCL11A erythroid enhancer editing. Two separate chromosomal translocation events encompassing both loci were detected in each double-edited arm and quantified using digital droplet PCR. Both were more frequent in simultaneous (mean of 1.0% and 0.6%) than sequential reactions (means <0.2%, n=3, p=0.0420 and p=0.0296). Analysis of single BFU-E colonies grown on methocult media revealed markedly different indel patterns at the 2 target loci. Concurrent hemoglobin fraction analysis of these clonal populations by HPLC demonstrated greatest HbF proportion in double-edited colonies. Where ≥60% editing was reported at both loci mean HbF was 71.3%, compared to 47.1% with ≥60% editing at HBG only, and 29.6% with ≥60% editing at BCL11A only (p=0.0373). Following transplant into mice, engraftment and lineage differentiation were comparable among all experimental arms. At necropsy, bone marrow populations of human CD45+ cells, CD34+CD38low HSPCs and, within these, HSC-enriched CD90+CD45RA- subpopulation, were present at comparable levels indicating that the dual editing approach did not impair engraftment. Translocation events were detected, albeit at frequencies of less than 0.25%, in the bone marrows of all 4 mice transplanted with simultaneously dual-edited cells and in 5 of 6 mice transplanted with sequentially dual-edited cells. Bone marrow cells cultured ex vivo demonstrated greatest HbF from mice transplanted with sequentially double-edited cells. In summary, we present evidence of maximized HbF reinduction with sequentially applied multiplex genome editing at BCL11A erythroid enhancer and HBG promoter, with no impairment of engraftment or differentiation. However, chromosomal translocation events were consistently seen in double-edited reactions, even when edits at each locus were applied sequentially, and persisted in vivo after engraftment, thus rendering this approach inappropriate for clinical applications. However, multiplex editing and targeting both the BCL11A erythroid enhancer and HBG promoter to maximize fetal hemoglobin induction may be a promising strategy for alternative platforms such as base editors which are expected to greatly reduce or completely eliminate the occurrence of translocations. Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Enochian: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees.

Pediatric Patients with Sickle Cell Disease at Harbor-UCLA & Early Experience of L-Glutamine Therapy

Background.Sickle cell disease (SCD) affects millions of people worldwide, including >100,000 Americans, and is often complicated by acute crises. Complex pathophysiology makes it challenging to ameliorate SCD severity, which can be influenced by hydration and disease-modifying agents such as hydroxyurea (HU). Due to the increased metabolic demands of chronic illness, optimal nutrition and presumably antioxidant L-glutamine (Gln) may further contribute to disease control. Thus, we hypothesized that better nutrition and Gln adherence may reduce occurrences of pain crises in SCD. Methods.An IRB-approved retrospective review of pediatric patients with SCD ≥1 y of age was conducted at our institution, along with voluntary nutrition surveys. Evaluations were carried out for associations between disease severity and clinical/laboratory variables representing nutrition, growth, hematologic parameters [fetal hemoglobin (HbF), mean corpuscular volume (MCV)], and treatment compliance (with HU and/or Gln). Results.Preliminary analyses included data from 50 SCD patients (25 F/25 M), with a mean age of 9.1 ±7 y and a mean BMI of 56.4 ±32.6% (±SDEV). Sixty percent of the patients had HbSS/Sß0 (32%-HbSC and 8%-HbSß+). The average number of annual pain crises (APC, 0.97 ±1.14) requiring hospital visits or admissions per patient rose with age. HbF expectedly dropped with age. HbF levels <10% in teenage and older patients were associated with a ≥2-fold higher APC, compared to younger patients (despite similar proportions of HbSS/Sß0). Proper hydration and nutrition were reported by 69% and 43% of 35 survey participants, respectively. APC values for 3 main groups were: - 0.87 ±1.1 for well hydrated/well nourished (n=14, 3.1 ±2.3 y) - 1.4 ±2 for well hydrated/poorly nourished (n=10, 11 ±6.4 y) - 1.97 ±2 for poorly hydrated/poorly nourished (n=8, 13 ±5.4 y). There were no correlations between caloric intake and BMI. However, 20% of surveyed patients (12 ±7.8 y) had Z-scores lower than -1 SD for both height and weight. Their mean APC was significantly higher (2.5 ±2.5vs.1 ±1.3,p=0.03 byt-test). Their prealbumin levels trended lower than normal for age, and were more diminished in older patients. Nearly 60% of our patients were on HU (80% with HbSS/Sß0, age 10.8 ±7.4 y). Good HU adherence was reported by 22 patients, typically ≥90%, which was associated with higher MCVs (92 ±9.6 vs. 74 ±10 f/L,p<0.01), a trend toward higher HbF levels, and younger ages (15 vs. 9% and 9.7 ±7 vs. 14 ±7.7 y,p=NS). The majority (83%) of our patients 5 y of age or older were prescribed Gln, and 70% of the prescriptions were dispensed. Some refused Gln due to abdominal pain or taste, and a quarter of patients reported <50% Gln intake while on both HU and Gln. Thus, overall Gln compliance in 23-months was ≥70% in 12 patients. Patients were excluded while on chronic transfusion. Of 10 evaluable patients, 6 (8.8 ±2.2 y) had a significantly lower APC with Gln (mean APC decrease 0.9 to 0.2,p=0.016 by pairedt-test) coupled with a 12% rise in prealbumin (14.1 to 15.8 mg/dL;p=0.1). Conclusions.Well-nourished/hydrated patients were younger and had a milder disease phenotype, whereas more severe disease was seen in poorly nourished older patients, often with suboptimal compliance. L-Glutamine can be a clinically meaningful addition to HU and should be further studied in pediatric SCD. Disclosures No relevant conflicts of interest to declare.

