In one form of ␤-thalassemia, a genetic blood disorder, a mutation in intron 2 of the ␤-globin gene (IVS2-654) causes aberrant splicing of ␤-globin pre-mRNA and, consequently, ␤-globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human ␤-globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human ␤-globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.␤-Thalassemia, a genetic blood disorder, affects a large number of people in the Mediterranean basin, Middle East, South East Asia, and Africa. Close to 100 thalassemic mutations causing defective ␤-globin gene expression and ␤-globin deficiency have been identified, but no more than 10 mutations are responsible for Ϸ90% of cases worldwide (1). Of the frequently occurring mutations, the ones that cause aberrant splicing of intron 1 of the human ␤-globin gene are predominant in South Eastern Europe, Cyprus, Lebanon (mutation IVS1-110), India, Malaysia, and Indonesia (IVS1-5). Additional splicing mutations in intron 1 (IVS1-6) as well as in intron 2 of the ␤-globin gene (IVS2-745) are also common in the above countries, while IVS2-654 is frequent among ␤-thalassemia patients in China and Thailand (1-8). All of these mutations activate aberrant splice sites and change the splicing pathway even though the correct splice sites remain potentially functional. We hypothesized that blocking of the aberrant splice sites or other sequence elements involved in splicing with antisense oligonucleotides may force the splicing machinery to reselect the correct splice sites and induce the formation of ␤-globin mRNA and polypeptide, hence restoring the gene function.Although we have previously effected correction of splicing by antisense oligonucleotides in cell-free extracts from HeLa cells (9), it was not at all clear whether the oligonucleotides delivered into the cell could enter the nucleus, hybridize to the aberrant splice sites in competition with the splicing factors, and promote the formation of the spliceosome and subsequent splicing at the correct splice site. Here we report that correct splicing was efficiently restored when phosphorothioate 2Ј-Omethyl-oligoribonucleotides were targeted to the aberrant splice sites of IVS2-654 pre-mRNA expressed in mammalian cells stably transformed with this mutated human ␤-globin gene. This is a novel approach since antisense oligonucleotides have been used mostly as sequence specific down-regulators of gene expression (10). MATERIALS AND METHODSCells. Human ␤-globin gene carrying a thalassemic mutation IVS2-654 w...

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...

In vitro splicing of human beta-globin pre-mRNA can be fully inhibited by treatment of the splicing extract with polyclonal antibodies against hnRNP core proteins prior to the addition of pre-mRNA. Inhibition of the first step in the splicing pathway, cleavage at the 5' splice site and lariat formation, requires more antibodies than inhibition of the second step, cleavage at the 3' splice site and exon ligation. The anti-hnRNP antibodies can also inhibit the splicing reaction after the formation of the active nucleoprotein splicing complex which is known to occur during the initial lag period. Thus, hnRNP core proteins appear to be present in the complex that performs pre-mRNA splicing.

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A HeLa cell line stably expressing the human beta-globin gene carrying thalassemic mutations beta(E)/IVS1-6 served as a thalassemia model for repair of aberrant splicing of beta(E)-globin pre-mRNA with antisense oligonucleotides. Treatment of beta(E)/IVS1-6 HeLa cells with a morpholino oligonucleotide targeted immediately upstream of the aberrant 5' splice site activated by the mutations resulted in an increase in the amount of correctly spliced beta(E)-globin mRNA in a dose-dependent and sequence-specific fashion. The repaired beta(E)-globin mRNA was stable and could be translated into full-length beta(E)-globin polypeptide. Application of the same oligonucleotide to erythroid progenitor cells from two beta-thalassemia/HbE patients resulted in an approximately 70% increase in correct beta(E)-globin mRNA and 36% increase in hemoglobin E. The erythroid progenitor cells represent the actual targets for the clinical application of antisense repair of defective pre-mRNAs.

Correct human ␤-globin mRNA has been restored in erythroid cells from transgenic mice carrying the human gene with ␤-globin IVS2-654 splice mutation and from thalassemia patients with the IVS2-654/␤ E genotype. This was accomplished in a dose-and time-dependent manner by free uptake of morpholino oligonucleotide antisense to the aberrant splice site at position 652 of intron 2 in ␤-globin pre-mRNA. Under optimal conditions of oligonucleotide uptake, the maximal levels of correct human ␤-globin mRNA and hemoglobin A in patients' erythroid cells were 77 and 54%, respectively. These levels of correction were equal to, if not higher than, those obtained by syringe loading of the oligonucleotide into the cells. Comparison of splicing correction results with the cellular uptake of fluorescein-labeled oligonucleotide indicated that the levels of mRNA and hemoglobin A correlate well with the nuclear localization of the oligonucleotide and the degree of erythroid differentiation of cultured cells. Similar but not as pronounced results were obtained after the oligonucleotide treatment of bone marrow cells from IVS2-654 mouse. The effectiveness of the free antisense morpholino oligonucleotide in restoration of correct splicing of IVS2-654 pre-mRNA in cultured erythropoietic cells from transgenic mice and thalassemic patients suggests the applicability of this or similar compounds in in vivo experiments and possibly in treatment of thalassemia.

A series of HeLa cell lines which stably express b-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 59 splice sites and activate a common 39 cryptic splice site upstream leading to aberrantly spliced b-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 59 splice sites affected sensitivity of both the 59 and 39 splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 59 splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.

A novel shuttle vector, pBH140, has been constructed that allows stable maintenance of large genomic inserts as human artificial episomal chromosomes (HAECs) in mammalian cells. The vector, essentially a hybrid BAC-HAEC, contains an F-based replication system as in a bacterial artificial chromosome (BAC) and the Epstein-Barr virus (EBV) latent origin of replication system, oriP, for replication in human cells. A 185-kb DNA insert containing the entire human beta-globin locus, including its locus control region (LCR), was retrofitted into this vector. The resulting beta-globin BAC-HAEC clone, p148BH, was transfected into human cells and analyzed for episomal maintenance and expression of the beta-globin gene. FISH revealed an association of the vector with different human chromosomes but no integration. The beta-globin BAC-HAECs were present at an average copy number of 11-15 per nucleus in the stably transformed human cells. After 1 year of continuous in vitro cultivation, the HAECs persisted as structurally intact 200-kb episomes. While no beta-globin transcription could be detected in the parental D98/Raji cells, correctly spliced RT-PCR products were produced at significant levels in long-term cultures of the BAC-HAEC-transduced cells. The wide availability of BAC and PAC libraries, the ease in manipulating cloned DNA in bacteria, and the episomal stability of the pBH140 vector make this system ideal for studies on gene expression and other genomic functions in human cells. The potential significance of large, functionally active episomes for gene therapy is discussed.