Aideen O'Doherty

Purification of adenovirus and adeno-associated virus: comparison of novel membrane-based technology to conventional techniques

Adenovirus (Ad) and Adeno-associated virus (AAV) are efficient gene delivery systems; manipulation of the wild-type genome allows their use as vectors for the overexpression of desirable transgenes. Generation and purification of such viral vectors can be labour intensive, costly and require specialized equipment, but a new generation of membrane-mediated ion exchange kits for purification of recombinant virus may facilitate this process. Here, we examine the yields, transgene expression and purity of preparations of Ad and AAV purified using commercially available kits in comparison to other established techniques for purification of recombinant viral vectors. We demonstrate comparable results for Ad and AAV respectively in all parameters investigated, with a substantial reduction in purification time for the kit-based technology. Such approaches are attractive methods for small-scale purification of recombinant Ad and AAV viral vectors.

Development and characterisation of a novel rat model of Parkinson's disease induced by sequential intranigral administration of AAV-α-synuclein and the pesticide, rotenone.

Modeling Parkinsons disease remains a major challenge for preclinical researchers, as existing models fail to reliably recapitulate all of the classic features of the disease, namely, the progressive emergence of a bradykinetic motor syndrome with underlying nigrostriatal α-synuclein protein accumulation and nigrostriatal neurodegeneration. One limitation of the existing models is that they are normally induced by a single neuropathological insult, whereas the human disease is thought to be multifactorial with genetic and environmental factors contributing to the disease pathogenesis. Thus, in order to develop a more relevant model, we sought to determine if administration of the Parkinsons disease-associated pesticide, rotenone, into the substantia nigra of rats overexpressing the Parkinsons disease-associated protein, α-synuclein, could reliably model the triad of classic features of the human disease. To do so, rats underwent stereotaxic surgery for unilateral delivery of the adeno-associated virus (AAV)-α-synuclein into the substantia nigra. This was followed 13 weeks later by delivery of rotenone into the same site. The effect of the genetic and environmental insults alone or in combination on lateralised motor performance (Corridor, Stepping, and Whisker Tests), nigrostriatal integrity (tyrosine hydroxylase immunohistochemistry), and α-synucleinopathy (α-synuclein immunohistochemistry) was assessed. We found that rats treated with either AAV-α-synuclein or rotenone developed significant motor dysfunction with underlying nigrostriatal neurodegeneration. However, when the genetic and environmental insults were sequentially administered, the detrimental impact of the combined insults on motor performance and nigrostriatal integrity was significantly greater than the impact of either insult alone. This indicates that sequential exposure to relevant genetic and environmental insults is a valid approach to modeling human Parkinsons disease in the rat.

The behavioural and neuropathological impact of intranigral AAV-α-synuclein is exacerbated by systemic infusion of the Parkinson's disease-associated pesticide, rotenone, in rats

Despite the widely held belief that Parkinsons disease is caused by both underlying genetics and exposure to environmental risk factors, it is still widely modelled in preclinical models using a single genetic or neurotoxic insult. This single-insult approach has resulted in a variety of models that are limited with respect to their aetiological, construct, face and/or predictive validity. Thus, the aim of the current study was to investigate the interplay between genes and the environment as an alternative approach to modelling Parkinsons disease. To do so, rats underwent stereotaxic surgery for unilateral delivery of the Parkinsons disease-associated gene, α-synuclein, into the substantia nigra (using AAV vectors). This was followed 13 weeks later by subcutaneous implantation of an osmotic minipump delivering the Parkinsons disease-associated pesticide, rotenone (2.5mgkg(-1)day(-1) for 4 weeks). The effect of the genetic and environmental insults alone or in combination on lateralised motor performance (Corridor, Stepping and Whisker Tests), nigrostriatal integrity (tyrosine hydroxylase immunohistochemistry) and α-synucleinopathy (α-synuclein immunohistochemistry) was assessed. We found that exposing AAV-α-synuclein-treated rats to rotenone led to a model in which the classical Parkinsons disease triad of progressive motor dysfunction, nigrostriatal neurodegeneration and α-synucleinopathy was evident. However, delivering rotenone systemically was also associated with bilateral motor dysfunction and loss of body weight. Thus, although we have shown that Parkinsons disease can be modelled in experimental animals by combined exposure to both genetic and environmental risk factors, this approach is limited by systemic toxicity of the pesticide rotenone. Direct intracerebral delivery of rotenone may be more useful in longer-term studies as we have previously shown that it overcomes this limitation.

