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Sean K Wang, Yunlu Xue, Parimal Rana, Christin Hong, Constance Cepko; Soluble CX3CL1 gene therapy improves cone survival and function in mouse models of retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5175. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In retinitis pigmentosa (RP), early loss of rod photoreceptors is followed by the eventual death of cones, culminating in the deterioration of high acuity vision. It has been postulated that this non-cell autonomous cone degeneration may involve the body’s own immune cells, specifically resident macrophages of the retina known as microglia. We hypothesized that adeno-associated virus (AAV)-mediated overexpression of microglia "quiescence" signals could promote cone survival and improve vision in genetic mouse models of RP.
AAVs were generated expressing variants of two known microglia "quiescence" signals (CD200 and CX3CL1) and administered via subretinal injection into RP mouse strains (rd1, rd10,or Rho-/-) at birth. A cone-specific GFP virus (AAV-GFP) was co-injected to visualize cones. To assay cone survival, retinas were isolated from rd1 mice (N=35) at postnatal day 50 (P50), rd10 (N=9) at P100, and Rho-/- (N=8) at P150. Following fixation, retinas were flat-mounted, imaged with a widefield fluorescence microscope, and quantified for GFP+ cones using an automated ImageJ module. To measure visual function, rd10 mice were subjected to electroretinography (ERG) at P40 (N=16) and optomotor recordings every 5 days from P45-60 (N=21) by a blinded observer. To transcriptionally profile microglia, CD11b+ Ly6C- Ly6G- retinal microglia were collected from rd10 mice (N=8) at P70 using fluorescence-activated cell sorting (FACS) and analyzed by Smart-Seq2 RNA sequencing. Two-tailed Student's t-test and two-way ANOVA were used for statistical analyses.
An AAV overexpressing soluble CX3CL1 (AAV-sCX3CL1) was found to significantly increase cone survival in retinas from rd1 (p<0.0001), rd10 (p<0.01),and Rho-/- (p<0.001) mice compared to retinas receiving AAV-GFP only. Functionally, AAV-sCX3CL1 improved ERG (p<0.05) and optomotor (p<0.01) responses in rd10 mice. RNA sequencing of microglia from AAV-sCX3CL1 treated retinas demonstrated paradoxical upregulation of lysosomal pathways (p<0.001) as well as previously characterized microglia activation markers.
We identify AAV-sCX3CL1 as a promising gene therapy candidate for RP and other inherited retinal dystrophies. The mechanism of AAV-sCX3CL1-mediated cone rescue may involve microglia activation rather than quiescence.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
P50 rd1 retina treated with AAV-GFP only
P50 rd1 retina treated with AAV-sCX3CL1 + AAV-GFP
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