Safety and Efficacy of CTX001 in Patients with Transfusion-Dependent β-Thalassemia and Sickle Cell Disease: Early Results from the Climb THAL-111 and Climb SCD-121 Studies of Autologous CRISPR-CAS9-Modified CD34+ Hematopoietic Stem and Progenitor Cells

Safety and Efficacy of CTX001 in Patients with Transfusion-Dependent β-Thalassemia and Sickle Cell Disease: Early Results from the Climb THAL-111 and Climb SCD-121 Studies of Autologous CRISPR-CAS9-Modified CD34+ Hematopoietic Stem and Progenitor Cells

Robust Pre-Clinical Results and Large-Scale Manufacturing Process for Edit-301: An Autologous Cell Therapy for the Potential Treatment of SCD

Sickle cell disease (SCD) is an inherited blood disorder affecting approximately 100,000 individuals in the United States. As fetal hemoglobin (HbF) has been shown to be protective against clinical manifestation of SCD, we are developing EDIT-301, an autologous cell therapy comprising CD34+ cells genetically modified using a Cas12a ribonucleoprotein (RNP) to promote HbF expression to treat SCD. Fetal hemoglobin induction for EDIT-301 is achieved by disrupting the HBG1 and HBG2 promoter distal CCAAT-box region where naturally occurring mutations are found to be associated with elevated HbF expression. Cas12a was selected over Cas9 due to the more productive and sustainable (NHEJ derived) indel profile, as well as high specificity. Using Cas12a RNP, on-target editing of ~90% was achieved in mobilized peripheral blood CD34+ cells (mPB-CD34+ cells) from both healthy and SCD donors at research scale with no detectable off targets. Editing of CD34+ cells led to an average of 43% and 54% of HbF expression in the erythroid progeny of normal donor and SCD donor cells respectively in a pancellular fashion (~93% population). The robust HbF induction in SCD red blood cells (RBCs) resulted in significant phenotypic and functional improvement including reduced sickling and increased deformability under hypoxia ex vivo. Using a microfluidic assay that replicated blood flow in microvasculature under varying oxygen conditions, SCD RBCs derived from RNP electroporated CD34+ cells showed improved rheological behavior. The rheology improvement under hypoxia was strongly correlated with the increased levels of HbF in each sample. Infusion of the modified CD34+ cells from normal donors into NBSGW mice resulted in long-term multi lineage and polyclonal reconstitution. Editing levels at 16 weeks post infusion were > 90% in all human lineages tested, demonstrating the efficient editing of SCID-repopulating hematopoietic stem cells (HSCs). Consistent with the high editing levels, human erythroid cells from the bone marrow of mice that received Cas12a-RNP treated cells demonstrated pancellular (~90% F+ RBCs) HbF expression averaging 40-50% of total hemoglobin compared to ~5% HbF observed in the control group. We have developed a consistent large-scale process using functionally closed, semi-automated systems suitable for use in clinical manufacturing. We have shown robust editing of normal donor CD34+ cells and SCID-repopulating HSCs with the clinical scale process. Editing levels of >90% were detected after long term engraftment in mice. In summary, we have demonstrated successful on-target editing of mPB CD34+ cells derived from both normal and SCD donors using a Cas12a RNP, which coincided with robust HbF induction and a phenotypic reduction of sickling in the SCD erythroid progeny, as well as improved rheological behavior. Editing of the HBG1 and HBG2 promoters using this RNP was highly specific with no measurable off-target. In vivo, cells from normal donors readily engrafted and reconstituted all blood cell lineages at levels comparable to unedited cells. Finally, a robust large-scale manufacturing process has been developed to supply material for the clinical setting. Based on these results, we are completing the activities required to assess EDIT-301 in the clinic as treatment for SCD. Disclosures De Dreuzy: Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Heath:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Sousa:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Janoudi:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. An:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Albright:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Teixeira:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Monesmith:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Chang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company.

