Isolation of more potent oncolytic paramyxovirus by bioselection.

Newcastle disease virus (NDV) is an oncolytic paramyxovirus with a nonsegmented single-stranded RNA genome. In this report, a recombinant oncolytic NDV was passaged in human tumor xenografts and reisolated and characterized after two rounds of bioselection. Several isolates could be recovered that differed from the parental virus with respect to virus spread in tumor cells and the ability to form syncytia in human tumor cells. Three isolates were identified that demonstrated superior oncolytic potency compared with the parental virus as measured by increased oncolytic potency in confluent tumor cell monolayers, in tumor cell spheroids and in a mouse xenograft tumor model. The surface proteins F and HN were sequence analyzed and characterized for fusogenicity. The present study demonstrates that in vivo NDV bioselection can enable the isolation of novel, oncolytic NDV and thus represents a powerful methodology for the development of highly potent oncolytic viruses.

An adenovirus E1A mutant that demonstrates potent and selective systemic anti-tumoral efficacy.

Replication-selective oncolytic viruses constitute a rapidly evolving and new treatment platform for cancer. Gene-deleted viruses have been engineered for tumor selectivity, but these gene deletions also reduce the anti-cancer potency of the viruses. We have identified an E1A mutant adenovirus, dl922-947, that replicates in and lyses a broad range of cancer cells with abnormalities in cell-cycle checkpoints. This mutant demonstrated reduced S-phase induction and replication in non-proliferating normal cells, and superior in vivo potency relative to other gene-deleted adenoviruses. In some cancers, its potency was superior to even wild-type adenovirus. Intravenous administration reduced the incidence of metastases in a breast tumor xenograft model. dl922-947 holds promise as a potent, replication-selective virus for the local and systemic treatment of cancer.

In vitro analysis of cidofovir and genetically engineered TK expression as potential approaches for the intervention of ColoAd1-based treatment of cancer.

We have generated a novel oncolytic Adenovirus (Ad), ColoAd1, with significantly increased potency ( approximately 100-fold) relative to its parent viruses, Ad11p and Ad3, or to the clinically tested oncolytic Ad, ONYX-015. Although this agent has a significant increase in its therapeutic window relative to ONYX-015 or its parent viruses, its ability to intervene and control virotherapy in treated patient is an important safety consideration for a novel biological therapy, such as ColoAd1. As there are no approved treatments for Ad infections, we sought to define whether antivirals being used to experimentally treat Ad infections (cidofovir (CDV), ribavirin) had any activity against ColoAd1. In addition, we incorporated a well-described pro-drug converting enzyme, the herpes simplex virus-thymidine kinase (HSV-TK) gene, into the viral genome to test whether the expression of this enzyme directly from the virus could be exploited as a safety valve for arresting the viral infection in the presence of the pro-drug, ganciclovir. Both the antiviral drug, CDV, and the incorporation of the pro-drug-converting TK enzyme were validated as effective approaches to controlling ColoAd1 infection, and this represents an important advancement in the development of ColoAd1 as an anticancer treatment.

Regulated expression of the interferon-beta gene in mice.

A single plasmid regulated expression vector based upon a mifepristone-inducible two plasmid system, termed pBRES, has been constructed and tested in mice using murine interferon-b (mIFNb) as the transgene. The expression of mIFNb in the circulation was followed by measuring the systemic induction of IP-10, a validated biomarker for mIFNb in mice. Long-term, inducible expression of mIFNb was demonstrated following a single intramuscular (i.m.) injection of the pBRES mIFNb plasmid vector into the hind limb of mice. Induction of mIFNb expression was achieved by administration of the small molecule inducer, mifepristone (MFP). Plasmid DNA and mIFNb mRNA levels in the injected muscles correlated with mIFNb expression as monitored by IP-10 over a 3-month time period. Renewable transgene expression was achieved following repeat administration of the plasmid at 3 months following the first plasmid injection. A dose-dependent increase in expression was demonstrated by varying the amount of injected plasmid or the amount of the inducer administered to the mice. Finally, the pBRES plasmid expressing mIFNb under control of the inducer, MFP, was shown to be efficacious in a murine model of experimental allergic encephalomyelitis, supporting the feasibility of gene-based therapeutic approaches for treating diseases such as multiple sclerosis.

