Treatment Of Sickle Cell Disease Research Articles

Optimization of Lentiviral ß-Globin Vectors in a Forward Orientation to Enhance Therapeutic Effectiveness for Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic hemoglobin (Hgb) disorder characterized by a single pointmutation in the β-globin gene which causes Hgb polymerization under low oxygen tension and sickling of red blood cells (RBCs) which in turn drives severe systemic injury. Gene addition technologies like hematopoietic stem cell (HSC) targeted lentiviral (LV) gene therapy are among the most promising curative strategies for SCD. To expand this approach, improvements in LV titer, transduction efficiency, along with suppression of native sickle-globin, are preferred to generate successful therapeutic outcomes. One such improvement targets LV orientation: the relationship between the DNA encoding the β-globin gene in the LV and the promoter driving healthy β-globin gene expression. We created a forward-oriented β-globin-expression vector with an improved titer that results in more efficient transduction of human CD34+ cells in vitro, in human reconstituting CD34+ cells in xenografted mice, and in rhesus reconstituting CD34+ cells(Uchida N. et al., 2019). We report here on further optimization of LVs that in addition to forward orientation, also express shRNA that decreases native β-globin and sickle-globin, which could enhance the assembly of corrected Hgb tetramers. Seven unique constructs which varied in anti-β-globin shRNA sequences, packaging elements, and regulatory elements, were transduced into MEL-BB88 cells and expanded for 2 weeks. gDNA was collected for vector copy number (VCN) analysis, and treated with 10 µM hemin to induce Hgb expression. Five days after hemin-induction, cells were harvested for benzidine staining and immunoblot analysis to validate globin expression. Sca-1+ HSC/progenitor (HSPC) BM cells were isolated from Towne's humanized SCD donor mice prior to stimulation for 48 h with cytokines, then transduced at an MOI of 24 twice, 24 h apart. To assess the in vivo engraftment potential of the transduced HSPCs, 2.5x10^6 cells were transplanted into lethally irradiated CD45.1 recipient mice. Peripheral blood (PB) was sampled at 2-week intervals for PBMC chimerism and lineage engraftment, Hgb electrophoresis, Lorrca OxygenScan and OsmoScan assays, and PB VCN. Whole BM from engrafted recipients was collected at 30 weeks and used for secondary transplantation assays which were again analyzed over 24 weeks for PB engraftment, VCN, and donor Hgb expression. LVs were produced at varying titers and all successfully transduced MEL-BB88 cells with VCNs between 1 and 5 that were maintained following hemin induction. Interestingly, variations in RRE elements dramatically affected human globin expression in vitro. Furthermore, the transplanted mice engrafted fully with HSPCs transduced with multiple vectors. Notably, mice transplanted with HSPCs transduced with vector CX0066p (LCR-Enhancer-P-HBB-shmiR-3g1.223 + 3g2.30a-R1) maintained a VCN of 0.2-0.4 in PB for the 30-week duration of the primary transplant. Cellulose acetate gels of RBCs from PB showed ~40% HbA in a background of HbS. The P50 right-shifted towards normal, RBC counts increased, WBC counts decreased, and OxygenScan parameters were all corrected towards controls and away from those values obtained from mice transplanted with mock transduced HSPCs. EI max and EI min increased while pO2@95% decreased. Lastly, the RBC sickling was corrected in an in vitro sickling assay. In conclusion, based on LV titer, MEL cell expression, transduction efficiency, engraftment of modified cells, in vivo globin expression, anti-sickling activity, and oxygen transport, these constructs provide outstanding Hgb expression and phenotype correction. Consequently, these candidates serve as a solid foundation for future human gene therapy products in the treatment of SCD.

Inclacumab Reduce Preexisting Red Blood Cell Adhesion to Activated Endothelial Cells: In-Vitro Assessment of the Microfluidic Platform Endothelium-on-a-Chip

Background: In 2019 FDA approved the use of an antibody anti P-selectin (Crizanlizumab) for the treatment of sickle cell disease (SCD) to reduce the vaso occlusive crisis (VOCs). New anti-adhesive treatments are being developed recently. Global Blood Therapeutics, Inc., acquired by Pfizer Inc. in October 2022, acquired Inclacumab from Roche and started two clinical trials now in phase 3 to evaluate the capacity of this humanized IgG4 monoclonal antibody anti P-selectin to reduce the VOCs in patients with SCD. In our recent work presented at EHA 23 in Frankfurt Germany, we showed that Inclacumab prevents the adhesion of RBCs to chronically and acutely heme activated human umbilical vein endothelial cells (HUVECs) with a statistically significant higher effect in comparison to Crizanlizumab. In this work we used a similar approach to test the capacity of Inclacumab in the reduction of preexisting adhesion events on acutely heme activated HUVECs. Methods:Whole blood EDTA samples from six SCD subjects all with HbSS genotypes were collected at University Hospital Cleveland Medical Center, Cleveland, OH, USA. To standardize the samples, we isolated RBCs by centrifugation and subsequential washes in Phospahte buffer s of the whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. HUVECs (Lonza, Morristown, NJ, USA) were introduced and maintained within the endothelialized microfluidic channels at physiological flow for at least 48-72 hours prior to experiments. We performed an acute short-term activation, where the blood samples were supplemented with 40 µM heme +/- 100 µg/ml injected through the microfluidic channels for 15 minutes, after the adhesion took place we washed the non-adherent cells with Inclacumab and Crizanlizumab and we compared with untreated washing buffer (both compounds were provided by Global Blood Therapeutics, Inc., a wholly owned subsidiary of Pfizer Inc. South San Francisco, CA, USA) A phase-contrast images of the remaining RBCs were acquired with an inverted microscope (DMi8 Leica Microsystems Inc. Deerfield, IL, USA) and quantified based on previous published methods (Figure 1A). Paired t-test was used to calculate statistical significance. Results: Acute short-term heme activation induces high level of RBCs adhesion. Both compounds Inclacumab and Crizanlizumab showed a reduction of the number of adherent RBCs to the activated heme, but only Inclacumab results statistically a significant higher reduction compared to untreated RBCs (Figure 1B). Conclusions: Inclacumab is able not only to prevent the adhesion of RBCs to acutely and chronically activated HUVECs but is able to remove the preexisting adhesion and results in significant higher reduction compared to untreated. RBCs adhesion assay on Endothelium-on-a-chip is a novel and efficient way to study the efficacy of the new antiadhesive drug and could be used to monitor patient response to new therapies in SCD. Figure1 : Effect of Inclacumab vs Crizanlizumab on removal of SCD RBC adhesion to heme activated HUVECs. A) Schematic of adhesion experiment and inverted microscope contrast of phase images 10X of adherent RBCs at acutely heme activated HUVECs. B) Inclacumab has statistically significant effect on remove adherent RBCs to acutely heme activated HUVECs (p=0.009, N=7) For all experiments, the heme concentrations were 40 µM and Inclacumab and Crizanlizumab were 100 µg/ml. Scale bare 20 µm. P-values were based on paired t-test. Error bars represent the standard error of the mean (SEM)

