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

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.

Selective Upregulation of SIRT1 Expression in Retinal Ganglion Cells by AAV-Mediated Gene Delivery Increases Neuronal Cell Survival and Alleviates Axon Demyelination Associated with Optic Neuritis

“…To increase AAV7m8 transduction efficiency to RGCs and achieve therapeutic effects in these cells, AAV7m8 was further modified to drive transgene expression by an RGC-selective SNCG promotor ( Figure 1 A). In preliminary experiments, we demonstrated the potential of AAV7m8.SNCG.eGFP, which carries SNCG promoter-driven eGFP to transduce RGCs in the mouse retina to a similar degree as in prior studies [ 25 ]. AAV7m8.SNCG.eGFP was injected intravitreally into 4-week-old mice, and the reporter transgene expression in RGCs was analyzed four weeks later ( Figure 1 B).…”

“…The use of the SNCG promoter allowed a highly efficient targeted transfer and translation of the functional transgene cassette into RGCs with greater than 90% cell selectivity. A previous study showed similar RGC-selective expression of eGFP using this construct in a traumatic injury model [ 25 ] although this study did not describe the expression pattern of the therapeutic human SIRT1 transgene. The selection of the SNCG promoter was derived from previous studies showing that the fraction of GCL cells expressing SNCG (45%) matched the estimated fraction of RGCs in the mouse GCL (41–44%), indicating that the SNCG promoter is active in nearly all RGCs [ 31 ].…”

“…This further supports the utility of this vector in preclinical and potentially clinical applications. Secondly, AAV7m8.SNCG.SIRT1, which drives transgene expression selectively in RGCs under the control of the SNCG promoter, was more efficient in transducing RGCs than reported in a prior study using a cytomegalovirus/chicken beta actin (CAG) hybrid promoter, which indiscriminately drives transgene expression in all cell types [ 25 ]. The use of the SNCG promoter allowed a highly efficient targeted transfer and translation of the functional transgene cassette into RGCs with greater than 90% cell selectivity.…”

“…Histologic and immunohistochemical staining and scoring were performed as previously described [ 18 , 19 , 20 , 25 ]. Optic nerves were collected, fixed in 4% paraformaldehyde (PFA), and embedded in paraffin.…”

“…Here, we examined the neuroprotective effects of AAV-mediated SIRT1 expression selectively in RGCs in the mouse model of EAE. We used a synthetic AAV2-derived AAV7m8.SNCG.SIRT1 vector, which was designed to drive optimal human SIRT1 expression selectively in RGCs under the control of the gamma-synuclein (SNCG) promoter [ 24 , 25 ] and demonstrated potential protective effects from RGC trauma [ 25 ]. We found that intravitreal injection of AAV7m8.SNCG.SIRT1 vector reduced RGC loss and demyelination of the optic nerve and provided effective neuroprotection in the model of EAE.…”