Effect of viral dose on neutralizing antibody response and transgene expression after AAV1 vector re-administration in mice.

Neutralizing antibodies (nAB) at the time of administration hamper the effectiveness of adeno-associated virus (AAV) as a clinical DNA delivery system. The present study was designed to investigate if AAV re-administration in muscle tissue is dependent on the nAB titer. Recombinant (r)AAV serotype 1, as a promising candidate for targeting skeletal muscle, was used for gene delivery. C57Bl/6 mice were infected intramuscularly with doses between 1 x 10(9) and 5 x 10(10) virus particles (vp) of AAV1-expressing luciferase (AAV1-luc) or human interferon-beta (AAV1-hIFNbeta). Increasing transgene expression was observed over the first 2 months and anti-AAV1 nAB titers peaked between weeks 4 and 8. Six months after the first administration, 5 x 10(10) vp of AAV1-IFNbeta were re-administered. Following re-administration, nAB titers increased but did not significantly affect transgene expression from the AAV vector that had been administered first. In contrast, hIFNbeta expression originating from the second vector administration was significantly diminished and reflected the nAB titer present at the day of re-administration. The present study extends earlier observations that preexisting nAB affects AAV1 re-administration. The level of nAB is proportional to the virus dose used for the first injection and transgene expression following re-administration is dependent on preexisting nAB titer.

Identification and function probing of an antithrombin IIIß conformation-specific antibody.

UNLABELLED: ESSENTIALS: Antithrombin III (AT)ß binds heparin with higher affinity than ATα. A conformation-specific antibody against ATß, TPP2009, was made to investigate ATß in hemostasis. TPP2009 bound specifically to heparin-ATß and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATß in hemostasis. BACKGROUND: Antithrombin III (AT)ß is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. OBJECTIVES: To characterize a conformation-specific antibody against ATß and begin to investigate the role of ATß in maintaining hemostasis. METHODS: Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATß (ATß*H) by the use of phage display. RESULTS: SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATß*H and glycosaminoglycan-associated ATß on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATß and heparin. In purified systems with ATß and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies. CONCLUSIONS: TPP2009 specifically targets a unique conformational epitope on ATß*H and blocks ATß-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATß in hemostasis.

Adenovirus proteins that subvert host defenses.

Adenovirus encodes numerous products that counteract host defenses. A virus-encoded RNA, VA RNA1, prevents interferon-mediated shut-off of protein synthesis. Other protein products inhibit interferon-induced gene transcription, prevent cell killing by cytotoxic T cells or block apoptosis, and three sets of proteins independently block the cytolysis and inflammation induced by tumor necrosis factor. Studies of these factors are providing insights into viral pathogenesis.

Lung-specific expression of adenovirus E3-14.7K in transgenic mice attenuates adenoviral vector-mediated lung inflammation and enhances transgene expression.

Herein, we report that the adenovirus E3-14.7K protein inhibits the inflammatory response to adenovirus in transgenic mice in which the E3-14.7K gene was selectively expressed in the respiratory epithelium, using the human surfactant protein C (SP-C) promoter. E3-14.7K mRNA and protein were detected specifically in the lungs of SPC/E3-14.7K transgenic mice. Responses of the transgenic mice to Av1Luc1, an E1-E3-deleted Ad vector encoding the luciferase reporter gene, were examined, including vector transgene expression and lung inflammation. In wild-type mice, luciferase activity declined rapidly and was lost 14 days following Av1Luc1 administration. The loss of luciferase activity was associated with pulmonary infiltration by macrophages and lymphocytes. In heterozygous SPC/E3-14.7K mice, luciferase activity was increased by 7 days compared with control littermates, and pulmonary infiltration by macrophages was decreased. In homozygous (+/+) SPC/E3-14.7K mice, luciferase activity was increased 7, 14, and 21 days following administration compared with wild-type mice, and lung inflammation was markedly reduced. After Av1Luc1 administration, PCNA staining of bronchiolar and alveolar respiratory epithelial cells was decreased in SPC/E3-14.7K transgenic mice, indicating decreased epithelial cell proliferation, a finding consistent with the observed reduction in inflammation. CD4 and CD8 lymphocyte populations were only mildly altered, while humoral responses to adenoviral vectors were unchanged in the SPC/E3-14.7K mice. The E3-14.7K protein expressed selectively in respiratory epithelial cells suppresses Ad-induced pulmonary epithelial cell cytotoxicity and lung inflammation in vivo and prolongs reporter gene expression.

