Comparison of SNCG and NEFH Promoter-Driven Expression of Human SIRT1 Expression in a Mouse Model of Glaucoma.
O'Neill Nuala, Meng Miranda, Chaqour Brahim, Dine Kimberly, Sarabu Neha, Pham Jennifer C, Shindler Kenneth S, Ross Ahmara G
AI Summary
Researchers found that smaller NEFH and SNCG promoters equally drive neuroprotective gene delivery to retinal ganglion cells in a glaucoma mouse model, improving RGC survival and vision. This allows for packaging larger therapeutic genes.
Abstract
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.
MeSH Terms
Shields Classification
Key Concepts5
The eGFP transduction efficiency of AAV2-eCMV-NEFH was similar to that of AAV2-eCMV-SNCG and AAV2-DiATOH7-SNCG in C57BL/6J mice.
When combined with the SNCG promoter, a larger ATOH7 enhancer was less efficient than the shorter DiATOH7 enhancer in driving gene expression in RGCs in C57BL/6J mice.
The hSIRT1 efficiency of AAV2-eCMV-NEFH was similar to that of AAV2-eCMV-SNCG in C57BL/6J mice subjected to microbead (MB)-induced intraocular pressure (IOP) elevation.
AAV2-eCMV-NEFH and AAV2-eCMV-SNCG were equally efficient in increasing RGC survival and improving visual function in a mouse model of MB-induced IOP elevation.
SNCG and NEFH promoters represent two equally efficient and comparable RGC selective promoter sequences, with the NEFH promoter offering a smaller packaging size.
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