Abstract
Purpose :
The current treatment for AMD is limited to the direct injection of antibody fragments against VEGF, which requires repeated administration and is not always effective. This genetic and functional study has identified that HTRA1 is a crucial mediator in the development and progression of AMD. We plan to therapeutically target HTRA1 by generating an AAV vector, which can specifically deliver the neutralizing antibody fragment to RPE for long term expression.
Methods :
An AAV backbone vector containing a VMD2 promoter region driving RPE-specific expression of anti-HTRA1 scFv and a secretory signal for soluble expression in the eye was transfected into ARPE19 cells to examine the expression and secretion of scFv. The inhibitory function of the scFv to the HTRA1 activity was evaluated with protease activity assay. The in vivo expression and anti-HTRA1 scFv in mouse retina were evaluated by qPCR, Western blotting, and immune-fluorescent staining.
Results :
The AAV-VMD2-anti-HTRA1 cassette was confirmed with restriction enzymatic analysis and sequencing. The plasmid was then transfected into human ARPE19 cells. Secretory anti-HTRA1 was collected from cell supernatant and confirmed its affinity to and inhibitory effect on HTRA1. The plasmid was then packaged into AAV particles, which were used for the transfection of ARPE19 cells in vitro and mice eyes in vivo via intravitreal injection. Western blot result confirmed a distinct band showing AAV-mediated anti-HTRA1 expression in ARPE19 cells as well as adult mice eyes. IHC identified anti-HTRA1 expression location in adult mice eye, which is not limited to the RPE layer but across all retinal layers as well. qPCR results on adult mice eye extract demonstrated a high level of anti-HTRA1 mRNA production.
Conclusions :
We have constructed an AAV vector that can specifically deliver the neutralizing anti-HTRA1 fragment into RPE cells and produce significant titer of soluble scFv in the culture medium. Characterization of its bioactivity showed that the AAV-produced scFv-anti-HTRA1 can effectively inhibit the protease activity of HTRA1. Intravitreal injection of AAV demonstrated the retinal and RPE expression of anti-HTRA1 in the mouse. These data show the therapeutic potential of delivering engineered antibody fragment for RPE specific expression with rAAV vector and targeting HTRA1 activity for the treatment of AMD.
This is a 2020 ARVO Annual Meeting abstract.