Sickle Cell Disease Phenotype Research Articles

Bone-marrow-homing lipid nanoparticles for genome editing in diseased and malignant haematopoietic stem cells.

Therapeutic genome editing of haematopoietic stem cells (HSCs) would provide long-lasting treatments for multiple diseases. However, the in vivo delivery of genetic medicines to HSCs remains challenging, especially in diseased and malignant settings. Here we report on a series of bone-marrow-homing lipid nanoparticles that deliver mRNA to a broad group of at least 14 unique cell types in the bone marrow, including healthy and diseased HSCs, leukaemic stem cells, B cells, T cells, macrophages and leukaemia cells. CRISPR/Cas and base editing is achieved in a mouse model expressing human sickle cell disease phenotypes for potential foetal haemoglobin reactivation and conversion from sickle to non-sickle alleles. Bone-marrow-homing lipid nanoparticles were also able to achieve Cre-recombinase-mediated genetic deletion in bone-marrow-engrafted leukaemic stem cells and leukaemia cells. We show evidence that diverse cell types in the bone marrow niche can be edited using bone-marrow-homing lipid nanoparticles.

Azathioprine/hydroxyurea preconditioning prior to nonmyeloablative matched sibling donor hematopoietic stem cell transplantation in adults with sickle cell disease: A prospective observational cohort study.

Nonmyeloablative, matched sibling donor hematopoietic stem cell transplantation with alemtuzumab/total body irradiation (TBI) conditioning is a curative therapy with low toxicity for adults with sickle cell disease (SCD). However, relatively low donor chimerism levels and graft rejection remain important challenges. We hypothesized that adding azathioprine/hydroxyurea preconditioning will improve donor chimerism levels and reduce graft failure rate. In this prospective cohort study, we enrolled consecutive adult patients with SCD undergoing matched sibling donor transplantation at the Amsterdam UMC. Patients received azathioprine 150 mg/day and hydroxyurea 25 mg/kg/day for 3 months prior to alemtuzumab 1 mg/kg and 300 cGy TBI conditioning. Twenty patients with SCD (median age 26 years [range 19-49], 13 females) were transplanted. Median follow-up was 46.0 months (IQR 21.8-57.9). One-year overall survival and event-free survival (graft failure or death) were both 95% (95% confidence interval 86-100). Mean donor myeloid and T-cell chimerism 1-year post-transplant were 95.2% (SD ±10.6) and 67.3% (±15.3), respectively. One patient (5%) experienced graft failure without autologous regeneration, resulting in infections and death. All other patients had a corrected SCD phenotype and were able to discontinue sirolimus. Three patients were successfully treated with alemtuzumab (1 mg/kg) after the transplant because of declining donor chimerism and cytopenias to revert impending graft rejection. Toxicity was mostly related to sirolimus and alemtuzumab. One patient developed steroid-responsive grade II intestinal acute graft-versus-host disease. Collectively, preconditioning with azathioprine/hydroxyurea prior to nonmyeloablative matched sibling donor transplantation resulted in excellent event-free survival and robust donor T-cell chimerism, enabling the successful withdrawal of sirolimus. ClinicalTrials.gov: NCT05249452.

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPC). This LVV contains locus control region cores expressing an anti-sickling βAS3-globin gene and two microRNA adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently reducing sickle hemoglobin (HbS). The reduction in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to reduce costs and help improve accessibility to gene therapy for SCD.

Sickle Cell Disease Phenotypes and Obstructive Sleep Apnea; Are They Related?

This study aims to compare the polysomnographic features between Arab-Indian and Benin phenotypes of sickle cell disease (SCD). This prospective cross-sectional study was conducted in the Children's Hospital at King Fahad MedicalCity, in Riyadhwhere childrenwere recruited fromthe pediatric hematology clinic and pediatric sleepmedicine. All families were approached and patients who met the inclusion criteria and agreed to participate were included in the study. Eighty four children (37 of whom were females) with SCD were included in the study. Their median (interquartile) age was 9 (6.65, 11) years and their body mass index z score was -1.45 (-2.195, -1.45). The evidence of obstructive sleep apnea (OSA) was more prominent in the Benin phenotype (66.7%) in comparison to those of the Arab-Indian (35.2%) phenotype ( p = 0.006). Additionally, 56.7% of Benin had moderate to severe OSA whereas Arab-Indian had 18% with a ( p = 0.0003). Controlling for other factors, the odds ratio (confidence interval) of having OSA in Benin phenotype was 4.68 (1.42-15.38) times higher as compared to Arab-Indian phenotype. The risk of having OSA as well as the severity of OSA is higher in Benin phenotype as compared to Arab-Indian phenotype which indicates the presence of potential OSA risk factors other than the SCD itself.

Clinical Biomarkers of Acute Vaso-Occlusive Sickle Cell Crisis.

