RESUMEN
Purpose: Adeno-associated virus (AAV) demonstrates promise in delivering therapeutic genes to retinal ganglion cells (RGCs). Delivery of neuroprotective genes is constrained by packaging size and/or cell selectivity. This study compares the ability of the RGC-selective gamma-synuclein (SNCG) promoter and the smaller RGC-selective neurofilament heavy chain (NEFH) promoter, as well as portions of the RGC-selective atonal bHLH transcription factor 7 (ATOH7) enhancer, to drive gene expression in RGCs. Methods: AAV2 constructs with green fluorescent protein (GFP) or human sirtuin 1 (hSIRT1) driven by cytomegalovirus (CMV) enhancer and NEFH promoter (AAV2-eCMV-NEFH) or distal active sequences of the ATOH7 enhancer (DiATOH7) with the SNCG promoter (AAV2-DiATOH7-SNCG) were intravitreally injected into C57BL/6J mice. RGCs were immunolabeled with Brn3a antibodies and counted. AAV constructs with the utmost transduction efficiency were used to test the therapeutic efficacy of the hSIRT1 gene in 12-week-old C57BL/6J mice subjected to microbead (MB)-induced intraocular pressure (IOP) elevation. Visual function was measured using optokinetic responses (OKRs). Results: The eGFP transduction efficiency of AAV2-eCMV-NEFH was similar to that of AAV2-eCMV-SNCG and AAV2-DiATOH7-SNCG. When combined with the SNCG promoter, a larger ATOH7 enhancer was less efficient than the shorter DiATOH7 enhancer. Similarly, the hSIRT1 efficiency of AAV2-eCMV-NEFH was similar to that of AAV2-eCMV-SNCG. The latter two vectors were equally efficient in increasing RGC survival and improving visual function in the mouse model of MB-induced IOP elevation. Conclusions: SNCG and NEFH promoters represent two equally efficient and comparable RGC selective promoter sequences; however, the NEFH promoter offers a smaller packaging size. Translational Relevance: Smaller enhancer-promoter combinations can be used to deliver larger genes in human cells and for treatment in optic neuropathies including glaucoma.
Asunto(s)
Dependovirus , Modelos Animales de Enfermedad , Glaucoma , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Células Ganglionares de la Retina , Sirtuina 1 , gamma-Sinucleína , Animales , Sirtuina 1/genética , Sirtuina 1/metabolismo , Ratones , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Humanos , Glaucoma/genética , Glaucoma/terapia , Glaucoma/metabolismo , Dependovirus/genética , gamma-Sinucleína/genética , gamma-Sinucleína/metabolismo , Inyecciones Intravítreas , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Presión Intraocular/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión GénicaRESUMEN
Purpose: Retinal ganglion cell (RGC) loss provides the basis for diagnosis and stage determination of many optic neuropathies, and quantification of RGC survival is a critical outcome measure in models of optic neuropathy. This study examines the accuracy of manual RGC counting using two selective markers, Brn3a and RBPMS. Methods: Retinal flat mounts from 1- to 18-month-old C57BL/6 mice, and from mice after microbead (MB)-induced intraocular pressure (IOP) elevation, are immunostained with Brn3a and/or RBPMS antibodies. Four individuals masked to the experimental conditions manually counted labeled RGCs in three copies of five images, and inter- and intra-person reliability was evaluated by the intraclass correlation coefficient (ICC). Results: A larger population (approximately 10% higher) of RGCs are labeled with RBPMS than Brn3a antibody up to 6 months of age, but differences decrease to approximately 1% at older ages. Both RGC-labeled populations significantly decrease with age. MB-induced IOP elevation is associated with a significant decrease of both Brn3a- and RBPMS-positive RGCs. Notably, RGC labeling with Brn3a provides more consistent cell counts than RBPMS in interpersonal (ICC = 0.87 to 0.11, respectively) and intra-personal reliability (ICC = 0.97 to 0.66, respectively). Conclusions: Brn3a and RBPMS markers are independently capable of detecting significant decreases of RGC number with age and in response to IOP elevation despite RPBMS detecting a larger number of RGCs up to 6 months of age. Brn3a labeling is less prone to manual cell counting variability than RBPMS labeling. Overall, either marker can be used as a single marker to detect significant changes in RGC survival, each offering distinct advantages.
Asunto(s)
Enfermedades del Nervio Óptico , Células Ganglionares de la Retina , Animales , Ratones , Envejecimiento , Anticuerpos , Ratones Endogámicos C57BL , Reproducibilidad de los Resultados , Proteínas de Unión al ARNRESUMEN
SIRT1 prevents retinal ganglion cell (RGC) loss in several acute and subacute optic neuropathy models following pharmacologic activation or genetic overexpression. We hypothesized that adeno-associated virus (AAV)-mediated overexpression of SIRT1 in RGCs in a chronic ocular hypertension model can reduce RGC loss, thereby preserving visual function by sustained therapeutic effect. A control vector AAV-eGFP and therapeutic vector AAV-SIRT1 were constructed and optimized for transduction efficiency. A magnetic microbead mouse model of ocular hypertension was optimized to induce a time-dependent and chronic loss of visual function and RGC degeneration. Mice received intravitreal injection of control or therapeutic AAV in which a codon-optimized human SIRT1 expression is driven by a RGC selective promoter. Intraocular pressure (IOP) was measured, and visual function was examined by optokinetic response (OKR) weekly for 49 days following microbead injection. Visual function, RGC survival, and axon numbers were compared among control and therapeutic AAV-treated animals. AAV-eGFP and AAV-SIRT1 showed transduction efficiency of ~ 40%. AAV-SIRT1 maintains the transduction of SIRT1 over time and is selectively expressed in RGCs. Intravitreal injections of AAV-SIRT1 in a glaucoma model preserved visual function, increased RGC survival, and reduced axonal degeneration compared with the control construct. Over-expression of SIRT1 through AAV-mediated gene transduction indicates a RGC-selective component of neuroprotection in multiple models of acute optic nerve degeneration. Results here show a neuroprotective effect of RGC-selective gene therapy in a chronic glaucoma model characterized by sustained elevation of IOP and subsequent RGC loss. Results suggest that this strategy may be an effective therapeutic approach for treating glaucoma, and warrants evaluation for the treatment of other chronic neurodegenerative diseases.