Validation of decoy oligodeoxynucleotide drug candidates in experimental heart failure

Coordinator:		Prof. Dr. Markus Hecker
Institution:		Institute of Physiology and Pathophysiology, Heidelberg University
Homepage:		www.medizinische-fakultaet-hd.uni-heidelberg.de

Subproject 2 also had the task to establish an in vivo model for the proof-of-efficacy of the various decoy ODN in the mouse. For this purpose, left ventricular hypertrophy was induced in C57BL/6 mice by the continuous administration of angiotensin II via implanted osmotic mini-pumps. To allow for a better assessment of the efficacy of a therapeutic treatment with the decoy ODNs in mice, the calsarcin-1 gene was introduced using a gene therapy approach with adeno-associated viruses (AAV9) with the aim to specifically overexpress it in cardiomyocytes. To enhance the angiotensin II-induced left ventricular hypertrophy and to increase the effectiveness of the gene therapy approach at the same time, the same study was conducted with calsarcin-1 knockout mice. However, despite clear effects of the calsarcin-1 gene transfer on different parameters of cardiac function as well as on the program of pro-hyperthrophic gene expression and fibrosis in the myocardium, no significant expression of the transgene could be detected in the transfected animals. In a final series of experiments still open questions in this context are currently investigated.    Subproject 2 successfully provided the aimed-for proof-of-concept for the optimized decoy ODNs directed against the three target transcription factors AP-1, NFATc1 to 4 and NF-IL6 in neonatal rat fibroblasts and cardiomyocytes. Here, the expression of a set of target genes significantly regulated by these transcription factors was suppressed by at least 60%, in some cases even over 90%. Due to the surprisingly poor transport of the decoy ODNs through the membrane of the rat cells, the nucleic acids were initially transfected into cells using common reagents. Under these conditions surprisingly low concentrations of the decoy ODNs were sufficient for a generally very robust biological effect. However, since transfection reagents are not applicable in humans, the microbubble technique primarily established in vivo was adapted so that it would reproducibly function with non-adherent as well as adherent cells in vitro.

Subproject 2 also had the task to establish an in vivo model for the proof-of-efficacy of the various decoy ODN in the mouse. For this purpose, left ventricular hypertrophy was induced in C57BL/6 mice by the continuous administration of angiotensin II via implanted osmotic mini-pumps. To allow for a better assessment of the efficacy of a therapeutic treatment with the decoy ODNs in mice, the calsarcin-1 gene was introduced using a gene therapy approach with adeno-associated viruses (AAV9) with the aim to specifically overexpress it in cardiomyocytes. To enhance the angiotensin II-induced left ventricular hypertrophy and to increase the effectiveness of the gene therapy approach at the same time, the same study was conducted with calsarcin-1 knockout mice. However, despite clear effects of the calsarcin-1 gene transfer on different parameters of cardiac function as well as on the program of pro-hyperthrophic gene expression and fibrosis in the myocardium, no significant expression of the transgene could be detected in the transfected animals. In a final series of experiments still open questions in this context are currently investigated.