Therapeutic single-stranded oligonucleotides in gene repair and cancer.

摘要

The therapeutic response of human cells to single-stranded oligonucleotides is the central focus of the projects documented in this dissertation. Currently, activation of DNA repair, recombination and replication pathways are the most effective means to elevate the frequency of oligonucleotide-directed gene repair to the level needed for therapeutic benefit. Higher frequencies of targeted gene repair in human cells can be achieved by inducing indirect or direct DSBs. MMS is an alkylating agent capable of inducing DNA lesions and subsequent SSBs and DSBs, activating the NHEJ and HR DNA repair pathways. In mammalian cells, the conversion frequency of a mutant base pair to wild-type in an integrated enhanced green fluorescent protein gene is increased by pretreatment with MMS, correlating to the level of DSB induced by MMS treatment. Bleomycin is an anticancer drug capable of efficient induction of direct double strand breaks in mammalian genomic DNA. Treatment with bleomycin results in a comparable, dose-dependent increase for gene repair in DLD-1 cells, and it is only necessary to administer this treatment at the time of oligonucleotide transfection. These data suggest that both MMS- and bleomycin-induced DNA damage elicits a cellular response that stimulates gene repair in mammalian cells and provides a direct method for elevating levels of gene correction. In addition, it is advantageous to develop methods to increase the activity of DNA repair proteins without causing the harmful effects of DNA damage. Selenomethionine, an antioxidant amino acid, can induce a DNA repair response and enhance repair-complex formation by modulating activity of the p53 protein. The data presented here illustrate the crucial role that p53 plays in the targeted nucleotide exchange reaction and serves to advance a more complete understanding of the mechanism of gene repair.; Based on an observation carried over from the work in oligonucleotide-directed gene repair, quadruplex oligonucleotides have been developed here for use as an anti-proliferation aptamer in tumorigenic cells. The specialized oligonucleotide contains a sequence of only guanosine and can specifically induce apoptosis in the malignant esophageal cell line, OE19, both in cell culture and in a NODscid mouse model. The reaction is dose dependent and appears to rely on the capacity of the G-rich oligonucleotide to adopt a very stable G-quartet conformation. Importantly, nonmalignant esophageal cells or normal human lung fibroblasts are not impeded in their cell cycle progression when incubated with the G-rich oligonucleotides. These data suggest that a selective killing of esophageal tumor cells is directed by G-rich oligonucleotides. The therapeutic potentials for both gene repair and G-quadruplex DNA oligonucleotides are substantial, considering the numerous advantages that small nucleotides have over other vectors and drug molecules.