Adrian M. Timmers

Restoration of cone vision in a mouse model of achromatopsia

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.

Anti-apoptotic effects of CNTF gene transfer on photoreceptor degeneration in experimental antibody-induced retinopathy.

Autoantibodies against recoverin are found in the sera of patients with cancer-associated retinopathy syndrome, a paraneoplastic disease associated with retinal degeneration. We have previously shown that anti-recoverin autoantibodies induced photoreceptor apoptotic cell death after injection into the vitreous of Lewis rats. Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of a number of neuronal cell types, including photoreceptors. In this study, we examined whether an adeno-associated virus (AAV)-mediated delivery of gene encoding the human CNTF protected photoreceptor cells from anti-recoverin antibody-induced death. One month after subretinal injection of the AAV-CNTF gene into one eye and a control vector into the other eye, an anti-recoverin antibody was injected to induce retinal cell death in Lewis rats. Subretinal administration of the virus led to an efficient transduction of photoreceptors, as indicated by immunostaining of retinas with anti-CNTF. Histological examination of the corresponding retinas showed that photoreceptor cells were significantly protected from apoptotic death in the CNTF-treated eyes. CNTF treatment of the retinas resulted in a time-dependent activation of STAT 3. The present study shows that an AAV-mediated delivery of CNTF may protect photoreceptors from antibody-induced cell death through the activation of STAT3 and the suppression of caspase 3 activity, a key caspase leading to apoptosis. Thus, CNTF may be a useful treatment for human antibody-mediated retinal degeneration.

Cone-specific expression using a human red opsin promoter in recombinant AAV

PURPOSE To determine the feasibility of targeting gene expression specifically to cone photoreceptors using recombinant adeno-associated virus (rAAV) as the vector. METHODS An rAAV vector was constructed that contains a 2.1kb upstream sequence of the human red opsin gene to direct green fluorescent protein (GFP) expression. A control construct containing a 472bp mouse rod opsin promoter, previously shown to drive photoreceptor-specific expression, was also used. Each recombinant virus was injected into the subretinal space of rat, ferret or guinea pig eyes. GFP expression was analyzed 4-6 weeks after injection microscopically. RESULT The human 2.1kb cone opsin gene upstream sequence targeted GFP expression only to a subset of photoreceptors. Cone-specific expression was shown by co-localization of GFP fluorescence and cone-specific opsin antibody staining. Additionally, in rats, expression was specific for L/M-cones whereas no S-cones exhibited GFP fluorescence. The efficiency of rAAV mediated cone transduction surrounding the injection site was high since every L/M-cone antibody-staining cone was also positive for GFP expression. CONCLUSION The human red/green opsin gene promoter used in this study is sufficient to direct efficient cone-specific gene expression in several mammalian species, suggesting that key cell-type specific regulatory elements must be broadly conserved in mammals. These observations have significance in devising gene therapy strategies for retinal dystrophies that primarily affect cones and point toward a way to functionally dissect the cone opsin promoter in vivo.

Early expression and localization of rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) in the developing fetal bovine retina

Differentiation and maturation of the photoreceptor outer segments are key steps in the development of the visual system. Morphological studies presented here show that the cow and human are nearly identical in the timing of outer segment appearance during fetal development, implying that the bovine retina is a good model system for the final stages of human photoreceptor development. To study photoreceptor maturation, rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) were quantified by ELISA in a developmentally staged series of fetal bovine retinas. In addition, their localization within these retinas was determined by immunogold electron microscopy. Rhodopsin, as detected by antibodies directed against either the N- or C-terminal portions of the molecule, is first found at about 5.5 months gestation. It is first detected on the plasma membrane of the immature cilia and on the earliest emergent outer segment membrane, even before organized disk membranes are apparent. In contrast, whereas rhodopsin levels and outer segments are nearly undetectable before 5 months gestation, IRBP accumulates to a significant level (4-5% of the adult) as early as 3 months gestation. Immunogold electron microscopy confirmed this finding, with localization of IRBP predominantly in the subretinal space.

