Ad Replication Research Articles

Abstract 472: Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus vaccine vectors.

Abstract Cancer vaccines continue to be of global importance. In the HIV/SIV non-human primate models replicating adenovirus has shown promise as a vaccine vector. Priming with replicating recombinant host-range mutant adenovirus (Ad5hr) followed by envelope (Env) protein boosting, elicits strong cellular, humoral and mucosal immunity, and protection in non-human primates. Based on the results using Ad5hr in the HIV/SIV non-human primate models replicating Ad4 vaccines are being developed for HIV/AIDS, and Influenza. These vectors may also be of value for cancer. A limitation of replicating Ad vectors is the transgene carrying capacity which is about 3000 base pairs (bp). Using an Ad5hr encoding full-length single-chain HIVBal gp120 linked to rhesus CD4 D1D2 domains (rhFLSC; DeVico et al., PNAS 2007) we systematically deleted the genes encoding E4-orf1 to E4-orf4. The latter virus with a 1100 bp deletion expands the total carrying capacity to ∼4000 bp. Using plaque assays we were unable to determine any differences in viral progeny production that could be attributed to the deletions. We assessed the virus-host interactions by evaluating the cell cycle profile of infected cells over time. Here too no differences could be attributed to the deletions. The Ad replication cycle is divided into early and late phases with regard to viral DNA synthesis. Western blots confirmed that the parental and deleted viruses expressed similar transgene levels in a temporally regulated manner similar to Ad late proteins. Splenocytes and sera from mice vaccinated with the parental, the E4-deleted variants, or with an Ad5hr empty vector at 0 and 4 weeks were collected at week 6 and used to evaluate whether the deletions altered transgene or vector immunogenicity. Levels of Env-specific cytokines responses by T-cells were negligible for mice immunized with the empty vector yet positive for those immunized with the parental and E4-deleted viruses. Ad responses were similar in all 6 groups. Interestingly, while the 5 immunization groups yielded similar binding antibody titers against rhFLSC and gp120 the sera showed differential binding patterns for Ad antigens. Thus while little to no host-cell or cellular immunogenicity differences distinguish the E4 deleted viruses from the parent, the differential binding patterns for Ad antigens opens the possibility that the E4 gene products may influence Ad antigen recognition. These differences may prove useful in the design of replicating Ad vaccine vectors. Citation Format: Michael A. Thomas, Rui Song, Marjorie Robert-Guroff. Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus vaccine vectors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2013-472

Oncolytic adenoviral therapy enhanced by targeting cyclin E overexpression and inducing autophagy

Oncolytic virotherapy is a new potential approach in cancer therapy. Here we aim to enhance the efficacy of this drug platform with two approaches. First, we used the cyclin E promoter to control the E1a gene of a tumor‐specific oncolytic adenovirus (Ad). So far, most tumor‐specific promoters are relatively weak and only Ad dl1520 with its native E1a promoter has been approved for the cancer treatment. We have shown that the activity of cyclin E promoter is high in cancer cells and further enhanced by Ad infection. Thus, we constructed a novel oncolytic Ad‐cycE which specifically targets cancer cells and self‐enhance during the viral replication process. Ad‐cycE is competent to induce oncolysis in multiple cancer cells comparable to the wild‐type Ad and Ad dl1520. Our animal studies also illustrated the promising efficacy of Ad‐cycE on repressing lung tumors growth. Secondly, we applied autophagy to improve the oncolytic Ad‐cycE replication. We have demonstrated that Ad‐induced autophagy helps Ad replication and is related to cell death. Here we further revealed that an autophagy inducer rapamycin increased adenoviral E1A expression of Ad‐cycE and increases virus production in the human lung cancer cells. Our studies provide a new insight in vector development and demonstrate the novel roles of autophagy in adenovirus replication that may benefit future combination virotherapy. Supported by NIH Grant R01 CA129975.

