Prevalence of Sickle Cell Anemic Subjects from Gadchiroli District, Maharashtra, India

Sickle cell disease is caused by Mutations in the HBB gene. Hemoglobin consists of four protein subunits, typically, two subunits called alpha-globin and two subunits called beta-globin. People with sickle cell anemia suffers with high morbidity and with many intercurrent infections, people of study district are with high economic burden, terminate fatality in childhood state and have the emotional and psychological trauma including the family members the exact magnitude of the problem in the study district is still obscure. The study conducted from April 2009 to April 2012 to know the prevalence of sickle cell anemia by month long survey and by visiting all PHC’S and RH of district and data collected to know the prevalence of sickle cell trait and sickle cell disease total 7763 cases were recorded in present study and age wise, gender wise and caste wise distribution recorded and the data was analyzed statistically. Keywords: Sickle, anemia, Gadchiroli, Haemoglobin, beta-globin

Preclinical Development of EDIT301, an Autologous Cell Therapy Comprising AsCas12a-RNP Modified Mobilized Peripheral Blood-CD34 + Cells for the Potential Treatment of Transfusion Dependent Beta Thalassemia

Beta thalassemia is one of the most common recessive hematological disorders in the world with more than 200 mutations identified to date. These mutations reduce or completely abrogate beta globin expression. As beta globin pairs with alpha globin to form adult hemoglobin (HbA, α2β2), reduced or absent beta globin results in excessive alpha globin chains, which form toxic aggregates. These aggregates cause maturation blockade and premature death of erythroid precursors, and hemolysis of red blood cells (RBC), leading to varying degrees of anemia. Patients with the most severe form of beta thalassemia, namely beta thalassemia major, are transfusion-dependent, i.e., requiring life-long RBC transfusions accompanied by the burden of iron chelation therapy. EDIT-301 is an experimental autologous cell therapy in which CD34 + cells are genetically modified to promote gamma globin expression. EDIT-301 is currently in clinical development for sickle cell disease, and IND enabling stage for transfusion-dependent beta thalassemia (TDT). Gamma globin decreases the alpha to beta globin chain imbalance in beta thalassemia by pairing with the over-abundant alpha globin chains to form fetal hemoglobin (HbF, α2γ2). Gamma globin induction, and consequently HbF induction, for EDIT-301 is achieved through AsCas12a ribonucleoprotein (RNP)-mediated editing of the distal CCAAT box region of the HBG1 and HBG2 promoters, where naturally occurring hereditary persistence of fetal hemoglobin (HFPH) mutations exist. We chose this target over BCL11A based on previous preclinical data demonstrating that BCL11A editing reduces erythroid output in NBSGW mice. An engineered AsCas12a RNP edits the HBG1 and HBG2 promoter distal CCAAT box with high efficiency and specificity. We have previously shown that on-target editing of >80% was achieved in mobilized peripheral blood (mPB) CD34 + cells from normal donors with no detectable off-target editing both at research scale and at clinical manufacturing scale. Edited normal donor CD34 + cells led to long-term, polyclonal, multilineage engraftment without lineage skewing in immunocompromised mice and sustained robust HbF production in their erythroid progeny. To test whether EDIT-301 may be an efficacious therapy for TDT, mPB CD34 + cells from individuals with TDT were electroporated with the engineered AsCas12a RNP targeting the HBG1 and HBG2 promoters. AsCas12a RNP edited mPB CD34 + cells from individuals with TDT as efficiently as CD34 + cells from normal donors. Importantly, EDIT-301 has the potential to address the underlying pathophysiology of TDT, i.e., the maturation blockade and premature death of erythroid precursors. Erythroid differentiation of edited beta thalassemia CD34 + cells showed significant improvement in erythroid maturation and health. Specifically, ~70% edited erythroblasts reached late erythroblast stage compared to ~53% unedited erythroblasts; ~56% edited erythroid cells underwent terminal maturation and enucleated compared to ~28% of unedited erythroid cells; and non-viable erythroblasts decreased from ~33% to ~22% after editing. The improved erythropoiesis was accompanied by significantly increased total hemoglobin content per cell. These data strongly support that editing of the HBG1 and HBG2 promoter CCAAT box using engineered AsCas12a RNP can reverse the dyserythropoiesis associated with beta thalassemia and increase the hemoglobin production. In summary, we have provided strong preclinical data supporting the development of EDIT-301 for the treatment of TDT. Edited mPB CD34 + cells retained their ability to engraft without lineage skewing, resulted in robust HbF induction long-term, improved erythropoiesis, and increased hemoglobin content in TDT erythroid cells. These data support that a single administration of EDIT-301 may have the potential to safely and effectively reverse dyserythropoiesis and ameliorate anemia in individuals with TDT long-term. Clinical studies to demonstrate the safety and efficacy of EDIT-301 in the treatment of TDT are currently being planned. Disclosures Sousa: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Janoudi: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. deDreuzy: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Status research Thalassemia carier among children of ethnic Tay, Dao in Tuyen Quang province

