Current Gene Therapy (v.13, #3)
Microbubble-Assisted p53, RB, and p130 Gene Transfer in Combination with Radiation Therapy in Prostate Cancer by Rounak Nande, Adelaide Greco, Michael S. Gossman, Jeffrey P. Lopez, Luigi Claudio, Marco Salvatore, Arturo Brunetti, James Denvir, Candace M. Howard, Pier Paolo Claudio (163-174).
Combining radiation therapy and direct intratumoral (IT) injection of adenoviral vectors has been explored as ameans to enhance the therapeutic potential of gene transfer. A major challenge for gene transfer is systemic delivery ofnucleic acids directly into an affected tissue. Ultrasound (US) contrast agents (microbubbles) are viable candidates to enhancetargeted delivery of systemically administered genes.Here we show that p53, pRB, and p130 gene transfer mediated by US cavitation of microbubbles at the tumor site resultedin targeted gene transduction and increased reduction in tumor growth compared to DU-145 prostate cancer cellxenografts treated intratumorally with adenovirus (Ad) or radiation alone. Microbubble-assisted/US-mediated Ad.p53 andAd.RB treated tumors showed significant reduction in tumor volume compared to Ad.p130 treated tumors (p<0.05). Additionally,US mediated microbubble delivery of p53 and RB combined with external beam radiation resulted in the mostprofound tumor reduction in DU-145 xenografted nude mice (p<0.05) compared to radiation alone. These findings highlightthe potential therapeutic applications of this novel image-guided gene transfer technology in combination with externalbeam radiation for prostate cancer patients with therapy resistant disease.
Progress and Prospects: The Use of 3D Spheroid Model as a Relevant Way to Study and Optimize DNA Electrotransfer by Laure Gibot, Marie-Pierre Rols (175-181).
The use of electrotransfer to deliver therapeutic agents such as cytotoxic drugs and nucleic acids to cells andtissues has been successfully developed over the last decade. This strategy is promising for the systemic secretion oftherapeutic proteins, vaccination and gene therapy. The safe and efficient use of this physical method for clinical purposesrequires knowledge of the mechanisms underlying the DNA electrotransfer and expression phenomena. Despite the factthat the pioneering work on plasmid DNA electrotransfer to cells was initiated 30 years ago, many of the underlyingmechanisms remain elucidated. While efficient in vitro, the method faces a lack of efficiency in packed tissues. Until now,the great majority of studies have been performed on cells in 2D cultures in Petri dishes or in suspension. However, thesestudies cannot get access to the tissue-specific architecture and organization present in 3D living tissues. In this context,3D cell culture models are more relevant concerning in vivo cell organization since cell-cell contacts are present as well asextracellular matrix. The aim of this review is to describe the relevance of using spheroid as a model to address and improvethe electrotransfer processes.
Prevalence of Neutralizing Factors Against Adeno-Associated Virus Types 2 in Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy by Yong Cheng, Lvzhen Huang, Xiaoxin Li, Huijun Qi, Peng Zhou, Wenzhen Yu, Yide A. Jiang, Samuel Wadsworth, Abraham Scaria (182-188).
Adeno-associated virus type 2 (AAV2) mediated gene therapy providing a potential treatment in the eye. However,immune responses can limit virally mediated gene transfer and therapy. To assess preexisting AAV2 neutralizingfactors (NF) titers in peripheral blood and the vitreous in patients with age-related macular degeneration (AMD) andpolypoidal choroidal vasculopathy (PCV). 130 subjects were enrolled: 50 with neovascular AMD, 30 with PCV, and 50controls. The serum and the vitreous were obtained for AAV2 NF assay. We found AAV2 NF are present in all of AMD,PCV patients and controls we tested. There were no significant differences in prevalence of NAb in serum between AMD,PCV and controls (P =0.999). There was no correlation between NF in serum and in vitreous (P>0.05), and NF in vitreouswas significantly less than in serum. Our results for the first time showed in Chinese population, NF against AAV2 waspresent in serum of all the patients with AMD or PCV and controls, and there were no significant differences among thesegroups. Therefore, it demonstrated there were no correlations between AAV2 NF titer and these diseases. We found NF invitreous was considerably less than in serum in all groups. We also found no direct correlation between NF in vitreous andin serum suggesting serum antibody levels may not be used to predict their counterparts in the vitreous. Our results willprovide crucial information for future clinical studies in the development of new therapies based on AAV2 mediated genedelivery in the eye.
