Abstract 552: Sustained Inhibition Of High Mobility Box 1 By Antisense Oligonucleotide

Abstract

Background: High-mobility group box 1 (HMGB1), a highly conserved nonhistone DNA-binding nuclear protein, may contribute to vascular diseases including atherosclerosis, pulmonary hypertension, and abdominal aortic aneurysm. Since whole-body genetic deletion of HMGB1 is embryonic lethal, pharmacological approaches, such as neutralizing antibodies and functional inhibitors, have been used to manipulate HMGB1 in mice. However, it remains desirable to genetically manipulate HMGB1 for further understanding its role. In the current study, we assessed the efficacy of a novel antisense oligonucleotides (ASOs) approach to deplete HMGB1 in mice. Methods and Results: First, abundance of HMGB1 mRNA was assessed by qPCR in major organs and tissues of male C57BL/6J mice (N=6). Hmgb1 mRNA was expressed ubiquitously, but it was most highly abundant in the lung, while least abundant in the testes. Next, we examined the efficacy of ASOs to reduce HMGB1 protein abundance at selected intervals. Either ASOs (25 mg/kg/day) or phosphate-buffered saline (PBS) were injected intraperitoneally into male C57BL/6J mice (8-10-week-old) at day 0 and 3 in the initial week and then once a week during the remainder of the study. Mice were terminated at either 2, 6, or 12 weeks after the initial injection of ASOs (N=5/group). Subsequently, mRNA and protein abundance of HMGB1 were determined in the lungs. Since previous studies have shown that systemic administration of ASOs mainly targets kidney and liver, HMGB1 mRNA or protein abundance was also examined in these organs. We also assessed HMGB1 protein abundance in the heart. In lungs, kidney, and liver, ASO administration resulted in a consistent >90% decrease of Hmgb1 mRNA abundance at 2 weeks of ASO injection (P>0.001). The decreased Hmbg1 mRNA abundance was also consistent at both weeks 6 and 12 (P>0.001). ASOs decreased HMGB1 protein abundance significantly at 6- and 12-week time intervals. Heart tissue also indicated a consistent Hmgb1 mRNA decrease in response to ASO administration. Conclusion: ASO approach decreased HMGB1 at mRNA and protein abundance for 12 weeks in the liver, kidney, lungs, and heart. This mode may provide more clear insights into understanding the biological functions of HMGB1.