Lentiviral Vector Tropism In Degenerating Retinas

Abstract

Introduction: : In normal mice, lentiviral vector (LV) shows a great efficiency to infect the RPE cells, but transduces retinal neurons more efficiently during development. Here, we investigated the tropism of LV in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the interphotoreceptor matrix (IPM). We tested two different LV-pseudotypes using the VSVG and the Mokola envelopes.

Methods: : Subretinal injections were performed in wild-type (C57/Bl6) and rhodopsin knockout (Rho^-/-) mice. We injected LV-VSVG-EFS-GFPII into 3.3-4.9 month old mice and LV-VSVG-Rho-GFP into 1-1.4 month old mice to target the photoreceptors (PR). LV-MOK-CMV-GFP was injected into 2.4-3.3 months old mice. We sacrificed the animals one week post injection, used immunohistochemistry to identify the transduced cells, and investigated the OLM integrity.

Results: : Using LV-VSVG-EFS-GFPII into 3.3-4.9 months mice, we observed significant retinal and RPE transduction in Rho^-/- mice. However, the retinas showed transduction mainly at the injection’s site. We mostly observed GFP+ cells having a Müller cell morphology. Using LV-MOK-CMV-GFP into 2.4-3.3 months mice, we evidenced the same pattern of viral infection, but with more Müller cells targeted by the virus. Using LV-VSVG-Rho-GFP into 1-1.4 month old mice, we don’t note any difference between Rho^-/- and wild-type mice for transduced cells. The IPM stained with ZO1 appears irregular into the 4.9 months old Rho^-/- mice; for the youngest mice (Rho^-/- and C57/Bl6), there is no modification of the IPM.

Conclusions: : The degeneration improves retinal cells transduction due to the alteration of the IPM in old Rho^-/- mice. Müller cells seem (by morphological evidences) to be the principal cells expressing the transgene. The LV with Mokola envelope can transduce Müller cells in a degenerating retina with an intact IPM. In 1 month old mice, the degeneration doesn’t enhance the transduction in rod PR probably because the IPM is not yet altered. The possibility to target photoreceptors at a later stage of the degeneration is under investigation.

Keywords: gene transfer/gene therapy • retina • Muller cells