Bone Morphogenic Protein-7 (BMP7) Gene Therapy Inhibits Fibrosis by Up-regulating Smad1/5/8 in Rabbit Cornea in vivo

Abstract

Purpose: : BMP7 is known to modify TGFβ-driven Smad signaling leading to fibrosis. This study tested the potential of targeted BMP7 gene therapy for corneal fibrosis delivered into rabbit keratocytes in vivo with AAV5 or polyethylenimine conjugated gold nanoparticles (PEI2-GNP) using a rabbit model.

Methods: : New Zealand White rabbits were used and divided into 3 groups. Corneal haze was produced with -9 diopter photorefractive keratectomy (PRK) with excimer laser. Animals of first group received 50μl of naked-vector, second group received 50μl AAV5-BMP7 (3.0x10¹²vg/ml) and third groups received 100μl PEI2-GNP-BMP7 transfection solution topically for 5 minutes immediately after PRK via a custom defined delivery technique. Nanoparticle transfection solution was prepared by incubating PEI2-GNP with plasmid in PBS containing 10% glucose for 30 min at 37^oC. The contralateral eyes were naïve controls. Animals were sacrificed 4-weeks after the vector application. Slitlamp-, and stereo-biomicroscopy monitored health of the eyes and level of corneal haze in live rabbits. Fibrosis was quantified by Immunohistochemistry and western blotting techniques to compare all 3 groups. Inflammation and toxicity was measured with CD11b, F4/80 and TUNEL assay.

Results: : Slitlamp biomicroscopy quantification showed that single BMP7 gene therapy given via AAV or nanoparticle decreased corneal haze up to 61% in rabbit eyes. Histological analysis showed significant reduction in fibrosis parameters (alpha smooth muscle actin, fibronectin and f-actin) levels in BMP7-delivered corneas compared to undelivered cornea. Real-time PCR and southern blotting confirmed delivery of BMP7 gene in the rabbit corneas. AAV vector showed no toxicity whereas PEI2-GNP showed mild inflammation and coloration in the cornea at tested N/P ratio of 180 in slitlamp biomicroscopy. Histological examination of toxicity in nanoparticle- or AAV-applied corneas with TUNEL assay and inflammatory markers (CD11b and F4/80) are underway cells. Statistical analysis is pending.

Conclusions: : This study provides evidence that hybrid gold nanoparticle-mediated BMP7 gene therapy has potential to treat corneal fibrosis in the rabbit cornea in vivo with no major side effects.

Keywords: cornea: stroma and keratocytes • gene transfer/gene therapy • wound healing