Gene therapy restores vision in a canine model of childhood blindness.

The relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (RPE) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. The molecular bases for both primary photoreceptor and RPE diseases that cause blindness have been identified. Gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and RPE in a large-animal model of human disease has not been reported. Here we study one of the most clinically severe retinal degenerations, Leber congenital amaurosis (LCA). LCA causes near total blindness in infancy and can result from mutations in RPE65 (LCA, type II; MIM 180069 and 204100). A naturally occurring animal model, the RPE65-/- dog, suffers from early and severe visual impairment similar to that seen in human LCA. We used a recombinant adeno-associated virus (AAV) carrying wild-type RPE65 (AAV-RPE65) to test the efficacy of gene therapy in this model. Our results indicate that visual function was restored in this large animal model of childhood blindness.

Growth and Nutritional Outcomes in Children Post-Haematopoietic Stem Cell Transplant without Exposure to Total Body Irradiation.

AIMS: Poor growth in childhood cancer survivors who undergo haematopoietic stem cell transplant (HSCT) without exposure to radiation is reported anecdotally, although literature to support this is limited. The aims of this study were to assess the change in height standard deviation score (SDS) and the final adult height (FAH) in children who underwent chemotherapy-only conditioned HSCT and to identify predictors of poor growth. MATERIALS AND METHODS: We conducted a retrospective hospital medical record review (1984-2010) of children (1-10 years) who underwent chemotherapy-only conditioned HSCT, noting anthropology measurements at cancer diagnosis, HSCT, 10 years old and FAH. RESULTS: The median age at HSCT of the 53 patients was 4.5 years, 75% had a haematological malignancy and 25% a solid tumour. Half of the cohort underwent allogenic HSCT and most (89%) conditioned with busulphan. The mean change in height SDS from primary cancer diagnosis to FAH was -1.21 (±1.18 SD), equivalent to 7-8.5 cm loss, with a mean FAH of -0.91 SDS (±1.10 SD). The greatest height loss occurred between diagnosis and HSCT (-0.77 SDS, 95% confidence interval -1.42, -0.12, P = 0.01), with no catch-up growth seen by FAH. Patients with solid tumours had the greatest height loss. Overall body mass index SDS did not change significantly over time, or by cancer type. CONCLUSIONS: Chemotherapy-only conditioned HSCT during childhood can impact FAH, with the greatest height loss occurring prior to HSCT and no catch-up growth after treatment finishes. Children transplanted for a solid tumour malignancy seem to be more at risk, possibly due to intensive treatment regimens, both pre-transplant and during conditioning.

Photoreceptor cell rescue in retinal degeneration (rd) mice by in vivo gene therapy.

Mutations in the beta subunit of the cGMP phosphodiesterase gene (beta PDE) can cause a recessively inherited retinal degeneration in several species, including mice, dogs and humans. We tested the possibility of altering the course of retinal degeneration in the rd mouse through subretinal injection of a recombinant replication-defective adenovirus that contains the murine cDNA for wild-type (beta PDE, Ad.CMV beta PDE. Subretinal injection of Ad.CMV beta PDE results in beta PDE transcripts and increased PDE activity and delays photoreceptor cell death by six weeks. The findings demonstrate cell rescue by in vivo gene transfer, thus supporting the feasibility of treating an inherited retinal degeneration by somatic gene therapy.

Additional transduction events after subretinal readministration of recombinant adeno-associated virus.

Subretinal delivery of recombinant adeno-associated virus (rAAV) results in a systemic humoral response in the adult immunocompetent mouse. We characterized this response and determined whether it is possible to readminister rAAV to the subretinal space despite the presence of antibodies to the vector. A systemic humoral response to rAAV capsid proteins was induced by either unilateral subretinal injection or by intradermal administration of 1 x 10(9) infectious units of rAAV carrying the cDNA encoding green fluorescent protein (GFP), rAAV-GFP. Experiments were performed in cohorts of adult C57BL/6 mice. Assessment of systemic humoral response to viral capsid protein was performed through enzyme-linked immunoabsorbent assay (ELISA) and infection inhibition studies of serum samples 3 weeks after virus delivery. The rAAV-GFP virus was readministered by subretinal injection. GFP expression after subretinal administration was evaluated ophthalmoscopically throughout the course of the experiment and histologically at the termination of the experiment. We observed significant systemic humoral responses to viral capsid protein after subretinal delivery of rAAV. Intradermal injection resulted in a larger humoral response (with a higher percentage of neutralizing antibodies) than subretinal injection. Additional transduction events were observed after readministration of rAAV despite the presence of strong humoral response to the vector.

