Ad Gene Research Articles

Adenovirus types 11p and 35 attach to and infect primary lymphocytes and monocytes, but hexon expression in T-cells requires prior activation

Hematopoietic cells are attractive targets for gene therapy, but the conventional adenovirus (Ad) vectors, based on Ad5, transduce these cells inefficiently. One reason for low permissiveness of hematopoietic cells to infection by species C Ads appears to be inefficient attachment. Vectors pseudotyped with species B fibers are clearly more efficient at transducing hematopoietic cells than Ad5. To evaluate which Ad species B type(s) would be the most efficient vector(s) for primary T-cells, B-cells and monocytes, attachment to and entry of the species B1 serotypes 3p and 7p and the species B2 serotypes 11p and 35 into primary PBMCs was studied. Ad11p and Ad35 were the only serotypes to show efficient binding and for which uptake by PBMCs could be detected. Infection of PBMCs by Ad5, Ad11p and Ad35 was compared. Expression of Ad hexons was detected in stimulated PBMCs, most frequently in T-cells, and in unstimulated monocytes, although B-cells appear to be refractory to productive infection. Replication of Ad DNA was severely restricted in most PBMCs. Neither hexon expression nor genome replication could be detected in unstimulated lymphocytes, but FISH and a real-time PCR-based assay suggested that Ad11p and Ad35 DNA reach the nucleus. Activation thus appears to be required for T-cells to be permissive to Ad gene expression. In summary, there are substantial differences between Ad3p and Ad7p on the one hand and Ad11p and Ad35 on the other, in their ability to interact with PBMCs. Ad11p and Ad35 probably represent vectors of choice for these cell types.

980. In Vivo, High Throughput Analysis Expands the Known Anti-Ad Innate Immune Response Profile, and Implicates TLR Pathways in Ad Cellular Immune Response

We exploited high titer adenoviral (Ad) vectors to more fully investigate the innate humoral and cellular responses to Ad gene transfer in vivo. As a result of this high throughput analysis, we have delineated a broader, and more potent innate humoral and cellular transcriptome response to Ad than has been previously reported. For example, gene array analysis revealed significant dysregulation in 20% of 12,000 RNA transcripts derived from the livers of Ad infected mice within hours of Ad injection.

1108. High Rate of Anti-Tumor Immune Responses in a Phase 1 Clinical Trial of Adenoviral-Interferon-Beta Gene Transfer for Malignant Mesothelioma (MM) and Malignant Pleural Effusions (MPE)

Our group recently completed a Phase 1 trial of intrapleural interferon-beta gene transfer of Ad.huIFNbeta (Biogen-IDEC) in 10 patients with MM and MPE. Vector doses of 9x1011 and 3x1012 viral particles were used. Here, we report the anti-tumor immunologic responses observed.

Long-term Follow-up of Patients with Malignant Pleural Mesothelioma Receiving High-Dose Adenovirus Herpes Simplex Thymidine Kinase/Ganciclovir Suicide Gene Therapy

Delineation of the long-term follow-up data on a series of patients with malignant mesothelioma, who received a single intrapleural dose of a nonreplicative adenoviral (Ad) vector encoding the herpes simplex virus thymidine kinase "suicide gene" (Ad.HSVtk) in combination with systemic ganciclovir. This report focuses on the 21 patients receiving "high-dose" therapy, defined by an intrapleural dose of vector (> or =1.6 x 10(13) viral particles), where transgene-encoded tk protein was reliably identified on immunohistochemical staining. In 13 patients, the vector was deleted in the E1 and E3 regions of the Ad; in the other eight patients, the vector had deletions in the Ad genes E1 and E4. Safety, immunologic responses, transgene expression, and clinical responses were evaluated. Both the E1/E3-deleted vector and the E1/E4-deleted vector were well tolerated and safe, although production of the E1/E4 vector was more difficult. Posttreatment antibody responses against the tumors were consistently seen. Interestingly, we observed a number of clinical responses in our patients, including two long-term (>6.5 year) survivors, both of whom were treated with the E1/E4-deleted vector. Intrapleural Ad.HSVtk/ganciclovir is safe and well tolerated in mesothelioma patients and resulted in long-term durable responses in two patients. Given the limited amount of gene transfer observed, we postulate that Ad.HSVtk may have been effective due to induction of antitumor immune responses. We hypothesize that approaches aiming to augment the immune effects of Ad gene transfer (i.e., with the use of cytokines) may lead to increased numbers of therapeutic responses in otherwise untreatable pleural malignancies.