It is known that an inherited blood condition called sickle cell disease (SCD) is a result of one gene. A number of blood and urine biomarkers have been determined in association with lab and clinical history for SCD patients. SCD has numerous interacting pathways associated with it, which have been identified by biomarkers. These mechanisms consist of some examples, such as endothelial vasodilation response, hypercoagulability, hemolysis, inflammation, oxidative stress, vascular dysfunction, and reperfusion injury among others. To effectively manage SCD, a comprehensive panel of validated blood and urine biomarkers must be established. Despite its monogenic inheritance, the complex nature of the SCD phenotype has impeded progress in its treatment. However, significant strides have been made in clinical biotechnology, paving the way for potential breakthroughs. In SCD, a panel of verified blood and urine biomarkers must be established, however. Despite monogenic inheritance, the great complexity of the SCD phenotype has hindered progress in its management. With few exceptions, clinical biomarkers of illness severity have been found through epidemiological investigations; nevertheless, systematic integration of these biomarkers into clinical treatment algorithms has not occurred. Furthermore, sickle cell crisis, the primary acute consequence of SCD, has been difficult to diagnose with the biomarkers now in use. Inadequate care and a lack of appropriate outcome measures for clinical research are the consequences of these diagnostic constraints. A new chapter in SCD customized treatment has begun with recent advancements in molecular and imaging diagnostics. Strategies in precision medicine are especially relevant now that molecular therapies are within reach. The significance of biochemical indicators linked to clinical manifestation and sub-phenotype identification in SCD is reviewed in this research.

Simvastatin-Mediated Nrf2 Activation Induces Fetal Hemoglobin and Antioxidant Enzyme Expression to Ameliorate the Phenotype of Sickle Cell Disease.

Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. Here, we report that the FDA-approved oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient primary erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammatory stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of the antioxidant reduced glutathione, and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of the critical histone methyltransferase enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant gene regulatory regions in an antioxidant response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression, modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD, and represents a therapeutic strategy for further development.

Mortality in adults with sickle cell disease: Results from the sickle cell disease implementation consortium (SCDIC) registry.

The cause of death in people affected by sickle cell disease (SCD) is often challenging to define as prior studies have used retrospective or administrative data for analysis. We used a prospective longitudinal registry to assess mortality and clinical co-morbidities among subjects enrolled in the Sickle Cell Disease Implementation Consortium (SCDIC) registry. At enrollment, we collected the following data: patient-reported demographics, SCD phenotype, baseline laboratory values, comorbidities, and current medications. Subjects were followed for a median of 4.7 years before the present analysis. The relationship of clinical co-morbidities (at time of enrollment) to mortality was determined using survival analysis, adjusting for SCD phenotype and gender. There was a total of 2439 people with SCD enrolled in the SCDIC registry. One hundred and twenty-eight participants (5%) died during the observation period (2017-2022). Six people died from trauma and were excluded from further analysis. Proximate cause of death was unwitnessed in 17% of the deaths, but commonest causes of death include cardiac (18%), acute chest or respiratory failure (11%), sudden unexplained death (8%). Enrollment characteristics of the individuals who died (n = 122) were compared to those of survivors (n = 2317). Several co-morbidities at enrollment increased the odds of death on univariate analysis. All co-morbidities were included in a multivariable model. After backward elimination, iron overload, pulmonary hypertension, and depression, remained statistically significant predictors of the risk of death. SCD reduces life expectancy. Improved comprehensive and supportive care to prevent end-organ damage and address comorbidities is needed for this population.

Neurological abnormalities among pediatric patients with sickle cell disease in Saudi Arabia: a single-center retrospective study.

Sickle cell disease (SCD) is a common inherited blood disorder characterized by the production of abnormal sickle-shaped red blood cells. SCD can lead to various complications including neurological issues. Early detection and treatment are crucial for preventing these complications. This study aimed to describe the neurological manifestations, radiological findings, and neurological diagnosis related to SCD in Saudi children with the aim of contributing to the formulation of population-based guidelines for screening and treating SCD-related neurological complications. This descriptive retrospective study included pediatric patients aged < 14 years diagnosed with SCD who were regularly followed up at the hematology clinic in KAMC, Jeddah, Saudi Arabia, from January 2008 to January 2022. Demographic and clinical data were collected from the clinical charts of 101 participants. This study included 101 patients with SCD with a mean age of 23 months at diagnosis. Among these, 59% had SCD and high fetal hemoglobin (HbF) levels. Neurological sequelae, including seizures, stroke, and other abnormalities, were observed in 26.7% of patients. There were no significant differences in the onset of neurological issues between the patients with SCD-high HbF and those with other SCD phenotypes. This study highlights the increased risk of brain injury and neurocognitive deficits in children with SCD. The occurrence of neurological sequelae in many patients emphasizes the need for early detection and intervention. Some patients experience neurological complications despite having high HbF levels, suggesting that further interventions are needed. This study has some limitations, including its small sample size and retrospective nature. Early detection and intervention are crucial for neurological complications in patients with SCD. This study emphasizes the need for further research and effective treatment strategies considering the presence of neurological complications despite the presence of high HbF levels. Large-scale studies and population-specific guidelines are warranted for better understanding and management of SCD-related neurological complications in the Saudi population.

