Sihong Song

CMV-β-Actin Promoter Directs Higher Expression from an Adeno-Associated Viral Vector in the Liver than the Cytomegalovirus or Elongation Factor 1α Promoter and Results in Therapeutic Levels of Human Factor X in Mice

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 beta-actin promoter results in 9.5-fold higher expression after portal vein injection than an AAV vector with the EF1 alpha 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-beta-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-beta-actin promoter might correct hemophilia due to hFX deficiency.

Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors.

Previous work from our group showed that recombinant adeno-associated virus (rAAV) vectors mediated long-term secretion of therapeutic serum levels of human alpha-1 antitrypsin (hAAT) after a single injection in murine muscle. We hypothesized that hepatocyte transduction could be even more efficient, since these cells represent the natural site of AAT production and secretion. To test this hypothesis, rAAV vectors containing the hAAT cDNA driven by either the human elongation factor 1 alpha promoter, the human cytomegalovirus immediate–early promoter (CMV), or the CMV-chicken beta actin hybrid (CB) promoter were injected into the portal or tail veins of adult C57Bl/6 mice. Potentially therapeutic serum levels of hAAT (600 μg/ml) were achieved after portal vein injection of doses of 4 × 109 infectious units (IU), a 10-fold lower dose than that required for similar levels of expression via the i.m. route. Serum levels greater than 1 mg/ml were achieved at doses of 3 × 1010 IU. Southern blotting of liver DNA revealed the presence of circular episomal vector genomes. Immunostaining showed that transgene expression was scattered throughout the liver parenchyma. Similar results were obtained with a rAAV-CB-green fluorescent protein (GFP) vector. There was no evidence of hepatic toxicity. These data indicate that liver-directed rAAV-based gene therapy is effective in the murine model, and hence might be feasible for treatment of human AAT deficiency.

Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice

The development of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice provides for their use as a model of human type 1 diabetes. To test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (rAAV) vectors containing murine cDNAs for immunomodulatory cytokines IL-4 or IL-10. Skeletal muscle transduction of female NOD mice with IL-10, but not IL-4, completely abrogated diabetes. rAAV-IL-10 transduction attenuated the production of insulin autoantibodies, quantitatively reduced pancreatic insulitis, maintained islet insulin content, and altered splenocyte cytokine responses to mitogenic stimulation. The beneficial effects were host specific, as adoptive transfer of splenocytes from rAAV IL-10-treated animals rapidly imparted diabetes in naive hosts, and the cells contained no protective immunomodulatory capacity, as defined through adoptive cotransfer analyses. These results indicate the utility for rAAV, a vector with advantages for therapeutic gene delivery, to transfer immunoregulatory cytokines capable of preventing type 1 diabetes. In addition, these studies provide foundational support for the concept of using immunoregulatory agents delivered by rAAV to modulate a variety of disorders associated with deleterious immune responses, including allergic reactions, transplantation rejection, immunodeficiencies, and autoimmune disorders.

Systemic Overexpression of IL-10 Induces CD4+CD25+ Cell Populations In Vivo and Ameliorates Type 1 Diabetes in Nonobese Diabetic Mice in a Dose-Dependent Fashion

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.

Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver

Human pancreatic islet cells and hepatocytes represent the two most likely target cells for genetic therapy of type I diabetes. However, limits to the efficiency of rAAV serotype 2 (rAAV2)-mediated gene transfer have been reported for both of these cell targets. Here we report that nonserotype 2 AAV capsids can mediate more efficient transduction of islet cells, with AAV1 being the most efficient serotype in murine islets, suggesting that receptor abundance could be limiting. In order to test this, we generated rAAV particles that display a ligand (ApoE) that targets the low-density lipoprotein receptor, which is present on both of these cell types. The rAAV/ApoE viruses greatly enhanced the efficiency of transduction of both islet cells ex vivo and murine hepatocytes in vivo when compared to native rAAV2 serotype (220- and four-fold, respectively). The use of receptor-targeted rAAV particles may circumvent the lower abundance of receptors on certain nonpermissive cell types.

Effect Of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle

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 α1-antitrypsin (rAAV-hAAT) vectors were delivered by intramuscular injection to either C57BL/6 (DNA-PKcs+) or C57BL/6-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 C57BL/6 strain. These results indicate that DNA-PK is involved in the formation of circular rAAV episomes.

Alpha1-antitrypsin protects beta-cells from apoptosis.

β-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 α1-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 β-cell apoptosis. Specifically, strong antiapoptotic activities for AAT (Prolastin, human) were observed when murine insulinoma cells (MIN6) were exposed to tumor necrosis factor-α. In a second model system involving STZ-induced β-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 β-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.

α1-Antitrypsin Protects β-Cells From Apoptosis

β-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 α1-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 β-cell apoptosis. Specifically, strong antiapoptotic activities for AAT (Prolastin, human) were observed when murine insulinoma cells (MIN6) were exposed to tumor necrosis factor-α. In a second model system involving STZ-induced β-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 β-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.

DNA-dependent PK inhibits adeno-associated virus DNA integration

Recent studies have shown that recombinant adeno-associated virus (rAAV) can persist in episomal form; however, factors affecting rAAV persistence are poorly understood. DNA-dependent PK (DNA-PK) is a DNA repair enzyme, which we previously found played an important role in determining the molecular fate of the rAAV genome in mouse skeletal muscle. In the present study, we tested the effect of DNA-PK on AAV serotype 2 integration in vitro and in vivo in mouse liver. In an in vitro integration system, addition of DNA-PK decreased AAV integration, whereas antibody against DNA-PKcs increased integration. In vivo, matched doses of a recombinant AAV serotype 2 vector were injected into the portal vein of either C57BL/6 (DNA-PKcs+/+) or severe combined immunodeficient (DNA-PKcs-/-) mice. After partial hepatectomy to stimulate hepatocyte proliferation, retention of vector genomes and of transgene expression was substantially higher in severe combined immunodeficient mice, indicating that in the absence of DNA-PKcs, a greater proportion of genomes integrated into the cellular genome. In summary, we have provided evidence that DNA-PK inhibits AAV integration both in vitro and in vivo.

Recombinant adeno-associated virus-mediated alpha- 1 antitrypsin gene therapy prevents type I diabetes in NOD mice

Type I diabetes results from an autoimmune destruction of the insulin-producing pancreatic β cells. Although the exact immunologic processes underlying this disease are unclear, increasing evidence suggests that immunosuppressive, immunoregulatory and anti-inflammatory agents can interrupt the progression of the disease. Alpha 1 antitrypsin (AAT) is a multifunctional serine proteinase inhibitor (serpin) that also displays a wide range of anti-inflammatory properties. To test the ability of AAT to modulate the development of type I diabetes, we performed a series of investigations involving recombinant adeno-associated virus vector (rAAV)-mediated gene delivery of human alpha-1 antitrypsin (hAAT) to nonobese diabetic (NOD) mice. Recombinant AAV-expressing hAAT (rAAV2-CB-AT) was administered intramuscularly to 4-week-old female NOD mice (1 × 1010 i.u./mouse). A single injection of this vector reduced the intensity of insulitis, the levels of insulin autoantibodies, and the frequency of overt type I diabetes (30% (3/10) at 32 weeks of age versus 70% (7/10) in controls). Transgene expression at the injection sites was confirmed by immunostaining. Interestingly, antibodies against hAAT were present in a majority of the vector-injected mice and circulating hAAT was undetectable when assessed 10 weeks postinjection. This study suggests a potential therapeutic role for AAT in preventing type I diabetes as well as the ability of AAV gene therapy-based approaches to ameliorate disease effectively.