Administration Of Ad Research Articles

Markedly enhanced intratumoral spread and antitumor effect of oncolytic adenovirus expressing decorin

AbstractWith the aim of improving viral distribution and tumor penetration, we have engineered decorin expressing replication-incompetent (dl-LacZ-DCNG) and -competent (Ad-[DELTA]E1B-DCNG) adenoviruses. In both tumor spheroids and established solid tumors in vivo, administration of dl-LacZ-DCNG resulted in greater transduction efficiency and viral spread throughout the tumor mass. Ad-[DELTA]E1B-DCNG also enhanced viral distribution and tumor spread, leading to an increased anti-tumor effect and survival advantage. Upon histological analysis, Ad-[DELTA]E1B-DCNG also elicited greater percentage of apoptotic cells and extensive necrosis compared to those from untreated or control virus-treated tumors. Furthermore, Ad-[DELTA]E1B-DCNG substantially decreased extracellular matrix components within the tumor tissue, while normal tissue adjacent to the tumor was not affected. Finally, intratumoral administration of Ad-[DELTA]E1B-DCNG did not enhance but inhibited the formation of pulmonary metastases of B16BL6 melanoma cells in mice. Taken together, these data demonstrate the utility of decorin as a dispersion agent and suggest its utility and potential in improving the efficacy of replicating adenovirus-mediated cancer gene therapy.

Vascular damage and anti-angiogenic effects of tumor vessel-targeted adenovirus-mediated herpes simplex virus thymidine kinase gene

To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase (HSV-tk) targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. Recombinant adenovirus containing kinase domain insert with receptor (KDR) or cytomegalovirus (CMV) promoter-controlled HSV-tk gene (AdKDR-tk and AdCMV-tk) was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells (HUVEC) and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir (GCV), the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups: ganciclovir group (I), Ad group (II), AdCMV-tk group (III) and AdKDR-tk group (IV). Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir (GCV) was given at a dose of 100 mg kg(-1) d(-1) (ip) started on the following day and lasted 10 d. Microvessel density (MVD) of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and after the last GCV dose. Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk-transfected HUVEC and HepG2 decreased from 100% to (28.94 +/- 5.67)% and (75.45 +/- 2.91)% at proper order, respectively (P < 0.01), while the survival rate of AdCMV-tk-transfected HUVEC and HepG2 declined from 100% to (17.56 +/- 2.48)% and (23.15 +/- 5.72)%, respectively (P > 0.05). Compared with group I, there was a decrease of tumor weight by 14.7% in group III and by 23.6% in group IV. And there was a distinct difference between group III and IV (P < 0.05). The median MVD for all groups was 37.4 +/- 8.6, 30.6 +/- 7.8, 27.6 +/- 7.1, and 10.7 +/- 4.1 (microvessels/mm(2)) in group I, II, III and IV, respectively. And there was a marked difference between group III and II (P < 0.05), IV and II (P < 0.01), and IV and III (P < 0.01). KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.

Renal Pathophysiology After Systemic Administration of Recombinant Adenovirus: Changes in Renal Cytochromes P450 Based on Vector Dose

Recombinant adenovirus (Ad) significantly alters hepatic cytochrome P450 (CYP). Because changes in renal function can alter hepatic CYP, the effect of Ad on renal CYPs 4A1, 4A2, 4F1, and 2E1 was evaluated. Male Sprague-Dawley rats were given one of six intravenous doses (5.7x10(6)-5.7x10(12) viral particles/kg [VP/kg]) of Ad expressing beta-galactosidase or saline. CYP protein, activity, gene expression, and serum creatinine (SCr) were evaluated 0.25, 1, 4, and 14 days later. Doses of 5.7x10(11) and 5.7x10(12) VP/kg increased CYP4A protein within 24 hr by 35 and 48%, respectively (p<0.05). A similar trend was observed on day 4. CYP4A1 mRNA doubled 6 hr after doses of 5.7x10(10)-10(12) VP/kg (p<0.01). Similar effects were observed 1 day after each dose tested. CYP4A2 gene expression was 20% above control 1 day after treatment with 5.7x10(10)-10(12) VP/kg and remained high through day 14. CYP4F1 expression was unaffected by all doses (p=0.08). CYP2E1 activity and gene expression were significantly suppressed 24 hr after administration of all doses and began to normalize by day 14 (p<0.01). SCr was significantly reduced (approximately 50%) throughout the study for doses at and below 5.7x10(11) VP/kg. SCr was increased by a factor of 3 by 5.7x10(12) VP/kg and glomerular filtration was significantly reduced (p<0.01). This suggests that changes in renal CYP and corresponding arachidonic acid metabolites may play a role in the documented toxicity associated with the systemic administration of recombinant Ad.

