Suppression of Choroidal Neovascularization in Mice by Subretinal Delivery of Multigenic Lentiviral Vectors Encoding Anti-Angiogenic MicroRNAs

Abstract

Lentivirus-based vectors have been used for the development of potent gene therapies. Here, application of a multigenic lentiviral vector (LV) producing multiple anti-angiogenic microRNAs following subretinal delivery in a laser-induced choroidal neovascularization (CNV) mouse model is presented. This versatile LV, carrying back-to-back RNApolII-driven expression cassettes, enables combined expression of microRNAs targeting vascular endothelial growth factor A (Vegfa) mRNA and fluorescent reporters. In addition, by including a vitelliform macular dystrophy 2 (VMD2) promoter, expression of microRNAs is restricted to the retinal pigment epithelial (RPE) cells. Six days post injection (PI), robust and widespread fluorescent signals of eGFP are already observed in the retina by funduscopy. The eGFP expression peaks at day 21 PI and persists with stable expression for at least 9 months. In parallel, prominent AsRED co-expression, encoded from the VMD2-driven microRNA expression cassette, is evident in retinal sections and flat-mounts, revealing RPE-specific expression of microRNAs. Furthermore, LV-delivered microRNAs targeting the Vegfa gene in RPE cells reduced the size of laser-induced CNV in mice 28 days PI, as a consequence of diminished VEGF levels, suggesting that LVs delivered locally are powerful tools in the development of gene therapy-based strategies for treatment of age-related macular degeneration.

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

“…Both methods verified vector-encoded expression of PEDF in transfected melanoma cells, and, furthermore, western blot analysis revealed secretion of a 46-kDa protein into the medium, consistent with the size of PEDF. Together with previous studies,34, 36 these observations verified functionality of the expression cassettes, and vectors were packaged in AAV5 capsids.…”

“…Multigenic LVs encoding antiangiogenic miRNAs (miR5,B,7) and proteins have been engineered and thoroughly described in our previous studies 34, 35, 36. Specifically, correct processing of the three miRNAs (miR5, miRB, and miR7) from the intron region of our expression cassette, resulting in ∼21- to 23-nt-long mature miRNAs, was verified by northern blotting 34 .…”

“…To verify that the detected reduction in CNV area coincided with the suppression of VEGFA, western blotting was performed on whole-eye cellular extracts obtained from mice likewise injected with 4.2 × 10 9 vg AAV/miR(Irr)-AsR-PE, AAV/miR(5,B,7)-AsR-PE, and AAV/miR(5,B,7)-PEDF-PE. To verify changes in VEGF expression, ten laser burns were applied in each injected eye outside the transduced area 36, 47, 48, 49. 3 days after laser rupture of Bruch’s membrane, mice were sacrificed and western blotting was performed (Figure 5D).…”

“…Cell-specific, robust, and stable expression was obtained in RPE cells for up to 9 months following a single injection of LVs encoding therapeutic anti-VEGFA miRNAs expressed from the RPE-specific vitelliform macular dystrophy 2 (VMD2) promoter. Remarkably, significant VEGFA silencing resulted in reduced choroidal neovascularization (CNV) size in the laser-induced CNV mouse model following subretinal delivery of the multigenic vector, 36 suggesting that virus-based gene delivery is a viable option for sustained, combinational treatment of retinal neovascular diseases.…”

c-Jun-mediated microRNA-302d-3p induces RPE dedifferentiation by targeting p21Waf1/Cip1

“…Compared to traditional therapeutic strategies, miRNA-based treatments have more advantages in drug efficiency and delivery 16 . Gene therapies targeting miRNAs in treating CNV have also been well developed 43 , 44 . Therefore, miR-302d-3p inhibitors with high efficiency, including tough decoys (TuDs) and small guide RNA with CRISPR/Cas9 system, are prospective in suspending or even preventing AMD disease course 45 , 46 .…”