Gene Editing of KLF1 to Cure Sickle Cell Disease

Sickle cell disease (SCD) affects millions of people worldwide and represents the most common monogenic disease of mankind (1). It is due to a homozygous T to A transversion in the β-globin gene that results in an amino acid variant - G6V - and production of HbS, which polymerises in red blood cells (RBCs) under hypoxic conditions. This generates irreversibly sickled cells that fail to traverse the microcirculation, resulting in micro-infarcts, hypoxia and pain, or 'sickle cell crises'. During gestation RBCs utilise different sets of globin genes to produce embryonic and fetal hemoglobins (HbF), so it is not until after birth when adult hemoglobin (HbA) is first produced that the first signs of SCD become apparent. This process termed 'hemoglobin switching' has been the focus of research efforts for decades because it offers an opportunity to reactivate HbF in adult cells of patients with hemoglobinopathies. A number of transcription factors, including Krüppel-like factor 1 (KLF1), play critical roles in hemoglobin switching. KLF1 is an essential erythroid transcription factor that co-ordinates the expression of more than a thousand genes critical to the formation of adult RBCs. KLF1 directly binds the β-globin gene promoter to up regulate its expression, whilst regulating the expression of additional factors like BCL11A and LRF that directly repress γ-globin expression (HbF). Heterozygosity for loss of function mutations in KLF1 leads to a significant increase in HbF that is beneficial to patients with β-thalassemia. We propose this can be recreated by advanced gene editing techniques to provide an effective therapy for SCD. We have employed CRISPR-based gene editing to knockout the expression of KLF1 in human cells. We designed two separate sgRNAs with corresponding HDR templates to target the second exon of KLF1 and ablate its function. We optimised transfection protocols and tested the on-target specificity of our sgRNAs achieving >90% efficacy in all cell types assayed. Using HUDEP-2 cells (2), a conditionally immortalised erythroid cell line which harbors three copies of KLF1 (3), we have demonstrated that these cells require at least one copy (>1/3) for survival; heterozygous cells (+/-/- or +/+/-) proliferate at a reduced rate, but are able to differentiate normally. Using RNA-seq, we identified some genes, including ICAM-4 and BCAM, which are down-regulated accordingly in a KLF1 gene dosage-dependent manner. ICAM-4 and BCAM are cellular adhesion molecules implicated in triggering vaso-occlusive episodes (4; 5), so it is anticipated their reduced expression may provide additional benefit in treating SCD. Gamma-globin is upregulated 10-fold, BCL11A down-regulated 3-fold, and HbF+ RBCs generated at ~20% of total RBCs in KLF1 +/-/- HUDEP-2 cell lines. We also engineered the ablation of KLF1 in CD34+ cells harvested from the peripheral blood of SCD patients undergoing exchange transfusions. Following transfection of the two guides, we performed directed differentiation using an erythroid differentiation medium and analysed the levels of HbF. We observed HbF at levels of between 40-60% of total Hb by HPLC, and HbF+ cells of ~50% by FACS. There was no measurable block in erythroid differentiation by FACS. We documented the types of gene editing using a high throughout NGS assay (6). We compared efficiencies of CRISPR repair of the HbS mutation with CRIPSR damage of the KLF1 gene. Lastly, we transplanted gene-edited CD34 cells into NSGW41 mice (where human erythropoiesis is established) to determine the efficiency and safety of editing long term HSCs from SCD patients. We will report on the results of these xenotransplantation assays. Taken together these results reveal the potential utility in targeting KLF1 to cure SCD.

Control of Fetal Hemoglobin Levels By NFI Transcription Factors

Sickle cell disease and some forms of b-thalassemia are disorders, which can be improved by therapies that elevate HbF, the fetal form of hemoglobin. It is currently thought that in adult type erythroid cells, silencing of the fetal type b-globin-like genes HBG1 and HBG2 is accomplished predominantly by two major transcription factors: BCL11A and ZBTB7A. However, it is unknown whether there are additional transcription factors that contribute to the repression of the HBG1/2 genes. Here, using a DNA-binding domain focused CRISPR-Cas9 screening approach, we identified NFIA as a novel regulator of HbF expression. NFIA belongs to the NFI transcription factor family, which is composed of NFIA, NFIB, NFIC and NFIX. NFIA and NFIX are the predominantly expressed forms in human erythroid cells, and their expression is higher in adult erythroblast cells when compared to fetal erythroid cells. CRISPR-Cas9 mediated disruption of NFIA in an immortalized human umbilical cord blood-derived erythroid progenitor cell line 2 (HUDEP2) or CD34+ hematopoietic stem and progenitor cell-derived primary erythroblast cells led to a modest reactivation of HBG1/2 mRNA expression, whereas disruption of NFIX had little effect. However, combined NFIA and NFIX disruption produced a substantial increase in HBG1/2 expression, suggesting that these factors function in a partially redundant manner. ChIP-seq and RNA-seq studies showed that NFIA and NFIX have comparable chromatin binding and activity profiles in human erythroid cells. ChIP-seq failed to detect NFI protein occupancy at or near the HBG1/2 genes. However, given the known difficulty in detecting repressor molecules by ChIP at the silent HBG1/2 genes [Martyn et al., Nature Genetics 2018; Liu et al., Cell 2018], we tested a direct involvement of NFI proteins by disrupting putative NFI binding sites near the HBG1/2 genes. Perturbation of one such NFI motif residing upstream of the transcription start site in the HBG1/2 promoters both in HUDEP2 and primary human erythroid cells markedly increased HBG1/2 mRNA levels, comparable to those achieved by combined disruption of NFIA and NFIX. Mutation of another putative NFI motif within intron1 in the HBG1/2 gene also significantly raised HbF levels. While these results implicate NFI proteins in the direct silencing of the HBG1/2 genes, the identity of the bound factors at the NFI motifs remains to be established. Studies are currently ongoing that use alternative approaches such as Cleavage Under Target & Release Using Nuclease (CUT & RUN) to map the chromatin occupancy of NFI factors at the HBG1/2 genomic region. We will also discuss results from ongoing studies of NFI factors in NBSGW mice models. In sum, we uncovered NFI transcription factors as a novel HbF regulator suggesting that the silencing of HbF involves a transcription factor network that is more complex than previously appreciated. Disclosures Weiss: Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company. Blobel:Pfizer: Research Funding; Fulcrum Therapeutics: Consultancy.

