Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-␣ promoter (AAV-E-AT) were examined. In vitro in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 ؋ 10 13 particles into skeletal muscles of several mouse strains (C57BL͞6, BALB͞c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 g͞ml in SCID and over 400 g͞ml in C57BL͞6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions. Alpha-1-antitrypsin (AAT) deficiency is the second most common monogenic lung disease, accounting for 3% of all early deaths due to obstructive pulmonary disease. AAT is produced in the liver, secreted into the serum, and circulated to the lung where it protects elastin fibers and other connective tissue components of the alveolar wall from degradation by neutrophil elastase. Current therapy for AAT deficiency includes avoidance of cigarette smoke exposure and weekly i.v. infusions of recombinant human AAT (hAAT) protein (1). Attempts at gene augmentation have been limited by the short duration of expression and by the high circulating levels of AAT, which are required for therapeutic effect (800 g͞ml) (2).Several groups have demonstrated that adeno-associated virus (AAV) vectors are capable of stable in vivo expression (3-5) and are less immunogenic than other viral vectors (6). AAV is a nonpathogenic human parvovirus whose life cycle includes a mechanism for long-term latency. In the case of wild-type AAV (wtAAV), this persistence is due to sitespecific integration into a site on human chromosome 19 (AAVS1) (7), whereas with recombinant AAV (rAAV) vectors, persistence occurs by both episomal persistence and integration into non-chromosome 19 locations (8-9). rAAV latency also differs from that of wtAAV in that wtAAV is rapidly converted to double-stranded DNA in the absence of helper virus (e.g., Ad) infection, whereas rAAV-leading st...

Pompe disease is an autosomal recessive disorder caused by mutations in the acid-α glucosidase (GAA) gene. Lingual dysfunction is prominent but does not respond to conventional enzyme replacement therapy (ERT). Using Pompe (Gaa(-/-)) mice, we tested the hypothesis that intralingual delivery of viral vectors encoding GAA results in GAA expression and glycogen clearance in both tongue myofibers and hypoglossal (XII) motoneurons. An intralingual injection of an adeno-associated virus (AAV) vector encoding GAA (serotypes 1 or 9; 1 × 10(11) vector genomes, CMV promoter) was performed in 2-month-old Gaa(-/-) mice, and tissues were harvested 4 months later. Both serotypes robustly transduced tongue myofibers with histological confirmation of GAA expression (immunochemistry) and glycogen clearance (Period acid-Schiff stain). Both vectors also led to medullary transgene expression. GAA-positive motoneurons did not show the histopathologic features which are typical in Pompe disease and animal models. Intralingual injection with the AAV9 vector resulted in approximately threefold more GAA-positive XII motoneurons (P < 0.02 versus AAV1); the AAV9 group also gained more body weight over the course of the study (P < 0.05 versus AAV1 and sham). We conclude that intralingual injection of AAV1 or AAV9 drives persistent GAA expression in tongue myofibers and motoneurons, but AAV9 may more effectively target motoneurons.

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The conducting airways are the primary target for gene transfer in cystic fibrosis (CF), yet the inflammation associated with CF lung disease could potentially pose a significant barrier to gene transfer vectors, such as recombinant adeno-associated virus (rAAV). In order to investigate this possibility, aliquots of bronchoalveolar lavage (BAL) fluid from eight individuals with CF were tested for their in vitro inhibitory effects on rAAV transduction, along with BAL from non-CF individuals. While the non-CF BAL fluid was not inhibitory, seven of eight CF BAL samples had significant inhibitory activity, resulting in a five- to 20-fold reduction in transduction events. Inhibition of rAAV transduction by CF BAL could be reversed by alpha-1-antitrypsin (AAT), but not by DNase. When neutrophil elastase and neutrophil alpha defensins (human neutrophil peptides, HNP) were measured in these samples, they were elevated by 500- and 10,000-fold, respectively. The levels of HNP correlated inversely with the amount of rAAV transduction. Furthermore, rAAV transduction could be blocked by purified HNP in an AAT-reversible manner at HNP concentrations within the range measured in these fluids. We conclude that products of inflammation in CF BAL fluid are inhibitory to rAAV transduction, and that these effects may be reversible by AAT.