Suppression of Choroidal Neovascularization by AAV-Based Dual-Acting Antiangiogenic Gene Therapy

Anne Louise Askou,Sidsel Alsing,Josephine N.E Benckendorff,Andreas Holmgaard,Jacob Giehm Mikkelsen,Lars Aagaard,Toke Bek,Thomas J Corydon

doi:10.1016/j.omtn.2019.01.012

Abstract

Vascular endothelial growth factor A (VEGFA) is involved in the pathogenesis of vasoproliferative retinal diseases, such as exudative age-related macular degeneration (AMD). The objective of this study was to investigate whether dual-acting therapy based on the simultaneous expression of anti-VEGFA microRNAs (miRNAs) and the secreted, antiangiogenic protein pigment endothelial-derived factor (PEDF) delivered by adeno-associated virus (AAV) vectors provides improved protection against choroidal neovascularization (CNV). To investigate this, a multigenic AAV vector allowing retina pigment epithelium (RPE)-specific expression of anti-VEGFA miRNAs and PEDF was engineered. Robust expression of PEDF, driven by the RPE-specific vitelliform macular dystrophy 2 promoter, was observed in human cells and in mouse retina. A significant reduction in CNV was observed in a laser-induced CNV mouse model 57 days post-injection of the AAV5 particles conveying either anti-VEGFA miRNA and PEDF dual therapy or anti-VEGFA miRNA monotherapy. Overall, CNV reduction was most prominent in animals receiving dual-acting therapy. In both cases, the reduction in CNV was accompanied by a significant attenuation of VEGFA. In conclusion, the presented data reveal that gene therapy targeting VEGFA via multigenic AAV vectors displays combined efficacy, suggesting that dual-acting therapy is an important tool in future eye gene therapy for the treatment of neovascular ocular diseases, including AMD.

Highlights

For the production of AAV5 particles, the two back-to-back RNA polymerase II (Pol II)-driven expression cassettes were cloned into the pAAV/small interfering RNA (siRNA) shuttle vector, resulting in the three plasmids entitled p/miR(Irr)-AsR-PE, p/miR(5,B,7)-AsR-PE, and p/miR(5,B,7)-Pigment endothelial-derived factor (PEDF)-PE (Figure 1A)
We show that incorporation of the multigenic expression cassette into the associated virus (AAV) plasmid leads to the following: (1) expression of multiple proteins, including PEDF, in vitro following the transfection of relevant mammalian cells; (2) a high level of reporter gene expression in retina pigment epithelium (RPE) cells following a single subretinal injection in mice; (3) co-expression of miRNA(5,B,7) and PEDF in eyes injected with multigenic AAV/miR(5,B,7)-PEDF-PE vectors; (4) reduced choroidal neovascularization (CNV) formation in eyes treated with AAV vectors encoding therapeutic cassettes (AAV/miR(5,B,7)-AsR-PE and AAV/miR(5,B,7)-PEDFPE); and (5) an increased, albeit not statistically significant, efficacy due to combined expression of multiple antiangiogenic molecules from the same vector
The most remarkable finding was a significant reduction of CNV area by 34% and 45% in the eyes of mice treated with AAV/miR(5,B,7)

Summary

Introduction

Adeno-associated virus (AAV)-based vectors are popular and wellaccepted gene delivery vehicles for a wide variety of tissues,[1,2,3,4,5,6] and they represent promising tools for the treatment of retinal degenerations.[1,2,3,7,8,9,10] The non-pathogenic nature, long-term transgene expression in the absence of genome integration in both dividing and non-dividing cells, low immunogenicity, and ease of capsid swapping for the targeting of specific tissues are attractive features of recombinant AAV (rAAV) vectors.[11,12,13] These advances have made rAAV the vector of choice for most gene therapy applications to the eye, illustrated by several clinical trials[2,7,10,14] utilizing rAAV particles to ferry genes to specific retinal cells. The anatomical advantages, including shape, Gene therapy has been applied to acquired retinal diseases, such as neovascular age-related macular degeneration (nAMD) (ClinicalTrails.gov: NCT00109499, NCT01494805, NCT01024998, NCT01301443, NCT00363714, NCT00713518). nAMD is the leading cause of blindness in the western world, and the disease is currently treated by repetitive, often monthly intraocular injections of antivascular endothelial growth factor (VEGF) drugs (e.g., antibodies or traps) to maintain vision.[24,25,26] nAMD is a complex and

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