Genetic Correction of Sickle Cell Anemia and β-Thalassemia: Progress and New Perspective

Gene therapy for β-globinopathies, particularly β-thalassemia and sickle cell anemia, holds promise for the future as a definitive corrective approach for these common and debilitating disorders. Correction of the β-globinopathies using lentivirus vectors carrying the β- or γ-globin genes and elements of the locus control region has now been well established in murine models, and an understanding of "what is required to cure these diseases" has been developed in the first decade of the 21st century. A clinical trial using one such vector has been initiated in France with intriguing results, while other trials are under development. Vector improvements to enhance the safety and efficiency of lentivirus vectors are being explored, while new strategies, including homologous recombination in induced pluripotent cells, for correction of sickle cell anemia have shown proof-of-concept in vitro. Here, a review is provided of the current substantial progress in genetic correction of β-globin disorders

The 3′ Region of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to Its Core and Is Required for Full Insulator Activity

Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the “core”, which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5′ core with a novel 3′ 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors

p190-B RhoGAP and intracellular cytokine signals balance hematopoietic stem and progenitor cell self-renewal and differentiation

The mechanisms regulating hematopoietic stem and progenitor cell (HSPC) fate choices remain ill-defined. Here, we show that a signalling network of p190-B RhoGAP-ROS-TGF-β-p38MAPK balances HSPC self-renewal and differentiation. Upon transplantation, HSPCs express high amounts of bioactive TGF-β1 protein, which is associated with high levels of p38MAPK activity and loss of HSC self-renewal in vivo. Elevated levels of bioactive TGF-β1 are associated with asymmetric fate choice in vitro in single HSPCs via p38MAPK activity and this is correlated with the asymmetric distribution of activated p38MAPK. In contrast, loss of p190-B, a RhoGTPase inhibitor, normalizes TGF-β levels and p38MAPK activity in HSPCs and is correlated with increased HSC self-renewal in vivo. Loss of p190-B also promotes symmetric retention of multi-lineage capacity in single HSPC myeloid cell cultures, further suggesting a link between p190-B-RhoGAP and non-canonical TGF-β signalling in HSPC differentiation. Thus, intracellular cytokine signalling may serve as 'fate determinants' used by HSPCs to modulate their activity

Effective hematopoietic stem cell-based gene therapy in a murine model of hereditary pulmonary alveolar proteinosis

Hereditary pulmonary alveolar proteinosis due to GM-CSF receptor deficiency (herPAP) constitutes a life-threatening lung disease characterized by alveolar deposition of surfactant protein secondary to defective alveolar macrophage function. As current therapeutic options are primarily symptomatic, we have explored the potential of hematopoietic stem cell-based gene therapy. Using Csf2rb−/− mice, a model closely reflecting the human herPAP disease phenotype, we here demonstrate robust pulmonary engraftment of an alveolar macrophage population following intravenous transplantation of lentivirally corrected hematopoietic stem and progenitor cells. Engraftment was associated with marked improvement of critical herPAP disease parameters, including bronchoalveolar fluid protein, cholesterol and cytokine levels, pulmonary density on computed tomography scans, pulmonary deposition of Periodic Acid-Schiff+ material as well as respiratory mechanics. These effects were stable for at least nine months. With respect to engraftment and alveolar macrophage differentiation kinetics, we demonstrate the rapid development of CD11c+/SiglecF+ cells in the lungs from a CD11c–/SiglecF+ progenitor population within four weeks after transplantation. Based on these data, we suggest hematopoietic stem cell-based gene therapy as an effective and cause-directed treatment approach for herPAP

Significance of Serum Sodium and Disorders on Altering its Concentration in Chronic Renal Failure Patients

ABSTRACT Sodium is widely distributed in food materials, more in animal sources than plants. Sodium ion concentration is very important in regulation of osmotic pressure of body fluids, preservation of normal irritability of muscles and the permeability of the cells. Most of the sodium is found in the extracellular fluid. Almost all the blood sodium is found in the plasma, there is very little in the red cells. In chronic cases the plasma concentration may be only slightly lower. A negative balance of sodium or potassium may cause renal failure. Changes in sodium concentration largely lead to severe diarrhea, vomiting, diabetes, acute pathological processes involving the brain, diuretics and hyponatraemia, lead to a salt deficiency and hence to low plasma sodium values which may be exaggerated if water losses are replaced without adequate salt therapy. Hyponatraemia results when the water content of the body is increased absolutely and relative to the sodium content so that the osmoiality of body fluid falls. The most common pathogenesis of hyponatraemia is mixed salt and water depletion. The present paper includes the study of serum sodium levels of 200 patients (according to age group and sex) with chronic renal failure (CRF) before and after the process of treatment and it has been compared with 50 normal healthy individuals comprising the control group

Strategies for precise gene edits in mammalian cells

CRISPR-Cas technologies have the potential to revolutionize genetic medicine. However, work is still needed to make this technology clinically efficient for gene correction. A barrier to making precise genetic edits in the human genome is controlling how CRISPR-Cas-induced DNA breaks are repaired by the cell. Since error-prone non-homologous end-joining is often the preferred cellular repair pathway, CRISPR-Cas-induced breaks often result in gene disruption. Homology-directed repair (HDR) makes precise genetic changes and is the clinically desired pathway, but this repair pathway requires a homology donor template and cycling cells. Newer editing strategies, such as base and prime editing, can affect precise repair for relatively small edits without requiring HDR and circumvent cell cycle dependence. However, these technologies have limitations in the extent of genetic editing and require the delivery of bulky cargo. Here, we discuss the pros and cons of precise gene correction using CRISPR-Cas-induced HDR, as well as base and prime editing for repairing small mutations. Finally, we consider emerging new technologies, such as recombination and transposases, which can circumvent both cell cycle and cellular DNA repair dependence for editing the genome

Publication History

Metal complexes of the type ML2X2 (where M = Cu (II), Co (II) and Ni (II), L = 2-amino-4-(p-hydroxy phenyl)thiazole and X = cl-, CH3COO-) were prepared and characterized with the help of their elemental analysis, IR, electronic and magnetic susceptibility studies. IR studies have shown that nitrogen of the amino group and sulphur of the thiazole ring took part in co-ordination. Magnetic and electronic spectral studies have shown that all the complexes are having octahedral geometry. These newly synthesized complexes were also screened for their antifungal activity against different fungi at different concentrations. The activity decreases with decrease of concentration and the metal complexes are less toxic than the parent ligand. Key Words: Thiazole, Fungicidal activity, Ligand