Improving Treatment Adherence in Patients with Sickle Cell Disease

BACKGROUND: Crizanlizumab is approved for treatment of sickle cell disease (SCD) patients with frequent pain crises and hospital admissions. Here, we address systemic factors that have led to sub-optimal adherence to monthly crizanlizumab infusions at our institution. Due to 340B status limitation, the drug was originally offered at a site affiliated to our hospital with little SCD team presence. In 2020, UW hospital (primary site of SCD care) acquired 340B status, but transferring crizanlizumab infusions to UW hospital was not prioritized. In 2022, our institution's crizanlizumab adherence was 22% 1. Patients receiving crizanlizumab were interviewed to identify adherence barriers. Top barriers included location (44%), scheduling (44%), and low knowledge about the medication (67%). A detailed analysis with SCD team input revealed barriers such as restriction on opioids for day treatment, scheduling delays after hospital admissions, onerous paper orders process, different EMRs, and poor communication/coordination between sites (Fig 1). METHODS: The hospital leadership was appraised of these barriers in a series of meetings by the SCD team and approval to administer crizanlizumab at UW hospital infusion center was granted. UW health pharmacy and infusion staff planned a transition phase with 5 patients transitioned each month between March-April 2023. Crizanlizumab orders were updated in the EMR, and infusion nursing staff received training about the drug. Smart phrases were created for patient education in after visit summaries (AVS). Day treatment with IV fluids and pain management was offered with infusions. SCD care coordinator and social worker helped with scheduling, transport, and childcare. Providers aimed to discuss treatment goals and pros/cons during visits. Goal was set to assess adherence at months 3 and 12 after implementation. Average rate of adherence was defined as percent of prescribed treatments taken by the patient over a specified time period. In case of hospitalization, adherence was defined as rescheduling and receiving the treatment. We aimed to improve adherence to 50% at 3 months and 75% at 12 months if crizanlizumab FDA approval is maintained. RESULTS: By mid-July 2023, 11 patients were transitioned to UW Hospital for crizanlizumab infusions. 10/11 patients received the drug during this period (1 patient taken off due to pregnancy). The average adherence rate increased from 22% to 71% at 3 months, exceeding the original goal. 5/10 patients had an adherence rate of 75% or more. Additionally, 70% received at least one visit from the SCD social worker (SW) during treatment and 70% saw their provider concurrently with at least one visit. All patients received day treatment with supportive IVFs and/or pain medications during the infusion visit (Fig. 2). Only 2/10 patients had 25% or less adherence which occurred due to scheduling errors that will be addressed moving forward. DISCUSSION: Our study aims to address both intentional and unintentional barriers to crizanlizumab adherence that included infusion location, scheduling, and patient education. Our team advocated for the transition of infusions to the main hospital where they receive all other SCD care. This provided patients with direct access to their SCD team during infusions. Care coordinator and social work involvement helped consolidate treatment-related orders to one EMR, standardize nursing and patient education, organize scheduling, and address social barriers. The adherence rate increased from 22% to 71% at 3 months after implementation. Although the current approval of crizanlizumab is under review, we believe the barriers addressed in this project can be extended to current SCD treatments and upcoming novel therapies. Splitting care sites due to insurance and logistical reasons can create barriers for this vulnerable population due to complex care coordination and psychosocial needs. The rapid improvement in crizanlizumab adherence displayed here is a testament to the impact of having a dedicated SCD team at one leading site to advocate for and address the unique barriers to healthcare experienced by people with SCD. Sources 1. Nixon G, Fallon M, Fulcer K, Curran W, Nazeef M, Pradhan R. How can infusion therapy adherence by improved in Sickle Cell Disease. American Society of Health-System Pharmacists; Dec. 2022. Las Vegas, NV.

FORTITUDE: A Phase 2 Open-Label Study in Progress to Evaluate Etavopivat for the Treatment of Anemia in Patients with Lower-Risk Myelodysplastic Syndromes

Background: Most patients with lower-risk myelodysplastic syndrome (LR-MDS) develop anemia and a high proportion of patients become dependent on red blood cell (RBC) transfusions. MDS-related anemia is associated with low hemoglobin (Hb) levels, iron overload and poor quality of life. Erythropoiesis-stimulating agents and other drugs, including lenalidomide, luspatercept and imetelstat (an investigational product), can lead to RBC transfusion independence and improve Hb levels in many patients; however, responses are generally transient and novel treatments are needed for this population. Etavopivat is an investigational, once-daily, selective erythrocyte pyruvate kinase (PKR) activator in development for the treatment of sickle cell disease (SCD) and anemia in patients with LR-MDS. In a phase 1 study in adult and adolescent patients with SCD by Saraf et al. HemaSphere. (2022), etavopivat 400 mg once daily was considered well tolerated and showed sustained Hb increases up to 12 weeks for most patients. Additional studies in healthy volunteers and patients with SCD support an acceptable safety profile together with improved RBC health and function during continuous treatment with etavopivat. In patients with LR-MDS, ineffective hematopoiesis may be due, in part, to insufficient ATP production, leading to increased erythroid lineage apoptosis. By activating PKR, etavopivat may improve RBC health and function by stimulating energy production (increasing ATP levels), increasing Hb levels (decreasing anemia), and decreasing erythroid lineage apoptosis. Aims:Describe the design of a multicenter, open-label, phase 2 study (FORTITUDE; NCT05568225) evaluating hematologic improvements in patients with very low-, low- and intermediate-risk MDS during treatment with etavopivat. Study design:Key eligibility criteria include age ≥18 years; very low-, low- and intermediate-risk MDS per the Revised International Prognostic Scoring System; and anemia defined by mean Hb <10.0 g/dL and <3 RBC transfusions <16 weeks prior to first dose for non-transfusion-dependent (NTD) patients or ≥3 units of RBCs for anemia <16 weeks prior to first dose for transfusion-dependent (TD) patients. Key exclusion criteria include MDS associated with del 5q cytogenetic abnormality and known TP53 abnormality, history of acute myeloid leukemia, and an absolute neutrophil count <0.5 × 10 9/L. Additional exclusion criteria include prior treatment with azacitidine; decitabine; erythropoietin, other hematopoietic growth factor treatment or lenalidomide within 30 days of Day 1 or anticipated to be required during the study; and luspatercept within 30 days of Day 1 for NTD patients and within 16 weeks of Day 1 for TD patients. Patients will receive etavopivat 400 mg once daily for at least 48 weeks. Treatment may be continued beyond 48 weeks in patients demonstrating clinical benefit. The primary objective of the study is to assess hematologic improvements based on an erythroid response over any continuous period ≥8 weeks within 24 weeks on etavopivat treatment in patients with LR-MDS. The primary endpoint will be based on the combined incidence of: ≥1.5 g/dL increase in Hb from baseline maintained for ≥8 consecutive weeks for NTD patients; absence of any transfusion for ≥8 consecutive weeks for low transfusion-burden patients; and reduction by ≥50% of RBC units for ≥8 consecutive weeks for high transfusion-burden patients. Safety, tolerability, pharmacokinetic, and pharmacodynamic properties of etavopivat will be evaluated. FORTITUDE is open for enrollment in Canada, France, Germany, and the USA. Planned enrollment includes approximately 15 patients in each of the three transfusion requirement-based groups (Figure). Statistical analysis: The primary endpoint (pooled incidence) will be tested with a one-sided Fisher's exact test at the significance level of 0.025 where 0.2 is the assumed null proportion. Summary:Etavopivat is a novel, investigational, once-daily, selective PKR activator with the potential to improve RBC health and lifespan. This phase 2 study will assess the safety of etavopivat and its impact on Hb levels and RBC transfusion burden in patients with LR-MDS and anemia.