Molecular Basis for Viral Selective Replication in Cancer Cells: Activation of CDK2 by Adenovirus-Induced Cyclin E

Adenoviruses (Ads) with deletion of E1b55K preferentially replicate in cancer cells and have been used in cancer therapies. We have previously shown that Ad E1B55K protein is involved in induction of cyclin E for Ad replication, but this E1B55K function is not required in cancer cells in which deregulation of cyclin E is frequently observed. In this study, we investigated the interaction of cyclin E and CDK2 in Ad-infected cells. Ad infection significantly increased the large form of cyclin E (cyclin EL), promoted cyclin E/CDK2 complex formation and increased CDK2 phosphorylation at the T160 site. Activated CDK2 caused pRb phosphorylation at the S612 site. Repression of CDK2 activity with the chemical inhibitor roscovitine or with specific small interfering RNAs significantly decreased pRb phosphorylation, with concomitant repression of viral replication. Our results suggest that Ad-induced cyclin E activates CDK2 that targets the transcriptional repressor pRb to generate a cellular environment for viral productive replication. This study reveals a new molecular basis for oncolytic replication of E1b-deleted Ads and will aid in the development of new strategies for Ad oncolytic virotherapies.

Computationally designed adeno-associated virus (AAV) Rep 78 is efficiently maintained within an adenovirus vector

Adeno-associated virus (AAV) is a single-stranded parvovirus retaining the unique capacity for site-specific integration into a transcriptionally silent region of the human genome, a characteristic requiring the functional properties of the Rep 78/68 polypeptide in conjunction with AAV terminal repeat integrating elements. Previous strategies designed to assemble these genetic elements into adenoviral (Ad) backbones have been limited by the general intolerability of AAV Rep sequences, prompting us to computationally reengineer the Rep gene by using synonymous codon pair recoding. Rep mutants generated by using de novo genome synthesis maintained the polypeptide sequence and endonuclease properties of Rep 78, while dramatically enhancing Ad replication and viral titer yields, characteristics indistinguishable from adenovirus lacking coexpressed Rep. Parallel approaches using domain swaps encompassing WT and recoded genomic segments, coupled with iterative computational algorithms, collectively established that 3' cis-acting Rep genetic elements (and not the Rep 78 polypeptide) retain dominant-acting sequences inhibiting Ad replication. These data provide insights into the molecular relationships of AAV Rep and Ad replication, while expanding the applicability of synonymous codon pair reengineering as a strategy to effect phenotypic endpoints.

Development of an adenovirus vector lacking the expression of virus-associated RNAs

A major limitation of the use of adenovirus (Ad) vectors is the innate immune response, which causes inflammatory cytokine production and tissue damages. To overcome this limitation, it is necessary to develop safer Ad vectors that are less likely to induce innate immunity. The Ad genome encodes two non-coding small RNAs, virus-associated (VA)-RNA I and VA-RNA II, which are transcribed by RNA polymerase III and promote Ad replication. Recently, we reported that VA-RNAs are produced in the cells transduced with a conventional first-generation (E1-deleted) Ad vector (FG-Ad) and trigger innate immune responses through intracellular nucleic acid sensors. In the present study, we have developed a VA-RNA-deleted Ad (AdΔVR) vector, in which the transcriptional control elements of the VA-RNA-expression were deleted. Although conventional HEK293 cells did not support the propagation of the AdΔVR vectors, HEK293 transformants inducibly expressing VA-RNA I (VR293 cells) with appropriate induction of VA-RNA I expression allowed the propagation of the AdΔVR vector. The AdΔVR vector showed high transduction efficiency comparable to that of the conventional FG-Ad vector in the cultured cells. The AdΔVR vector may be a safer alternative to the FG-Ad vector.

Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model

Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow-derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response.

Proteasome-dependent degradation of Daxx by the viral E1B-55K protein in human adenovirus-infected cells.

The death-associated protein Daxx found in PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) is involved in transcriptional regulation and cellular intrinsic antiviral resistence against incoming viruses. We found that knockdown of Daxx in a nontransformed human hepatocyte cell line using RNA interference (RNAi) techniques results in significantly increased adenoviral (Ad) replication, including enhanced viral mRNA synthesis and viral protein expression. This Daxx restriction imposed upon adenovirus growth is counteracted by early protein E1B-55K (early region 1B 55-kDa protein), a multifunctional regulator of cell-cycle-independent Ad5 replication. The viral protein binds to Daxx and induces its degradation through a proteasome-dependent pathway. We show that this process is independent of Ad E4orf6 (early region 4 open reading frame 6), known to promote the proteasomal degradation of cellular p53, Mre11, DNA ligase IV, and integrin alpha3 in combination with E1B-55K. These results illustrate the importance of the PML-NB-associated factor Daxx in virus growth restriction and suggest that E1B-55K antagonizes innate antiviral activities of Daxx and PML-NBs to stimulate viral replication at a posttranslational level.

