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AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury.

ABSTRACT: Gene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome.Double-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types.Successful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Müller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve.Damaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.

SUBMITTER: Nickells RW

PROVIDER: S-EPMC5716181 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury.

Nickells Robert W RW Schmitt Heather M HM Maes Margaret E ME Schlamp Cassandra L CL

Investigative ophthalmology & visual science 20171201 14

<h4>Purpose</h4>Gene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome.<h4>Methods</h4>Double-transgenic mice carrying a Rosa26-(Lox ...[more]

PMID: 29204649

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Project description:PurposeTo investigate the optic nerve and macular morphology in patients with optic nerve hypoplasia (ONH) using spectral-domain optical coherence tomography (SD OCT).DesignProspective, cross-sectional, observational study.SubjectsA total of 16 participants with ONH (10 female and 6 male; mean age, 17.2 years; 6 bilateral involvement) and 32 gender-, age-, ethnicity-, and refraction-matched healthy controls.MethodsHigh-resolution SD OCT (Copernicus [Optopol Technology S.A., Zawiercie, Poland], 3 μm resolution) and handheld SD OCT (Bioptigen Inc [Research Triangle Park, NC], 2.6 μm resolution) devices were used to acquire horizontal scans through the center of the optic disc and macula.Main outcome measuresHorizontal optic disc/cup and rim diameters, cup depth, peripapillary retinal nerve fiber layer (RNFL), and thickness of individual retinal layers in participants with ONH and in controls.ResultsPatients with ONH had significantly smaller discs (P < 0.03 and P < 0.001 compared with unaffected eye and healthy controls, respectively), horizontal cup diameter (P < 0.02 for both), and cup depth (P < 0.02 and P < 0.01, respectively). In the macula, significantly thinner RNFL (nasally), ganglion cell layer (GCL) (nasally and temporally), inner plexiform layer (IPL) (nasally), outer nuclear layer (ONL) (nasally), and inner segment (centrally and temporally) were found in patients with ONH compared with the control group (P < 0.05 for all comparisons). Continuation of significantly thicker GCL, IPL, and outer plexiform layer in the central retinal area (i.e., foveal hypoplasia) was found in more than 80% of patients with ONH. Clinically unaffected fellow eyes of patients with ONH showed mild features of underdevelopment. Visual acuity and presence of septo-optic dysplasia were associated with changes in GCL and IPL. Sensitivity and specificity for the detection of ONH based on disc and retinal optical coherence tomography (OCT) parameters were >80%.ConclusionsOur study provides evidence of retinal changes in ONH. In addition to thinning of retina layers mainly involving the RNFL and GCL, signs reminiscent of foveal hypoplasia were observed in patients with ONH. Optic nerve and foveal parameters measured using OCT showed high sensitivity and specificity for detecting ONH, demonstrating their useful for clinical diagnosis.

Project description:Glaucoma is a neurodegenerative disease that is characterized by the loss of retinal ganglion cells (RGC) and optic nerve fibers. Increased age and intraocular pressure (IOP) elevation are the main risk factors for developing glaucoma. Mice that are heterozygous (HET) for the mega-karyocyte protein tyrosine phosphatase 2 (PTP-Meg2) show chronic and progressive IOP elevation, severe RGCs loss, and optic nerve damage, and represent a valuable model for IOP-dependent primary open-angle glaucoma (POAG). Previously, evidence accumulated suggesting that glaucomatous neurodegeneration is associated with the extensive remodeling of extracellular matrix (ECM) molecules. Unfortunately, little is known about the exact ECM changes in the glaucomatous retina and optic nerve. Hence, the goal of the present study was to comparatively explore ECM alterations in glaucomatous PTP-Meg2 HET and control wild type (WT) mice. Due to their potential relevance in glaucomatous neurodegeneration, we specifically analyzed the expression pattern of the ECM glycoproteins fibronectin, laminin, tenascin-C, and tenascin-R as well as the proteoglycans aggrecan, brevican, and members of the receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) family. The analyses were carried out in the retina and optic nerve of glaucomatous PTP-Meg2 HET and WT mice using quantitative real-time PCR (RT-qPCR), immunohistochemistry, and Western blot. Interestingly, we observed increased fibronectin and laminin levels in the glaucomatous HET retina and optic nerve compared to the WT group. RT-qPCR analyses of the laminins α4, β2 and γ3 showed an altered isoform-specific regulation in the HET retina and optic nerve. In addition, an upregulation of tenascin-C and its interaction partner RPTPβ/ζ/phosphacan was found in glaucomatous tissue. However, comparable protein and mRNA levels for tenascin-R as well as aggrecan and brevican were observed in both groups. Overall, our study showed a remodeling of various ECM components in the glaucomatous retina and optic nerve of PTP-Meg2 HET mice. This dysregulation could be responsible for pathological processes such as neovascularization, inflammation, and reactive gliosis in glaucomatous neurodegeneration.

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AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury.

Publications

AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury.

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