Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood-derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies

Near Infrared Imaging Survey of Bok Globules: Density Structure

On the basis of near-infrared imaging observations, we derived visual extinction (Av) distribution toward ten Bok globules through measurements of both the color excess (E_{H-K}) and the stellar density at J, H, and Ks (star count). Radial column density profiles for each globule were analyzed with the Bonnor-Ebert sphere model. Using the data of our ten globules and four globules in the literature, we investigated the stability of globules on the basis of xi_max, which characterizes the Bonnor-Ebert sphere as well as the stability of the equilibrium state against the gravitational collapse. We found that more than half of starless globules are located near the critical state (xi_max = 6.5 +/- 2). Thus, we suggest that a nearly critical Bonnor-Ebert sphere characterizes the typical density structure of starless globules. Remaining starless globules show clearly unstable states (xi_max > 10). Since unstable equilibrium states are not long maintained, we expect that these globules are on the way to gravitational collapse or that they are stabilized by non-thermal support. It was also found that all the star-forming globules show unstable solutions of xi_max >10, which is consistent with the fact that they have started gravitational collapse. We investigated the evolution of a collapsing gas sphere whose initial condition is a nearly critical Bonnor-Ebert sphere. We found that the column density profiles of the collapsing sphere mimic those of the static Bonnor-Ebert spheres in unstable equilibrium. The collapsing gas sphere resembles marginally unstable Bonnor-Ebert spheres for a long time. We found that the frequency distribution of xi_max for the observed starless globules is consistent with that from model calculations of the collapsing sphere.Comment: Accepted for publication in the Astronomical Journal. 39 pages in preprint format, including 10 figures. The version with higher resolution figures can be obtained at the following site (http://alma.mtk.nao.ac.jp/~kandori/preprint/

PLGA-Nanoparticles for Intracellular Delivery of the CRISPR-Complex to Elevate Fetal Globin Expression in Erythroid Cells

Ex vivo gene editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) offers great opportunities to develop new treatments for a number of malignant and non-malignant diseases. Efficient gene-editing in HSPCs has been achieved using electroporation and/or viral transduction to deliver the CRISPR-complex, but cellular toxicity is a drawback of currently used methods. Nanoparticle (NP)-based gene-editing strategies can further enhance the gene-editing potential of HSPCs and provide a delivery system for in vivo application. Here, we developed CRISPR/Cas9-PLGA-NPs efficiently encapsulating Cas9 protein, single gRNA and a fluorescent probe. The initial ‘burst’ of Cas9 and gRNA release was followed by a sustained release pattern. CRISPR/Cas9-PLGA-NPs were taken up and processed by human HSPCs, without inducing cellular cytotoxicity. Upon escape from the lysosomal compartment, CRISPR/Cas9-PLGA-NPs-mediated gene editing of the γ-globin gene locus resulted in elevated expression of fetal hemoglobin (HbF) in primary erythroid cells. The development of CRISPR/Cas9-PLGA-NPs provides an attractive tool for the delivery of the CRISPR components to target HSPCs, and could provide the basis for in vivo treatment of hemoglobinopathies and other genetic diseases

Genome-wide association study of red blood cell traits in Hispanics/Latinos: The Hispanic Community Health Study/Study of Latinos

Prior GWAS have identified loci associated with red blood cell (RBC) traits in populations of European, African, and Asian ancestry. These studies have not included individuals with an Amerindian ancestral background, such as Hispanics/Latinos, nor evaluated the full spectrum of genomic variation beyond single nucleotide variants. Using a custom genotyping array enriched for Amerindian ancestral content and 1000 Genomes imputation, we performed GWAS in 12,502 participants of Hispanic Community Health Study and Study of Latinos (HCHS/SOL) for hematocrit, hemoglobin, RBC count, RBC distribution width (RDW), and RBC indices. Approximately 60% of previously reported RBC trait loci generalized to HCHS/SOL Hispanics/Latinos, including African ancestral alpha- and beta-globin gene variants. In addition to the known 3.8kb alpha-globin copy number variant, we identified an Amerindian ancestral association in an alpha-globin regulatory region on chromosome 16p13.3 for mean corpuscular volume and mean corpuscular hemoglobin. We also discovered and replicated three genome-wide significant variants in previously unreported loci for RDW (SLC12A2 rs17764730, PSMB5 rs941718), and hematocrit (PROX1 rs3754140). Among the proxy variants at the SLC12A2 locus we identified rs3812049, located in a bi-directional promoter between SLC12A2 (which encodes a red cell membrane ion-transport protein) and an upstream anti-sense long-noncoding RNA, LINC01184, as the likely causal variant. We further demonstrate that disruption of the regulatory element harboring rs3812049 affects transcription of SLC12A2 and LINC01184 in human erythroid progenitor cells. Together, these results reinforce the importance of genetic study of diverse ancestral populations, in particular Hispanics/Latinos

Deficiency of ribosomal protein S26, which is mutated in a subset of patients with Diamond Blackfan anemia, impairs erythroid differentiation

Introduction: Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by defective maturation of the erythroid progenitors in the bone marrow, for which treatment involves steroids, chronic transfusions, or hematopoietic stem cells transplantation. Diamond Blackfan anemia is caused by defective ribosome biogenesis due to heterozygous pathogenic variants in one of 19 ribosomal protein (RP) genes. The decreased number of functional ribosomes leads to the activation of pro-apoptotic pathways and to the reduced translation of key genes for erythropoiesis.Results and discussion: Here we characterized the phenotype of RPS26-deficiency in a cell line derived from human umbilical cord blood erythroid progenitors (HUDEP-1 cells). This model recapitulates cellular hallmarks of Diamond Blackfan anemia including: imbalanced production of ribosomal RNAs, upregulation of pro-apoptotic genes and reduced viability, and shows increased levels of intracellular calcium. Evaluation of the expression of erythroid markers revealed the impairment of erythroid differentiation in RPS26-silenced cells compared to control cells.Conclusions: In conclusion, for the first time we assessed the effect of RPS26 deficiency in a human erythroid progenitor cell line and demonstrated that these cells can be used as a scalable model system to study aspects of DBA pathophysiology that have been refractory to detailed investigation because of the paucity of specific cell types affected in this disorder

RUNX3 levels in human hematopoietic progenitors are regulated by aging and dictate erythroid-myeloid balance

Healthy bone marrow progenitors yield a co-ordinated balance of hematopoietic lineages. This balance shifts with aging toward enhanced granulopoiesis with diminished erythropoiesis and lymphopoiesis, changes which likely contribute to the development of bone marrow disorders in the elderly. In this study, RUNX3 was identified as a hematopoietic stem and progenitor cell factor whose levels decline with aging in humans and mice. This decline is exaggerated in hematopoietic stem and progenitor cells from subjects diagnosed with unexplained anemia of the elderly. Hematopoietic stem cells from elderly unexplained anemia patients had diminished erythroid but unaffected granulocytic colony forming potential. Knockdown studies revealed human hematopoietic stem and progenitor cells to be strongly influenced by RUNX3 levels, with modest deficiencies abrogating erythroid differentiation at multiple steps while retaining capacity for granulopoiesis. Transcriptome profiling indicated control by RUNX3 of key erythroid transcription factors, including and These findings thus implicate RUNX3 as a participant in hematopoietic stem and progenitor cell aging, and a key determinant of erythroid-myeloid lineage balance