Relationship Between Neutralizing Antibodies Against Adeno-Associated Virus in the Vitreous and Serum: Effects on Retinal Gene Therapy.

PURPOSE: We determine the prevalence of neutralizing antibodies (NAbs) to adeno-associated virus (AAV) in the vitreous humor and serum of patients with vitreoretinal diseases and investigate the relationship between NAb titers in the vitreous humor and serum. METHODS: We analyzed NAbs to AAV serotypes 2, 5, 8, and 9 via in vitro neutralization in the vitreous humor and serum from 32 patients requiring vitrectomy for vitreoretinal diseases. The blood-retinal barrier (BRB) was evaluated for integrity based on preoperative examinations, with vitreous hemorrhage (VH) on funduscopy or dye leakage on fluorescein angiography observed indicating disruption. RESULTS: NAb levels were much lower in the vitreous humor than in the serum regardless of serotype. Patients with VH had higher levels of NAbs in the vitreous humor than those without VH. The NAb ratio (ratio between NAb titers in the serum and vitreous humor) was much lower in patients with epiretinal membrane with than in those without leakage. A significantly lower NAb ratio was noticed in patients with than in those without BRB disruptions. CONCLUSIONS: The NAb ratio between levels in serum and vitreous humor varies according to the condition of the BRB. Therefore, in addition to measuring the serum NAb level, physicians should examine BRB integrity when planning retinal gene therapy. TRANSLATIONAL RELEVANCE: This study provides substantial basis for retinal gene therapy using AAVs and how maintenance of BRB integrity in target diseases should be considered.

Development of a Pde6b Gene Knockout Rat Model for Studies of Degenerative Retinal Diseases.

Purpose: To describe the phenotypes of a newly developed Pde6b-deficient rat model of retinal degeneration. Methods: Pde6b knockout rats were produced by CRISPR-Cpf1 technology. Pde6b knockout rats were evaluated for ocular abnormalities by comparison with wild-type eyes. Eyes were imaged using fundus photography and optical coherence tomography (OCT), stained by hematoxylin and eosin (H&E), and examined by TUNEL assay. Finally, eyes were functionally assessed by electroretinograms (ERGs). Results: Pde6b knockout rats exhibited visible photoreceptor degeneration at 3 weeks of postnatal age. The fundus appearance of mutants was notable for pigmentary changes, vascular attenuation with an irregular vascular pattern, and outer retinal thinning, which resembled retinitis pigmentosa (RP) in humans. OCT showed profound retinal thinning in Pde6b knockout rats; the outer nuclear layer (ONL) was significantly thinner in Pde6b knockout rats, with relative preservation of the inner retina at 3 weeks of postnatal age. H&E staining confirmed extensive degeneration of the ONL, beginning at 3 weeks of postnatal age; no ONL remained in the retina by 16 weeks of postnatal age. Retinal sections of Pde6b knockout rats were highly positive for TUNEL, specifically in the ONL. In ERGs, Pde6b knockout rats showed no detectable a- or b-waves at 8 weeks of postnatal age. Conclusions: The Pde6b knockout rat exhibits photoreceptor degeneration. It may provide a better model for experimental therapy for RP because of its slower progression and larger anatomic architecture than the corresponding mouse model. Further studies in this rat model may yield insights into effective therapies for human RP.

In Vivo Fluorescence Retinal Imaging Following AAV2-Mediated Gene Delivery in the Rat Retina.

PURPOSE: The purpose of this study was to evaluate longitudinal gene expression patterns by retinal imaging using a modified custom-built confocal laser-scanning microscope in experimental rats after intravitreal injection of recombinant adeno-associated virus 2 (rAAV2-green fluorescent protein [GFP]). METHODS: Ten 9-week-old Wistar rats were divided into two groups: experimental group (group 1) that received a rAAV2-GFP intravitreal injection and control group (group 2) that received a vehicle. After anesthesia using a Zoletil intraperitoneal injection, 8 µL rAAV2-GFP in group 1 or vehicle in group 2 was injected intravitreally using a 33-G Hamilton syringe. In vivo fluorescence retinal images were acquired under anesthesia at 2, 4, 6, and 13 days after rAAV or vehicle delivery. RESULTS: Differences in GFP fluorescence were identified starting from day 2 after the intravitreal injection of rAAV2-GFP in group 1. Between days 4 and 6, the intensity and area of fluorescence in the retina began to increase and peaked at day 13. Based on the pattern of GFP expression, the axon of the nerve fiber layer ganglion cell was identified. In group 2, eyes treated with the vehicle showed a small amount of autofluorescence in a limited area for up to 2 weeks, with no increase in intensity during this period. CONCLUSIONS: In vivo retinal imaging confirmed gene expression within 2 weeks after the intravitreal injection of rAAV2-GFP. Gene transfer and expression in the rat retina occurs quickly in 2 days and appears to peak within 2 weeks of gene delivery. In vivo retinal imaging may be a useful noninvasive tool to continuously monitor gene expression in the retina over time.

Prenatal maternal distress affects atopic dermatitis in offspring mediated by oxidative stress.

BACKGROUND: Recent evidence suggests that prenatal maternal distress increases the risk of allergic diseases in offspring. However, the effect of prenatal maternal depression and anxiety on atopic dermatitis (AD) risk remains poorly understood. OBJECTIVE: We investigated whether prenatal maternal distress is associated with AD risk in offspring and whether the mechanism is mediated by reactive oxygen species. METHODS: Two general population-based birth cohorts formed the study. One cohort (Cohort for Childhood Origin of Asthma and Allergic Diseases [COCOA]) consisted of 973 mother-baby dyads, and the other (Panel Study on Korean Children [PSKC]) consisted of 1531 mother-baby dyads. The association between prenatal distress and AD was assessed by using Cox proportional hazards and logistic regression models. In COCOA placental 11ß-hydroxysteroid dehydrogenase type 2 and glutathione levels and serum IgE levels in 1-year-old children were measured. RESULTS: In COCOA and PSKC AD occurred in 30.6% (lifetime prevalence) and 11.6% (1 year prevalence) of offspring, respectively. Prenatal maternal distress increased the risk of AD in offspring, both in COCOA (hazard ratio for depression, 1.31 [95% CI, 1.02-1.69]; hazard ratio for anxiety, 1.41 [95% CI, 1.06-1.89]) and PSKC (odds ratio for distress, 1.85 [95% CI, 1.06-3.25]). In COCOA both prenatal maternal depression and anxiety scores were positively related to the predicted probability of AD (P < .001 in both). Prenatal distress decreased placental glutathione to glutathione disulfide ratios (P = .037) and, especially in those who later had AD, decreased placental 11ß-hydroxysteroid dehydrogenase type 2 levels (P = .010) and increased IgE levels at 1 year of age (P = .005). CONCLUSION: Prenatal maternal depression and anxiety promote risk of AD in offspring. Maternal distress increases the predicted probability of AD. The mechanism might involve chronic stress, abnormal steroid levels, and reactive oxygen species.