Abstract
Purpose :
Basic levels of visual restoration have been achieved following delivery of light sensitive proteins to the surviving cells of the degenerate retina (optogenetics). Restricting delivery to specific cell populations demonstrates functional advantage over ‘non-specific,’ high efficiency promotor-based systems but cell specific promoters can often be too large for adeno-associated viral vector (AAV) envelopes. Here we repurposed a compressed promoter complex termed ‘L7-6’ derived from the L7 (pcp2) promoter to test the hypothesis that it can be used to restrict delivery of a functional optogenetic tool to retinal ON-bipolar cells using AAV in a mouse model.
Methods :
At P45, retina-degenerate mice lacking native melanopsin (Pderd1/rd1 Opn4-/-) received intravitreal injections of saline or AAV2.2 containing the human melanopsin gene (hOpn4) driven by the L7-6 promotor construct (n=8 per group, 4 male, 4 female). Flat mount immunohistochemistry (IHC) (n=4 per group) and multiple electrode array (MEA) recordings of retinal ganglion cell (RGC) light responses (n=4 per group) were performed on intact retina explants 8 weeks post injection with a range of intensities of light. Comparisons of means were performed by unpaired t-test.
Results :
L7-6.hOPN4 treated retinae demonstrated IHC staining for hOPN4 restricted to 17.75±3.54% of L7+ bipolar cells in the inner nuclear layer & 1.44±0.14% of all cells in the GCL (N=4, n=16) compared to 0.00±0.00% cells in either layer in the saline group (p<0.0001). On MEA, 38 electrodes responded to a 480nm light at 1015 photons cm-2 s-1 in the L7-6.hOPN4 treated (n=0 saline, p<0.0001). A sigmoidal irradiance-response relationship was seen in the treatment group with an EC50 of 13.30±0.08 photons cm-2 s-1.
Conclusions :
The L7-6 promotor can be used to deliver a functional melanopsin to retinal ON-bipolar cells using intravitreally delivered AAV vector. This provides a further method to replicate the power of cell-specific melanopsin optogenetics previously demonstrated in transgenic L7.Cre.lox mouse models.
This is a 2021 ARVO Annual Meeting abstract.