Anne David

Interleukin-10 produced by recombinant adenovirus prolongs survival of cardiac allografts in rats

Interleukin-10 (IL-10) and interleukin-4 (IL-4), two Th2-derived cytokines, are molecules with anti-inflammatory and immunodeviating properties whose direct expression in allografts may prolong graft survival. Recombinant adenoviruses represent efficient vectors for gene tranfer in quiescent cells in vivo. Adenoviral vectors encoding rat IL-10 (AdIL-10), rat IL-4 (AdIL-4) or β-galactosidase (AdlacZ) or without transgene (Addl324) were injected directly into rat hearts at the time of transplantation in order to test their potential to prolong heart allograft survival. Expression of vectorized sequences was confirmed in heart biopsies, and kinetic analysis of β-galactosidase showed transient expression. Cardiac allograft survival was significantly prolonged after administration of 109 p.f.u. of AdIL-10 (16.6 ± 3.2 days, P < 0.05), but not adil-4 (9.8 ± 1.6 days), compared with addl324-treated (9.3 ± 3.3 days) or untreated groups (7.8 ± 1.5 days). immunohistochemical analysis of allografts after gene transfer of il-10 showed that leukocyte infiltration was quantitatively equivalent to that seen in control groups but with a strong tendency towards lower levels of cd8+ cells. Importantly, adenovirus-derived IL-10 modified the functional status of leukocytes by inducing a significant decrease in IFN-γ production but significantly increased transforming-growth factor β1 (TGF-β1) expression within the grafts compared with those treated with Addl324. These results show that expression of IL-10 by rat hearts after gene transfer mediated by an adenoviral vector decreases allogeneic immune responses and allows prolongation of allograft survival.

Lethal Hepatitis After Gene Transfer of IL-4 in the Liver Is Independent of Immune Responses and Dependent on Apoptosis of Hepatocytes: A Rodent Model of IL-4-Induced Hepatitis

The putative role of IL-4 in human and animal models of hepatitis has not yet been directly determined. We now report that direct expression of IL-4 in the liver of rats or mice using recombinant adenoviruses coding for rat or mouse IL-4 (AdrIL-4 and AdmIL-4, respectively) results in a lethal, dose-dependent hepatitis. The hepatitis induced by IL-4 was characterized by hepatocyte apoptosis and a massive monocyte/macrophage infiltrate. IL-4-induced hepatitis was independent of T cell-mediated immune responses. Hepatitis occurred even after gene transfer of IL-4 into nude rats, CD8-depleted rats, cyclosporine A-treated rats, or recombinase-activating gene 2−/− immunodeficient mice. Peripheral depletion of leukocytes using high doses of cyclophosphamide, and/or the specific depletion of liver macrophages with liposome-encapsulated dichloromethylene diphosphonate in rats did not block lethal IL-4-induced hepatitis. Direct transduction of hepatocytes with adenoviruses was not essential, since injection of AdrIL-4 into the hind limb induced an identical hepatitis. Finally, primary rat hepatocytes in culture also showed apoptosis when cultured in the presence of rIL-4. IL-4-dependent hepatitis was associated with increases in the intrahepatic levels of IFN-γ, TNF-α, and Fas ligand. Administration of AdmIL-4 to IFN-γ, TNF-α receptor type I, or TNF-α receptor type II knockout mice also resulted in lethal hepatitis, whereas a moderate protection was observed in Fas-deficient lpr mice. IL-4-dependent hepatocyte apoptosis could be abolished by treatment with caspase inhibitory peptides. Our results thus demonstrate that IL-4 causes hepatocyte apoptosis, which is only partially dependent on the activation of Apo-1-Fas signaling and is largely independent of any immune cells in the liver.

A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer

Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of β-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases β-galactosidase expression 90-fold in SMC in vitro and ≈40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies.

Adenovirus expression of IL-1 and NF-κB inhibitors does not inhibit acute adenoviral-induced brain inflammation, but delays immune system-mediated elimination of transgene expression

