UVB-induced cyclobutane pyrimidine dimers (CPDs) are considered to be the main cause of acute sunburn and epidermal carcinogenesis. In humans, these lesions are repaired by nucleotide excision repair, but marsupials and lower organisms present photolyase enzyme which rapidly removes CPDs in a visible light-dependent process (photoreactivation). Previously we have established a method in which human keratinocyte cell lines are transfected of with pseudouridine-modified mRNA encoding CPD-specific photolyase, and this approach was suitable to avoid UVB-induced apoptosis. 
According to clinical experiences, there is a need to diminish the deleterious effects of sunburn when the first symptoms UVB injuries appear. Our aim was to determine the therapeutic window following UVB exposure when apoptosis of keratinocytes is still preventable.
Normal human epidermal keratinocytes were transfected with lipofectamine-complexed, in vitro transcribed mRNA encoding CPD-specific photolyase. Cells were irradiated with 60 mJ/cm2 UVB. After UVB, at 0, 6, 8 or 12 hours cells were either exposed to visible light (photoreactivation) or kept in dark. Cell viability was measured by dual staining with Annexin V and propidium iodide followed by flow cytometry.  The relative amount of intracellular CPDs was measured by CPD-specific ELISA.
Photolyase-mediated CPD-removal restored cell viability close to the baseline conditions upto 6 hours after UVB treatment. The effect of CPD removal on keratinocyte survival began to diminish at 8 hours after the UVB damage. In mRNA-transfected and photoreactivated cells, 80% of CPDs were removed at 6 hours post-irradiation. 8 to 12 hours after UVB 40-60% of the CPDs were eliminated. 
Our results suggest that UV-induced keratinocyte apoptosis can be prevented within 6-8 hours after the UVB injury by the elimination of CPD photolesions. At later time point the effect of CPD removal on cell viability is less effective.

Time-dependence of UVB induced cellular mechanisms in cultured human keratinocytes