Docetaxel increases antitumor efficacy of oncolytic prostate-restricted replicative adenovirus by enhancing cell killing and virus distribution.

We explored multiple molecular mechanisms of the combination of docetaxel and an oncolytic prostate-restricted replication competent adenovirus (Ad) (PRRA) in advanced prostate cancer (PCa) models. The combinational therapy has potential to overcome the therapeutic limitations of poor virus distribution inside solid tumors. We evaluated the effect of docetaxel on the antitumor efficacy and efficiency of virus transduction, transgene expression and virus distribution of PRRA in a prostate-specific antigen/prostate-specific membrane antigen-positive tumor xenograft model. We also evaluated the effect of docetaxel on apoptosis induction, cell killing and the efficiency of transgene expression and virus replication in vitro. Tumor growth inhibition was significantly enhanced when docetaxel was administrated before intratumor injection of PRRA. In vivo dual-photon microscopy and ex vivo fluorescence microscopy and immunohistochemistry showed that docetaxel increased transgene expression and expanded virus distribution. The combination of docetaxel and PRRA also increased cell apoptosis. In vitro, docetaxel significantly increased cell killing in PRRA-treated PCa cells. Docetaxel significantly increased Ad-mediated trangene expression independent of Ad binding receptors and replication capability. Docetaxel increased the activity of cytomegalovirus (CMV) promoter but not of a chimeric prostate-specific enhancer, resulting in higher transgene expression. The enhanced CMV promoter activity resulted from activation of p38 mitogen-activated protein kinase (MAPK) because inhibition of p38 MAPK blocked the docetaxel-induced increase in CMV promoter activity. Combining docetaxel with an oncolytic PRRA improved therapeutic potential by expanding virus distribution and enhancing cell apoptosis and killing. These studies suggested a novel mechanism for enhancing the effect of therapeutic genes delivered by a PRRA.

Abstract 586: Targeted cancer gene-therapy using second-generation HIF-activated oncolytic adenoviruses with increased replication, oncolytic, and anti-tumor efficacy.

Abstract A clinically important target for anti-tumor therapy is hypoxia-inducible factor (HIF). HIF is a transcription factor that is expressed in a wide range of primary tumors, metastases, and cancer stem cells, whereas it is undetectable in normal healthy tissues. HIF expression in tumors is associated with (i) resistance to apoptosis, chemotherapy, and radiotherapy and (ii) increased invasion/metastasis, angiogenesis, and patient mortality. Oncolytic adenoviruses (Ad) are a cancer therapy which utilize the cytolytic replication cycle of the virus to specifically kill tumor cells (oncolysis). A wild-type Ad cannot be used for cancer therapy because it lacks tumor-specificity and is toxic to normal healthy tissues. We exploited the differential activation of HIF-dependent gene expression in tumors versus normal tissue for the design of targeted HIF-activated oncolytic Ads. In the first-generation HIF-activated oncolytic Ad called HYPR-Ad, we placed the E1A gene which is essential for Ad replication under the regulation of a HIF-inducible promoter. Our published studies with HYPR-Ad provided critical proof that a HIF-activated oncolytic Ad can specifically kill HIF-active tumor cells and inhibit tumor growth. While therapeutically efficacious, HYPR-Ad exhibited attenuated replication and oncolytic activity when compared to a wild-type Ad in vitro. To overcome these deficiencies and improve anti-tumor efficacy, we created a second-generation HIF-activated oncolytic Ad, called HIF-Ad, which has two key changes: (i) a modified HIF-responsive promoter to regulate the E1A replication gene and (ii) insertion of the E3 gene region. To augment the anti-tumor activity of HIF-Ad beyond the killing of HIF-active tumor cells, it was modified to express interleukin-4 (HIF-Ad-IL4). The IL4 cytokine was selected because it has potent multimodal anti-tumorigenic activities. Using normal and tumor cell lines in vitro, we found that the HIF-Ads have HIF-dependent E1A expression, replication, and oncolytic activity. Importantly, the replication and oncolytic efficacy of the HIF-Ads was similar to a wild-type Ad and superior to the HYPR-Ads. The HIF-Ads demonstrated strong anti-tumor activity against subcutaneous human tumor xenografts. Notably, HIF-Ad-IL4 treatment led to tumor regressions and its anti-tumor efficacy was up to 6-fold greater than HYPR-Ad-IL4, HIF-Ad, and wild-type Ad treatment. These studies demonstrate that the HIF-Ads have improved anti-tumor activity compared to the HYPR-Ads and that treatment with a HIF-activated oncolytic Ad leads to a measurable therapeutic response. The novel design of the HIF-Ads represents a significant improvement compared to first-generation oncolytic Ads and has great potential to increase the anti-tumor efficacy of this cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 586.