Control of adenovirus packaging

The results of studies of Adenovirus have contributed to our basic understanding of the molecular biology of the cell. While a great body of knowledge has been developed concerning Ad gene expression, viral replication, and effects on the infected host, the molecular details of the assembly process of Adenovirus particles are largely unknown. In this article, we would like to propose a theoretical model for the packaging and assembly of Adenovirus and present an overview of the studies that have contributed to our present understanding. In particular, we will summarize the molecular details of the process for packaging of viral DNA into virus particles and highlight the events in packaging and assembly that require further study.

Brain activation in offspring of AD cases corresponds to 10q linkage

Previously, we reported evidence of genetic heterogeneity in late-onset familial Alzheimer's disease, based on sex of affected parent, demonstrating linkage to chromosome 10q, a region identified by other groups and implicated as a quantitative trait loci for Abeta levels, in families with an affected mother. Using functional magnetic resonance imaging and a memory encoding task, we now show differential brain activation patterns among asymptomatic offspring which correspond to the previous linkage finding. These results suggest the possibility that activation patterns may prove useful as a preclinical quantitative trait related to the putative familial late-onset AD gene in this chromosome 10 region.

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

The safety of gene therapy vectors is a major concern when novel viral or nonviral therapeutics are proposed for applications in humans. Adenovirus (Ad) vectors have been extensively used as efficient gene delivery vehicles in vitro over the last two decades. However, upon i.v. application, they elicit robust innate and inflammatory responses that may be fatal for the host. To date, the primary cytokines and chemokines involved in the initiation of these host responses remain illusive. In this study, we demonstrate that IL-1 is a major mediator involved in the initiation of immediate host responses toward i.v. applied Ad vectors. Using mice in which IL-1 signaling was genetically eliminated (IL-1RI-KO), or wild-type animals for which signaling was blocked by anti-IL-1 Abs, we found that i.v. applied Ad vectors elicited dramatically reduced acute inflammatory responses when compared with control animals. Importantly, the efficiency of Ad gene transfer in vivo was not significantly affected by interfering with IL-1 signaling. Using an in situ hybridization technique, we found that hepatocytes and Kupffer cells trigger IL-1 transcription in liver tissue after i.v. Ad vector administration. We also found that expression of the MIP-2 chemokine gene (which is responsible for recruitment of neutrophils to the liver) depends on IL-1 activation. Our data indicate that immediate innate and inflammatory host responses toward i.v. applied Ad vectors can be pharmacologically controlled through interference with IL-1 signaling pathways.

67. Protective Immunity Against Yersinia pestis in Mice Immunized with an Ad-Based Vaccine Vector Expressing the Y. pestis F1 and V Antigens

The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, an extremely virulent and rapidly fatal disease that develops within days post-infection. In the context of a bioterror threat, an effective anti-plague vaccine must elicit rapid, robust and long acting protective immunity after a single dose. At present, no plague vaccines are available for use in the USA. Two subunits of Y. pestis, fraction 1 (F1) and V antigen have been identified as vaccine candidates based on: (1) F1 antigen is a temperature-regulated capsular protein of Y. pestis with anti-phagocytic activity in the host; (2) V antigen has local anti-inflammatory effects and influences the function of important virulence factors such as the Yersinia outer membrane proteins (Yops). It is known that protective immunity against Y. pestis challenge can be induced by multiple immunizations with recombinant F1 and V proteins and immediate protection can be achieved by passive transfer of immune sera against these antigens. Based on the knowledge that adenovirus (Ad) gene transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, replication-defective Ad gene transfer vectors encoding either the Y. pestis V antigen or the F1 protein (AdsecV and AdsecF1, respectively) were constructed. The F1 and V antigen genes were synthesized by an overlap PCR strategy using mammalian preferred codons, and fused to the human Ig signal sequence for extracellular secretion. We tested the hypothesis that AdsecV and AdsecF1 can stimulate strong protective immune responses without a requirement for repeat administration. To test the efficacy of the vaccines, BALB/c mice were immunized with a single intramuscular administration of either vector at a dose of 108 to 1011 particles units (pu). Four wk after immunization, mice were challenged in a BSL3 experimental animal facility (n=10/group) by the intranasal route with 3103 cfu of the fully virulent Yersinia pestis strain, CO92. At challenge time, anti-V antigen IgG reciprocal titers from AdsecV–immunized mice (n=10/group) were 666137 (108 pu), 3045735 (109 pu), 97032193 (1010 pu), and 7611215914 (1011 pu). In contrast, anti-F1 antigen IgG reciprocal titers from AdsecF1-immunized mice (n=10/group) were much lower with 122 (108 pu), 9135 (109 pu), 1154138 (1010 pu), and 939157 (1011 pu). All (10/10) of the animals survived the Y. pestis challenge in the group vaccinated with 1011 pu AdsecV, and 40% (4/10) survived in the 1010 pu AdsecV dose, while in all the other experimental groups immunized with AdsecV and in the control groups the mice died within 2 to 5 days after challenge. No mice vaccinated with AdsecF1 survived the Y. pestis challenge. These observations suggest that an Ad-based vaccine encoding for the V antigen of Y. pestis (AdsecV) is an excellent candidate as a effective single dose vaccine against the plague.