In vivo measurement of RBC survival in patients with sickle cell disease before or after hematopoietic stem cell transplantation

AbstractStable, mixed-donor–recipient chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with sickle cell disease (SCD) is sufficient for phenotypic disease reversal, and results from differences in donor/recipient–red blood cell (RBC) survival. Understanding variability and predictors of RBC survival among patients with SCD before and after HSCT is critical for gene therapy research which seeks to generate sufficient corrected hemoglobin to reduce polymerization thereby overcoming the red cell pathology of SCD. This study used biotin labeling of RBCs to determine the lifespan of RBCs in patients with SCD compared with patients who have successfully undergone curative HSCT, participants with sickle cell trait (HbAS), and healthy (HbAA) donors. Twenty participants were included in the analysis (SCD pre-HSCT: N = 6, SCD post-HSCT: N = 5, HbAS: N = 6, and HbAA: N = 3). The average RBC lifespan was significantly shorter for participants with SCD pre-HSCT (64.1 days; range, 35-91) compared with those with SCD post-HSCT (113.4 days; range, 105-119), HbAS (126.0 days; range, 119-147), and HbAA (123.7 days; range, 91-147) (P<.001). RBC lifespan correlated with various hematologic parameters and strongly correlated with the average final fraction of sickled RBCs after deoxygenation (P<.001). No adverse events were attributable to the use of biotin and related procedures. Biotin labeling of RBCs is a safe and feasible methodology to evaluate RBC survival in patients with SCD before and after HSCT. Understanding differences in RBC survival may ultimately guide gene therapy protocols to determine hemoglobin composition required to reverse the SCD phenotype as it relates directly to RBC survival. This trial was registered at www.clinicaltrials.gov as #NCT04476277.

Renal outcomes in pediatric patients with sickle cell disease: a single center experience in Saudi Arabia.

Sickle cell nephropathy (SCN) is a significant complication of sickle cell disease (SCD) with an asymptomatic onset in childhood and potential progression to chronic kidney disease (CKD). The clinical findings of SCN include hyposthenuria, hematuria, proteinuria, hyperfiltration, and CKD. Data on renal manifestation among patients with SCD in Saudi Arabia is lacking. Therefore, this study aimed to evaluate renal outcomes in patients with SCD who visited a hematology clinic at the National Guard Hospital, Jeddah. We conducted a retrospective chart review of renal complications in patients with SCD who are within 0-14 years of age and on regular follow-ups at the pediatric hematology clinic in King Abdulaziz Medical City-Jeddah, Saudi Arabia. Among the 140 patients with SCD, 99 met the inclusion criteria. The median age at diagnosis was 18 (1-108) months. Two SCD phenotypes were observed, with 82 (83%) patients having sickle cell anemia (HbSS) and 17 (17%) having HbS/B+ thalassemia. Of the total patients, 92 (93%) were administered hydroxyurea (HU), with a median starting age of 48 (9-168) months. The most common renal complication observed during routine urinalysis was hematuria (38%), followed by proteinuria (11%). After stratifying the sample into four age groups (0-3 years old, 4-7 years old, 8-11 years old, and 12-14 years old), the mean glomerular filtration rate (GFR) values were 96.16, 101.36, 112.69, and 120.11 ml/min/1.73 m2 respectively. Renal imaging revealed abnormal findings in 27 (29%) patients. The most common abnormality observed on US was increased echogenicity (43%). SCN is a significant complication of SCD. In this study, we assessed the renal outcomes in pediatric patients with SCD. After analyzing the clinical findings of SCN, we concluded that the presence of renal complications in pediatric patients presented a progressive pattern.

Pharmacologic Reduction of Platelet Count Attenuates Whole Blood Hypofibrinolysis in a Mouse Model of Sickle Cell Disease

