Abstract
BackgroundAdeno associated virus (AAV) is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV’s ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC) of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc) genomes in the anterior segment of the eye.Methodology/Principle FindingsAC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE), iris and chamber angle including trabecular meshwork, with scAAV2(Y444F) and scAAV2(triple) being the most efficient.Conclusions/SignificanceThis is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene-based therapies for glaucoma and acquired or inherited corneal anomalies.
Highlights
Adeno associated virus (AAV)- mediated gene delivery has been used successfully to improve vision in animal models of inherited retinal disease and its safety/efficacy has been proven in clinical trials [1,2,3,4,5,6,7,8]
Care was taken to avoid any irritation of ocular tissues, we assume that the cornea of 1 mouse and 1 rat eye may have been disturbed during the injection procedure, leading to inadvertent intrastromal delivery of AAV vectors and transduction of corneal stromal keratinocytes
At four weeks post injection, eyes injected with scAAV2-based vectors exhibited green fluorescent protein (GFP) expression in the corneal endothelium, the ciliary non-pigmented epithelium (NPE), the iris and the chamber angle including the TM (Fig 2and Fig 3)
Summary
Adeno associated virus (AAV)- mediated gene delivery has been used successfully to improve vision in animal models of inherited retinal disease and its safety/efficacy has been proven in clinical trials [1,2,3,4,5,6,7,8]. Targets of interest in the cornea include carbohydrate sulfotransferase 6 (CHST6), keratin 3 (KRT3), keratin 12 (KRT12), a FYVE finger containing phosphoinositide kinase (PIK5K3), sodium bicarbonate transporter-like protein (SLC4A11), tumor associated calcium signal transducer 2 (TACSTD2), transforming growth factor, beta induced, 68 kDA (TGFBI) and UbiA prenyltransferase domain-containing protein 1 (UBIAD1) [18, 19]. Gene delivery to these tissues will require both a reliable injection technique and vectors that can efficiently transduce target cells in vivo. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC) of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc) genomes in the anterior segment of the eye
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.