Third strand-mediated psoralen-induced correction of the sickle cell mutation on a plasmid transfected into COS-7 cells.
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 beta-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 re-isolation 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.
Varganov
Y
Y
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
Amosova
O
O
Fresco
J R
JR
D000755:Anemia, Sickle Cell / Q000097:blood / Q000628:therapy*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D018736:DNA Adducts; D004260:DNA Repair; D011564:Furocoumarins / Q000235:genetics*; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005914:Globins / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000494:pharmacology*; D017354:Point Mutation; D014162:Transfection / Q000379:methods*
D018736:DNA Adducts; D011564:Furocoumarins; D005914:Globins