The goal of this proposal is to develop recombinant adeno-associated virus (rAAV) vector transduction of hepatocytes as a strategy for gene therapy for alpha-1-anti trypsin (AAT) deficiency. AAT deficiency is a common monogenic disease that can lead to pulmonary emphysema in most affected patients and, in addition, to liver failure in a significant minority of patients. AAT is the major serum serine protease inhibitor (serpin) in humans; individuals with deficient (Z or S) or null AAT alleles can develop severe emphysema in early adulthood due to destruction of pulmonary elastin fibers by neutrophil elastase; cathespin G, and other neutrophil products. Current therapy to prevent lung disease in AAT- deficient patients consists of weekly intravenous infusions of a serum- derived AAT preparation, while in mice, rAAV-AAT vectors have been shown to express therapeutic serum levels after a single intramuscular injection. The efficiency of expression and secretion could be improved by targeting hepatocytes rather than skeletal myocytes, but earlier reports indicate that the efficiency of hepatocyte transduction may be limited to 5%. We propose to evaluate each of the rate limiting steps in hepatocyte transduction with 2 goals in mind. In the case of AAT lung disease, the goal is to increase the overall expression level, in terms of total output of AAT into the serum. The liver disease, however, is due to polymerization of the mutant protein, and so the goal will be to down-regulate expression of the mutant protein in as high a percentage of hepatocytes as possible. These two goals will be addressed in the following specific aims: 1. Delivery: The ability of rAAV-hAAT vectors to efficiently transduce hepatocytes after portal venous or systemic venous delivery will be assessed and enhanced, via the insertion of new targeting ligands into the rAAV capsid. 2. Molecular State of rAAV vector: The integration state of rAAV-hAAT in hepatocytes will be assessed serially over time using Southern blot hybridization and FISH to determine whether there is a limitation to the percentage of hepatocytes capable of long-term retention rather than short-term uptake of rAAV genomes. 3. Transcriptional Efficiency: The level and specificity of rAAV-hAAT expression within hepatocytes will be enhanced at the transcriptional level by optimizing promoter strength and by using a newly described system for amplifying tissue-specific expression by the use of a potent transcriptional transactivator. 4. Down-regulation of Mutant mRNA: Hammerhead ribozyme vectors will be designed to down-regulate the expression of the endogenous mutant forms of AAT and these will be tested in vitro and in vivo in transgenic mouse models. It is anticipated that these studies will also lay the groundwork for future clinical trials of rAAV-based gene therapy for AAT deficient patients.
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