Alpha 1-antitrypsin (alpha 1 AT) represents the first line of defense to preserve the delicate elastin fiber network of the lung parenchyma against damage by neutrophil elastase (NE), to which it is constantly exposed. Alpha 1 AT deficiency is a common autosomal recessive disorder which predisposes patients to early onset of pulmonary emphysema, particularly if they are also exposed to cigarette smoke. While intravenous protein replacement is available and is generally safe, it involves weekly repeated infusions which can be painful and costly. Genetic therapy for alpha 1 AT wold appear to be feasible since the protein is biologically active when secreted from any of a number of sites including liver (its natural source), skeletal muscle, and airway epithelium. However, vectors which have been used in previous preclinical experiments have been generally transient in their expression. Adeno-associated virus (AAV) vectors may have some advantages for alpha 1 AT gene therapy since their expression is stable. Recent data from a number of groups indicates that AAV vectors may be particularly useful in skeletal muscle, where stable reporter expression has been found for over 1 year from a single injection. Furthermore, the secretionof a biologically active protein, human erythropoietin (h-epo) from an AAV vector caused physiologic effects (Increased hematocrit) which were stable over many months. 50.91 propose to evaluate AAV transduction of skeletal muscle as a depot for sustained secretion of alpha 1 AT, and to pursue this strategy as potential gene therapy for patients with alpha1 AT-deficiency. The major remaining questions regarding this approach are (1) Will immunologic responses develop to the vectors of the transgenes which could limit the efficacy of the AAV/skeletal muscle depot approach? And(2) Will the level of expression be high enough to prevent the development of the pulmonary disease phenotype (i.e., to achieve the threshold protective serum level of > 11 muM)? These questions are addressed in four specific aims: (1) An AAV-CMV-alpha1 AT vector will be evaluated for its potential ability to express human-alpha1 AT in vitro fromthe murine myoblast line C2C12; (2) The level and duration of AAV-CMV-alpha1 AT expression will be evaluated in murine skeletal muscle in vivo; (3) In order to more thoroughly address the potential immunologic consequences of expression of a foreign transgene from an AAV vector in muscle, similar in vivo transduction experiments will be performed in a number of relevant strains of genetically defective mice; (4) Once the feasibility of AAV/muscle platform for h-alpha1 AT replacement has been established, we propose to complete preclinical primate testing of the AAV-CMV-alpha1 AT vector in anticipation of phase I trails in alpha1 AT patients with emphysema.
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