Dataset Information

Restoration of correct ?IVS2-654-globin mRNA splicing and HbA production by engineered U7 snRNA in ?-thalassaemia/HbE erythroid cells.

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

SUBMITTER: Nualkaew T

PROVIDER: S-EPMC6529457 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Restoration of correct β<sup>IVS2-654</sup>-globin mRNA splicing and HbA production by engineered U7 snRNA in β-thalassaemia/HbE erythroid cells.

Nualkaew Tiwaporn T Jearawiriyapaisarn Natee N Hongeng Suradej S Fucharoen Suthat S Kole Ryszard R Svasti Saovaros S

Scientific reports 20190521 1

A cytosine to thymine mutation at nucleotide 654 of human β-globin intron 2 (β<sup>IVS2-654</sup>) 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 wo ...[more]

PMID: 31113996

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Project description:BackgroundLow HbF expression in HbE-β+-thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the β- and α-globin genes and the modifying factors related to HbF expression in patients with an Hb phenotype similar to that of HbE heterozygotes. Furthermore, screening tools for differentiating HbE-β+-thalassemia from HbE heterozygotes have been investigated.Participants and methodsA total of 2133 participants with HbE and HbA with varying HbF levels were recruited. Polymerase chain reaction-based DNA analysis and sequencing were performed to characterize β- and α-globin genes. DNA polymorphism at position -158 nt 5' to Gγ-globin was performed by XmnI restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA2, HbE, and HbF levels for the differentiation of HbE-β+-thalassemia from HbE heterozygotes were determined.ResultsFive β+-thalassemia mutations trans to βE-gene (β-87(C>A), β-31(A>G), β-28(A>G), β19(A>G), and β126(T>G)) were identified in 79 patients. Among these, 54 presented with low HbF levels, and 25 presented with high HbF levels. ROC curve analysis revealed an excellent AUC of 1.000 (95% confidence interval:1.000-1.000) for HbE levels, and a cut-off point of ≥35.0% had 100.0% sensitivity, specificity, and Youden's index for differentiating HbE-β+-thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-β+-thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α2-globin genes were identified. The genotype and allele of the polymorphism at -158 nt 5' to Gγ-globin was found to be negatively associated with HbF expression.ConclusionsHbE-β+-thalassemia cannot be disregarded until appropriate DNA analysis is performed, and the detection of α-thalassemia mutations should always be performed under these conditions. An HbE level ≥35.0% may indicate screening of samples for DNA analysis for HbE-β+-thalassemia diagnosis.

Dataset Information

Restoration of correct ?IVS2-654-globin mRNA splicing and HbA production by engineered U7 snRNA in ?-thalassaemia/HbE erythroid cells.

Publications

Restoration of correct β<sup>IVS2-654</sup>-globin mRNA splicing and HbA production by engineered U7 snRNA in β-thalassaemia/HbE erythroid cells.

Similar Datasets

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