Generation of an adenoviral vaccine vector based on simian adenovirus 21

Adenoviral vectors can be used to generate potent humoral and cellular immune responses to transgene products. Use of adenoviral vectors based on non-human isolates may allow for their utilization in populations harbouring neutralizing antibodies to common human serotypes. A vector chimera was constructed using simian adenovirus 22 (a serotype belonging to the species Human adenovirus E) and simian adenovirus 21 (a serotype belonging to the species Human adenovirus B) expressing the Ebola (Zaire) virus glycoprotein (Ad C5/C1-ZGP). This chimeric adenovirus vector was used as a model to test its efficacy as a genetic vaccine and comparisons were made to a vector based on the commonly used human adenovirus C serotype 5 (Adhu5-ZGP). Ebola glycoprotein-specific T- and B-cell responses were measured in B10BR mice vaccinated with either Adhu5-ZGP or Ad C5/C1-ZGP vectors. Both vectors resulted in Ebola glycoprotein-specific gamma interferon-expressing T cells, although the Ad C5/C1-ZGP vector appeared to induce lower frequencies with kinetics slower than those elicited by the Adhu5-ZGP vector. The total immunoglobulin G response to Ebola glycoprotein was similar in sera from mice vaccinated with either vector. Two rhesus macaques vaccinated with the Ad C5/C1-ZGP vector were found to mount T-cell and antibody responses to the Ebola glycoprotein. It was found that a single administration of the chimeric Ad C5/C1-ZGP vector protected mice against a lethal challenge with a mouse-adapted strain of the Ebola (Zaire) virus.

Virology- and immunology-based gene therapy for cancer.

Current strategies for cancer gene therapy consist mainly of direct inhibition of tumor cell growth and activation of systemic host defense mechanisms. Conventional chemotherapy and radiotherapy, even considered to be temporally suppressing tumor growth, suppress immune responses; therefore, we examined potential clinical feasibility of virus-mediated tumor destruction, which can rather enhance immunity. We showed that human tumors were more susceptible to adenoviruses (Ad) in which the E1A expression was controlled by a putative tumor promoter than normal cells, and that a replication of the Ad was greater in tumor cells than in normal cells. We also demonstrated that the intratumoral injection of the Ad bearing a tumor promoter inhibited the subsequent tumor growth in vivo. The E1A expression was detected in the tumors injected with the Ad but not in non-tumorous tissues of the same mice. The Ad modified to show the regulated E1A expression is thereby oncolytic in nature. Antitumor immune responses are initiated after the acquisition of putative tumor antigen(s) by dendritic cells (DCs); therefore, enhanced antigen presentation is a crucial step for the early phase of cell-mediated immunity. Destruction of tumors can release the tumor antigens and DCs come to recognize them thereafter. We found that the stimulation of Fas expressed on DCs with Fas ligand (FasL) did not induce apoptosis of DCs but rather enhanced the antigen presentation. Activation of DCs induced production of a number of cytokines, and we showed that the interleukin-12 family secreted from tumors could induce systemic antitumor immunity. We presume that the administration of oncolytic Ad, which can destroy local tumors and subsequently make the putative tumor antigen(s) released from the tumors, stimulation of DCs with the Fas/FasL signal pathway and secretion of DCs-derived cytokines coordinately produce synergistic antitumor effects and that a combinatory application of these procedures can be a possible therapeutic strategy for cancer treatment.

72. Intravascular Interaction between Adenovirus and Platelets Promotes Adenovirus Sequestration, Degradation and Toxicity

Intravascular (iv) delivery of adenovirus (Ad) vectors is hindered by acute toxicity that is triggered by trapping of virus in the reticuloendothelial system (RES) within minutes of administration, and subsequent production of pro-inflammatory cytokines/chemokines. While Ad localizes to tissue macrophages and endothelial cells of the liver, spleen and lung within minutes of iv delivery, the mechanism of this reticuloendothelial targeting is unknown. We have identified a novel pathway by which platelets mediate trapping of Ad in the RES. Rapidly after iv administration fiber independent interaction between Ad and circulating platelets can be seen by Southern blot, by transmission electron microscopy (TEM) of isolated platelets and blood cell fractionation of mice receiving radiolabeled Ad. Blood platelet levels drop after Ad administration, while serum levels of the platelet activation marker soluble CD62p rise. Within 5 minutes of administration, Ad and platelets co-localize in the liver sinusoids along with endothelial and Kupffer cells. TEM showed Ad particles inside degranulated platelets and platelet aggregates within the liver sinusoids. Ad particles could also be seen inside Kupffer cells. Immunohistochemistry (IHC) showed widespread co-labelling of platelets (CD41), Kupffer cells (F4/80) and Ad (hexon) throughout the liver. In the spleen, Ad genomes were found within 5 minutes and Ad particles were detected in CD41 and F4/80 rich regions of the marginal zone by IHC. In the lung, Ad genomes were found within 5 minutes. Platelet accumulation was seen by IHC after delivery of Ad vectors, with platelet-endothelial interactions visible in the pulmonary vasculature by TEM. To determine the contribution of Ad-platelet interactions towards toxicity, mice were pre-injected with a platelet depleting antibody prior to virus delivery. At 6 hours post virus delivery, vector genomes in liver, lung and spleen were detected by Southern blot, and circulating serum cytokine levels were determined. Serum levels of the pro-inflammatory cytokines/chemokines IFN-|[gamma]|, IL-6 and MCP- 1 were lower in platelet depleted mice. The effect of Ad neutralizing antibodies on Ad-platelet interactions was also determined in mice that had been pre-immunized with the same vector prior to iv Ad vector administration. Early after vector administration significantly lower levels of Ad genomes were associated with circulating platelets in pre-immunized mice. While the levels of Ad deposition in the liver were unchanged in pre-immunized mice, there was reduced Ad deposition within the lungs of pre-immunized mice. Taken together these data suggest that rapid interaction between Ad and platelets occurs after iv administration. Platelet-Ad interactions apparently play a role in RES sequestration and degradation of Ad, and subsequent toxicity. We are currently attempting to identify the structural components involved in Ad-platelet interactions in vivo and investigating the contribution of this interaction to sequestration, degradation and toxicity, after Ad administration.

