Breaching the barrier with matrix biology.

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Basement membranes (BMs) are non-cellular protein layers that simultaneously provide parenchymal cell-attachment sites and also barriers between tissue compartments. The inner limiting membrane (ILM) is a special BM that prevents movement into the retina from the vitreous as such it is a formidable barrier to intravitreal viral-mediated gene transfer or stem cell transplantation. Current methods to circumvent the ILM involve its physical or enzymatic removal with limited effectiveness. The consequence has been that successful cell- and gene- based therapies have targeted a subretinal approach, an effective approach for the outer retina, but of extremely limited use for the inner retina. The goal of this exploratory project is to manipulate the molecular structure of the ILM to promote integration of neural stem cells or viral transfection. ILM formation is dependent on the deposition of laminins, particularly those containing the β2 subunit. We propose two different manipulations of the laminin polymer forming the backbone of the ILM: one, genetic and a second, employs a small biomolecule to disrupt the laminin polymer. Our first approach will be to remove genetically a key component of the ILM, Lamb2. Our prior studies show this disrupts the sheet-like nature of the ILM. We will test if this molecular rearrangement of the ILM renders these retinae more permeant to cell and virus entry. Second approach will be to disrupt laminin polymerizing; netrins, small matrix molecules with laminin homology, are known to disrupt laminin polymerization during normal development and in experimental conditions. We will produce netrin-induced focal disruptions of the ILM and assay for facilitated gene and cell integration. We will perform our experiments, in vitro using organotypic cultures of retina as well as in vivo murine models. The in vitro approach has the advantage that we can add back laminin and reconstitute the ILM and thereby proving that laminin manipulation is the key step. The in vivo approach is more clinically relevant. While in this exploratory project, we will use murine models future successor applications could employ more clinically relevant animal models. Together, the experiments in this project will assess the barrier that the ILM presents to biologic-based engraftment therapies. They will determine if specifically disrupting the laminin backbone is enough to promote engraftment. These experiments if successful will provide a powerful tool to promote advanced matrix-based approach to manipulation of retinal BMs to promote gene-based and cell-based therapeutics in the inner retinal disease.