Increased risk of venous thrombosis is a hallmark of sickle cell disease (SCD). We previously reported that SCD dramatically alters the properties of venous clots, including resistance to tissue plasminogen activator (tPA)-mediated fibrinolysis. Using an internal fibrinolysis assay (tPA added to whole blood before clot formation), clot lysis time (CLT) was significantly (p&amp;lt;0.01) prolonged in clots formed ex vivo from whole blood of SCD patients (SS) (651±34 min) compared to race-matched controls (AA) (370±75). We then investigated how therapeutic RBC exchange affects CLT in SCD patients. Interestingly, despite hemoglobin A levels of approximately 50%, CLT for the pre-exchange SS clots (639±81 min) was similar to CLT recorded for blood clots from SS patients who did not undergo RBC exchange (see above). However, CLT for post-exchange SS clots was significantly shortened (240±34; p&amp;lt;0.05 vs pre-exchange) to the level observed for clots from AA controls. We noticed that the RBC exchange procedure not only changes the proportion of AA and SS RBCs, but also reduces both platelet counts and serum plasminogen activator inhibitor-1 (PAI-1) levels by approximately 50-60%. Importantly, exvivo restoration of the platelet number in post-exchange samples to the level observed in pre-exchange blood resulted in restoration of the hypofibrinolytic phenotype and a partial restoration of serum PAI-1 levels. Changes in platelet count after RBC exchange are quickly normalized. Therefore, in the current study, we investigated the effect of sustained reduction of platelet count on whole blood hypofibrinolysis in SCD mice. Using an external fibrinolysis assay (2.5 nM tPA added after clot formation) as described by Bonnard et al. (Sci Rep, 2017) we determined if hypofibrinolysis is also observed in Townes sickle cell mice (SS). Consistent with human SCD, the CLT was profoundly increased in ex vivo clots formed from whole blood of SS mice (n=13) compared to non-sickle (AA) control mice (n=12) (Figure 1A; first two bars). Next, we demonstrated that the cellular fraction of SS blood was responsible for resistance of the clots to tPA-mediated fibrinolysis, regardless of whether the plasma was from SS or AA mice (Figure 1A; last four bars). We then used a pharmacologic strategy to reduce platelet counts. Thrombopoietin (TPO) is a growth factor that promotes maturation of hematopoietic stem cells into megakaryocytes and thereby increases platelet production. We previously showed that silencing liver TPO expression using a liver-specific antisense oligonucleotide (TPO-ASO) resulted in a sustained 50% reduction in both plasma TPO levels and platelet counts without affecting hemostasis in both baboons and mice. In the present study, TPO-ASO (20 mg/kg subcutaneous) or vehicle control were administered to 3-month old AA and SS mice (n=3-6) on days 0, 3, 5 and 7, followed by weekly maintenance injections for the next 3 weeks. 4 weeks after initiation of treatment, mice were euthanized, and blood was collected to evaluate plasma levels of TPO and external whole blood fibrinolysis. Platelet, but no other cell count was significantly reduced in both AA (by 62%, p&amp;lt;0.05) and SS (by 47%, P&amp;lt;0.05) mice treated with TPO-ASO. Baseline plasma levels of TPO were reduced in SS mice (101±20 pg/ml) compared to AA controls (182±10; p&amp;lt;0.05). TPO-ASO treatment resulted in significant reduction of TPO plasma levels in both AA (21±16; p&amp;lt;0.01) and SS (17±9; p&amp;lt;0.05) mice. Interestingly, TPO-ASO-mediated reduction of platelet count did not affect CLT of ex vivo clots formed from whole blood of AA mice (Figure 1B). In contrast, TPO-ASO treatment dramatically reduced CLT in SS clots (p&amp;lt;0.05) (Figure 1B). In conclusion, we demonstrated that: (i) the resistance of ex vivo formed clots to tPA-mediated fibrinolysis observed in SCD patients can be reproduced in a mouse model of the disease; (i) the external fibrinolysis assay provides a robust and reliable model for studying whole blood hypofibrinolysis in SCD; (iii) hypofibrinolysis in SCD appears to be predominantly driven by the cellular fraction of blood; and (iv) platelets play an important role in the hypofibrinolytic phenotype in SCD, demonstrated by mitigation of the hypofibrinolytic phenotype in SS mice subjected to long-term reduction of platelet count. Further studies investigating the effect of platelet count reduction on venous thrombosis and vascular stasis in SS mice are currently ongoing.

Prevalence of Duffy Null Status and Its Impact on Hydroxyurea Dosing in Young Children with Sickle Cell Disease in the United States

