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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.

Two sets of eucaryotic expression vectors encoding trans-acting hammerhead ribozymes and frans-acting hairpin ribozymes were constructed. In one set of vectors ribozyme RNA transcription was placed under the control of a mouse mammary tumor virus long terminal repeat (MMTV-LTR). In the other set ribozyme expression was controlled by a metallothionein IIA (Mt-IIA) promoter. Each ribozyme was directed to the first target sequence in the Alzheimer amyloid peptide precursor mRNA (βAPP mRNA), 5′↓GUC↓3′. Ribozyme RNA transcribed from these vectors, which should cleave all six alternatively spliced forms of βAPP mRNA as well as βAPP pre-mRNA, was shown to cleave a βAPP RNA substrate analog in vitro. Stably transfected COS-7 cell lines bearing both vector types were prepared. Steady-state levels of βAPP mRNA were reduced 25–30% in cells containing either active or mutant hammerhead ribozyme vectors driven by the MMTV-LTR promoter grown in the presence of glucocorticoids. In cell lines bearing Mt-IIA driven ribozymes steady-state levels of βAPP mRNA were reduced 67–80% in both hammerhead and hairpin ribozyme containing cell lines following promoter induction by glucocorticoids. These levels correlate with the appearance of low levels of induced ribozyme RNA. In contrast, steady-state α-actin mRNA and G3PDH mRNA levels in these cells remained constant. Western blotting of cell extracts revealed that all forms of βAPP were correspondingly reduced. Neither the RNA nor protein decreases observed in ribozyme transfected cell lines were observed in stably transfected control cells bearing the vector alone. These results suggest that ribozyme-mediated degradation of βAPP mRNA in COS-7 cells does not depend on ribozyme cleavage.

The sequences surrounding the first 5'GUC3' in the mRNA encoding the Alzheimer amyloid peptide pre–cursor (βSAPP) were used to construct a pair of transacting hammerhead ribozymes. Each ribozyme contained the conserved core bases of the hammerhead motif found in the positive strand of satellite RNA of tobacco ringspot virus [(+)sTRSV] and two stems, 7 and 8 bases long, complementary to the target, βAPP mRNA. However, one of the ribozyme cleaving strands was lengthened at its 3′ end to include the early splicing and polyadenylation signal sequences of SV40 viral RNA. This RNA, therefore, more closely mimics transcripts produced by RNA polymerase II from eucaryotic expression vectors In vivo. RNA, prepared by run-off transcription of cDNA oligonucleotide or plasmid constructs containing a T7 RNA polymerase promoter was used to characterize several properties of the cleavage reaction. In the presence of both ribozyme cleaving strands magnesium-Ion dependent cleavage of a model 26 base βAPP substrate RNA or full-length βAPP—751 mRNA was observed at the hammerhead consensus cleavage site. Neither ribozyme was active against non-message homologs of βAPP mRNA, nor was cleavage detected when point mutations were made in the conserved core sequences. However, the kcatKm at 37°C in 10 mM Mg+2 of the longer ribozyme was reduced twenty-fold when model and full-length substrates were compared. The use of short deoxyoligonucleotides (13—17 mere) that bind upstream of the ribozyme was found to enhance the rate of cleavage of the full-length but not βAPP model substrate RNAs. The rate of enhancement depended on both the length of the deoxyoligonucleotide used as well as its site of binding with respect to the ribozyme. These data demonstrate the utility of ribozymes to cleave target RNAs In a catalytic, site-specific fashion In vitro. Direct comparison of the efficiency of different ribozyme constructs and different modulating activities provide an experimental strategy for designing more effective ribozymes for therapeutic purposes.