Romain Da Costa1, Carsten Röger1, Jasmin Segelken2, Maya Barben3, Christian Grimm4, John Neidhardt5. 1. Institute of Human Genetics, Faculty of Medicine and Health Sciences, University of Oldenburg, Oldenburg, Germany. 2. Visual Neuroscience, Faculty of Medicine and Health Sciences, University of Oldenburg, Oldenburg, Germany. 3. Laboratory for Retinal Cell Biology, Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland 4Neuroscience Center Zurich (ZNZ), University of Zurich, Zurich, Switzerland. 4. Laboratory for Retinal Cell Biology, Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland 4Neuroscience Center Zurich (ZNZ), University of Zurich, Zurich, Switzerland 5Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland. 5. Institute of Human Genetics, Faculty of Medicine and Health Sciences, University of Oldenburg, Oldenburg, Germany 6Research Center Neurosensory Science, University Oldenburg, Oldenburg, Germany.
Abstract
PURPOSE: Gene therapies to treat eye disorders have been extensively studied in the past 20 years. Frequently, adeno-associated viruses were applied to the subretinal or intravitreal space of the eye to transduce retinal cells with nucleotide sequences of therapeutic potential. In this study we describe a novel intravitreal injection procedure that leads to a reproducible adeno-associated virus (AAV)2/8-mediated transduction of more than 70% of the retina. METHODS: Prior to a single intravitreal injection of a enhanced green fluorescent protien (GFP)-expressing viral suspension, we performed an aspiration of vitreous tissue from wild-type C57Bl/6J mice. One and one-half microliters of AAV2/8 suspension was injected. Funduscopy, optical coherence tomography (OCT), laser scanning microscopy of retinal flat mounts, cryosections of eye cups, and ERG recordings verified the efficacy and safety of the method. RESULTS: The combination of vitreous aspiration and intravitreal injection resulted in an almost complete transduction of the retina in approximately 60% of the eyes and showed transduced cells in all retinal layers. Photoreceptors and RPE cells were predominantly transduced. Eyes presented with well-preserved retinal morphology. Electroretinographic recordings suggested that the new combination of techniques did not cause significant alterations of the retinal physiology. CONCLUSIONS: We show a novel application technique of AAV2/8 to the vitreous of mice that leads to widespread transduction of the retina. The results of this study have implications for virus-based gene therapies and basic science; for example, they might provide an approach to apply gene replacement strategies or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in vivo. It may further help to develop similar techniques for larger animal models or humans.
PURPOSE: Gene therapies to treat eye disorders have been extensively studied in the past 20 years. Frequently, adeno-associated viruses were applied to the subretinal or intravitreal space of the eye to transduce retinal cells with nucleotide sequences of therapeutic potential. In this study we describe a novel intravitreal injection procedure that leads to a reproducible adeno-associated virus (AAV)2/8-mediated transduction of more than 70% of the retina. METHODS: Prior to a single intravitreal injection of a enhanced green fluorescent protien (GFP)-expressing viral suspension, we performed an aspiration of vitreous tissue from wild-type C57Bl/6J mice. One and one-half microliters of AAV2/8 suspension was injected. Funduscopy, optical coherence tomography (OCT), laser scanning microscopy of retinal flat mounts, cryosections of eye cups, and ERG recordings verified the efficacy and safety of the method. RESULTS: The combination of vitreous aspiration and intravitreal injection resulted in an almost complete transduction of the retina in approximately 60% of the eyes and showed transduced cells in all retinal layers. Photoreceptors and RPE cells were predominantly transduced. Eyes presented with well-preserved retinal morphology. Electroretinographic recordings suggested that the new combination of techniques did not cause significant alterations of the retinal physiology. CONCLUSIONS: We show a novel application technique of AAV2/8 to the vitreous of mice that leads to widespread transduction of the retina. The results of this study have implications for virus-based gene therapies and basic science; for example, they might provide an approach to apply gene replacement strategies or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in vivo. It may further help to develop similar techniques for larger animal models or humans.
Authors: Mariana S Dias; Victor G Araujo; Taliane Vasconcelos; Qiuhong Li; William W Hauswirth; Rafael Linden; Hilda Petrs-Silva Journal: Gene Ther Date: 2019-09-27 Impact factor: 5.250
Authors: Sanford L Boye; Shreyasi Choudhury; Sean Crosson; Giovanni Di Pasquale; Sandra Afione; Russell Mellen; Victoria Makal; Kaitlyn R Calabro; Diego Fajardo; James Peterson; Hangning Zhang; Matthew T Leahy; Colin K Jennings; John A Chiorini; Ryan F Boyd; Shannon E Boye Journal: Mol Ther Date: 2020-04-11 Impact factor: 12.910