Rapid cohort generation and analysis of disease spectrum of large animal model of cone dystrophy.

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients.

Gene therapy regenerates protein expression in cone photoreceptors in Rpe65(R91W/R91W) mice.

Cone photoreceptors mediate visual acuity under daylight conditions, so loss of cone-mediated central vision of course dramatically affects the quality of life of patients suffering from retinal degeneration. Therefore, promoting cone survival has become the goal of many ocular therapies and defining the stage of degeneration that still allows cell rescue is of prime importance. Using the Rpe65(R91W/R91W) mouse, which carries a mutation in the Rpe65 gene leading to progressive photoreceptor degeneration in both patients and mice, we defined stages of retinal degeneration that still allow cone rescue. We evaluated the therapeutic window within which cones can be rescued, using a subretinal injection of a lentiviral vector driving expression of RPE65 in the Rpe65(R91W/R91W) mice. Surprisingly, when applied to adult mice (1 month) this treatment not only stalls or slows cone degeneration but, actually, induces cone-specific protein expression that was previously absent. Before the intervention only part of the cones (40% of the number found in wild-type animals) in the Rpe65(R91W/R91W) mice expressed cone transducin (GNAT2); this fraction increased to 64% after treatment. Correct S-opsin localization is also recovered in the transduced region. In consequence these results represent an extended therapeutic window compared to the Rpe65(-/-) mice, implying that patients suffering from missense mutations might also benefit from a prolonged therapeutic window. Moreover, cones are not only rescued during the course of the degeneration, but can actually recover their initial status, meaning that a proportion of altered cones in chromophore deficiency-related disease can be rehabilitated even though they are severely affected.

Remaining rod activity mediates visual behavior in adult Rpe65-/- mice.

PURPOSE: C57/Bl6, Cpfl1(-/-) (cone photoreceptors function loss 1; pure rod function), Gnat1a(-/-) (rod α-transducin; pure cone function), and Rpe65(-/-);Rho(-/-) double-knockout mice were studied to distinguish the respective contributions of the different photoreceptor (PR) systems that enable light perception and mediate a visual reflex in adult Rpe65(-/-) mice, with a simple behavioral procedure. METHODS: Visual function was estimated using a rotating automated optomotor drum covered with vertical black-and-white stripes at spatial frequencies of 0.025 to 0.5 cycles per degree (cyc/deg) in both photopic and scotopic conditions. Mouse strains with different luminances were tested to evaluate the contribution and the light-intensity threshold of each PR system. RESULTS: Stripe rotation elicited head movements in the wild-type (WT) animals in photopic and scotopic conditions, depending on the spatial frequency, whereas the Cpfl1(-/-) mice show a reduced activity in the photopic condition and the Gnat1a(-/-) mice an almost absent response in the scotopic condition. A robust visual response was obtained with Rpe65(-/-) knockout mice at 0.075 and 0.1 cyc/deg in the photopic condition. The residual rod function in the Rpe65(-/-) animals was demonstrated by testing Rpe65(-/-);Rho(-/-) mice that present no response in photopic conditions. CONCLUSIONS: The optomotor test is a simple method of estimating the visual function and evaluating the respective contributions of rod and cone systems. This test was used to demonstrate that in Rpe65(-/-) mice, devoid of functional cones and of detectable 11-cis-retinal protein, the rods mimic cone function in part, by mediating vision in photopic conditions.