Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

Fig. 2.Transformants releasing E␤C suffered less damage than control lines when EPNs were present. (A) Root damage measured on plants that had received neither WCR eggs nor nematodes was minimal, and there was no difference between transformed and nontransformed plants (n ϭ 5, P ϭ 0.87). (B) Root damage on plants that received only WCR eggs, but no nematodes, was substantial. Again, no significant difference was found between the transformed and nontransformed plants (n ϭ 5, P ϭ 0.18). (C) In plots that received WCR eggs and H. megidis, roots from transformed plants (pooled) had significantly less damage than roots from control lines (n ϭ 30, P ϭ 0.007). Approximately one-quarter of the transformed plants were found not to emit E␤C. Removing these plants from the statistical analysis did not significantly affect the results. The letters above the bars indicate significant differences within a graph. Error bars indicate standard errors.

Soluble tumor necrosis factor receptors (TNFRs) are important modulators of TNF bioactivity. Proteolytic cleavage of the 28-kDa ectodomain of TNFR1 has been recognized as the mechanism by which soluble TNFR is shed. We now describe the release of exosome-like vesicles as a mechanism for the generation of soluble, full-length 55-kDa TNFR1. We found unexpectedly that the predominant form of soluble TNFR1 in human serum and lung epithelial lining fluid is a full-length 55-kDa protein. Furthermore, supernatants from human vascular endothelial cells contain only full-length 55-kDa TNFR1 that can be sedimented by high-speed centrifugation, floated on sucrose gradients at a density of 1.1 g͞ml, and associated with vesicles that range in diameter from 20 nm to 50 nm. We conclude that the release of TNFR1 exosome-like vesicles represents a previously unrecognized mechanism by which constitutive production of soluble cytokine receptors may be regulated, independent of ectodomain cleavage by receptor sheddases. Binding of tumor necrosis factor (TNF) to the 55-kDa, type I TNF receptor (TNFR1, TNFRSF1A, CD120a, p55) activates signaling pathways that regulate inflammatory, immune, and stress responses, as well as host defense and apoptosis (1). TNF signaling is negatively regulated at two levels to prevent excessive or inappropriate immune or inflammatory responses. First, constitutive TNFR1 signaling is prevented by the binding of silencer of death domains (SODD) to the TNFR1 intracytoplasmic domain (2). TNF binding to TNFR1 releases SODD, thereby allowing the formation of an active TNFR1 signaling complex. The second regulatory mechanism is shedding of cell surface TNF receptors to function as soluble TNF binding proteins that inhibit TNF bioactivity by competing with cell surface TNF receptors for free ligand. Soluble TNF receptors may also reversibly bind to and stabilize trimeric TNF, thereby prolonging its half-life and serving as a slow release reservoir for TNF when levels are low (3).Soluble 27-to 30-kDa TNF-binding proteins, corresponding to the TNFR1 extracellular domain, were originally purified and isolated from human urine and serum (4-8). The demonstration by ELISA of TNFR1 molecules in culture supernatants from Chinese hamster ovary cells transfected with TNFR1 cDNA suggested that the soluble form is generated by proteolytic cleavage of the extracellular domain of cell surface receptors, rather than by alternative splicing (7). Sequence analysis identified the major TNFR1 cleavage site to be in the spacer region adjacent to the transmembrane domain between Asn-172 and Val-173, with a minor site between . Further, the ability of hydroxamic-acid based metalloprotease inhibitors to block TNFR1 shedding suggested that proteolytic cleavage of cell surface TNFR1 receptors is mediated by a zinc metalloprotease (11). Consistent with this, TNF-␣ converting enzyme (TACE, ADAM 17), a member of the metalloproteasedisintegrin (ADAM) family of zinc metalloproteases, was identified as mediating TNFR1 shedding. This conclusio...

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