Influence of vector design and host cell on the mechanism of recombination and emergence of mutant subpopulations of replicating retroviral vectors

<p>Abstract</p> <p>Background</p> <p>The recent advent of <it>murine leukaemia virus </it>(MLV)-based replication-competent retroviral (RCR) vector technology has provided exciting new tools for gene delivery, albeit the advances in vector efficiency which have been realized are also accompanied by a set of fresh challenges. The expression of additional transgene sequences, for example, increases the length of the viral genome, which can lead to reductions in replication efficiency and in turn to vector genome instability. This necessitates efforts to analyse the rate and mechanism of recombinant emergence during the replication of such vectors to provide data which should contribute to improvements in RCR vector design.</p> <p>Results</p> <p>In this study, we have performed detailed molecular analyses on packaged vector genomes and proviral DNA following propagation of MLV-based RCR vectors both in cell culture and in pre-formed subcutaneous tumours <it>in vivo</it>. The effects of strain of MLV, transgene position and host cell type on the rate of emergence of vector recombinants were quantitatively analysed by applying real-time PCR and real-time RT-PCR assays. Individual mutants were further characterized by PCR, and nucleotide sequence and structural motifs associated with these mutants were determined by sequencing. Our data indicate that virus strain, vector design and host cell influence the rate of emergence of predominating vector mutants, but not the underlying recombination mechanisms <it>in vitro</it>. In contrast, however, differences in the RNA secondary structural motifs associated with sequenced mutants emerging in cell culture and in solid tumours <it>in vivo </it>were observed.</p> <p>Conclusion</p> <p>Our data provide further evidence that MLV-based RCR vectors based on the Moloney strain of MLV and containing the transgene cassette in the 3' UTR region are superior to those based on Akv-MLV and/or containing the transgene cassette in the U3 region of the LTR. The observed discrepancies between the data obtained in solid tumours <it>in vivo </it>and our own and previously published data from infected cells <it>in vitro </it>demonstrates the importance of evaluating vectors designed for use in cancer gene therapy <it>in vivo </it>as well as <it>in vitro</it>.</p

Mouse Mammary Tumor Virus Promoter-Containing Retroviral Promoter Conversion Vectors for Gene-Directed Enzyme Prodrug Therapy are Functional In Vitro and In Vivo

Gene directed-enzyme prodrug therapy (GDEPT) is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1) metabolizes the prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon) vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC50 values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model

Molecularly Characterised Xenograft Tumour Mouse Models: Valuable Tools for Evaluation of New Therapeutic Strategies for Secondary Liver Cancers

To develop and evaluate new therapeutic strategies for the treatment of human cancers, well-characterised preclinical model systems are a prerequisite. To this aim, we have established xenotransplantation mouse models and corresponding cell cultures from surgically obtained secondary human liver tumours. Established xenograft tumours were patho- and immunohistologically characterised, and expression levels of cancer-relevant genes were quantified in paired original and xenograft tumours and the derivative cell cultures applying RT-PCR-based array technology. Most of the characteristic morphological and immunohistochemical features of the original tumours were shown to be maintained. No differences were found concerning expression of genes involved in cell cycle regulation and oncogenesis. Interestingly, cytokine and matrix metalloproteinase encoding genes appeared to be expressed differentially. Thus, the established models are closely reflecting pathohistological and molecular characteristics of the selected human tumours and may therefore provide useful tools for preclinical analyses of new antitumour strategies in vivo

Expression of the oncogenes mil and ras abolishes the in vivo differentiation of mammary epithelial cells

Three carcinoma-associated oncogenes, two of which have been strongly implicated in human mammary tumorigenesis, have been introduced into a novel mouse mammary epithelial cell line, EF43, that retains many differentiated functions. The effect of oncogene expression upon classical transformation parameters as well as parameters specific for mammary epithelial cells such as growth in three-dimensional collagen matrices and the ability to repopulate the cleared mammary fat pad and to form alveolar structures in vivo has been investigated. Expression of v-myc in EF43 cells results in no obvious phenotypic changes, and does not confer tumorigenic potential upon the cells. Expression of v-Ha-ras confers upon EF43 cells the ability to grow rapidly, grow in an anchorage-independent manner, results in tumor formation in nude and syngeneic animals, abolishes their ability to repopulate the mammary gland and, instead, results in rapid induction of anaplastic tumors. The v-mil oncogene, an avian homolog of the mouse v-mht and human c-raf oncogenes, previously thought to be non-transforming in the absence of a co-operating oncogene, transforms EF43 cells, allowing them to grow in an anchorage-independent manner, form tumors in nude mice and abolishes their ability to repopulate the cleared mammary fat pad. In contrast to v-ras, however, the tumors arising from v-mil expression have a differentiated morphology, typical of adenocarcinomas. Thus, different oncogenes show varying degrees of inhibition of the differentiation of mammary epithelial cells in viv

Dogmatisch-historische Beleuchtung des alten Judenthums

von Aaron Günzbur