Auswahl der wissenschaftlichen Literatur zum Thema „SS-thalassemia“

SummaryIt has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

Abstract Disease severity and healthcare utilization varies widely among persons with sickle cell disease (SCD). Hydroxyurea (HU) has been demonstrated to reduce rates of pain and acute chest syndrome, the leading causes of inpatient utilization in patients with Hb SS and S β°-thalassemia in clinical trials. We recently reported that HU was clinically effective in reducing rates of pain and acute chest syndrome in patients who initiate treatment. Use of hydroxyurea in SS/S β°-thalassemia has increased markedly since 2010. Thus we sought to detemine trends of hospital utilization for acute illness during an 8 yr in which HU utilization increased markedly (2010-2017). Data from years 2010-201 were obtained from the SCD database and patient records at Children's Healthcare of Atlanta (CHOA). Utilization data were restricted to acute care admissions. Admissions for elective procedures, non-SCD related discharge diagnoses, rare SCD genotypes, and patients who had undergone bone marrow transplant were excluded. Patients were compared based on number of hospitalizations, age, sex, SCD genotype (SS/S β°-thalassemia vs Hb SC/S β+thalassemia), and discharge diagnosis. A total of 3,116 patients had at least one encounter between 2010 and 2017; 2,947 patients met inclusion criteria. From 2010-2017 the total number of active patients per year increased from 1,546 patients to 1,789 patients (+16%), while the total number acute care admissions increased from 1,295 admissions to 1,609 admissions (+24%). There were no significant differences in the proportion of patients with genotypes SS/S β° thalassemia genotypes (67.0% vs 63.9%, p=0.06). Overall patients with SS/S β° thalassemia had higher admission rates compared to SC/S β+ thalassemia patients (0.94 vs 0.57 admissions per patient per year). During the study period. overall admission rates in SCD (acute illness hospitalizations/patient/yr) increased from a low of 0.74 in 2011 to a high of 0.90 in 2017. The proportion of admissions attributed to SS/S β°-thalassemia patients decreased (79.2% in 2010 vs 72.3% in 2017, p<0.0001). However, admission rate in SC/S β+ thalassemia increased (0.53 to 0.69 admissions per patient per year). Overall, over 60% of patients were not admitted in any given year, and the proportion of patients with 0-1 admissions in a given year remained unchanged. However, the proportion of super high hospital utilizers (SHHU), patients with 8 or more admissions in a given year, increased by 185%. In 2001 this group made up 0.6% of all patients and accounted for 7.3% of admissions; in 2017 SHHU had increased 1.8% of patients and 24.3% of admissions. There was no difference in genotype or sex between SHHU and non-SHHU patients. SHHU were older (>90% of patients over age 8 years), and had greater percentages of admissions for pain and acute chest syndrome then non-SHHU. In conclusion, during a period in which HU utilization in SS/S β°-thalassemia increased significantly, hospital utilization for acute illness in SS/S β° thalassemia decreased as expected. However, during the same period there was an unexpected increase in overall hospital utilization for acute illness in SCD. This increase in hospital utilization was the result of 1) a marked increase in SHHU and 2) an overall increased utilization in SC/S β+ thalassemia. Disclosures Dampier: Pfizer: Research Funding.

Abstract alpha Thalassemia modifies the hematologic expression of homozygous sickle cell (SS) disease, resulting in increased total hemoglobin and HbA2 and decreased HbF, mean cell volume, reticulocytes, irreversibly sickled cells, and bilirubin levels. The age at which these changes develop in children with SS disease is unknown. Ascertainment of globin gene status in a large representative sample of children with SS disease has afforded an opportunity to study the hematologic indices in nine children homozygous for alpha thalassemia 2 (two-gene group), 90 children heterozygous for alpha thalassemia 2 (three-gene group), and 167 children with a normal alpha globin gene complement (four-gene group). The two-gene group had significantly lower mean cell volumes from birth, higher red cell counts from one month, lower reticulocytes from three months, and higher HbA2 levels from one year, as compared with the four-gene group. Children with three genes had intermediate indices but resembled more closely the four-gene group. Differences in total hemoglobin or in fetal hemoglobin between the groups were not apparent by eight years of age. The most characteristic differences of the two-gene group were the raised proportional HbA2 level and low mean cell volume, the latter having some predictive value for alpha thalassemia status at birth.

