875. Ribozyme Approaches towards Down-Regulation of Pi*Z Mutant Human A-1 Anti-Trypsin
Authors: Thomas J. Conlon, Stuart G. Beattie, Chris W. Snyder, Alfred S. Lewin, Terry R. Flotte
Journal: Molecular Therapy (2004)

Alpha 1-antitrypsin (AAT), a 52kDa serine protease inhibitor, is normally secreted from hepatocytes and circulates in the plasma, protecting lung elastin from degradation by neutrophil elastase and related proteases. Deficiency of AAT can lead to pan-acinar emphysema due to destruction of pulmonary interstitial elastin if serum levels fall below ~800mg/ml. Human AAT deficiency is most commonly due to a single base mutation (Glu342Lys) that causes a defect in secretion of the mutant protein from hepatocytes. The PI*Z mutation eliminates a critical salt bridge between Glu342 and Lys290, which results in polymerization of AAT in the endoplasmic reticulum (ER). Approx 10% patients with AAT deficiency exhibit intracellular liver pathology, resulting from the mutant protein accumulating in the ER of the hepatocyte. This is due to slow or improper folding within the chaperone system of the ER, followed by aggregation of the mutant protein. This intracellular pathology causes progressive cirrhosis and eventual liver failure, and increases the risk of hepatocellular carcinoma. In our laboratory, recombinant adeno-associated viral (AAV) vectors have been shown to mediate long-term secretion of therapeutic serum levels of human AAT after a single injection in murine muscle. Similarly, serum levels greater than 1mg/ml were achieved after portal vein injection. While this mode of gene delivery may be useful as a therapy for the lung disease, the minority of patients who also suffer from the liver disease will require increased transduction of hepatocytes and may therefore require down-regulation and/or repair of the mutant PI*Z mRNA. We hypothesized that selectively reduced expression of the PI*Z-AAT protein can be achieved by a group I ribozyme targeted to anneal to and cleave AAT mRNA at various positions, including the 5' UTR and the PI*Z mutation. These ribozymes did not cleave replacement vector-encoded AAT because of a silent “hardening” mutation that will preserve the original amino acid sequence (demonstrated in a cell-free system). Stable transfected IB-3 fibroblast clones expressing PI*Z or PI*M (normal) AAT cDNAs were transduced with AAV expressing either a non-functional control ribozyme (Ctl Rz) or a ribozyme designed to cleave PI*Z AAT (Rz WAR). Our results indicate that Rz WAR is effective at cleaving PI*Z and PI*M AAT mRNA transcripts and reducing levels of secreted AAT protein by 12–30 %, relative to Ctl Rz (as determined by RT-PCR and ELISA). Immunohistochemical staining for intracellular AAT supported our hypothesis that Rz WAR can reduce the amount of intracellularly retained PI*Z AAT. No significant dose response trends were observed, indicating that the effects of Rz WAR reach a plateau at a relatively low MOI. Moreover, Rz WAR was not specific for PI*Z AAT mRNA and appears to actually cleave PI*M AAT more effectively than PI*Z, but again Rz WAR was not able to cleave the hardened PI*M AAT. Future studies include evaluation of these AAV-Rz reagents in human PI*Z AAT-transgenic mice.