Enhanced Integration of Transplanted Photoreceptor Precursors Following Molecular and Genetic Disruption of the Outer Limiting Membrane

Abstract

Purpose: : Retinal diseases culminating in photoreceptor loss are leading causes of untreatable blindness. Although photoreceptor transplantation is technically feasible and can restore the light pupil constriction reflex, a far greater number of cells would most likely be required to have significant impact on visual acuity. It is essential to identify factors that impede or enhance cell integration. The outer limiting membrane (OLM) is a natural barrier between the subretinal space and the outer nuclear layer (ONL), formed by adherens junctions between Müller glia and photoreceptors. Two proteins important for the formation of these junctions are Zona Occludens (ZO-1) and Crumbs homologue-1 (CRB1). Mutations in the human CRB-1 gene can cause Leber's congenital amaurosis and retinitis pigmentosa. Here, we sought to determine if integration of transplanted photoreceptor precursors could be increased by molecular and genetic manipulation of the OLM.

Methods: : Transient molecular disruption was induced by subretinal injections of small interfering (si)RNA targeted against ZO-1 prior to subretinal injection of dissociated early post-natal neural retinal cells. 3 weeks post injection, the number of integrated, differentiated photoreceptor cells present in the ONL of siRNA-treated eyes was compared to sham-injected, contralateral eyes. The Crb1^rd8 mouse provides a genetic model of OLM disruption; it carries a single base deletion in Crb-1, and displays a progressive fragmentation of the OLM and retinal degeneration. 3, 6 or 12 week old Crb1^rd8 mice received subretinal injections of donor cells. 3 weeks post injection, the number of integrated photoreceptor cells present in the ONL of Crb1^rd8 mice was compared to wildtype controls.

Results: : siRNA against ZO-1 induced a short-lived and reversible knockdown of the adherens junction complex, leading to disruption of the OLM. When combined with cell transplantation, this disruption led to markedly enhanced levels of donor cell integration (7-fold increase). Integration was also significantly higher in Crb1^rd8 mice, compared with wildtype controls.

Conclusions: : The OLM represents a significant barrier to cell transplantation, as demonstrated by both genetic and molecular manipulation. However, it can be transiently disrupted using siRNA, leading to enhanced integration.

Keywords: transplantation • photoreceptors • cell adhesions/cell junctions