Restoration of cone vision in a mouse model of achromatopsia

Abstract

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...

Targeting gene expression to cones with human cone opsin promoters in recombinant AAV

“…19,20 When a truncated version of the upstream sequences was incorporated into a recombinant adenoassociated viral vector (rAAV) of serotype-5 (rAAV5), this promoter targeted green fluorescent protein (GFP) expression to cones in the mouse, rat, ferret, guinea pig and squirrel monkey. [21][22][23] Glushakova et al 24 showed that elements of the human S-opsin promoter can preferentially target cones in rats, although specificity is limited and expression is promiscuous.…”

“…Different promoters previously shown to confer cone photoreceptor expression were used to target GFP expression to the dog L/M and S cones. [19][20][21][22][23][24] Our results demonstrate poor expression levels, and lack of specificity with the human S-opsin promoter. In contrast, the red cone opsin promoters conferred a high degree of specificity, and expression robustness was dependent on the promoter length and content.…”

Clinical and genetic investigation of a large Tunisian family with complete achromatopsia: identification of a new nonsense mutation in GNAT2 gene

“…Recently, both cone-mediated ERG response and visual acuity in a GNAT2 mouse model cpfl3 were rescued using targeted adeno-associated virus gene therapy. 33 Unraveling the genetic basis of the disease in the reported family is an essential step for future access to gene therapy. The GNAT2 mutations in nuclear families segregating with achromatopsia.…”

C1q enhances cone photoreceptor survival in a mouse model of autosomal recessive retinitis pigmentosa

“…[3][4][5] In addition, therapeutic modalities have been demonstrated for other genetic subtypes of RP and LCA in a growing number of animal models, both of recessive and dominant forms of disease. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] Notwithstanding such progress, the genetic complexity of this group of diseases represents a formidable logistic and economic hurdle in developing viable methods of prevention. Given this caveat, parameters effecting photoreceptor survival that are independent of the primary genetic lesion are critically important to identify.…”