Intrahepatic injection of adenovirus reduces inflammation and increases gene transfer and therapeutic effect in mice

Recombinant adenoviruses (Ad) are among the most extensively used vectors for liver gene transfer. One of the major limitations for the clinical application of these vectors is the inflammatory immune response associated with systemic administration of high dose of virus. We evaluated the effect of Ad administration route on the inflammatory immune response and liver transgene expression. We compared direct intrahepatic injection (IH) with the systemic administration via tail vein (IV). IH injection of Ad resulted in a lower inflammatory response and a higher transgene expression. When a relatively low dose of virus was used, IV administration resulted in no detectable protein expression but production of proinflammatory cytokines. In contrast, IH administration induced high levels of transgene expression and no inflammation, although we detected a transient hypertransaminemia, which fully resolved within days. Furthermore, IH injection resulted in a faster protein expression being more intense at the site of injection, whereas IV administration caused slower but diffuse liver expression. IH injection also reduced the spreading of the virus to other organs. Independently of the route, depletion of Kupffer cells significantly enhanced the transduction efficiency of Ad. This effect was stronger when using IV injection, indicating that IH injection partially overcomes Kupffer cell phagocytic activity. Moreover, the antitumor efficacy of high-capacity-Ad encoding murine interleukin-12 (IL-12) was significantly greater when the vector was administered by IH injection than when given IV. In conclusion, IH injection of adenovirus represents a safe and efficient administration route for clinical applications of gene therapy targeting the liver.

Adenovirus Induced Thrombocytopenia: The Role of P-Selectin and von Willebrand Factor in Virus- Mediated Platelet Clearance.

Acute thrombocytopenia has been consistently reported following IV administration of adenoviral vectors (Ad) but the mechanism responsible for this phenomenon has not been elucidated. Thrombocytopenia appears 24 hours after IV administration of Ad and is vector dose dependent. In this study, we have assessed the potential roles of the adhesive proteins P-selectin and von Willebrand Factor (VWF) on the aggregation and clearance of platelets following virus administration. We have addressed the question of whether the thrombocytopenia is due to a direct effect of the virus on platelets or an indirect effect related to interaction of platelets with other proteins or cells modified by the virus. We assessed platelet count in a group of Balb/c and C57Bl/6 mice over 1 week period following Ad administration and performed a detailed examination of the events within the first 24 h after Ad injection, the period that precedes the appearance of thrombocytopenia. We examined the effect of Ad on expression of the platelet activation marker P-selectin and the formation of platelet leukocyte aggregates (PLA) by means of flowcytometry after incubation of adenovirus with mouse platelets in vitro, and following Ad administration in vivo. To assess the role of VWF in Ad-induced thrombocytopenia we measured plasma VWF levels one hour after injection of Ad. Further investigations involved comparison of platelet counts, platelet activation, and the formation of PLA in a group of VWF KO mice. All studies have been performed with a replication deficient E1/E3-deleted Ad 1x 1011 viral particles/mouse. Our in vitro studies have shown that Ad directly activates mouse platelets as shown by increased expression of P-selectin. The average index of platelet activation for platelets stimulated by Ad was 2519.4 compared to 128.2 for resting platelets (n=5, p<0.02). Flow cytometric analysis of CD41 (platelets) and CD45 (leucocytes) double stained positive events indicated that Ad stimulation induced PLA when compared to the unstimulated samples. Our in vivo studies have confirmed the development of significant thrombocytopenia in both Balb/c as well as C57Bl/6 WT mice (n=8, p=0.00001, n= 6, p=0.002) 24 hours following Ad administration. Significant P-selectin expression was documented in both strains (n=4,p=0.0003; n=3, p=0.0008 respectively) as well as significant PLA one hour following Ad (n=4, p=0.01; n=3, p=0.007). The VWF KO mice showed non-significant thrombocytopenia (n= 6, p=0.063) at 24 hours following Ad, significant P-selectin expression (n=3, p=0.0003), but no significant PLA formation at one hour (n=3 p=0.12) relative to pre-injection levels. Plasma VWF levels were significantly elevated in both Balb/c and C57Bl/6 WT mice one hour following administration of the virus (n= 3, p=0.02; n= 3, p= 0.001). The average plasma VWF levels were 48.1 U/mL at 1h compared to 5.7 U/mL pre injection in Balb /c mice and 85.9 U/mL compared to 6.1 U/mL in C57Bl/6 mice. These studies have shown that Ad can act as an inducer of mouse platelet activation and as a promoter for platelet-leukocyte association both in vitro and in vivo. We have demonstrated a role for Ad in stimulating VWF release from the endothelium, and have shown that VWF has a critical role in platelet activation and clearance following Ad administration. We conclude that P-selectin and VWF proteins are directly involved in interactions between endothelial cells, platelets and leukocytes, a complex interaction that can explain at least in part the mechanisms underlying Ad-mediated thrombocytopenia.