DNA Variants Involved in Regulation of Fetal Hemoglobin in Mexican Patients with Sickle Cell Disease. a Preliminary Analysis

HbS (HBB:c.20A>T, beta 6 Glu>Val) is a frequent Hb variant in Mexico, in particular in some regions close to the Pacific and Atlantic Oceans, with carrier frequencies from 0.5 to 12.8%, therefore Sickle Cell Disease patients are often seen in those regions. It is well known that increased levels of Fetal Hemoglobin (HbF) ameliorates the clinical complications of sickle cell disease. Several genetic studies have identifiedBCL11A,HBS1L-MYB(intergenic region),HBG2andHBBP1genes, among others, to be involved in HbF regulation; DNA variants in theselociare associated to elevated HbF. The aim of present study was to analyze 15 variants in HbF regulatorylociin Mexican patients with Sickle Cell Disease. About 10 mL of peripheral blood was collected in EDTA for hematological and molecular testing from 24 sickle cell anemia patients (S/S) and 15 sickle cell trait carriers (S/A) from Southern Mexico, 13 were from the state of Guerrero and 26 from Chiapas. All subjects voluntarily agreed to participate in this study and gave signed informed consent; underaged patients' consent was obtained from their parents; all procedures were performed according to the ethical principles of the Declaration of Helsinki. Hemoglobin S genotype was determined by Sanger sequencing; DNA variants genotyping was performed by qPCR using commercial Taqman probes for the following variants: inBCL11Ars11886868, rs4671393, rs7557939, rs1427407, rs766432, rs6706648, rs7599488; inHBS1L-MYBrs7776054, rs28384513, rs9399137, rs4895441, rs9402686, rs1320963, inHBG2rs7482144 and inHBBP1rs10128556. Hematological and clinical data were analyzed by IBM SPSS v24®. A total of 39 sickle cell patients were studied, twenty-one patients were male and 18 were female (53.8% and 46.2% respectively). Thirty-two were pediatric age patients and 7 adults. Anemia was observed in all S/S patients, eight had severe anemia (<8 g/dL); on the other hand, only 5/15 S/A patients had anemia (10 - 12 g/dL); HbF over 5% was observed in 23/24 S/S patients and in 13/15 S/A had HbF from 2% - 5%. Nineteen S/S patients were treated with hydroxyurea and presented less severe phenotype and more elevated HbF, however, the statistical analysis showed no significant differences (12.9%vs8.9%p=0.248) (Table 1). Genotype and allele frequencies are displayed in Table 2. All minor alleles were observed in frequencies over 0.05, the mo2st commonly observed minor allele wasBCL11Ars1427407 (0.69), and the less frequently observed wasHBBP1rs101028556 (0.08). The allele frequencies of four HbF regulating variants were significant different from those reported for Mexican ancestry population (MXL) in 1000 Genome database (rs11886868 C>T, rs4671393 A>G, rs7599488 C>T and rs10128556 C>T), however, the analyzed sample in this study is undersized and is not representative of the global Mexican population. No differences were observed when comparing allele frequencies from GuerrerovsChiapas patients, nor S/SvsS/A. The correlation analysis of minor allele count (MAC) and HbF demonstrated no association when comparing it to HbF levels. Likewise, the analysis was performed onBCL11AandHBS1L-MYBvariants and showed no significant correlations. There was also no correlation between MAC and the other hematological parameters (RBC, Hb, PCV, MCV and MCH). We report the first study of HbF regulating DNA variants in Mexican sickle cell disease patients. The preliminary analysis of HbFvsminor allele count showed no relation of total MAC and increased HbF, neither with the variants ofBCL11AorHBS1L-MYB. Disclosures No relevant conflicts of interest to declare.

