Ixovex-1, a novel oncolytic E1B-mutated adenovirus.

There is a great demand for improved oncolytic viruses that selectively replicate within cancer cells while sparing normal cells. Here, we describe a novel oncolytic adenovirus, Ixovex-1, that obtains a cancer-selective replication phenotype by modulating the level of expression of the different, alternatively spliced E1B mRNA isoforms. Ixovex-1 is a recombinant adenovirus that carries a single point mutation in the E1B-93R 3' splice acceptor site that results in overexpression of the E1B-156R splice isoform. In this paper, we studied the characteristics of this novel oncolytic adenovirus by validating its in vitro behaviour in a panel of normal cells and cancer cells. We additionally studied its anti-tumour efficacy in vivo. Ixovex-1 significantly inhibited tumour growth and prolonged survival of mice in an immune-deficient lung carcinoma tumour implantation model. In complementation experiments, overexpression of E1B-156R was shown to increase the oncolytic index of both Ad5wt and ONYX-015. In contrast to prior viruses of similar type, Ixovex-1 includes a functional E3B region for better in vivo efficacy. Throughout this study, the Ixovex-1 virus has been proven to be superior in competency compared to a virus with multiple deletions.

A longitudinal study of serum insulin-like growth factor-I levels over 6 years in a large cohort of children and adolescents with type 1 diabetes mellitus: A marker reflecting diabetic retinopathy.

OBJECTIVE: To evaluate longitudinal serum insulin-like growth factor-I (IGF-I) in a large cohort of children and adolescents with type 1 diabetes in relation to hemoglobin A1c (HbA1c), age, diabetes duration, and body mass index (BMI), its association to height and retinopathy, and in comparison with healthy subject references. METHODS: A total of 2683 serum IGF-I values were obtained from 806 children and adolescents with T1DM, from annual blood samples for up to 6 consecutive years. RESULTS: In a multiple regression analysis IGF-I values were negatively correlated to HbA1c and diabetes duration, and positively correlated to BMI (P < .001, P < .001, and P < .001, respectively, adjusted r2 = 0.102). Children and adolescents with T1DM had lower mean IGF-I levels and reference interval limits compared to healthy subjects. In boys, mean (SD) IGF-I SD score (SDS) levels were -1.04 (±1.3) calculated from the healthy reference. IGF-I peaked at 15 years of age, similarly to healthy controls, but with markedly lower levels in late puberty. Girls were more affected at later stages of puberty but with a slightly less depressed overall mean IGF-I SDS of -0.69 (±1.2). In a subgroup of 746 subjects with fundus photography, a negative correlation was seen between individual mean IGF-I SDS and preproliferative retinopathy (P = .004, adjusted r2 = 0.021). In another subgroup of 84 adolescents, no correlation was seen between individual mean IGF-I SDS and target height SDS or distance to target height SDS. CONCLUSION: Poor metabolic control and diabetes duration impact negatively on serum IGF-I levels. A low individual mean IGF-I level was associated with progression of retinopathy independently of HbA1c, age, gender, and diabetes duration. Disease, sex and age related IGF-I SDS may become clinical helpful as a supplement to HbA1c in predicting the long-term outcome for children and adolescents with T1DM.

Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus.

Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (Kd = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.

Vaccinia virus induces programmed necrosis in ovarian cancer cells.

The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events.

Tissue-derived mesenchymal stromal cells used as vehicles for anti-tumor therapy exert different in vivo effects on migration capacity and tumor growth.