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. The efficiency of this system to date has been disappointing, and therefore its utility for therapeutic application limited. We demonstrate here that efficiency and cellular specificity of trans-splicing is dependent on selection of the appropriate AAV serotype. Efficiency of transgene expression resulting from trans-splicing in skeletal muscle approaches that obtained when delivering the intact transgene when using AAV2 vectors packaged with AAV5 capsids (AAV2/5). This expands the potential of AAV vectors for retinal gene therapy. The use of AAV2/5 also increases the efficiency of trans-splicing in photoreceptors. Selection of the appropriate AAV serotype is likely to affect efficiency of trans-splicing in other organ systems as well.

Adenovirus-mediated delivery of catalase to retinal pigment epithelial cells protects neighboring photoreceptors from photo-oxidative stress.

Oxidative stress is involved in the pathogenesis of many diseases. Overexpression of antioxidant enzymes by gene therapy may protect tissues from oxidative damage. Because the reactive oxygen species hydrogen peroxide can diffuse across cell membranes, we hypothesized that overexpression of the antioxidant catalase within certain cells might protect neighboring cells. To test this hypothesis, we transduced retinal pigment epithelial (RPE) cells in vitro and in vivo with adenovirus carrying the catalase gene (Ad.CMV.catalase). After transduction of only a subset of RPE cells in vitro, all cells in the culture were protected from exogenous hydrogen peroxide. Similarly, in vivo, eyes injected with Ad. CMV. catalase had high catalase levels in the RPE, which protected the adjacent photoreceptors from light damage and reduced photoreceptor oxidative stress as measured by the markers 4-hydroxynonenal and nitrotyrosine. Both in vitro and in vivo, gene therapy with Ad. CMV. catalase protected neighboring cells from oxidative stress-induced terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) positivity. The data provide a paradigm for antioxidant gene therapy with catalase, designed to protect not only transduced cells, but also neighboring cells.

Cell-mediated immune response and stability of intraocular transgene expression after adenovirus-mediated delivery.

PURPOSE: To evaluate the role of cell-mediated immunity in the stability of ocular adenovirus-mediated transgene expression. METHODS: Adenovirus (4 x 10(6) pfu) containing lacZ (Ad.CMVlacZ) was injected intravitreally or subretinally into one or both eyes of immunocompetent (+/+) and immunocompromised (nu/nu) CD-1 mice. Control eyes received injections of saline. Additional +/+ mice received subretinal injections of Ad.CMVlacZ with coadministration of 200 microg of human immunoglobulin (Ig) G or CTLA4Ig by intraperitoneal, intravitreal, or subretinal injection. The mice were killed at various times after injection, and their eyes were examined histologically and immunohistochemically. RESULTS: LacZ expression was extended from 1 week to more than 5 weeks in the corneal endothelium, iris, and trabecular meshwork of nu/nu mice compared with time of expression in +/+ mice when adenovirus was administered intravitreally. In contrast, subretinal injection resulted in only a minimal increase in transgene stability in nu/nu mice compared with that in +/+ mice. Neither systemic nor intraocular administration of IgG or CTLA4Ig affected the stability of lacZ expression in the retina or retinal pigment epithelium after subretinal injection in +/+ mice. Immunoglobulin G and CTLA4Ig enhanced the stability of transgene expression in the trabecular meshwork. CONCLUSIONS: A T-cell-mediated immune response appears to play a role in the loss of adenovirus-mediated lacZ expression after intravitreal but not after subretinal injection. This result implies that the subretinal space is an immune-privileged site and a favorable site for gene transfer.

Real-time, noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction.

PURPOSE: To evaluate the efficiency, cell specificity, stability, and toxicity of recombinant adeno-associated virus (rAAV)-mediated retinal transduction in vivo in the adult immunocompetent mouse. To assess the usefulness of green fluorescent protein (GFP) for real-time, noninvasive monitoring of retinal transgene expression in vivo. METHODS: Assessment of ocular GFP expression was performed in cohorts of mice for 11 weeks after subretinal injection of a recombinant adeno-associated virus carrying the complementary DNA (cDNA) for GFP. Examinations were performed in vivo by direct observation of fluorescence by ophthalmoscopy, using excitation-barrier filters. Histologic analyses of retinal tissue were used to identify transduced cells and to assess inflammation. RESULTS: Retinal GFP expression can be monitored in vivo using real-time, noninvasive imaging. Recombinant AAV efficiently transduces a variety of cells of the neural retina and of the retinal pigment epithelium (RPE). Transgene expression was not observed until 1 week after infection. The number of GFP-expressing cells increased over 3 weeks, and expressing photoreceptors and RPE, cells persisted at least through 11 weeks (the termination of the experiment). There was no clinical or histologic evidence of inflammatory response. CONCLUSIONS: Retinal gene transfer mediated by rAAV is stable and efficient and is associated with no clinically or histologically detectable toxicity or immune reaction. Green fluorescent protein allows noninvasive assessment of the extent and location of retinal transgene expression as a function of time and promises to be useful alone and as a tag for other transgenes delivered experimentally or therapeutically to the retina.

