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Laurel Clare Chandler, Alun R Barnard, Maria In?s Patrício, Kanmin Xue, Robert E MacLaren; Hydroxychloroquine enhances the efficacy of retinal gene therapy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):6005.
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© ARVO (1962-2015); The Authors (2016-present)
Gene therapies for inherited retinal dystrophies using adeno-associated viral (AAV) vectors have shown early signs of efficacy in clinical trials. We have previously reported the activation of intracellular immune responses to AAV in the retina, and postulated that these may limit viral transduction and therefore therapeutic effects. Here we demonstrate that the use of a putative inhibitor of innate immunity, hydroxychloroquine (HCQ), during gene therapy could significantly enhance the efficacy of transgene expression in vitro and in vivo.
Mouse embryonic fibroblasts (MEFs), primary non-human primate (NHP) retinal pigment epithelial (RPE) cells, and human retinal explants were treated with HCQ prior to transduction with an AAV2 vector expressing green fluorescent protein (GFP) as a reporter for successful transduction. On day 3 GFP expression was quantified by flow cytometry in the MEFs and by reverse transcription qPCR in the NHP RPE cells, and on day 11 by mean fluorescence grey value in the retinal explants. To test the safety and efficacy of HCQ in vivo, subretinal injections of AAV2.GFP with or without the addition of HCQ were performed in matched eyes of 7-week old C57BL/6J mice. Confocal scanning laser ophthalmoscopy with retinal autofluorescence (AF) imaging was performed at 2, 4 and 8 weeks post-injection, retinal thickness was measured using optical coherence tomography.
Compared with vector alone, the use of HCQ increased AAV transduction in vitro/ex vivo. GFP expression was increased by 3.4-fold in MEFs (one-way ANOVA: n=4, p=0.0013), 3-fold in NHP RPE cells (n=3, p=0.0253), and 2-fold in human retinal explants (n=6, p=0.0213). In vivo, mean retinal AF, which represents GFP expression, demonstrated a 2-fold increase in eyes that received gene therapy with HCQ at 4 and 8 weeks, relative to paired eyes injected with AAV vector only (two-way ANOVA: n=5, p=0.0057). No differences in retinal thickness (one-way ANOVA: n=5, p=0.4306) or architecture were seen between eyes injected with and without HCQ, indicating absence of toxicity.
HCQ significantly enhances AAV transduction across species. Subretinal injection of a single pulse of HCQ together with the gene therapy vector appears to be safe and leads to sustained increase in transgene expression. While the precise mechanism of action remains unclear, adjunctive use of HCQ can potentially enhance the therapeutic effects of gene therapy.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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