alpha Thalassemia modifies the hematologic expression of homozygous sickle cell (SS) disease, resulting in increased total hemoglobin and HbA2 and decreased HbF, mean cell volume, reticulocytes, irreversibly sickled cells, and bilirubin levels. The age at which these changes develop in children with SS disease is unknown. Ascertainment of globin gene status in a large representative sample of children with SS disease has afforded an opportunity to study the hematologic indices in nine children homozygous for alpha thalassemia 2 (two-gene group), 90 children heterozygous for alpha thalassemia 2 (three-gene group), and 167 children with a normal alpha globin gene complement (four-gene group). The two-gene group had significantly lower mean cell volumes from birth, higher red cell counts from one month, lower reticulocytes from three months, and higher HbA2 levels from one year, as compared with the four-gene group. Children with three genes had intermediate indices but resembled more closely the four-gene group. Differences in total hemoglobin or in fetal hemoglobin between the groups were not apparent by eight years of age. The most characteristic differences of the two-gene group were the raised proportional HbA2 level and low mean cell volume, the latter having some predictive value for alpha thalassemia status at birth.

Abstract Chronic hyperbilirubinemia is common in patients with sickle cell disease (SCD), frequently resulting in the formation of gallstones. This hyperbilirubinemia (predominantly unconjugated) is attributed to hemolysis that exceeds the conjugating capacity of the hepatic UDP-glucuronosyltransferase (UGT1A) enzyme. Previous studies have shown that genetic variants ([TA]n repeats) in the promoter region of the UGT1A gene have a major influence on the levels of bilirubin and gallstones. Alpha-thalassemia, which is associated with reduced hemolysis, has also been shown to affect bilirubin levels. Another potential modulating factor is heme-oxygenase, a rate-limiting enzyme in the heme catabolic pathway that results in the production of bilirubin. While the severity of jaundice and cholelithiasis in patients with SCD is predisposed by the inheritance of certain variants of the UGT1A gene, inconsistencies have been observed. We propose that some of these inconsistencies may be explained by the modulating effects of genetic variants of HO1 and α-thalassemia. A total of 263 patients with SCD attending specialist clinics in two hospitals were studied: King’s 116 SS, 5 Sβ0 and 59 SC; St Thomas’ 83 SS. 81 ethnically matched subjects were recruited as controls (HbAA). Groups were age and sex matched. Cholelithiasis data, ascertained by liver ultrasound, was available for a subset of SCD patients (76 SS, 4 SC). Samples were genotyped for variants of UGT1A, HO1 and α-thalassemia. The different genetic allele distributions were statistically similar between groups. Data was analysed according to the sum of [TA] repeats on both alleles of UGT1A. A range of 10–15 [TA] repeats was observed. For HO1, the median sum of repeats on both alleles was 63, so samples were grouped as &lt;63 or ≥ 63. α-thalassemia genotypes were as follows: SS, 127 αα/αα, 55 αα/α-, 11 α-/α-; SC, 37 αα/αα, 20 αα/α-; controls 51 αα/αα, 22 αα/α-, 4 α-/α-. Median bilirubin levels varied as expected between groups according to β-globin genotype and were as follows: King’s SS 42 (15–269.5); St Thomas’ SS 52.5 (15.5–696.5); King’s SC 22 (10–81.8); AA Controls 10 (5–24), median (range) mmol/L. Regression analysis showed that serum bilirubin levels were strongly associated with UGT1A repeat length in all subjects (p&lt;0.0001). Furthermore, the increase in serum bilirubin (21.3% mean for SS/Sβ0, 20.5% for SC) and cholelithiasis risk odds (86.5% mean for SS/Sβ0, 67.6% for SC) could be quantified per [TA] repeat. HO1 genotype did not affect serum bilirubin in SCD patients or the control cohort. The presence of α- thalassemia correlated (negatively) with serum bilirubin in SCD patients (p&lt;0.0001) but not controls. This is the first time the relationship between UGT1A [TA] repeat length, serum bilirubin and cholelithiasis has been shown quantitatively. Additionally, co-existing α-thalassemia appears to moderate bilirubin levels but HO1 variants do not.