HbF Inducing Eed Inhibitor Ftx-6058 Selectively and Reversibly Impacts Bone Marrow in Wild-Type Mice

Sickle cell disease (SCD) is a genetic disorder characterized by red blood cell sickling, hemolysis, vaso-occlusive crises (VOCs), and other complications resulting from a mutation in the HBB gene. Increasing fetal hemoglobin (HbF) levels has shown promise in mitigating disease-related pathophysiology. In individuals with SCD who exhibit hereditary persistence of fetal hemoglobin (HPFH) and have HbF levels ranging from 20% to 30%, a remarkable reduction in symptoms is observed, highlighting the protective effect of increased HbF in SCD. Preclinical and clinical findings indicate that compounds inhibiting the embryonic ectoderm development (EED) component of polycomb repressive complex 2 (PRC2) leads to elevations in HbF to therapeutic levels. These results suggest the potential for a substantial benefit for individuals living with SCD. To further understand the effects of PRC2 inhibition, this study aims to characterize the onset and reversibility of EED inhibition by FTX-6058. Results from in vivo studies in wild-type mice demonstrated upregulation of the mouse fetal hemoglobin ortholog mRNA (Hbb-bh1) as well as target engagement in bone marrow though quantification of H3K27 trimethylation during seven days of active dosing of FTX-6058. Target engagement and differential gene expression profiles in the bone marrow subsequently returned to baseline shortly after the dosing period. These findings demonstrate that inhibition of EED through FTX-6058 leads to a minimal, transient impact on the bone marrow in mice, and any transcriptional changes are rapidly reversible following cessation of dosing. A small molecule EED inhibitor has the potential to provide a new treatment for sickle cell disease without irreversibly altering the bone marrow.

The Need to Consider Pro-Active Treatment for Cerebral Artery Conditional Blood Velocity Determined By Transcranial Doppler Ultrasonography (TCD) in Sickle Cell Disease (SCD)

Introduction Cerebral artery peak systolic blood velocity (PSV) or timed average mean of the maximum blood velocity (TAMMV) determined by TCD in children of age 2-16 years with SCD is normal (NV) if TAMMV <170 cm/sec or PSV <200 cm/sec; abnormal (AV) if TAMMV > 200cm/s or PSV > 250 cm/sec; and conditional (CV) if TAMMV is 170-199cm/sec or PSV 200-249 cm/sec. Internal carotid (ICA) or middle cerebral (MCA) artery AV is associated with a stroke risk of 10% /year, CV a risk of 3-4% /year [Adams et al. Long-Term stroke risk in children with SCD screened with TCD. Ann Neurol 1997; 42: 699-704]. Blood transfusion or hydroxycarbamide is used to treat AV, but active treatment is currently not considered the standard of care for conditional velocity, although it could progress to abnormal values. ObjectiveTo evaluate the need for pro-active treatment of cerebral artery conditional blood velocity in children of age 2-16 years with SCD. Methods Following IRB approval and informed consent/assent, 308 children with SCD (126 girls and 182 boys, age 2-16 years) had TCD to determine PSV (n=193) or TAMMV (n=115) in the MCA, ICA and anterior cerebral arteries (ACA). This design was to make the research findings relevant to hospitals with TCD equipment that measure either PSV or TAMMV. Exclusion criteria were blood transfusion in the previous 4 months, therapy with hydroxycarbamide, omega-3 fatty acids or other agents that affect the severity of SCD, pregnancy, breast feeding, chronic disorders unrelated to SCD (such as diabetes mellitus) and stroke. Children with AV were started on a blood transfusion program. Those with conditional PSV were randomized in a 2:1 ratio to receive either standard care of SCD and active treatment with omega-3 fatty acids and potassium thiocyanate, or standard care only. After 3 months, measurement of PSV in the ICA, MCA and ACA was repeated. Study data were analysed with the Statistical Package for Social Sciences version 23 (IBM, USA). ResultsIn the TAMMV cohort of 115 children, 96 (84%) had NV, 7 (6%) AV, and 12 (10%) CV. Figure 1 shows the cumulative frequency of TAMMV in the MCA where most AV and CV occurred. In the PSV cohort of 193 children, 150 (77.7%) had NV, 7 (3.6%) AV, and 36 (18.7%) CV. There were no significant differences in gender or age distribution between the PSV and TAMMV cohorts. Altogether, cerebral artery blood velocity was normal in 246/308 children (80%), abnormal in 14 (4.5%) and conditional in 48 (15.5%). The PSV in 21 patients randomized to active treatment of conditional blood velocity or standard SCD care are shown in Table 1. After 3 months, PSV reduced to normal (<200cm/sec) in a significantly higher proportion (11/14 or 79%) of children on active treatment compared with 3/7 (43%) on standard care only, p = 0.04. Change in the right MCA median PSV showed a significant difference of 54cm/sec between children on active treatment who had a reduction from 201.5 cm/sec to 169.5 cm/s, and the standard care group with a rise from 184cm/s to 206 cm/sec (p = 0.018). One of 7 patients on standard care (14%) but none of 14 on active treatment (0%) had progression of PSV from conditional to abnormal. Reduction in conditional velocity following active treatment has been reported from other studies [Lagunju et al, Pediatr Blood Cancer 2015;62:1587-91; Estepp et al. Br J Haematol 2021;194:463-8]. Although not statistically significant, pre/post-treatment median steady state Hb level in actively treated patients showed an upward trend [6.8/7.3g/dl]; and the median platelet count a downward trend: 278/235 x10 9/l. The standard care group had opposite, but also not significant, trends: 7.45/7.0 g/dl, 224/261 x10 9/l. Discussion and Conclusion The 15.5% prevalence of conditional velocity translates to 15,500 /100,000 children born annually with SCD in our country, or 46,500 worldwide, of whom 1,860 (4%) may have stroke. If 14% of CV progress to AV, that is 6,510/year globally. If all are not treated, a reality in resource-limited places, any number up to 651 (10%) may develop stroke. The annual incidence, cumulative prevalence and progression rates of CV, and the significant difference in the proportion that revert to NV on active treatment compared to standard care only, suggest that there is need to consider CV for active treatment. This will prevent the cumulation of more than 1,860 children who develop stroke every year with its health, psychosocial and economic burden to the patients, their families and society.

