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Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing betathalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent proteinbeta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells.beta-thalassemia ͉ gene correction ͉ triplex-forming oligonucleotides ͉ gene targeting M utations in the beta-globin gene that affect any stage in beta-globin biogenesis can cause beta-thalassemia. Identified mutations include single base pair changes that lead to frameshift mutations or changes in canonical sequences that affect mRNA stability and processing (1). As monogenic disorders, betathalassemia and sickle cell anemia have attracted substantial efforts directed at gene therapy by gene replacement, and there has been ongoing progress in this regard. In one approach specific to the thalassemias in which the genetic defect affects mRNA splicing, antisense oligonucleotides have been used to manipulate the splice site choice in beta-globin premRNA to prevent aberrant splicing. Restoration of proper beta-globin splicing has been demonstrated in human erythroid cells derived from beta-thalassemic patients, and in transgenic mouse models containing splicing mutations in the beta-globin gene (2, 3).In this study, we use an antigene approach to correct a thalassemia-causing splice-site mutation at the level of chromosomal DNA in cultured cells, generating heritable, site-specific modification of the beta-globin gene. We have used peptide nucleic acids (PNAs), a class of triplex-forming molecules shown to be effective at provoking recombination and repair at chromosomal sites near PNA binding sites (4). PNAs contain standard nucleobases linked to a peptide-like backbone, and their advantages include resistance to nucl...

Early diagnosis of esophageal perforations following LACA may allow temporary esophageal stenting with successful esophageal healing. Prompt chest CT scans with oral and i.v. contrast should be considered in any patient with sub-sternal chest pain or dysphagia following LACA.

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