Abstract Osteoporosis represents an important cause of morbidity in adult patients with thalassemia. Its pathogenesis is multifactorial, and includes mainly bone marrow expansion, endocrine dysfunction and iron overload. Bone metabolism is altered in thalassemia. Osteoclast function is elevated, while osteoblast activity seems to be reduced and thus the balance of bone remodeling is in favor of bone loss. The exact mechanisms of osteoblast dysfunction have not been fully clarified to-date. Wingless-type (Wnt) signaling is an important pathway for osteoblast differentiation. Dickkopf-1 (Dkk-1) protein is an inhibitor of Wnt pathway and is implicated in the pathogenesis of several bone disorders. Collagen type-I is the main structural protein of the bone. The collagen type-I alpha (COLIA)-1 specific protein (Sp)-1 polymorphism has been related to osteoporosis in thalassemia. The aim of this study was to evaluate the serum levels of Dkk-1 in patients with thalassemia-induced osteoporosis who receive therapy with zoledronic acid (ZOL) and evaluate possible correlations with clinical and laboratory data, including the COLIA-1 Sp1 polymorphism. Sixty-six patients (21M/45F; median age 35.5 years) with thalassemia and osteoporosis were studied. Patients were blindly randomized to receive ZOL at a dose of 4 mg, iv, in 15 min infusion, every 6 months (group A, n=23) or every 3 months (group B, n=21), or to receive placebo every 3 months (group C, n=22) for a period of one year. All patients received oral calcium (500 mg) during the treatment period. Dkk-1 was measured at baseline and after 12 months of therapy using ELISA methodology (Biomedica Medizinprodukte, Wien, Austria) along with a series of serum bone remodeling indices: bone resorption markers [C-telopeptide of type-I collagen (CTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b)], bone formation markers [bone-specific alkaline phosphatase (bALP), osteocalcin, and C-terminal propeptide of collagen type-I (CICP)], and osteoclast regulators [receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG), and osteopontin]. The above bone markers were also evaluated in 30, age- and gender-matched, healthy controls. The G-->T mutation at base 1 of intron 1 at the binding site of the Sp1 transcription factor of the COLIA-1 gene was detected by polymerase chain reaction using mutagenesis primers followed by restriction enzyme analysis in all patients. BMD of the lumbar spine (L1-L4), femoral neck (FN) and wrist (W) was determined using DEXA, before and 12 months after treatment. At baseline, all patients had increased serum levels of Dkk-1 (mean±SD: 39±17.1 pmol/L) compared to controls (27.4±9.7 pmol/L; p<0.0001). Furthermore, thalassemia patients had increased values of CTX (p<0.0001), bALP (p<0.001), CICP (p=0.003), sRANKL (p=0.02), and OPG (p=0.001) compared to controls. Results for the COLIA-1 Sp1 polymorphism were available for 53 patients. Seventeen patients (32%) were G/T heterozygotes at the polymorphic Sp1 site (Ss), while 3 (5.6%) were T/T homozygotes (ss). Dkk-1 serum levels correlated with L1-L4 BMD (r=−0.290, p=0.022) and W-BMD (r=−0.415. p=0.001), but also with TRACP-5b (r=0.310, p=0.011) and bALP levels (r=−0.289, p=0.018). Ss and ss patients tended to have lower L1-L4 BMD compared with SS patients (p=0.09). No significant correlations were observed between Ss and ss patients with the measured bone markers or the response to ZOL. As reported previously, patients of group B experienced an increase of L1-L4 BMD, while no other alterations in BMD were observed in the 3 studied groups after 12 months of ZOL administration. Interestingly, patients of groups A+B showed a strong reduction of Dkk-1 after 12 months of ZOL (from 39.6±16.6 to 28.9±16.3 pmol/L; p=0.004); indeed they almost normalized Dkk-1 levels (no difference from control values). In contrast, patients of group C showed a borderline increase of Dkk-1 (from 33.1±16.8 to 40.1±23.2 pmol/L, p=0.08). These results show for the first time in the literature that Dkk-1 is increased in the serum of patients with thalassemia and osteoporosis, correlates with their BMD and is reduced post-ZOL therapy. This Dkk-1 elevation may be at least partially responsible for osteoblast dysfunction in thalassemia and reveal a novel possible target for the development of new agents for the management of bone loss in thalassemia patients.