Growth inhibition of an established A431 xenograft tumor by a full-length anti-EGFR antibody following gene delivery by AAV.

Therapeutic monoclonal antibodies continue to achieve clinical success for the treatment of many different diseases, particularly cancer. However, the production and purification of antibodies continues to be a time and labor-intensive process with considerable technical challenges. Gene-based delivery of antibodies may address this, via direct production within the host that achieves therapeutic levels. In this report, we validate the feasibility that gene-based delivery is a viable approach for efficacious delivery of antibodies in the preclinical and, presumably, clinical setting. We demonstrate high and sustained in vivo expression of the murine antihuman epidermal growth factor receptor antibody 14E1 following intramuscular delivery by adeno-associated virus (AAV) 2/1. Incorporating the Furin/2A technology for monocistronic expression of both heavy and light chains, we achieved sustained serum levels of full-length 14E1 peaking over 1 mg ml(-1) in athymic nude mice. In the A431 xenograft tumor model, 14E1 was capable of significantly inhibiting tumor growth and prolonging survival when AAV was administered prior to tumor challenge. Furthermore, 14E1 demonstrated significant antitumor efficacy against well-established tumors (approximately 400 mm(3)) when AAV was administered up to 20 days after tumor challenge. Here we demonstrate for the first time growth inhibition of a well-established tumor by a full-length antibody following delivery by AAV.

High-performance liquid chromatography method for rapid assessment of viral particle number in crude adenoviral lysates of mixed serotype.

Accurate adenovirus (Ad) quantification requires labor- and time-intensive viral stock purification. While crude viral lysates can be titered by plaque assay, this cell-based assay is neither rapid nor accurate. Consequently, a method for quantification of crude, unpurified viral culture lysates is needed. Given growing interest in alternative Ad serotypes (different from well-studied and characterized serotype Ad5) for basic research and for therapeutic applications, such a method should also apply to alternative serotypes. Using a Q Sepharose XL (QSXL) column-based method, we describe a robust quantification method resulting in efficient retention of viral particles of all serotypes, while non-viral components of crude infected cultures remain largely in the flow-through. The high-performance liquid chromatography-QSXL method allows rapid, accurate adenoviral quantification in crude lysates as well as identification of the various serotypes present in mixed-serotype crude lysates. We also report on conditions that efficiently strip and regenerate the column, extending its functional life.

The effect of hypoxia on the uptake, replication and lytic potential of group B adenovirus type 3 (Ad3) and type 11p (Ad11p).

Replicating, tumor selective viruses are being tested as potential treatments for human cancers. Hypoxia is a pathophysiological cancer condition that alters the lytic potential of the replication-competent adenovirus serotype 5 (Ad5) virus by a mechanism independent of receptor levels or internalization rates. We extend these initial studies to examine the potential effects of hypoxia on the group B adenoviruses (Ads), adenovirus type 3 (Ad3) (group B1) and adenovirus type 11p (Ad11p) (group B2). Receptor expression (CD46) is not altered by hypoxia. However, the lytic potential is compromised in a cell-dependent fashion. Consequently, our study suggests that group B replicating Ad-based treatments, like the group C Ad-5-based viruses, will need to be modified in order to effectively treat hypoxic components of human tumors.

Effect of hypoxia on Ad5 infection, transgene expression and replication.

