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.

An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector.

Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 x 10(10) viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007.

Anticancer activity of oncolytic adenovirus vector armed with IFN-alpha and ADP is enhanced by pharmacologically controlled expression of TRAIL.

We have previously described oncolytic adenovirus (Ad) vectors KD3 and KD3-interferon (IFN) that were rendered cancer-specific by mutations in the E1A region of Ad; these mutations abolish binding of E1A proteins to p300/CBP and pRB. The antitumor activity of the vectors was enhanced by overexpression of the Adenovirus Death Protein (ADP, E3-11.6K) and by replication-linked expression of IFN-alpha. We hypothesized that the anticancer efficacy of the KD3-IFN vector could be further improved by expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). E1-deleted Ad vectors were constructed carrying reporter genes for enhanced green fluorescent protein or secreted placental alkaline phosphatase (SEAP) and a therapeutic gene for TRAIL under control of the TetON system. Expression of the genes was increased in the presence of a helper virus and the inducer doxycycline such that up to 231-fold activation of expression for the TetON-SEAP vector was obtained. Coinfection with TetON-TRAIL augmented oncolytic activity of KD3 and KD3-IFN in vitro. Induction of TRAIL expression did not reduce the yield of progeny virus. Combination of TetON-TRAIL and KD3-IFN produced superior antitumor activity in vivo as compared with either vector alone demonstrating the efficacy of a four-pronged cancer gene therapy approach, which includes Ad oncolysis, ADP overexpression, IFN-alpha-mediated immunotherapy, and pharmacologically controlled TRAIL activity.

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.

Recombinant avian adenovirus CELO expressing the human interleukin-2: characterization in vitro, in ovo and in vivo.

In our study, a recombinant adenovirus based on the avian adenovirus CELO genome, has been constructed that contains the human interleukin-2 gene. We have shown the production of biologically active recombinant interleukin-2 in vitro (LMH and 293 cells) and in ovo (chicken embryos) infected with recombinant virus CELO-IL2. An increase in the median survival time of C57BL/6 mice carrying B16 melanoma tumors has been demonstrated after multiple intra-tumors injections of the recombinant adenovirus CELO-IL2.

[Eukaryotic vectors of Celo avian adenovirus genome, carrying GFP and human IL-2 genes]. / Eukarioticheskie vektory na osnove genoma adenovirusa ptits Celo, nesushchie geny GFP i IL-2 cheloveka.

Recombinant CELO avian adenoviruses carrying green fluorescent protein (GFP) and and human interleukin-2 (IL-2) genes were obtained by homologous recombination in cell culture. The resultant recombinant CELO viruses are reproduced in chick embryos in the renal tubular and chorionic allantoic membrane cells. The ability of CELO vectors to transduce human and animal cells was studied in vitro (in cell cultures) and in vivo (in laboratory animals). GFP gene delivery and expression in recombinant CELO virus in tumors in C57BL/6 mice were for the first time demonstrated for B16 melanoma. Human IL-2 gene expression and protein accumulation in allantoic fluid of chick embryos infected with CELO-IL-2 vector were detected for the first time.

[Delivery of secreted placental alkaline phosphatase (SEAP) gene in vitro and in vivo as a component of recombinant avian adenovirus (CELO)]. / Dostavka in vitro i in vivo gena sekretiruemoi platsentarnoi shchelochnoi fosfatazy (SEAP) v sostave rekombinantnogo adenovirusa ptits (CELO).

Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.

Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.

Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy.

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.

Tumor-specific, replication-competent adenovirus vectors overexpressing the adenovirus death protein.

We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting the adp gene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutant dl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G(0) to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus, dl1101/1107, or wild-type Ad. They are also more efficient than dl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1(-) E3(-) replication-defective vector expressing beta-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10(-4) PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected with dl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.

Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic.

Adenoviruses encode proteins that block responses to interferons, intrinsic cellular apoptosis, killing by CD8(+) cytotoxic T lymphocytes and killing by the death ligands TNF, Fas ligand and TRAIL. The viral proteins are believed to prolong acute and persistent adenovirus infections. The proteins may prove useful in protecting adenovirus gene therapy vectors and transplanted cells from the immune system.

[Early transcription of FAV-1 (CELO) avian adenovirus genome]. / Ranniaia transkriptsiia genoma adenovirusa ptits FAV-1 (CELO).

The patterns of RNA from cells infected with avian adenovirus chicken embryo lethal orphan (CELO) virus were analyzed by Northern hybridization method. Early RNAs are specifically hybridized with several fragments of the Eco-RI restriction map: A (49-81%), B (0.2-20.3%), D (81-92.5%), and E fragments of the Bam-HI CELO DNA restriction map. Early RNAs were not hybridized with C (37.8-49%), G (31-37.8%), and E (20.3-31%) fragments of the Eco-RI DNA CELO restriction map. At least 19 types of virus-specific RNA molecules homologous to 4 different regions of the virus genome are produced in CELO-infected permissive cells. Several classes of virus-specific RNAs were identified in CELO virus-transformed cell strains.