Effect of Enhanced Prostacyclin Synthesis by Adenovirus‐Mediated Transfer on Lipopolysaccharide Stimulation in Neuron‐Glia Cultures

Prostacyclin (PGI2) is known as a short-lived, potent vasodilator and platelet anti-aggregatory eicosanoid. This work attempts to selectively augment PGI2 synthesis in neuron-glia cultures by adenoviral (Ad) gene transfer of PGI synthase (PGIS) or bicistronic cyclooxygenase 1 (COX-1)/PGIS and examines whether PGI2 confers protection against lipopolysaccharide (LPS) stimulation. Cultures released low levels of eicosanoids. Upon Ad-PGIS or Ad-COX-1/PGIS infection, cultures selectively increased prostacyclin release. Both PGIS- and COX-1/PGIS-overexpressed cultures contained fewer microglial numbers. Further, they significantly attenuated LPS-induced iNOS expression and lactate, nitric oxide, and TNF-alpha production. Taken together, enhanced prostacyclin synthesis in neuron-glial cultures reduced microglia number and suppressed LPS stimulation.

528. Infectivity Enhancement of a Genetic Fiber Mosaic Adenovirus Vector in Ovarian Cancer Cells

Despite aggressive surgical debulking and chemotherapy, the 5-year survival rate for advanced ovarian cancer is very low. Thus, gene therapy is a promising treatment alternative; however, clinical cancer gene therapy trials with recombinant human adenovirus (Ad) serotype 5 have seen limited success. The efficiency of Ad gene transfer to ovarian cancer cells has been limited by low expression of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR). Thus, development of novel Ad vectors providing CAR-independent tropism may lead directly to therapeutic gain. We have previously shown that reovirus |[sigma]|1, a fiber-like attachment protein, incorporated into Ad5 virions, contributed to achieve infectivity enhancement in ovarian cancer cells via CAR-independent manner. Also we have reported that ovarian cancer cells possess a primary receptor for Ad3, which is independent from CAR. We hypothesized that an Ad5 vector containing both reovirus and Ad3 fibers would result in expanded tropism based on cellular expression of CAR independent receptors, thereby providing increased gene delivery to ovarian cancer cells. We have constructed a mosaic fiber Ad5 vector, designated Ad5/3-|[sigma]|1, encoding a luciferase reporter gene and two fibers. The first fiber is the chimeric fiber possessing the reovirus |[sigma]|1 and the second fiber is the chimeric fiber possessing the Ad3 fiber knob. Virus particles were purified and characterized by standard manners. Ad5/3-|[sigma]|1 gene delivery, as measured by luciferase transgene activity, was analyzed in a panel of ovarian cancer cell lines and primary ovarian cancer tissues. Characterization of Ad5/3-|[sigma]|1 confirmed the incorporation of both fibers in the same virus particle. Functionally, Ad5/3-|[sigma]|1 was able to utilize primary receptors for both reovirus and Ad3 for attachment to cells. Ad5/3-|[sigma]|1 provided 28 to 62-fold greater gene transfer versus the isogenic control Ad5 vector in a panel of ovarian cancer cell lines, known for relatively low CAR expression. Additionally, Ad5/3-|[sigma]|1 yielded 4.5-fold higher gene transfer to primary ovarian cancer tissue slices. We developed an infectivity enhanced Ad5 vector to address the key issue of limited Ad5 vector efficiency. The expanded tropism of the Ad5/3-|[sigma]|1 represents a crucial attribute for an Ad-based gene therapy vector for ovarian cancer, and may represent an agent with future clinical utility.

