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The accumulation of unbound α-globin chains in red blood cells is a crucial pathophysiology of β-thalassemia. IOX1 (5-carboxy-8-hydroxyquinoline) is a broad-spectrum 2-oxoglutarate (2OG)-dependent oxygenase inhibitor that can reduce α-globin mRNA expression in human cord blood erythroid progenitor cells. Therefore, IOX1 has been proposed as a potential compound for β-thalassemia treatment through the decrease in α-globin chain synthesis. However, there is no empirical evidence regarding the consequences of IOX1 in β-thalassemia. In this study, the therapeutic effects of IOX1 were investigated in β

MUi028-A human induced pluripotent stem cell (hiPSC) line was generated from normal fetal skin fibroblasts using a non-integrative reprogramming method. Reprogramming factors OCT4, SOX2, KLF4, L-MYC, and LIN28, and TP53 shRNA in three episomal vectors were delivered by electroporation. The MUi028-A cell line had embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of in vitro differentiation into the three germ layers. This iPSC line can be used as a control to study disease mechanisms.

Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of β-thalassemia disease by compensating for adult hemoglobin deficiency in patients. Previously, microarray analysis revealed that zinc finger protein (ZNF)802 (also known as Juxta-posed with another zinc finger gene-1 (JAZF1)) was upregulated in human erythroblasts derived from adult peripheral blood compared with fetal liver-derived cells, implying a potential role as a HbF repressor. However, deficiency in ZNF802 induced by lentiviral shRNA in β

The Krüppel-like factor (KLF) family dominates highly conserved three zinc finger DNA binding domains at the C-terminus and variable transactivation domains at the N-terminus. Humans possess 18 KLF genes that are differentially expressed in various tissues. Several KLFs recognize a specific CACCC DNA motif that is commonly found within hematopoietic-specific promoters. To investigate those KLFs that are involved in human hemoglobin (Hb) switching, the present study analyzed a previous microarray data set from fetal and adult erythroid cells and validated the mRNA expression levels of 18 KLFs by reverse transcription-quantitative PCR (RT-qPCR). KLF with a decreased expression level in the fetuses was selected for a functional study in human erythroid progenitor cells using lentiviral-based short hairpin RNA knockdown. The fetuses demonstrated a lower level of KLF4 mRNA expression when compared with the adults. Downregulation of KLF4 in erythroid progenitor cells from healthy individuals and individuals with β(0)-thalassemia/HbE evidenced the increasing embryonic and fetal globin mRNA expression with neither significant cytotoxicity nor gene expression alteration of the examined globin regulators, KLF1, B-cell lymphoma/leukemia 11A and lymphoma/leukemia-related factor. These findings demonstrate that the downregulation of KLF4 is associated with increased embryonic and fetal globin gene expression in human erythroid progenitor cells. Moreover, identifying putative compounds or molecular approaches that effectively downregulate KLF4 and further induce embryonic globin expression may provide an alternative therapeutic strategy for α-globin substitution in severe α-thalassemia.

The 13q deletion syndrome is a rare chromosomal disorder caused by loss of the long arm of chromosome 13, and usually entails developmental delay, intellectual disability, behavioral problems and distinctive facial features. In this study, we successfully generated a human iPSC line (MUi015-A) from skin fibroblasts of a patient who had large deletion of chromosome 13, del(13)(q14q22). The MUi015-A line exhibited embryonic stem cell characteristics with consistent pluripotency marker expression and the capability of differentiating into three germ layers. The cell line provides a good tool in studying pathophysiology of the tumors, drug testing and gene therapy.

Targeting the BCL11A erythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A +58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2 promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A +58 editing alone, 29.1%±3.9%; BCL11A +55 editing alone, 34.8±5.1%; HBG1/2 promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A +58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed. Together these results suggest that combined BCL11A +58 and +55 erythroid enhancer editing produces highly efficient on-target allelic disruption, erythroid-specific BCL11A downregulation, heightened HbF induction capacity compared to alternative approaches, preserved long-term multilineage engraftment potential by both human xenotransplant and rhesus autotransplant assays, and absence of evident genotoxicity, under clinically relevant SpCas9 RNP electroporation conditions. Disclosures No relevant conflicts of interest to declare.

Hemoglobin Constant Spring (HbCS, HBA2 : c.427T > C) is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34 + hematopoietic progenitors of a 52 year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling.