Foveolar choroidal blood flow in age-related macular degeneration.

PURPOSE: To compare measurements of the foveolar choroidal blood circulation in subjects with nonexudative, age-related macular degeneration (AMD) with those of control subjects. METHODS: Laser Doppler flowmetry was used to assess relative choroidal blood velocity (ChBVel), volume (ChBVol), and flow (ChBFlow) in the center of the fovea. Measurements were obtained in 20 eyes of 20 subjects with 10 or more large drusen, visual acuity of 20/32 or better, and no evidence of choroidal neovascularization. Findings obtained in these subjects were compared with those of 10 eyes of 10 age- and blood pressure-matched control subjects with no large drusen. Foveolar choroidal blood flow measurements were obtained by asking the study participants to fixate on a probing laser beam. RESULTS: No significant differences in average age, blood pressure, or intraocular pressure were observed between subjects with AMD and control subjects. In subjects with AMD, average ChBVol was 0.24 +/- 0.08 (+/- 1 SD) arbitrary units (AU); this value was 33% lower than that of control subjects (0.36 +/- 0.11 AU; two-tailed, independent Student's t-test, P = 0.005). Average ChBVel, conversely, was not significantly different from normal (0.44 +/- 0.07 AU) in subjects with AMD (0.44 +/- 0.10 AU). Average ChBFlow in subjects with AMD (8.7 +/- 3.1 AU) was 37% lower than that of control subjects (13.7 +/- 3.5 AU) (P = 0.0005). Average blood flow pulsatility was 6% higher in subjects with AMD (0.71 +/- 0.15) than in control subjects (0.66 +/- 0.14), but this difference was not statistically significant (P = 0.42). CONCLUSIONS: Average ChBFlow in the nonexudative stages of AMD is lower than that of age-matched controls, and the effect is caused mainly by a decrease in ChBVol. Further studies are needed to elucidate whether decreased ChBFlow plays a role in the development of choroidal neovascularization, and whether ChBFlow measurements may help identify subjects with AMD at risk for developing choroidal neovascularization.

Disease expression of RP1 mutations causing autosomal dominant retinitis pigmentosa.

PURPOSE: To determine the disease expression in heterozygotes for mutations in the RP1 gene, a newly identified cause of autosomal dominant retinitis pigmentosa (adRP). METHODS: Screening strategies were used to detect disease-causing mutations in the RP1 gene, and detailed studies of phenotype were performed in a subset of the detected RP1 heterozygotes using electroretinography (ERG), psychophysics, and optical coherence tomography (OCT). RESULTS: Seventeen adRP families had heterozygous RP1 changes. Thirteen families had the Arg677ter mutation, whereas four others had one of the following: Pro658 (1-bp del), Ser747 (1-bp del), Leu762-763 (5-bp del), and Tyr1053 (1-bp del). In Arg677ter RP1 heterozygotes, there was regional retinal variation in disease, with the far peripheral inferonasal retina being most vulnerable; central and superior temporal retinal regions were better preserved. The earliest manifestation of disease was rod dysfunction, detectable as reduced rod ERG photoresponse maximum amplitude, even in heterozygotes with otherwise normal clinical, functional, and OCT cross-sectional retinal imaging results. At disease stages when cone abnormalities were present, there was greater rod than cone dysfunction. Patients with the RP1 frameshift mutations showed similarities in phenotype to those with the Arg677ter mutation. CONCLUSIONS: Earliest disease expression of RP1 gene mutations causing adRP involves primarily rod photoreceptors, and there is a gradient of vulnerability of retinopathy with more pronounced effects in the inferonasal peripheral retina. At other disease stages, cone function is also affected, and severe retina-wide degeneration can occur. The nonpenetrance or minimal disease expression in some Arg677ter mutation-positive heterozygotes suggests important roles for modifier genes or environmental factors in RP1-related disease.

Lens-sparing vitreoretinal surgery in infants.