BACKGROUND: Mesenchymal stem cells (MSCs) have been promoted as an attractive option to use as cellular delivery vehicles to carry anti-tumor agents, owing to their ability to home into tumor sites and secrete cytokines. Multiple isolated populations have been described as MSCs, but despite extensive in vitro characterization, little is known about their in vivo behavior.The aim of this study was to investigate the efficacy and efficiency of different MSC lineages derived from five different sources (bone marrow, adipose tissue, epithelial endometrium, stroma endometrium, and amniotic membrane), in order to assess their adequacy for cell-based anti-tumor therapies. Our study shows the crucial importance of understanding the interaction between MSCs and tumor cells, and provides both information and a methodological approach, which could be used to develop safer and more accurate targeted therapeutic applications. METHODS: We first measured the in vivo migration capacity and effect on tumor growth of the different MSCs using two imaging techniques: (i) single-photon emission computed tomography combined with computed tomography (SPECT-CT), using the human sodium iodine symporter gene (hNIS) and (ii) magnetic resonance imaging using superparamagnetic iron oxide. We then sought correlations between these parameters and expression of pluripotency-related or migration-related genes. RESULTS: Our results show that migration of human bone marrow-derived MSCs was significantly reduced and slower than that obtained with the other MSCs assayed and also with human induced pluripotent stem cells (hiPSCs). The qPCR data clearly show that MSCs and hiPSCs exert a very different pluripotency pattern, which correlates with the differences observed in their engraftment capacity and with their effects on tumor growth. CONCLUSION: This study reveals differences in MSC recruitment/migration toward the tumor site and the corresponding effects on tumor growth. Three observations stand out: 1) tracking of the stem cell is essential to check the safety and efficacy of cell therapies; 2) the MSC lineage to be used in the cell therapy needs to be carefully chosen to balance efficacy and safety for a particular tumor type; and 3) different pluripotency and mobility patterns can be linked to the engraftment capacity of the MSCs, and should be checked as part of the clinical characterization of the lineage.

The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells.

At late times during a lytic infection human adenovirus type 5 produces ∼10(8) copies per cell of virus-associated RNA I (VA RNAI). This short highly structured RNA polymerase III transcript has previously been shown to be essential for lytic virus growth. A fraction of VA RNAI is processed by Dicer into small RNAs, so-called mivaRNAIs, which are efficiently incorporated into the RNA-induced silencing complex. Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the mivaRNAI duplex. The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells. Collectively, our results suggest that either strand of the mivaRNAI duplex does not have target mRNA interactions that are critical for the establishment of virus growth under lytic conditions. Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.

A mouse mammary epithelial cell line permissive for highly efficient human adenovirus growth.

Although a few immunocompetent animal models to study the immune response against human adenoviruses (HAdV) are available, such as Syrian hamsters and cotton rats, HAdV replication is several logs lower compared to human control cells. We have identified a non-transformed mouse epithelial cell line (NMuMG) where HAdV-2 gene expression and progeny formation was as efficient as in the highly permissive human A549 cells. HAdV from species, D and E (HAdV-37 and HAdV-4, respectively) also caused a rapid cytopathic effect in NMuMG cells, while HAdV from species A, B1, B2 and F (HAdV-12, HAdV-3, HAdV-11 and HAdV-41, respectively) failed to do so. NMuMG cells might therefore be useful in virotherapy research and the analysis of antiviral defense mechanisms and the determination of toxicity, biodistribution and specific antitumour activity of oncolytic HAdV vectors.

Adenovirus-mediated sensitization to the cytotoxic drugs docetaxel and mitoxantrone is dependent on regulatory domains in the E1ACR1 gene-region.

Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922-947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922-947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics.

Single molecule analysis of combinatorial splicing.

Alternative splicing creates diverse mRNA isoforms from single genes and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns along transcripts into a library of binary strings of nucleic acid tags that encode the exon composition of individual mRNA molecules. The exon inclusion pattern of each analyzed transcript is thus represented as binary data, and the abundance of different splice variants is registered by counts of individual molecules. The technique is illustrated in a model experiment by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits many genes to be analyzed in parallel and it will be valuable for elucidating the complex effects of combinatorial splicing.

Improved potency and selectivity of an oncolytic E1ACR2 and E1B19K deleted adenoviral mutant in prostate and pancreatic cancers.