BackgroundA key event in human development is the establishment of erythropoietic progenitors in the bone marrow, which is accompanied by a fetal-to-adult switch in hemoglobin expression. Understanding of this event could lead to medical application, notably treatment of sickle cell disease andβ-thalassemia. The changes in gene expression of erythropoietic progenitor cells as they migrate from the fetal liver and colonize the bone marrow are still rather poorly understood, as primary fetal liver (FL) tissues are difficult to obtain.MethodsWe obtained human FL tissue and adult peripheral blood (AB) samples from Thai subjects. Primary CD34+cells were culturedin vitroin a fetal bovine serum-based culture medium. After 8 days of culture, erythroid cell populations were isolated by flow cytometry. Gene expression in the FL- and AB-derived cells was studied by Affymetrix microarray and reverse-transcription quantitative PCR. The microarray data were combined with that from a previous study of human FL and AB erythroid development, and meta-analysis was performed on the combined dataset.ResultsFL erythroid cells showed enhanced proliferation and elevated fetal hemoglobin relative to AB cells. A total of 1,391 fetal up-regulated and 329 adult up-regulated genes were identified from microarray data generated in this study. Five hundred ninety-nine fetal up-regulated and 284 adult up-regulated genes with reproducible patterns between this and a previous study were identified by meta-analysis of the combined dataset, which constitute a core set of genes differentially expressed between FL and AB erythroid cells. In addition to these core genes, 826 and 48 novel genes were identified only from data generated in this study to be FL up- and AB up-regulated, respectively. Thein vivorelevance for some of these novel genes was demonstrated by pathway analysis, which showed novel genes functioning in pathways known to be important in proliferation and erythropoiesis, including the mitogen-activated protein kinase (MAPK) and the phosphatidyl inositol 3 kinase (PI3K)-Akt pathways.DiscussionThe genes with upregulated expression in FL cells, which include many novel genes identified from data generated in this study, suggest that cellular proliferation pathways are more active in the fetal stage. Erythroid progenitor cells may thus undergo a reprogramming during ontogenesis in which proliferation is modulated by changes in expression of key regulators, primarily MYC, and others including insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), neuropilin and tolloid-like 2 (NETO2), branched chain amino acid transaminase 1 (BCAT1), tenascin XB (TNXB) and proto-oncogene, AP-1 transcription factor subunit (JUND). This reprogramming may thus be necessary for acquisition of the adult identity and switching of hemoglobin expression.

β-Thalassemias are groups of inherited blood disorders caused by reduced or absent production of β-globin chains of adult hemoglobin (HbA; α2β2). The severely affected patients with β-thalassemia require lifelong blood transfusion and chelation therapy. Reactivation of fetal hemoglobin (HbF; α2γ2) could ameliorate the symptoms of patients via increased total hemoglobin production and reduced imbalanced α- to non-α-globin synthesis. Hemoglobin switching during human development requires activities of several transcription factor proteins, including those bind to the globin gene promoters. However, the regulation of fetal to adult hemoglobin switching is not fully understood. Previously, we have carried out the microarray analysis comparing human erythroblasts derived from therapeutic aborted fetal liver and adult peripheral blood. The results showed that ZNF802 mRNA is among the transcripts that up-regulated in adult erythroblast of greater than 2-fold change. ZNF802, also called JAZF1, is a 27 kDa protein containing two zinc finger motifs that acting as transcriptional repressor by binding to the direct repeat (DR) elements on DNA. In this study, quantitative RT-PCR was performed in order to validate the microarray data. We observed 13-fold up-regulation of ZNF802 in adult erythroid cells compared to those cells derived from fetal liver. To further elucidate the role of ZNF802 in reduction of HbF expression in adult erythroids, the lentivirus carrying ZNF802 specific shRNA and non-targeted shRNA were designed and constructed for transduction in erythroblasts. Hematopoietic CD34+ progenitor cells were isolated from β-thalassemia/HbE patients (n=9) and subjected to ex vivo two-phase liquid erythroid culture. The results showed the reduction of greater than 90% of ZNF802 transcript and nearly undetectable level of ZNF802 protein in cells transduced with the ZNF802 shRNA lentivirus. As expected, the level of γ-globin transcript and levels of HbF were increased in ZNF802 knockdown erythroid cells in β-thalassemia/HbE (5-fold changed and HbF 26.9±7.4% vs 18.5±6.8% in non-targeted shRNA (p-value = 0.04), respectively). Interestingly, we found up-regulation of the embryonic ε-globin and ζ-globin transcripts in knockdown ZNF802 cells compared to non-targeted shRNA control (15-fold and 10-fold changed (p-value = 0.01), respectively). To note, the diminishing of ZNF802 effect on the proliferation and differentiation pattern of erythriod cells was not found. Altogether, our findings suggested that ZNF802 may play a role as the transcriptional repressor of γ-globin gene in human adult erythroid cells, thus served as a possible new therapeutic target for treatment of β-thalassemia. The molecular mechanisms underlying the ZNF802 mediated HbF reduction are underway. Disclosures No relevant conflicts of interest to declare.