Lensectomy is routinely performed in infant eyes undergoing vitreoretinal surgery. Using a two-port system, we performed pars plicata vitrectomy and membrane peeling without removal of the crystalline lens. Surgical manipulations were done in the postequatorial region in eyes with areas of attached peripheral retina. Eight of 10 eyes showed no evidence of postoperative cataract formation. In no instance was a peripheral retinal break created. In selected cases, vitreoretinal surgery can be performed in infant eyes without the need for lens removal.

Scleral loop fixation for posteriorly dislocated intraocular lenses. Operative technique and long-term results.

Surgical management of a posteriorly dislocated intraocular lens requires vitrectomy techniques to remove adherent vitreous and prevent traction on the retina. The lens implant may be repositioned in the posterior chamber by ciliary sulcus fixation or by transscleral or iris suture fixation. Previous suture fixation techniques require multiple needle penetrations through the sclera, cornea, or both. We described a simplified technique for repositioning a posteriorly dislocated intraocular lens in which sclerotomy incisions are utilized for fixation. Six patients are described, with long-term follow-up demonstrating stable anatomic status and favorable visual outcome.

Retinal pigment epithelial tears through the fovea with preservation of good visual acuity.

We describe two patients with spontaneous retinal pigment epithelial tears through the fovea who have maintained at least 20/40 visual acuity for 1 year and 3 years following the rip. Both patients had long-standing serous detachments of the retinal pigment epithelium associated with age-related macular degeneration prior to the development of the tear. Each tear was at least five disc areas in size and centered on the fovea. Foveal fixation was documented despite the presumed absence of pigment epithelium. This observation suggests either that there may be remaining or redundant pigment epithelium or that pigment epithelium directly beneath the central macula is not required for maintenance of 20/40 visual acuity.

Cancer-associated retinopathy.

Patients with systemic cancer may have a variety of ocular complaints. Most commonly these are metastases or adverse effects of therapy. Paraneoplastic syndromes, like cancer-associated retinopathy, rarely cause ophthalmic symptoms. We describe a patient with a malignant mixed mullerian tumor and cancer-associated retinopathy who had circulating serum antibodies to recoverin and cells positive for recoverin in the tumor. We discuss the typical clinical symptoms as well as the pathophysiology of this uncommon disorder.

Hypopyon uveitis in patients with acquired immunodeficiency syndrome treated for systemic Mycobacterium avium complex infection with rifabutin.

OBJECTIVE: Iridocyclitis has been identified as a dosage-dependent side effect in patients with the acquired immunodeficiency syndrome (AIDS) who are treated for Mycobacterium avium complex (MAC) infection with systemic rifabutin. We reviewed cases of acute hypopyon uveitis occurring in patients with AIDS to establish whether there was an association. DESIGN: Retrospective case series. SETTING: Outpatient clinic and inpatient hospital-based ophthalmology referral practice and infectious disease specialty service. PATIENTS: Seven patients with AIDS, aged 10 to 40 years, presenting with acute unilateral hypopyon mimicking infectious endophthalmitis. MAIN OUTCOME MEASURES: Findings from complete ophthalmological evaluation and ancillary laboratory testing. RESULTS: At the time of presentation, all seven patients were receiving treatment for MAC infection with rifabutin (dosage range, 300 to 600 mg/d) and clarithromycin. Results of microbiological investigations in five patients were negative. Iridocyclitis became bilateral in all seven patients, and hypopyon developed in the contralateral eye in five of seven patients. Hypopyon resolved rapidly with intensive topical corticosteroid therapy. Residual inflammation responded to topical corticosteroids with or without reduction of the rifabutin dosage. CONCLUSIONS: Concomitant use of rifabutin, clarithromycin, and fluconazole may precipitate hypopyon uveitis in patients with AIDS being treated for MAC infection.

Retinal hemodynamics in retinitis pigmentosa.

