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

Abstract

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

RNA Splicing and Disease: Animal Models to Therapies

“…IVS2–654 β-thalassemia is one of the most common of these splicing mutations, causing a C to T change in the position 654 of the IVS2 intron in the β-hemoglobin gene ( HBB ) to generate an aberrant 5′-donor splice site at position 652 and activates a cryptic 3′acceptor splice site at position 579. This allows for the aberrant inclusion of an intron and the translation of a non-functional β-globin protein [75]. Expression of this non-functional protein leads to low oxygen levels and a shortage of red blood cells, which can lead to life threatening anemia.…”

<p>Nucleic Acid Therapy for β-Thalassemia</p>

“…SSOs modulate RNA splicing by redirecting alternative splicing that can be categorized in four mechanisms: exon skipping, exon retention, restoration of correct splicing and displacement of splicing factors from triplets repeats which have been reviewed extensively. 28 The SSOs have been used to correct aberrant splicing in β-thalassemia alleles including the mutations IVS1-110, 34 , 35 IVS2-654, 36 – 38 IVS2-705, 39 IVS2-745, 40 and βE. 36 SSOs successfully induced correct splicing and increased normal β-globin production in cell-free extracts, stable cell lines with a mutated β-globin gene, erythroid mononuclear cells from peripheral blood of patients, thalassemic mouse erythroid progenitors, human iPSCs and β-thalassemic mouse model.…”

“… 36 SSOs successfully induced correct splicing and increased normal β-globin production in cell-free extracts, stable cell lines with a mutated β-globin gene, erythroid mononuclear cells from peripheral blood of patients, thalassemic mouse erythroid progenitors, human iPSCs and β-thalassemic mouse model. 36 – 38 , 40 , 41 Among patients with β IVS2−654 thalassemia, erythroid cells, free uptake and syringe load of SSOs into cells increased correct β-globin mRNA up to 77% and HbA production up to 54%. 36 Moreover, in the β IVS2−654 -thalassemia mouse model, intravenous injection of SSOs restored 12% of correct β-globin mRNA in mice peripheral blood or increased 6-fold compared with untreated controls.…”

“…37 The latest construct could restore approximately 80% of correctly spliced β IVS2−654 - thalassemic human iPSCs. 38 Not only in β-thalassemia, but the modified U7 snRNAs has also been extensively explored in several RNA mis-splicing diseases including DMD and SMA. 46 – 52 …”

Induced Pluripotent Stem Cell-Derived Red Blood Cells and Platelet Concentrates: From Bench to Bedside

“…Apart from transfusion applications, understanding RBCs re-differentiation from iPS cells in vitro is also fundamental to recapitulate defects in erythroid differentiation and boost drug discovery [ 49 ] or to test the effectiveness of gene-edited iPS cell-derived HSCs [ 50 , 51 ].…”