811. ACE-Targeted eNOS and BMPR2 Gene Therapy Attenuates Pulmonary Hypertension in a Chronic Hypoxia Rat Model

Idiopathic pulmonary arterial hypertension is a fatal disease characterized by vascular remodeling and right ventricular failure. Modern vasodilator therapies improve prognosis but ultimately most patients succumb or require heart-lung transplantation. We previously developed targeted adenoviral (Ad) strategies for pulmonary vascular gene delivery based on the use of a bispecific conjugate (Fab-9B9) that targets Ad infection to angiotensin converting enzyme (ACE) expressed on pulmonary vascular endothelium. In the current study we determined whether targeting achieved therapeutic gains when using Ad gene delivery of endothelial nitric oxide synthase (eNOS), then evaluated a more novel approach using targeted delivery of the gene for bone morphogenetic protein receptor type 2, based on the knowledge that mutations in this receptor and defects in BMPR2 signaling account for many of the inherited forms of IPAH. Rats (n=6) were injected with 5|[times]|109 pfu of either irrelevant virus, AdCMVeNOS, AdCMVeNOS+Fab9B9 or saline (PBS), then exposed to 10% oxygen atmosphere for three weeks, then right ventricular systolic pressure (RVSP), mean pulmonary artery pressure (PAP) and right ventricular hypertrophy (Fulton Index, FI) were determined. A concurrent group of rats maintained in normoxic conditions was used as a control. Hypoxia lead to increased RVSP (42.2|[plusmn]|4.6mmHg), PAP (34.5|[plusmn]|2.8 mmHg) and FI (0.48|[plusmn]|0.02) vs normoxic controls (22.3|[plusmn]|0.8, 19.4|[plusmn]|1.2 and 0.26|[plusmn]|0.01 respectively). AdCMVeNOS attenuated RVSP(33.9|[plusmn]|1.9), PAP(27|[plusmn]|2.3) and FI(0.41|[plusmn]|.03), with a further therapeutic gain seen with Fab-9B9 targeting (RVSP 28.0|[plusmn]|1.8). A separate experiment using irrelevant virus+Fab-9B9, AdCMVBMPR2+Fab-9B9 or PBS showed that BMPR2 gene delivery attenuated the hypoxia-induced increase in RVSP, PAP and FI by 27%, 34% and 50% respectively. Irrelevant Ad alone or with Fab-9B9 had no effect. Thus, the novel findings are that Ad-mediated eNOS delivery attenuates chronic hypoxia-induced PHT, and this effect is improved with targeting. This is also the first demonstration that upregulation of BMPR2 signaling attenuates pulmonary hypertension, suggesting a therapeutic strategy for patients with BMPR2 mutations.

412. Renal Pathophysiology after Systemic Administration of a Recombinant Adenovirus: Changes in Drug Metabolism Based upon Vector Dose