Mutations in MYH9 gene is one of the major causes of inherited thrombocytopenia resulted from nonfunctional myosin-9 protein. We have generated a human induced pluripotent stem cell line MUi010-A from skin fibroblasts of a patient who had a point mutation c.2104C>T (p.R702C) in the exon 16 of MYH9 gene using a non-integrative reprogramming method. The MUi010-A exhibited embryonic stem cell-like characteristics with consistent pluripotent markers expression, was capable of all three embryonic germ layers differentiation, and had a normal karyotype.

The MUi019-A human induced pluripotent stem cell line was generated from peripheral blood CD34+ hematopoietic progenitors of a healthy woman using a non-integrative reprogramming method. Episomal vectors carrying reprogramming factors OCT4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 and EBNA-1 were delivered using electroporation. The iPSC line can be used as a control in studying disease mechanisms. Furthermore, gene editing approaches can be used to introduce specific mutations into the MUi019-A to model disease while the cell type affected by the disease is inaccessible.

Although many crustacean neuroendocrine hormones have been reported, the enzymes responsible for post-translational modification of neuroendocrine hormones have rarely been characterized. A prohormone convertase 2 (PC2)-like enzyme has been isolated from the optic lobe of the giant tiger shrimp, Penaeus monodon and referred as PmPC2. The full length cDNA sequence of PmPC2 has been identified and found to resemble evolutionarily conserved PC2 enzymes of vertebrates and invertebrates. PmPC2 was expressed in all larval developmental stages and in neuroendrocrine cells in the adult optic lobe. Its expression was found to be negatively related with shrimp body weight by qPCR (P0.05). Immunohistochemistry results using an anti-rPmPC2 antibody with adult shrimp revealed high staining intensity in specific neurosecretory cells including the sinus gland, the organ of Hanström (also referred to as the medullar terminalis X-organ) and the organ of Bellonci (also referred to as the sensory or X-organ). By using the yeast two hybrid technique, PmPC2 was found to bind with P. monodon hyperglycemic hormone (Pem-CHH1) that plays an important role in glucose metabolism. Since PmPC2 is a subtilisin-like serine proteinase, it is expected to cleave the synthetic substrate, pyr-RTKR-MCA, but the expressed recombinant catalytic domain of PmPC2 (rPmPC2-cat) showed no enzymatic activity as expected. In vivo injection of dsRNA-PmPC2 resulted in reduced transcripts for both PmPC2 and Pem-CHH1 on day 3 post injection, but there was no accompanying reduction of glucose level in the hemolymph. Taken together, PmPC2 localization, expression and activity suggest that it has a function(s) in the shrimp neuroendrocrine system and that it may not only activate Pem-CHH1 but also affect its expression. However, there is no obvious explanation for the negative correlation between PmPC2 expression level and shrimp body weight.

