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Thilo Matthias Buck, Peter M Quinn, Celso Alves, Elon Van Dijk, Charlotte Ohonin, Camiel J F Boon, Jan Wijnholds; Potency assay for AAV-gene vectors in human iPSCs-derived retinas and donor retinas. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4093.
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© ARVO (1962-2015); The Authors (2016-present)
Crumbs proteins (CRB1 and CRB2) localization at the subapical region adjacent to adherens junctions at the outer limiting membrane in the retina, critical to cell adhesion between photoreceptor cells (PRCs) and Müller Cells (MGCs), varies between species. It is unclear which AAV capsid can efficiently infect human MGCs and which AAV gene therapy promoter expresses in human MGCs. Hence, a reliable human retina potency assay is helpful in selecting the appropriate promoters and AAV serotypes for clinical studies. We set up screening assays for testing several AAV capsids and promoters on cultured human retinas (iPSCs-derived retinas and human donor retina explants).
We infected maturating human iPSCs-derived retinas and adult human donor retina explants with different AAV serotypes (AAV9, AAV5, ShH10Y), titres (108-109 genomic copies/μl), promoters (minimal and full length CMV, hGrk1, hRLBP1) and incubation time (7-21 days). We quantified GFP expressing cells/cell type in the human retina by immunohistochemistry using CLSM. The potency assay includes four components: (1) cell-specific expression, (2) promoter activity, (3) time-specific expression and (4) cross-validation in two models.
Healthy maturating human iPSCs-derived retina cultures can be used in infection and expression potency assays for at least one year, whereas human donor retinal explant cultures can be used up to 21 days because of progressive degeneration of the retina. AAV5, AAV9 as well as ShH10Y serotypes can efficiently transduce, and CMV promoters can efficiently express in, MGCs in adult human donor retinal explants and maturating iPSCs-derived retinas.
Our results demonstrate that an AAV2/9-CMV vector, previously used in a proof-of-concept study in Crb2 and Crb1Crb2 KO mice, can be used to rescue Crumbs expression in human MGCs. Further, the human retina assay is useful for testing novel capsids and promoters for gene rescue studies. We can now evaluate limits and possibilities between human in vitro models and animal in vivo models of an AAV-based gene therapy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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