Arginine Methylation of Human Adenovirus Type 5 L4 100-Kilodalton Protein Is Required for Efficient Virus Production

The adenovirus type 5 (Ad5) late region 4 (L4) 100-kDa nonstructural protein (L4-100K) mediates inhibition of cellular protein synthesis and selective translation of tripartite leader (TL)-containing viral late mRNAs via ribosome shunting. In addition, L4-100K has been implicated in the trimerization and nuclear localization of hexon protein. We previously proved that L4-100K is a substrate of the protein arginine methylation machinery, an emergent posttranslational modification system involved in a growing list of cellular processes, including transcriptional regulation, cell signaling, RNA processing, and DNA repair. As understood at present, L4-100K arginine methylation involves protein arginine methyltransferase 1 (PRMT1), which asymmetrically dimethylates arginines embedded in arginine-glycine-glycine (RGG) or glycine-arginine-rich (GAR) domains. To identify the methylated arginine residues and assess the role of L4-100K arginine methylation, we generated amino acid substitution mutations in the RGG and GAR motifs to examine their effects in Ad-infected and plasmid-transfected cells. Arginine-to-glycine exchanges in the RGG boxes significantly diminished L4-100K methylation in the course of an infection and substantially reduced virus growth, demonstrating that L4-100K methylation in RGG motifs is an important host cell function required for efficient Ad replication. Our data further indicate that PRMT1-catalyzed arginine methylation in the RGG boxes regulates the binding of L4-100K to hexon and promotes the capsid assembly of the structural protein as well as modulating TL-mRNA interaction. Furthermore, substitutions in GAR, but not RGG, regions affected L4-100K nuclear import, implying that the nuclear localization signal of L4-100K is located within the GAR sequence.

INGN 007, an oncolytic adenovirus vector, replicates in Syrian hamsters but not mice: comparison of biodistribution studies

Preclinical biodistribution studies with INGN 007, an oncolytic adenovirus (Ad) vector, supporting an early stage clinical trial were conducted in Syrian hamsters, which are permissive for Ad replication, and mice, which are a standard model for assessing toxicity and biodistribution of replication-defective (RD) Ad vectors. Vector dissemination and pharmacokinetics following intravenous administration were examined by real-time PCR in nine tissues and blood at five time points spanning 1 year. Select organs were also examined for the presence of infectious vector/virus. INGN 007 (VRX-007), wild-type Ad5 and AdCMVpA (an RD vector) were compared in the hamster model, whereas only INGN 007 was examined in mice. DNA of all vectors was widely disseminated early after injection, but decayed rapidly in most organs. In the hamster model, DNA of INGN 007 and Ad5 was more abundant than that of the RD vector AdCMVpA at early times after injection, but similar levels were seen later. An increased level of INGN 007 and Ad5 DNA but not AdCMVpA DNA in certain organs early after injection, and the presence of infectious INGN 007 and Ad5 in lung and liver samples at early times after injection, strongly suggests that replication of INGN 007 and Ad5 occurred in several Syrian hamster organs. There was no evidence of INGN 007 replication in mice. In addition to providing important information about INGN 007, the results underscore the utility of the Syrian hamster as a permissive immunocompetent model for Ad5 pathogenesis and oncolytic Ad vectors.

