Abstract: Integration of viral vectors into a host genome is associated with insertional mutagenesis and subjects in clinical gene therapy trials must be monitored for this adverse event. Several PCR based methods such as ligase-mediated (LM) PCR, linear-amplification-mediated (LAM) PCR and non-restrictive (nr) LAM PCR were developed to identify sites of vector integration. Coupling the power of next-generation sequencing technologies with various PCR approaches will provide a comprehensive and genome-wide profiling of insertion sites and increase throughput. In this bioinformatics study, we aimed to develop and apply quality metrics to viral insertion data obtained using next-generation sequencing. We developed five simple metrics for assessing next-generation sequencing data from different PCR products and showed how the metrics can be used to objectively compare runs performed with the same methodology as well as data generated using different PCR techniques. The results will help researchers troubleshoot complex methodologies, understand the quality of sequencing data, and provide a starting point for developing standardization of vector insertion site data analysis.
Abstract: Oncolytic virotherapy has advanced since the days of its conception but therapeutic efficacy in the clinics does not seem to reach the same level as in animal models. One reason is premature oncolytic virus clearance in humans, which is a reasonable assumption considering the immune-stimulating nature of the oncolytic agents. However, several studies are beginning to reveal layers of restriction to oncolytic virotherapy that are present before an adaptive neutralizing immune response. Some of these barriers are present constitutively halting infection before it even begins, whereas others are raised by minute cues triggered by virus infection. Indeed, we and others have noticed that delivering viruses to tumors may not be the biggest obstacle to successful therapy, but instead the physical make-up of the tumor and its capacity to mount antiviral defenses seem to be the most important efficacy determinants. In this review, we summarize the constitutive and innate barriers to oncolytic virotherapy and discuss strategies to overcome them.
Abstract: Cancer has been, from the beginning, a target of intense research for gene therapy approaches. Currently, more than 60% of all on-going clinical gene therapy trials worldwide are targeting cancer. Indeed, there is a clear unmet medical need for novel therapies. This is further urged by the fact that current conventional cancer therapies are frequently troubled by their toxicities. Different gene therapy strategies have been employed for cancer, such as pro-drug activating suicide gene therapy, anti-angiogenic gene therapy, oncolytic virotherapy, gene therapy-based immune modulation, correction/compensation of gene defects, genetic manipulation of apoptotic and tumor invasion pathways, antisense, and RNAi strategies. Cancer types, which have been targeted with gene therapy, include brain, lung, breast, pancreatic, liver, colorectal, prostate, bladder, head and neck, skin, ovarian, and renal cancer. Currently, two cancer gene therapy products have received market approval, both of which are in China. In addition, the stimulation of the host’s immune system, using gene therapeutic approaches, has gained vast interest. The intention of this review is to point out the most commonly viral and non-viral vectors and methods used in cancer gene therapy, as well as highlight some key results achieved in clinical trials.
Abstract: Helper-dependent adenoviral (HDAd) vectors that are devoid of all viral coding sequences are promising non-integrating vectors for gene therapy because they efficiently transduce a variety of cell types in vivo, have a large cloning capacity, and drive long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd vectors is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration and result in acute toxicity, the severity of which is dose dependent. Intense efforts have been focused on elucidating adenoviral vector–host interactions and the factors involved in the acute toxicity. This review focuses on the recent acquisition of data on such interactions and on strategies investigated to improve the therapeutic index of HDAd vectors.
Abstract: Adenoviruses (Ads) infect a broad range of tissue types, and derived vectors have been extensively used for gene therapy. Helper-dependent Ad vectors (HDAds), devoid of viral coding sequences, allow for insertion of large or multiple transgenes in a single vector and have been preclinically used for the study of genetic disorders. However, the clinical application of Ad vectors including HDAds for genetic disorders has been hampered by an acute toxic response. This characteristic, while disadvantageous for gene replacement therapy, could be strategically advantageous for the activation of an immune response if HDAds were used as an adjunct treatment in cancer. Cancer treatments including immunotherapy are frequently limited by the inhibitory environment produced by both tumors and their stroma, each of which express numerous inhibitory molecules. Hence, multiple inhibitory mechanisms must be overcome for development of anti-tumor immunity. The large coding capacity of HDAds can accommodate multiple immune modulating transgenes that could produce a combined effect to overcome tumor-derived inhibition and ensure intratumoral effector T-cell proliferation and function. In this review, we discuss the potential advantages of HDAds to cancer immunotherapy based on potent host immune responses to Ads.
Abstract: Humans are unable to synthesise ascorbate (Vitamin C) due to the lack of a functional gulonolactone oxidase (Gulo), the enzyme that catalyses the final step in the biosynthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including as a cofactor for metalloenzymes that regulate the transcription factor hypoxia-inducible factor-1 (HIF-1), which governs cell survival under hypoxia. In most animals, ascorbate is made in liver cells. This study aimed to restore ascorbate synthesis to human hepatocellular carcinoma HepG2 cells and determine the effect of internally produced ascorbate on HIF-1 activation. HepG2 cells were gene-modified with a plasmid encoding the mouse Gulo cDNA, tested for genomic incorporation by PCR and ascorbate synthesis by high performance liquid chromatography. Levels of HIF-1 protein were measured using Western blotting. Gulo-modified HepG2 cells showed increased adherence compared to control HepG2 cells. A PCR-positive clone synthesised ascorbate when the Gulo substrate, l-gulono-1,4-lactone, was supplied. Intracellular ascorbate concentrations reached 5% of saturation levels (6 nmol/106 cells). Addition of ascorbate or gulonolactone reduced HIF-1 accumulation in the Gulo clone, but also in parental HepG2 cells. Our data confirm the requirement for a number of factors in addition to Gulo in the ascorbate biosynthesis pathway in human cells.