Rapid and Automated Quantitation of Dense Red Blood Cells: A Robust Biomarker of Therapeutic Response to Early Initiation of Hydroxyurea in Young Children with Sickle Cell Anemia

Introduction: Hydroxyurea has well-described clinical and laboratory benefits in sickle cell anemia (SCA). Traditionally, hemoglobin concentration (Hb) and fetal hemoglobin (HbF) are used to assess treatment response, but these aggregate parameters do not fully capture the range of erythrocyte abnormalities in SCA. Dense red blood cells (DRBCs) are defined as RBCs with density >1.11 g/mL as measured by density-gradient fractionation methods, which are tedious and require technical expertise. This subpopulation of dehydrated RBCs has been associated with increased SCA severity but has not been extensively studied in children. Here we describe the utility of rapid, automated DRBC quantitation and show that the percent of DRBCs (%DRBCs) is a robust biomarker of hydroxyurea response in children with SCA. Methods: The Therapeutic Response Evaluation and Adherence Trial (TREAT, NCT02286154) was a single-center prospective study that demonstrated individualized, pharmacokinetic (PK)-guided hydroxyurea dosing in young patients (mean age 12.1 months at initiation) results in robust and sustained HbF levels >30-40% for most adherent patients. The longitudinal follow-up phase of TREAT aims to comprehensively evaluate hematologic parameters and organ function to demonstrate the long-term benefits of this treatment strategy. During clinic visits (every 3-6 months), TREAT participants had complete blood counts (CBCs) with the ADVIA® 2120i system. This automated analyzer quantifies hyperchromic RBCs within seconds by directly measuring cell hemoglobin concentration, where DRBCs are known to correspond to RBCs with a measured MCHC >41 g/dL. Data were analyzed at baseline and after at least 6 months of hydroxyurea therapy (M≥6). α- and β-globin genotypes were determined for all patients. Results: Thirty-three TREAT participants had ADVIA data available for analysis (Table). All had homozygous SCA except one with sickle-β0-thalasemia; 17 (51.5%) were male. The median age at hydroxyurea initiation was 10.3 months (mean 26.1 months; range 6 months - 17 years). The median duration of hydroxyurea therapy was 27.7 months (mean 31.5). At baseline, median %DRBCs was 2.1%. Baseline %DRBCs were directly correlated with age at initiation of hydroxyurea (p=0.022) and inversely correlated with baseline HbF (p=0.001). After initiation of hydroxyurea (M≥6), there was a 47.6% reduction in %DRBCs (p=0.001). Median %DRBCs at M≥6 was directly correlated with absolute reticulocyte (ARC) (p=0.002), absolute neutrophil (ANC) (p=0.003) and platelet counts (p<0.001) and inversely correlated with Hb (p=0.013) and hematocrit (Hct) (p=0.019), consistent with the laboratory benefits of hydroxyurea. In bivariate analysis, the change in %DRBCs from baseline was directly correlated with baseline %DRBCs (p<0.001) and inversely correlated with baseline HbF (p=0.005). In multivariate analysis, baseline %DRBCs was the only biologic parameter that independently predicted %DRBC change (β=-0.86, p<0.001, model r2=0.91; Figure). Even when baseline %DRBCs were only mildly increased (as low as 1.5%), participants had a marked decline in %DRBC with hydroxyurea. When baseline %DRBCs were very low at baseline (<1.5%), they remained low after treatment. Of note, there was no correlation between %DRBC change and α-thalassemia status. Conclusions: DRBCs can be rapidly quantified without the tedium of classical density-gradient fractionation methods using the automated ADVIA 2120i hematology analyzer, which directly measures hyperchromic RBCs. These data from TREAT demonstrate the feasibility of serial %DRBC measurements in clinical trials and clinical practice. TREAT participants had low baseline DRBCs compared to untreated adult SCA patients (reported mean ≈12%), reflecting the young age of these patients. Nevertheless, there was a significant decline in %DRBCs with treatment that correlated strongly with the known laboratory benefits of hydroxyurea. In conclusion, automated DRBC measurements are a robust biomarker that can be incorporated in a panel of laboratory measurements of response to hydroxyurea (and severity of SCA). A larger, multicenter study of PK-guided hydroxyurea therapy (HOPS, NCT03789591) is currently underway in 11 US sickle cell centers and will provide generalizable data on DRBC responses with optimal hydroxyurea therapy. Disclosures Malik: Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties. Kalfa:Agios Pharmaceuticals, Inc: Consultancy, Research Funding; Forma Therapeutics, Inc: Research Funding.

