Targeted Gene Correction Mediated By Oligonucleotide And The Application Of This Strategy In β-Thalassemia Gene Therapy Research

Gene therapy is a kind of new treatment for genetic disorders, cancer and infectious diseases by using genetic materials such as DNA or RNA to cure the diseases. Targeted gene therapy, which includes tissue-specific gene transfer, site-specific integration and therapeutic gene expression in a regulated way, is an ideal strategy for clinical trials. In recent years, many investigators have devoted themselves in this field and got some new proceedings. DNA repair approach has focused on gene targeting or gene replacement whereby the defective gene is removed and a normal gene is inserted at the same position in the chromosome and so it maintains the corrected gene's expression in the natural context. Homologous recombination can direct the replacement process and the efficiency can be improved in mouse embryonic stem cells (ES) by the selective system. However, the extremely low efficiency of this process (10^-6—10^-5) and the unsuitable selective system to mammalian somatic cells impede its application in gene therapy research.Chimeraplasty is a new kind of technology, which is developed with the findings that RNA can facilitate the homologous pairing during gene transcription, so a RNA/DNA chimeric oligonucleotide named chimera was designed and synthesized. When the chimera was transfected into cells it can find the homologous region in the genomic DNA and pair with its counterpart efficiently and then a mismatch base emerge in the pairing region. This mismatch pair can be recognized by the mismatch repair machinery of the cells to induce a single mutation in the target site. The targeted nucleotide conversion efficiency is much higher than the homologous recombination (10³-10⁶) and it was found to be effective both in vitro and in vivo.Now a cell-free extract system is developed to elucidate the reactions that enable the correction and several significant observations demonstrated that the all-DNA strand of the chimera directs the initial correction once binding to its complementary target strand; the...