In addition to its ability to maintain water and electrolyte balance and efficiently remove waste products from the circulation, the kidney actively metabolizes many drugs, hormones and xenobiotics through the cytochrome P450 (CYP) enzyme system. Considering that adenovirus (Ad) significantly alters the expression and function of specific CYP enzymes in the liver, and that slight changes in renal function can adversely affect hepatic CYP activity and expression, we investigated the effect of a model recombinant Ad on the expression of the renal CYP isoforms: 4A1, 4A2, 4F1, and 2E1. CYP4A and 4F convert arachidonic acid to active metabolites responsible for regulation of epithelial transport and vascular function. CYP2E1 oxidizes many small lipophilic compounds and metabolizes carcinogens and cytotoxic agents. Male Sprague-Dawley rats were given one of six doses (5.7 |[times]| 106 - 5.7 |[times]| 1012 virus particles/kilogram (vp/kg)) of Ad expressing E. coli beta-galactosidase or saline (vehicle control) intravenously and sacrificed 0.25, 1, 4, and 14 days after injection. CYP protein expression was measured by Western blot and gene expression by RT-PCR. Serum creatinine levels (SCr) were assessed to profile changes in kidney function. The CYP4A family of proteins were significantly induced within 24 hours of administration of 5.7 |[times]| 1011 vp/kg by 35% and by 47% after 5.7 |[times]| 1012 vp/kg (P|[le]|0.01). A similar increase was detected at the 4 day timepoint. RT-PCR revealed that CYP4A1 gene expression doubled 6 hours after administration of doses above 1010. Gene expression remained induced (70% above control) 1 day after administration of doses, which exceeded 106 vp/kg (P|[le]|0.001). Animals treated with 5.7 |[times]| 1012 vp/kg began to recover at 14 days. However, animals treated with 5.7 |[times]| 1010 and 5.7 |[times]| 1011 vp/kg remained elevated (41% and 67%, P|[le]|0.05). CYP4A2 gene expression was also significantly induced (18%, 24%, and 15%) above control at 1 day in animals treated with 5.7 |[times]| 1010 - 5.7 |[times]| 1012 vp/kg, respectively. This isoform was elevated throughout the duration of the study. CYP4F1 gene and protein expression was unaffected by any of the treatments (P|[ge]|0.05). CYP2E1 gene expression was significantly suppressed by all treatments throughout the study (P|[le]|0.05). In general, suppression peaked within 24 hours (approximately 50% of control) for all doses, but trended towards baseline at 14 days. SCr was significantly decreased at all time points for Ad doses at and below 5.7 |[times]| 1011 vp/kg. SCr was increased by a factor of 3 in animals treated with 5.7 |[times]| 1012 vp/kg 4 days after administration (P|[le]|0.01). This data indicates that systemic Ad treatment can alter renal CYP expression and function up to 14 days after injection. This is of particular importance as Ad genomes were only detected by quantitative real-time PCR in kidneys of animals given the 1011 and 1012 vp/kg doses. This suggests that the aberrations in renal CYPs may not be related to the physical presence of viral particles. This reduction in metabolism should be considered when evaluating the pharmacokinetics of drugs included in clinical protocols involving Ad gene transfer for any application.

140. Primary Adenovirus Specific Cytotoxic T Lymphocyte Response Occurs after Viral Clearance and Liver Enzyme Elevation

Top of pageAbstract The relative contribution of the specific cytotoxic T lymphocyte (CTL) response and innate immune response to the clearance of adenovirus (Ad) and its transgene in the context of gene therapy has been a subject of debate. Transient transgene expression was believed due to the elimination of Ad transduced cells by CTLs. To determine the relative contribution of specific CTL killing of Ad-infected hepatocytes, we have utilized a wild-type Ad5 to study viral clearance from the liver and its associated liver enzyme elevation during a primary immune response. Human Ad5 replicates poorly in mice due to species restriction but induces a dominant CTL response to the E1B peptide (AA192 VNIRNCCYI) associated with mouse class I MHC antigen, H2-Db, in C57BL/6 (B6) mice. Thus, Ad5 was used to mimic a low replicative Ad vectors used in humans and an MHC-I tetramer that contains the E1Bp, H2-Db and b2-microglubulin (Db-E1B), was developed and used to detect the Ad-specific CTLs in Ad5-primed mice. After Ad5 infection, the Db-E1B+CD8+T cells were undetectable in the spleen, liver and lung until day 5, and peaked at day 8. At the peak of the CTL response, 8 days post infection, 10% of CD8+ cells in the spleen. Surprisingly, 45% in the liver and 55% in the lung of the CD8+ T cells were Db-E1B+, indicating that most CD8+ T cells in target tissue are specifically recruited to these sites. Furthermore, nearly 100% of Db-E1B+CD8+ cells were IFN|[gamma]| and granzyme-B positive with intracellular staining, indicating that the recruited CD8+CTL exhibit potent killing activity. The E1B-specific in vivo CTL response was detected by the transfer of CFSE-labeled, E1B192 peptide-pulsed target cells from H-2b mice into Ad5-primed syngeneic mice. The magnitude of the in vivo CTL response correlated with the number of Db-E1B+ CTLs in the spleen and liver (r2=0.96, p<0.01). Interestingly, viral DNA genome and E1B mRNA peaked at day 2 and reduced rapidly by day 6 and then maintained at a stable level without significant change 200 days after infection. Viral clearance correlated with the peak liver enzyme response that occurred on day 4, before CTLs became detectable. In contrast, there was no further change in the level of viral DNA genome and E1B mRNA in the liver or lung as a result of the peak CTL response at day 8. Administration of Ad5 to athymic SCID mice induced no CD8 T cells or in vivo specific CTL response against E1B192 peptide, yet viral clearance and liver enzyme response in SCID mice were comparable to those of B6 mice. Taken together, our results suggest that the innate response, but not the CTL response, plays a dominant role in elimination of Ad during a primary response. Stable long-term expression of lower levels of virus and transgene persist for 200 days despite the presence of potent CD8+ CTLs, indicating that they reside in liver cells that are resistant to CTL elimination. These findings will be useful in developing efficient and safe Ad gene therapy protocols.

