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...

1␤-Cell apoptosis appears to represent a key event in the pathogenesis of type 1 diabetes. Previous studies have demonstrated that administration of the serine proteinase inhibitor ␣1-antitrypsin (AAT) prevents type 1 diabetes development in NOD mice and prolongs islet allograft survival in rodents; yet the mechanisms underlying this therapeutic benefit remain largely unclear. Herein we describe novel findings indicating that AAT significantly reduces cytokine-and streptozotocin (STZ)-induced ␤-cell apoptosis. Specifically, strong antiapoptotic activities for AAT (Prolastin, human) were observed when murine insulinoma cells (MIN6) were exposed to tumor necrosis factor-␣. In a second model system involving STZ-induced ␤-cell apoptosis, treatment of MIN6 cells with AAT similarly induced a significant increase in cellular viability and a reduction in apoptosis. Importantly, in both model systems, treatment with AAT completely abolished induced caspase-3 activity. In terms of its activities in vivo, treatment of C57BL/6 mice with AAT prevented STZ-induced diabetes and, in agreement with the in vitro analyses, supported the concept of a mechanism involving the disruption of ␤-cell apoptosis. These results propose a novel biological function for this molecule and suggest it may represent an effective candidate for attempts seeking to prevent or reverse type 1 diabetes. Diabetes 56:1316-1323, 2007 T ype 1 diabetes is an autoimmune disease resulting from destruction of the insulin-producing islet ␤-cells (1). Multiple lines of evidence indicate that antigen-presenting cells (APCs), especially dendritic cells, are pathologically active in orchestrating the process of insulitis (2). APCs within islets likely respond to micro-environmental triggers, including ␤-cell death and apoptosis, and initiate the insulitis process by migrating out of the islet and into the peripheral pancreatic lymph nodes. These APCs trigger the activation and proliferation of ␤-cell-reactive T-cells, which destroy islet cells at a rate that eventually results in type 1 diabetes. It has been shown that both direct cytotoxic (T-cell mediated) and indirect cytokine-dependent (e.g., interleukin-1, tumor necrosis factor-␣ [TNF-␣], and ␥-interferon) mechanisms are responsible for ␤-cell apoptosis (3).The direct cytotoxic mechanisms appear to involve the release of cytotoxic granule contents (e.g., perforin and granzymes) by cytotoxic T-cells (4). Granzymes play a critical role in triggering apoptotic cell death through mitochondrial pathways or by the activation of cellular caspases. Indeed, caspases (e.g., caspase-3) are key players in controlling the events leading to cellular apoptosis. Caspases are synthesized as inactive zymogens, which can be cleaved and activated by proteinases, including granzymes, cathepsins, and calpains. Granzymes and cathepsins are also members of a class of molecules known as serine proteinase inhibitor (serpin) proteinases. As for indirect mechanisms for ␤-cell apoptosis involving cytokines, signal transduction activities ...

Our findings suggest that inhibition of structural alveolar cell apoptosis by alpha1-antitrypsin represents a novel protective mechanism of the serpin against emphysema. Further elucidation of this mechanism may extend the therapeutic options for emphysema caused by reduced level or loss of function of alpha1-antitrypsin.

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (104, 106, 108, and 109 infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (109 IU), or saline. Transduction with rAAV-IL-10 at 109 IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 108 IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.

A major hurdle in most current gene therapy modalities is the ability to transduce target tissues at very high efficiencies that ultimately lead to therapeutic levels of transgene expression. We have developed a novel method of recombinant adeno-associated virus 2 (rAAV) delivery that results in increased vector transduction efficiencies using microspheres reversibly conjugated to rAAV vectors. We hypothesize that conjugation to microspheres should result in a higher effective concentration of vector as well as longer relative exposure time of vector to target cells as it moves through the tissue vasculature. In vitro experiments demonstrate that the same level of transduction seen with free vector can be achieved using 1% of vector when conjugated to microspheres. In addition, using magnetic microspheres, the region of infection can be targeted. In vivo, we demonstrate that microsphere-mediated delivery of rAAV vector results in higher transduction efficiencies than delivery with free vector alone when administered either intramuscularly or intravenously. Furthermore, we demonstrate targeting of transgene expression to specific tissues by retention of microsphere-bound vector in the capillary bed. These studies demonstrate a novel method to deliver rAAV vectors more effectively that could prove to be a successful alternative mode of virus-mediated human gene therapy.

