SUNY at Albany
June 19-23, 2001
Triplex-Based Repair Of The Sickle Cell Mutation
The ability to target photochemical adducts to specific genomic DNA sequences provides a potential basis for a mode of gene therapy. We have exploited triplex-mediated psoralen modification of T residues for correcting the mutation that underlies Sickle Cell Anemia, which results from A_T transversion in the beta-globin gene located on chromosome 11. Correction of this mutation at the somatic level can be triggered via recognition and excision of the psoralen adduct by the cellular DNA repair system. A high degree of such directed psoralen modification can ultimately result in replacement of the mutant base pair by a wild type one with sufficient efficiency to restore satisfactory levels of normal beta-globin synthesis. A_T transvertion mutation occurs immediately upstream of 24 bp purine-rich sequence that consists of two adjacent regions located on opposite strands, the longer downstream one containing four inverted base pairs. This difficult target required the design of a bifunctional third strand that couples third strand binding to target DNA duplex with strand invading element. Specific third-strand binding and photoadduct formation, the first step towards the desired goal, has already been demonstrated in vitro using a 40 bp linear DNA model of the mutant sequence of the Sickle beta-globin gene. This binding was successfully extended to a plasmid containing the beta-globin target sequence. Third strand binding has been studied and optimized for physiological conditions by varying triplex motifs and using modified base analogs in the third strand. Efficiency and specificity of psoralen photomodification was analyzed for various irradiation conditions and different psoralen linker sizes. The experiments are now in progress on enzymatic repair of the photomodified plasmid both in vitro and in vivo. This technology could then be exploited in human hematopoietic stem cells ex vivo.
Olga Amosova, Nina G. Dolinnaya, Steven L. Broitman, and Jacques R.Fresco
Department of Molecular Biology, Princeton University,