Gene transfer using the mature form of VEGF-D reduces neointimal thickening through nitric oxide-dependent mechanism

Gene transfer to the vessel wall using vascular endothelial growth factors (VEGFs) has shown therapeutic potential for the treatment of restenosis. In this study, we evaluated the effect of catheter-mediated adenoviral (Ad) gene transfer of the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) in balloon-denuded cholesterol-fed rabbit aorta. AdLacZ was used as a control. Transduced VEGF-D(DeltaNDeltaC) mRNA was detectable in the arterial wall with RT-PCR at 6, 14 and 28 days. Gene transfer efficiency as detected with X-gal staining 6 days after the AdLacZ transduction was 1.91 +/- 1.32% in intima. AdVEGF-D(DeltaNDeltaC) gene transfer led to 52% reduction in intima/media ratio (I/M) as compared to the AdLacZ controls at 14 days time point. At 6 days there were no differences in I/M, but the number of macrophages in the vessel wall was 85% lower in the AdVEGF-D(DeltaNDeltaC) group as compared to the controls. The therapeutic effect was no longer detectable 28 days after the gene transfer. The therapeutic effect of VEGF-D(DeltaNDeltaC) was nitric oxide (NO)-dependent as the feeding of NO synthase inhibitor, L-NAME, blocked the reduction in intimal thickening. It is concluded that AdVEGF-D(DeltaNDeltaC) gene transfer reduces intimal thickening and macrophage influx into the vessel wall in balloon-denuded rabbit aortas.

Induction and Inhibition of Innate Inflammatory Responses by Adenovirus Early Region Proteins

First-generation adenovirus (Ad) gene therapy vectors deleted for the E1A, E1B, and E3 regions and carrying foreign genes under the control of strong foreign promoters induce high-level innate inflammatory responses within the first 24 hrs after transduction. Both uptake of the capsid and expression of gene products encoded by the vector contribute to the innate inflammatory response. Natural infections by Ad are frequently asymptomatic, suggesting that Ad has potent methods of inhibiting inflammation. The inability of Ad vectors to counter inflammatory responses suggests that the products of the Ad genes deleted in vector construction play critical roles in inhibiting these responses. Genetic analysis of the roles of Ad early region gene functions in vivo demonstrated that a virus made replication-incompetent by deletion of the preterminal protein gene and deleted for the transcriptional activation function of E1A effectively inhibits the innate inflammatory processes induced by Ad vectors. The mechanism(s) by which the Ad early region proteins inhibit inflammation is complex, as certain early region proteins can promote as well as inhibit inflammation, depending on the genetic context of the virus. Understanding of the roles of the Ad gene products in the induction and inhibition of innate inflammatory functions offers potential for the development of non-inflammatory vectors as well as for understanding of the mechanisms by which inflammation is regulated.

Adenovirus-Based Genetic Vaccines for Biodefense

The robust host responses elicited against transgenes encoded by (E1-)(E3-) adenovirus (Ad) gene transfer vectors can be used to develop Ad-based vectors as platform technologies for vaccines against potential bioterror pathogens. This review focuses on pathogens of major concern as bioterror agents and why Ad vectors are ideal as anti-bioterror vaccine platforms, providing examples from our laboratories of using Ad vectors as vaccines against potential bioterror pathogens and how Ad vectors can be developed to enhance vaccine efficacy in the bioterror war.