Abstract Free circulating DNA has been shown to be present in the plasma of healthy subjects and elevated in conditions characterised by increased cell death, such as cancer and physical trauma. In sickle cell disease (SCD) an increased cell turnover can be expected through hemolysis and recurrent episodes of vaso-occlusion and inflammation, leading to cell death and organ damage. We propose that circulating DNA levels would be higher in patients with SCD, that these elevations would increase with acute crises and that the extent of increase may serve as a prognostic marker of disease severity. Plasma samples were collected from patients with SCD attending the specialist clinic at King’s College Hospital (KCH). Over a 2 year period (April 2003 - May 2005) a total of 442 samples from 154 patients (105 HbSS, 46 HbSC, 3 HbS/β0 thalassemia) in steady state at each visit to the KCH outpatient sickle clinic was collected. Samples were also obtained from 21 of these 154 patients during an acute crisis, as defined by hospital admission for sickle-related pain. Control subjects consisted of 55 healthy Afro-Caribbean and West African individuals (53 HbAA, 2 HbAS). Plasma DNA concentration was measured by real-time quantitative PCR using a probe specific for the β-globin gene. As the distribution of plasma DNA levels was not gaussian, data was normalised by log-transformation. The median plasma DNA levels in genome equivalents (GE)/ml were as follows: 933 (range 144 to 19370) in controls; 841 (range 60.1 to 16070) in all sickle patients (SS, HbSC and S/β0 thalassemia) during steady state; 970 (range 92 to 16070) in SS and S/β0 thalassemia during steady state; 719 (range 60.1 to 14650) in SC during steady state. Median DNA levels for crisis samples were 10070 (range 444 to 57910) in all SCD and 12000 (range 444–57910) in the SS and S/β0 thalassemia group. There was no significant difference in plasma DNA levels between controls and SCD patients during steady state. Differences between steady state and crisis did not reach significance in the SC group due to the small number of crisis samples (n=3). However, mean plasma DNA levels for sickle patients during steady state, and those in crisis were highly significantly different (by Student’s t test) for all sickle patients (p&lt;0.0001) and for the SS and S/β0 thalassemia group (p&lt;0.0001). Circulating DNA levels correlated with CRP levels in the SS/Sβ0 (r = 0.24, p&lt;0.005) and SC groups (r = 0.31, p &lt;0.05) but not in the controls. DNA levels correlated with WBC in the SS/Sβ0 group only (r = 0.25, p&lt;0.05). However, plasma DNA showed no correlations with hemoglobin, reticulocyte count, red blood cell count or LDH levels. Our preliminary studies show that, unexpectedly, circulating DNA levels are not elevated in steady state SCD despite ongoing organ damage and hemolysis. However, DNA concentration may be a reliable biomarker in SCD crisis. We are currently carrying out longitudinal studies to explore the value of serial measurement of plasma DNA levels and their association with organ damage in SCD.