Heat Shock Protein 70 Reduces α-Synuclein-Induced Predegenerative Neuronal Dystrophy in the α-Synuclein Viral Gene Transfer Rat Model of Parkinson's Disease

It has become increasingly evident that the nigrostriatal degeneration associated with Parkinsons disease initiates at the level of the axonal terminals in the putamen, and this nigrostriatal terminal dystrophy is either caused or exacerbated by the presence of α‐synuclein immunopositive neuronal inclusions. Therefore, strategies aimed at reducing α‐synuclein‐induced early neuronal dystrophy may slow or halt the progression to overt nigrostriatal neurodegeneration. Thus, this study sought to determine if adeno‐associated virus (AAV) mediated overexpression of two molecular chaperone heat shock proteins, namely Hsp27 or Hsp70, in the AAV‐α‐synuclein viral gene transfer rat model of Parkinsons disease could prevent α‐synuclein‐induced early neuronal pathology.

Comparison of Viral and Nonviral Vectors for Gene Transfer to Human Endothelial Progenitor Cells

BACKGROUND/AIMS The ability of endothelial progenitor cells (EPCs) to home to sites of neoangiogenesis makes them attractive candidates for use in the field of gene therapy. The efficacy of this approach depends on the efficiency of the vector used for transgene delivery. METHODS/RESULTS In this study, we have compared the efficiency of adenovirus, five serotypes of AAV2, VSVG-pseudotyped lentivirus, and nonviral plasmid/liposome DNA vectors to deliver the green fluorescence protein reporter gene to human early EPCs to determine efficacy and vector-related cell toxicity. Adenovirus proved most effective with efficiencies of up to 80% with low levels of cell death. Lower levels of expression were seen with other vectors. Electroporation proved unsuitable at the parameters tested. We have also identified at least two distinct subpopulations that exist in the heterogeneous parent EPC culture, one of which is amenable to transduction with adenovirus and one that is not. In addition, adenoviral transduction did not disrupt the ability of the cells to incorporate into endothelial structures in vitro. CONCLUSION We have found adenovirus to be the most efficient of the vector systems tested for gene delivery to EPCs, an effect that is mediated almost entirely by one of two identified subpopulations.

Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5.

Vascular gene therapy requires safe and efficient gene transfer in vivo. Recombinant adeno‐associated virus (AAV) is a promising viral vector but its use in the vasculature has produced conflicting results and serotypes other than AAV2 have not been intensively studied. We investigated the efficiency of alternative AAV serotypes for vascular gene delivery in vitro and in vivo.

Differentiation of Vascular Stem Cells Contributes to Ectopic Calcification of Atherosclerotic Plaque

The cellular and molecular basis of vascular calcification (VC) in atherosclerosis is not fully understood. Here, we investigate role of resident/circulating progenitor cells in VC and contribution of inflammatory plaque environment to this process. Vessel‐derived stem/progenitor cells (VSCs) and mesenchymal stem cells (MSCs) isolated from atherosclerotic ApoE−/− mice showed significantly more in vitro osteogenesis and chondrogenesis than cells generated from control C57BL/6 mice. To assess their ability to form bone in vivo, cells were primed chondrogenically or cultured in control medium on collagen glycosaminoglycan scaffolds in vitro prior to subcutaneous implantation in ApoE−/− and C57BL/6 mice using a crossover study design. Atherosclerotic ApoE−/− MSCs and VSCs formed bone when implanted in C57BL/6 mice. In ApoE−/− mice, these cells generated more mature bone than C57BL/6 cells. The atherosclerotic in vivo environment alone promoted bone formation by implanted C57BL/6 cells. Un‐primed C57BL/6 VSCs were unable to form bone in either mouse strain. Treatment of ApoE−/− VSC chondrogenic cultures with interleukin (IL)‐6 resulted in significantly increased glycosaminoglycan deposition and expression of characteristic chondrogenic genes at 21 days. In conclusion, resident vascular cells from atherosclerotic environment respond to the inflammatory milieu and undergo calcification. IL‐6 may have a role in aberrant differentiation of VSCs contributing to vascular calcification in atherosclerosis. Stem Cells 2016;34:913–923