Immunosuppression Enhances Oncolytic Adenovirus Replication and Antitumor Efficacy in the Syrian Hamster Model

We recently described an immunocompetent Syrian hamster model for oncolytic adenoviruses (Ads) that permits virus replication in tumor cells as well as some normal tissues. This model allows exploration of interactions between the virus, tumor, normal organs, and host immune system that could not be examined in the immunodeficient or nonpermissive animal models previously used in the oncolytic Ad field. Here we asked whether the immune response to oncolytic Ad enhances or limits antitumor efficacy. We first determined that cyclophosphamide (CP) is a potent immunosuppressive agent in the Syrian hamster and that CP alone had no effect on tumor growth. Importantly, we found that the antitumor efficacy of oncolytic Ads was significantly enhanced in immunosuppressed animals. In animals that received virus therapy plus immunosuppression, significant differences were observed in tumor histology, and in many cases little viable tumor remained. Notably, we also determined that immunosuppression allowed intratumoral virus levels to remain elevated for prolonged periods. Although favorable tumor responses can be achieved in immunocompetent animals, the rate of virus clearance from the tumor may lead to varied antitumor efficacy. Immunosuppression, therefore, allows sustained Ad replication and oncolysis, which leads to substantially improved suppression of tumor growth.

Gene medicine for cancer treatment: Commercially available medicine and accumulated clinical data in China

Loss of p53 function compromises genetic homeostasis, which induces deregulated DNA replication, damages DNA, and subsequently results in increased resistance to anticancer agents. Pharmacological approaches using recombinant adenoviruses (Ad) have been developed to restore the p53 functions. Another approach for gene medicine is to modify Ad replication in a tumor-specific manner, which induces tumor cell death without damaging normal tissues in the vicinity. The Ad-derived gene medicines, Ad expressing the wild-type p53 gene and replication-competent Ad defective of the E1B-55kDa gene, have been tested for their clinical feasibility and became commercially available in China. These agents demonstrated their antitumor activities as a monotherapy and in combination with conventional chemotherapeutic agents. In this article, we summarize the outcomes of clinical trials in China, most of which have been published in domestic Chinese journals, and discuss potential directions of cancer gene therapy with these agents.

Hexadecyloxypropyl-cidofovir, CMX001, prevents adenovirus-induced mortality in a permissive, immunosuppressed animal model

Adenoviruses (Ads) cause a wide array of end-organ and disseminated diseases in severely immunosuppressed patients. For example, approximately 20% of pediatric allogeneic hematopoietic stem cell transplant recipients develop disseminated Ad infection, and the disease proves fatal in as many as 50-80% of these patients. Ad infections are a serious problem for solid-organ transplant recipients and AIDS patients as well. Unfortunately, there are no antiviral drugs approved specifically to treat these infections. A suitable animal model that is permissive for Ad replication would help in the discovery process. Here we identify an animal model to study Ad pathogenesis and the efficacy of antiviral compounds. We show that human serotype 5 Ad (Ad5) causes severe systemic disease in immunosuppressed Syrian hamsters that is similar to that seen in immunocompromised patients. We also demonstrate that hexadecyloxypropyl-cidofovir (CMX001) rescues the hamsters from a lethal challenge with Ad5. The antiviral drug provided protection both prophylactically and when given up to 2 days after i.v. exposure to Ad5. CMX001 acts by reducing Ad replication in key target organs. Thus, the immunosuppressed Syrian hamster is a powerful model to evaluate anti-Ad drugs, and its use can facilitate the entry of drugs such as CMX001 into clinical trials.

Efficacy of Topical Immunoglobulins against Experimental Adenoviral Ocular Infection

Presently, there is no U.S. Federal Drug Administration (FDA)-approved antiviral therapy for the treatment of adenoviral (Ad) ocular infections. The goal of the present study was to determine the antiviral efficacy of human immunoglobulin (Ig), a preparation of highly purified and concentrated immunoglobulin (IgG) antibodies isolated from a large pool of human plasma donors, in vitro and on acute Ad replication in the Ad5 New Zealand White (NZW) rabbit ocular model. The antiviral activity of human Ig against multiple wild-type and human ocular isolates of adenovirus serotypes was investigated in vitro by using neutralizing assays in different human epithelial cell lines. In vivo bilateral topical ocular toxicity and antiviral efficacy were evaluated with established Ad5/NZW rabbit ocular models. In vivo Ig antiviral results were compared with those obtained with topical 0.5% cidofovir and saline. In three different epithelial cell lines, <or=6.25 mg/mL of the Ig neutralized several wild-type adenoviral serotypes that cause ocular infections. A dose of <or=10 mg/mL neutralized 88% of ocular isolates of the adenovirus serotypes. After treatment of infected animals, adenovirus-positive cultures per total cultures (days 1-14; P = 0.021), the duration of Ad5 shedding, (P = 0.008), and the mean combined ocular viral titer during the early (days 1-5; P = 0.0001) and the late (days 7-14; P = 0.013) phases of infection were significantly lower in Ig-treated animals than in saline-treated animals and were similar to those in cidofovir-treated animals. Ig demonstrated antiviral properties against multiple adenoviral serotypes in vitro and in the Ad5/NZW rabbit ocular model. Further studies are needed to advance topical immunoglobulin for treatment and prophylaxis of ocular infections.

