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Transduction of the inner mouse retina using AAVrh8 and AAVrh10 via intravitreal injection.

ABSTRACT: Adeno-associated virus (AAV) is a proven, safe and effective vector for gene delivery in the retina. There are over 100 serotypes of AAV, and AAV2 through AAV9 have been evaluated in the retina. Each AAV serotype has different cell tropism and transduction efficiency. Intravitreal injections of AAV into the eye tend to transduce cells in the ganglion cell layer (GCL), while subretinal injections tend to transduce retinal pigment epithelium and photoreceptors. Efficient transduction of the inner retina beyond the GCL is not well established with the current methodologies and serotypes used to date. In this study, we compared the cellular tropism of AAVrh8 and AAVrh10 vectors encoding enhanced green fluorescent protein (EGFP) using intravitreal injections. We found that AAVrh8 largely transduced cells in the GCL and also amacrine cells in the inner nuclear layer (INL), as well as Müller and horizontal cells. Inner retinal transduction with AAVrh10 was similar to AAVrh8, but AAVrh10 appeared to also transduce bipolar cells. The transduction efficiency as measured by the intensity of EGFP signal was 3.5 fold higher in horizontal cells transduced with AAVrh10 than AAVrh8. Glial fibrillary accessory protein (GFAP) levels were increased in Müller cells in transduced areas for both serotypes. The results of this study suggest that AAVrh8 and AAVrh10 may be excellent vector candidates to deliver genetic material to the INL, particularly for amacrine and horizontal cells, however they may also cause cellular stress as shown by increased glial GFAP expression.

SUBMITTER: Giove TJ

PROVIDER: S-EPMC2962726 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Transduction of the inner mouse retina using AAVrh8 and AAVrh10 via intravitreal injection.

Giove Thomas J TJ Sena-Esteves Miguel M Eldred William D WD

Experimental eye research 20100817 5

Adeno-associated virus (AAV) is a proven, safe and effective vector for gene delivery in the retina. There are over 100 serotypes of AAV, and AAV2 through AAV9 have been evaluated in the retina. Each AAV serotype has different cell tropism and transduction efficiency. Intravitreal injections of AAV into the eye tend to transduce cells in the ganglion cell layer (GCL), while subretinal injections tend to transduce retinal pigment epithelium and photoreceptors. Efficient transduction of the inner ...[more]

PMID: 20723541

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Project description:Effective intravitreal gene delivery to cells of the central retina (i.e., photoreceptors) would be of substantial benefit for treating patients with retinal diseases, such as achromatopsia, where retinal detachment from a subretinal may be harmful. Previous studies demonstrated that mutation of the recombinant adeno-associated virus (rAAV) capsid through introduction of peptide insertions or amino acid substitutions dramatically alters vector tropism. Herein, we evaluate the photoreceptor transduction efficiency of three rAAV2/2-based capsid mutant vectors: rAAV2/2[7m8], rAAV2/2[QuadYF+TV], and a chimeric vector incorporating both mutations (termed rAAV2/2[MAX]) following intravitreal delivery in mice. Furthermore, we evaluate the transduction efficiency of rAAV2/2[MAX] using explanted human central retinal samples to address clinical translatability.Vectors containing a GFP or mCherry reporter gene were intravitreally injected into C57BL/6J or Nrl-EGFP mice, respectively. Transduction was assessed in vivo utilizing a custom multiline confocal scanning laser ophthalmoscope. Injected Nrl-EGFP mouse retinas were used to quantify transduced photoreceptors using flow cytometry. Postmortem human retinal tissue was cultured following administration of rAAV2/2[MAX]. C57BL/6J retinas and human explants were cryosectioned to determine vector tropism.The chimeric vector rAAV2/2[MAX] transduced significantly higher proportions of the retina than did either single mutant serotypes following intravitreal delivery in murine retina, including inner retinal cells and photoreceptors. Vector rAAV2[MAX] demonstrated transduction of human photoreceptors and ganglion cells.Transduction observed via rAAV2/2[MAX] indicates that combining mutations with complementary mechanisms of action in a single vector results in enhanced transduction. rAAV2/2[MAX] also presented the ability to transduce human photoreceptors and ganglion cells, indicating potential for efficient intravitreal vector delivery.

Project description:The glial protein S100B, which belongs to a calcium binding protein family, is up-regulated in neurological diseases, like multiple sclerosis or glaucoma. In previous studies, S100B immunization led to retinal ganglion cell (RGC) loss in an experimental autoimmune glaucoma (EAG) model. Now, the direct degenerative impact of S100B on the retina and optic nerve was evaluated. Therefore, 2 ?l of S100B was intravitreally injected in two concentrations (0.2 and 0.5 ?g/?l). At day 3, 14 and 21, retinal neurons, such as RGCs, amacrine and bipolar cells, as well as apoptotic mechanisms were analyzed. Furthermore, neurofilaments, myelin fibers and axons of optic nerves were evaluated. In addition, retinal function and immunoglobulin G (IgG) level in the serum were measured. At day 3, RGCs were unaffected in the S100B groups, when compared to the PBS group. Later, at days 14 and 21, the RGC number as well as the ?-III tubulin protein level was reduced in the S100B groups. Only at day 14, active apoptotic mechanisms were noted. The number of amacrine cells was first affected at day 21, while the bipolar cell amount remained comparable to the PBS group. Also, the optic nerve neurofilament structure was damaged from day 3 on. At day 14, numerous swollen axons were observed. The intraocular injection of S100B is a new model for a glaucoma like degeneration. Although the application site was the eye, the optic nerve degenerated first, already at day 3. From day 14 on, retinal damage and loss of function was noted. The RGCs in the middle part of the retina were first affected. At day 21, the damage expanded and RGCs had degenerated in all areas of the retina as well as amacrine cells. Furthermore, elevated IgG levels in the serum were measured at day 21, which could be a sign of a late and S100B independet immune response. In summary, S100B had a direct destroying impact on the axons of the optic nerve. The damage of the retinal cell bodies seems to be a consequence of this axon loss.

Dataset Information

Transduction of the inner mouse retina using AAVrh8 and AAVrh10 via intravitreal injection.

Publications

Transduction of the inner mouse retina using AAVrh8 and AAVrh10 via intravitreal injection.

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