Rods and cones subserve mouse vision over a 100 million-fold range of light intensity (Ϫ6 to 2 log cd m ). Mouse rod and cone sensitivities are similar to those of human. This parametric study characterizes the functional properties of the mouse visual system, revealing the rod and cone contributions to contrast sensitivity and to the temporal processing of visual stimuli.

Whereas the mammalian retina possesses a repertoire of factors known to establish general retinal cell types, these factors alone cannot explain the vast diversity of neuronal subtypes. In other CNS regions, the differentiation of diverse neuronal pools is governed by coordinately acting LIM-homeodomain proteins including the Islet-class factor Islet-1 (Isl1). We report that deletion of Isl1 profoundly disrupts retinal function as assessed by electroretinograms and vision as assessed by optomotor behavior. These deficits are coupled with marked reductions in mature ON-and OFF-bipolar (Ͼ76%), cholinergic amacrine (93%), and ganglion (71%) cells. Mosaic deletion of Isl1 permitted a chimeric analysis of "wild-type" cells in a predominantly Isl1-null environment, demonstrating a cell-autonomous role for Isl1 in rod bipolar and cholinergic amacrine development. Furthermore, the effects on bipolar cell development appear to be dissociable from the preceding retinal ganglion cell loss, because Pou4f2-null mice are devoid of similar defects in bipolar cell marker expression. Expression of the ON-and OFF-bipolar cell differentiation factors Bhlhb4 and Vsx1, respectively, requires the presence of Isl1, whereas the early bipolar cell marker Prox1 initially did not. Thus, Isl1 is required for engaging bipolar differentiation pathways but not for general bipolar cell specification. Spatiotemporal expression analysis of additional LIM-homeobox genes identifies a LIMhomeobox gene network during bipolar cell development that includes Lhx3 and Lhx4. We conclude that Isl1 has an indispensable role in retinal neuron differentiation within restricted cell populations and this function may reflect a broader role for other LIM-homeobox genes in retinal development, and perhaps in establishing neuronal subtypes.

Loss of cone function in the central retina is a pivotal event in the development of severe vision impairment for many prevalent blinding diseases. Complete achromatopsia is a genetic defect resulting in cone vision loss in 1 in 30,000 individuals. Using adeno-associated virus (AAV) gene therapy, we show that it is possible to target cones and rescue both the cone-mediated electroretinogram response and visual acuity in the Gnat2 cpfl3 mouse model of achromatopsia.The human retina has approximately 6 million cone photoreceptors, concentrated predominantly in the central retina, which are responsible for high-resolution and color vision. Complete achromatopsia is a disease involving cones that results in permanent central vision loss, deficient cone-mediated electroretinogram (ERG) signal and color blindness, with affected individuals usually having a visual acuity of 20/200 or less 1-3 . Because these individuals only have functioning rods, they experience extreme light sensitivity and daytime blindness 1-3 , owing to the fact that the rods become light saturated. In humans, mutations in cyclic nucleotide-gated channel β-3 (or α-3) and guanine nucleotide α-transducin (GNAT2) genes can give rise to this phenotype 1-3 . The Gnat2 cpfl3 homozygous mouse 4 has a single base substitution inducing a missense mutation (D200N) in cone α-transducin, which results in little or no light-adapted (cone-mediated) ERG response and a normal dark-adapted ERG response, similar to observations in the human form of complete achromatopsia 5 . The GNAT2 mutant form of achromatopsia in humans, and Gnat2 cpfl3 mice, results in a disruption of heterotrimeric G-protein signaling 2,4 , which couples light-activated cone visual pigments to the visual transduction cascade.To develop an AAV vector system that could effectively transduce cones, we first needed a promoter that would efficiently target expression in cones. Previous studies 6,7 have demonstrated that mice transgenic for sequences upstream of the red/green opsin genes, containing a core promoter with a locus control region 6,8,9 , can direct the expression of a reporter gene to both classes of cones in the mouse retina. On the basis of these considerations, we selected a 2.1-kb human red/green opsin promoter construct (PR2.1) to provide cone specificity for our vector. PR2.1 is composed of bases spanning −4,564 to −3,009 and −496 to 0 of the human red cone opsin gene 6 . When incorporated into an AAV vector and packaged in serotype-5 capsids (AAV5-PR2.1-GFP), this promoter targeted green fluorescent protein (GFP) expression to mouse cones when injected subretinally, as shown by the colocalization of GFP with mouse cone arrestin (Fig. 1a). Subsequently, we created an AAV5 vector in which the wild-type mouse cone α-transducin cDNA (Gnat2) was under control of the PR2.1 promoter (AAV5-PR2.1-Gnat2).We then injected 4 × 10 10 vector genome-containing particles of AAV5-PR2.1-Gnat2 into the subretinal space of Gnat2 cpfl3 mouse eyes, from two litters of 10 and 11 mice, on postna...

The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the β subunit of rod cGMP-phosphodiesterase (PDEβ). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEβ was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEβ expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEβ-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEβ-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration.

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