Levels Of Beta-globin Research Articles

In Vivo HSC Transduction and Selection Results in Long-Term, High-Level Expression of Human Gamma Globin in Peripheral Blood Erythrocytes of Mice

Current protocols for HSC gene therapy, involving the transplantation of ex vivo lentivirus vector-transduced HSCs into myeloablated recipients, are complex and not without risk for the patient. We have developed a new approach for in vivo gene delivery into HSCs that does not require myeloablation and HSC transplantation. It involves injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. Transgene integration is achieved (in a random pattern) using a hyperactive Sleeping Beauty transposase (SB100x). We demonstrated in adequate mouse models, using GFP as a transgene, that primitive HSCs transduced in the periphery home back to the bone marrow where they persist and stably express GFP long-term (Blood 128:2206 (2016). To achieve high level transgene marking in differentiated peripheral blood cells, we combined our in vivo HSC transduction approach with in vivo selection of transduced HSCs using the mgtmP140K mutant and low dose O6BG/BCNU treatment. Here were tested the potential of this approach for the therapy of hemoglobinopathies. Our HDAd5/35++ vector contained the human-gamma globin gene under the control of a 6kb version of the beta-globin LCR (containing HS1 to HS4 and the beta globin promoter) and the mgmtP140K gene under the control of the PGK promoter. Mobilized immunocompetent hCD46 transgenic mice were injected IV with the integrating vector system and subjected to three rounds of treatment with low-dose O6BG/BCNU followed by immunosuppression to avoid immune responses against the human transgene product. The procedure was safe and resulted in stable human gamma globin expression in >95% of erythrocytes in the peripheral blood of in vivo transduced cells mice. Human gamma globin levels were 100% of endogenous mouse beta globin levels in erythrocytes measured by HPLC. Analysis of bone marrow at week 16 after in vivo transduction showed that >60% of bone marrow LSK cells contained the integrated transgene cassette and that the beta globin LCR efficiently restricted gamma globin expression to erythroid cells. After transplantation of unsorted, lineage-depleted bone marrow cells into lethally irradiated recipients, long-term gamma-globin expression at levels seen in the primary in vivo treated mice was demonstrated. The efficacy of our approach achieved in this study indicates that it would be curative in thalassemia models or patients. Our new approach avoids the collection of HSCs, their transduction ex vivo, and subsequent transplantation into myeloablated recipients. It therefore could greatly simplify HSC gene therapy of common diseases. DisclosuresKiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Homeobox A5 and A9 expression and beta-thalassemia

ABSTRACT Background and aim: The pathogenesis of β-thalassemia has been attributed to ineffective erythropoiesis. The function of Hox genes in normal haematopoiesis has been widely studied using gene expression analysis. The aim of this study is to evaluate the expression of HoxA9, and HoxA5 genes in beta-thalassemia. Materials and methods: Children with thalassemia major, thalassemia intermediate, and age and sex-matched healthy controls (n = 50/group) were enrolled. Detection of HoxA5 and HoxA9 mRNA expression was performed by real-time polymerase chain reaction (RT-PCR). Results: Expression of HoxA9 increased in a direct linear trend (median 0.5 in controls, 2.4 in intermediate disease, 4.1 in major disease, p = 0.001) and generally correlated with the red cell count, haematocrit, ferritin and levels of beta-globin. In those with thalassemia major, the relative change of HoxA9 was linked to transfusion history, the white blood cell count, ferritin, and beta-globin (all r > 0.5, p < 0.001). Levels of HoxA9 were superior to HoxA5 in differentiating controls from thalassemia intermedia, whilst both differentiated major from the intermediate disease. Conclusion: This study highlights the importance of HoxA genes in early identification of patients, at high risk of developing complications, as it allows specific measures to delay the progression of the disease. HoxA gene expression is a promising diagnostic and prognostic marker in patients with β-thalassemia.

Transmembrane Protease, Serine 6 (TMPRSS6) Antisense Oligonucleotide (IONIS-TMPRSS6-LRX) Reduces Plasma Iron Levels of Healthy Volunteers in a Phase 1 Clinical Study