Dexamethasone Promotes Transgene Expression and Supresses the Early Immune /Inflammatory Response in Adenovirus- Mediated Gene Delivery to Hemophilic Mice.

The major limitation of adenovirus-mediated gene therapy is the generation of immune/inflammatory responses that result in host toxicity and impair gene transfer efficiency. Adenovirus vectors (Ad) activate the innate arm of the immune response resulting in systemic inflammation and cytotoxicity to transduced tissues. In addition to these systemic effects, endothelial cell interactions play a critical role in the host response to Ad vectors. Dexamethasone (DEX) is one of the long acting corticosteroid preparations that acts as a potent anti-inflammatory and immune suppression agent with a wide variety of clinical applications. We investigated the effects of Dexamethasone as an anti-inflammatory agent on the outcome of adenoviral gene delivery of FVIII to a group of Balb/C hemophilic mice. We have used an E1/E3 deleted Ad vector expressing B domain deleted version of the canine FVIII gene under the transcriptional control of a liver-restricted promoter. All mice received 1 x 10 11 viral particles/ mouse. Dexamethasone at 1.5 mg/Kg was administered SC, 24h before adenovirus injection and the treatment regimen was continued twice a week for 4 weeks post virus administration. We compared the outcome of gene therapy in mice treated with dexamethasone (DEX+Ad group) to a group of mice that received adenovirus only (Ad only group) as well as saline and DEX only controls. We assessed the outcome of the gene therapy by measuring FVIII levels, acute liver injury by means of Alanine aminotransferase enzyme (ALT), the principal cytokine TNF alpha, platelet count and Hb levels. Compared to the Ad only group, the DEX+ Ad group showed significant elevation of FVIII expression levels at day 2, 1w, 2w and 4w post Adenovirus administration (n=3, p= 0.05, 0.002, 0.0002 and 0.005 respectively). DEX resulted in 2.7, 1.6, 2.7and 2.5 fold increase in transgene expression in these time points over the Ad only group. There was a significant reduction of TNF alpha levels 5 hours post Ad administration in the DEX+Ad group (n=3, p< 0.002) when compared to the Ad only group. This corresponded to 4-fold reduction in cytokine release. With regards acute hepatotoxicity, ALT levels were significantly reduced at 1w post administration in the DEX+ Ad group Vs Ad only group. The average ALT levels represented a 5.3 fold elevation and only 1.4 fold elevation relative to the pre vector administration levels in the Ad only group Vs DEX+Ad. Dexamathasone did not appear to have a significant effect on the acute transient thrombocytopenia. In each of the DEX+Ad group and Ad only group, the reduction in platelet count at 24 h post Ad injection was significant when compared to the pre injection level (n=3, p<0.01, p< 0.003 respectively). Monitoring HB level over 4 weeks did not show any significant alteration with respect to the use of DEX when compared the Ad only treated mice (n=3, p: 0.09–0.4 throughout).Our results indicate a beneficial effect of coverage with Dexamethasone to prevent some of the early adverse host effects that are associated with adenovirus gene therapy. This study provides a rationale for the further assessment of this anti-inflammatory agent as a useful tool in adenovirus- based gene transfer protocols.

148. The Transgene Cassette Is Not Fully Responsible for Alterations of Renal Cytochrome P450 Expression after Systemic Administration of Recombinant Adenovirus

Gene transfer to the kidney has shown promise in treating severe renal disease and prevention of acute renal graft rejection in several animal models. To date, recombinant adenovirus (Ad) is the most efficient vector for gene delivery to the kidney. Although Ad does not infect the kidney efficiently when administered systemically, transgene products secreted from the liver can reach the kidney at therapeutic levels. We have found that systemic administration of recombinant Ad significantly alters the expression of the cytochrome P450 (CYP) enzymes, largely responsible for the clearance of toxicants and the metabolism of xenobiotics and endogenous compounds, in the kidney. In order to determine if the transgene cassette could be partially responsible for this effect, Sprague-Dawley rats received one of four treatments via a surgically implanted catheter: 0.5 ml of phosphate-buffered saline (control) or 5.7|[times]|1012 viral particles/kg of a first generation Ad expressing either E.coli beta-galactosidase (AdlacZ), murine erythropoietin (Epo) or no transgene (Null). Animals were sacrificed 0.25, 1, 4 and 14 days after injection. Renal CYP protein expression and mRNA levels were analyzed by Western blot and RT-PCR. All treatments significantly induced protein levels of the CYP4A family. Following AdlacZ administration, CYP4A increased by 50%, 32% and 64% above control at 0.25, 1 and 4 days, respectively (p<0.01). The vector expressing Epo raised CYP4A protein levels by 42%, 24% and 64% and the Null vector produced elevations of 74%, 25% and 49% above the control at the same timepoints (p<0.01). CYP4A protein expression recovered by 14 days in each group. RT-PCR revealed that mRNA levels of CYP4A2, the primary CYP4A isoform responsible for renal fatty acid metabolism, increased by 16.8% and 19% above control 24 hours after administration of AdlacZ and Epo, respectively (p<0.01). CYP4F1, an isoform responsible for metabolism of fatty acids, prostaglandins and leukotrienes, was unaffected in all treatment groups. In contrast, renal CYP2E1, which metabolizes small lipophilic compounds and carcinogenic agents, was significantly reduced throughout the study. CYP2E1 mRNA levels in animals treated with AdlacZ were 74%, 78%, 69% and 66% of control at 0.25, 1, 4 and 14 days, respectively (p<0.01). Similar reductions were seen with Epo and Null vectors although levels form animals treated with Epo began to recover by 14 days. Real time quantitative PCR revealed that viral genomes (|[sim]|850/100 ng DNA) were initially present in the kidney and declined throughout the study for all treatments. These results suggest that systemic administration of Ad significantly altered the transcription and translation of several key CYP enzymes in the kidney regardless of the transgene expression cassette. This should be considered for accurate drug monitoring when employing recombinant Ad vectors in the clinic. Additional mechanistic studies assessing the effect of expression of Ad early genes and local and systemic eicosanoid biosynthesis on the expression of renal CYP will allow further explanation of these results.