PURPOSE: Replication-selective oncolytic adenoviruses are a promising class of tumor-targeting agents with proven safety in hundreds of patients. However, clinical responses have been limited and viral mutants with higher potency are needed. Here, we report on the generation of a novel set of mutants with improved efficacy in prostate and pancreatic carcinoma models. Currently, no curative treatments are available for late-stage metastatic prostate or rapidly progressing pancreatic cancers. EXPERIMENTAL DESIGN: Adenovirus type 5 mutants were created with deletions in the E1ACR2 region for tumor selectivity and/or the E1B19K gene for attenuated replication in vivo; all constructs retain the E3 genes intact. Cell-killing efficacy, replication, and cytotoxicity in combination with chemotherapeutics were investigated in normal cells (PrEC and NHBE), seven carcinoma cell lines, and human (PC3 and DU145) and murine (TRAMPC, CMT-64, and CMT-93) tumor models in vivo. RESULTS: The double-deleted AdDeltaDelta (DeltaE1ACR2 and DeltaE1B19K) mutant had high cell-killing activity in prostate, pancreatic, and lung carcinomas. Replication was similar to wild-type in all tumor cells and was attenuated in normal cells to levels less than the single-deleted AdDeltaCR2 mutant. AdDeltaDelta combined with the chemotherapeutics docetaxel and mitoxantrone resulted in synergistically enhanced cell killing and greatly improved antitumor efficacy in prostate xenografts in vivo. In murine immunocompetent in vivo models efficacy was greater for mutants with the E3B genes intact even in the absence of viral replication, indicating attenuated macrophage-dependent clearance. CONCLUSIONS: These data suggest that the novel oncolytic mutant AdDeltaDelta is a promising candidate for targeting of solid tumors specifically in combination with chemotherapeutics.

Oncolytic adenoviral mutants with E1B19K gene deletions enhance gemcitabine-induced apoptosis in pancreatic carcinoma cells and anti-tumor efficacy in vivo.

PURPOSE: Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal. We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. EXPERIMENTAL DESIGN: Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdDeltaE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. RESULTS: The DeltaE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdDeltaE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. CONCLUSIONS: Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways. These findings imply that less toxic doses than currently practiced in the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants.

Pulmonary edema and hemoptysis after breath-hold diving at residual volume.

To simulate pressure effects and experience thoracic compression while breath-hold diving in a relatively safe environment, competitive breath-hold divers exhale to residual volume before diving in a swimming pool, thus compressing the chest even at depth of only 3-6 m. The study was undertaken to investigate whether such diving could cause pulmonary edema and hemoptysis. Eleven volunteer breath-hold divers who regularly dive on full exhalation performed repeated dives to 6 m during a 20-min period. The subjects were studied with dynamic spirometry, video-fibernasolaryngoscopy, and single-breath diffusion capacity of carbon monoxide (Dl(CO)). The duration of dives with empty lungs ranged from 30 to 120 s. Postdiving forced vital capacity (FVC) was reduced from mean (SD) 6.57 +/- 0.88 to 6.23 +/- 1.02 liters (P < 0.05), and forced expiratory volume during the first second (FEV(1.0)) was reduced from 5.09 +/- 0.64 to 4.59 +/- 0.72 liters (P < 0.001) (n = 11). FEV(1.0)/FVC was 0.78 +/- 0.05 prediving and 0.74 +/- 0.05 postdiving (P < 0.001) (n = 11). All subjects reported a (reversible) change in their voice after diving, irritation, and slight congestion in the larynx. Fresh blood that originated from somewhere below the vocal cords was found by laryngoscopy in two subjects. Dl(CO)/alveolar ventilation (Va) was 1.56 +/- 0.17 mmol.kPa(-1).min(-1).l(-1) before diving. After diving, the Dl(CO)/Va increased to 1.72 +/- 0.24 (P = 0.001), but 20 min later it was indistinguishable from the predive value: 1.57 +/- 0.20 (n = 11). Breath-hold diving with empty lungs to shallow depths can induce hemoptysis in healthy subjects. Edema was possibly present in the lower airways, as suggested by reduced dynamic spirometry.

A downstream polyadenylation element in human papillomavirus type 16 L2 encodes multiple GGG motifs and interacts with hnRNP H.