National Long-Term Follow-up of Children with Sickle Cell Disease Diagnosed By Newborn Screening in the Netherlands: Overview of the Morbidity and Mortality

Background: Sickle cell disease (SCD) is a genetic red blood cell disorder associated with increased morbidity and mortality. Newborn screening for SCD was introduced in January 2007 in the Netherlands to enable early diagnosis and initiation of preventive measures. The objective of this study was to assess the effect of newborn screening on the morbidity and mortality of children living with SCD in the Netherlands, 16 years after its implementation. Methods: In this national cohort study all children with SCD diagnosed by newborn screening were included. Patients were followed between 1 January 2007 and 31 March 2023 until end of follow-up, death or successful stem cell transplantation. Following informed consent, data were collected from medical files on SCD genotype, treatment, mortality and development of SCD-related complications. The incidence rate of SCD-complications was presented as lifetime-event per 100 person-years for the total duration of follow-up and for the age categories: 0-4, 5-11, and 12-16 years. Results: In total, 391 out of 540 eligible patients (72%) with SCD were included in all hospitals with patients with SCD under treatment, accounting for 3047 person-years. The mean age at the end of follow-up was 7.8 years (SD±4.5). The majority of patients had SCD genotype HbSS (58%), followed by HbSC (28%). As for the mortality, five patients died during follow-up, primarily at or below the age of 2 years (4/5, 80%). In three patients, the cause of death was related to SCD, including infection (n=2; aged 1.2 and 2.0 years) and acute splenic sequestration (n=1; 2.0 years). The SCD-related complications are presented in Table 1. The most common SCD complication in this cohort was vaso-occlusive crisis (including dactylitis) with hospitalization. SCD-related complications that mainly developed within the first 4 years of life, were vaso-occlusive crisis, acute hemolytic crisis, aplastic crisis, severe infections, splenic sequestration and ischemic cerebral infarction. Acute chest syndrome occurred equally in all age categories. Other SCD-related complications developed to a lesser extent in our cohort, and at a later age in adolescence or adulthood. Conclusion: In this cohort of neonatally screened children with SCD, a substantial number of SCD-complications occurred in the first 4 years of life. This study underlines the importance of parental education and 24/7 availably, high level acute specialized clinical care for these children from birth on. Further analysis to identify risk factors at young age for a severe phenotype will be conducted, to enable more targeted interventions in a timely manner. Hopefully, this will lead to reduction of disease burden, improving the quality of life in children living with SCD in the Netherlands.

Pharmacokinetic-Guided Hydroxyurea to Reduce Transfusion Requirements in Ugandan Children with Sickle Cell Anemia: Baseline Data from the Alternative Dosing and Prevention of Transfusions (ADAPT) Trial

Background: Children are the primary recipients of blood transfusions in sub-Saharan Africa where the demand for safe blood far exceeds the supply. Without disease-modifying therapy, sickle cell anemia (SCA) causes excess morbidity, mortality, and healthcare resource utilization, specifically requiring a high reliance on blood transfusions. Hydroxyurea is a safe and effective medication for the treatment of SCA that decreases blood transfusion use in this population, which may offset the costs of improving hydroxyurea access. The widespread use of hydroxyurea in Africa is limited in part by challenges determining an individual's optimal dose; pharmacokinetic (PK) dosing of hydroxyurea may streamline the dosing process, maximizing the clinical benefits and minimizing toxicities. We designed the Alternative Dosing And Prevention of Transfusions (ADAPT) trial to analyze the reduction in transfusion use with hydroxyurea and as a pilot evaluation of the feasibility of PK-guided hydroxyurea dosing in Uganda. Methods: ADAPT (NCT05662098) is a prospective, single-center, open-label cohort study at Jinja Regional Referral Hospital in Uganda. Children 1-10 years old with HbSS were eligible to enroll; those already receiving hydroxyurea or scheduled blood transfusions, or with malignancy, HIV, tuberculosis, or other chronic illnesses were excluded, and those who received a transfusion within 30 days were temporarily excluded. All participants completed a 3-month screening period during which they underwent baseline evaluations and hydroxyurea PK testing. For PK testing, a 500 mg hydroxyurea test dose was administered, and serum was collected 30, 60, and 180 minutes after the dose. Hydroxyurea concentrations were determined via chemical derivatization and high-performance liquid chromatography in Uganda. Area-under-time-concentration-curves (AUC) and Bayesian estimation were then used to calculate the optimal hydroxyurea dose based on target medication exposure. Those for whom hydroxyurea PK evaluation was completed successfully with a dose recommendation between 15-35 mg/kg/day started on their PK-guided hydroxyurea dose; otherwise, participants started on the default dose of 20 mg/kg/day. All participants will be treated on hydroxyurea for a minimum of 12 months during which time both treatment groups will follow the same laboratory monitoring and dose adjustment protocol to achieve the maximum tolerated dose. The primary study endpoint is the incidence rate ratio of blood transfusion use during screening versus treatment. Secondary endpoints include characterization of blood transfusions (indications, risk factors, adverse reactions), feasibility of PK testing, and laboratory and clinical adverse events comparing children started on PK-guided versus default dosing. Results: From June 2022 to January 2023, 106 participants were enrolled, exceeding the projected enrollment pace and goal. The average age (mean ± SD) at enrollment was 4.6 ± 2.3 years, and 50% were female. Per family-reported history, most participants had previously received a blood transfusion (78%) with 15% reporting at least 6 lifetime transfusions. Five percent had a prior stroke. A majority also had a history of pain events (86%), malaria (79%), and dactylitis (74%). Only 2% had previously used hydroxyurea. At enrollment, the median weight-for-height z-score was -0.19 (interquartile range -1.03-0.32), and baseline exam demonstrated a palpable spleen in 20%. Abdominal ultrasonography was completed on all participants, and the spleen was identified in 75% of participants with a mean volume of 132 ± 99 mL. Transcranial Doppler exams were completed on 105 participants (99%) during screening with 13% conditional and 6% abnormal results. Baseline labs (mean ± SD) documented hemoglobin = 7.7 ± 1.3 g/dL and HbF = 10.3 ± 7.1 %. Of 106 children enrolled, 1 died from acute chest syndrome in screening and 2 were withdrawn after moving away from the clinic; 103 successfully initiated hydroxyurea treatment. Conclusion: ADAPT has fully completed enrollment and will prospectively evaluate the effectiveness of hydroxyurea treatment on reducing transfusion use in Ugandan children with SCA, a finding with both individual and public health implications. ADAPT will also provide pilot data regarding the feasibility of PK-guided dosing of hydroxyurea to facilitate safe, effective hydroxyurea use in Africa.