991. Adenovirus-Mediated IL-12 Gene Transfer Increase Virus Specific CD8 T Cell Response in Aged Mice

Top of pageAbstract Respiratory viral infection is a major cause of morbidity and mortality in the elderly population. IL-12 and other Th1 cytokines can prevent the age-related defect in the cytotoxic T lymphocyte (CTL) response by blocking the age-associated down modulation of CD28 and immune senescence of CD8+ T cells. Gene therapy is one method to restore defective Th1 cytokines and reverse immune senescence. However, the effect of IL-12 on the generation and effector function of the viral epitope-specific cytotoxic CD8 T cells (CTLs) after delivery by an adenovirus vector has been difficult to dissect due to the lack of appropriate tetramers capable of identifying the virus-specific CTL response. This limitation can now be overcome by the use of our recently developed MHC class I tetramer containing the adenovirus (Ad) epitope peptide E1Bp, referred to as Db-E1Bp, in combination with the use of an in vivo killing assay to analyze the virus specific CD8 T cell response in young and old mice after Ad administration. To determine the effect of IL-12 on the generation and effector function of the virus-specific CTLs, we utilized an recombinant Ad vector to deliver the IL-12 gene into aged (16-mo-old) and young (2-mo-old) C57BL/6 (B6; H-2b) mice before infected with a wild-type Ad5. The primary CTL response was assayed by the Db-E1Bp tetramer and E1Bp-specific in vivo killing assay. Our results indicate that there was a 58% decrease in the production of virus-specific Db-E1Bp+CD8+ T cells in aged mice at day 8 after Ad5 infection, compared with that from young mice (p<0.01; Table). This was associated with a 30% decrease in the specific killing activity by these CTLs in aged mice (p<0.01). In vivo pretreatment of aged mice with Ad-IL12 resulted in an increase in serum IL-12, from 18|[plusmn]|7 ng/ml before pretreatment to 96|[plusmn]|12 ng/ml at the time of Ad5 infection 3 days later. Ad-IL12 pretreatment also induced strong IFNg production of 34|[plusmn]|11 ng/ml at the time of Ad5 injection compared to the base line of 0.5|[plusmn]|0.2 ng/ml. There was only a moderate increase of these cytokines in young mice. However, the serum IL-12 correlated with IFNg production in both old (r2=0.889; p<0.001) and young (r2=0.946; p<0.001) mice. At day 8 after Ad5 infection, both the Db-E1Bp+CD8+ /total CD8+ T cells and E1Bp specific lysis efficiency in the spleen and liver of aged mice increased to levels comparable to those of young mice (Table). This was associated with an increased thymocyte proliferative response mediated through IL-2 and IL-7 in aged mice. Our results indicate that aged mice have a defect in the CTL response to Ad infection, and that Ad-IL12 treatment significantly enhances the Ad-specific CTL response in aged mice, partially by induction of IFN|[gamma]|.

1017. Selective Induction of Tumor-Associated Antigens in Murine Pulmonary Vasculature Using Double-Targeted Adenoviral Vectors

Targeted therapies directed to tumor-associated antigens are being investigated for the treatment of cancer. However, there are few suitable animal models for testing the ability to target these tumor markers. Therefore, we have exploited mice transgenic for the human coxsackie and adenovirus receptor (hCAR) to establish a new model for transient expression of human tumor-associated antigens in the pulmonary vasculature. Systemic administration of Ad in hCAR mice resulted in an increase in transgene expression in the lungs compared to wild type mice, as determined using a luciferase reporter gene. To reduce transgene expression in the liver, the predominant organ of ectopic Ad localization and transgene expression following systemic administration, we utilized the endothelial-specific flt-1 promoter, which resulted in a further increased lung-to-liver ratio of luciferase expression. Administration of an adenoviral vector encoding the tumor-associated antigen carcinoembryonic antigen (CEA) under transcriptional control of the flt-1 promoter resulted in selective expression of this antigen in the pulmonary vasculature of hCAR mice. Feasibility of targeting to expressed CEA was subsequently demonstrated using adenoviral vectors pre-incubated with a bifunctional adapter molecule recognizing this tumor-associated antigen, thus demonstrating utility of this transient transgenic animal model.

