Next generation tyrosine-mutant AAV8 shows high mouse corneal endothelial transduction in vivo [abstract]

Purpose: Recently, point mutations in surface-exposed tyrosine residues have been shown to increase AAV transduction significantly. We examined the efficacy of conventional AAV8 and next generation tyrosine-mutant AAV8 vectors to deliver genes into corneal endothelium in vivo. Methods: The study, which used female C57BL/6 mice, was approved by the institutional animal care and use committee and experiments were performed inaccordance with the tenets of the ARVO statement for the use of animals. Ketamine and Xylazine hydrochloride were administered for anesthesia. Two microliters of AAV8 titer (~1010 vg/ml) was injected into the anterior chamber of the eye from four different clock-positions with the Hamilton Microinjection Syringe System under an operating microscope. Eyes were then washed with water and sponge-dried. Slit lamp microscopy was utilized for clinical eye exam. Stereomicroscopy and immunocytochemistry techniques measured the levels and location of transgene. Delivered-transgene expression was quantified with real-time PCR and western blot analyses. Results: Both, conventional and tyrosine-mutant AAV8 vectors successfully transduced mouse corneal endothelium in vivo. Mutant AAV8 showed significantly higher transgene delivery into corneal endothelium than the conventional AAV8. Injections performed from 12 or 3 o'clock position showed highest targeted transgene delivery into corneal endothelium. Low-to-mild transgene delivery was also detected in the stroma. The tested AAV8 showed no apparent side effects or toxicity. Conclusions: Tested AAV8 vectors are efficient to treat corneal endothelial dystrophies via gene therapy. Future studies will determine optimal doses of conventional and tyrosine-mutant AAV8 vectors to deliver therapeutic genes into corneal endothelial in vivo

SMAD-signaling inhibition : potential for developing newer treatments for corneal fibrosis [abstract]

Transforming growth factor [beta] (TGFb) is known to cause fibrosis in the cornea following injury and/or infection. Effective reduction in corneal fibrosis has been reported by inhibiting TGFb activity. However, associated molecular mechanism is still unknown. The aim of study was to test the hypothesis that the alteration in SMAD signaling is a novel approach for treating corneal fibrosis using an established in vitro model

AAV Serotype Influences Gene Transfer in Corneal Stroma \u3cem\u3ein vivo\u3c/em\u3e

This study evaluated the cellular tropism and relative transduction efficiency of three AAV serotypes, AAV6, AAV8 and AAV9, for corneal gene delivery using mouse cornea in vivo and donor human cornea ex vivo. The AAV6, AAV8 and AAV9 serotypes having AAV2 plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Viral vectors (109 vg/μl) were topically applied onto mouse cornea in vivo and human cornea ex vivo after removing the epithelium. Human corneas were processed for transgene delivery at day 5 after viral vector application. Mouse corneas were harvested at 4, 14 and 30 days after vector application for AP staining. Transduction efficiency was calculated by quantifying pixels of AP-stained area using Image J software and also confirmed by functional AP enzyme activity in the corneal lysates. Cellular toxicity of the three AAV serotypes was tested with TUNEL assay. Inflammatory response was detected by immunostaining for CD11b and F4/80. All three AAV serotypes successfully transduced mouse and human corneas. The order of transduction efficiency was AAV9\u3eAAV8\u3eAAV6. The transduction efficiency of AAV9 was 1.1–1.4 fold higher (p\u3e0.05) as compared to AAV8 and 3.5–5.5 fold higher (p\u3c0.01) as compared to AAV6. The level of transgene expression for all the three serotypes was greater at 14 days compared to 4 days and this high level of transgene expression was maintained up to the tested time point of 30 days. Corneas exposed to any of the three AAV serotypes did not show significant TUNEL positive cells or any inflammatory response as tested by CD11b or F4/80 staining suggesting that tested AAV serotypes do not induce cell death or inflammation and are safe for corneal gene therapy

Gene transfer technology : a tool for studying gene function and role in corneal pathogenesis [abstract]

Transforming growth factor β (TGFb) is associated with many corneal pathologies, diseases and dystrophies. The function of TGFb in adult corneas cannot be studied using conventional transgenic approach because TGFb1 and TGFb2 deficient transgenic animals suffer multiple inflammatory diseases, severe developmental defects, and death by 3-4 weeks of age. This study tested the hypothesis that selective tissue-targeted gene transfer approaches will permit examination of TGFb gene function in the adult cornea without altering TGFb expression in vital organs