Effect of Nicotinamide, 1-Methylnicotinamide, and N’-Methylnicotinamide on Erythroid Colony Formation and γ-Globin Expression in Cultured Baboon CD34+ Cells

Pharmacological treatments designed to increase Fetal Hemoglobin (HbF) levels offer great promise to alleviate the symptoms and improve the lifespan of the vast numbers of patients afflicted with sickle cell disease (SCD) and β-thalassemia. Hydroxyurea can increase HbF, but a large fraction of patients with SCD do not respond to the drug. DNMT1 and LSD1 inhibitors are the most powerful drugs to increase HbF but are limited by side effects that include neutropenia, thrombophilia and/or thrombocytopenia. The development of new, more effective, and safer pharmacological strategies to augment HbF levels in the blood thus continues to be an important goal. Previous studies have shown that γ-globin gene expression is dynamically regulated during erythroid differentiation (Papayannopoulou et al PNAS 74:2923-2927, 1977). The proportion of γ-globin gene expression is higher at earlier stages (BFUe) of erythroid differentiation than at more advanced stages (CFUe). Therefore, we suggest the hypothesis that expansion of primitive, less differentiated progenitors might favor increased γ-globin, particularly when combined other HbF-inducing drugs. To investigate this hypothesis, we have tested whether nicotinamide (NAM), the major NAM metabolite 1-methylnicotinamide (1-mNAM), and N'-methylnicotinamide (N'-mNAM), a chemical derivative of NAM, can foster expansion of erythroid colony-forming cells (BFUe and CFUe) and increase γ-globin expression of cultured baboon CD34+ cells. Previous observations have shown that NAM facilitates in vitro expansion of cord blood CD34+ cells and enhanced long term engraftment in transplanted recipients (Horwitz et al J Clin Invest 124:3121, 2014). Contrasting effects of NAM, 1-mNAM, and N'-mNAM on differentiation and proliferation of the murine erythroleukemia cell line (MEL) have been previously reported (Terada et al PNAS 76:6414, 1979; Kuykendall et al Toxicol In Vitro 21:1656, 2007). While both NAM and N'-mNAM induced MEL cell differentiation, N'-mNAM was far more potent. In contrast, 1-mNAM increased cell proliferation, reduced spontaneous differentiation, and blocked differentiation induced by NAM and N'-mNAM. The effect of all three forms of NAM was examined using bone marrow (BM) CD34+ cells from a pre-clinical non-human primate large animal model. To test the effect of NAM, 1-mNAM, and N'-mNAM on expansion of erythroid colony-forming cells, the agents (5mM) were added to liquid cultures of baboon CD34+ bone marrow cells previously expanded for 5 days in serum-free expansion media (SFEM). Colony assays were performed on d8. In two experiments total erythroid colonies (BFUe and CFUe) were 2 fold higher in cultures treated with 1-mNAM compared to untreated controls (p<0.05) while no effect was observed in cultures treated with NAM. No colonies were observed in cultures treated with N'-mNAM (Figure 1A). Observation of Wright's stained cytospin preparations showed extensive erythroid differentiation on d8 in cells treated with N'-mNAM (Figure 1B). The effect of NAM, 1-mNAM, and N'-NAM on γ-globin expression was tested in baboon CD34+ cells grown in co-culture with the AFT024 cell line. NAM, 1-mNAM, or N'-mNAM (5mM) were added to cultures on d7. Expression of γ- and β-globin mRNA was measured by RT-PCR on d17. Increased γ-globin expression (0.57±0.04 γ/γ+β)) was observed in cells treated with N'-mNAM on d7 compared to untreated controls (0.20±0.09 γ/γ+β; p<0.001). NAM and 1-mNAM had no significant effect on γ-globin gene expression (Figure 1C). These results thus show that while erythroid colonies are increased by 1-mNAM, N'-mNAM is a potent inducer of erythroid differentiation and increases γ-globin expression in primary cultures of baboon CD34+ cells. In conclusion, 1-mNAM and N'-mNAM have contrasting effects on erythroid differentiation in primary baboon CD34+ cell cultures, confirming previous experiments in the MEL cell line. Future experiments are planned to test the effect of these agents on HbF in the baboon. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Fetal Hemoglobin Mediates the Effect of Beta Globin Gene Polymorphisms on Neurocognitive Functioning in Sickle Cell Disease