When using mRNA from gills of normal whiteleg shrimp Penaeus (Litopenaeus) vannamei as the tester and mRNA from yellow head virus (YHV)-infected shrimp as the driver, subtractive suppression hybridization (SSH) revealed that a novel EST clone of 198 bp with a putative C-type lectin-like domain (CTLD) was downregulated in YHV-infected shrimp. The clone nucleotide sequence had 99% identity with one contig MGID1052359 (1,380 bp) reported in an EST database of P. vannamei, and the presence of this target in normal shrimp was confirmed by RT-PCR using primers designed from the MGID1052359 sequence. Analysis of the primary structure of the deduced amino acid (a.a.) sequence of the contig revealed a short portion (40 a.a. residues) at its N-terminus with high similarity to a low density lipoprotein receptor (LDLR) class A domain and another 152 a.a. residues at its C-terminus with high similarity to a C-type lectin domain. Thus, the clone was named LvCTLD and three recombinant proteins (LvCTLD, the LDLR domain and the CTLD domain) were synthesized in a bacterial system based on its sequence. An in vitro encapsulation assay revealed that Sepharose 4B beads coated with rLvCTLD were encapsulated by shrimp hemocytes and that melanization followed by 24 h post-encapsulation. The encapsulation activity of rLvCTLD was inhibited by 100 mM galactose, but not mannose or EDTA. In vivo injection of rLvCTLD or rLvCTLD plus YHV resulted in a significant elevation of PO activity in the hemolymph of the challenged shrimp when compared to shrimp injected with buffer, suggesting that rLvCTLD could activate the proPO system. An ELISA test revealed that rLvCTLD could bind to YHV particles in the presence of shrimp hemolymph. Phylogenetic analysis suggested that the LvCTLD sequence was more closely related to an antiviral gene found in Penaeus monodon (PmAV) than to other reported shrimp lectins. Taken together, we conclude that a novel shrimp LvCTLD is a host recognition molecule involved in the shrimp defense mechanism against YHV via recruitment of hemocytes, probably at the site of viral infection, and via activation of the proPO system.

The optic lobe of the female-shrimp eyestalk was selected as the target tissue for suppressive subtractive hybridization (SSH) because it is a place for synthesis of many hormones and peptides/enzymes involved in molting and growth. SSH was performed to screen genes differentially expressed in the optic lobe between large female (LF; body weight > 90 percentile of weight distribution curve) and small female (SF; body weight E- value − 4 ) indicating little available molecular information on the optic lobe. Among the EST with relatively high homology to GenBank records, 5 candidate ESTs had homology to records for genes involved in cell differentiation/proliferation, cell cycle and hormone processing. These included genes resembling cyclophilin, cyclophilin A, fibrillarin, SPARC and PC2. These candidate ESTs were selected to confirm the reproducibility of the SSH data using semi-quantitative RT-PCR normalized with EF-1α. Pearson's correlation analysis confirmed that the index of relative cyclophilin, SPARC and fibrillarin-like expression was negatively correlated with body weight ( p 2 ) based on the index of relative cyclophilin-like expression in the optic lobe of female P. monodon .

Of 23 strains of halotolerant (up to 12% w/v NaCl) photosynthetic bacteria isolated from various sources, one isolate, SH5, accumulated intracellular 5-aminolevulinic acid (ALA) at 0.45 microg/g dry cell wt (DCW) growing aerobically in the dark. The strain was identified as Rhodobacter sphaeroides using 16S rDNA sequencing. Biosynthesis of ALA was enhanced to 14 microg/g DCW using modified glutamate/glucose (50 mM) medium with the addition of 10 mM levulinic acid after 24 h cultivation. Addition of 30 microM Fe(2+) to this medium increased the yield to 226 microg/g DCW.

Intracellular fatty acid-binding proteins (FABPs) are small members of the superfamily of lipid-binding proteins, which occur in invertebrates and vertebrates. Included in this superfamily are the cellular retinoic acid-binding proteins and retinol-binding proteins, which seem to be restricted to vertebrates. Here, we report the cDNA cloning and characterization of two FABPs from hemocytes of the freshwater crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon. In both these proteins, the binding triad residues involved in interaction with ligand carboxylate groups are present. From the sequence and homology modeling, the proteins are probably FABPs and not retinoic acid-binding proteins. The crayfish transcript (plFABP) was detected at high level in hemocytes, hepatopancreas, intestine and ovary and at low level in hematopoietic tissue and testis. Its expression in hematopoietic cells varied depending on the state of the crayfish from which it was isolated. Expression was 10-15 times higher in cultures isolated from crayfish with red colored plasma, in which hemocyte synthesis was high, if retinoic acid was added to the culture medium. In normal colored crayfish, with normal levels of hemocytes, no increase in expression of p1FABP was detected. Two other putative plFABP ligands, stearic acid and oleic acid, did not have any effect on plFABP expression in hematopoietic cells. These results suggest that retinoic acid-dependent signaling may be present in crustaceans.