Sage Journals: Discover world-class research

Abstract

Restoration of correct splicing of β^IVS2-654-globin pre-mRNA was previously accomplished in erythroid cells from β-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 β^IVS2-654 mutation. While correction of β^IVS2-654 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.

Get full access to this article

View all access options for this article.

References

Higgs

, Engel

, Stamatoyannopoulos

. Thalassaemia. Lancet, 2012; 379:373–383.

Rund

. Thalassemia 2016: modern medicine battles an ancient disease. Am J Hematol, 2016; 91:15–21.

Coquerelle

, Ghardallou

, Rais

, et al. Innovative curative treatment of beta thalassemia: cost-efficacy analysis of gene therapy versus allogenic hematopoietic stem-cell transplantation. Hum Gene Ther, 2019; 30:753–761.

Ikawa

, Miccio

, Magrin

, et al. Gene therapy of hemoglobinopathies: progress and future challenges. Hum Mol Genet, 2019; 28:R24–R30.

Cheng

, Orkin

, Antonarakis

, et al. β-Thalassemia in Chinese: use of in vivo RNA analysis and oligonucleotide hybridization in systematic characterization of molecular defects. Proc Natl Acad Sci U S A, 1984; 81:2821–2825.

Havens

, Hastings

. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res, 2016; 44:6549–6563.

Hoy

. Nusinersen: first global approval. Drugs, 2017; 77:473–479.

Syed

. Eteplirsen: first global approval. Drugs, 2016; 76:1699–1704.

Lacerra

, Sierakowska

, Carestia

, et al. Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients. Proc Natl Acad Sci U S A, 2000; 97:9591–9596.

10.

Suwanmanee

, Sierakowska

, Lacerra

, et al. Restoration of human β-globin gene expression in murine and human IVS2-654 thalassemic erythroid cells by free uptake of antisense oligonucleotides. Mol Pharmacol, 2002; 62:545–553.

11.

Svasti

, Suwanmanee

, Fucharoen

, et al. RNA repair restores hemoglobin expression in IVS2-654 thalassemic mice. Proc Natl Acad Sci U S A, 2009; 106:1205–1210.

12.

Nualkaew

, Jearawiriyapaisarn

, Hongeng

, et al. Restoration of correct β^IVS2-654-globin mRNA splicing and HbA production by engineered U7 snRNA in β-thalassaemia/HbE erythroid cells. Sci Rep, 2019; 9:7672.

13.

Lewis

, Yang

, Kim

, et al. A common human β globin splicing mutation modeled in mice. Blood, 1998; 91:2152–2156.

14.

Yang

, Shi

, Chu

, et al. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: useful for making iPS cells and transduction of primary cells. Biotechnol Lett, 2016; 38:1631–1641.

15.

De Angelis

, Sthandier

, Berarducci

, et al. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48–50 DMD cells. Proc Natl Acad Sci U S A, 2002; 99:9456–9461.

16.

Eckenfelder

, Tordo

, Babbs

, et al. The cellular processing capacity limits the amounts of chimeric U7 snRNA available for antisense delivery. Mol Ther Nucleic Acids, 2012; 1:e31.

17.

Preedagasamzin

, Nualkaew

, Pongrujikorn

, et al. Engineered U7 snRNA mediates sustained splicing correction in erythroid cells from β-thalassemia/HbE patients. Biochem Biophys Res Commun, 2018; 499:86–92.

18.

Le Guiner

, Montus

, Servais

, et al. Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients. Mol Ther, 2014; 22:1923–1935.

19.

Odermatt

, Trüb

, Furrer

, et al. Somatic therapy of a mouse SMA model with a U7 snRNA gene correcting SMN2 splicing. Mol Ther, 2016; 24:1797–1805.

20.

Will

, Luhrmann

. Spliceosomal UsnRNP biogenesis, structure and function. Curr Opin Cell Biol, 2001; 13:290–301.

21.

Suter

, Tomasini

, Reber

, et al. Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human β-thalassemic mutations. Hum. Mol. Genet, 1999; 8:2415–2423.

22.

Vacek

, Ma

, Gemignani

, et al. High-level expression of hemoglobin A in human thalassemic erythroid progenitor cells following lentiviral vector delivery of an antisense snRNA. Blood, 2003; 101:104–111.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.01 MB

Engineered U7 Small Nuclear RNA Restores Correct β-Globin Pre-mRNA Splicing in Mouse β IVS2-654 -Thalassemic Erythroid Progenitor Cells

Abstract

Get full access to this article

References

Supplementary Material