Abstract Objectives: Hemoglobin A2 (HbA2) is elevated in the presence of beta thalassemia trait and it is used as an indicator of its presence. High-Performance Liquid Chromatography (HPLC) overestimates HbA2 in patients with Sickle Cell Disease (SCD) due to the co-elution of HbS in HbA2 column. The optimal cut-off level of HbA2 to indicate the presence of beta thalassemia trait has not been well established in patients with SCD. We aim to define the optimal cut-off level of HbA2 to differentiate between SS and S/Beta SCD genotype variants. Methods: In this cross-sectional study, we included 241 patients with SCD who have either SS or S/Beta genotype based on their HPLC and the Sickledex test®. The diagnoses were confirmed by the direct sequencing of PCR amplified products of all exons, exon-intron junctions and promoter region of the beta globin gene. We retrieved the following clinical and laboratory variables from the electronic health records: age, gender, Hemoglobin (Hb), Mean Corpuscular Volume (MCV), Red Blood Cell (RBC) count and HbA2. We used the receiver operating curve (ROC) analysis to obtain the optimal cut-off level of HbA2 using the maximum sensitivity and specificity (Youden criteria). All descriptive and analytical statistics were performed using R program. The ROC analysis was performed with the "OptimalCutpoints" package available in R program. Results: Among the 241 patients included in the analysis, SS and S/Bthal patients were 81% and 19% respectively. Male to female ratio was 126:115. Fifty-two percent were using hydroxyurea. The median HbA2 level was 4.5% (Range: 0.0-6.5) in the SS group and 6.5% (Range: 3.5-8.2) in the S/Bthal group. The median Hb, MCV and RBC count was 9.4 g/dL (Range: 5.3 - 15.0), 75 fL (55 - 111) and 3.8 *1012/L (1.9 - 6.3) respectively for the SS group; while it was 9.7 g/dL (6.6 - 12.3), 68 fL (57 - 86) and 4.2 *1012/L (2.5 - 5.4) respectively for the S/Bthal group. The optimal cut-off level for HbA2 was estimated to be 5.7% using a sensitivity of 91% and a specificity of 92%. The positive and negative predictive values were 75% and 98% respectively. The discrimination estimated using the Area Under the Curve (AUC) was 0.936 (95% Confidence Interval: 0.878-0.994). Conclusions: The optimal cut-off HbA2 level to differentiate SCD with the S/Bthal genotype from the SS genotype is 5.7% with a high sensitivity, specificity and discrimination. The unexpected overlap in the MCV and the RBC count is likely related to the high rate of Alpha thalassemia trait in the analyzed population. Incorporation of the presence of alpha thalassemia trait in the analysis may improve the discrimination of MCV and RBC count. Disclosures No relevant conflicts of interest to declare.

Key Points The algorithms have high sensitivity and specificity to identify patients with hemoglobin SS/Sβ0 thalassemia and acute care pain encounters. Codes conforming to common data model are provided to facilitate adoption of algorithms and standardize definitions for EHR-based research.

