Abstract
Purpose :
Pharmacologic activators and genetic overexpression of sirtuin 1 (SIRT1) promote neuroprotective effects in multiple neurodegenerative disease models, including optic neuropathies. AAV2-SIRT1 mediated overexpression in mouse retina partially attenuates experimental optic neuritis despite only a 21% transduction efficiency for retinal ganglion cells (RGCs). Viral vectors with increased transfection efficiency are needed.
Methods :
AAV7m8 vectors were designed to contain the putative RGC-specific gamma-synuclein gene (SNCG) promoter and either the cDNA encoding enhanced protein (eGFP) or a codon optimized human SIRT1 . Gene expression was optimized in ARPE-19 cells, and SIRT1 expression was determined by RT-qPCR. Vectors were delivered by intravitreal injection in 4 week old C57/Bl6 mice and the RGC transduction profile following intravitreal delivery was tested at 4 weeks post injection using immunohistochemistry techniques. The RGCs were identified with Brn3a staining, and were tested for transduction efficiency by co-localization using the 3xFLAG tag on the vectors.
Results :
The AAV7m8 vector expressing human SIRT1 driven by the SNCG promotor demonstrated enhanced expression of SIRT1 similar to its AAV2 predecessor in vivo. However, intravitreal injections using both vector showed a superior AAV transduction profile by the novel AAV7m8 construct. The AAV7m8-SIRT1 vector showed >50% transfection efficiency for RGCs compared with only 21% by AAV2-SIRT1, and AAVm8-SIRT1 drives expression almost exclusively in RGCs whereas AAV2-SIRT1 overexpression is enhanced in multiple retinal layers.
Conclusions :
The AAV7m8-SIRT1 vector represents a clinically viable, RGC-specific, and efficient vector for delivering SIRT1 in the retina. This represents an encouraging therapy to enhance visual outcomes and RGC preservation in multiple models of optic neuropathy. Additionally, the development of this vector has the potential to be used as a backbone for gene delivery of other novel gene candidates suggested to enhance neuroprotection that can be designed in the future.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.