Background: Sickle cell disease (SCD) affects approximately 100,000 Americans and causes many morbidities mediated by inflammation. Hydroxyurea (HU), the primary SCD therapy, demonstrates superior outcomes with dose-escalation. While HU induces fetal hemoglobin, it also reduces absolute neutrophil counts (ANC) and inflammation. However, ANC is used to determine if HU can be dose-escalated. Thus, a lower baseline ANC has potential to limit dose escalation and outcomes. The Duffy null phenotype, ubiquitous among West Africans, reduces ANC through neutrophil egress from the blood into tissues.While this does not increase infection risk, its prevalence and impact on ANC, HU dosing, and outcomes among children with SCD in the U.S. are unknown. Thus, the primary objective of this study was to determine prevalence of Duffy null among children with SCD (any genotype) receiving care and who had erythrocyte phenotyping performed at a large, Midwestern SCD center between 2010-2022. We also aimed to compare baseline ANC of children with SCD and Duffy null and to children with SCD and Duffy positive phenotypes at their one-year hematology visit (overall and by genotype severity). Finally, in those who initiated HU, we aimed to compare ANC, HU dosing, and the frequency of acute healthcare utilization for SCD complications between groups during the first year of HU use. Methods: This was a retrospective study of children with SCD. Subjects were identified using a list of those who had phenotyping performed at the center and confirmed to have SCD in their electronic health records. Individuals with Fy a-Fy b- were considered to be Duffy null. Data abstracted included demographics (e.g., age, race) and laboratory studies (e.g., hemoglobin (Hb), ANC). HU prescribing information and emergency department and/or admissions among those who used HU for ≥1year were also recorded. Descriptive statistics (median and interquartile range (IQR)) were used and Chi-squared, Fisher's exact, and Wilcoxon rank sum were used to compare categorical and continuous groups; p-value &amp;lt;0.05 was considered statistically significant. Results: Of 187 children with SCD who had phenotyping; 147 (78.6%) had Duffy null (Table 1) and were compared to Duffy positive children with SCD (Figure 1). A subset of 72 children with Duffy null and 22 with Duffy positive initiated HU;59 with Duffy null and 15 with Duffy positive remained on HU for ≥1 year. Median HU doses at initiation (p=0.72) and at one year (p=0.35) did not significantly differ between groups. Median ANC at HU initiation was 4.0 mm 3 (IQR 3.3.-5.9) in the Duffy null group and 5.0 mm 3 (IQR 3.2-6.8) in the Duffy positive group but this difference was not significantly different (p=0.21), nor was median ANC at one year between groups (2.0 mm 3, IQR 1.9-4.2 vs. 3.7 mm 3, IQR 2.8-4.9, p=0.07). Median number of acute visits did not differ between the Duffy null (0, IQR 0-1) and Duffy positive (0, IQR 0-1) groups (p=0.65). Discussion: We identified that Duffy null, while not ubiquitous, was present in more than three fourths of young children with SCD in the Midwest and was associated with having a lower ANC at one year of life. Given that HU dose-escalation improves outcomes, it was reassuring that those with Duffy null on HU did not have a significantly lower ANC and were not prescribed lower dose HU than Duffy positive children at either point. However, it is notable that these may be because few children in both groups had ANC low enough to preclude HU initiation or escalation based on dosing guidelines and because acute visits were infrequent. Limitations include the retrospective design, small number of those who initiated and remained on HU for ≥1 year, and short time interval that clinical and laboratory outcomes were compared. Also, it is possible that some did not have phenotyping completed, but standard practice at our center is to complete phenotyping on all with SCD. In conclusion, Duffy null is highly prevalent in young children with SCD in the U.S. While this does not appear to have an impact on HU dosing of young children with SCD early in their HU course, subsequent longitudinal studies of older children are needed to explore if Duffy null has an impact on longer-term HU dosing and outcomes.

Initial Results in a Phase 1b Trial of PB-04 in Sickle Cell Disease Demonstrate Fetal Hemoglobin Induction, Additive Activity with Hydroxyurea, and Improved Red Blood Cell Sickling Parameters

Extensive evidence has shown fetal hemoglobin (HbF) is a major modulator of sickle cell disease (SCD) phenotype. Clinical benefit with any increment of HbF was shown in the NIH-sponsored Natural History Study and the Multicenter Study of Hydroxyurea (HU), with mean HbF increases of 3.6% above baseline (BL), to improve survival, reduce painful vaso-occlusive crises, and recently stroke prevention and improved cardiac and neurocognitive function with HU are reported. As some patients do not tolerate optimal HU doses due to cytopenias, additional inducers suitable for combined use are desirable. Proportions of F-cells and HbF-quantity/cell have been cited as important. PB-04, a repurposed drug with a well-known safety profile, is an oral fetal globin stimulant identified in high throughput screening, which suppresses 4 repressors of the HMG gene promoter, ( BCL-11A, LSD-1, HDAC-3, KLF-1). PB-04 induces fetal globin in nonhuman primates, transgenic mice, with additive effect when combined with HU in SCD erythroid progenitors, and was shown to reduce infarcts in multiple organs with preserved splenic function in transgenic sickle mice. An ongoing Phase 1b trial in beta thalassemia intermedia (BTI) ( NCT04432623) of 3 doses (1,3, or 5 mg/kg QD) given 3 (TIW) or 5 times/week (Wk) for 12 or 24 Wks in 18 courses in 10 subjects demonstrates a favorable safety profile, dose-proportional PK with 5 &amp;gt;3 mg/kg doses and 5 &amp;gt;3 doses/Wk, Median fold-increases from baseline (BL) in F-cells of 4.4x BL (range 2.3-7x), 2.8x F-reticulocytes (1.6-14.8x), and 6.5x (range 3-11x) increases in mean fluorescence intensity (MFI, representing amount of HbF/RBC). Mean increases in % HbF were 6.5%. Evaluation of PB-04 has now begun in 7 subjects with SCD, 19-51 yrs; 3 were receiving HU with HbF responses (12-22%). Adverse events, F-cells, F-reticulocytes, MFI (amount of HbF/RBC or HbF/reticulocyte) by FACS and ImageStream Analysis and % HbF are assessed Q 4 wks; flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM) and sickling kinetics are assessed q 12 wks. In 4 subjects on 3 mg/kg QD TIW for 24 wks, changes observed from BL values included mean increases of 6% F-cells and 27% F-reticulocytes (2-fold) . In 3 ongoing subjects receiving 5 mg/kg QD TIW for 12 wks mean changes above BL are currently 13% F-cells, 15% F-reticulocytes; HbF/cell or HbF/reticulocyte have increased up to 10-fold. Mean % HbF has increased above HU effects in 2/3 pts on the second dose by 5-12.6% (mean 8.8%), producing 81-95% F-cells, and 35-38% HbF. Decline in FA-WB-VCAM) was observed ± HU and with both doses in 3 subjects. Correlation of HbF quantity/cell with RBC survival and sickling dynamics is being explored. These early results with not-yet-optimized doses of PB-04 suggest additive activity with HU, producing significant HbF and F-cell levels, and support further PK-optimized studies in patients with SCD.

