Gene therapy to facilitate healing of damaged adult-mammalian tissues that have very limited regenerative potential is significantly limited by our inability to target transgene expression to the edge of the defect, in a timely manner, while limiting transduction of the uninjured-healthy tissue. As an example, we have been working towards a gene therapy for articular and meniscal cartilage defects, which are extremely prevalent (>80% of people >50yrs) due to simple sports related injuries, and are known to be the leading cause of osteoarthritis and the need for joint replacement surgery20,21
. Based on the biology of cartilage repair in vertebrae-regenerate species, which is highlighted by precise temporal-spatial gene expression at the edge of the defect immediately following injury to establish a morphogenic gradient and blastema formation22,23
, we envisioned a gene therapy performed during standard arthroscopy with potential to achieve this requisite gene expression profile. In this surgery, mechanical debridement is performed to remove the fragmented cartilage and stabilize the margins of the defect. This surgical procedure re-establishes the opportunity to initiate formation of new cartilage via creation of morphogenic gradient of chondrogenic factors (i.e. GDF-5) via site-specific gene therapy. LAGT represents one approach that is compatible with standard arthroscopy, and we have previously demonstrated safe and effective LAGT with 325nm UVA in a rabbit articular cartilage defect model16
. However, several significant improvements are needed to maximize the potential of LAGT therapy. These improvements require greater understanding of action spectra at the UVB-UVA boundary, which is the focus of the current study.
Biological pathways related to UV damage have been shown to be wavelength dependent 24
. The two basic mechanisms for UV induced cellular DNA damage are direct damage and indirect damage through alternative intracellular photosensitizing molecules. For irradiation close to the UVC range (250nm to 300nm), the dominant form of DNA damage is direct excitation that can be traced through pyrimidine dimerization. With longer wavelengths approaching the UVA range (>300nm), damage tends to occur indirectly through the excitation of intracellular chromophores. These chromophores can either be stimulated to damage DNA directly via generation of singlet oxygen species (type I photoreaction from O2
radicals), or via superoxide/hydroxyl radical formation through Fenton reaction (type II photoreaction)25
. This oxidative DNA damage is primarily repaired through the action of DNA glycosylases, AP endonucleases, and general nucleotide excision repair mechanisms including methylguanine DNA methyl transferase, MGMT activation26
. Interestingly, these ROS including superoxide, singlet oxide, and peroxide radicals have been shown to induce activation of ATM kinase and p53 transcription, which are known to play a major role in DNA repair processes without causing DNA damage directly.27
Thus, UVA-induced type I and type II photoreactions provide therapeutic potential if a significant window between cytotoxicity-genotoxicty and efficacy exists, and efforts to identify the empirical windows for various action spectra at the UVB-UVA boundary are warranted.
Consistent with the known direct DNA damage induced by short wavelength UVB, here we demonstrate that 288nm LAGT and cytotoxicity is directly associated with high levels of pyrimidine dimers without ROS generation. In contrast, 311nm effects are associated with significant ROS levels without pyrimidine dimer formation. Furthermore, 311nm failed to induce macroscopic DNA damage as assessed by a Comet assay, demonstrating that these exposures do not induce type I or type II DNA damage either. Given that the empirical cutoff of UVB-induced LAGT independent of DNA damage must fall within the 288nm and 311nm systems, we interpolated this cutoff to be 299±3nm as follows. First, there is a rapid drop off in DNA absorbance at UV wavelengths focusing at 299nm, leading to the complete absence of DNA absorbance above 302nm.28
Since this rapid drop of DNA absorbance coincides with the 299nm border wavelength between the 288nm irradiation system that produced high DNA damage with low ROS, and the 311nm irradiation system that produced low DNA damage with high ROS, and since the irradiation peaks centered at 288nm and 311nm do not overlap, we conclude that the empirical boundary of the action spectra of DNA damage dependent vs. DNA damage independent LAGT to be at 299±3nm.
A major disappointment with the 325nm HeCd laser system we used previously is its modest LAGT effects (8-fold)16
. This prompted us to evaluate shorter UVA wavelengths more closely. Remarkably, both 311nm and 320nm UVA achieved 100-fold greater LAGT effects at their peak fluences. Moreover, the 4-fold increased LAGT effect observed in C3H vs. 293 cells is consistent with a greater net activation of host DNA polymerases in the less transfectable cell line. As this effect would be even greater in nondividing cells, the peak LAGT effect in articular chondrocytes could be several thousand fold, and warrants future investigation.
As with most experimental research, our studies produced several results whose explanation is beyond the scope of our current understanding. Most notably are the sharp loss of the LAGT effect at doses 3-fold and 1.5-fold above the peak for 311nm and 320nm UV respectively. As this was not due to a commensurate increase in cytotoxicity, this biphasic response to short wavelength UVA suggests that there may be negative cellular feedback mechanisms that are triggered prior to cell death. Our other enigmatic observation that begs further investigation is the observation that greater cytotoxicity and DNA damage was observed at 320nm compared to 311nm and 330nm. This suggests that a yet to be identified cellular chromophore with a narrow excitation spectrum between 316nm and 325nm exists.
Collectively, our results clearly indicated that the 311nm UVA system is the most ideal light source for LAGT that we have tested to date, based on its ability to generate high levels of ROS with minimal DNA damage, and cause more than a 800-fold increase in transgene expression in C3H cells. Moreover, we find this 311nm system to be a clinically relevant option, due to the 311nm peak in the emission spectrum of Hg lamps, which are an inexpensive and widely used light source. Thus, future studies with this system to assess LAGT therapy in vivo are warranted to assess the true potential of this technology.