Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography.

BACKGROUND: Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. METHODS: HEK-293 cells were cultured in ten-layer CellStacks() and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP). Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader. RESULTS: Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF-AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX-GF) resulted in a vector yield of 2.3 x 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 10(7) pfu/cm(2) for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. CONCLUSIONS: We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation.

Recovery of cardiac function mediated by MSC and interleukin-10 plasmid functionalised scaffold.

Stem cell transplantation has been suggested as a treatment for myocardial infarction, but clinical studies have yet to demonstrate conclusive, positive effects. This may be related to poor survival of the transplanted stem cells due to the inflammatory response following myocardial infarction. To address this, a scaffold-based stem cell delivery system was functionalised with anti-inflammatory plasmids (interleukin-10) to improve stem cell retention and recovery of cardiac function. Myocardial infarction was induced and these functionalised scaffolds were applied over the infarcted myocardium. Four weeks later, stem cell retention, cardiac function, remodelling and inflammation were quantified. Interleukin-10 gene transfer improved stem cell retention by more than five-fold and the hearts treated with scaffold, stem cells and interleukin-10 had significant functional recovery compared to the scaffold control (scaffold: -10 ± 7%, scaffold, interleukin-10 and stem cells: +7 ± 6%). This improved function was associated with increased infarcted wall thickness and increased ratios of collagen type III/type I, decreased cell death, and a change in macrophage markers from mainly cytotoxic in the scaffold group to mainly regulatory in scaffold, stem cells and interleukin-10 group. Thus, treatment of myocardial infarction with stem cells and interleukin-10 gene transfer significantly improved stem cell retention and ultimately improved overall cardiac function.

Generation of antioxidant adenovirus gene transfer vectors encoding CuZnSOD, MnSOD, and catalase.

Replication-deficient adenovirus gene transfer vectors are very useful for the experimental delivery of genes into cells and are widely used both in vitro and in vivo to determine the effects of transgene expression. Having a broad cell tropism, these vectors allow efficient transduction of many cell types and permit transfer of large amounts of DNA with resulting high expression levels within the target cell. Manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD) and catalase are all known antioxidants whose over-expression can result in amelioration of pathology brought about by an excess of reactive oxygen species within a cell. Their use has been suggested as therapies for many conditions, including cardiovascular disease, arthritis, diabetes, cancer, and damage to central nervous system cells. This chapter describes the methodology commonly used for production of replication-deficient adenovirus vectors encoding MnSOD, CuZnSOD, and catalase.

Adenoviral-mediated gene transfer of nitric oxide synthase isoforms and vascular cell proliferation.

OBJECTIVE: Many vascular diseases are associated with reduced nitric oxide (NO) bioavailability. Nitric oxide synthase (NOS) gene therapy to the vasculature is a possible treatment for vascular disease as a means of increasing NO bioavailability, and this may be achieved using any of the NOS isoforms. The aim of our study was to compare the effects of adenoviral-mediated overexpression of the most commonly used NOS isoforms eNOS and iNOS on vascular cell proliferation. METHODS: Human coronary artery smooth muscle cells (HCSMCs) and human umbilical vein endothelial cells (HUVECs) were transduced with adenoviral vectors encoding eNOS or iNOS at a multiplicity of infection of 100. Control cells were exposed to AdNull (empty vector) or diluent alone. Transgene expression was sought by Western blotting. The Greiss assay was used to measure nitrite levels. Cell proliferation was assessed by cell counting on days 0, 3 and 6. Apoptosis was sought using FACS analysis. Angiogenesis was measured using a commercially available in vitro kit. RESULTS: Expression of both isoforms was detected in transduced cells by Western blot at all three time points. NOS transduction resulted in increased nitrite levels with higher levels seen in iNOS- compared to eNOS-transduced cells. Cell proliferation was diminished in AdeNOS- and AdiNOS-transduced cells compared with non-transduced cells on days 3 and 6 in both HCSMCs and HUVECs. Apoptosis was not detected in either cell line with either of the isoforms at any timepoint studied. Both eNOS and iNOS gene transfer caused a reduction in angiogenesis. CONCLUSIONS: NOS gene transfer to both endothelial and vascular smooth muscle cells is antiproliferative and antiangiogenic. The biological effect is identical with both isoforms and there is no evidence to support a differential effect on endothelial and vascular smooth muscle cell biology.