Impact of PLK-1 Silencing on Endothelial Cells and Cancer Cells of Diverse Histological Origin by Carla P. Gomes, Ligia C. Gomes-da-Silva, Jose S. Ramalho, Maria C.P. de Lima, Sergio Simoes, Joao N. Moreira (189-201).
The main goal of this work was to assess in vitro the potential of Polo-like kinase gene (PLK-1) as a moleculartarget within the tumor microenvironment, namely in both cancer cells of tumors of different histological origin and endothelialcells from angiogenic blood vessels, upon silencing with anti-PLK-1 siRNA. In addition, the effect of Plk-1 downregulationon the cancer cells chemosensitization to paclitaxel was further assessed.Downregulation of Plk-1 reduced cancer cells viability from 40 to 85% and up to 59% in endothelial cells. Regarding thelatter, it compromised their ability to form new tube-like structures, decreasing the formation of network projections up to46%. This suggested for the first time, PLK-1 as a valuable angiogenic molecular target. In combination with paclitaxel,anti-PLK-1 siRNA chemosensitized non-small cell lung cancer (NSCLC) and prostate carcinoma cell lines, leading up toa 2-fold increase in the drug cytotoxic effect. Moreover, the sequential incubation of anti-PLK-1 siRNA and paclitaxel ledto a decrease in the IC50 of the latter up to 2.7- and 4.1-fold, in A-549 and PC-3 cells, respectively. The combination ofanti-PLK-1 siRNA with paclitaxel led to cell cycle arrest, increasing the number of cells at the G2/M and S phases to 1.5and 1.3-fold in PC-3 cells, and to 1.6 and 1.4-fold in A-549 cells, respectively. Overall, it has been demonstrated thatPLK-1 silencing with siRNA can impact multiple cellular players of tumor aggressiveness, thus enabling the opportunityto interfere with different hallmarks of cancer, in tumors with diverse histological origin.
Thymidine Kinase-Mediated Shut Down of Bone Morphogenetic Protein-4 Expression Allows Regulated Bone Production by Barbara Lombardo, Teresa Rocco, Maria T. Esposito, Bruno Cantilena, Sara Gargiulo, Adelaide Greco, Donatella Montanaro, Arturo Brunetti, Lucio Pastore (202-210).
Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis thathave generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has beensuccessful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previouslyshown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drivebone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also beenimplicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral(FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidinekinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expressiondoes not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration ofganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateralmuscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activityof TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir.We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blockingunwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuousbone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor.
Insertional Mutagenesis by Retroviral Vectors: Current Concepts and Methods of Analysis by Sean Knight, Mary Collins, Yasuhiro Takeuchi (211-227).
Retroviral vectors derived from gammaretroviruses or lentiviruses have now been used extensively in clinicalgene therapy trials for several diseases including primary immunodeficiencies, beta thalassaemia and adrenoleukodystrophy.Their utility in this setting has been readily demonstrated by the largely favourable outcomes in recent clinical trials,however this success has been marred by the emergence of malignancies in some trials. These malignancies were a consequenceof perturbation of cellular proto-oncogene expression by the integrated retroviral vectors, the process of which isreferred to as 'insertional mutagenesis' (IM). In this review, the origins of our understanding of IM are reviewed and appliedto the clinical gene therapy trials conducted with retroviral vectors. Old and new methods for assessing this phenomenonare discussed with a view to provide a comprehensive account of this emerging field.