A Genotype Validated Bimodal Method for the Large-Scale Identification and Phenotyping of Persons with Sickle Cell Disease Using Electronic Health Record Data

Introduction Clinical trials for using hematopoietic stem cell transplant (HSCT) with gene therapies to cure sickle cell disease (SCD) began in 2019. The Food and Drug Administration (FDA) requires that 1) all gene therapy participants receive at least 15 years of ongoing surveillance and 2) their outcomes be compared to a contemporaneous cohort that did not receive gene therapy. Previously we developed an automated contemporaneous cohort of children and adults with SCD from electronic health record (EHR) data (Cronin RM et al. 2023, Blood Adv). Our work was built upon previously explored algorithms utilizing ICD codes and laboratory data to identify persons with sickle cell anemia (SCA), a severe form of SCD (Singh et al. 2018, Blood Adv). Still, our algorithm additionally classifies other predicted genotypes. However, we could not test the SCD phenotypes against the SCD genotypes due to the absence of beta-globin gene sequence data. Here we tested the hypothesis that our Vanderbilt-EHR algorithm for identifying a contemporaneous cohort at Vanderbilt can classify their predicted genotype compared to beta-globin gene sequencing, the gold standard for SCD diagnosis, in the cohort. We subsequently tested the Vanderbilt-EHR algorithm in an established cohort of children from St. Jude Children's Research Hospital that have been deeply phenotyped. Methods We applied the Vanderbilt-EHR algorithm to a cohort of 275 samples from Vanderbilt's biorepository of DNA, BioVU, linked to de-identified medical records in a data warehouse called the Synthetic Derivative. These 275 samples were previously submitted for whole genome sequencing as part of the Genetic Variation of Heart, Lung, and Kidney Disease in Sickle Cell Disease: Pre- and Post-Curative Therapies TOPMed project (HLK-SCD phs002617). The returned genotypes were validated using the hemoglobin variant database HbVar (Giardine BM et al. 2021, Nucleic Acids Res) (Table 1). We applied the hemoglobinopathy genotype classification portion of the algorithm to the Sickle Cell Clinical Research and Intervention Program (SCCRIP) cohort from St. Jude Children's Research Hospital, which includes putative genotype diagnoses derived from a comprehensive evaluation of clinical data for each member. We performed statistical analyses of the identification and classification results of the Vanderbilt cohort and the SCCRIP cohort's classification results. Results Of the 275 genotyped samples, the algorithm correctly predicted 255 SCD cases and 10 non-SCD cases. There were three cases predicted to be false positives; however, a comprehensive analysis showed that all have laboratory values indicative of SCA (high hemoglobin S levels &amp;gt;60%, absent or negligible hemoglobin A levels, elevated reticulocytes, and low mean corpuscular volume) suggesting that there may be a genotyping error. The remaining 7 cases were either indeterminate in the prediction or in the genotype (Table 1). Given the small number of indeterminate results, we performed a sensitivity analysis to identify the range of performance for SCD identification. The method performed very well with over 98% sensitivity and over 96% positive predictive value (PPV) (Table 2). The lower specificity and negative predictive value (NPV) could be largely attributed to the disproportionately high number of SCD cases in the dataset compared to non-SCD cases. The SCD classification algorithm performed similarly in both the VUMC and SCCRIP cohorts (Table 2). The algorithm performs best in identifying SC and SCA types of SCD with nearly 100% accuracy for SC and over 94% accuracy for SCA. Transfusions can confound the diagnosis of SCA vs. S beta thalassemia +, but the latter has a much lower prevalence consistent with our results (Table 2). Discussion and Conclusions Larger SCD cohorts will be identified using these approaches in singular EHR databases and in de-identified data warehouses that EHR companies have developed by aggregating data across multiple EHR systems (e.g., EPIC Cosmos and Cerner Real-World Data). The ongoing need for contemporaneous comparison cohorts of persons with SCD to understand outcomes related to treatment and phenotype can be greatly assisted by accurate automated approaches. The Vanderbilt-EHR algorithm is the first to comprehensively phenotype SCD and other hemoglobinopathies involving variant hemoglobin beta alleles.

Pain Frequency and Health Care Utilization Patterns in Women with Sickle Cell Disease Experiencing Menstruation-Associated Pain Crises.