Background: Fetal hemoglobin (HbF) is the most influential modifier of the clinical and hematologic phenotype of sickle cell disease (SCD) and is highly heritable. Low HbF is independently associated with increased white matter changes on brain imaging and poorer performance on neurocognitive measures (Ruffiuex, Child Neuropsychology, 2013). Our previous work has shown that 11 SNPs in three genes (BCL11A, MYB, and β-globin), contribute over 20% of the variance in HbF (Rampersaud, Kang. et al., 2020, under review). Clinically, these HbF-associated SNPs are associated with disease severity and frequency of pain events. However, the neurocognitive implications of these SNPs have yet to be explored. As part of a prospective longitudinal cohort study, the Sickle Cell Clinical Research and Intervention Program (SCCRIP), we evaluated the relationship between HbF-associated SNPs and neurocognitive functioning in SCD patients. Methods: We included 257 patients with SCD (69% HbSS/HbSβ0-thalassemia). The median age of participants was 13 years (range 4 - 25). We extracted genotypes for the 11 HbF-associated SNPs from whole genome sequencing data and analyzed them based on an additive genetic model. Following informed consent, patients completed a battery of gold-standard neurocognitive measures, supervised by a psychologist, assessing IQ, verbal and perceptual reasoning, working memory, processing speed, sustained attention, and executive functioning skills. HbF was the average value of measurements collected within 3 months of neurocognitive assessment, or the closest value. In univariate analyses, Kruskal-Wallis rank sum test was used to assess the associations of the 11 HbF-associated SNPs with neurocognitive measures. Linear regression was used to examine these associations adjusting for age, sickle genotype, hydroxyurea exposure, socioeconomic status (index of social vulnerability) and 5 principal components to adjust for population stratification. Benjamini and Hochberg false discovery rate (FDR) was used for multiple corrections and FDR adjusted p (pFDR)<0.05 was considered significant for all analyses. Mediation analyses (Imai, Psychological Methods, 2010) were used to evaluate the indirect effects of HbF-associated SNPs on neurocognitive outcomes via HbF. Results: One SNP in the β-globin gene, rs968857, was associated with performance on measures of IQ, verbal reasoning, working memory, and executive functioning by Kruskal-Wallis rank sum test (Figure 1) and by linear regression adjusting for covariates listed above at pFDR <0.05. HbF expression was positively associated with IQ and verbal reasoning scores at pFDR <0.05 using linear regression adjusting for age, sickle genotype, hydroxyurea exposure, and socioeconomic status. HbF mediated the relationship between rs968857 and scores on IQ and verbal reasoning measures at pFDR <0.05 (Figure 2). The direct effect of rs968857 on neurocognitive functioning remained significant following inclusion of HbF at pFDR <0.05, indicating partial mediation. Full linear regression models with rs968857, HbF, and covariates accounted for 10.8%, 14.5%, 6.4%, and 8.3% of the variance in IQ, verbal reasoning, working memory, and executive functioning measures, respectively. Conclusions: This is the first study to demonstrate a relationship between genetic modifiers of SCD and neurocognitive functioning. Beyond findings on neuroimaging and sociodemographic factors, there is little known about risk for neurocognitive deficits in SCD. The present results suggest an influence of SNP rs968857 on cognitive function, which was partially mediated by expression of HbF. rs968857, is one of four SNPs that defines almost all β-globin gene cluster haplotypes and influences HbF levels in SCD. How this SNP effects HbF and neurocognition is unknown and requires further investigation of its mechanism. The highly heritable nature of HbF may allow for future use of precision medicine. SCD patients could be stratified according to risk for neurocognitive deficits to utilize early intervention strategies, informed by genetic polymorphisms. Correlation with neuroimaging will help further elucidate the relationship between genetic modifiers and neurocognitive functioning. Future trials with larger samples are needed to validate our findings and investigate if the observed relationships differ by age or hydroxyurea treatment status. Disclosures Estepp: Daiichi Sankyo, Esperion, Global Blood Therapeutics: Consultancy; Global Blood Therapeutics, Forma Therapeutics, Pfizer, Eli Lilly and Co: Research Funding; ASH, NHLBI: Research Funding. King:Amphivena Therapeutics: Research Funding; Bioline: Consultancy; Celgene: Consultancy; Cell Works: Consultancy; Incyte: Consultancy; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novimmune: Research Funding; RiverVest: Consultancy; Tioma Therapuetics: Consultancy; WUGEN: Current equity holder in private company. Hankins:Novartis: Research Funding; National Heart, Lung, and Blood Institute: Honoraria, Research Funding; UptoDate: Consultancy; Global Blood Therapeutics: Consultancy, Research Funding; MJH Life Sciences: Consultancy, Patents & Royalties; American Society of Pediatric Hematology/Oncology: Honoraria; LINKS Incorporate Foundation: Research Funding.

Significance of heme oxygenase-1(HMOX1) gene on fetal hemoglobin induction in sickle cell anemia patients

Though the patients with sickle cell anemia (SCA) inherit same genetic mutation, they show considerable phenotypic heterogeneity. It has been observed that patients with elevated fetal hemoglobin (HbF) levels have a relatively mild clinical course. There is sparse literature on the association of higher HbF levels leading to reduction in the oxidative stress in SCA patients. Hence in this study, the significance between the HMOX1 gene polymorphisms and the HbF levels has been studied. Preliminary screening was carried out. Genotyping of 3 variants in the HMOX1 gene was performed in 90 SCA patients and 50 healthy controls by PCR–RFLP, GeneScan and direct DNA sequencing. It was observed that SCA patients with higher HbF levels, showed improved hematological indices with an inverse effect on HbS levels. The TT genotype of rs2071746 (A→T) polymorphism was found to be associated with elevated HbF levels (P: 0.012). Also, the long form (> 25 GT repeats) of rs3074372 (GT)n repeats was found to be linked with increased HbF levels. We could not find any association of rs2071749 (A→G) polymorphism with the HbF levels. As, the sickle cell anemia patients show significant oxidative stress due to hemolysis, the study of polymorphisms in the HMOX1 gene may act as a potential independent marker for elevated HbF levels.