ENHANCED ENDOCHONDRAL OSSIFICATION IN VESSEL DERIVED STEM CELLS BY ATHEROSCLEROTIC ENVIRONMENT

Vascular calcification, far from being a degenerative passive process, is increasingly being understood as an active, highly organised cell controlled event. The presence of a number of stem progenitor niches and/or lineages in vasculature has been postulated and their possible role in vascular calcification is heavily under investigation. Pericytes are such poorly defined cell populations. Although traditionally considered as supporting cells, they have recently been proposed to play a more active role in the repair and pathogenesis of various vascular diseases. In this study, we hypothesised that a pericyte-like stem cell population, termed vessel derived stem cells or VSCs with chondrogenic and osteogenic potential exist in the vessel wall and in presence of the inflammatory cytokines seen in atherosclerotic environment, contribute, along with the circulating mesenchymal stem cells (MSCs), to the calcification of atherosclerotic plaques vi a endochondral ossification. VSCs from aortae of ApoE−/− mice and background C57BL/6 mice were isolated and characterised for cell surface markers by flow cytometry and immunocytochemistry. MSCs from bone marrow of these mice were also isolated and characterised. To assess the ability of VSCs and MSCs from normal and ApoE−/− mice to form bone, cells were seeded onto collagen glycosaminoglycan scaffolds and primed chondrogenically in vitro followed by subcutaneous implantation for 8 weeks. Chondrogenically primed constructs from both cell types (VSCs and MSCs) showed the ability to form bone by endochondral ossification in vivo in both ApoE−/ and C57BL/6 mice. Assessment of quantity and quality of bone formed is currently being performed.

8 Ossification of atherosclerotic plaque: the role of vessel derived stem cells

Introduction Pericytes, although traditionally considered as supporting cells, have recently been proposed to have a more active role in the repair and pathogenesis of various vascular diseases. In this study, we hypothesised that a pericyte-like stem cell population, termed vessel derived stem cells (VSCs), with chondrogenic and osteogenic potential exists in the vessel wall and in the presence of the inflammatory cytokines seen in atherosclerotic environment contributes, along with the circulating mesenchymal stem cells, to the calcification of atherosclerotic plaque which occurs through the endochondral pathway. Methods VSCs from aortae of ApoE−/− mice and control C57BL/6 mice were isolated and characterised for cell surface markers by flow cytometry and immunocytochemistry. MSCs from the bone marrow of these mice were also isolated and characterised. Chondrogenic potential of these cells was investigated in presence or absence of inflammatory cytokines such as IL-6 and IFN-γ. Results and Discussion Isolated VSCs were strongly positive for Sca-1, CD44 and negative for CD31 and CD34. A sub-population of VSCs also expressed 3G5, a specific pericyte marker. Differentiation assays demonstrated the ability of the cells to undergo osteogenesis and chondrogenesis. VSCs had significantly higher GAG/DNA ratio than MSCs indicating comparatively increased chondrogenesis. That both MSCs and VSCs from the ApoE−/− atherosclerotic mice generate a more mature hypertrophic chondrocyte than cells from the C57BL/6 mice is interesting and suggests that the atherosclerotic environment may modulate the stem cell phenotype. Collagen-type II and aggrecan expression and effect of oxidised LDL on these cells in vitro will be investigated to further test this hypothesis.