Efficient and Stable Makassar Base Editing in Nonhuman Primates

Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin chain. This nucleotide can be modified using adenine base editors (ABEs) to convert the defective SCD β-globin gene (HBB S) into the non-pathogenic Makassar β-globin (HBB M) variant. Proof-of-concept for this editing strategy in human and mouse hematopoietic stem cells (HSCs) has been previously demonstrated by transplantation after ex vivo modification as well as by in vivo adenoviral editing followed by selection. Here, we evaluated the feasibility, long-term efficiency, and safety of autologous ex vivo Makassar base editing in the preclinical nonhuman primate (NHP) large animal model to pave the way towards clinical applications in humans. As NHPs don't carry the sickle mutation (position A7 in the protospacer), bystander editing at position A9 was monitored as a surrogate. NHP CD34 + hematopoietic stem and progenitor cells (HSPCs) were enriched, CD34 +CD90 + HSCs FACS-purified as previously reported to increase the editing efficiency of short- and long-term engrafting cells, primed over-night, and ABE8e-NRCH base editor mRNA was delivered by electroporation. Base-edited HSCs were cultured overnight and then combined with unmodified CD34 +CD90 - cells and infused into animals after myeloablative total body irradiation. The infusion product was quality controlled by flow-cytometry, next generation sequencing (NGS), and assessing the mono-/bi-allelic base editing efficiency on a single cell level from colony-forming cell (CFC) assays. Animals were followed for up to 200 days analyzing the editing efficiency in peripheral blood (PB) white blood cells (WBCs), FACS-purified lineages from the PB, bone marrow (BM) WBCs, as well as FACS-purified BM HSPCs. Comprehensive bioinformatics analysis was performed on the generated NGS data to determine the frequency of editing over time as well as the on- and off-target editing within the protospacer at all four target locations. Finally, chromosomal integrity after base editing was confirmed performing Kromatid karyotyping assays on gene-edited BM-derived HSPCs. Ex vivo base editing of CD34 +CD90 + was highly reproducible at the Makassar locus reaching more than 60% efficiency within bulk HSCs. No impact of base editing on the myeloid and erythroid differentiation of HSCs was seen in CFC assays. Clonal analysis of colonies revealed that more than 90% of HSCs in the infusion product were edited with > 20% of cells showing biallelic editing. Gene-edited HSCs combined with unmodified progenitors rapidly engrafted in myeloablated animals without any noticeable delay in the recovery of neutrophils, red blood cells, as well as platelets and multilineage reconstitution was established within 3-6 months. Most importantly, stable editing of >20% was observed in PB WBCs and across all lineages throughout the entire follow-up. Similarly, the BM stem cell compartment fully recovered to baseline within 6-month post-transplant. Engrafted HSPCs demonstrated normal erythro-myeloid colony-formation potential. Assessment of editing in colonies showed persistence of HSCs with mono- and bi-allelic Makassar editing 6-month post-transplant. Closely matching the editing efficiency in the PB, all BM lineages, HSPC subsets, and colonies demonstrated >20% editing efficiency confirming successful editing of long-term persisting multipotent HSCs. Finally, comprehensive assessment of NGS data from all tissues in conjunction with the Kromatid assay revealed no unwanted off-target effects or chromosomal rearrangements. Editing was primarily observed at positions A9 and A12 confirming highly specific on-target editing. In summary, we demonstrate efficient, persistent, and safe base editing of NHP HSCs for the treatment of SCD. The editing efficiency of the bystander adenine in NHP HSCs is almost identical to the previously observed editing in human patient HSCs to generate the Makassar HBB. Base edited HSCs showed no impairment in homing, long-term engraftment, or multilineage differentiation in the NHP. These findings have major implications for the application of base editing in patients with hematological disease and disorders.

Availability and Cost of Basic Drugs for Sickle Cell Disease in 13 African Countries

Background Sub-Saharan African countries are the hardest hit by sickle cell disease (SCD). Several studies have demonstrated the remarkable efficacy of hydroxyurea (HU) in African sickle-cell populations. As a result, many specialists recommend widespread use of this drug in Africa to reduce the disease's high morbidity/mortality. Nevertheless, in those countries health insurance mechanisms do not exist or and many families affected by SCD do not have enough income to pay for the care. In addition to cost, frequent supply chain disruptions lead to low drugs availability. The cost of basic treatments for SCD and their actual availability in different African countries is not yet known. Yet this information is essential for guiding health policies or the action of non-governmental organizations, and for planning the cost of assistance that could be provided to these patients. The aim of this study was to determine the availability, frequency of supply disruptions and cost of basic treatments for SCDin sub-Saharan African countries. Methods A cross-sectional online international survey was carried out from 24 March to 08 May 2023 among health professionals from French-speaking sub-Saharan African countries following SCD patients and registered in the database of the non-governmental organisation (NGO) Drep-Afrique. Participants completed questions covering the availability (on a scale from 0 (drug not available) to 10 (no availability concern) and the cost of basic drugs in their centres: HU, penicillin V, folic acid, morphine and vaccines. Results 120 health professionals, mainly physicians (91%), from 13 French-speaking Sub-Saharan African countries took part in this study. 58% of the participants worked in SCD referral centres and 36% in university hospitals. 91,000 homozygous SCD patients were reported as being followed in their centres. The median availability score for hydroxyurea and morphine were respectively 5 (IQR 3-8) and 5 (IQR 2-7), compared with 10 (IQR 10-10) for folic acid, 8 (IQR 4-10) for penicillin V, 3 (IQR 1-8) for anti-pneumococcal and 5 (IQR 2-8) for anti-meningococcal vaccines. Frequent supply chain disruptions were declared: 78% for HU, 77% for anti-pneumococcal vaccine, 66% for morphine, 45 % for Penicillin V, while they were rare for folic acid (8%). The median price of HU for one month's treatment of a 30 kg child (mean dose 20 mg/kg/day) was $19.3 (IQR 13.5- 25.3) and varied greatly from country to country (from $38 in Guinea to $10.1 in Togo (p=0.0002) (figure 1). The median price of pneumococcal vaccine was $35.3 (IQR 16.9- 58.7), and thant of meningococcal vaccine $31.4 (IQR 11- 57.5), also with significant differences between countries. The cost of pneumococcal vaccine was 1.6 times higher in centres where the supply chain disrupted than when there is no disruption (p=0.02). Conclusion This study shows that the cost of HU varies from one country to another in French-speaking Africa. Although not very high, it remains unaffordable for the most patients' families. For example, in 2023, the median monthly salary is 238 $ in the Democratic Republic of Congo (the country with the highest prevalence of SCD in the world with Nigeria), compared with a median monthly cost of HU of 25 $ in this country for a child in our study. The high cost of vaccines, beyond the first years of life when a free national program is available in most of African countries, is also a problem. In addition to cost, availability and frequent supply disruptions prevent regular intake of hydroxyurea and, to a lesser extent, of penicillin V. International solidarity and the governments of these countries should work to bring down the cost of hydroxyurea and improve the distribution circuit for these essential medicines.

