Attenuation of corneal myofibroblast development through nanoparticle-mediated soluble transforming growth factor-β type II receptor (sTGFβRII) gene transfer

Purpose: To explore (i) the potential of polyethylenimine (PEI)-DNA nanoparticles as a vector for delivering genes into human corneal fibroblasts, and (ii) whether the nanoparticle-mediated soluble extracellular domain of the transforming growth factor–β type II receptor (sTGFβRII) gene therapy could be used to reduce myofibroblasts and fibrosis in the cornea using an in vitro model. Methods: PEI-DNA nanoparticles were prepared at a nitrogen-to-phosphate ratio of 30 by mixing linear PEI and a plasmid encoding sTGFβRII conjugated to the fragment crystallizable (Fc) portion of human immunoglobulin. The PEI-DNA polyplex formation was confirmed through gel retardation assay. Human corneal fibroblasts (HCFs) were generated from donor corneas; myofibroblasts and fibrosis were induced with TGFβ1 (1 ng/ml) stimulation employing serum-free conditions. The sTGFβRII conjugated to the Fc portion of human immunoglobulin gene was introduced into HCF using either PEI-DNA nanoparticles or Lipofectamine. Suitable negative and positive controls to compare selected nanoparticle and therapeutic gene efficiency were included. Delivered gene copies and mRNA (mRNA) expression were quantified with real-time quantitative PCR (qPCR) and protein with enzyme-linked immunosorbent assay (ELISA). The changes in fibrosis parameters were quantified by measuring fibrosis marker α-smooth muscle actin (SMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Cytotoxicity was determined using cellular viability, proliferation, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: PEI readily bound to plasmids to form nanoparticular polyplexes and exhibited much greater transfection efficiency (p<0.01) than the commercial reagent Lipofectamine. The PEI-DNA-treated cultures showed 4.5×10[superscript 4] plasmid copies/µg DNA in real-time qPCR and 7,030±87 pg/ml sTGFβRII protein in ELISA analyses, whereas Lipofectamine-transfected cultures demonstrated 1.9×10[superscript 3] gene copies/µg DNA and 1,640±100 pg/ml sTGFβRII protein during these assays. The PEI-mediated sTGFβRII delivery remarkably attenuated TGFβ1-induced transdifferentiation of corneal fibroblasts to myofibroblasts in cultures, as indicated by threefold lower levels of SMA mRNA (p<0.01) and significant inhibition of SMA protein (up to 96±3%; p<0.001 compared to no-gene-delivered cultures) in immunocytochemical staining and immunoblotting. The nanoparticle-mediated delivery of sTGFβRII showed significantly better antifibrotic effects than the Lipofectamine under similar experimental conditions. However, the inhibition of myofibroblast in HCF cultures by sTGFβRII overexpression by either method was significantly higher than the naked vector transfection. Furthermore, PEI- or Lipofectamine-mediated sTGFβRII delivery into HCF did not alter cellular proliferation or phenotype at 12 and 24 h post-treatment. Nanoparticles treated with HCF showed more than 90% cellular viability and very low cell death (2–6 TUNEL+ cells), suggesting that the tested doses of PEI-nanoparticles do not induce significant cell death. Conclusions: This study demonstrated that PEI-DNA nanoparticles are an attractive vector for the development of nonviral corneal gene therapy approaches and that the sTGFβRII gene delivery into keratocytes could be used to control corneal fibrosis in vivo.National Institutes of Health (U.S.) (RO1EB000244

BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10[superscript 4] gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.National Institutes of Health (U.S.) (RO1EB000244)Mason Eye Institute (Research to Prevent Blindness Unrestricted Grant

BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis \u3cem\u3ein vivo\u3c/em\u3e

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2- GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [a-smooth muscle actin (aSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (26104 gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.6860.31 compared to 3.260.43 in control corneas; p,0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of aSMA (4665% p,0.001) and fibronectin proteins (4865% p,0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (.88%; p,0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFb demonstrated significantly enhanced pSmad-1/5/8 (95%; p,0.001) and Smad6 (53%, p,0.001), and decreased aSMA (78%; p,0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFb1-mediated profibrotic Smad signaling

Targeted AAV5-Smad7 Gene Therapy Inhibits Corneal Scarring \u3cem\u3ein vivo\u3c/em\u3e

Corneal scarring is due to aberrant activity of the transforming growth factor β (TGFβ) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFβ signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFβ signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision

Targeted AAV5-Smad7 gene therapy inhibits corneal scarring in vivo.

Corneal scarring is due to aberrant activity of the transforming growth factor β (TGFβ) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFβ signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFβ signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision

Corneal Gene Therapy: Basic Science and Translational Perspective

Corneal blindness is the third leading cause of blindness worldwide. Gene therapy is an emerging technology for corneal blindness due to the accessibility and immune-privileged nature of the cornea, ease of vector administration and visual monitoring, and ability to perform frequent noninvasive corneal assessment. Vision restoration by gene therapy is contingent upon vector and mode of therapeutic gene introduction into targeted cells/tissues. Numerous efficacious vectors, delivery techniques, and approaches have evolved in last decade for developing gene-based interventions for corneal diseases. Maximizing the potential benefits of gene therapy requires efficient and sustained therapeutic gene expression in target cells, low toxicity, and a high safety profile. This review describes the basic science associated with many gene therapy vectors and the present progress of gene therapy carried out for various ocular surface disorders and diseases

Western blot (A) and fluorescence microscopy image (B) showing delivered BMP7 protein expression and localization of delivered transgene in rabbit corneas collected 4 weeks after a single 5 min topical application transfection solution made of PEI2-GNPs and plasmid expressing BMP7 (A) or GFP gene (B).

<p>Scale bar denotes 100 µm.</p

Real-time PCR data showing quantification of delivered BMP7 gene copies in rabbit corneas after a single 5 min topical application of PEI2-GNPs-BMP7 transfection solution.

<p>Real-time PCR data showing quantification of delivered BMP7 gene copies in rabbit corneas after a single 5 min topical application of PEI2-GNPs-BMP7 transfection solution.</p

Quantification of corneal haze in live animals (A), and SMA+ (B) and fibronectin+ (C) cells in corneal tissues 4-week after laser ablation and +/− BMP7 gene transfer.

<p>PEI2-GNPs mediated BMP7 gene transfer significantly reduced (p<0.05) in corneal haze, and decreased SMA+ cells (p<0.001) and fibronectin-immunostained area (p<0.01).</p