Drépanocytose et bêta-thalassémie sont dues à des mutations affectant la production de la chaîne de globine β. La sévérité clinique est atténuée par des mutations augmentant la quantité d'hémoglobine fœtale (HbF), une condition appelée persistance héréditaire d'HbF à l'âge adulte. La transplantation autologue de cellules souches/progénitrices hématopoïétiques (CSPH) génétiquement corrigées est prometteuse. Une approche CRISPR/Cas9 pour réprimer BCL11A, un répresseur de la globine γ, a été approuvée mais comporte des risques de génotoxicité dues aux cassures double brin (CDB). Nous avons ciblé les sites de liaison (SL) des activateurs transcriptionnels GATA1 et ATF4, présents dans les régions +58-kb et +55-kb, respectivement, des enhancers érythroïdes de BCL11A, avec des éditeurs de base (BEs) pour introduire des mutations ponctuelles précises sans créer de CDBs. Des ARN guides (ARNg) ont été testés dans des cellules souches hématopoïétiques (CSH) de patients drépanocytaires en combinaison avec des BEs, générant différents nucléotides dans les motifs de liaison avec une efficacité d'édition atteignant 90 %, avec peu ou pas d'indels induits par les CDB. La réactivation d'HbF a été observée dans tous les échantillons édités, mesurée par HPLC, mais pas suffisante pour un sauvetage complet du phénotype dans les cellules érythroïdes issues des CSPHs drépanocytaires éditées. Nous avons donc ciblé simultanément les SL de GATA1 et ATF4 pour augmenter les niveaux d'HbF. Les cellules éditées au niveau des enhancers +58 et +55 ont montré une augmentation de l'expression d'HbF par rapport aux cellules recevant des ARNg individuels, dépassant les niveaux atteints avec la stratégie CRISPR/Cas9. Enfin, la réactivation de l'HbF était suffisante pour permettre un sauvetage substantiel du phénotype malade, diminuant le nombre de cellules drépanocytaires à 16,4%. Pour évaluer les effets hors cibles, nous avons utilisé le GUIDE-seq couplé au séquençage ciblé, le séquençage de l'exome entier et le RNA-seq, tandis que les effets indésirables présent dans les sites cibles ont été évalués par séquençage Long read. L'efficacité du BE pour repeupler les CSH a été démontrée en transplantant des CSH éditées dans des souris immunodéficientes, prouvant l'efficacité de l'édition simultanée des éléments transrégulateurs de la globine γ pour la réactivation de l'HbF. Cette preuve de concept permettra le développement préclinique et clinique de CSH modifiées pour le traitement des β-hémoglobinopathies. Cependant, des travaux récents montrent que les BEs peuvent également générer de larges délétions ou indels (Antoniou et al. 2022). Ainsi, une nouvelle stratégie basée sur l'édition de l'épigénome a été développée pour moduler l'expression des gènes sans modifier la séquence d'ADN sous-jacente. Nous avons analysé les marques épigénétiques dans deux régions cis-régulatrices clés, les promoteurs de HBG et les enhancers de BCL11A, dans des cellules érythroïdes dérivées de CSPH. Des marques épigénétiques répressives, telles que la méthylation de l'ADN, ont été détectées au niveau des promoteurs de HBG dans des cellules érythroïdes adultes n'exprimant pas la globine γ. En revanche, la déméthylation de l'ADN et les marques épigénétiques activatrices telles que l'acétylation de la lysine 27 de l'histone 3 (H3K27ac) et la triméthylation de la lysine 4 (H3K4me3) ont été détectées au niveau des promoteurs de HBG dans les cellules érythroïdes exprimant la globine γ. La forte expression de BCL11A dans les cellules érythroïdes adultes est associée à de faibles niveaux de méthylation de l'ADN au niveau des enhancers de BCL11A. L'inactivation des enhancers de BCL11A est associée à une augmentation de la méthylation de l'ADN. Ces données nous ont permis de concevoir des modificateurs de l'épigénome pour manipuler l'architecture épigénétique des promoteurs de HBG et des enhancers de BCL11A afin d'atteindre des niveaux thérapeutiques d'HbF