New Insights into VOC Incidence By Analyzing the Single Cell Quantification of HbF per RBC and Mchbs

Vaso-occlusive crisis (VOC), the main complication of sickle cell disease (SCD), is responsible for more than 50% of the associated deaths. The effect of hydroxyurea, the cornerstone treatment for SCD, on the occurrence of VOC is mainly mediated by increasing HbF levels, which is a protective factor by inhibiting hemoglobin S (HbS) polymerization. 1,2 However, HbF level is variable between patients, and its expression is heterogeneous among the red blood cell (RBC) population. 3 As a result, while an increase in HbF levels is associated with better survival, %HbF only partially explains patients' clinical manifestations. The protective effect of HbF is also related to the HbS content; the more HbS, the more HbF is needed to inhibit its polymerization. In this study, we wondered if the single-cell quantification of HbF that we developed could better explain the VOC incidence of SCD compared to %HbF. Patients and method We analyzed data from the FCDREP1 protocol, aiming to obtain a predictive score for VOC at 1 year. Inclusion criteria were SS and S- S-b 0 Thal SCD patients, more than 1 month after a vaso-occlusive crisis, 3 months after a transfusion, and on a stable dose of hydroxyurea (HU) for at least 3 months. After giving their consent (IRB no. 00003835) during a routine blood sampling visit, an additional sample was taken for HbF single cell quantification, providing HbF/cell distribution. VOC were collected prospectively for 1 year. HbF single cell quantification was measured by flow cytometry using a previously published protocol, 4 data are expressed as histograms representing % RBC in each HbF range in pg for the whole cluster. K-means clustering was performed on different combination of variables containing ranges of HbF/cell distribution versus %HbF, with MCHbS (MCH x %HbS) using UMAP for dimensionality reduction (into 2), after we applied Min-Max scaling to preprocess the features, with python 3 on sklearn and umap library. This clustering approach can group data points based on their similarity, providing insights into distinct patterns or subgroups within the dataset. Results One hundred two patients were included with a mean age of 38 ± 11 years, a sex ratio F/M of 0.56, 63% of patients were on HU. Patients had a total of 74 VOC with a mean of 0.71 ± 1.4 VOC per patient in the year. Analyses were performed finding 4 different clusters in different 2 groups. Group 1: clusters identified by the association of HbF/cell distribution and MCHbS (HbF/Cell) and Group 2: clusters identified by %HbF and MCHbS (%HbF). Clusters were numbered according to increasing HbF levels (Figure 1A and B). There was no significant difference in age or sex ratio between the groups whereas the percentage of patients on HU differed between groups (Figure 1F). The number of patients by cluster was also similar. Cluster analysis of Group 1 showed a decrease in VOC incidence (Figure 1E, blue bars) associated with an increase in HbF level per RBC (Figure 1A). If clusters 1 and 2 are similar regarding the % of HU-treated patients, cluster 1 shows a lower HbF/cell content (Figure 1A) but lower MCHbS and MCH (Figure 1C and 1D), possibly explaining a similar VOC incidence in the 2 clusters. With a higher % of HU-treated (Figure 1F), Cluster 3 and 4 stand out from each other by different HbF expression patterns and VOC incidence (Figure 1A). The clusters of group 2 does not show the same increasing incidence of VOC as a function of median %HbF (Figure 1B and E). If cluster 4, mostly treated with HU, corresponds to patients with high %HbF and low VOC; cluster 1, with little HU treatment and very low %HbF, has few VOCs, not explained by MCHbS. The difference in severity between clusters 2 and 3, which have the same percentage of HU, could be explained by the greater difference in %HbF distribution, with median %HbF values of 11.8 [5-14] and 13.8 [11-15] (p=0.06) respectively, as well as by the low MCHbS of cluster 3. Conclusion : Single cell quantification of HbF in RBC in association with MCHbS could help improve to better understand VOC incidence.

Illness Perception and Stigma Among Children and Adolescents with Sickle Cell Disease

Background: Stigma and illness perceptions surrounding adolescents and young adults (AYA) with sickle cell disease (SCD) are not well-studied. The psycho-social and emotional factors may have implications on overall patient-reported outcomes, including health-related quality of life (HRQOL) and medication adherence behavior. Aims: (1) Evaluate the impact of patients' perceptions of SCD and their medications on HRQOL and adherence (2)Examine the relationship of illness perception and stigma among AYA with SCD Methods: In this longitudinal, single-institution study, 68 participants were enrolled from outpatient hematology clinic at Lurie Children's Hospital of Chicago. Patients were approached if they were between 10 to 25 years old, English-speaking, and taking hydroxyurea, voxelotor and/or crizanlizumab for SCD treatment. Participants completed the Brief-Illness Perception Questionnaire (B-IPQ), Patient Reported Outcomes Measurement Information System (PROMIS) HRQOL Domains, Measure of Sickle Cell Stigma Questionnaire (MoSCS), Visual Analogue Scale (VAS) and Adherence to Refills and Medications Scale 7 (ARMS-7) every 3-4 months. Patient statistics were evaluated utilizing Wilcoxon (Mann-Whitney) rank sum tests, spearmen rho correlations (r s), and chi-square analysis. The data presented in this abstract represent baseline analysis for all enrolled, active study participants. Results: Sixty-eight participants completed study assessments (90% Black; 53% Female) with a median age of 14 years old (IQR 11-17) and 84% HbSS, homozygous hemoglobin S. In Table 1, patients with less perceived personal control of SCD and more emotional response to SCD reported worse social isolation (r s= -0.32, p=0.01; r s =0.31, p<0.01), mobility (r s =0.34, p<0.01; r s = -0.27, p=0.03), anxiety (r s = -0.29, p=0.02; r s =0.38, p<0.01), depression (r s = -0.29, p=0.02; r s =0.42, p<0.001), and fatigue (r s = -0.41, p0.001; r s =0.3, p=<0.01), respectively. Further, more perceived personal control was associated with less physical stress (r s = -0.49, p<0.001), pain (r s = -0.26, p=0.03), fatigue (r s = -0.41, p= <0.001), and greater mobility (r s =0.34, p <0.01). Participants with more negative perceptions of SCD consequences and concerns reported more physical stress (r s=0.29, p=0.02; r s =0.27, p=0.03), depression (r s =0.32, p<0.01; r s =0.40, p=0.001), and fatigue (r s=0.33, p=0.01; r s=0.37, p<0.01) (Table 1). Additionally, the B-IPQ correlating with PROMIS baseline data indicated patients who had greater understanding of their SCD reported better self-efficacy in both managing their medication and symptoms (r s =0.29, p=0.01; r s =0.29, p=0.02). In Table 2, patients with higher self-reported medication adherence using VAS, had better treatment control (5 vs 4, p = <0.001) and less perceived SCD-related symptoms (2 vs 3, 0.03). Utilizing ARMS-7, patients with higher self-reported medication adherence perceived less SCD-related consequences (2 vs 3, p =<0.01), symptoms (5 vs 4, p =0.01), displayed better treatment control (10 vs 8, p=0.03), but greater emotional response (1 vs 3, p = 0.04) (Table 2). Most patients (82%) endorsed ≥1 statement related to stigma. Patients frequently indicated that they are very careful who they tell they have SCD (64%), telling someone they have SCD is risky (38%), no one knows that they have SCD (52%), and they worry about people discriminating against them because they have SCD (31%). Patients with greater levels of stigma reported worse social isolation (r s= 0.38, p<0.01) and self-efficacy managing symptoms (r s= -0.30, p=0.01). Summary/Conclusion: The multifaceted relationship between medication adherence, illness perception and stigma related to HRQOL in adolescents and young adults relies upon patients' perceived control over their illness. Future evaluation of influential factors involving patients' perceived control are needed to improve SCD-clinical outcomes.

