Directed evolution generates a novel oncolytic virus for the treatment of colon cancer.

Background: Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed.

Combination of tumor site-located CTL-associated antigen-4 blockade and systemic regulatory T-cell depletion induces tumor-destructive immune responses.

Accumulating data indicate that tumor-infiltrating regulatory T cells (Treg) are present in human tumors and locally suppress antitumor immune cells. In this study, we found an increased Treg/CD8 ratio in human breast and cervical cancers. A similar intratumoral lymphocyte pattern was observed in a mouse model for cervical cancer (TC-1 cells).

Adenovirus-platelet interaction in blood causes virus sequestration to the reticuloendothelial system of the liver.

Intravenous (i.v.) delivery of recombinant adenovirus serotype 5 (Ad5) vectors for gene therapy is hindered by safety and efficacy problems.

Baculovirus-based vaccination vectors allow for efficient induction of immune responses against plasmodium falciparum circumsporozoite protein.

Baculovirus vectors are able to transduce a large variety of mammalian cell types and express transgenes placed under the control of heterologous promoters. In this study, we evaluated the potential of baculovirus vectors for malaria vaccination. To induce efficient CD4(+) and CD8(+) T-cell responses, we produced a series of vectors that display the Plasmodium falciparum circumsporozoite (CS) protein in the virion envelope and/or allow for CS expression upon transduction of mammalian cells.

Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes.

Upon local delivery, adenovirus (Ad) serotype 5 viruses use the coxsackie and Ad receptor (CAR) for cell binding and alpha(v) integrins for internalization. When administered systemically, however, their role in liver tropism is limited because CAR-permissive and mutated viruses show similar biodistribution, a finding recently attributed to blood coagulation factor (F) IX or complement protein C4BP binding to the adenovirus fiber and "bridging" to either low-density lipoprotein receptor-related protein or heparan sulfate proteoglycans. Here, we show that hepatocyte transduction in vitro can be enhanced by the vitamin K-dependent factors FX, protein C, and FVII in addition to FIX but not by prothrombin (FII), FXI, and FXII.

Evaluation of adenovirus vectors containing serotype 35 fibers for vaccination.

In contrast to commonly used serotype 5-based adenovirus (Ad) vectors, Ad's containing fibers derived from B-group serotype 35 (Ad5/35) efficiently transduce human DCs ex vivo and appear to target antigen-presenting cells after intravenous injection into baboons. Based on this, Ad5/35 vectors could be valuable tools for immunotherapy and vaccination. On the other hand, a number of studies indicate that signaling through the B-group Ad receptor, CD46, can cause tolerance or immunosuppression.

Effect of adenovirus-mediated heat shock protein expression and oncolysis in combination with low-dose cyclophosphamide treatment on antitumor immune responses.

Heat shock proteins such as gp96 have the ability to chaperone peptides and activate antigen-presenting cells. In this study, we tested whether adenovirus-mediated overexpression of secreted or membrane-associated forms of gp96 in tumor cells would stimulate an antitumor immune response. Studies were carried out in C57Bl/6 mice bearing aggressively growing s.

Evaluation of biodistribution and safety of adenovirus vectors containing group B fibers after intravenous injection into baboons.

Vectors containing group B adenovirus (Ad) fibers are able to efficiently transduce gene therapy targets that are refractory to infection with standard Ad serotype 5 (Ad5) vectors, including malignant tumor cells, hematopoietic stem cells, and dendritic cells. Preliminary studies in mice indicate that, after intravenous injection, B-group fiber-containing Ads do not efficiently transduce most organs and cause less acute toxicity than Ad5 vectors. However, biodistribution and safety studies in mice are of limited value because the mouse analog of the B-group Ad receptor, CD46, is expressed only in the testis, whereas in humans, CD46 is expressed on all nucleated cells.

Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity.

Adenoviruses (Ad) are efficient vehicles for gene delivery in vitro and in vivo. Therefore, they are a promising tool in gene therapy, particularly in the treatment of cancer and cardiovascular diseases. However, preclinical and clinical studies undertaken during the last decade have revealed a series of problems that limit both the safety and efficacy of Ad vectors, specifically after intravenous application.

Interference with the IL-1-signaling pathway improves the toxicity profile of systemically applied adenovirus vectors.

The safety of gene therapy vectors is a major concern when novel viral or nonviral therapeutics are proposed for applications in humans. Adenovirus (Ad) vectors have been extensively used as efficient gene delivery vehicles in vitro over the last two decades. However, upon i.

Assessment of a combined, adenovirus-mediated oncolytic and immunostimulatory tumor therapy.

In this study, we identified murine breast cancer cell lines that support DNA replication of E1-deleted adenovirus vectors and which can be killed by an oncolytic adenovirus expressing adenovirus E1A and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in a replication-dependent manner (Ad.IR-E1A/TRAIL). We showed that systemic or intratumoral (i.

Development and assessment of human adenovirus type 11 as a gene transfer vector.