Inactivation of the Basigin gene impairs normal retinal development and maturation

5A11/Basigin is an immunoglobulin-like glycoprotein expressed on the surface of Müller cells, the apical and basal surfaces of the retinal pigmented epithelium, and photoreceptor cell bodies and their inner segments. Disruption of the 5A11/Basigin gene in the mouse results in photoreceptor degeneration and a corresponding decrease in electroretinogram amplitudes in mature mice. The purpose of this study was to examine the electrophysiology of the 5A11/Basigin null mouse retina at earlier ages than previously examined. Although the architecture of the 5A11/Basigin null mouse retina appears normal, the ERG amplitudes are severely depressed at eye opening, indicating failure in retinal maturation.

Alligator rhodopsin: Sequence and biochemical properties

We sequenced selected peptides of alligator rhodopsin that accounted for about half of the total protein. These sequences were confirmed when the total primary structure of alligator rhodopsin was deduced from the cDNA sequence. Differences in the amino-terminal region, compared to that of bovine rhodopsin, account for failure of cross-reactivity of several anti-bovine rhodopsin monoclonal antibodies. Differences in the carboxyl-terminal region give rise to limited antibody cross-reactivity and may also account for a slightly reduced ability of alligator rhodopsin to be phosphorylated by bovine rhodopsin kinase. Alligator rhodopsin regenerates much faster than bovine rhodopsin. The pseudo-first-order rate constant for alligator rhodopsin regeneration is approximately 25 times that of bovine. Phylogenetic analysis of 17 rhodopsin sequences indicates that the alligator is more closely related to the chicken than to the other species examined.

Topographical regulation of cone and rod opsin genes: parallel, position dependent levels of transcription

RNase protection assays were used to follow rhodopsin and red cone opsin mRNA levels during bovine fetal development as a function of retinal position. Following induction, an equivalent radial gradient of rod and cone opsin mRNA is present in the fetal retina. This gradient is maintained in the adult retina even though no corresponding gradient in rod or cone cell density is present. Since the mRNA expression gradient does not progress radially, position dependent levels of photoreceptor-specific transcription is suggested.

Disease mechanisms and gene therapy in a mouse model for X-linked retinoschisis.

X-linked retinoschisis (RS) is an inherited recessive macular degeneration that affects between 1 in 5000 and 1 in 25,000 males early in life (George et al., 1995; Sieving, 1998; Tantri et al., 2004). It is characterized by a loss in central vision, splitting of the retina with the appearance of spoke-like cystic cavities radiating from the parafoveal region of the retina, a loss in the b-wave of the electroretinogram (ERG), and progressive atrophy of the macula. In about 50% of the cases, bilateral schisis is observed in the peripheral retina with some loss in peripheral vision. During the course of the disease, complications can arise which include retinal detachment, vitreal hemorrhage and choroidal sclerosis.

The eyes have it.

The Jackson Laboratory’s huge resource of defined mouse strains, coupled with the genetic malleability of the mouse and the accessibility of the mouse eye to a variety of noninvasive analyses of retinal function, makes it clear that our understanding of human retinal disease will remain intimately tied to the mouse eye throughout the upcoming, functional genomics age of human gene medicine.

Toxicology and Pharmacology of an AAV Vector Expressing Codon-optimized RPGR in RPGR-deficient Rd9 Mice

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated AAV2tYF-GRK1-RPGRco, to treat retinitis pigmentosa (RP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (RPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1, GRK1) and is packaged in an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a safety and potency study of this vector administered by subretinal a injection in the naturally occurring RPGR-deficient Rd9 mouse model. Sixty Rd9 mice (20 per group) received a subretinal injection in the right eye of vehicle (control) or AAV2tYF-GRK1-RPGRco at one of two dose levels (4 × 108 or 4 × 109 vg/eye) and were followed for 12 weeks after injection. Vector injections were well tolerated, with no systemic toxicity. There was a trend towards reduced electroretinography b-wave amplitudes in the high vector dose group that was not statistically significant. There were no clinically important changes in hematology or clinical chemistry parameters and no vector-related ocular changes in life or by histological examination. Dose-dependent RPGR protein expression, mainly in the inner segment of photoreceptors and the adjacent connecting cilium region, was observed in all vector-treated eyes examined. Sequence integrity of the codon-optimized RPGR was confirmed by sequencing of PCR-amplified DNA, or cDNA reverse transcribed from total RNA extracted from vector-treated retinal tissues, and by sequencing of RPGR protein obtained from transfected HEK 293 cells. These results support the use of rAAV2tYF-GRK1-RPGRco in clinical studies in patients with XLRP caused by RPGR mutations.