Preferences for potential benefits and risks for gene therapy in the treatment of sickle cell disease

AbstractObjective of this study is to quantify benefit-risk tradeoffs pertaining to potential gene therapies among adults and parents/caregivers of children with sickle cell disease (SCD). A discrete-choice experiment survey was developed in which respondents selected their preferred treatment alternatives in a series of experimentally controlled pairs of hypothetical gene therapies and a “no gene therapy” option. Gene therapy alternatives were defined based on the chance of eliminating SCD symptoms, expected increases in life expectancy they could offer, treatment-related risk of death, and potential increases in lifetime cancer risk. Respondents made selections based on their current disease severity and in the context of expectations of worsened disease. Three clinical sites and 1 patient organization recruited 174 adult patients and 109 parents of children with SCD to complete the survey. Adult and parent respondents were generally willing to choose gene therapies, but the adults required higher expected levels of efficacy (ie, higher chance of eliminating symptoms) than parents to choose gene therapies that conferred mortality risks of ≥10%. When adults and parents of children with less severe symptoms were asked to consider scenarios of higher levels of disease severity, the increased risk tolerance, and the lowest acceptable level of efficacy for gene therapies with mortality risks dropped by >50%. Baseline SCD symptoms are a major driver of gene therapy acceptability. Adults and parents of patients with milder symptoms may prefer other treatment options; however, an expectation of symptoms deterioration triggers strong reassessment of the acceptable benefit-risk balance of this novel technology.

Shared decision-making between paediatric haematologists, children with sickle cell disease and their parents: an exploratory study

Children with sickle cell disease (SCD) face various healthcare choices to be made during the disease process that may impact their lives. Shared decision-making (SDM) could improve their health outcomes. We assessed if, and to what extent, paediatricians engage children with SCD and/or their parents in the decision-making process. In this observational cross-sectional study, paediatric SCD patients and their parents visiting the outpatient paediatrics clinic of a university hospital participated in a SDM baseline measurement. Two evaluators independently and objectively analysed the level of patient involvement in decision-making from the audio-recordings of the consultations using the OPTION-5 instrument, a 0–20-point scale from which scores are usually expressed as a percentage of ideal SDM. The level of SDM, as perceived by patients, parents and paediatricians, was appreciated using the SDM-Q-9 and SDM-Q-Doc questionnaires, respectively. Scores could range from 0% (no SDM) to 100% (exemplary SDM). Twenty-four consultations in which a decision needed to be made about SCD treatment were audiotaped and analysed; six were from each paediatrician. The group consisted of 17 male and 7 female patients from various cultural backgrounds between 2 and 17 years old, with a mean age of 9.4 years (SD 4.2). Median OPTION-5 scores were 25.0% [IQR] 20.0–40.0%; range 0–55%). Median SDM-Q-9 and SDM-Q-Doc scores were 56.7% (IQR 39.4–88.9%) and 68.9% (IQR 57.8–77.8%), respectively.Conclusion: Although subjective scores of SDM were fair, the objectively scored level of SDM among children suffering from SCD leaves room for improvement. This may be realized by increasing knowledge about the benefits of SDM, child-centred SDM interventions and SDM-training for paediatricians that takes into account the complexity of intercultural challenges and risk communication between stakeholders.What is Known:• Children that suffer from sickle cell disease (SCD) are more vulnerable to factors that negatively impact the care that they receive as well as suboptimal health outcomes.• Shared decision-making (SDM) can help children participate in a collaborative decision-making process about their preferred treatment options and improve their health outcomes.What is New:• The level of participation in the decision-making process for patients suffering from SCD and the families that they belong to leaves room for improvement. The impact of intercultural challenges and the quality and consistency of risk-communication between stakeholders in paediatric SDM needs further exploration.• Paediatricians are more confident about their ability to involve the child and parents compared to how children and their parents experience their level of involvement in a shared decision-making process.

Design, Synthesis, and Antisickling Investigation of a Thiazolidine Prodrug of TD-7 That Prolongs the Duration of Action of Antisickling Aromatic Aldehyde.

The synthetic allosteric effector of hemoglobin, TD-7 has been investigated as a potential therapeutic agent for the treatment of sickle cell disease. The pharmacologic activity of TD-7 is due to formation of a Schiff-base interaction between its aldehyde group and the two N-terminal αVal1 amines of hemoglobin, effectively inhibiting sickling of red blood cells. However, TD-7 faces a challenge in terms of poor oral bioavailability due to rapid in-vivo oxidative metabolism of its aldehyde functional group. To address this shortcoming, researches have explored the use of a L-cysteine ethyl ester group to cap the aldehyde group to form a thiazolidine aromatic aldehyde prodrug complex, resulting in the improvement of the metabolic stability of this class of compounds. This report details the synthesis of a thiazolidine prodrug of TD-7, referred to as Pro-7, along with a comprehensive investigation of Pro-7 functional and biological properties. In an in-vitro Hb modification and Hb oxygen affinity studies using normal whole blood, as well as erythrocyte sickling inhibition using sickle whole blood, Pro-7 exhibited a gradual onset but progressive increase in all activities. Additionally, in-vivo pharmacokinetic studies conducted with Sprague Dawley rats demonstrated that Pro-7 can undergo hydrolysis to release TD-7. However, the blood concentration of TD-7 did not reach the desired therapeutic level. These findings suggest that the incorporation of the L-cysteine ethyl ester group to TD-7 represents a promising strategy to enhance the metabolic stability of aromatic aldehydes that could lead to the development of a more effective drug for the treatment of sickle cell disease.

Sickle cell disease: combination new therapies vs. CRISPR-Cas9 potential and challenges - review article.

