Organization and annotation of the Xcat critical region: elimination of seven positional candidate genes.

Xcat mice display X-linked congenital cataracts and are a mouse model for the human X-linked cataract disease Nance Horan syndrome (NHS). The genetic defect in Xcat mice and NHS patients is not known. We isolated and sequenced a BAC contig representing a portion of the Xcat critical region.

Maculas affected by age-related macular degeneration contain increased chelatable iron in the retinal pigment epithelium and Bruch's membrane.

Objective: To investigate whether iron is involved in the pathogenesis of age-related macular degeneration (AMD).

Methods: Postmortem AMD-affected (nonexudative or exudative) and healthy maculas were studied using the 3,3'-diaminobenzidine-enhanced Perls Prussian blue stain. The Perls Prussian blue stain was quantified by computer-assisted analysis of digital images.

Proapoptotic bcl-2 family members, Bax and Bak, are essential for developmental photoreceptor apoptosis.

Purpose: Apoptosis has been implicated in retinal development and degeneration, but the specific apoptotic pathways used are incompletely understood. The purpose of this study was to characterize the roles in retinal development of the proapoptotic Bcl-2 family members Bax and Bak.

Methods: Eyes from mice at postnatal day (P)7, during the peak of developmental apoptosis in the retina, were processed for TdT-dUTP terminal nick-end labeling (TUNEL) to determine whether Bax knockout or double Bax/Bak knockout causes a defect in developmental apoptosis.

Gene therapy and animal models for retinal disease.

Those plagued by retinal diseases are often robbed of their vision, as often, effective treatments do not exist. Knowledge of the pathophysiology of retinal diseases stems from research on available animal models. Gene therapy may be useful for both genetic and acquired retinal diseases.

Delivery of adeno-associated virus vectors to the fetal retina: impact of viral capsid proteins on retinal neuronal progenitor transduction.

The development of fetal ocular gene transfer may be useful as a therapeutic tool for the prevention of retinal genetic disorders with congenital or early clinical manifestations. In this study we explored the neural progenitor transduction patterns of adeno-associated virus (AAV) vectors following delivery to the developing retina. Recombinant vectors with the same genome carrying the enhanced green fluorescent protein (EGFP) transgene packaged in capsids of differing serotypes (serotypes 1, 2, and 5, termed AAV2/1, AAV2/2, and AAV2/5, respectively) were created.

Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model.

Purpose: RNA interference mediated by small interfering RNAs (siRNAs) is a powerful technology allowing the silencing of mamalian genes with great specificity and potency. The purpose of this study was to demonstrate the feasibility of RNA interference mediated by siRNA in retinal cells in vitro and in the murine retina in vivo.

Methods: siRNAs specific for enhanced green fluorescent protein (EGFP) and murine and human vascular endothelial growth factor (VEGF) were designed.

Amyloid-beta is found in drusen from some age-related macular degeneration retinas, but not in drusen from normal retinas.

Purpose: Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. Increased understanding of the pathogenesis is necessary. Amyloid-beta (Abeta), a major extracellular deposit in Alzheimer's disease plaques, has recently been found in drusen, the hallmark extracellular deposit in AMD.

Increased expression of ceruloplasmin in the retina following photic injury.

Purpose: Oxidative stress plays a role in the photic injury model of retinal degeneration and in age-related macular degeneration. Our preliminary microarray analysis of retinal gene expression upon photic injury suggested increased expression of ceruloplasmin, a ferroxidase that could reduce retinal oxidative stress. Patients with acerul oplasminemia have retinal degeneration, indicating that ceruloplasmin is necessary for maintenance of retinal health.

Gene therapy for choroideremia: in vitro rescue mediated by recombinant adenovirus.

Choroideremia (CHM) is an X-linked retinal degenerative disease resulting from a lack of functional Rab Escort Protein-1 (REP-1). As a first step in developing gene-based therapies for this disease, we evaluated the feasibility of delivering functional REP-1 to defective lymphocytes and fibroblasts isolated from individuals with CHM. A recombinant adenovirus delivering the full-length human cDNA encoding REP-1 under the control of a cytomegalovirus promoter was generated.

Efficient trans-splicing in the retina expands the utility of adeno-associated virus as a vector for gene therapy.

Recombinant vectors based on adeno-associated virus (AAV) can efficiently transduce many different cell types, including cells of the retina, resulting in stable gene expression. A major shortcoming of this vector is its small packaging capacity. A trans-splicing approach, which reconstitutes gene expression from two independent AAV vectors, can be used to overcome the vector's packaging limitations.

Pigment epithelium-derived factor expression in the developing mouse eye.

Purpose: Pigment Epithelium-Derived Factor (PEDF) is a 50 kDa secretable protein with neuroprotective, neurotrophic, and antiangiogenic properties. Expression patterns in the human eye suggest that modulation of this protein over time and place may play a role in development of normal ocular vasculature. Because of the potential importance of normal PEDF expression patterns in controlling ocular blood vessel growth in health and disease, we characterized these patterns over the period of retinal vascular development in the mouse.

Phenotypic rescue after adeno-associated virus-mediated delivery of 4-sulfatase to the retinal pigment epithelium of feline mucopolysaccharidosis VI.

Background: Mucopolysaccharidosis VI (MPS VI), due to recessively inherited 4-sulfatase (4S) deficiency, results in lysosomal storage of dermatan sulfate in numerous tissues. Retinal involvement is limited to the retinal pigment epithelium (RPE). This study aimed to determine whether recombinant adeno-associated virus (AAV)-mediated delivery of 4S would reverse the RPE pathology seen in MPS VI cats.

Inhibition of retinal neovascularization by intraocular viral-mediated delivery of anti-angiogenic agents.

