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. 2021 May;28(5):256-264.
doi: 10.1038/s41434-021-00219-z. Epub 2021 Feb 15.

Rescue of retinal ganglion cells in optic nerve injury using cell-selective AAV mediated delivery of SIRT1

Ahmara G Ross  1   2 , Devin S McDougald  3 , Reas S Khan  4   3 , Thu T Duong  4   3 , Kimberly E Dine  4 , Puya Aravand  4   3 , Jean Bennett  4   3 , Venkata Ramana Murthy Chavali  4 , Kenneth S Shindler  4   3
Affiliations

Rescue of retinal ganglion cells in optic nerve injury using cell-selective AAV mediated delivery of SIRT1

Ahmara G Ross et al. Gene Ther. 2021 May.

Abstract

SIRT1 prevents retinal ganglion cell (RGC) loss in models of optic neuropathy following pharmacologic activation or genetic overexpression. The exact mechanism of loss is not known, prior evidence suggests this is through oxidative stress to either neighboring cells or RGC specifically. We investigated the neuroprotective potential of RGC-selective SIRT1 gene therapy in the optic nerve crush (ONC) model. We hypothesized that AAV-mediated overexpression of SIRT1 in RGCs reduces RGC loss, thereby preserving visual function. Cohorts of C57Bl/6J mice received intravitreal injection of experimental or control AAVs using either a ganglion cell promoter or a constitutive promoter and ONC was performed. Visual function was examined by optokinetic response (OKR) for 7 days following ONC. Retina and optic nerves were harvested to investigate RGC survival by immunolabeling. The AAV7m8-SNCG.SIRT1 vector showed 44% transduction efficiency for RGCs compared with 25% (P > 0.05) by AAV2-CAG.SIRT1, and AAV7m8-SNCG.SIRT1 drives expression selectively in RGCs in vivo. Animals modeling ONC demonstrated reduced visual acuity compared to controls. Intravitreal delivery of AAV7m8-SNCG.SIRT1 mediated significant preservation of the OKR and RGC survival compared to AAV7m8-SNCG.eGFP controls, an effect not seen with the AAV2 vector. RGC-selective expression of SIRT1 offers a targeted therapy for an animal model with significant ganglion cell loss. Over-expression of SIRT1 through AAV-mediated gene transduction suggests a RGC selective component of neuro-protection using the ONC model. This study expands our understanding of SIRT1 mediated neuroprotection in the context of compressive or traumatic optic neuropathy, making it a strong therapeutic candidate for testing in all optic neuropathies.

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Conflict of interest statement

JB is a founder of Gensight Therapeutics and Limelight Bio and a scientific founder of Spark Therapeutics. JB receives a grant from Limelight Bio, is on the SAB for Akouos, and holds intellectual property relevant to this study. AGR, DSM, and KSS hold intellectual property relevant to this study. The other authors whose names are listed immediately below (RSK, TTD, KED, PA, VRMC) certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Figures

Fig. 1
Fig. 1. Design and in vitro characterization of AAV2-CAG and AAV7m8 SNCG vectors.
A Outline of proviral expression cassettes used in the study. A1 and A2 illustrates comparative vector using AAV2 and CAG promoter with the cDNAs encoding eGFP or SIRT1 respectively. A3 and A4 Illustrates plasmid using the ganglion cell specific promoter, SNCG, driving cDNAs eGFP or SIRT1 respectively. B Fluorescent micrographs of human SIRT1 protein expression in iPS-RGCs transduced with AAV7m8.SNCG.hSIRT1-3XFLAG. Blue-DAPI nuclear stain, Red-BRN3A nuclear stain, green- 3XFlag Tag epitope showing cytoplasmic and nuclear expression from the vector and blue/red/green merged images.
Fig. 2
Fig. 2. AAV7m8 transduction profile and RGC transduction efficiency following intravitreal delivery.
A Representative micrograph of retinal flat mount following intravitreal injection of AAV7m8-SNCG.eGFP. RGCs are labeled with BRN3A (red). (inlet) Representative retinal flat mount used for calculating RGC transduction efficiency with AAV7m8. B Quantification of RGC transduction (n = 10, experiments performed in triplicate; retinal whole mounts) comparing AAV2-CAG.SIRT1 vector. C Representative cross-section of mouse retina following intravitreal injection of AAV2-CAG.SIRT1. RGCs are labeled with Brn3a (red) and localized to the ganglion cell layer(GCL). Cells expressing the SIRT1 transgene are labeled green and also largely localized to the GCL.Data represented as mean ± SEM.
Fig. 3
Fig. 3. Effect of AAV2 gene transfer on visual acuity and RGC in ONC.
OKR recordings demonstrate significantly decreased visual acuity in eyes of ONC mice treated with AAV2-CAG.eGFP (n = 10; experiments performed in tripicate). Treatment with AAV2- Mice treated with AAV2-CAG.SIRT1 (n = 12; experiments performed in tripicate) show no significant effect on A visual acuity or B RGC survival by day 7. C Representative RGC counts by nuclear BRN3A staining. Data represented as mean ± SEM. *P < 0.05, **P < 0.01 by 1-way ANOVA with Tukey’s HSD post-test.
Fig. 4
Fig. 4. Effect of AAV7m8 gene transfer on visual acuity and RGC in ONC.
OKR recordings demonstrate significantly decreased visual acuity in eyes of ONC mice treated with AAV2-eGFP (n = 10; experiments performed in triplicate) compared with Sham injured mice (n = 10; experiments performed in tripicate ***p = 0.002). A Treatment with AAV7m8-SIRT1 (n = 15; experiments performed in tripicate) showed a significant delay in loss of visual function in ONC (n = 10; experiments performed in tripicate) compared with Sham injured mice (n = 10; experiments performed in tripicate) (*p = 0.03) on visual acuity seen in peach area outlined in the graph. B RGC flat mount counts demonstrate significantly decreased numbers in eyes of ONC mice treated with AAV2-eGFP (n = 10; experiments performed in tripicate) compared with Sham injured mice (n = 10; experiments performed in triplicate; ***p = 0.001). Treatment with AAV7m8-SIRT1 (n = 10; experiments performed in tripicate) showed a significant increase in retinal ganglion cell counted per flat month function in ONC (n = 7) compared with control ONC mice (n = 10; experiments performed in tripicate) (*p = 0.03). C Representative RGC counts by nuclear BRN3A staining Data represented as mean ± SEM. *P < 0.05, **P < 0.01 by 1-way ANOVA with Tukey’s HSD post-test.
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