PURPOSE: To investigate the retinal hemodynamic changes occurring in patients with retinitis pigmentosa (RP). METHODS: Bidirectional laser Doppler velocimetry and monochromatic fundus photography were used to determine retinal venous diameter (D), maximum erythrocyte velocity (Vmax), and volumetric blood flow (Q) in the major retinal veins of eight patients with RP and eight age-matched normal controls. The retinal vascular regulatory responses to hyperoxia, defined as the percent decreases in D (RD), Vmax (RVmax), and Q (RQ) at four to six minutes of breathing 100% oxygen, were determined in eight normal subjects and five RP patients. RESULTS: Average D, Vmax, and Q +/- S.D. in the largest retinal vein of each subject were 106 +/- 14 microns, 1.01 +/- 0.20 cm/sec, and 3.5 +/- 1.3 microliters/min, respectively, in RP patients, and 166 +/- 12 microns, 1.79 +/- 0.14 cm/sec, and 14.7 +/- 2.6 microliters/min, respectively, in normal subjects. This corresponded to significant decreases from normal of 36% in D, 44% in Vmax, and 76% in Q in RP patients (Wilcoxon's rank sum test, P < .001). Average total retinal volumetric blood flow rate was 8.2 +/- 2.9 microliters/min in RP patients and 37 +/- 4.9 microliters/min in normal subjects, corresponding to a significant decrease from normal of 78% (Wilcoxon's rank sum test, P < .001). In RP patients, the regulatory responses to hyperoxia (RD, RVmax, and RQ) were similar to those observed in normal subjects. RESULTS: Retinal blood flow is significantly decreased in patients with RP, probably as a result of vascular remodeling in response to reduced metabolic demand. The regulatory responses to hyperoxia are similar to those of normal subjects. Measurements of retinal blood flow may help assess the progression of the disease and the effects of treatment.

Perilimbal anesthesia for pars plana vitrectomy.

We attempted to determine the efficacy of perilimbal anesthesia with Monitored Anesthesia Care (intravenous sedation with monitoring of the patients' vital signs by anesthesia) in patients undergoing pars plana vitrectomy. We selected 15 patients who were screened for any physical condition that would preclude them from lying supine and motionless for the duration of the surgery. Patients with complicated vitreoretinal abnormalities and patients who required reoperations were excluded. The patients ranged in age from 55 to 79 years and included three with nonclearing vitreous hemorrhage, seven with macular hole, three with macular pucker, one with proliferative diabetic retinopathy, and one with endophthalmitis. Total operating time ranged from 45 to 135 minutes (mean, 102.3 +/- 22.0 minutes). No complaints of discomfort were reported. All operations were completed without additional anesthetic. Our findings demonstrate that pars plana vitrectomy using perilimbal anesthesia with monitored anesthesia care is effective in selected patients. This form of anesthesia administration eliminates the risks of inadvertent globe perforation associated with retrobulbar and perilimbal anesthesia and the risks of general anesthesia.

Rapidly progressive optic disk neovascularization after diabetic papillopathy.

PURPOSE: To determine if diabetic papillopathy may be associated with optic disk neovascularization and visual loss. METHODS: We examined two young adults with bilateral diabetic papillopathy who had permanent loss of vision associated with rapidly progressive posterior segment neovascularization. RESULTS: Within three months both patients developed florid optic disk neovascularization. This neovascularization was slow to regress despite full panretinal photocoagulation. One patient developed a macular tractional retinal detachment. CONCLUSIONS: Diabetic papillopathy may be associated with rapid progression of diabetic retinopathy and, in particular, optic disk neovascularization. Patients should be monitored for this possibility.

One-year outcomes of panretinal photocoagulation in proliferative diabetic retinopathy.

PURPOSE: To describe the clinical features and complications of diabetic retinopathy, visual acuity, and number of repeat treatments after panretinal photocoagulation for proliferative diabetic retinopathy in a tertiary care center. METHODS: A cohort study was conducted with data collection from medical records of patients undergoing panretinal photocoagulation between 1985 and 1995 at the Scheie Eye Institute; 297 eyes of 186 patients were eligible for study. RESULTS: The presence of neovascularization of the disk at baseline, an earlier onset of diabetes, and a shorter duration of disease before panretinal photocoagulation were the strongest risk factors for needing an additional panretinal photocoagulation treatment. Sixty-two percent of eyes with poor visual acuity (< or =20/200) at baseline still had poor visual acuity at 1 year, and 76% with good visual acuity (> or =20/40) at baseline maintained good visual acuity at 1 year. Poor vision at baseline was the only risk factor for having poor vision at 1 year. Vitreous hemorrhage was present in 44% of eyes at baseline. New vitreous hemorrhage developed in 37% of eyes during the first year after panretinal photocoagulation. A traction retinal detachment was present in 4% of eyes at baseline and newly developed in 6% of eyes during follow-up. A repeat panretinal photocoagulation treatment was performed in 39% of eyes after initial treatment. A vitrectomy was performed in 10% of eyes from baseline through the 1-year follow-up visit. CONCLUSIONS: The data from this study are useful for counseling patients with respect to likely visual outcome, possibility of major complications from proliferative diabetic retinopathy, and the chance of undergoing additional laser treatment after panretinal photocoagulation.