In Vivo Ribozyme Targeting of βAPP+ mRNAs

In Alzheimer's disease (AD) and Down's syndrome (DS) patients, posttranscriptional alterations of sequences encoded by exon 9 and exon 10 of the β-amyloid precursor protein (βAPP) mRNA result in mutant proteins (βAPP⁺) that colocalize with neurofibrillary tangles and senile plaques. These aberrant messages may contribute to the development of sporadic or late-onset Alzheimer's disease; thus, eliminating them or attenuating their expression could significantly benefit AD patients. In the present work, self-cleaving hammerhead ribozymes targeted to βAPP exon 9 (Rz9) and βAPP⁺ mutant exon 10 (Rz10) were examined for their ability to distinguish between βAPP and βAPP⁺ mRNA. In transiently transfected A-204 cells, quantitative confocal fluorescence microscopy showed that Rz9 preferentially lowered endogenous βAPP. In contrast, in transient cotransfection experiments with βAPP⁺ mRNAs containing a wild-type exon 9 and mutant exon 10 (βAPP-9/βAPP-10+1), or a mutant exon 9 and wild-type exon 10 (βAPP-9+1/βAPP-10) we found that Rz9 and Rz10 preferentially reduced βAPP⁺-mutant exon 10 mRNA in a concentration and a ribozyme-dependent manner.