title: Assessing the potential of non-integrating lentiviral vectors for cardiovascular gene therapy
Creators: name: Chick, Helen Elizabeth
Publisher: name: University of Glasgow
Year: 2011
Subjects: subject: Non-integrating lentiviral vectors, acute vascular injury, vascular gene therapy, Nogo-B

description: Vascular smooth muscle cell (VSMC) migration and proliferation are important hallmarks in the development of the neointima formation (NIF) following acute vascular injury, contributing significantly to the pathogenesis of post-angioplasty restenosis, in-stent restenosis (ISR) and low lumen patency of coronary artery bypass grafting. At present there are a finite number of pharmacological treatments in use for the prevention of the NIF, such as drug-eluting stents (DES) for ISR and more are under development. Vascular gene delivery strategies have been extensively explored. There is a requirement to identify and provide a safe and effective gene transfer strategy for therapeutic gene delivery to attenuate NIF in the clinical setting. Lentiviral vector (LV)s are efficient in transducing vascular cells and have been considered as useful vectors for delivering therapeutic interventions to reduce the NIF following acute vascular injury in vivo. Non-integrating LV (NILV)s offer additional potential for LVs in vascular gene therapy. Nogo-B is a member of the reticulon 4 family of transmembrane proteins, and is expressed in vascular endothelial cell (VEC)s and VSMCs. Nogo-B is a regulator in vascular maintenance and remodelling. Nogo-B has a favourable profile for the prevention of the NIF, because this protein acts as a positive and negative regulator of VECs and VSMCs, respectively. This effect is facilitated by Nogo-B mediated chemo-attraction of VECs, and the antagonism of migration and proliferation of VSMCs in response to platelet derived growth factor (PDGF) and/or serum. Nogo-B is down-regulated following vascular injury, which correlates with the development of NIF. Previously, it was demonstrated that adenoviral-mediated Nogo-B gene transfer facilitated over-expression of Nogo-B, and rescued the injury-induced NIF in two distinct and appropriate in vivo models of acute vascular injury (the murine wire-injury model and the porcine vein grafting model).
 
The aim of this thesis was to assess the potential of NILVs for application to vascular gene therapy, by over-expression of Nogo-B. First, second generation self-inactivating (SIN) LVs were optimised (in terms of promoter alternatives, pseudotyping and integration-deficiency) for transgene expression in human vascular cells. Second, the effects of Nogo-B over-expression on human VSMC migration and proliferation, mediated by NILVs were assessed in vitro and compared to its integrating counterparts. Third, the mechanism of action of Nogo-B on human primary VSMCs was explored in vitro.

LVs were evaluated for efficiency and/or longevity in vascular cell gene transfer in vitro with regards to alternative internal heterologous promoters, pseudotyping and integration-proficiency versus integration-deficiency. Undoubtedly, both promoters, spleen focus forming virus (SFFV) and enhancer-less ubiquitously chromatin opening element (UCOE) evoked efficient and longevity of [vesicular stomatitis virus glycoprotein (VSV-g) pseudotyped] integrating-LV (int-LV)-mediated eGFP transgene expression in VECs and VSMCs. However in contrast to the UCOE promoter, the SFFV promoter was significantly higher in terms of transgene expression in VECs and VSMCs, and thereby was used in subsequent experiments. Interestingly, int-LVs pseudotyped with Rabies, Baculovirus glycoprotein 64 or Ebola Zaire glycoprotein envelope, demonstrated their potential usefulness in efficient VEC transduction and subsequent eGFP transgene expression. Importantly, VSV-g pseudotyped NILVs were efficient in mediating the expression of eGFP in VECs and VSMCs. Thereafter, these selected VSV-g pseudotyped NILVs under the control of the SFFV promoter were used for therapeutic gene transfer analysis in VSMCs in vitro. 

A new gene delivery vector, NILV expressing Nogo-B (NILV-Nogo-B) was constructed and analysed in vitro for its therapeutic potential in the prevention of NIF associated with acute vascular injury. The Nogo-B cDNA was cloned into a LV construct plasmid (consisting of the SFFV promoter) for the generation of int-LV expressing Nogo-B (int-LV-Nogo-B) and NILV-Nogo-B. Indeed, in contrast to their LV expressing eGFP (LV-eGFP) control vector and un-infected VSMCs (endogenous Nogo-B level), NILV-Nogo-B mediated efficient Nogo-B over-expression in human VSMCs, which led to significant phenotypic effects on migration and proliferation with similar effects to int-LV-Nogo-B. NILV-Nogo-B-mediated gene transfer was as effective as its integrating counterparts in reducing VSMC migration. Interestingly, NILV-Nogo-B was at least as good as int-LV-Nogo-B in inhibiting VSMC proliferation.
 
The mechanism of action of Nogo-B associated with phenotypic effects on VSMC migration and proliferation was evaluated in vitro. Previous reports have implicated Nogo-B as being a pro-apoptotic protein, and therefore the effect of LV-mediated gene transfer of Nogo-B on VSMC apoptosis was assessed. Evidently, neither int-LV-Nogo-B nor NILV-Nogo-B significantly induced VSMC apoptosis. Over-expression of Nogo-B mediated by int-LV was observed co-localised with the endoplasmic reticulum (ER) in VSMCs. A published report indicated that the over-expression of Nogo-B induced ER-stress mediated apoptosis. Results suggested that Nogo-B over-expression did not significantly induce ER-stress signalling in VSMCs. Interestingly, Nogo-B over-expression significantly reduced ERK 1 &amp; 2 activation in VSMCs, but not Akt signalling transduction pathway, and therefore this could have possible implications in phenotypic effects on VSMC migration and proliferation.

Taken together, this thesis demonstrated that SFFV-driven VSV-g pseudotyped NILVs are efficient for vascular cell gene transfer at least in vitro. Additionally, this thesis supports the use of NILV-Nogo-B as a candidate for therapeutic application for the prevention of NIF associated with acute vascular injury during revascularisation in vivo. In conclusion, this study addresses important implications for the use of NILVs as a potential therapeutic vector in vascular cell gene delivery studies.