Third strand-mediated psoralen-induced correction of the sickle cell mutation on a plasmid transfected into COS-7 cells

Abstract

A significant level of correction of the mutation responsible for sickle cell anemia has been achieved in monkey COS-7 cells on a plasmid containing a b-globin gene fragment. The plasmid was treated in vitro with a nucleic acid 'third strand' bearing a terminal photoreactive psoralen moiety that binds immediately adjacent to the mutant base pair. Following covalent attachment of the psoralen by monoadduct or diadduct formation to the mutant T-residue on the coding strand, the treated plasmid was transfected into the cells, which were then incubated for 48 h to allow the cellular DNA repair mechanisms to remove the photoadducts. Upon reisolation and amplification of the transfected plasmid, sickle cell mutation correction, as determined by sequence analysis of both complementary strands, was established in a full 1%. This result encourages extension of the approach to correct the mutation directly on the chromosome.

A radiation target method for size determination of supercoiled plasmid DNA

“…However, it is anticipated that DNA repair in COS-7 cells could resuscitate radiation damage in GFP plasmids, resulting in increased GFP production and diminished target size. The extent of COS-7 repair of photoadducts of plasmids was determined to be 1% [15]. If a similar degree of repair occurred in the GFP system used here, the effect would be too small to significantly alter the estimated target size.…”

Oligo/Polynucleotide-Based Gene Modification: Strategies and Therapeutic Potential

“…One approach to overcome this limitation has been to develop TFOs that have modified nucleotides such as locked nucleic acid or peptide nucleic acid residues to increase the affinity of the TFO for specific target sequences (Simon et al, 2008). Another approach has been linkage of the TFO to psoralens that bind to DNA upon UV light irradiation and both anchor the TFO to its target site and also stimulate DNA repair Luo et al, 2000;Vasquez et al, 2001a;Broitman et al, 2003;Varganov et al, 2007;Liu et al, 2009b). Although the use of psoralens might be useful in a laboratory setting, it may not meet safety standards because of its ability to enhance DNA damage and potentiate carcinogenesis (Pathak et al, 1959;Pathak and Joshi, 1984;Tamaro et al, 1986).…”

Induced cross-linking reactions to target genes using modified oligonucleotides

“…[23][24][25] Psoralen-modified TFOs have also been used for photoinduced directed mutagenesis of specific sites in duplex DNA and targeted gene knock out in cells. [26][27][28] Thus, psoralen-conjugated ONs have been widely applied to several in vitro and in vivo studies related to antisense and antigene methods. Recently, psoralen has been conjugated at the adenosine 2¢-O-position through alkoxy methylene linkers 29 and directly at the sugar 1¢-position 30 to improve its flexibility when conjugated at the ON 5¢ end (Fig.2).…”