Objectives: To determine the prevalence of thalassemia carrier, genotype and hematological parameters among children bearing the thalassemia gene in Tuyen Quang. Methodology: A descriptive study was conducted from January to March 2017. 505 ethnic minority children in 6 districts and Tuyen Quang City, Tuyen Quang province were registered voluntarily by the family in the study. MCV index <80fL combined with the DCIP test were used for screening thalassemia and HbE. Hemoglobin electrophoresis and DNA analysis of mutations in the globin alpha gene was performed for all cases positive with screening tests. Results: The prevalence of thalassemia common for ethnic minority children in Tuyen Quang was 28,1%. Four types of single-gene mutations in the alpha globin gene were identified, following types --SEA, -α3.7; -αCS; -α4.2. Conclusion: The general prevalence of thalassemia gene among the Tay and Dao children in Tuyen Quang is 28.1%. Six phenotypic groups carrying thalassemia gene were detected with 10 mutant genotypes. Mutation - SEA accounts for the highest proportion of single allele mutations (72.09%). Keywords: Thalassemia carrier, children, ethnic Tay, ethnic Dao, Tuyen Quang

Hemoglobin Interacts with Endothelial Nitric Oxide Synthase to Regulate Vasodilation in Human Resistance Arteries

ABSTRACT Background: In small arteries, constriction of vascular smooth muscle triggers local release of nitric oxide from the adjacent endothelial cell. This feedback vasodilation is a homeostatic mechanism that opposes vasoconstriction. Here, we investigate the role of endothelial alpha globin as a regulator of directed nitric oxide signaling across the myoendothelial junction. Methods: Human omental arteries 100-200µm in diameter were microdissected from omentum samples obtained during clinically indicated abdominal operations on NIH protocol 13-C-0176 (NCT01915225). Each artery was cannulated, perfused free of blood, and preserved for analysis or subjected to pressure myography. Preserved arteries underwent RNA extraction for gene expression; protein extraction for co-immunoprecipitation and Western blot; or immunostaining for multiphoton microscopy. Bio-layer interferometry quantified the binding of alpha globin to endothelial nitric oxide synthase (eNOS). Ex vivo pressure myography characterized arterial vasoreactivity before and after disruption of eNOS-Hb binding with an alpha globin mimetic peptide. Results: HBA1, HBA2, HBB, and NOS3 transcripts were abundant in RNA from the artery wall, and the blood cell gene SLC4A1 was not. Beta globin and eNOS co-immunoprecipitated with alpha globin in protein extracted from human omental artery segments, suggesting an eNOS-hemoglobin complex. Biolayer interferometry studies estimated alpha globin to bind to the oxidase domain of eNOS with an equilibrium dissociation constant of 1.3 x 10-6 M. Multiphoton microscopy of intact arteries revealed alpha globin, beta globin, and eNOS to co-localize within distinct punctates in a plane defined by the internal elastic lamina that separates endothelial cells from vascular smooth muscle. Forster resonance energy transfer confirmed close physical proximity of alpha globin to eNOS in situ. Omental arteries constricted to 39.1 ± 3.2 % of baseline diameter in response to phenylephrine. After treatment with an alpha globin mimetic peptide, the same arteries constricted to 64.6 ± 1.6% of baseline (p < 0.01). Inhibition of NOS with L-NAME restored vasoconstriction in the mimetic peptide-treated arteries to 41.9 ± 2.0% (p < 0.0001). Conclusion: Alpha globin and beta globin are expressed in the endothelium of human resistance arteries, form a complex with eNOS at the myoendothelial junction, and limit the release of nitric oxide triggered by alpha-1-adrenergic stimulation.

