AAV-mediated expression targeting of rod and cone photoreceptors with a human rhodopsin kinase promoter.

PURPOSE: Gene therapy for retinal degeneration requires well-defined promoters that drive expression in rod and cone photoreceptors. This study was undertaken to develop short, active derivatives of the human rhodopsin kinase (RK) gene promoter for targeting transgene expression in rods and cones. RK, also known as G protein-coupled receptor kinase 1 (GRK1), is a component of the light adaptation pathway expressed in rods and cones. METHODS: Human RK (hRK) promoter and its concatemers or derivatives extending into the conserved 5' untranslated region (5'-UTR) were assayed for promoter activity in WERI retinoblastoma or Crx/Sp1-supplemented HEK-293 cells. The derivative displaying the highest activity was linked to a GFP reporter and packaged in a pseudotyped adenoassociated viral vector (AAV2/5). The AAV vector was tested in vivo by subretinal injections in wild-type mice, in the all-cone Nrl(-/-) mice, and in the cone-rich diurnal Nile grass rat (Arvicanthis niloticus). Control eyes received a similar AAV2/5 vector carrying a mouse rod opsin (mOps) promoter-controlled GFP reporter. RESULTS: The hRK promoter with the full 5' untranslated sequence (-112 to +180) was the most active in cell culture. Delivered by the AAV2/5 vector, RK promoter drove GFP expression specifically in photoreceptors. In rods, hRK promoter-mediated expression was as efficient as, but appeared more uniform than, mOps promoter-mediated expression. In cones, the hRK promoter drove expression, whereas the mOps promoter did not. CONCLUSIONS: The hRK promoter is active and specific for rod and cone photoreceptors. Because of its small size and proven activity in cones, it is a promoter of choice for somatic gene transfer and gene therapy targeting rods and cones.

Conserved structure and spatiotemporal function of the compact rhodopsin kinase (GRK1) enhancer/promoter.

PURPOSE: To demonstrate that the crucial elements responsible for the spatial and temporal expression patterns of rhodopsin kinase (Rk) are contained within a narrow conserved segment immediately flanking the Rk transcription start sites. METHODS: Sequences upstream of the mouse Rk gene were compared to the human sequence to identify areas of conservation. Transgenic mice carrying a segment of the conserved human DNA sequence linked upstream of the green fluorescent protein (GFP) gene were examined by fluorescence microscopy and RT-PCR to localize GFP expression in retina and pineal gland. Rk and GFP temporal expression patterns were further compared by immunostaining and real-time RT-PCR in transgenic eyes during development. RESULTS: Comparison of the mouse and human 5' flanking sequence revealed only a small island of conserved sequence upstream of the respective Rk start sites. Uniform GFP expression was supported by a 0.2 kb fragment of the conserved human sequence in the transgenic mouse rods, cones, and pinealocytes. Developmental studies revealed an exponential rise in Rk and GFP transcripts in the first ten day postnatal period followed by a plateau later extending to adulthood. Rk and GFP proteins were first detected after postnatal day 10 and rose in parallel afterwards, overlapping in time with the maturation of photoreceptor outer segments and eye opening. CONCLUSIONS: The conserved short enhancer/promoter immediately upstream of the Rk gene contains the key elements required for appropriate response to spatial and temporal cues during photoreceptor cell differentiation and fate determination. The above studies narrow the core sequences that govern gene expression in photoreceptors in vivo.

A short, highly active photoreceptor-specific enhancer/promoter region upstream of the human rhodopsin kinase gene.

PURPOSE: Rhodopsin kinase (Rk or GRK1) is a photoreceptor-specific enzyme that mediates adaptation of photoreceptors to light and protects these cells against light-induced injury. This study examined the transcriptional mechanisms that maintain physiologic levels of this essential enzyme in photoreceptors. METHODS: The 2.0-kb region flanking the 5' end of the human Rk gene was isolated, mapped, and sequenced. The sequence was fused upstream of the luciferase gene and was tested for promoter activity in retinoblastoma cells by transient transfection. Transcriptionally active segments were identified by deletion and site-directed mutagenesis. Transgenic mice were generated that carried the immediate 5' flanking segment linked upstream of the enhancerless green fluorescent protein (GFP) gene. GFP expression was analyzed by RT-PCR, fluorescence microscopy, and immunohistochemistry. RESULTS: Mapping and sequence analysis uncovered a TATA-less promoter with several recognizable elements concentrated proximally. A conserved putative homeodomain response element H1 and a GC- and a GA-rich motif were noted within a 0.11-kb region. Another putative homeodomain binding site, H2, and a stretch of C-rich repeats were present distally. Mutagenesis in conjunction with transient transfection in retinoblastoma cells identified the 0.11-kb region and H1 sequence as the key active enhancer-promoter sequences. The distal sequences were inhibitory. Transgenic mice that carried the 0.11-kb DNA segment with the GFP gene linked downstream showed GFP transcript, fluorescence, and immunoreactivity that were restricted to photoreceptors. CONCLUSIONS: The experiments defined a short, highly active photoreceptor-specific enhancer-promoter region upstream of the Rk gene. The H1 element contributed substantially but not exclusively to the transcriptional activity of the region. The findings support a transcriptional basis for photoreceptor-specific expression of Rk.