Targeting Interferon-α Increases Antitumor Efficacy and Reduces Hepatotoxicity of E1A-mutated Spread-enhanced Oncolytic Adenovirus

Novel approaches are needed to improve the antitumor potency and to increase the cancer specificity of oncolytic adenoviruses (Ad). We hypothesized that the combination of interferon-alpha (IFN-α) expression with a specific mutation in the e1a gene of Ad could target vector replication to genetic defects in the IFN-α pathway resulting in both improved antitumor efficacy and reduced toxicity. The conditionally replicative Ad vector KD3-IFN carries the dl1101/1107 mutation in the e1a gene that eliminates binding of E1A proteins to p300/CBP and pRb. KD3-IFN expresses human IFN-α in concurrence with vector replication and overexpresses the adenovirus death protein (ADP; E3-11.6K). The antitumor activity of KD3-IFN was significantly higher than that of a control vector in established human hepatocellular carcinoma tumors in immunodeficient mice and in hamster kidney cancer tumors in immunocompetent Syrian hamsters. The dl1101/1107 mutation rendered Ad replication sensitive to the antiviral effect of IFN-α in normal as opposed to cancer cells. These results translated to reduced vector toxicity upon systemic administration to C57BL/6 mice. The combination of Ad oncolysis, ADP overexpression, and IFN-α-mediated immunotherapy represents a three-pronged approach for increasing the anticancer efficacy of replicative Ads. Exploiting the dl1101/1107 mutation provides a mechanism for additional selectivity of IFN-α-expressing replication-competent Ads.

Adeno-associated virus rep protein-mediated inhibition of transcription of the adenovirus major late promoter in vitro.

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four AAV replication proteins (Rep78, Rep68, Rep52, and Rep40) are pleiotropic effectors of virus integration, replication, transcription, and virion assembly. These proteins exert effects on Ad gene expression and replication. In transient plasmid transfection assays, Rep proteins inhibit gene expression from a variety of transcription promoters. We have examined Rep protein-mediated inhibition of transcription of the Ad major late transcription promoter (AdMLP) in vitro. Rep78/68 are the strongest transcription suppressors and the purine nucleotide binding site in the Rep proteins, and by implication, the ATPase activity or conformational change induced by nucleotide binding is required for full repression. Rep52 has modest effects, and Rep40 exerts no significant effect on transcription. Rep78/68 and their N-terminal 225-residue domain bind to a 55-bp AdMLP DNA fragment in gel shift assays, suggesting that protein-DNA interactions are required for inhibition. This interaction was confirmed in DNase I protection assays and maps to a region extending from the TATA box to the transcription initiation site. Gel shift, DNase I, and chemical cross-linking assays with TATA box-binding protein (TBP) and Rep68 indicate that both proteins interact with each other and with the promoter at adjacent sites. The demonstration of Rep interaction with TBP and the AdMLP suggests that Rep78/68 alter the preinitiation complex of RNA polymerase II transcription. These observations provide new insight into the mechanism of Rep-mediated inhibition of gene expression.

651. VRX-007, an Oncolytic Adenovirus Vector, Replicates in Syrian Hamsters but Not Mice: Comparison of Biodistribution Studies Performed in the Syrian Hamster and Mouse

