Coordinated ß-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for ß-thalassemia.

A primary challenge in lentiviral gene therapy of ß-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVß-shα2, that allows coordinated expression of the therapeutic ßA-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in ß-thalassemia. We demonstrate that LVß-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and ßA-T87Q-globin gene expression identical to the parent vector. Compared with the first ßA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVß-shα2 shows 1.7-fold greater potency to improve α/ß ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of ß-thalassemia and provide an improved benefit/risk ratio regardless of the ß-thalassemia genotype.

SLN124, a GalNac-siRNA targeting transmembrane serine protease 6, in combination with deferiprone therapy reduces ineffective erythropoiesis and hepatic iron-overload in a mouse model of ß-thalassaemia.

Beta-thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co-receptor for the bone morphogenetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in ß-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in ß-thalassaemia mice (Hbbth3/+ ). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of ß-thalassaemia and related disorders.

UNC0638 induces high levels of fetal hemoglobin expression in ß-thalassemia/HbE erythroid progenitor cells.

Increased expression of fetal hemoglobin (HbF) improves the clinical severity of ß-thalassemia patients. EHMT1/2 histone methyltransferases are epigenetic modifying enzymes that are responsible for catalyzing addition of the repressive histone mark H3K9me2 at silenced genes, including the γ-globin genes. UNC0638, a chemical inhibitor of EHMT1/2, has been shown to induce HbF expression in human erythroid progenitor cell cultures. Here, we report the HbF-inducing activity of UNC0638 in erythroid progenitor cells from ß-thalassemia/HbE patients. UNC0638 treatment led to significant increases in γ-globin mRNA, HbF expression, and HbF-containing cells in the absence of significant cytotoxicity. Moreover, UNC0638 showed additive effects on HbF induction in combination with the immunomodulatory drug pomalidomide and the DNMT1 inhibitor decitabine. These studies provide a scientific proof of concept that a small molecule targeting EHMT1/2 epigenetic enzymes, used alone or in combination with pomalidomide or decitabine, is a potential therapeutic approach for HbF induction. Further development of structural analogs of UNC0638 with similar biological effects but improved pharmacokinetic properties may lead to promising therapies and possible clinical application for the treatment of ß-thalassemia.

Engineered U7 snRNA mediates sustained splicing correction in erythroid cells from ß-thalassemia/HbE patients.

Repair of a splicing defect of ß-globin pre-mRNA harboring hemoglobin E (HbE) mutation was successfully accomplished in erythroid cells from patients with ß-thalassemia/HbE disorder by a synthetic splice-switching oligonucleotide (SSO). However, its application is limited by short-term effectiveness and requirement of lifelong periodic administration of SSO, especially for chronic diseases like thalassemias. Here, we engineered lentiviral vectors that stably express U7 small nuclear RNA (U7 snRNA) carrying the splice-switching sequence of the SSO that restores correct splicing of ßE-globin pre-mRNA and achieves a long-term therapeutic effect. Using a two-step tiling approach, we systematically screened U7 snRNAs carrying splice-switching SSO sequences targeted to the cryptic 5' splice site created by HbE mutation. We tested this approach and identified the most responsive element for mediating splicing correction in engineered U7 snRNAs in HeLa-ßE cell model cell line. Remarkably, the U7 snRNA lentiviral vector (U7 ßE4+1) targeted to this region effectively restored the correctly-spliced ßE-globin mRNA for at least 5 months. Moreover, the effects of the U7 ßE4+1 snRNA lentiviral vector were also evident as upregulation of the correctly-spliced ßE-globin mRNA in erythroid progenitor cells from ß-thalassemia/HbE patients treated with the vector, which led to improvements of pathologies in erythroid progenitor cells from thalassemia patients. These results suggest that the splicing correction of ßE-globin pre-mRNA by the engineered U7 snRNA lentiviral vector provides a promising, long-term treatment for ß-thalassemia/HbE.

Interplay between α-thalassemia and ß-hemoglobinopathies: Translating genotype-phenotype relationships into therapies.

α-Thalassemia represents one of the most important genetic modulators of ß-hemoglobinopathies. During this last decade, the ongoing interest in characterizing genotype-phenotype relationships has yielded incredible insights into α-globin gene regulation and its impact on ß-hemoglobinopathies. In this review, we provide a holistic update on α-globin gene expression stemming from DNA to RNA to protein, as well as epigenetic mechanisms that can impact gene expression and potentially influence phenotypic outcomes. Here, we highlight defined α-globin targeted strategies and rationalize the use of distinct molecular targets based on the restoration of balanced α/ß-like globin chain synthesis. Considering the therapies that either increase ß-globin synthesis or reactivate γ-globin gene expression, the modulation of α-globin chains as a disease modifier for ß-hemoglobinopathies still remains largely uncharted in clinical studies.

Engineered U7 Small Nuclear RNA Restores Correct ß-Globin Pre-mRNA Splicing in Mouse ßIVS2-654-Thalassemic Erythroid Progenitor Cells.

Restoration of correct splicing of ßIVS2-654-globin pre-mRNA was previously accomplished in erythroid cells from ß-thalassemia/HbE patients by an engineered U7 small nuclear RNA (snRNA) that carried a sequence targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP+623 snRNA. In this study, this approach was tested in thalassemic mice carrying the ßIVS2-654 mutation. While correction of ßIVS2-654 pre-mRNA splicing was achieved in erythroid progenitors transduced with a lentiviral vector carrying the U7.BP+623 snRNA, a high level of truncated U7.BP+623 snRNA was also observed. The discrepancy of processing of the modified U7 snRNA in human and mouse constructs hamper the evaluation of pathologic improvement in mouse model.