Neovascularization characterizes diabetic retinopathy and choroidal neovascularization associated with age-related macular degeneration, the most common causes of severe visual loss in the developed world. Gene transfer to the eye using adeno-associated viral (AAV) vectors is a promising new treatment for inherited and acquired ocular diseases. We used an AAV vector with rapid onset and high levels of gene expression in the retina to deliver three anti-angiogenic factors (pigment epithelium-derived factor, tissue inhibitor of metalloproteinase-3, and endostatin) to the eyes of mice in a mouse model of retinopathy of prematurity.

Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate.

Purpose: Vascular endothelial growth factor (VEGF) is a potent ischemia-upregulated angiogenic protein that has been implicated in diabetic retinopathy. Intravitreal VEGF injections have not previously been shown to produce preretinal neovascularization. The purpose of this study was to further characterize the angiopathic changes that occur after intravitreal injections in a nonhuman primate and determine if preretinal neovascularization develops.

Functionally rodless mice: transgenic models for the investigation of cone function in retinal disease and therapy.

Two genetically engineered strains of mice were used to characterize murine cone function electroretinographically, without interference of rod-driven responses: (1) mice with a deletion of the gene for the rod transducin alpha-subunit (transducin alpha-/-), and (2) mice with rod arrestin deleted (arrestin -/-). In the first three months of age, both strains have a normal complement of rods and normal rod structure, but transducin alpha-/- mice have no rod-driven responses to light, while rod-driven activity of arrestin -/- mice can be suppressed by a single intense flash for hours. In response to intense flashes the electroretinograms of these strains of mice showed a readily identifiable, pure-cone a-wave of approximately 10 microV saturating amplitude.

A deviant immune response to viral proteins and transgene product is generated on subretinal administration of adenovirus and adeno-associated virus.

The immune response after ocular exposure to foreign antigens varies substantially from that of a typical systemic response. Anterior chamber associated immune deviation (ACAID) has been well documented. The immune response of the subretinal space has not been studied in as much detail.

The nuclear receptor NR2E3 plays a role in human retinal photoreceptor differentiation and degeneration.

Normal human retinal development involves orderly generation of rods and cones by complex mechanisms. Cell-fate specification involves progenitor cell lineage and external signals such as soluble factors and cell-cell interactions. In most inherited human retinal degenerations, including retinitis pigmentosa, a mutant gene causes loss of visual function, death of mature rods, and eventually death of all cone subtypes.

Identification and subcellular localization of the RP1 protein in human and mouse photoreceptors.

Purpose: Mutations in the RP1 gene account for 6% to 10% of autosomal dominant retinitis pigmentosa (adRP). Previous studies have shown that the RP1 gene is expressed specifically in photoreceptor cells. So far, little is known about the RP1 protein or how mutations in RP1 lead to photoreceptor cell death.

Long-term protection of retinal structure but not function using RAAV.CNTF in animal models of retinitis pigmentosa.

The present study aimed to determine whether intravitreal administration of an adeno-associated virus (AAV) carrying ciliary neurotrophic factor (CNTF) can achieve long-term morphological and physiological rescue of photoreceptors in animal models of retinitis pigmentosa, and whether injection of this virus after degeneration begins is effective in protecting the remaining photoreceptors. We injected rAAV.CNTF.

Gene therapy and retinitis pigmentosa: advances and future challenges.

It may be possible, one day, to use gene therapy to treat diseases whose genetic defects have been discerned. Because many genes responsible for inherited eye disorders within the retina have been identified, diseases of the eye are prime candidates for this form of therapy. The eye also has the advantage of being highly accessible with altered immunological properties, important considerations for easy delivery of virus and avoidance of systemic immune responses.

Pathways to photoreceptor cell death in inherited retinal degenerations.

The mutations that cause many forms of inherited retinal degenerations have been identified, yet the mechanisms by which these mutations lead to death of photoreceptor cells of the retina are not completely understood. Investigations of the pathways from mutation to retinal degeneration have focused on spontaneous and engineered animal models of disease. Based on the studies performed to date, four major categories of degeneration mechanism can be identified.

Localization of pigment epithelium derived factor (PEDF) in developing and adult human ocular tissues.

Purpose: To localize pigment epithelium-derived factor (PEDF) in developing and adult human ocular tissues.

Methods: PEDF was localized in fetal and adult eyes by immunofluorescence with a polyclonal antibody (pAb) against amino acids 327-343 of PEDF, or a monoclonal antibody (mAb) against the C-terminal 155 amino acids of PEDF. Specificity of the antibodies was documented by Western blotting.

AAV-mediated delivery of ciliary neurotrophic factor prolongs photoreceptor survival in the rhodopsin knockout mouse.

Retinitis pigmentosa (RP), an inherited retinal degenerative disease causing blindness, is characterized by progressive apoptotic death of photoreceptors. Therapeutic modification of photoreceptor apoptosis may provide an effective therapy for this disorder. Ciliary neurotrophic factor (CNTF) has been shown to promote survival of a number of different neuronal cell types, including photoreceptors.

Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina.

Recombinant adeno-associated virus (rAAV) is a promising vector for therapy of retinal degenerative diseases. We evaluated the efficiency, cellular specificity, and safety of retinal cell transduction in nonhuman primates after subretinal delivery of an rAAV carrying a cDNA encoding green fluorescent protein (EGFP), rAAV. CMV.

Mutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pigmentosa.

The autosomal dominant retinitis pigmentosa (RP) locus, designated RP1, has been mapped through linkage studies to a 4-cM interval at 8q11-13. Here we describe a new photoreceptor-specific gene that maps in this interval and whose expression is modulated by retinal oxygen levels in vivo. This gene consists of at least 4 exons that encode a predicted protein of 2,156 amino acids.