Hypoxia-Induced Alpha-Globin Expression in Syncytiotrophoblasts Mimics the Pattern Observed in Preeclamptic Placentas

Preeclampsia (PE) is a pregnancy disorder associated with placental dysfunction and elevated fetal hemoglobin (HbF). Early in pregnancy the placenta harbors hematopoietic stem and progenitor cells (HSPCs) and is an extramedullary source of erythropoiesis. However, globin expression is not unique to erythroid cells and can be triggered by hypoxia. To investigate the role of the placenta in increasing globin levels previously reported in PE, flow cytometry, histological and immunostaining and in situ analyses were used on placenta samples and ex vivo explant cultures. Our results indicated that in PE pregnancies, placental HSPC homing and erythropoiesis were not affected. Non-erythroid alpha-globin mRNA and protein, but not gamma-globin, were detected in syncytiotrophoblasts and stroma of PE placenta samples. Similarly, alpha-globin protein and mRNA were upregulated in normal placenta explants cultured in hypoxia. The upregulation was independent of HIF1 and NRF2, the two main candidates of globin transcription in non-erythroid cells. Our study is the first to demonstrate alpha-globin mRNA expression in syncytiotrophoblasts in PE, induced by hypoxia. However, gamma-globin was only expressed in erythrocytes. We conclude that alpha-globin, but not HbF, is expressed in placental syncytiotrophoblasts in PE and may contribute to the pathology of the disease.

Endothelial alpha globin is a nitrite reductase

Small artery vasodilation in response to hypoxia is essential for matching oxygen supply to tissue oxygen demand. One source of hypoxic dilation via nitric oxide (NO) signaling is nitrite reduction by erythrocytic hemoglobin (α2β2). However, the alpha subunit of hemoglobin is also expressed in resistance artery endothelium and localized to myoendothelial junctions, a subcellular domain that contacts underlying vascular smooth muscle cells. We hypothesized that nitrite reduction mediated by endothelial alpha globin may occur at myoendothelial junctions to regulate hypoxic vasodilation. To test this concept, we created two novel mouse strains: one lacking alpha globin specifically in endothelium (EC Hba1Δ/Δ) and one where alpha globin is mutated such that its inhibitory association with endothelial NO synthase (eNOS) is prevented (Hba1WT/Δ36-39). In EC Hba1Δ/Δ or Hba1WT/Δ36-39 mice hemoglobin levels, hematocrit and erythrocyte counts were unchanged from littermate controls. Loss of the full alpha globin protein from the endothelium in the EC Hba1Δ/Δ model was associated with decreased exercise capacity and decreased intracellular nitrite utilization in hypoxic conditions. These effects were not seen in Hba1WT/Δ36-39 animals. Hypoxia induced vasodilation was decreased by 60% in isolated thoracodorsal arteries from EC Hba1Δ/Δ, while infusion of erythrocytes only partially rescued the dilatory response. Lastly, unlike other models where blood pressure is decreased, EC Hba1Δ/Δ blood pressure was not altered in response to hypoxia. Overall, we conclude that alpha globin in the resistance artery endothelium can act as a nitrite reductase to provide a local vasodilatory response to hypoxia.