Role of Transforming Growth Factor Beta in Corneal Function, Biology and Pathology

Transforming growth factor-beta (TGFβ) is a pleiotropic multifunctional cytokine that regulates several essential cellular processes in many parts of the body including the cornea. Three isoforms of TGFβ are known in mammals and the human cornea expresses all of them. TGFβ1 has been shown to play a central role in scar formation in adult corneas whereas TGFβ2 and TGFβ3 have been implicated to play a critical role in corneal development and scarless wound healing during embryogenesis. The biological effects of TGFβ in the cornea have been shown to follow SMAD dependent as well as SMAD-independent signaling pathways depending upon cellular responses and microenvironment. Corneal TGFβ expression is necessary for maintaining corneal integrity and corneal wound healing. On the other hand, TGFβ is perhaps the most important cytokine in the pathogenesis of fibrotic disease in the cornea. Although the transformation of keratocytes to myofibroblasts induced by TGFβ is largely believed to cause corneal fibrosis or scarring, the precise molecular mechanism(s) involved in this process is still unknown. Currently no drugs are available to treat corneal scarring effectively without causing significant side effects. Many approaches to treat TGFβ-mediated corneal scarring are under investigation. These include blocking of TGFβ, TGFβ receptor, TGFβ function and/or TGFβ maturation. Other strategies such as modulating keratocyte proliferation, apoptosis, transcription and DNA condensation are also being investigated. The potential of gene therapy to neutralize the pathologic effects of TGFβ has also been demonstrated recently

AAV5-mediated targeted decorin gene therapy : effective and safe for corneal fibrosis [abstract]

Corneal fibrosis is 3rd leading cause of global blindness according to WHO report. At present, no agents are proven to clinically reduce corneal fibrosis without causing significant side effects. It was hypothesized that decorin gene delivered into keratocytes prevents corneal fibrosis in the cornea in vivo by blocking transforming growth factor β (TGFb), which converts keratocyte to myofibroblasts and cause fibrosis

Tissue-selective controlled decorin gene delivery in the rabbit cornea significantly retards corneal angiogenesis in vivo [abstract]

Recent studies have shown that decorin gene therapy inhibits neovascularization in many non-ocular tissues. We tested the efficacy of decorin gene delivery into stroma with AAV5 to impede vascular endothelial growth factor (VEGF)-induced angiogenesis in rabbit cornea in vivo

Targeted Decorin Gene Therapy Delivered with Adeno-Associated Virus Effectively Retards Corneal Neovascularization \u3cem\u3ein vivo\u3c/em\u3e

Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 μl; 5x10^12 vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p\u3c0.05), 66% (p\u3c0.001), and 63% (p\u3c0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57–65, p\u3c0.5), and CD31 immunoblotting (62–67%, p\u3c0.05) supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic) and up-regulated PEDF (anti-angiogenic) genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5– mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients

SAHA : FDA approved histone deacetylase inhibitor demonstrates exceptionally high inhibition of corneal haze following PRK surgery in rabbit model [abstract]

TGF[beta] induces the transformation of corneal keratocytes into fibroblasts and myofibroblasts resulting in the formation of corneal haze (scar) following injury. We investigated whether epigenetic modifications can prevent development of corneal haze in vivo using a rabbit model

N/P ratio in the PEI2-GNP-DNA complex affects transgene delivery in the human cornea in vitro

Recently, we discovered that polyethylenimine-conjugated gold nanoparticles (PEI2- GNP) could be used as gene therapy vector for the cornea. It was hypothesized that DNA concentration, incubation timing and PEI monomer amount in transfection solution affect gene transfer efficiency and toxicity. The aims of this study were to test whether molar ratio of PEI2 nitrogen (N) and phosphate (P) of DNA in PEI2-GNP transfection solution regulates transgene delivery in human corneal fibroblasts in vitro, and examine PEI2-GNP toxicity, uptake and clearance for the cornea in vivo."National Eye Institute, NIH, Bethesda for RO1EY017294 (RRM), Diversity (RRM) and Veteran Health Affairs Merit (RRM) grants and Unrestricted grant from Research to Prevent Blindness, New York