Iron overload is the major cause of morbidity and mortality in beta-thalassemia patients. The low levels of beta-globin and ineffective erythropoiesis in these patients result in the suppression of hepcidin. The inappropriately low levels of hepcidin trigger an increased absorption of dietary iron and increased iron release from storage, causing iron overload. Expression of hepcidin, which is predominately produced in the liver, is negatively regulated by the transmembrane protease, serine 6 (TMPRSS6). Mouse and human genetic data indicated that lowering TMPRSS6 expression could up-regulate hepcidin and ameliorate many of the disease symptoms associated with beta-thalassemia. Previously we identified a highly specific and potent antisense oligonucleotide (ASO) targeting either the murine (Guo et al. J Clin Invest. 2013; 123(4):1531-41) or the human (Aghajan et al, Blood 2016; 128:1013) TMPRSS6 mRNA. Downregulation of TMPRSS6 with ASO treatment resulted in dose-dependent hepcidin upregulation, leading to dramatic reductions in serum iron and transferrin saturation in animal models This in turn ameliorated the anemia and iron overload phenotypes in a mouse model of beta-thalassemia (th3/+ mice), which recapitulates beta-thalassemia intermedia in humans.Herein, we are reporting the initial clinical safety and pharmacodynamics of IONIS-TMPRSS6-LRX. This GalNAc-conjugated, TMPRSS6 ASO was evaluated in a placebo-controlled, double-blind, randomized, single-center Phase 1 clinical study enrolling healthy volunteers. During an 8-week period, placebo or IONIS-TMPRSS6-LRX was administered subcutaneously four times (Weeks 1, 4, 6 and 8) at doses of 20, 40 or 60 mg. At doses of 20 and 40 mg, mean (±SEM) levels of serum iron were reduced 34±10% and 49±7% on week 10 (The 60 mg treatment cohort is ongoing). Consistent with the reduction of plasma iron, the mean (+/-SEM) percent transferrin saturation was reduced from baseline levels of 28±3% and 30±1% to 14±2% and 13±2%, for 20 and 40 mg groups, respectively, at Week 10. Furthermore, plasma hepcidin levels were increased from 2.1±0.6 and 2.5±0.6 nM to 2.7±0.6 and 6.7±0.9 nM, respectively. During this time period, there were small reductions in Hgb (-9±2%), reticulocyte Hgb (-13±2%) at the 40 mg dose. There were no serious adverse events in the study and the treatment-emergent adverse events were generally mild. In summary, IONIS-TMPRSS6-LRX, a novel antisense oligonucleotide targeting TMPRSS6, effectively reduces plasma iron levels and has the potential as a therapeutic for patients with beta-thalassemia and related disorders. The safety profile of IONIS-TMPRSS6-LRX supports further development. DisclosuresMcCaleb:Ionis Pharmaceuticals, INC: Employment, Equity Ownership. Lickliter:Nucleus Network: Employment. Dibble:Ionis Pharmaceuticals, INC: Employment. Schneider:Ionis Pharmaceuticals, INC: Employment, Other: shareholders. Aghajan:Ionis Pharmaceuticals, Inc: Employment. Guo:Ionis Pharmaceuticals, Inc: Employment. Hughes:Ionis Pharmaceuticals, INC: Employment, Other: shareholders. Geary:Ionis Pharmaceuticals, INC: Employment, Other: shareholders. Monia:Ionis Pharmaceuticals, Inc: Employment, Other: Intellectual property rights.

Monitoring of transplanted liver health by quantification of organ-specific genomic marker in circulating DNA from receptor.

BackgroundHealth assessment of the transplanted organ is very important due to the relationship of long-term survival of organ transplant recipients and health organ maintenance. Nowadays, the measurement of cell-free DNA from grafts in the circulation of transplant recipients has been considered a potential biomarker of organ rejection or transplant associated complications in an attempt to replace or reduce liver biopsy. However, methods developed to date are expensive and extremely time-consuming. Our approach was to measure the SRY gene, as a male organ biomarker, in a setting of sex-mismatched female recipients of male donor organs.MethodsCell-free DNA quantization of the SRY gene was performed by real-time quantitative PCR beforehand, at the moment of transplantation during reperfusion (day 0) and during the stay at the intensive care unit. Beta-globin cell-free DNA levels, a general cellular damage marker, were also quantified.ResultsBeta-globin mean values of patients, who accepted the graft without any complications during the first week after surgery, diminished from day 0 until patient stabilization. This decrease was not so evident in patients who suffered some kind of post-transplantation complications. All patients showed an increase in SRY levels at day 0, which decreased during hospitalization. Different complications that did not compromise donated organs showed increased beta-globin levels but no SRY gene levels. However, when a donated organ was damaged the patients exhibited high levels of both genes.ConclusionDetermination of a SRY gene in a female recipient's serum is a clear and specific biomarker of donated organs and may give us important information about graft health in a short period of time by a non-expensive technique. This approach may permit clinicians to maintain a close follow up of the transplanted patient.