Selective induction of tumor-associated antigens in murine pulmonary vasculature using double-targeted adenoviral vectors

Targeted therapies directed to tumor-associated antigens are being investigated for the treatment of cancer. However, there are few suitable animal models for testing the ability to target these tumor markers. Therefore, we have exploited mice transgenic for the human coxsackie and adenovirus receptor (hCAR) to establish a new model for transient expression of human tumor-associated antigens in the pulmonary vasculature. Systemic administration of Ad in hCAR mice resulted in an increase in transgene expression in the lungs compared to wild-type mice, as determined using a luciferase reporter gene. To reduce transgene expression in the liver, the predominant organ of ectopic Ad localization and transgene expression following systemic administration, we utilized the endothelial-specific flt-1 promoter, which resulted in a further increased lung-to-liver ratio of luciferase expression. Administration of an adenoviral vector encoding the tumor-associated antigen carcinoembryonic antigen (CEA) under transcriptional control of the flt-1 promoter resulted in selective expression of this antigen in the pulmonary vasculature of hCAR mice. Feasibility of targeting to expressed CEA was subsequently demonstrated using adenoviral vectors preincubated with a bifunctional adapter molecule recognizing this tumor-associated antigen, thus demonstrating utility of this transient transgenic animal model.

Protective Immunity Against α-Cobratoxin Following a Single Administration of a Genetic Vaccine Encoding a Non-Toxic Cobratoxin Variant

Venomous snakebites result in almost 125,000 deaths per year worldwide. We present a new paradigm for the development of vaccines to protect against snakebite, using knowledge of the structure and action of specific toxins combined with a gene-based strategy to deliver a toxin gene modified to render it non-toxic while maintaining its three-dimensional structure and hence its ability to function as an immunogen. As a model for this approach, we developed a genetic vaccine to protect against alpha-cobratoxin (CTX), a potent, post-synaptic neurotoxin that is the major toxic component of the venom of Naja kaouthia, the monocellate cobra. To develop the vaccine, substitutions in the CTX cDNA were introduced at two residues critical for binding to the nicotinic acetylcholine receptor (Asp27 to Arg, Arg33 to Gly). The mutated CTX expression cassette was delivered in the context of a replication deficient adenovirus vector (AdmCTX). To assess whether expression of the mutated CTX in vivo leads to the development of protective immunity, BALB/c mice were challenged by IV administration of 2 microg of alpha-cobratoxin protein 21 or 63 days after administration of AdmCTX or Ad- Null (as a control; both, 10(9) particle units). Animals receiving AdmCTX but no alpha-cobratoxin challenge suffered no ill effects, but > or =80% of naive animals or those receiving the AdNull control vector died within 10 min from the alpha-cobratoxin challenge. In contrast, 100% of animals receiving a single dose of AdmCTX 21 or 63 days prior to alpha-cobratoxin challenge survived. The data demonstrates that an adenovirus-based vaccine can be developed to protect against lethal challenge with a potent snake venom. The effectiveness of this approach might serve as a basis to consider the development of a global public health program to protect those at risk for death by snakebite.

Evaluation of toxicity from high-dose systemic administration of recombinant adenovirus vector in vector-naive and pre-immunized mice.

Toxicity associated with in vivo administration of adenovirus (Ad) vectors has been linked to activation of both innate and adaptive immune responses. Pre-existing immunity to the prevalent Ad serotypes, acquired by the majority of the human population as a result of natural infections, has the potential to modulate vector efficacy and safety. Previously, we evaluated some aspects of toxicity from systemic Ad vector in vector-naive and pre-immunized rhesus monkeys. In this report, we summarize data from several studies analyzing toxic effects from systemically administered E1/E3-deleted Ad vector in vector-naive and pre-immunized C57BL/6 mice. Our results indicate that pre-immunization can be associated with increased mortality shortly after systemic administration of Ad. Transient leukopenia and thrombocytopenia were observed early post vector infusion in both vector-naive and pre-immunized animals. Pre-exposure to the vector did not prevent induction of pro-inflammatory cytokines; however, pre-immunized mice showed less tissue toxicity. Growth of bone marrow myeloid and erythroid progenitors was transiently inhibited in pre-immunized animals, but only the myeloid progenitors were affected in vector-naive animals. In summary, pre-existing immunity to Ad vector substantially modifies host immune responses to systemic Ad vector.

Considerations for Use of Recombinant Adenoviral Vectors: Dose Effect on Hepatic Cytochromes P450

Recombinant adenovirus (Ad) serotype 5 is a vector commonly used for gene delivery. Although this vector has a natural tropism for the liver, there is a limited understanding of how Ad administration affects one of the primary hepatic processes, drug metabolism. The effects of systemic administration of a model recombinant adenoviral vector on two hepatic cytochrome P450 (P450) enzymes, CYP3A2 and 2C11, were investigated. Sprague-Dawley rats were treated with one of six vector doses, ranging from 5.7 x 10(6) to 5.7 x 10(12) virus particles (vp)/kg. Hepatic P450 protein expression, catalytic activity, and mRNA levels were measured over 14 days. Ad administration (5.7 x 10(10)-5.7 x 10(12) vp/kg) reduced CYP3A2 over the duration of the study. Six hours after administration of 5.7 x 10(12) vp/kg, CYP3A2 activity and mRNA levels were suppressed by 45 and 65%, respectively (P < or = 0.01). This continued throughout the study with levels dropping to 36 and 45% of controls by 14 days, respectively (P < or = 0.01). A similar trend was detected for CYP2C11 within this dosing range. Administration of 5.7 x 10(6), 5.7 x 10(8), and 5.7 x 10(9) vp/kg of Ad significantly increased both CYP2C11 protein expression by 86, 71, and 107% and activity 110, 118, and 53%, respectively, above those of animals treated with saline (P < or = 0.01). These results clearly indicate that a single dose of adenovirus significantly alters key drug metabolizing enzymes for an extended period of time and should be investigated further in the context of the design and implementation of clinical trial protocols.