Adenovirus vectors based on human serotype 5 (Ad5) have successfully been used as gene transfer vectors in many gene therapy-based approaches to treat disease. Despite their widespread application, many potential therapeutic applications are limited by the widespread prevalence of vector-neutralizing antibodies within the human population and the inability of Ad5-based vectors to transduce important therapeutic target cell types. In an attempt to circumvent these problems, we have developed Ad vectors based on human Ad serotype 11 (Ad11), since the prevalence of neutralizing antibodies to Ad11 in humans is low.

Xenograft models for liver metastasis: Relationship between tumor morphology and adenovirus vector transduction.

The improvement of initial tumor cell transduction with viral vectors is a major task in tumor gene therapy. We have developed mouse tumor models with hepatic metastases to study transduction of tumor cells after systemic adenovirus vector application. The tumor models were established by intraportal transplantation of human tumor cell lines into immunodeficient mice.

Analysis of adenovirus sequestration in the liver, transduction of hepatic cells, and innate toxicity after injection of fiber-modified vectors.

After intravenous administration, adenovirus (Ad) vectors are predominantly sequestered by the liver. Delineating the mechanisms for Ad accumulation in the liver is crucial for a better understanding of Ad clearance and Ad-associated innate toxicity. To help address these issues, in this study, we used Ad vectors with different fiber shaft lengths and either coxsackievirus-Ad receptor (CAR)-interacting Ad serotype 9 (Ad9) or non-CAR-interacting Ad35 fiber knob domains.

A tumor-targeted and conditionally replicating oncolytic adenovirus vector expressing TRAIL for treatment of liver metastases.

We have constructed a new capsid-modified adenovirus (Ad) vector that specifically replicates in tumor cells and expresses TNF-related apoptosis-inducing ligand (TRAIL). The Ad capsid contains short-shafted fibers derived from Ad serotype 35, which allow for efficient infection of malignant tumor cells, and largely avoids innate toxicity after intravenous application. Replication-dependent homologous recombination in Ad genomes was used to achieve tumor-specific expression of Ad E1a (to mediate viral replication) and TRAIL (to mediate apoptosis and enhance release of progeny virus from infected cells).

The effect of sequestration by nontarget tissues on anti-tumor efficacy of systemically applied, conditionally replicating adenovirus vectors.

Avoiding transduction of normal tissue after intravenous application of oncolytic adenoviruses (Ad) is an important strategy to improve the safety and efficacy of these vectors in gene therapy. As a model for a targeted vector, we used Ad vectors with type 35 fibers (Ad5/35), which efficiently transduce human cervical carcinoma cells but not liver cells. In an in vitro model of liver metastases, in which small nests of HeLa cells were surrounded by mouse hepatocytes, we showed that an Ad5/35-based conditionally replicating vector regulated by DNA replication-dependent recombination conferred increased gene transfer to tumor cells and enhanced viral replication and tumor cell lysis compared to the nontargeted Ad5 vector.

Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors.

We previously reported that the HS-4 insulator, derived from the chicken beta-globin locus, was able to shield a downstream inducible promoter from viral enhancers or silencers present in the genome of adenovirus vectors. In this study, we constructed two recombinant adenoviruses (Ad) that express an alkaline phosphatase (AP) reporter gene driven by an alpha-fetoprotein (AFP) enhancer/promoter with and without HS-4 insulator (Ad.HS4.

Enzyme-activated Prodrug Therapy Enhances Tumor-specific Replication of Adenovirus Vectors.

Adenoviruses (Ads) that selectively replicate in tumor cells have shown promising preliminary results in clinical trials, especially in combination with chemotherapy. Here, we describe a system that combines the antitumor synergy of Ads and chemotherapeutic agents with the benefits of enzyme-activated prodrug therapy. In this system, a functional transgene expression cassette is created by homologous recombination during adenoviral DNA replication.

A new type of adenovirus vector that utilizes homologous recombination to achieve tumor-specific replication.

We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.

Dimerizer-induced proliferation of genetically modified hepatocytes.

Stable gene transfer to the liver by viral vectors is inefficient. In an attempt to stimulate expansion of retrovirally transduced hepatocytes, we employed a synthetic drug (AP20187) that can reversibly dimerize and activate fusion proteins that contain a growth factor receptor signaling domain. Signaling domains derived from receptors for interleukin-6 (gp130), hepatocyte growth factor (c-met), epithelial growth factor (EGF-R), and thrombopoietin (mpl) triggered monkey hepatocytes to enter the cell cycle.

Targeting of adenovirus vectors to tumor cells does not enable efficient transduction of breast cancer metastases.

Targeting of oncolytic adenoviruses to tumors can potentially increase their efficacy and safety profile after systemic application. We have developed recently a capsid-modified vector containing the adenovirus serotype 35 fiber shaft and knob inserted into an Ad5 capsid. This Ad5/35 vector infects cells via a coxsackievirus adenovirus receptor-independent pathway.