Correction of Hemoglobin Levels in a Heterozygous Humanized Mouse Model of Thalassemia after Fetal Gene Therapy

BackgroundBeta thalassaemia is a genetic blood disease that causes life-threatening anemia. Hematopoietic stem cell (HSC) transplantation successfully cures the disease but in only 30% of patients. We hypothesized that in utero gene therapy (IUGT) to the fetal HSC compartment with the corrected beta globin gene might cure the disease before birth.MethodsA humanized mouse model of thalassemia (Cooley’s anemia; CA) was used in which heterozygous animals are affected by anemia, splenomegaly and extra-medullary hematopoiesis. At E13.5 a “GLOBE” vector (HIV-2 based lentiviral vector that incorporates a mini hemoglobin beta gene, the beta-globin promoter and HS2/3 β-LCR element) was injected into the liver of each fetus (n=12). At 12 weeks of age, recipient blood, liver, spleen and bone marrow were collected for complete blood count, blood film, as well as RNA and DNA isolation. Extra-medullary hematopoiesis was examined in the spleen and liver using flow cytometry (CD71+/Ter119+ cells) and histo-pathological analysis. Gene expression of human and mouse alpha and beta globins, as well as human gamma globin was assessed by quantitative polymerase chain reaction (qPCR). High performance liquid chromatography (HPLC) was used to quantify the presence of human beta globin in the peripheral blood of treated animals. Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the ethical committee on animal experimentation at University College London.ResultsCompared to non-injected heterozygous pups (control), IUGT increased hemoglobin levels [11.3±0.4g/dl (n=6) vs. 7.6±0.6g/dl (n=8); p<0.01], red blood cell count [9.3±0.3*1012/L vs. 6.2±0.5*1012/L; p<0.01), and hematocrit [41.2±2.2% vs. 27.2±2.0; p<0.01]. Moreover, treated CA animals had reduced spleen weight [130±5mg vs. 310±21mg; p <0.01], as well as reduced extra-medullary hematopoiesis in the liver [0.7±0.1% (n=4) vs. 6.0±0.9% (n=5); p<0.01] and spleen [6.6±1.8 (n=4) vs. 23.1±1.4 (n=3); p<0.05]. qPCR analysis demonstrated increased gene expression of human beta globin (figure A) and reduced expression of human gamma globin in blood and bone marrow of IUGT offspring. HPLC analysis confirmed these findings at protein level (figure B). The average vector copy number in the liver was 0.1.ConclusionsIUGT resulted in phenotypic normalization in a heterozygous humanized mouse model of CA. Increased levels of beta globin and associated down-regulation of gamma globin is consistent with a switch from fetal to adult human hemoglobin, confirming successful prenatal correction of the genetic defect. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clonal Effect Of Lenalidomide On Akt Activation In Low-Risk MDS Patients With Del(5q)

Lenalidomide is an immunomodulating drug currently used in the treatment of del(5q) low-risk MDS patients, where it can suppress the del(5q) clone and restore a normal erythropoiesis. The exact molecular mechanisms underlying the effect of Lenalidomide in del(5q) MDS are not completely clear, although Akt phosphorylation is inhibited in Lenalidomide-sensitive del(5q) cell lines (Gandhi et al, 2006). On the other hand, the activation of the Akt/mTOR pathway has been demonstrated in CD34+ cells from high-risk MDS (Follo et al, 2007), which show alterations on stem cell proliferation, differentiation and apoptosis. These processes are important also in low-risk MDS, that usually show a stable disease but can evolve towards a worse clinical status, characterized by an increased cell proliferation.In this study we firstly investigated the effect of Lenalidomide in 6 patients with del(5q) MDS (IPSS: Low or Int-1). Given the limited number of cells, we analyzed bone marrow total mononuclear cells. As for Akt phosphorylation, we analyzed its localization along with RPS14, in order to specifically detect the del(5q) clone. On the other hand, by Real-Time PCR analyses, we assessed the expression of Globin genes, to evaluate the effect of the drug on erythropoiesis. In addition, we analyzed the effect of Lenalidomide on two cell lines with a different 5q status, one bearing a normal 5q chromosome and one showing the 5q deletion, to further investigate the effect of this drug on cell cycle, erythroid differentiation and inositide signalling pathways.Clinically, 4/6 del(5q) MDS patients showed a favourable response to Lenalidomide. At a molecular level, these cases showed an activation of erythropoiesis, in that Beta-Globin levels increased, as compared with baseline. Moreover, these subjects also displayed a specific phosphorylation of Akt. Interestingly, Akt resulted to be specifically activated in cells not showing the 5q deletion, whereas it was down-regulated in del(5q) cells. The two non responder patients early discontinued Lenalidomide for adverse events, and for these patients neither a clinical assessment of Lenalidomide effect, nor a molecular analysis, were possible. As for cell lines, ongoing analyses are showing that Lenalidomide specifically inhibits the growth of the del(5q) clone, blocking cells in G1 phase. On the other hand, Akt phosphorylation specifically increases in cells with a normal 5q chromosome.Taken together, our data show a specific activation of erythropoiesis in del(5q) low-risk MDS patients responding to Lenalidomide. In addition, our results indicate that Akt is specifically phosphorylated in normal cells without the del(5q), leading to hypothesize that Lenalidomide has a double effect: it can induce apoptosis in clonal del(5q) cells, but it also supports the proliferation and erythroid differentiation of normal cells, as also described in non-del(5q) MDS (Ebert et al, 2008). Therefore, our findings might contribute to elucidate the molecular mechanisms of Lenalidomide and possibly pave the way for the development of innovative therapeutic targeted strategies in MDS. Disclosures:No relevant conflicts of interest to declare.

