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VCAM-1 targeted AAV6 gene therapy for atherosclerosis treatment
thesis
posted on 2017-03-22, 01:26 authored by Hannah PearceCardiovascular
disease is a major cause of death and disability worldwide, and a key
underlying factor in this is atherosclerosis. This involves the build-up of
plaque in arteries, due in part to an inflammatory response by the immune
system. A key molecule involved in this process is vascular cell adhesion
molecule-1 (VCAM-1), which is highly expressed at the inflamed lesion site and
participates in the recruitment of immune cells to plaques. Given the long term
and progressive nature of atherosclerosis, a long lasting therapeutic approach
such as gene therapy would be desirable. However the most clinically relevant
gene therapy vector; adeno associated virus (AAV), does not transduce the
vasculature efficiently, and the transduction is also limited by background
uptake in various tissues.
Targeting AAV towards VCAM-1 expressed on the inflamed endothelium could allow efficient delivery of gene therapies to atherosclerotic lesions, and reduce off target transduction. To achieve this, AAV serotype 6 (AAV6) is employed, along with an antibody single chain variable fragment (scFv) that binds specifically to mouse VCAM-1.
With the view of therapeutic relevance a fusion construct, CD7/VCAM, which inhibits VCAM-1 and thus immune cell recruitment in vitro, was also trialled with AAV6. This thesis however focuses more on the production methods and conjugations strategies in order to produce and test the AAV6 targeted towards VCAM-1, without the CD7/VCAM therapeutic gene.
The anti VCAM-1 scFv was initially produced in a bacterial expression system, before an improved production method in an insect cell expression system was developed, enabling pure scFv to be produced with yields of 1-5mg/L culture. The scFv bound specifically to VCAM-1 expressing SVEC4-10 cells in vitro and contained an LPETG peptide tag at its C terminus, allowing enzymatic Sortase conjugations. The AAV6 was then genetically engineered to contain the matching Sortase conjugation tag; GGGWW, at the externally localised N terminus of its redundant VP2 capsid protein. Unfortunately, when produced the entire VP2 protein was largely absent from the capsid, and the modified AAV6 displayed additionally impaired endothelial cell transduction.
Following this, a different approach was employed, using methylglyoxal (MGO) to form adducts with arginine residues on the exterior of the AAV6, abolishing some of the native tropism. It also allowed attachment of an azide group using azide-MGO (N3MGO). Anti VCAM-1 scFv that had been Sortase conjugated to the azide reactant group BCN could then be attached to the exterior of the AAV6 using click chemistry, as shown with western blotting. Native AAV6 had significantly lower transduction in endothelial SVEC4-10 cells compared to control cells, however following scFv attachment the uptake in SVEC4-10 cells was enhanced relative to controls, particularly with reduced incubations. This was entirely inhibited by VCAM-1 competitive inhibition, indicating specificity. The system developed with this conjugation strategy thus enables retargeting of the AAV6 gene therapy vector to VCAM-1 expressing cells and warrants further testing. Overall, as all the components are interchangeable, a highly flexible platform technology for gene transfer could be established.
Targeting AAV towards VCAM-1 expressed on the inflamed endothelium could allow efficient delivery of gene therapies to atherosclerotic lesions, and reduce off target transduction. To achieve this, AAV serotype 6 (AAV6) is employed, along with an antibody single chain variable fragment (scFv) that binds specifically to mouse VCAM-1.
With the view of therapeutic relevance a fusion construct, CD7/VCAM, which inhibits VCAM-1 and thus immune cell recruitment in vitro, was also trialled with AAV6. This thesis however focuses more on the production methods and conjugations strategies in order to produce and test the AAV6 targeted towards VCAM-1, without the CD7/VCAM therapeutic gene.
The anti VCAM-1 scFv was initially produced in a bacterial expression system, before an improved production method in an insect cell expression system was developed, enabling pure scFv to be produced with yields of 1-5mg/L culture. The scFv bound specifically to VCAM-1 expressing SVEC4-10 cells in vitro and contained an LPETG peptide tag at its C terminus, allowing enzymatic Sortase conjugations. The AAV6 was then genetically engineered to contain the matching Sortase conjugation tag; GGGWW, at the externally localised N terminus of its redundant VP2 capsid protein. Unfortunately, when produced the entire VP2 protein was largely absent from the capsid, and the modified AAV6 displayed additionally impaired endothelial cell transduction.
Following this, a different approach was employed, using methylglyoxal (MGO) to form adducts with arginine residues on the exterior of the AAV6, abolishing some of the native tropism. It also allowed attachment of an azide group using azide-MGO (N3MGO). Anti VCAM-1 scFv that had been Sortase conjugated to the azide reactant group BCN could then be attached to the exterior of the AAV6 using click chemistry, as shown with western blotting. Native AAV6 had significantly lower transduction in endothelial SVEC4-10 cells compared to control cells, however following scFv attachment the uptake in SVEC4-10 cells was enhanced relative to controls, particularly with reduced incubations. This was entirely inhibited by VCAM-1 competitive inhibition, indicating specificity. The system developed with this conjugation strategy thus enables retargeting of the AAV6 gene therapy vector to VCAM-1 expressing cells and warrants further testing. Overall, as all the components are interchangeable, a highly flexible platform technology for gene transfer could be established.
