Development of a WNT-selective oncolytic adenovirus for imaging the therapy of colorectal cancers.

PhDIntroduction: The concept of oncolytic adenoviruses has been validated in preclinical studies but clinical trials have demonstrated that the virus spread remains limited and the virus fails to infect all cancer cells in a tumour. Arming an oncolytic virus with a therapeutic trans gene would enhance the antitumour effect of these viruses by killing adjacent non-infected cells. The aim of this thesis is to test armed oncolytic adenoviruses targeting a constitutive activation of the Wnt signalling pathway in pre-clinical models of colorectal cancer. Methods: The Nail symporter was inserted into the genome of Wnt-selective oncolytic adenoviruses to visualise adenoviral spread in the tumour and assess image-guided radiotherapy. Results: In vitro testing of the virus has demonstrated that the Wnt-selectivity of the virus remains intact. The virus we generated has an equal or greater cytopathic effect than wild type adenovirus in Wnt-expressing cancer cell lines. The ability of the infected cells to take up iodine has been confirmed by iodine uptake assays. The virus has been injected into subcutaneous human tumour implants in nude mice. Images obtained with a SPECT/CT camera have demonstrated that viral propagation can be visualised in vivo. Finally, we have used the imaging data to determine the correct timing for the administration of therapeutic doses of 131 I. Conclusion: We have validated a non invasive method to image viral propagation and transgene expression in a preclinical model of colonic cancer. Sequential imaging can provide information on the ideal time point for therapeutic intervention. In pilot experiments, the aim was to exploit the potential of the Nail symporter for the concentration of radioactive iodine, but it did not lead to increased therapeutic efficacy in vivo in preclinical models. There is strong evidence that if these experiments were repeated, therapeutic efficacy could he demonstrated

Therapeutic Potential of Replication-Selective Oncolytic Adenoviruses on Cells from Familial and Sporadic Desmoid Tumors

PURPOSE: Constitutive activation of the Wnt signaling pathway is a hallmark of many cancers and has been associated with familial and sporadic desmoid tumors. The aim of the present study is to assess the therapeutic potential of oncolytic adenoviruses selectively replicating in cells in which the Wnt signaling pathway is active on primary cells from desmoid tumors. EXPERIMENTAL DESIGN: Primary cells extracted from familial (n=3) or sporadic (n=3) desmoid tumors were cultured short term. Cancer cell survival and viral replication were measured in vitro upon infection with two different oncolytic adenoviruses targeting a constitutive activation of the Wnt signaling pathway. Adenoviral infectivity was also assessed. RESULTS: Although cells extracted from one sporadic desmoid tumor responded very well to the oncolytic action of the adenoviruses (less than 20% of viable cells upon infection at a multiplicity of infection of 10), cells from two tumor samples were totally resistant to the viral action. Cells from the remaining samples showed intermediate sensitivity to the oncolytic viruses. These effects were correlated to the level of infectivity of the cells. Finally, in responder cells, evidences of viral replication was observed. CONCLUSIONS: Our experimental data suggest that the response of desmoid tumor cells to oncolytic adenovirus is neither correlated to the type of mutation activating the Wnt signaling pathway nor to the familial or sporadic nature of the tumor. In addition, they highlight the variability of infectivity of individual tumors and predict a great variability in the response to oncolytic adenoviruses

Targeted Radionuclide Therapy Using a Wnt-Targeted Replicating Adenovirus Encoding the Na/I Symporter

International audienceThe Na/I symporter (hNIS) promotes concentration of iodine in cells. In cancer gene therapy, this transgene has potential as a reporter gene for molecular imaging of viral biodistribution and as a therapeutic protein promoting (131)I-mediated radiotherapy. Here, we combined the imaging and therapeutic potential of hNIS in an oncolytic adenoviruses targeting colorectal cancer cells

Inhibition of repair of radiation-induced DNA damage enhances gene expression from replication-defective adenoviral vectors

Radiation has been shown to up-regulate gene expression from adenoviral vectors in previous studies. In the current study, we show that radiation-induced dsDNA breaks and subsequent signaling through the mitogen-activated protein kinase (MAPK) pathway are responsible, at least in part, for this enhancement of transgene expression both in vitro and in vivo. Inhibitors of ataxia-telangiectasia–mutated, poly(ADP-ribose) polymerase–mutated, and DNA-dependent protein kinase (DNA-PK)–mediated DNA repair were shown to maintain dsDNA breaks (γH2AX foci) by fluorescence-activated cell sorting and microscopy. Inhibition of DNA repair was associated with increased green fluorescent protein (GFP) expression from a replication-defective adenoviral vector (Ad-CMV-GFP). Radiation-induced up-regulation of gene expression was abrogated by inhibitors of MAPK (PD980059 and U0126) and phosphatidylinositol 3-kinase (LY294002) but not by p38 MAPK inhibition. A reporter plasmid assay in which GFP was under the transcriptional control of artificial Egr-1 or cytomegalovirus promoters showed that the DNA repair inhibitors increased GFP expression only in the context of the Egr-1 promoter. In vivo administration of a water-soluble DNA-PK inhibitor (KU0060648) was shown to maintain luciferase expression in HCT116 xenografts after intratumoral delivery of Ad-RSV-Luc. These data have important implications for therapeutic strategies involving multimodality use of radiation, targeted drugs, and adenoviral gene delivery and provide a framework for evaluating potential advantageous combinatorial effects

Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles

International audienceThe lack of safe and efficient systemic gene delivery vectors has largely reduced the potential of gene therapy in the clinic. Previously, we have reported that polypropylenimine dendrimer PPIG3/DNA nanoparticles are capable of tumor transfection upon systemic administration in tumor-bearing mice. To be safely applicable in the clinic, it is crucial to investigate the colloidal stability of nanoparticles and to monitor the exact biodistribution of gene transfer in the whole body of the live subject. Our biophysical characterization shows that dendrimers, when complexed with DNA, are capable of forming spontaneously in solution a supramolecular assembly that possesses all the features required to diffuse in experimental tumors through the enhanced permeability and retention effect. We show that these nanoparticles are of sizes ranging from 33 to 286 nm depending on the DNA concentration, with a colloidal stable and well-organized fingerprint-like structure in which DNA molecules are condensed with an even periodicity of 2.8 nm. Whole-body nuclear imaging using small-animal nano-single-photon emission computed tomography/computer tomography scanner and the human Na/I symporter (NIS) as reporter gene shows unique and highly specific tumor targeting with no detection of gene transfer in any of the other tissues of tumor-bearing mice. Tumor-selective transgene expression was confirmed by quantitative reverse transcription-PCR at autopsy of scanned animals, whereas genomic PCR showed that the tumor sites are the predominant sites of nanoparticle accumulation. Considering that NIS imaging of transgene expression has been recently validated in humans, our data highlight the potential of these nanoparticles as a new formulation for cancer gene therapy