Sustained Muscle Expression of Dystrophin from a High-Capacity Adenoviral Vector with Systemic Gene Transfer of T Cell Costimulatory Blockade

Adenoviral vector (Ad)-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD). However, cellular and humoral immune responses induced by vector gene transfer limit the application of this approach. Blockade of the costimulatory interaction between naïve T cells and antigen-presenting cells has proven to be a successful means to diminish immunity induced by gene transfer. In this study we explore the potential of supplementing dystrophin gene delivery to dystrophin-deficient Dmd mouse skeletal muscle with systemic gene delivery of CTLA4Ig and CD40Ig molecules to effect costimulatory blockade. We found that systemic administration of a high-capacity Ad (HC-Ad) vector carrying murine CTLA4Ig (AdmCTLA4Ig) either alone or codelivered with an HC-Ad vector carrying murine CD40Ig (AdmCD40Ig) provided sustained expression of recombinant full-length murine dystrophin from an HC-Ad vector carrying the dystrophin cDNA (AdmDys). The level of AdmDys vector genomes remained stable in animals cotreated with systemic delivery of vectors carrying molecules to block costimulation. In addition, muscle CD4+ and CD8+ T cell infiltrates and Th1 cytokine production by splenocytes were reduced. The production of neutralizing antibody against Ad vector was significantly inhibited in mice receiving systemic codelivery of both AdmCTLA4Ig and AdmCD40Ig, but not in the mice treated with AdmCTLA4Ig alone. The results suggested that coblockade of both CD28/B7 and CD40L/CD40 costimulatory pathways is required for effective inhibition of the Ad vector-induced humoral immune response in Dmd mice, whereas blockade of CD28/B7 alone by murine CTLA4Ig would be sufficient for prolonged dystrophin expression in treated muscle.

1010. Interference with the IL-1 Signaling Pathway Significantly Improves the Toxicity Profile of Systemically Applied Adenovirus Vectors

Adenovirus vectors (Ad) are the second largest group of viral vectors that are extensively used in clinical trials in the US as prospective therapeutics against numerous inborn and acquired human diseases, including cancer. Most recently, interest in Ad has further expanded due to its potential as a vector for vaccination against life threatening infectious agents such as anthrax. It is currently recognized that manifestations of virus-related systemic toxicity greatly impairs safety and efficacy of Ad vectors. To better understand the molecular mechanisms and primary mediators involved in the initiation of the innate host response toward systemically applied Ad, and to analyze the feasibility of pharmacological management of Ad-mediated toxicity, we administered Ad5-based vectors intravenously into both wild type C57BL/6 and genetically manipulated mice knocked out for specific signaling pathways involved in activating and maintaining an innate immune response. At different times post virus injection, the hepatic mRNAs and plasma levels of different cytokines/chemokines were evaluated using an RNAse protection assay and mouse inflammatory cytometric bead arrays. These analyses revealed that immediately following systemic Ad application, the transcription of IL-1 and MIP-2 genes in the liver was up-regulated more than 30 times over their baseline. Although TNFα gene transcription was slightly up-regulated at 30 min post virus injection, its maximum levels were not reached before 6 h post virus administration. Importantly, although TNFα signaling was clearly involved in the host response toward systemically applied Ad, our data demonstrate that TNF signaling mediates a delayed rather than an immediate response, and that TNFα is not a primary mediator, initiating anti-Ad host inflammatory responses. Thus, pre-injection of wild type mice with anti-TNFα antibody prior to virus administration could neither reduce the levels of immediate IL-1 and MIP-2 gene up-regulation nor alleviate liver pathology seen following Ad administration. Using capsid-modified Ad vectors which have a reduced capacity to transduce liver cells in vivo, we found that the level of MIP-2 gene up-regulation directly correlates with the level of IL-1 gene transcription. Furthermore, we found that in wild type mice, the level of IL-1 gene up-regulation depends on the level of Ad hepatocyte transduction. Interference with IL-1 signaling pathways by pre-injection of mice with anti-IL-1α/β antibody dramatically reduced both the levels of immediate cytokine gene up-regulation and liver damage caused by systemic Ad administration. Importantly, interference with IL-1 signaling pathways did not affect the ability to Ad vectors to efficiently transduce liver cells in vivo. Our data provide new insights into molecular mechanisms involved in host innate immune and inflammatory responses toward systemically applied Ad. They also provide a rational approach for pharmacological management of anti-Ad host responses, allowing for significant improvement of the safety profiles of existing Ad vectors while preserving their gene transfer efficiency.

