| Disease phenotypes that can be traced to point mutations are amenable to gene therapy by direct repair of the disease-causing allele on the chromosome. Here, the Sickle Cell allele is used as a demonstration case of gene repair using third strand-directed DNA damage with the photomutagen psoralen. To this end, an in vivo dual reporter system was designed and its inputs tested.;A dual reporter construct was designed and integrated into the genome of mouse embryonic stem cells (mESCs). This construct consists of a translational fusion of monomeric red fluorescent protein (mRFP) with enhanced green fluorescent protein (EGFP), where mRFP and EGFP are separated by the Sickle Cell allele target sequence that overlaps with an opal stop codon. Thus, EGFP expression requires third strand-mediated psoralen photoadduct damage to be mutagenically repaired. As the dual reporter was integrated as a single copy per cell, the expression of mRFP not only indicates the potential for EGFP expression, but also monitors for large deletion events that arise from psoralen photoadduct repair.;Prior to using this dual reporter to monitor gene repair, however, the inputs to the system were examined. First, the lipofection of a fluorescently labeled, psoralen-modified deoxyoligonucleotide was monitored using flow cytometry of isolated nuclei. These studies established parameters for quantitative and efficient lipofection, and demonstrated that such an oligonucleotide reaches the nucleus and persists there for at least two cell doublings. Second, since repair of the stop codon in the dual reporter sequence requires mutagenic repair of a psoralen photoadduct, detection of such a DNA lesion was sought using single-strand ligation PCR (sslig-PCR). No photoadducts were detected under the studied conditions. The detection of photoadducts was not hindered by cellular repair of such lesions, as mESCs do not repair third strand-directed psoralen photoadducts on a plasmid within 24 hours of introduction into the cell. Copy number of the third strand target and the transcriptional activity of the reporter translational fusion gene did not influence psoralen photoadduct yield either. Since recent research indicates that mESCs do not efficiently repair DNA interstrand crosslinks (such as psoralen photoadducts), and instead undergo apoptosis, human embryonic stem cells (hESCs) were also examined for their photoadduct-forming potential. In the initial trials performed here, no in vivo third strand-directed psoralen photoadducts were detected in hESCs either. Work with hESCs was far from exhaustive, and several variables should be explored further.;Should creation and mutagenic repair of third strand-directed psoralen photoadduct lesions be observed, corrected stem cells can be selected and expanded. Such corrected cells can be partially differentiated into hematopoietic progenitor cells, and these progenitor cells used for therapeutic purposes. In a clinical setting, patient-specific induced pluripotent stem cells (iPS cells) would be used, so that ex vivo corrected and differentiated cells could be reintroduced into the patient without fear of graft rejection. While much work remains to be done before this therapy realizes its eventual clinical use, advances have been made here in understanding its mechanism at the molecular level. Results obtained here also demonstrate the need for further experiments to elucidate the exact rules of psoralen intercalation and in vivo photoadduct formation. |