Production of human papillomavirus type 16 (HPV-16) virus particles is totally dependent on the differentiation-dependent induction of viral L1 and L2 late gene expression. The early polyadenylation signal in HPV-16 plays a major role in the switch from the early to the late, productive stage of the viral life cycle. Here, we show that the L2 coding region of HPV-16 contains RNA elements that are necessary for polyadenylation at the early polyadenylation signal. Consecutive mutations in six GGG motifs located 174 nucleotides downstream of the polyadenylation signal resulted in a gradual decrease in polyadenylation at the early polyadenylation signal. This caused read-through into the late region, followed by production of the late mRNAs encoding L1 and L2. Binding of hnRNP H to the various triple-G mutants correlated with functional activity of the HPV-16 early polyadenylation signal. In addition, the polyadenylation factor CStF-64 was also found to interact specifically with the region in L2 located 174 nucleotides downstream of the early polyadenylation signal. Staining of cervix epithelium with anti-hnRNP H-specific antiserum revealed high expression levels of hnRNP H in the lower layers of cervical epithelium and a loss of hnRNP H production in the superficial layers, supporting a model in which a differentiation-dependent down regulation of hnRNP H causes a decrease in HPV-16 early polyadenylation and an induction of late gene expression.

A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein.

We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3' UTR) in HPV-16 gene expression. We found that deletion of the early 3' UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3' half of the early 3' UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3' splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.

HPV-16 L1 genes with inactivated negative RNA elements induce potent immune responses.

Introduction of point mutations in the 5' end of the human papillomavirus type 16 (HPV-16) L1 gene specifically inactivates negative regulatory RNA processing elements. DNA vaccination of C57Bl/6 mice with the mutated L1 gene resulted in improved immunogenicity for both neutralizing antibodies as well as for broad cellular immune responses. Previous reports on the activation of L1 by codon optimization may be explained by inactivation of the regulatory RNA elements. The modified HPV-16 L1 DNA that induced anti-HPV-16 immunity may be seen as a complementary approach to protein subunit immunization against papillomavirus.

Mutational inactivation of two distinct negative RNA elements in the human papillomavirus type 16 L2 coding region induces production of high levels of L2 in human cells.

Here we show that the 5' end and the middle region of the L2 coding sequence of human papillomavirus type 16 contain strong inhibitory RNA sequences termed inhibitory regions I and II. This is in contrast to L1, which contains one inhibitory region in the 5' end of the coding region. Inhibitory regions I and II acted in cis to reduce L2 mRNA levels and to inhibit the use of the mRNA. In tandem, the two regions reduced L2 mRNA production to undetectable levels. Specific mutational inactivation of the two inhibitory elements in the 5' end and in the middle region of L2 by the introduction of nucleotide substitutions that changed the nucleotide sequence but not the protein sequence resulted in production of high levels of L2 mRNA and protein. In contrast to L2, a partial L1 mutant in which only the first one third of L1 was mutated produced levels of L1 mRNA and protein similar to those in a full L1 mutant. In addition, the constitutive transport element of simian retrovirus type 1 overcomes the effect of the inhibitory sequences of L1 but not L2.

Specific inactivation of inhibitory sequences in the 5' end of the human papillomavirus type 16 L1 open reading frame results in production of high levels of L1 protein in human epithelial cells.

The expression of human papillomavirus type 16 late genes encoding virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We wish to understand the molecular mechanisms that determine the levels of expression of the human papillomavirus type 16 late genes. We have previously shown that the L1 coding region contains inhibitory sequences. Here we extend previous findings to show that the 5' end of the L1 gene contains strong inhibitory sequences but that the 3' end does not. We show that the first 514 nucleotides of the L1 coding region contain multiple inhibitory elements that act independently of one another and that the major inhibitory element is located within the first 129 nucleotides of the L1 gene. Introduction of point mutations in the inhibitory elements in the 5' end of the L1 gene which altered the RNA sequence without affecting the protein sequence specifically inactivated the inhibitory elements and resulted in production of high levels of human papillomavirus type 16 L1 mRNA and protein in human epithelial cells. Furthermore, we show that inhibitory sequences are present in the L1 coding regions of multiple human papillomavirus types, demonstrating that these elements are conserved among the human papillomaviruses, and suggest that they have an important function in the viral life cycle.