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

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

Mycobacterium tuberculosis Lineage Influences Innate Immune Response and Virulence and Is Associated with Distinct Cell Envelope Lipid Profiles

The six major genetic lineages of Mycobacterium tuberculosis are strongly associated with specific geographical regions, but their relevance to bacterial virulence and the clinical consequences of infection are unclear. Previously, we found that in Vietnam, East Asian/Beijing and Indo-Oceanic strains were significantly more likely to cause disseminated tuberculosis with meningitis than those from the Euro-American lineage. To investigate this observation we characterised 7 East Asian/Beijing, 5 Indo-Oceanic and 6 Euro-American Vietnamese strains in bone-marrow-derived macrophages, dendritic cells and mice. East Asian/Beijing and Indo-Oceanic strains induced significantly more TNF-α and IL-1β from macrophages than the Euro-American strains, and East Asian/Beijing strains were detectable earlier in the blood of infected mice and grew faster in the lungs. We hypothesised that these differences were induced by lineage-specific variation in cell envelope lipids. Whole lipid extracts from East Asian/Beijing and Indo-Oceanic strains induced higher concentrations of TNF-α from macrophages than Euro-American lipids. The lipid extracts were fractionated and compared by thin layer chromatography to reveal a distinct pattern of lineage-associated profiles. A phthiotriol dimycocerosate was exclusively produced by East Asian/Beijing strains, but not the phenolic glycolipid previously associated with the hyper-virulent phenotype of some isolates of this lineage. All Indo-Oceanic strains produced a unique unidentified lipid, shown to be a phenolphthiocerol dimycocerosate dependent upon an intact pks15/1 for its production. This was described by Goren as the ‘attenuation indictor lipid’ more than 40 years ago, due to its association with less virulent strains from southern India. Mutation of pks15/1 in a representative Indo-Oceanic strain prevented phenolphthiocerol dimycocerosate synthesis, but did not alter macrophage cytokine induction. Our findings suggest that the early interactions between M. tuberculosis and host are determined by the lineage of the infecting strain; but we were unable to show these differences are driven by lineage-specific cell-surface expressed lipids

Use of cell therapy as a means of targeting chemotherapy to inoperable pancreatic cancer

Although approved for the treatment of pancreatic cancer, the chemotherapeutic agent ifosfamide is not an effective therapy for this type of tumour. Ifosfamide must be activated by cytochrome P450 (P450) enzymes in the liver, initially to a short lived intermediate and then to toxic metabolites that are subsequently distributed by the circulatory system. Particularly for pancreatic cancer, this liver-mediated conversion results in relatively high systemic toxicities and poor therapeutic concentrations at the liver-distant site of the tumour. Activation of ifosfamide at the site of the tumour may allow lower doses to be used, while increasing the therapeutic index due to the resultant active concentrations generated locally. A cell-based therapy has been conceived where encapsulated, 293-derived cells genetically modified to overexpress a cytochrome P450 enzyme, are implanted near solid tumours. The cells are encapsulated in polymers of cellulose sulphate in order to provide a means of immunoprotection in vivo as well as to physically constrain them to the vicinity of the tumour. A major advantage of this strategy is that it allows one standard cell line to be applied to all patients and this approach can be extended to the treatment of other tumour types. After proof of principle studies in animal models, a phase I/II clinical trial was initiated in patients with stage III/IV nonresectable pancreatic cancer. Encapsulated cells were angiographically placed into the tumour vasculature of 14 patients and followed by systemic low dose ifosfamide treatment. Angiographic delivery of encapsulated cells proved feasible in all but one patient, and was well tolerated with no capsule or ifosfamide treatment-related adverse events. Four of the treated patients showed tumour regressions after capsule delivery and ifosfamide treatment in computer-tomography scans. The other 10 patients showed no further tumour growth (i.e. stable disease) during 20 weeks observation period. The median life expectancy of the patient collective was extended two fold as compared to age and status matched historical controls, with a 3-fold improvement in one year survival being attained. Evidence for a clinical benefit of the treatment was also obtained on the basis of standard parameters for quality of life. This approach has been evaluated by the European Medicines Evaluation Agency (EMEA) and orphan drug status has been granted. A pivotal clinical trial is now being planned with the help of the EMEA. Taken together, the data from this clinical trial suggest that encapsulated cytochrome P450-expressing cells combined with chemotherapy may be useful for the local treatment of a number of solid tumours and support the performance of further clinical studies of this new treatment

Oncogenic Viruses and Breast Cancer: Mouse Mammary Tumor Virus (MMTV), Bovine Leukemia Virus (BLV), Human Papilloma Virus (HPV), and Epstein–Barr Virus (EBV)

BackgroundAlthough the risk factors for breast cancer are well established, namely female gender, early menarche and late menopause plus the protective influence of early pregnancy, the underlying causes of breast cancer remain unknown. The development of substantial recent evidence indicates that a handful of viruses may have a role in breast cancer. These viruses are mouse mammary tumor virus (MMTV), bovine leukemia virus (BLV), human papilloma viruses (HPVs), and Epstein–Barr virus (EBV-also known as human herpes virus type 4). Each of these viruses has documented oncogenic potential. The aim of this review is to inform the scientific and general community about this recent evidence.The evidenceMMTV and human breast cancer—the evidence is detailed and comprehensive but cannot be regarded as conclusive. BLV and human breast cancer—the evidence is limited. However, in view of the emerging information about BLV in human breast cancer, it is prudent to encourage the elimination of BLV in cattle, particularly in the dairy industry. HPVs and breast cancer—the evidence is substantial but not conclusive. The availability of effective preventive vaccines is a major advantage and their use should be encouraged. EBV and breast cancer—the evidence is also substantial but not conclusive. Currently, there are no practical means of either prevention or treatment. Although there is evidence of genetic predisposition, and cancer in general is a culmination of events, there is no evidence that inherited genetic traits are causal.ConclusionThe influence of oncogenic viruses is currently the major plausible hypothesis for a direct cause of human breast cancer