Alpha-1-antitrypsin Deficiency

Terry Flotte, M.D.

Alpha 1-antitrypsin (AAT) deficiency (A1AD) is characterized by a marked decrease in the circulating levels of the major serum antiprotease, AAT, with a subsequent compromise of pulmonary elastin resulting in a susceptibility to emphysema. The most common AAT mutation associated with this disease, the PI*Z allele (Glu342Lys), accounts for approximately 95% of cases. A minority of AAT deficient individuals will also develop a liver disease which is characterized by polymerization of the Z-form of AAT (Z-AT) within the endoplasmic reticulum (ER) of hepatocytes, which triggers hepatocellular injury, and ultimately cirrhosis. While the pulmonary disease associated with AAT deficiency can be treated by replacing serum levels of the protein up to a known threshold (11 M), the proximate goals of therapy for A1AD liver disease are less clear. Since the pathology appears to relate to a gain-of-function by the abnormal protein, down-regulation of expression of the mutant endogenous alleles might be considered. However, heterozygotes appear much less susceptible to disease than PI*Z homozygotes, which may indicate either a protective effect of the wild-type (PiM) version of the protein or a gene dosage effect. Furthermore, recent reports indicate that chemical chaperones, such as 4-phenylbutyric acid (4-PBA), can assist the proper folding of Z-AT, perhaps suggesting that an alternate therapeutic approach by augmenting chaperone function at a molecular level. The goal of this application is to sort out these various therapeutic options using recombinant AAV vectors that can stably insert therapeutic molecules without significant toxicity. This will be accomplished in the following specific aims:

Aim 1: To utilize gene transfer vectors in order to define cellular endpoints for therapy of A1AD-liver disease. Our hypothesis is that a 50% decrease in Z-AT by rAAV-ribozyme vectors will be sufficient to prevent cellular pathology due to Z-AT polymerization. We will confirm this by treating stably transfected CHO cell lines that constitutively express Z-AT with transcriptionally-controlled anti-AAT ribozymes already in use in our laboratory. The endpoints for these studies will be ER polymerization and accumulation of Z-AT as judged by EM and immunofluorescent staining combined with confocal microscopy. The potential need for augmentation with PiM AAT (M-AT) in facilitating this correction will also be examined.

Aim 2: To define the role of augmenting Hsp70 function as therapy for conformational disease. Previous studies with chemical chaperones suggest that augmentation of Hsp70 with rAAV-Hsp70 vectors will increase folding of mutant AAT and CFTR in a native configuration and facilitate degradation of the misfolded protein. In the case of the Z-AT-expressing CHO cell lines, this should result in an increase in secretion of the mutant protein into the supernatant media, as well as correction of the accumulation of mutant protein in the ER.

Aim 3: To evaluate the potential roles for rAAV-hAAT, rAAV-Hsp70, and rAAV-Rz, vectors to ameliorate liver pathology in mouse models. In a companion NIDDK-sponsored program, our laboratory has been actively working to increase the efficiency of rAAV-mediated transduction of hepatocytes in vivo as a potential means to deliver therapeutic molecules in the context of A1AD liver disease. In this final aim, we will combine those molecular strategies that appear most fruitful in the cell culture models with those vector improvements that result in the greatest enhancement of rAAV-mediated transduction. The resultant vectors will be used in one of two kinds of models: (1) a Z-AT overexpressing transgenic mouse model that has previously been published; or (2) mice that are stably expressing human AAT from previous portal vein injection of rAAV-hAAT vectors. The former model will be useful for all three therapies, while the latter will only be useful for ribozyme experiments.