523. Deoxyribonuclease I and Endonuclease G Are Host Defense Enzymes Responsible for Inactivation of Foreign DNA in Kidney Tubular Epithelial Cells
Authors: Timea Buzder, Xiaoyan Yin, Xiaoying Wang, Gaspar Banfalvi, Alexei G. Basnakian
Journal: Molecular Therapy (2006)

Gene delivery to kidney cells is essential for the development of gene therapy of nephropathy. Maintaining DNA stability is crucial for successful gene delivery. It is known that DNases play a major role in degrading "foreign" DNA before and after it is consumed by the host cell, however what these DNases are and whether inactivation of these enzymes can improve gene delivery have not been examined. We have recently shown that two pro-apoptotic endonucleases, deoxyribonuclease I (DNase I) and endonuclease G (EndoG) provide more than 90% of the total DNase/endonuclease activity in the mouse kidney. Since primary cells are notoriously resistant to transfections, we studied DNA stability during gene delivery to primary renal tubular epithelial cells isolated from mice. When compared to immortalized mouse tubular epithelial (TKPTS) cells, primary cells had a higher total endonuclease activity (25±2 units/μg protein in primary cells vs. 7±3 units/μg protein in TKPTS cells, n=3-6, p<0.001) as measured using the pBR322 plasmid incision assay in the presence of Ca2+ and Mg2+ ions, which are cofactors of most cellular DNases. Endonuclease activity was present both in total cellular protein extracts (DNase I, EndoG and other DNases) and in culture media (mainly DNase I). Pre-treatment with Lipofectamine did not protect plasmid DNA against in vitro digestion by endonucleases. We next used primary tubular epithelial cells isolated from DNase I or EndoG knockout mice to examine the role of these endonucleases in the degradation of plasmid DNA during uptake by renal tubular epithelial cells. As determined by real-timePCR, Western blotting and activity assays, homozygous DNase I knockout (CD-1) mice had 99-100% of DNase I inactivated, while heterozygous EndoG knockout (129xB6) mice had 60-70% of EndoG inactivated. The decrease of Ca/Mg-dependent (DNase I-like) endonuclease activity in DNase I knockout cells was associated with Mn-dependent endonuclease (EndoG) becoming the major endonuclease, indicating that EndoG is the second most active endonuclease in primary kidney cells. To determine the effects of endonuclease inactivation on DNA stability, cells were transiently transfected with Lipofectamine-packed pECFP-N1 plasmid expressing cyan fluorescent protein. The efficiency of transfection was determined by quantitative fluorescent microscopy 48 hours post-transfection. DNase I and EndoG knockout cells showed a significantly higher rate of transfection than wild-type cells (22±3% vs. 13±3% transfected cells, n=3, p<0.03; 19±4% vs. 7±2% transfected cells, n=3, p<0.001, respectively). These data demonstrate the important role of DNase I and EndoG in host cell defense against gene delivery in primary tubular epithelial cells. Future studies may determine whether these two endonucleases cooperate in destroying "foreign" DNA. Specific inhibition of these endonucleases may provide new modalities to improve DNA stability during gene delivery. Gene delivery to kidney cells is essential for the development of gene therapy of nephropathy. Maintaining DNA stability is crucial for successful gene delivery. It is known that DNases play a major role in degrading "foreign" DNA before and after it is consumed by the host cell, however what these DNases are and whether inactivation of these enzymes can improve gene delivery have not been examined. We have recently shown that two pro-apoptotic endonucleases, deoxyribonuclease I (DNase I) and endonuclease G (EndoG) provide more than 90% of the total DNase/endonuclease activity in the mouse kidney. Since primary cells are notoriously resistant to transfections, we studied DNA stability during gene delivery to primary renal tubular epithelial cells isolated from mice. When compared to immortalized mouse tubular epithelial (TKPTS) cells, primary cells had a higher total endonuclease activity (25±2 units/μg protein in primary cells vs. 7±3 units/μg protein in TKPTS cells, n=3-6, p<0.001) as measured using the pBR322 plasmid incision assay in the presence of Ca2+ and Mg2+ ions, which are cofactors of most cellular DNases. Endonuclease activity was present both in total cellular protein extracts (DNase I, EndoG and other DNases) and in culture media (mainly DNase I). Pre-treatment with Lipofectamine did not protect plasmid DNA against in vitro digestion by endonucleases. We next used primary tubular epithelial cells isolated from DNase I or EndoG knockout mice to examine the role of these endonucleases in the degradation of plasmid DNA during uptake by renal tubular epithelial cells. As determined by real-timePCR, Western blotting and activity assays, homozygous DNase I knockout (CD-1) mice had 99-100% of DNase I inactivated, while heterozygous EndoG knockout (129xB6) mice had 60-70% of EndoG inactivated. The decrease of Ca/Mg-dependent (DNase I-like) endonuclease activity in DNase I knockout cells was associated with Mn-dependent endonuclease (EndoG) becoming the major endonuclease, indicating that EndoG is the second most active endonuclease in primary kidney cells. To determine the effects of endonuclease inactivation on DNA stability, cells were transiently transfected with Lipofectamine-packed pECFP-N1 plasmid expressing cyan fluorescent protein. The efficiency of transfection was determined by quantitative fluorescent microscopy 48 hours post-transfection. DNase I and EndoG knockout cells showed a significantly higher rate of transfection than wild-type cells (22±3% vs. 13±3% transfected cells, n=3, p<0.03; 19±4% vs. 7±2% transfected cells, n=3, p<0.001, respectively). These data demonstrate the important role of DNase I and EndoG in host cell defense against gene delivery in primary tubular epithelial cells. Future studies may determine whether these two endonucleases cooperate in destroying "foreign" DNA. Specific inhibition of these endonucleases may provide new modalities to improve DNA stability during gene delivery.