Background: Pain crises in sickle cell disease (SCD) lead to high rates of health care utilization. Historically, women have reported higher pain burdens than men, with recent studies showing a temporal association between pain crisis and menstruation. However, health care utilization patterns of SCD women with menstruation-associated pain crises have not been reported. We studied the frequency, severity, and health care utilization of menstruation-associated pain crises in SCD women. Materials and Methods: A multinational, cross-sectional cohort study of the SCD phenotype was executed using a validated questionnaire and medical chart review from the Consortium for the Advancement of Sickle Cell Research (CASiRe) cohort. Total number of pain crises, emergency room/day hospital visits, and hospitalizations were collected from a subcohort of 178 SCD women within the past 6 months and previous year. Results: Thirty-nine percent of women reported menstruation-associated pain crises in their lifetime. These women were significantly more likely to be hospitalized compared with those who did not (mean 1.70 vs. 0.67, p = 0.0005). Women reporting menstruation-associated pain crises in the past 6 months also experienced increased hospitalizations compared with those who did not (mean 1.71 vs. 0.75, p = 0.0016). Forty percent of women reported at least four menstruation-associated pain crises in the past 6 months. Conclusions: Nearly 40% of SCD women have menstruation-associated pain crises. Menstruation-associated pain crises are associated with high pain burden and increased rates of hospitalization. Strategies are needed to address health care disparities within gynecologic care in SCD.

Improved Biorepository to Support Sickle Cell Disease Genomics and Clinical Research: A Practical Approach to Link Patient Data and Biospecimens from Muhimbili Sickle Cell Program, Tanzania.

In Africa, sickle cell disease phenotypes' genetic contributors remain understudied due to the dearth of databases that pair biospecimens with demographic and clinical details. The absence of biorepositories in these settings can exacerbate this issue. This article documents the physical verification process of biospecimens in the biorepository, connecting them to patient clinical and demographic data and aiding in the planning of future genomic and clinical research studies' experience from the Muhimbili Sickle Cell Program in Dar es Salaam, Tanzania. The biospecimen database was updated with the current biospecimen position following the physical verification and then mapping this information to its demographic and clinical data using demographic identifiers. The biorepository stored 74,079 biospecimens in three -80°C freezers, including 63,345 from 5159 patients enrolled in the cohort between 2004 and 2016. Patients were identified by a control (first visit), entry (when confirmed sickle cell homozygous), admission (when hospitalized), and follow-up numbers (subsequent visits). Of 63,345 biospecimens, follow-ups were 46,915 (74.06%), control 8067 (12.74%), admission 5517 (8.71%), and entry 2846 (4.49%). Of these registered patients, females were 2521 (48.87%) and males were 2638 (51.13%). The age distribution was 1-59 years, with those older than 18 years being 577 (11.18%) and children 4582 (88.82%) of registered patients. The notable findings during the process include a lack of automated biospecimen checks, laboratory information management system, and tubes with volume calibration; this caused the verification process to be tedious and manual. Biospecimens not linked to clinical and demographic data, date format inconsistencies, and lack of regular updating of a database on exhausted biospecimens and updates when biospecimens are moved between positions within freezers were other findings that were found. A well-organized biorepository plays a crucial role in answering future research questions. Enforcing standard operating procedures and quality control will ensure that laboratory users adhere to the best biospecimen management procedures.

Genetic Variation and Sickle Cell Disease Severity

Sickle cell disease (SCD) is a monogenic disorder, yet clinical outcomes are influenced by additional genetic factors. Despite decades of research, the genetics of SCD remain poorly understood. To assess all reported genetic modifiers of SCD, evaluate the design of associated studies, and provide guidelines for future analyses according to modern genetic study recommendations. PubMed, Web of Science, and Scopus were searched through May 16, 2023, identifying 5290 publications. At least 2 reviewers identified 571 original, peer-reviewed English-language publications reporting genetic modifiers of human SCD phenotypes, wherein the outcome was not treatment response, and the comparison was not between SCD subtypes or including healthy controls. Data relevant to all genetic modifiers of SCD were extracted, evaluated, and presented following STREGA and PRISMA guidelines. Weighted z score meta-analyses and pathway analyses were conducted. Outcomes were aggregated into 25 categories, grouped as acute complications, chronic conditions, hematologic parameters or biomarkers, and general or mixed measures of SCD severity. The 571 included studies reported on 29 670 unique individuals (50% ≤ 18 years of age) from 43 countries. Of the 17 757 extracted results (4890 significant) in 1552 genes, 3675 results met the study criteria for meta-analysis: reported phenotype and genotype, association size and direction, variability measure, sample size, and statistical test. Only 173 results for 62 associations could be cross-study combined. The remaining associations could not be aggregated because they were only reported once or methods (eg, study design, reporting practice) and genotype or phenotype definitions were insufficiently harmonized. Gene variants regulating fetal hemoglobin and α-thalassemia (important markers for SCD severity) were frequently identified: 19 single-nucleotide variants in BCL11A, HBS1L-MYB, and HBG2 were significantly associated with fetal hemoglobin (absolute value of Z = 4.00 to 20.66; P = 8.63 × 10-95 to 6.19 × 10-5), and α-thalassemia deletions were significantly associated with increased hemoglobin level and reduced risk of albuminuria, abnormal transcranial Doppler velocity, and stroke (absolute value of Z = 3.43 to 5.16; P = 2.42 × 10-7 to 6.00 × 10-4). However, other associations remain unconfirmed. Pathway analyses of significant genes highlighted the importance of cellular adhesion, inflammation, oxidative and toxic stress, and blood vessel regulation in SCD (23 of the top 25 Gene Ontology pathways involve these processes) and suggested future research areas. The findings of this comprehensive systematic review and meta-analysis of all published genetic modifiers of SCD indicated that implementation of standardized phenotypes, statistical methods, and reporting practices should accelerate discovery and validation of genetic modifiers and development of clinically actionable genetic profiles.