Oxygen deprivation (hypoxia) is a common feature of various human maladies, including cardiovascular diseases and cancer; however, the effect of hypoxia on Ad-based gene therapies has not been described. In this study, we evaluated how hypoxia (1% pO(2)) affects different aspects of Ad-based therapies, including attachment and uptake, transgene expression, and replication, in a series of cancer cell lines and primary normal cells. We found that hypoxia had no significant effect on the expression or function of the Ad5 attachment (Coxsackievirus and Adenovirus Receptor) and internalization (alpha(v) integrins) proteins, nor on the human cytomegalovirus-driven expression of an exogenous gene carried by a replication-incompetent Ad. Viral replication, however, was compromised by hypoxic conditions. Our studies revealed hypoxia-induced reductions in E1A levels that were mediated at the post-transcriptional level. E1A drives cells into the viral replication optimal S phase of the cell cycle; consequently, the combination of reduced E1A protein and hypoxia-induced G1 arrest of cells may be responsible for the lack of efficient viral replication under hypoxic conditions. Consequently, while traditional replication-incompetent Ad-based vectors appear to be viable delivery systems for hypoxia-associated disease indications, our studies suggest that Oncolytic Ads may need additional factors to efficiently treat hypoxic regions of human tumors.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the ADP region.

Genetically modified replication-selective human adenoviruses are currently undergoing testing in the clinical setting as anticancer agents. Coupling the lytic function of these viruses with virus-mediated transgene delivery represents a powerful extension of this treatment. We have designed a unique system for gene delivery from the replicating virus. It takes advantage of the endogenous gene expression control sequences (promoter, splicing, polyadenylation signals) to efficiently and predictably deliver transgenes from the non-essential E3 transcription unit while still maintaining the expression of the remaining E3 genes in the multi-gene transcription unit. In this article, we engineered restriction enzyme sites into the virus genome selectively to delete the ADP gene and replace it with the therapeutic transgenes CD and TNFalpha. We demonstrate that: (1) transgene expression from this region mirrors the substituted ADP gene; (2) the loss of ADP in these viruses results in infected cells with extended viability and protein synthesis when compared with a wild-type Ad5 infected cell; and (3) expression of surrounding E3 genes can be maintained in such a system. The potential advantages of delivering transgenes from the ADP region of the replicating adenovirus are discussed.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the E3B region.

Successful therapies for cancer need to deal with the complexity associated with the human tumor. Studies of tumor and viral biology have progressed to a point where replicating viruses are now being engineered as potential treatments for human cancers. The complex nature of human cancers dictates that successful treatments will require combination therapies. To this end, we have focused on developing the gene delivery capacity of the replicating adenovirus, using the non-essential E3 region transcription unit as a target site for therapeutic transgene insertions. Utilizing the endogenous expression machinery of the E3 region (promoter, splicing, polyA) we show that a therapeutic transgene, TNF, is efficiently expressed from the E3B region and with exclusive late gene expression kinetics. Potential clinical applications are discussed.

A cis-acting element in the major immediate-early (IE) promoter of human cytomegalovirus is required for negative regulation by IE2.

The major immediate-early promoter (MIEP) of human cytomegalovirus (CMV) contains a number of different enhancer elements in both repetitive and nonrepetitive sequences that influence the level of downstream transcription. This report describes a cis-acting element in the MIEP that responds to negative regulation by the IE2 gene product. Deletion analysis demonstrated that the cis-acting repressor element is located between the TATA box and the transcription initiation site from -13 to -1. The DNA sequence of the repressor element is 5'-CGTTTAGTGAACC-3'. The sequence is found in both the human and simian CMV MIEPs but not the murine CMV MIEP or in several other enhancer-containing promoters. The repressor element was isolated in a DNA fragment from -13 to +3 and was found to be functional in either orientation. It could be transferred to a heterologous enhancer-containing promoter and was functional when placed between the TATA box and the transcription initiation site. The element did not function when placed downstream of the transcription initiation site. Therefore, the cis-acting repressor element is position dependent. The role of the repressor element and the IE2 gene product in human CMV productive or latent infection is discussed.

Human cytomegalovirus immediate-early two protein region involved in negative regulation of the major immediate-early promoter.

The immediate-early two (IE2) gene products of human cytomegalovirus negatively regulate gene expression from the major immediate-early promoter in permissive human fibroblasts. A mutational analysis of the IE2 proteins indicated that the carboxyl-terminal region is required for negative regulation. The IE2 proteins that lack amino acid residues 365 to 519, or the carboxyl-terminal amino acids failed to negatively regulate. Most of the amino-terminal portion of the IE2 protein was not required for negative regulation. A possible explanation of the negative effect on downstream expression by the IE2 proteins is discussed.