Late-onset autosomal dominant macular dystrophy with choroidal neovascularization and nonexudative maculopathy associated with mutation in the RDS gene.

PURPOSE: To examine the molecular genetic basis and phenotypic characteristics of an unusual late-onset autosomal dominant macular dystrophy with features of age-related macular degeneration (AMD) in a large family (SUNY901), by using linkage and mutation analyses. METHODS: Blood samples were collected from 17 affected members, 17 clinically unaffected members, and 5 unrelated spouses. Clinical analyses included a review of medical history and standard ophthalmic examination with fundus photography, fluorescein angiography, and electroretinography. Linkage and haplotype analyses were performed with microsatellite markers. Mutation analysis was performed by amplification of exons followed by sequencing. RESULTS: A wide spectrum of clinical phenotypes including exudative and nonexudative maculopathy was observed, with onset in the late fifth decade. Linkage analysis excluded most of the previously known maculopathy loci. Markers D6S1604 (Z(max) of 3.18 at theta = 0), and D6S282 (Z(max) of 3.18 at theta = 0) gave significant positive LOD scores and haplotype analysis localized the disease gene to a 9-centimorgan (cM) interval between markers D6S1616 and D6S459. Mutation analysis excluded the GUCA1A and GUCA1B genes and revealed a missense mutation in the RDS/peripherin gene leading to a Tyr141Cys substitution. A phenotype and haplotype comparison between this and a separate family with the Tyr141Cys mutation suggested the presence of a common ancestral haplotype. CONCLUSIONS: The RDS mutation in codon 141 is associated with an unusual AMD-like late-onset maculopathy. An apparent selective bias was noted favoring the transmission of the mutant allele. These observations broaden the spectrum of phenotypes associated with RDS gene mutations.

Variation in rhodopsin kinase expression alters the dim flash response shut off and the light adaptation in rod photoreceptors.

PURPOSE: Rod photoreceptors are exquisitely sensitive light detectors that function in dim light. The timely inactivation of their light responses is critical for the ability of rods to reliably detect and count photons. A key step in the inactivation of the rod transduction is the phosphorylation of the rod visual pigment, rhodopsin, catalyzed by G-protein-dependent receptor kinase 1 (GRK1). Absence of GRK1 greatly prolongs the photoreceptors' light response and enhances their susceptibility to degeneration. This study examined the light responses from mouse rods expressing various levels of GRK1 to evaluate how their function is modulated by rhodopsin inactivation. METHODS: Transretinal and single-cell rod electrophysiological recordings were obtained from several strains of mice expressing GRK1 at 0.3- to 3-fold the wild-type levels. The effect of GRK1 expression level on the function of mouse rods was examined in darkness and during background adaptation. RESULTS: Altering the expression of GRK1 from 0.3- to 3-fold that in wild-type rods had little effect on the single photon response amplitude. Notably, increasing the expression level of GRK1 accelerated the dim flash response shut off but had no effect on the saturated response shut off. Additionally, GRK1 excess abolished the acceleration of saturated responses shut off during light adaptation. CONCLUSIONS: These results demonstrate that rhodopsin inactivation can modulate the kinetics of recovery from dim light stimulation. More importantly, the ratio of rhodopsin kinase to its modulator recoverin appears critical for the proper adaptation of rods and the acceleration of their response shut off in background light.

Effect of g protein-coupled receptor kinase 1 (Grk1) overexpression on rod photoreceptor cell viability.

PURPOSE: Photoreceptor rhodopsin kinase (Rk, G protein-dependent receptor kinase 1 [Grk1]) phosphorylates light-activated opsins and channels them into an inactive complex with visual arrestins. Grk1 deficiency leads to human retinopathy and heightened susceptibility to light-induced photoreceptor cell death in the mouse. The goal of this study was to determine whether excess Grk1 activity is protective against photoreceptor cell death. METHODS: Grk1-overexpressing transgenic mice (Grk1(+)) were generated by using a bacterial artificial chromosome (BAC) construct containing mouse Grk1, along with its flanking sequences. Quantitative reverse transcription-PCR, immunoblot analysis, immunostaining, and activity assays were combined with electrophysiology and morphometric analysis, to evaluate Grk1 overexpression and its effect on physiologic and morphologic retinal integrity. Morphometry and nucleosome release assays measured differences in resistance to photoreceptor cell loss between control and transgenic mice exposed to intense light. RESULTS: Compared with control animals, the Grk1(+) transgenic line had approximately a threefold increase in Grk1 transcript and immunoreactive protein. Phosphorylated opsin immunochemical staining and in vitro phosphorylation assays confirmed proportionately higher Grk1 enzyme activity. Grk1(+) mice retained normal rod function, normal retinal appearance, and lacked evidence of spontaneous apoptosis when reared in cyclic light. In intense light, Grk1(+) mice showed photoreceptor damage, and their susceptibility was more pronounced than that of control mice with prolonged exposure times. CONCLUSIONS: Enhancing visual pigment deactivation does not appear to protect against apoptosis; however, excess flow of opsin into the deactivation pathway may actually increase susceptibility to stress-induced cell death similar to some forms of retinal degeneration.

Conserved interactions of a compact highly active enhancer/promoter upstream of the rhodopsin kinase (GRK1) gene.

Rhodopsin kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.