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Laurel Clare Chandler, Michelle E McClements, Alun R Barnard, Maria In�s Patr�cio, Robert E MacLaren, Kanmin Xue; Photoreceptor-specific AAV gene replacement enhanced by the adjunctive use of hydroxychloroquine in vivo. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3391.
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© ARVO (1962-2015); The Authors (2016-present)
One of the major challenges of gene therapy for retinal diseases using adeno-associated viral (AAV) vectors is how to achieve sufficient levels of gene replacement within photoreceptors at a safe vector dose. Methods to improve efficacy of AAV transduction, without increasing vector dose, could be key in achieving a greater treatment effect. We previously reported that adjunctive use of hydroxychloroquine (HCQ) could improve AAV transduction with a ubiquitous reporter construct. Here, we further investigate whether HCQ can specifically enhance transgene expression in photoreceptor cells in vivo and whether the effect is applicable to different AAV serotypes.
Subretinal injection of 1x108 genome copies (gc) of an AAV serotype 8 (Y733F) vector expressing GFP under the human photoreceptor-specific rhodopsin kinase promoter (GRK1) were performed in SVEV mice with or without the addition of 3.13 or 18.75 µM HCQ in paired eyes. Six weeks post-injection, retinal thickness was measured by in vivo optical coherence tomography (OCT) and retinal GFP protein expression assessed by western blot; analysis was performed blind. For comparison, C57BL/6J mice were subretinally injected with 1x108 gc of an AAV serotype 2 vector expressing GFP under the ubiquitous CAG promoter with or without 18.75 µM HCQ in paired eyes. Eight weeks post-injection retinal GFP protein expression was measured.
Compared with AAV vector alone, a 6.6 and 4.6-fold increase in retinal GFP protein expression was seen in mice injected with 18.75 µM HCQ together with AAV8(Y733F).GRK1.GFP (Wilcoxon matched-pairs signed rank test: p=0.0391; n=9) or AAV2.CAG.GFP (p=0.0034; n=12), respectively. No significant increase in GFP protein expression was found in eyes treated with 3.13 µM adjunctive HCQ compared with vector alone. OCT showed no difference in retinal thickness or laminar structure in eyes injected with AAV and 3.13 or 18.75 µM HCQ.
Adjunctive use of HCQ significantly increased retinal transgene expression in vivo following retinal gene therapy using either AAV8 or AAV2 vectors containing photoreceptor-specific or ubiquitous promoters. This potentially provides a means of achieving clinically efficacious levels of transgene expression in photoreceptors without a need to increase viral dose. Further work is needed to assess the mechanism of action of HCQ and its effects in the degenerate retina.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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