Anti-pan Cytokeratin Anticorpo [C-11] (PE) (A86444)

Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in-depth preclinical assessment of novel therapeutics and may serve as a platform for further testing of current, novel gene delivery approaches.

Acidic mammalian chitinase (AMCase) is produced during and plays an important role in the pathogenesis of Th2-mediated diseases and antiparasite responses. However, the effector responses of AMCase in these settings have not been adequately defined and the relationship(s) between its chitinolytic and other biologic properties have not been investigated. In these studies, we demonstrate that AMCase protects airway epithelial cells from Fas ligand- and growth factor withdrawal-induced apoptosis. This cytoprotection was associated with Akt phosphorylation and abrogated when the PI3K/Akt pathway was inhibited. Comparable cytoprotection was also seen in experiments comparing wild-type AMCase and mutant AMCase that lacked chitinolytic activity. Importantly, the apoptosis-inhibiting effect of enzymatically active and inactive AMCase was abrogated by treatment with allosamidin. These studies demonstrate that secreted AMCase feeds back in an autocrine and/or paracrine manner to protect pulmonary epithelial cells from growth factor withdrawal- and Fas ligand-induced apoptosis. They also demonstrate that the cytoprotection is mediated via a PI3K/Akt-dependent and allosamidin-sensitive pathway that is independent of the chitinolytic activity of this chitinase.

Background: Adeno-associated virus (AAV) vectors have significant potential as gene delivery vectors for cancer gene therapy. However, broad AAV2 tissue tropism results in nonspecific gene expression.

Materials and methods: We investigated use of the C-X-C chemokine receptor type 4 (CXCR4) promoter to restrict AAV expression to tumour cells, in subcutaneous MCF-7 xenograft mouse models of breast cancer and in patient samples, using bioluminescent imaging and flow cytometric analysis.

Results: Higher transgene expression levels were observed in subcutaneous MCF-7 tumours relative to normal tissue (muscle) using the CXCR4 promoter, unlike a ubiquitously expressing Cytomegalovirus promoter construct, with preferential AAVCXCR4 expression in epithelial tumour and CXCR4-positive cells. Transgene expression following intravenously administered AAVCXCR4 in a model of liver metastasis was detected specifically in livers of tumour bearing mice. Ex vivo analysis using patient samples also demonstrated higher AAVCXCR4 expression in tumour compared with normal liver tissue.

Conclusion: This study demonstrates for the first time, the potential for systemic administration of AAV2 vector for tumour-selective gene therapy.