Adenovirus mediated transduction of the human DNA polymerase eta cDNA

Predominant role of DNA polymerase eta and p53-dependent translesion synthesis in the survival of ultraviolet-irradiated human cells

“…Moreover, NER-proficient XP-V cells were unable to activate the protective mechanism of a split-dose protocol, meaning that pol eta has a more relevant role in cell survival protection in a split-dose context than does DNA damage removal due to NER induction (XPC and DDB2). In contrast, the overexpression of pol eta alone by adenoviral transduction was not sufficient to support any increase in cell survival or DNA replication in NER-deficient XP-A SV40-transformed fibroblasts (38). Such result may indicate that arrested transcription complexes, generated in TCR-deficient cells, constitute a more problematic burden that can trigger cell death despite the fact that pol eta could be improving DNA replication at DNA lesions which are not encountered by transcription bubbles.…”

On the Search for Skin Gene Therapy Strategies of Xeroderma Pigmentosum Disease

“…XP cell populations infected with the respective virus were demonstrated to express high levels of the transduced gene product. DNA repair levels were recovered, and resistance to UVirradiation of transduced-XP cells was reverted to levels comparable to wild type cells, thus demonstrating functional correction of the XP phenotype [Muotri et al, 2002;Armelini et al, 2005;Lima-Bessa et al, 2006]. Moreover, although adenoviral transduction is usually just transient, sustainable XPA protein expression and parallel increased UVirradiation resistance were obtained even two months after cells had been infected with AdyXPA, the adenoviral vector carrying the human XPA cDNA [Muotri et al, 2002].…”

Recombinant Viral Vectors for Investigating DNA Damage Responses and Gene Therapy of Xeroderma Pigmentosum

“…It is well known that UV radiation promotes DNA elongation delay as a result of replication blockage by UV photolesions (Cleaver et al, 1983), which can be easily seen by running pulse-chase experiments in alkaline sucrose gradients. Using this approach, it has been possible to show that XP-V transduced cells were able to elongate nascent DNA on UVdamaged DNA templates as efficiently as wild type cells (Lima-Bessa et al, 2006), once again demonstrating the great potential of recombinant adenoviruses in the transduction and expression of functional proteins. One interesting conclusion came from the observation that even though XPA, XPC and XPD genes were over-expressed in all transduced cell lines when compared to NER proficient cells, this had no impact in the UV-resistance or NER capability, suggesting that neither of these proteins is limiting for NER in human cells.…”

“…Experiments using first generation recombinant adenoviral vectors have been successfully employed in the transduction of both SV40-transformed and primary fibroblasts derived from XP-A, XP-C, XP-D and XP-V patients (Armelini et al, 2007). The expression of the respective functional proteins in all transduced defective cell populations was significantly increased, reaching levels even higher than seen for wild type cells (Armelini et al, 2005;Lima-Bessa et al, 2006;Muotri et al, 2002). Moreover, different phenotypical analyses, including cell cycle, apoptosis and cell survival assays, have been carried out, all indicating that the protein expression mediated by the recombinant adenoviruses was clearly accompanied by the recovery of the DNA repair ability and increased resistance to UV radiation, thereby demonstrating functional correction of the XP phenotype.…”