Absence of unspecific innate immune cell activation by GATA-3-specific DNAzymes.

DNAzymes of the 10-23 family represent an important class of antisense molecules with implications for therapeutic treatment of diseases. These molecules are single-stranded oligodeoxynucleotides combining the high specificity of oligonucleotide base pairing with an inherent RNA-cleaving enzymatic activity. However, like other oligonucleotide-based molecules these substances might exert so-called off-target effects, which have not been investigated so far for this molecule class. Therefore, the present study investigates putative off-target effects of DNAzymes on innate immune mechanisms using GATA-3-specific DNAzymes that have recently been developed as novel therapeutic approach for the treatment of allergic diseases including allergic asthma. The conserved catalytic domain of 10-23 DNAzymes contains a CpG motif that may stimulate innate immune cells via Toll-like receptor 9 (TLR-9). Therefore, potential TLR-9-mediated as well as TLR-9 independent cell activation was investigated using TLR-9-transfected HEK293 cells, macrophage cell lines and primary innate immune cells. Furthermore, putative effects of GATA-3-specific DNAzymes on the activation of neutrophil granulocytes and degranulation of mast cells/basophils were analyzed. In summary, no innate immune cell-stimulating activities of the tested DNAzymes were observed in any of the systems. Consequently, use of GATA-3-specific DNAzymes may represent a novel and highly specific approach for the treatment of allergic diseases.

Effective prevention and therapy of experimental allergic asthma using a GATA-3-specific DNAzyme.

BACKGROUND: Allergic bronchial asthma is a chronic inflammatory disease of the airways. The transcription factor GATA-3 was shown to play an important role in TH2 cell activation, but also in the regulation of other cell types involved in bronchial asthma including mast cells, eosinophils, and epithelial cells. DNAzymes represent a new class of antisense molecules that combines the specificity of DNA base pairing with an inherent RNA-cleaving enzymatic activity. OBJECTIVE: To develop a GATA-3 mRNA-specific DNAzyme and analyze its allergy-preventing activity in murine models of experimental allergic asthma. METHODS: The most active DNAzyme (termed gd21) was selected by in vitro cleavage assays. Allergic airway inflammation was assessed by inflammatory cell and cytokine analysis within bronchoalveolar lavage. Lung histology, including goblet cell hyperplasia and lung function, was analyzed using head-out body-plethysmography. RESULTS: Intranasal administration of gd21 prevented airway inflammation and mucus production and inhibited development of airway hyperresponsiveness to methacholine in models of acute allergic airway inflammation. Similar effects were also detected in a model of chronic experimental asthma. Interestingly, gd21 was at least as effective as other antisense molecules, and off-target effects were not detected. Further experiments indicated that pulmonary surfactant may facilitate the cellular uptake of gd21 by acting as an endogenous transfectant. CONCLUSION: These results indicate that topical application of the GATA-3-specific DNAzyme is a promising novel approach for the treatment of allergic bronchial asthma.