Optimization of Clarin-1 AAV Gene Delivery Vectors to the Mouse Retina

Abstract

Purpose: Usher syndrome type III (USH3A) is a recessively inherited disorder resulting from mutations in the Clarin-1 (CLRN1) gene. Patients present with sensorineural hearing loss as well as progressive retinal degeneration. Previously, we have shown the AAV-clarin mediated overexpression can be toxic for the retina. This study will utilize various rAAV vectors carrying wild-type human CLRN1 to assess a safe optimal delivery method, titer, and promoter for USH3A gene therapy.

Methods: AAV quadruple capsid mutant (Y272F+Y444F+ Y500F+Y730F) vectors containing either a ubiquitous CBA promoter or GRK photoreceptor specific promoter driving human CLRN1 with a hemagglutinin (HA) tag at the C-terminal end were injected either subretinally or intravitreally into wild-type C57BL/6 mice. Full strength (viral titer of 8.43 x10¹² vg/ml) and serial dilutions of sc-smCBA-hCLRN1-HA were tested out to 1:1000 (viral titer of 8.43 x10⁹ vg/ml). The sc-GRK-hCLRN1-HA vector was used at a viral titer of 2.1 x10¹² vg/ml. Retinal function and morphology were analyzed up to 1 year post-injection by electroretinography (ERG) and histology using an anti-HA antibody.

Results: The sc-sm-CBA-hCLRN1-HA construct was toxic upon subretinal injection using a full strength viral titer. Scotopic ERG response amplitudes were significantly reduced in treated eyes, and histological analysis revealed that there was significant photoreceptor loss at 6 weeks post-injection. Strong human Clrn1 expression was seen in both RPE and photoreceptor cells. This toxicity was not seen following an intravitreal injection approach which targets ganglion, Muller, inner retina, and photoreceptor cells. There appeared to be less photoreceptor cell death using the GRK1 construct which targets Clrn1 specifically to photoreceptor cells. This could be due to the lower titer of this vector compared to the sc-smCBA-hCLRN1-HA, or to the fact that Clrn1 expression was restricted to photoreceptor cells thus avoiding potential toxicity from expression in the RPE.

Conclusions: The vector dose delivered is critical when using a CBA promoter to drive CLRN1 expression via a subretinal approach. Because endogenous CLRN1 is believed to be expressed at low levels within the retina, it is crucial to employ therapeutic vectors expressing CLRN1 at similar levels This could be achieved either by using a photoreceptor specific promoter, or an intravitreal method of delivery or by careful vector dosing.

Keywords: 538 gene transfer/gene therapy • 688 retina • 648 photoreceptors