RNA interference allows selective gene silencing, and is widely used for functional analysis of individual genes in vertebrate cells and represents an attractive therapeutic option for treating central nervous system diseases. However, growing evidence exists that the expression of short hairpin RNAs (shRNAs) can trigger cellular immune response resulting in unspecific cellular phenotypes and severe side effects. We found that lentiviral vector (LV)-mediated expression of shRNAs in primary cortical cultures resulted in strong expression of the interferon-stimulated gene oligoadenylate synthetase 1 (Oas1), which was accompanied by accelerated apoptosis and substantial net neuron loss. Modification of the shRNA construct by implementing features of the naturally occurring microRNA-30 (miR-30) precursor avoided Oas1 induction in transduced primary cultures, whereby modification of the passenger strand seems to be a crucial feature to circumvent interferon-stimulated gene expression. This work represents the first experimental study showing that an miR-30-based shRNA construct prevents Oas1 pathway associated off-target effects, which we consider as an essential prerequisite for shRNA use in future gene therapeutic approaches.