Despite their ability to provide long-term transgene expression in the central nervous system of naïve hosts, the use of first-generation adenovirus (Ad) vectors for the treatment of chronic neurological disorders is limited by peripheral immunization, which stimulates anti-adenovirus immune responses and causes severe inflammation in the central nervous system (CNS) and elimination of transgene expression. The purpose of this study was to investigate the roles of NF-kappaB and interleukin-1 (IL-1) during inflammatory responses to Ads in the CNS of naïve and preimmunized rats. We assessed activation of macrophages/microglia, up-regulation of MHC I expression, infiltration of leukocytes, and transgene expression following delivery of Ads to the rat striatum. After delivery of increasing doses of adenoviral vectors expressing various anti-inflammatory agents (e.g., NF-kappaB or IL-1 inhibitors) to naïve rats, no reduction in Ad-mediated CNS inflammation was seen 1 week after delivery of Ads, compared to a control Ad.hCMV.beta-galactosidase (RAd.35) virus. We then assessed CNS inflammation and transgene expression at a time when control transgene expression would be completely eliminated, i.e., 1 month post-vector injection into the brain. This would optimize the assessment of an anti-inflammatory agent expressed by an adenoviral vector that could either delay or diminish immune system-mediated elimination of transgene expression. As expected, at 1 month postinfection, control preimmunized rats receiving Ad.mCMV.beta-galactosidase (RAd.36)/saline or RAd.36/Ad.null (RAd.0) showed complete elimination of beta-galactosidase expression in the brain and levels of inflammation comparable to those of naïve animals. However, animals injected with RAd.36 in combination with Ads expressing NF-kappaB or IL-1 inhibitors showed a delayed elimination of beta-galactosidase compared to controls. As predicted, the extended presence of transgene expression was accompanied by increased levels of CNS inflammation. This suggests that blocking NF-kappaB or IL-1 delays, albeit partially, transgene elimination in the presence of a preexisting systemic immune response. Prolonged transgene expression is predicted to extend concurrent brain inflammation, as noted earlier. Taken together these data demonstrate a role for NF-kappaB and IL-1 in immune system-mediated elimination of Ad-mediated CNS transgene expression.

Adenoviras‐mediated expression of human CD59 on xenogeneic endothelial cells: Protection against human complement‐mediated lysis and induction of cellular activation by adenoviral transduction

Abstract: The expression of human regulators of complement activation in endothelium of a transplanted organ is an approach to overcoming the complement (C)‐mediated rejection of a xenograft. We designed a replication‐deficient adenovirus (Ad) containing the human CD59 cDNA (AdCD59) in order to induce expression of human CD59 on xenogeneic endothelial cells (EC) and confer protection against human C‐mediated lysis. In vitro transduction of rat EC with AdCD59 led to consistent levels of CD59 expression in all cells and significantly reduced EC lysis induced by human xenogeneic natural antibodies (XNA) binding and C activation. In addition, we analyzed whether Ad transduction had modified the phenotype of EC and the xenogeneic recognition of EC by human serum. For this, we first demonstrated that transduction of rat EC with AdCD59 or an Ad carrying the lacZ gene (AdlacZ) markedly enhanced the expression of some cell‐membrane markers of EC activation, including class I MHC antigens and rat CD59, to levels comparable to those obtained with TNFα Up‐regulation of class I MHC antigens was observed for both mRNA and protein expression. In contrast, AdCD59‐mediated gene transfer slightly increase intercellular adhesion molecule‐1 (ICAM‐1) and did not modify the expression of Crry, a rat complement regulatory molecule. Second, we showed that these phenotypic modifications of EC did not affect the expression of the Galαl‐3Gal epitope and the binding of human XNA. In addition, neither AdlacZ‐mediated transduction, nor TNFα treatment, modified the sensitivity of xenogeneic EC to human serum‐mediated lysis. In conclusion, this study demonstrated that transduction of human CD59 with an adenoviral vector conferred resistance to xenogeneic cultured EC against human C‐mediated lysis but is associated with cellular activation of transduced EC. This finding may have important implications for the in vivo protocols and strategies intended to generate safer Ad vectors.

In vitro and in vivo gene transfer in the rat: characterization of recombinant adenoviral vectors for rat interleukin-4 or interleukin-10 cDNA

Abstract Gene transfer into grafts of molecules with potentially immunomodulating actions may prolong allograft survival and give a method of analyzing the mechanisms of allograft rejection and tolerance. 1 Interleukin-4 (IL-4) and interleukin-10 (IL-10) have the capacity to drive the immune response from a Thl to a Th2 profile and inhibit macrophage activation, 2 making them candidates to be overexpressed in the local graft microenvironment. 3 The purpose of this study was to analyse the feasibility and conditions for efficient recombinant adenovirus (rAd)-mediated gene transfer into rat liver, kidney, and pancreatic islets, and to validate rAds as efficient vectors for localized cytokine overexpression.

Transient Transgenesis in The Endocrine System: Viral Vectors for Gene Delivery

Transgenic animals and knockout technology have been of great importance to elucidate the molecular basis of many endocrine processes. In both these technologies, changes are introduced into germline cells, and animals develop with the addition or lack of individual genes. Germline alterations have two major shortcomings. Some will be embryonic lethal mutations, whilst others will induce compensatory changes during development, which will then contribute to the phenotype of mutant animals.