Sequence of the avian adenovirus FAV 1 (CELO) DNA encoding the hexon-associated protein pVI and hexon.

The genomic region of the avian adenovirus FAV1 (CELO) encoding the precursor to virion structural protein VI (p VI) and the major capsid protein hexon has been sequenced. The 223-unit sequence of the CELO pVI protein has two potential Ad endoproteinase cleavage sites and a conserved C-terminal sequence including the Cys residue supposedly involved in endoproteinase activation. The CELO hexon gene sequence predicts a 942-residue protein (106.7 kDa). Multiple sequence alignment with other six known hexon protein sequences (human, bovine, murine, and avian) reveals high overall homology. The identity is highest in the regions corresponding to the pedestals which from the base of the hexon, and lowest in the regions corresponding to the loops which are exposed on the outer surface of the virion.

[Expression of a gene encoding the porcine rotavirus VP7 capsid using a nondefective recombinant adenoviral genome]. / Ekspressiia gena, kodiruiushchego blok kapsida VP7 rotavirusa svinei v sostave genoma nedefektnogo rekombinantnogo adenovirusa.

A nondefective recombinant human adenovirus 5 (Ad5) carrying the gene encoding for the porcine rotavirus outer capsid protein VP7 in the E3 region of the Ad5 genome has been obtained. mRNA for VP7 in recombinant Ad5KV14/VP7 infected cells was demonstrated by means of RT-PCR. Radioimmunoprecipitation of cell extracts infected with Ad5KV14/VP7 on early (12 h p.i.) and late (24 h p.i.) stages of adenovirus infection with rabbit polyclonal antiserum raised to purified rotavirus virions revealed a recombinant protein possessing the same electrophoretic mobility, 37 kDa, as the native rotavirus K VP7. One-dimensional peptide mapping also demonstrated the identity of the recombinant and native VP7.

The fowl adenovirus type 1 (CELO) virus-associated RNA-encoding gene: a new ribozyme-expression vector.

A new system for hammerhead ribozyme (Rz) expression was examined in which fowl adenovirus type 1 (CELO) virus-associated RNA (CELO VA RNA) was used as a vector for the incorporation of Rz to target the mRNA of secreted alkaline phosphatase (SEAP) both in vitro and in vivo. The Rz gene was integrated into the CELO VA RNA between the internal promoter boxes A and B; apparently this did not interfere with its transcription. Rz integrated into CELO VA RNA and, lacking the viral sequences, exhibited the same activity in vitro, Consequently, CELO VA RNA sequences did not inhibit the integrated Rz activity in vitro. In vivo experiments were carried out with human 293 cells by co-transfection with plasmids containing Rz and SEAP. Inhibition of enzyme activity was 50% in 48 h. We conclude that CELO VA RNA may be used for effective expression of hammerhead Rz.

[In vitro and in vivo study of the activity of secreted alkaline phosphatase mRNA ribozyme gene]. / Issledovanie aktivnosti ribozima KmRNK gena sekretiruemoi shchelochnoi fosfatazy in vitro i in vivo.

A ribozyme was constructed of the catalytic domain of tobacco ringspot virus satellite RNA and flanking sequences complementary to the target, secreted alkaline phosphatase (SEAP) mRNA. The ribozyme specifically cleaved the substrate in vitro; compared with that constant temperature. The relationship between specific endoribonuclease activity of the ribozyme and Mg2+ concentration was shown. The ribozyme was active in 293 cell line which was cotransfected with plasmids carrying the SEAP gene under control of RSV LTR promoter and the ribozyme gene under early HCMV promoter: SEAP activity reduced by half.

Expression of the gene encoding secreted placental alkaline phosphatase (SEAP) by a nondefective adenovirus vector.

A nondefective recombinant human adenovirus 5 (Ad5) carrying the SEAP gene, encoding human secreted placental alkaline phosphatase, in the E3 region of the Ad5 genome was obtained. The expression of SEAP at the early and late stages of Ad5 infection was demonstrated in permissive and semi-permissive cell cultures. The amount of SEAP in the culture medium of the 293 cells was 13.6% of the total protein.

[Preparation of a non-defective adenovirus vector based on the Ad5 genome with the E3 region replaced with plasmid DNA with a kanamycin- resistance gene]. / Poluchenie nedefektnogo adenovirusnogo vektora na osnove genoma Ad5 s E3-oblast'iu, zameshchennoi plazmidnoi DNK s genom ustoichivosti k kanamitsinu.

The plasmid pH5KV14 containing the right end of the Ad5 viral genome (45-100 map units) with deleted nonessential E3 region (78.5-84 map units) has been constructed. A helper-independent recombinant virus Ad5KV14 has been obtained as a result of in vivo transformation of the line 293 human cells by the pH5KV14 plasmid DNA and an EcoRI-A DNA fragment of the virus Ad5 and subsequent recombination. A nondefective recombinant virus Ad5KV17 containing the Ad5 genome with the E3 region substituted for the plasmid DNA fragment coding for the kanamycin-resistance has been constructed by a similar procedure.