In 2022, sickle cell disease (SCD) continues to affect the lives of millions of people, being one of the most frequently inherited blood disorders worldwide. Recently, several new therapies have been FDA approved for the treatment of SCD. The complexity of the pathophysiology of sickling has given opportunity to the evolution of several modalities of therapies. Nonetheless, the potential for complementary targeting of HbS polymerization, vasocclusion, and other inflammatory pathways remains controversial. None of these drugs can be considered a single curative line of treatment. With the advancement of CRISPR/Cas9 technology, autologous transplant of gene-edited hematopoietic stem cells could possibly provide a cure for most patients with SCD. The advantage of this approach over the conventional stem cell transplantation is that it decreases the need for immuno-suppressive drugs and the risk of graft-versus-host disease. In addition, recent technological advances can reduce the off-target effects, but long-term monitoring is needed to ensure the reliability of these methods in the clinical setting. This review explores the efficacy and safety of combination therapies and contrasting this alternative with the challenges that exist with sickle cell gene therapy using CRISPR.

Phytochemical Characterization and In Vitro Evaluation of the Anti-Sickle Cell Activity of Aqueous and Ethanolic Extracts of Two Medicinal Plants from Niger: Flueggea virosa (Roxb. ex Willd.) Royle and Kigelia africana (Lam.) Benth.

Sickle cell anaemia is a hereditary blood disorder that attacks the red blood cells and deforms them, giving them a sickle shape. Sickle cell anaemia is a serious health problem in the West African country of Niger. Moreover, the cost associated with medical care is very high. The main objective of this study is to contribute to the valorisation of Flueggea virosa (Roxb. ex Willd.) Royle (aerial part), Kigelia africana (lam), and Benth (leaves) from Niger were used to treat sickle cell disease using aqueous and ethanolic extracts of phytochemical compounds. To achieve this objective, the evaluation of anti-sickle cell activity was carried out in vitro using the Emmel technique through the normalisation rate. The analyses showed that the aqueous and ethanolic extracts contained various classes of bioactive substances known for their valuable biological activities. The chemical composition rich in bioactive compounds led to very good results in biological assays. Thus, from a dose of 0.05 mg/mL, the ethanolic extracts of the two plants normalised up to 75% of the sickle cells. As the rate of normalisation was shown to be dose-dependent, at a dose of 10 mg/mL, the ethanolic extracts showed the best rates of sickle cell normalisation, with 95% for F. virosa and 93% for K. africana. Phytochemical screening was used to correlate the secondary metabolite and anti-sickle cell activities of the extracts from the two plants. These results may justify the use of these two species in traditional medicine for the treatment of sickle cell disease in Niger. The inclusion of these plants in phytomedicines could provide significant relief to people suffering from sickle cell disease.

Targeted modification of furan-2-carboxaldehydes into Michael acceptor analogs yielded long-acting hemoglobin modulators with dual antisickling activities.

Sickle cell disease (SCD) is the most common genetic disorder, affecting millions of people worldwide. Aromatic aldehydes, which increase the oxygen affinity of human hemoglobin to prevent polymerization of sickle hemoglobin and inhibit red blood cell (RBC) sickling, have been the subject of keen interest for the development of effective treatment against SCD. However, the aldehyde functional group metabolic instability has severly hampered their development, except for voxelotor, which was approved in 2019 for SCD treatment. To improve the metabolic stability of aromatic aldehydes, we designed and synthesized novel molecules by incorporating Michael acceptor reactive centers into the previously clinically studied aromatic aldehyde, 5-hydroxymethylfurfural (5-HMF). Eight such derivatives, referred to as MMA compounds were synthesized and studied for their functional and biological activities. Unlike 5-HMF, which forms Schiff-base interaction with αVal1 nitrogen of hemoglobin, the MMA compounds covalently interacted with βCys93, as evidenced by reverse-phase HPLC and disulfide exchange reaction, explaining their RBC sickling inhibitory activities, which at 2 mM and 5 mM, range from 0% to 21% and 9% to 64%, respectively. Additionally, the MMA compounds showed a second mechanism of sickling inhibition (12%-41% and 13%-62% at 2 mM and 5 mM, respectively) by directly destabilizing the sickle hemoglobin polymer. In vitro studies demonstrated sustained pharmacologic activities of the compounds compared to 5-HMF. These findings hold promise for advancing SCD therapeutics.

Modeling the public health impact of voxelotor in the management of sickle cell disease in France.

Sickle cell disease (SCD) is an inherited blood disorder in which sickle hemoglobin (HbS) polymerizes, leading to red blood cell sickling and chronic hemolytic anemia, vaso-occlusive crises, and end-organ damage associated with early mortality. Despite standard of care, patients with SCD still experience complications and early mortality, highlighting remaining unmet treatment needs. Voxelotor is a first-in-class HbS polymerization inhibitor approved by the US Food and Drug Administration as a treatment for SCD and by the European Medicines Agency for hemolytic anemia due to SCD. In clinical studies, voxelotor has been shown to increase hemoglobin (Hb) and decrease hemolytic markers in patients with SCD. The objective of this study was to estimate the impact of voxelotor on the burden of SCD in France using a modeling approach, accounting for its anticipated adoption and diffusion over the next 5 years. We designed a sequential multi-cohort model to project and compare the cumulative incidence of SCD complications over a 20-year time horizon in a world with and without voxelotor. A distribution of patients was simulated across various levels of Hb response based on the phase 3 HOPE trial results, and relative risk reduction was adjusted using published meta-analysis results that projected risk reduction due to a 1 g/dL increase in Hb. In 6100 modeled patients with SCD treated with voxelotor, the model projected the number of deaths to decrease by 39.4%, with an increase of 1.8% in life-years gained. The model also projected life expectancy to increase by 15.8%, and incident cases of stroke, pulmonary hypertension, and chronic kidney disease to decrease by 19.8%, 24.5%, and 25.1%, respectively. The model suggests that improving Hb using a treatment such as voxelotor may have a positive public health impact by reducing the burden of SCD for patients and the healthcare system.

Provider prescription of hydroxyurea in youth and adults with sickle cell disease: A review of prescription barriers and facilitators.

Sickle cell disease (SCD) is an inherited red blood cell disorder associated with frequent painful events and organ damage. Hydroxyurea (HU) is the recommended evidence-based treatment of SCD. However, among patients eligible for HU, prescription rates are low. Utilizing a scoping review approach, we summarized and synthesized relevant findings regarding provider barriers and facilitators to the prescription of HU in youth and adults with SCD and provided suggestions for future implementation strategies to improve prescription rates. Relevant databases were searched using specified search terms. Articles reporting provider barriers and/or facilitators to prescribing HU were included. A total of 10 studies met the inclusion criteria. Common barriers to the prescription of HU identified by providers included: doubts around patients' adherence to HU and their engaging in required testing, concerns about side effects, lack of knowledge, costand patient concerns about side effects. Facilitators to the prescription of HU included beliefs in the effectiveness of HU, provider demographicsand knowledge. Findings suggest significant provider biases exist, particularly in the form of negative perceptions towards patients' ability to adhere to taking HU and engaging in the required follow-up. Improving provider knowledge and attitudes towards HU and SCD may help improve low prescription rates.