Effects of the Ad5 upstream E1 region and gene products on heterologous promoters

All recombinant adenovirus vectors contain the upstream region of the E1A gene comprising the viral origin of replication, encapsidation signal, and cis-acting regulatory elements for transcription of the E1A and other early genes. Using different reporter genes, some previous studies demonstrated the maintenance of heterologous promoter specificity in the adenoviral context, while others reported that adenoviral sequences interfere with promoter activity. Plasmid DNA-based luciferase reporter gene assays and adenovirus type 5 (Ad5) infection were combined to examine the effect of the Ad5 (nt 1-353) element and/or adenoviral gene products on tissue-specific (Midkine (MK) and COX-2), cell cycle associated (Ki-67 and E2F1) and viral promoters (Ad5 E1, Ad5 E4 and SV40). As a proof of concept, data were verified in the setting of recombinant replication-defective and replication-competent adenoviral vectors. Viral and E2F1 promoter activities were enhanced by the Ad5 (nt 1-353) segment by approximately 100% and 145%, respectively, regardless of its position. A polyadenylation sequence (polyA) upstream of the promoter had no effect, confirming an enhancer element within the Ad5 (nt 1-353) segment. Ad5 (nt 1-353) increased COX-2 promoter activity by 146% but was blocked by an upstream polyA, indicating a cryptic transcription start site. When placing the reporter gene cassette in a replication-defective adenovirus, similar data were obtained. In the plasmid vector-based system, adenoviral gene products transactivated the E2F1 and viral promoters by 194%, 19%, 67%, and 16%, respectively. Tissue-specific promoter activities were not significantly affected by the Ad5 (nt 1-353) segment, nor adenoviral gene products. In concert with these data, we were able to target replication-competent adenoviral vectors with the COX-2 promoter, but not with the cell cycle associated promotor. The adenovirus E1A upstream regulatory region and gene products interact with some but not all heterologous promoters. Often, the basal promoter activity can be reduced with an upstream polyA. Since the data obtained in our plasmid vector-based assay with internal control and infection with adenovirus could be confirmed in the adenoviral setting, our system might be suitable to speed up the identification of promoters which maintain their specificity in the adenoviral context and circumvent the problems associated with determining infectious adenovirus titers.

Adenovirus vectors deleted for genes essential for viral DNA replication

Adenovirus (Ad) gene therapy vectors made replication defective by deletion of the E1 region (first-generation vectors) induce high-level inflammation that leads to loss of both transduced gene expression and transduced cells. First-generation vectors were initially considered to be incapable of viral DNA replication, but it is necessary to delete one or more of the genes, all in the E2 transcription unit, that encode proteins essential for Ad DNA replication to completely eliminate viral DNA replication. Vectors deleted for one or more of the E2 genes (second-generation vectors) induce reduced levels of inflammation in certain animal models and offer promise for understanding the mechanisms by which adenovirus vectors induce inflammation and how inflammation can be inhibited. While first-generation vectors dominated the initial human gene therapy trials using adenovirus vectors, second-generation vectors may offer greater promise and greater safety for clinical studies.

Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd= 200 +/- 25 nM). A 38 bp, Rep68-protected region (5'-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3') was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

Parvalbumin gene delivery improves diastolic function in the aged myocardium in vivo

Abnormal relaxation of the heart, termed diastolic dysfunction, is a significant and growing problem that is a major cause of heart failure in the aged population. The potential of gene transfer of parvalbumin (Parv), a cytoplasmic calcium-binding protein, to improve diastolic function in the aged myocardium in vivo was evaluated. Despite evidence for an early developmental influence on the efficiency of Ad5 striated muscle transduction, results show that Ad5 gene transfer efficiency to adult cardiac myocytes in vitro is identical in young and old rats, suggesting that the basic processes of adenovirus binding and internalization are unaffected by aging. In contrast, Ad5-mediated Parv gene transfer to the myocardium in vivo is reduced in old rats compared to young rats. Nonetheless, Parv gene transfer and expression in vivo were sufficient to improve τ, a load-independent indicator of diastolic function, assessed using catheter-based micromanometry in the aged myocardium. These results suggest that expression of the calcium buffer Parv may represent an effective approach to functional correction of the failing heart in the aging.