Abstract
Purpose :
Many retinal diseases results in the loss of inner/outer segments (IS/OS) of photoreceptors (PRCs). PRCs can be infected with some recombinant adeno-associated virus (AAV) serotypes for gene augmentation therapy, CRISPR/Cas9 gene editing or CRISPR/Cas13 RNA base editing. AAVs can efficiently deliver up to 4.9 kb ssDNA to the retina/retinal pigment epithelium. Here, we tested in cultured human retinas the tropism and expression capacity of AAV vectors on photoreceptors with and without IS/OS.
Methods :
Different AAV.CMV.GFP vectors (serotypes AAV5, AAV9 or AAV6 variant ShH10Y445F) were applied to 7-8 month-old human iPSCs-derived retinal organoids (3D mini-retinas) or donor post-mortem adult human retinal explants with or without proper IS/OS PRC segments. The retinas were cultured post-infection for 14-21 days and collected for histology, immunohistochemistry and confocal laser scanning microscopy.
Results :
Infection of photoreceptors with AAV5, AAV9 and ShH10Y445F was inefficient in cultured human donor retinal explants lacking IS/OS. In these retinas, mainly Müller glial cells and to lesser extent ganglion cells expressed GFP. Infection of photoreceptors with AAV.CMV.GFP of serotypes AAV5 and AAV9 but not ShH10Y445F was efficient in human donor retinas containing IS/OS while competing for transduction of Müller glial cells. Infection was inefficient in cultured human iPS derived cone-rich 3D mini-retinas with very small rudimentary IS/OS. These data suggest that, similar as in human retinal explants lacking photoreceptor IS/OS, the iPS derived 3D mini-retinas could not be efficiently infected when cone photoreceptors only had rudimentary IS/OS. These tropism and expression data were confirmed by AAV.hGRK1.GFP vectors applied on human donor retinas with and without IS/OS. The photoreceptor-specific promoter (hGRK1) drove GFP expression only in photoreceptors with IS/OS but no GFP expression was found in retinas lacking IS/OS.
Conclusions :
We show that AAV5 and AAV9 infection of photoreceptors is strongly influenced by the presence or absence of the IS/OS in human ex vivo models. The data suggest that the AAV serotypes are most potent on photoreceptors in human retina in the presence of mature IS/OS.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.