Investigating the missing heritability of fetal haemoglobin level in Africa.

Investigating the missing heritability of fetal haemoglobin level in Africa.

Mild dyserythropoiesis and \u03b2-like globin gene expression imbalance due to the loss of histone chaperone ASF1B

The expression of the human β-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to β). The γ- to β-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (β-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to β-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to β-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

HbF Levels in Sickle Cell Disease Are Associated with Proportion of Circulating Hematopoietic Stem and Progenitor Cells and CC-Chemokines.

The concentration of circulating hematopoietic stem and progenitor cells has not been studied longitudinally. Here, we report that the proportions of Lin-CD34+38- hematopoietic multipotent cells (HMCs) and of Lin-CD34+CD38+ hematopoietic progenitors cells (HPCs) are highly variable between individuals but stable over long periods of time, in both healthy individuals and sickle cell disease (SCD) patients. This suggests that these proportions are regulated by genetic polymorphisms or by epigenetic mechanisms. We also report that in SCD patients treated with hydroxyurea, the proportions of circulating HMCs and HPCs show a strong positive and negative correlation with fetal hemoglobin (HbF) levels, respectively. Titration of 65 cytokines revealed that the plasma concentration of chemokines CCL2, CCL11, CCL17, CCL24, CCL27, and PDGF-BB were highly correlated with the proportion of HMCs and HPCs and that a subset of these cytokines were also correlated with HbF levels. A linear model based on four of these chemokines could explain 80% of the variability in the proportion of circulating HMCs between individuals. The proportion of circulating HMCs and HPCs and the concentration of these chemokines might therefore become useful biomarkers for HbF response to HU in SCD patients. Such markers might become increasingly clinically relevant, as alternative treatment modalities for SCD are becoming available.

Genetic research and clinical analysis of deletional Chinese G\u03b3+(A\u03b3\u03b4\u03b2)0 -thalassemia and Southeast Asian HPFH in South China

Chinese Gγ+(Aγδβ)0-thalassemia and SEA-HPFH are the most common types of β-globin gene cluster deletion in Chinese population. The aim of the study was to analyze clinical features of deletional Chinese Gγ+(Aγδβ)0-thalassemia and Southeast Asian hereditary persistence of fetal hemoglobin (SEA-HPFH) in South China. A total of 930 subjects with fetal hemoglobin (HbF) level ≥ 2% were selected on genetic research of Chinese Gγ+(Aγδβ)0-thalassemia and SEA-HPFH. The gap polymerase chain reaction was performed to identify the deletions. One hundred cases of Chinese Gγ+(Aγδβ)0-thalassemia were detected, including 90 cases of Chinese Gγ+(Aγδβ)0/βN-thalassemia, 7 cases of Chinese Gγ+(Aγδβ)0 /βN-thalassemia combined with α-thalassemia, 2 cases of Chinese Gγ+(Aγδβ)0-thalassemia combined with β-thalassemia, and 1 case of Chinese Gγ+(Aγδβ)0-thalassemia combined with β-gene mutation. One hundred nine cases of SEA-HPFH were detected, including 97 cases of SEA-HPFH/βN, 9 cases of SEA-HPFH/βN combined with α-thalassemia, 2 cases of SEA-HPFH combined with β-thalassemia, and 1 case of SEA-HPFH combined with β-gene mutation. Statistical analysis indicates significant differences in MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), and HbA2 and HbF levels between Chinese Gγ+(Aγδβ)0-thalassemia heterozygotes and SEA-HPFH heterozygotes (P < 0.001). There are statistical differences in hematological parameters between them. Clinical phenotypic analysis can provide guidance for genetic counseling and prenatal diagnosis.

Rapid Molecular Detection for Differentiation of Homozygous HbE and ß0-Thalassemia/HbE in Samples Related With HbE >80% and Variable HbF Levels.

To validate a novel rapid molecular testing method for differentiation of homozygous hemoglobin (Hb)E and HbE/β 0-thalassemia genotypes using multiplex melt curve combined with high-resolution melt (HRM) analysis in a single test tube. All 10 genotypes contained (β N/β N; n = 95), (β N/β 3.5-kb; n = 71), (β N/β 45-kb; n = 28), (β N/β E; n = 10), (β E/β 3.5-kb; n = 6), (β E/β 45-kb; n = 4), (β E/β 41/42; n = 28), (β E/β 17; n = 9), (β E/β IVSI#1; n = 6), and (β E/β E; n = 76) were recruited for validation. A proposed strategy for rapid differentiation of β 0-thalassemia/HbE disease and homozygous Hb E in specimens with HbE greater than 80% and variable HbF levels was demonstrated. In the validation method, all genotypes showed 100% concordance, compared with the conventional reverse dot blot (RDB) and gap-polymerase chain reaction (PCR) methods. Our newly developed method could be useful in routine laboratory settings. The method is rapid, simple, and cost effective; does not require a post-PCR step; and can be applied in routine settings.