Role of endogenous endonucleases and tissue site in transfection and CpG-mediated immune activation after naked DNA injection

DNA degradation is a fundamental problem for any gene therapy or genetic im munization approach, since destruction of incoming genes translates into lo ss of gene expression. To characterize the biology of DNA degradation after naked DNA injection, the location and levels of tissue nucleases were asse ssed. Extracts from the serum, kidney, and liver of mice had high levels of calcium-dependent endonuclease activity. High levels of acidic endonucleas e activity were identified in the spleen, liver, kidney, and skin with litt le activity in skeletal or cardiac muscle. Relatively little exonuclease ac tivity was observed in any tissue. The presence of endonucleases in the ski n and muscle mediated degradation of 99% of naked DNA within 90 min of inje ction. This degradation most likely occurred in the extracellular space ups tream of other cellular events. Despite this massive destruction, gross tis sue nuclease levels did not determine skin-to-muscle transfection efficienc y, or site-to-site transfection efficiency in the skin. While gross tissue nuclease levels do not appear to determine differences in transfection effi ciency, the presence of robust tissue nuclease activity still necessitates that massive amounts of DNA be used to overcome the loss of 99% of expressi ble DNA. In addition to destroying genes, the nucleases may play a second r ole in genetic immunization by converting large plasmids into small oligonu cleotides that can be taken up more easily by immune cells to stimulate CpG -dependent Th1 immune responses. For genetic immunization, vaccine outcome may depend on striking the right balance of nuclease effects to allow survi val of sufficient DNA to express the antigen, while concomitantly generatin g sufficient amounts of immunostimulatory DNA fragments to drive Th1 booste r effects. For gene therapy, all nuclease effects would appear to be negati ve, since these enzymes destroy gene expression while also stimulating cell ular immune responses against transgene-modified host cells.