742. A Therapeutic miRNA for Brain Disorders

Abstract

Introduction The use of TLR9 agonists as immunomodulators is supported by preclinical and clinical studies, showing their anti-tumor effect by enhancing both cellular and humoral immune responses. So far, two different families of DNA molecules containing non-methylated CG-motifs for TLR9 activation have been established: Dumbbell-shaped dSLIM (R) molecules are protected against exonucleolytic degradation by their covalently-closed, natural phosphodiester (PO) backbone. In contrast, single-stranded, oligodeoxynucleotides (CpG-ODN) are most commonly chemicallystabilized by phosphorothioates (PTO) in their phosphate moieties. PTO modification, however, produce off-target effects in immune cell populations and have resulted in an unfavorable risk-to-benefit ratio.Methods To avoid the off-target effects of PTO-modified CpG-ODN, linear single-stranded ODN were synthezised using L-deoxyribonucleotides at their 3'-ends, which are the natural enantiomers of Ddeoxyribonucleotides. The vast majority of deoxyribose in present organisms are D-deoxyribose, thus co-evolved nucleases are blind for L-deoxyribose thereby leaving L-protected ODN intact. We selected nucleotide sequences of such L-protected, CG-motif containing, single-stranded ODN, EnanDIM (R) , for high secretion of IFN-alpha and IP-10 from human peripheral blood mononuclear cells (PBMC). In a maximum feasible dose approach in CD-1 mice EnanDIM (R) doses of 10 to 50 mg per mice were injected subcutaneously to evaluate the acute toxicity and immunomodulatory properties of EnanDIM (R) molecules in vivo.Results EnanDIM581 and EnanDIM532 were chosen since they caused high secretion of IFN-alpha and IP-10 from human PBMC and resulted in a strong activation of monocytes, NK cells and plasmacytoid dendritic cells (pDC) in vitro. Notably, both showed a distinct immune activation pattern, with the highest secretion of IFNalpha by EnanDIM581 and the strongest maturation of TLR9-bearing pDC by EnanDIM532. EnanDIM744, comprising EnanDIM581 with additional 5'-end L-nucleotide protection and exhibiting an immune activation pattern similar to EnanDIM581, was selected as third EnanDIM (R) for in vivo studies. In the maximum feasible dose approach, safety assessments were performed throughout the study period and no mortality, clinical signs and body weight changes were observed, despite the fact that extremely high doses of app. 300 to 1700 mg/kg were used. A gross necropsy consisting of a macroscopic organ evaluation at day 15 revealed also no toxicity. Regarding immune activation, increased levels of IP-10 in serum were observed 24 hours after injection but not after 15 days confirming that L-nucleotides in EnanDIM (R) do not change the kinetic profile known from other DNA-based TLR9 agonists.Conclusions EnanDIM (R) , a new family of TLR9 agonists with conformation-mediated nuclease-resistance, broadly activates the immune system in vitro. Maximal feasible doses of EnanDIM (R) resulted in no signs of toxicity and confirmed immunomodulatory effects in vivo. Therefore En...