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Although AAV vectors show promise for hepatic gene therapy, the optimal transcriptional regulatory elements have not yet been identified. In this study, we show that an AAV vector with the CMV enhancer/chicken b -actin promoter results in 9.5-fold higher expression after portal vein injection than an AAV vector with the EF1a promoter, and 137-fold higher expression than an AAV vector with the CMV promoter/enhancer. Although induction of the acute-phase response with the administration of lipopolysaccharide (LPS) activated the CMV promoter/enhancer from the context of an adenoviral vector in a previous study, LPS resulted in only a modest induction of this promoter from an AAV vector in vivo. An AAV vector with the CMV-b -actin promoter upstream of the coagulation protein human factor X (hFX) was injected intravenously into neonatal mice. This resulted in expression of hFX at 548 ng/ml (6.8% of normal) for up to 1.2 years, and 0.6 copies of AAV vector per diploid genome in the liver at the time of sacrifice. Neonatal intramuscular injection resulted in expression of hFX at 248 ng/ml (3.1% of normal), which derived from both liver and muscle. We conclude that neonatal gene therapy with an AAV vector with the CMV-b -actin promoter might correct hemophilia due to hFX deficiency. OVERVIEW SUMMARYOptimization of gene expression from an AAV vector might allow higher levels of expression to be achieved, which will be necessary for effective gene therapy for some genetic deficiencies. It might also allow a lower dose of vector to be administered, which would reduce the risk of insertional mutagenesis or germ line transmission. Neonatal gene transfer might reduce the chance of inducing an immune response, and would lead to a more immediate correction of a genetic disease than would transfer into adults. We demonstrate here that the CMV-b -actin promoter is expressed well from an AAV vector in the liver. Neonatal intravenous administration of an AAV vector that expresses the coagulation protein factor X from this promoter results in therapeutic levels of factor X for more than 1 year in mice. Neonatal gene therapy with an AAV vector may allow effective gene therapy to be achieved for hemophilia.

We report here that the DNA-dependent protein kinase (DNA-PK) affects the molecular fate of the recombinant adeno-associated virus (rAAV) genome in skeletal muscle. rAAV-human ␣1-antitrypsin (rAAV-hAAT) vectors were delivered by intramuscular injection to either C57BL͞6 (DNA-PKcs ؉ ) or C57BL͞6-SCID [severe combined immunodeficient (SCID), DNA-PKcs ؊ ] mice. In both strains, high levels of transgene expression were sustained for up to 1 year after a single injection. Southern blot analysis showed that rAAV genomes persisted as linear episomes for more than 1 year in SCID mice, whereas only circular episomal forms were observed in the C57BL͞6 strain. These results indicate that DNA-PK is involved in the formation of circular rAAV episomes. R ecombinant adeno-associated virus (rAAV) vectors have been used increasingly for gene therapy because they are relatively nontoxic and only weakly immunogenic and because the duration of transgene expression with these vectors is prolonged (1, 2). Of the various cells and tissues in which rAAV has been used, skeletal muscle appears to be particularly permissive for efficient and stable expression (3-7). The molecular basis of rAAV persistence in muscle has been examined previously. Integrated and circular episomal forms of rAAV DNA have been found (5,6,8). However, long-term persistence of linear episomal forms of rAAV in muscle has not been examined. Furthermore, the effects of host cellular factors on the molecular fate of rAAV are poorly understood.The DNA-dependent protein kinase (DNA-PK), which is composed of a DNA-binding Ku70͞Ku86 heterodimer and a large catalytic subunit (DNA-PKcs), plays an important role in repair of double-stranded DNA breaks and in V(D)J recombination by nonhomologous end-joining (9). It has been reported recently that DNA-PKcs inhibits retrovirus integration (10) and that DNA-PK associated with adenovirus E4 gene products inhibits concatemer formation of adenovirus genome (11). To understand further the molecular fate of the rAAV genome in skeletal muscle and the role of DNA-PK in this process, rAAVhuman ␣1-antitrypsin (rAAV-hAAT) vectors were administered by intramuscular injection into either C57BL͞6 (DNAPKcs ϩ ) or C57BL͞6-SCID [severe combined immunodeficient (SCID), DNA-PKcs Ϫ ] mice. These strains differ only at the DNA-PKcs locus. Transgene expression was monitored up to 1 year after injection. Southern blot analysis was used to examine the molecular fate of the vector DNA in each strain. Materials and Methods rAAV Vector Preparation and Administration.A single rAAV vector containing both the hAAT coding sequence driven by the cytomegalovirus (CMV) immediate early promoter and the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase (TK) promoter was packaged by using a standard plasmid cotransfection technique followed by iodixanol-gradient purification (7,12). The physical titer (in DNase-resistant particles) and the biological titer (in infectious units) of each vector preparation was determined by ...