P-012 Clonal activation of Akt in low-risk MDS patients with del(5q) treated with lenalidomide

The activation of inositide signalling pathways, such as Akt/mTOR, has been demonstrated in high-risk MDS (1). Lenalidomide is currently used in the treatment of del(5q) low-risk MDS patients, where it may suppress the del(5q) clone and restore a normal erythropoiesis, via inhibition of Akt phosphorylation (2). Here, we studied the expression of inositide signalling molecules in 6 low-risk MDS patients who were given Lenalidomide by immunocytochemistry and Real-Time PCR. In our case series, 4 out of 6 del(5q) low-risk MDS patients responded to Lenalidomide and showed an activation of erythropoiesis, in that Beta-Globin levels increased, as compared with baseline. Moreover, these subjects also displayed an activation of PI-PLCgamma1 and Akt. Interestingly, Akt resulted to be specifically phosphorylated in cells not showing the 5q deletion, hinting at a clonal activation of this pathway. The 2 non responder patients early discontinued Lenalidomide for adverse events, and for these patients neither a clinical assessment of Lenalidomide effect, nor a molecular analysis, were possible. Our data show Akt/PI-PLCgamma1 activation during Lenalidomide treatment, and confirm the activation of erythropoiesis in responder patients. In addition, our results indicate that Akt is specifically phosphorylated in the 5q+ clone. Therefore, it is conceivable that Lenalidomide strengthens the proliferation of the 5q+ clone, whilst the del(5q) clone undergoes an apoptotic process, allowing the restoration of the normal erythropoiesis. This is extremely important, not only for MDS pathogenesis, but also for the development of innovative targeted therapies.

Diagnosis of invasive aspergillosis using bronchoalveolar lavage in haematology patients: influence of bronchoalveolar lavage human DNA content on real-time PCR performance

In order to improve invasive pulmonary aspergillosis (IPA) diagnosis, a real-time polymerase chain reaction (PCR) assay detecting Aspergillus spp. was developed. Its detection limit reached 2-20 conidia. The retrospective evaluation on 64 bronchoalveolar lavage (BAL) fluids from 57 patients at risk for IPA, including 20 probable and five proven IPA patients, revealed a 88% or 38% sensitivity in direct examination (DE)/culture-positive or culture-negative BAL, respectively, whereas galactomannan (GM) sensitivity reached 88% or 58%, respectively. Influence on the Aspergillus-PCR yield of BAL fluid volume, cellular count and DNA content (evaluated by human beta-globin quantification) was assessed. Significantly higher beta-globin levels were detected in Aspergillus PCR-positive (median 5,112 pg/microl) than negative (median 1,332 pg/microl) BAL fluids, suggesting that the beta-globin level could reflect BAL yields and DNA extraction. Using beta-globin for the interpretation of fungal PCR could improve the negative predictive value of this test.

Identification of Two New Synthetic Histone Deacetylase Inhibitors That Modulate Globin Gene Expression in Erythroid Cells from Healthy Donors and Patients with Thalassemia

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of gamma-globin and/or beta-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the gamma/(gamma+beta) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of gamma-globin mRNA and the gamma/(gamma+beta) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of gamma-globin and decreased by 2-fold that of beta-globin mRNA, increasing the gamma/(gamma+beta) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as gamma/(gamma+beta) mRNA inducers in erythroblasts obtained from patients with beta(0) thalassemia. Progenitor cells from patients with beta(0) thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low beta-globin levels but 10 times higher-than-normal levels of the alpha hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in beta(0) thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the gamma/(gamma+beta) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with beta(0) thalassemia.