Summary 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 β‐thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in β‐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 β‐thalassaemia and related disorders.

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The therapeutic use of patient‐specific induced pluripotent stem cells (iPSCs) is emerging as a potential treatment of β‐thalassemia. Ideally, patient‐specific iPSCs would be genetically corrected by various approaches to treat β‐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 β‐thalassemia. In this case, the aberration results from a mutation in the human β‐globin intron 2 causing an aberrant splicing of β‐globin pre‐mRNA and preventing synthesis of functional β‐globin protein. The iPSCs (derived from mesenchymal stromal cells from a patient with IVS2‐654 β‐thalassemia/hemoglobin (Hb) E) were transduced with a lentivirus carrying a modified U7 snRNA targeting an IVS2‐654 β‐globin pre‐mRNA in order to restore the correct splicing. Erythroblasts differentiated from the transduced iPSCs expressed high level of correctly spliced β‐globin mRNA suggesting that the modified U7 snRNA was expressed and mediated splicing correction of IVS2‐654 β‐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 β‐thalassemia. Stem Cells Translational Medicine 2017;6:1059–1069

A primary challenge in lentiviral gene therapy of b-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, LVb-sha2, that allows coordinated expression of the therapeutic b A-T87Q -globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the a2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of a-globin chain synthesis ameliorates disease severity in b-thalassemia. We demonstrate that LVb-sha2 reduces a2-globin mRNA expression in erythroid cells while keeping a1-globin mRNA levels unchanged and b A-T87Q -globin gene expression identical to the parent vector. Compared with the first b A-T87Q -globin lentiviral vector that has received conditional marketing authorization, BB305, LVb-sha2 shows 1.7-fold greater potency to improve a/b ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of b-thalassemia and provide an improved benefit/risk ratio regardless of the b-thalassemia genotype.

A cytosine to thymine mutation at nucleotide 654 of human β-globin intron 2 (β IVS2-654 ) is one of the most common mutations causing β-thalassaemia in Chinese and Southeast Asians. This mutation results in aberrant β-globin pre-mRNA splicing and prevents synthesis of β-globin protein. Splicing correction using synthetic splice-switching oligonucleotides (SSOs) has been shown to restore expression of the β-globin protein, but to maintain therapeutically relevant levels of β-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 β IVS2-654 -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 β IVS2-654 -thalassaemia/HbE patients, which showed restoration of correctly spliced β-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 β-thalassaemia.

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