Lysine-specific histone demethylase 1 inhibition enhances robust fetal hemoglobin induction in human ß0-thalassemia/hemoglobin E erythroid cells.

Induction of fetal hemoglobin (HbF) ameliorates the clinical severity of ß-thalassemias. Histone methyltransferase LSD1 enzyme removes methyl groups from the activating chromatin mark histone 3 lysine 4 at silenced genes, including the γ-globin genes. LSD1 inhibitor RN-1 induces HbF levels in cultured human erythroid cells. Here, the HbF-inducing activity of RN-1 was investigated in erythroid progenitor cells derived from ß0-thalassemia/ hemoglobin E (HbE) patients. The significant and reproducible increases in γ-globin transcript and HbF expression upon RN-1 treatment were demonstrated in erythroid cells with divergent HbF baseline levels, the average of HbF induction was 17.7±0.8%. RN-1 at low concentration did not affect viability and proliferation of erythroid cells, but decreases in cell number were observed in cells treated with RN-1 at high concentration. Delayed terminal erythroid differentiation was revealed in ß0-thalassemia/HbE erythroid cells treated with RN-1 as similar to other compounds that target LSD1 activity. Downregulation of repressors of γ- globin expression; NCOR1 and SOX6, was observed in RN-1 treatment. These findings provide proof of the concept that LSD1 epigenetic enzyme is a potential therapeutic target for ß0-thalassemia/HbE patients.

Development of DNA controls for detection of ß-thalassemia mutations commonly found in Asian.

INTRODUCTION: Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of ß-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe ß-thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for ß-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. METHODS: Four recombinant ß-globin plasmids, including a normal sequence and three different mutant panels covering 10 common ß-thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site-directed mutagenesis. These positive DNA controls were further validated by RDB analysis. RESULTS: We demonstrated the applicability of established positive DNA controls for ß-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant ß-globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant ß-globin plasmids, are required for detecting 10 common ß-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. CONCLUSION: The ß-globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of ß-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches.

Restoration of correct ßIVS2-654-globin mRNA splicing and HbA production by engineered U7 snRNA in ß-thalassaemia/HbE erythroid cells.

A cytosine to thymine mutation at nucleotide 654 of human ß-globin intron 2 (ßIVS2-654) is one of the most common mutations causing ß-thalassaemia in Chinese and Southeast Asians. This mutation results in aberrant ß-globin pre-mRNA splicing and prevents synthesis of ß-globin protein. Splicing correction using synthetic splice-switching oligonucleotides (SSOs) has been shown to restore expression of the ß-globin protein, but to maintain therapeutically relevant levels of ß-globin it would require lifelong administration. Here, we demonstrate long-term splicing correction using U7 snRNA lentiviral vectors engineered to target several pre-mRNA splicing elements on the ßIVS2-654-globin pre-mRNA such as cryptic 3' splice site, aberrant 5' splice site, cryptic branch point and an exonic splicing enhancer. A double-target engineered U7 snRNAs targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP + 623, was the most effective in a model cell line, HeLa IVS2-654. Moreover, the therapeutic potential of the vector was demonstrated in erythroid progenitor cells derived from ßIVS2-654-thalassaemia/HbE patients, which showed restoration of correctly spliced ß-globin mRNA and led to haemoglobin A synthesis, and consequently improved thalassaemic erythroid cell pathology. These results demonstrate proof of concept of using the engineered U7 snRNA lentiviral vector for treatment of ß-thalassaemia.

Enhancement of ß-Globin Gene Expression in Thalassemic IVS2-654 Induced Pluripotent Stem Cell-Derived Erythroid Cells by Modified U7 snRNA.

The therapeutic use of patient-specific induced pluripotent stem cells (iPSCs) is emerging as a potential treatment of ß-thalassemia. Ideally, patient-specific iPSCs would be genetically corrected by various approaches to treat ß-thalassemia including lentiviral gene transfer, lentivirus-delivered shRNA, and gene editing. These corrected iPSCs would be subsequently differentiated into hematopoietic stem cells and transplanted back into the same patient. In this article, we present a proof of principle study for disease modeling and screening using iPSCs to test the potential use of the modified U7 small nuclear (sn) RNA to correct a splice defect in IVS2-654 ß-thalassemia. In this case, the aberration results from a mutation in the human ß-globin intron 2 causing an aberrant splicing of ß-globin pre-mRNA and preventing synthesis of functional ß-globin protein. The iPSCs (derived from mesenchymal stromal cells from a patient with IVS2-654 ß-thalassemia/hemoglobin (Hb) E) were transduced with a lentivirus carrying a modified U7 snRNA targeting an IVS2-654 ß-globin pre-mRNA in order to restore the correct splicing. Erythroblasts differentiated from the transduced iPSCs expressed high level of correctly spliced ß-globin mRNA suggesting that the modified U7 snRNA was expressed and mediated splicing correction of IVS2-654 ß-globin pre-mRNA in these cells. Moreover, a less active apoptosis cascade process was observed in the corrected cells at transcription level. This study demonstrated the potential use of a genetically modified U7 snRNA with patient-specific iPSCs for the partial restoration of the aberrant splicing process of ß-thalassemia. Stem Cells Translational Medicine 2017;6:1059-1069.