Reduction of functional N-methyl-D-aspartate receptors in neurons by RNaseP-mediated cleavage of the NR1 mRNA

One approach to studying the functional role of individual NMDA receptor su bunits involves the reduction in the abundance of the protein subunit in ne urons. We have pursued a strategy to achieve this goal that involves the us e of a small guide RNA which can lead to the destruction of the mRNA for a specific receptor subunit. We designed a small RNP, molecule, termed 'exter nal guide sequence' (EGS), which binds to the NR1 mRNA and directs the endo nuclease RNase P to cleave the target message. This EGS has exquisite speci ficity and directed the RNase P-dependent cleavage at the targeted location within the NR1 mRNA. To improve the efficiency of this EGS, an in vitro ev olution strategy was employed which led to a second generation EGS that was 10 times more potent than the parent molecule. We constructed an expressio n cassette by flanking the EGS with self-cleaving ribozymes and this permit ted generation of the specified EGS RNA sequence from any promoter. Using a recombinant Herpes simplex virus (HSV), we expressed the EGS in neurons an d showed the potency of the EGS to reduce NR1 protein within neurons. In an excitotoxicity assay, we showed that expression of the EGS in cortical neu rons is neuroprotective. Our results demonstrate the utility of EGSs to red uce the expression of any gene (and potentially any splice variant) in neur ons.