Two biodistribution studies, one in Syrian golden hamsters (Mesocricetus auratus) and one in C57BL/6 mice, were performed to examine the dissemination and pharmacokinetics of VRX-007, a replication-competent (oncolytic) adenovirus (Ad) vector, following intravenous administration. The study in Syrian hamsters allowed us to examine the biodistribution of VRX-007 in a model that is permissive for VRX-007 (and Ad5) replication. In contrast, mice do not support replication of human Ads to an appreciable extent. Controls in the Syrian hamster study included wild-type Ad5 (replication-competent virus), AdCMVpA (a replication-defective vector), and vehicle only. Two quantitative real-time polymerase chain reaction (QPCR) assays were developed, one that specifically and exclusively detects VRX-007 vector genomic DNA (gDNA) and another that specifically detects both Ad5 and AdCMVpA gDNA. These assays were used to detect viral gDNA present in blood and various organs collected from Syrian hamsters (VRX-007 and Ad5/AdCMVpA assays) or C57BL/6 mice (VRX-007 assay only). In addition, the level of infectious virus/vector present in select Syrian hamster organs was titrated using a tissue culture infectious dose-50 (TCID50) assay. Both studies demonstrate that early after administration the virus/vector is distributed widely. In the Syrian hamster study, gDNA from the replication-competent vector/virus VRX-007 and Ad5 was more abundant than that of the replication-defective vector AdCMVpA on Days 2 and 7. On Days 29 and 92, the levels of VRX-007, Ad5, and AdCMVpA Gdna were roughly the same. In addition, there was an increase in VRX-007 and Ad5 copy number from Day 2 to Day 7 in heart, kidney, and adrenal glands (VRX-007 only) that was not seen with AdCMVpA. Infectious vector/virus was recovered from the livers, lungs, and testes of VRX-007- and Ad5-injected hamsters (Days 2 and 7 only), but not from any organs of AdCMVpA-injected hamsters. There was no detectable infectious virus on Day 29 or Day 92. The data in Syrian hamsters contrasts with that obtained in mice, where VRX-007 copy number was generally low on Day 2 (except in the liver), and in many organs decreased to undetectable levels over time. In addition, no infectious VRX-007 was recovered from mouse livers. From these studies we: 1) confirm and extend observations that Syrian hamsters are permissive for Ad replication, which occurred in the liver, lungs, and possibly other organs, 2) confirm that Ad does not replicate to an appreciable extent in C57BL/6 mice, 3) show that the liver is the primary organ of infection in Syrian hamsters and mice when virus/vector is delivered intravenously. The results of these studies will be discussed in light of data obtained from parallel toxicology studies in these two animal species.

642. Toxicological Findings with Oncolytic Adenovirus Vector VRX-007, Wild-Type Ad5, and a Replication-Defective Adenovirus Vector in Syrian Hamsters and C57BL/6 Mice

We have developed VRX-007, a fully replication-competent (RC) oncolytic adenovirus (Ad) vector that has a faster replication cycle and is more cytolytic than wild-type Ad5 due to overexpression of the E3 11.6K Adenovirus Death Protein (ADP). VRX-007 is very efficacious against various human cancer cell lines and against tumors in the nude mouse, cotton rat, and Syrian hamster. Tissues of the last two animals are permissive for human Ad replication, and these animals have an intact immune system.

Oncolytic adenovirus that overproduces ADP and replicates selectively in tumors due to hTERT promoter-regulated E4 gene expression

We have constructed a novel oncolytic adenovirus (Ad) vector, named VRX-011, in which the replication of the vector is targeted to cancer cells by the replacement of the wild-type Ad E4 promoter with the human telomerase reverse transcriptase (hTERT) promoter. Genes in the Ad E4 transcription unit are essential for Ad replication; therefore, VRX-011 will grow efficiently only in cells in which the hTERT promoter is active, that is, in a wide range of cancer and immortalized cells but not in most somatic cells. Consistent with these expectations, VRX-011 replicated efficiently in all cancer cell lines examined, while its growth was restricted in various primary and normal cells. VRX-011 overexpresses ADP (also known as E3-11.6K), an Ad protein required for efficient cell lysis and release of virions from cells at late stages of infection. This overexpression enhances cell-to-cell spread and could significantly increase antitumor efficacy. In a xenograft model in nude mice, both intratumoral and intravenous administration of VRX-011 effectively suppressed the growth of subcutaneous Hep3B human liver tumors. Also, intravenous delivery of VRX-011 greatly reduced the number and size of A549 human lung cancer cell nodules in a disseminated lung tumor model in nude mice. Importantly, tail vein administration of different doses of VRX-011 in C57BL/6 mice showed minimal liver toxicity. Considering its broad range of lytic replication in cancer cells, its attenuated phenotype in primary cells, its efficacy in suppressing xenografts, and its low toxicity in mouse liver, VRX-011 is a promising candidate for further evaluation as an anticancer therapeutic.