Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients

Abstract

Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant ␤-globin precursor mRNAs (premRNAs). The oligonucleotides restored correct splicing and translation of ␤-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654͞␤ E , IVS2-745͞ IVS2-745, and IVS2-745͞IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was Ϸ30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective ␤-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach. O ne of the most common genetic diseases of mankind is ␤-thalassemia. It affects large populations in the Mediterranean basin, Middle East, South East Asia, and Africa. Approximately 80 million people are carriers of the thalassemia trait and the percentage of carriers worldwide is increasing. Concomitant increases in the number of patients, presently numbering several hundred thousands, are held down by high infant mortality in underdeveloped countries, by population screening, genetic counseling, and abortions; the growing need for clinical treatment is evident (1).The disease is due to mutations causing defective ␤-globin gene expression and deficiency of ␤-globin and adult hemoglobin (Hb) A. Homozygotes or compound heterozygotes for severe defects are affected with thalassemia major or Cooley's anemia, lethal if untreated, and suffer pronounced anemia, bone deformities, and hepatomegaly and splenomegaly (2). Regular, lifelong transfusions combined with iron chelation constitute current treatment. Bone marrow transplantation, the only cure, is limited by the scarcity of suitable donors and facilities. Experimental protocols to stimulate synthesis of fetal hemoglobin by sodium butyrate or hydroxyurea (3-6) or ␤-globin gene repair or replacement (4,7,8) have not yet been fully tested at the clinical level. Clearly there is a need for alternative treatments to replace the costly and cumbersome transfusion regimen.More than 100 thalassemic mutations causing defective ␤-globin gene expression and ␤-globin deficiency have been identified, but the ones causing aberrant splicing are among the most common (9). They are found in intron 1 (IVS1-5, IVS1-6, and IVS1-110) and intron 2 (IVS2-654, IVS2-705, and IVS2-745) of the ␤-globin gene (9-15). A mutation in codon 26 of the gene also results in activation of an aberrant splicing pathway and a mutated ␤-globin protein (␤ E ) (16,17). A common pathogenic feature of these mutations is activation of aberrant splice sites and modification of the splicing pathways, even though the correct splice sites remain potentially functional.Aberrant splicing of several of the ␤-globin splicing mutants has been shown in cell free extracts (18), in transfected HeLa based cell lines (19-22), and in a transgenic mouse model (23).More importan...

siRNA-based approaches in cancer therapy

“…25 One such modification in oligomer chemistry has led to the development of the phosphorodiamidate morpholino oligomers (PMO) by AVI BioPharma Inc. (Portland, OR), which are non-ionic antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation in an RNaseH-independent manner. [26][27][28] PMO antisense agents have revealed excellent safety profile and efficacy in multiple disease models [29][30][31][32][33][34][35][36][37] including cancer preclinical studies. [38][39][40][41][42][43][44] Recent studies in our laboratory have characterized in vivo PMO bioavailability in solid tumors and pharmacokinetics of the PMO agents in human clinical trials, revealing the potential of these agents in gene-specific targeting.…”

RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

“…Work in this laboratory showed that splice-switching oligonucleotides (SSOs), which block aberrant splice sites in IVS2-654 and other pre-mRNAs (IVS1-5, IVS1-6, IVS1-110, IVS2-705, and IVS2-745) as well as in the coding sequence (HbE) of the ␤-globin gene, force the splicing machinery to reselect the existing correct splice sites, repairing the splicing pattern of ␤-globin pre-mRNA. This repair, which restores production of correctly spliced ␤-globin mRNA and protein, was accomplished in several in vitro systems and ex vivo in erythroid progenitor cells from thalassemic patients (6)(7)(8)(9)(10)(11)(12). In this study, we investigated the effectiveness in thalassemic splicing correction of a modified morpholino oligomer, SSO 654-P005, in a mouse model of IVS2-654 ␤-thalassemia.…”

Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

“…Antisense oligonucleotides have been used to restore pre-mRNA splicing in other disease models (1)(2)(3)(4)(5)(6)(7)(8); however, the therapeutic rationale for each varies considerably and, to our knowledge, none has addressed an intranuclear or DNA repair disorder. Furthermore, we believe that A-T offers several advantages for exploring the therapeutic potential of AMOs, such as (i) the availability of many surrogate markers for evaluating ATM function, ex vivo and in vivo, and (ii) a well characterized spectrum of ATM mutations supported by an extensive cell repository of lymphoblastoid cell lines (LCLs) derived from patients with those mutations (9)(10)(11)(12).…”