640. E1 Bp-Specific CD8+ T Lymphocytes Are Highly Induced in the Liver and Spleen Eight Days after Adenoviral Infection

Top of pageAbstract The use of Adenovirus (Ad) as a gene delivery vector is limited by transient gene expression due to the host cytotoxic T lymphocyte (CTL) response and innate responses. We and others have shown that the cytotoxic response to Ad is only partially inhibited by administration of Ad to mutant mice deficient in producing a cytotoxic immune response, or by treatment with TNF inhibitors. However, analysis of the specific CTL response to Ad has not been possible due to the lack of reagents that recognize the rearranged T cell receptor expressed on Ad-specific cytotoxic T cells. We therefore developed a tetramer that recognizes the immunodominant epitope of E1B (192 VNIRNCCYI), referred to as E1Bp, in the context of class Ib MHC. The tetramer consists of H2-Db, β2-microglobulin and the peptide epitope, and is referred to as Db-E1Bp. First, to test the specificity of this peptide-MHC complex, C57BL/6 (H-2b; B6) mice were injected with 8×108i.u. of wild type (wt) Ad5. As a control, wt Ad5 injected DBA/2 (H-2d; D2) were analyzed. Lymphocytes (105 )isolated 8 days later from spleen or liver were restimulated with E1Bp in vitro and analyzed for IFN-γ production by an ELISPOT assay. There was a significant increase in spots (6 and 63 spots, respectively) compared to cells stimulated with human influenza virus peptide M1(58 GILGFVFTL) (0 spot, p 98%) of the Db-E1Bp+CD8+ T cells are CD44+. No Db-E1Bp+CD8+ T cells were found in immunized D2 or naive B6 mice. To determine peptide-specific lysis of target cells in vivo, we labeled target cells from syngeneic mice with either high (2μM; CFSEHi) or low (0.2μM; CFSELo) concentration of CFSE and then pulsed CFSEHi cells with E1Bp and CFSELo cells remained unpulsed. Target cells were adoptively transferred into B6 or D2 mice that were challenged 8 days earlier with wt Ad5, and the percentage of specific killing was determined 6 hours later. As a peptide specificity control, CFSEHi cells pulsed with M1 peptide and unpulsed CFSELo cells were transferred to identically immunized B6 mice. E1Bp-specific lysis was significantly increased in B6 mice compared to control mice or immunized D2 mice (p<0.001). There was a significant correlation between E1Bp-specific lysis and the frequency of Db-E1Bp+ CD8+ T cells in B6 mice (p<0.01). We next used these methods to measure the decreased Ad-specific CTL response in aged mice. Compared with 2-mo-old mice, there was a 30% decrease in killing efficiency in 18-mo-old mice and a 58% decrease in Db-E1Bp+CD8+ T cells. Our results indicate that this tetramer is novel, sensitive and specific tool to detect and follow the E1Bp-specific CTL response against Ad infection.

23. T Cell Carriers for Gene Delivery to Tumors

Recurrent colorectal adenocarcinoma can be treated by surgery and chemotherapy, but effects on length of survival time are generally modest. Therefore, identification of new therapeutic modalities is of great importance to improve the survival of these patients. Adenoviral gene delivery systems have shown promise as cancer therapeutics, but insufficient or non-selective transduction of tumor cells remains an issue. Targeting adenoviral vectors (Ads) to tumor-associated antigens, such as carcinoembryonic antigen (CEA, which is overexpressed in colon carcinomas), may overcome this problem. Ads can be targeted using %targeting adapter molecules', such as sCAR-scFv fusion proteins. One part of these fusion proteins consists of the ectodomain of the Coxsackie and Adenovirus Receptor (sCAR, the primary receptor for Ad), which binds to the adenoviral particle. The other part consists of a CEA recognizing modality, such as a single chain antibody (scFv). In vitro, these fusion proteins have already shown great promise for targeting Ads to cancer cells. However, availability of animal models in which CEA is expressed is limited. This has hampered the subsequent in vivo characterization of these sCAR-scFv fusion proteins. We hypothesized that we could overcome these problems by using a transgenic hCAR mouse (a kind gift of Dr. S. Petterson, Sweden), in combination with Ads encoding CEA under transcriptional control of the endothelial specific flt-1 promoter to induce CEA in the pulmonary vasculature of these mice. In this manner we could define key Ad targeting issues, independent of specific tumor biology factors. First, Ad biodistribution in hCAR mice was determined by injecting a GFP labeled Ad (AdIXEGFP) in the inferior vena cava. Compared to wild type mice, hCAR mice showed an increased uptake of AdIXEGFP particles in the lungs. Second, transgene biodistribution was determined by administering a luciferase-encoding Ad (AdCMVLuc) in the tail vein. As expected from the change in biodistribution, hCAR mice showed an increase in luciferase activity in the lungs. However, luciferase expression in the liver, the main organ of ectopic transgene expression after systemic Ad administration, was high as well. To increase selectivity of transgene expression in the lungs, we employed the endothelial specific promoter flt-1. This resulted in significantly reduced liver luciferase expression, thereby increasing the lung/liver ratio by several orders of magnitude. Finally, AdfltCEA was administered to study cellular distribution of CEA using immunohistochemical techniques, showing that CEA transgene expression in the lungs was largely confined to pulmonary endothelial cells. Thus, selective expression of CEA in the pulmonary vasculature by use of the here-described in vivo system provides a valuable tool for transductional targeting studies. The targeting adapter molecule sCAR-scFv, recognizing CEA, can now be tested for its targeting potential in this %transient transgenic' mouse model. This is of vital importance before translation to a clinical setting for the treatment of colon carcinoma can be endeavored.