B-thalassemia and sickle cell disease (SCD) result from mutations that affect the synthesis or structure of adult hemoglobin. Historically, allogeneic hematopoietic stem cell (HSC) transplantation from a compatible donor was the only curative treatment. Transplantation of autologous, genetically modified HSCs offers a promising therapeutic alternative for patients lacking a suitable donor. The clinical severity in b-hemoglobinopathies is mitigated by co-inheritance of hereditary persistence of fetal hemoglobin (HPFH), a benign condition characterized by mutations occurring in the genes encoding the fetal y-globin chains, which lead to increased fetal hemoglobin (HbF, a2y2) expression, which can rescue the b-thalassemic and SCD phenotypes. HbF reactivation can be achieved by down-regulating BCL11A, encoding a key repressor of HbF. A CRISPR/Cas9 strategy targeting the GATA1 binding site (BS) within the +58-kb erythroid-specific enhancer of BCL11A has recently been approved as the first gene-editing therapy for b-thalassemia and SCD. Indeed, the targeting of the BCL11A erythroid-specific enhancer led to an efficient reduction of BCL11A in the erythroid cells, without impacting the differentiation of HSPCs in the other cell lineages. However, site-specific nucleases induce double strand breaks (DSBs), posing significant risks, such apoptosis and generation of large genomic rearrangements. In addition, to obtain an adequate number of corrected cells to transplant, several collections of HSCs are necessary to compensate for the cell loss due to DSB-induced apoptosis. Finally, the clinical study showed variability in the extent of HbF reactivation, still high HbS levels and modest correction of ineffective erythropoiesis. Novel CRISPR/Cas9 derived tools are currently available and can be used to develop therapeutic strategies associated with a low risk of DSB generation and increased HbF expression. In this project, we intend to develop universal, safe and efficacious therapeutic strategies for b-hemoglobinopathies aimed at modifying HSCs using base editors (BEs) and epigenome editors to reactivate HbF expression in their erythroid progeny. BEs are a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations with little DSB generation. In this work we used this technology to inactivate the GATA1 or the ATF4 transcriptional activator BS in the +58-kb and +55-kb BCL11A erythroid-specific enhancers through the insertion of point mutations. In particular, to reach levels of HbF sufficient to rescue the sickling phenotype, we performed simultaneous targeting of the two BS, achieving similar HbF levels compared to CRISPR/Cas9 nuclease-based approach. Additionally, we showed that BEs generated fewer DSBs and genomic rearrangements compared to the CRISPR/Cas9 nuclease approach. In parallel, we developed a novel epigenome-editing strategy aimed at modulating gene expression without altering the DNA sequence (e.g. without generating DSBs). We designed two approaches to upregulate HbF expression: a first strategy targeting and activating the y-globin promoters and a second approach downregulating BCL11A by targeting its erythroid-specific enhancers. We first identified the epigenetic marks in these trans- and cis-regulatory regions that are associated with active or inactive transcription in adult versus fetal erythroid cells. Then we used epigenome editors to deposit active histone modifications at the y-globin promoters and remove inactive marks such as DNA methylation. In parallel, we decorated the BCL11A enhancers with inactive epigenetic marks. Preliminary results demonstrated y-globin reactivation using both strategies, though the effects diminished over time, indicating the need for further optimization. In conclusion, we proposed two different editing approaches that allow to reduce DSB-associate issues as strategies to treat b-hemoglobinopathies

Many diseases have oxidative stress and inflammation as underlying pathological features, including metabolic and inflammatory/autoimmune disorders, diseases of the lung, liver, kidney, gastrointestinal tract, cardiovascular and nervous systems. A leading physiological mechanism for oxidative stress is the nuclear erythroid-related factor 2-like 2/antioxidant response element (Nrf2/ARE) signaling pathway. It maintains intracellular homeostasis and protects cells from oxidative damage by inducing phase II detoxifying and oxidative-stress responsive genes. Nrf2 transcription factor functions as the key controller of the redox homeostatic gene regulatory network, and is tightly controlled by the repressor protein, Kelch-like ECH-associated protein 1 (Keap1). Pharmacological agents to inhibit Keap1 and boost effectiveness of the Nrf2/ARE pathway have been developed and more are in development. This chapter elucidates the importance of hemodynamic laminar shear stress in oxidative homeostasis and examines hemodynamic induction of the shear stress (SS)/Krupple-like factor2 (KLF2) /Nrf2/ARE pathway as a means to combat oxidative stress through hemodynamics.