Effects of 2,3-DPG knockout on SCD phenotype in Townes SCD model mice.

Sickle cell disease (SCD) is a severe, multisystemic hematological disorder that impacts nearly every major organ in adults. The current approved treatments for SCD directly target mutant hemoglobin or address downstream disease pathology. Several compounds targeting reduction of 2,3-DPG by activation of Pyruvate Kinase-R are currently being evaluated in SCD patients. In this study, we genetically engineered a mouse lacking 2,3-DPG on the Townes SCD mouse model background and evaluated the effects of 2,3-DPG loss on disease pathology. Animals lacking 2,3-DPG showed improvements in hematological markers and reductions in RBC sickling relative to native Townes mice, however, minimal difference in organ damage was observed in 2,3-DPG deficient mice compared to native Townes animals. When animals lacking 2,3-DPG were dosed with a compound designed to increase hemoglobin oxygen affinity, oxygen delivery related toxicity was observed.

Natural Course of Albuminuria in Pediatric and Murine Sickle Cell Disease

Sickle cell disease (SCD), an inherited blood disorder, is associated with prevalent chronic kidney disease (CKD), evidenced by persistent albuminuria. It is critical to understand the natural history of albuminuria and albumin to creatinine (ACR) ratio variance to optimally identify, diagnose and treat SCD pediatrics prior to CKD. Thus, we investigated natural course of albuminuria in pediatrics and murine SCD, and identify ACR level predictive of persistent albuminuria.Basic science study: As hyperfiltration occurs at 12 weeks of age in male and 20 weeks of age in female humanized sickle cell (HbSS) mice, we performed metabolic cage studies in 1-week intervals for 4 weeks before (4 weeks old mice, n=21) and after hyperfiltration phase (males: 16 weeks (n=10), females: 20 weeks old (n=8)), and measured albuminuria using GenWay Biotech ELISA kit. Clinical study: We performed a retrospective study of the UAB Pediatric Sickle Cell Kidney Cohort. We identified all participants with HbSS and HbSB0 thalassemia ≥ 5 years of age with ACR measurements performed at a steady-state outpatient clinic visit. We identified the date and age of participants with at least 1 ACR level ≥100mg/g to compare outcomes for albuminuria and to determine the natural course of ACR after an ACR measurement &gt;100mg/g.We observed a significant difference in the trajectory of albuminuria over time prior and post hyperfiltration (p=0.02) in HbSS mice. Young HbSS mice presented with downward trend over time, with average values of albuminuria 47.6±27.2 (week 1), 32.6±20.3 (week 2), 32.0±24.3 (week 3), 31.3±16.1mg/24h (week 4), respectively. After hyperfiltration, older HbSS mice had an upward trend in albuminuria over time (week 1: 49.7±23.6, week 2: 74.5±66.9, week 3: 55.9±82.2, week 4: 73.0±86.4 mg/24h, respectively). Finally, prior to hyperfiltration the average albuminuria over a time of 4 weeks was 35.9±7.8 mg/24h (±21.7%), while following hyperfiltration it was 63.3±12.4 mg/24h (±19.6%). These findings mirror human data in which pediatric patients present with similar degree of natural variability in ACR. We identified 62 patients (17%) with persistent albuminuria, 46 (13%) with intermittent albuminuria, and 247 (70%) with not yet having albuminuria. Out of 107 patients with at least 1 abnormal ACR, 45 had 1 measurement &gt;100mg/g, and 43 of these participants were categorized with persistent albuminuria (50x higher odds ratio than patients with ACR&lt;100mg/g). Assessment of natural history of albuminuria after an initial ACR &gt;100mg/g showed significant variability. Regarding 1-year clinical trial design, the change in ACR showed a decline from 175 to 131 mg/g, accounting for 23% decline in albuminuria without any intervention.The natural course of albuminuria is an important characteristic of renal phenotype in SCD and should be considered while designing and assessing effectiveness of novel therapeutic approaches in preclinical and clinical studies targeting SCD nephropathy. Funded by NIH R25 DK 112731 and NHLBI–R00HL144817 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate β-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to β-globin exon-1 (βE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-βE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells invivo. Furthermore, PRR-βE1 gene editing is feasible without exvivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and β-thalassemia major. These findings imply that PRR-βE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for β-hemoglobinopathy gene therapy.