AAV cis-regulatory sequences are correlated with ocular toxicity.

Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1ß were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.

Animal Shelters' Response to the COVID-19 Pandemic: A Pilot Survey of 14 Shelters in the Northeastern United States.

Anecdotal reports indicate that many animal shelters experienced increased adoption and foster care rates during the COVID-19 pandemic, yet peer-reviewed evidence is lacking. In this pilot survey of 14 animal shelters in the Northeastern United States, we aimed to investigate the impact of the COVID-19 pandemic on animal intakes, foster care and five outcome types and describe operational changes reported by shelters in response to COVID-19. Paired sample t-tests and Wilcoxon signed-rank tests were used to compare intake, adoption, euthanasia and foster care rates and numbers between March-June 2019 and 2020. The number of dogs and cats that entered shelters was significantly lower during the COVID-19 pandemic compared with the same months of 2019 (t = 3.41, p = 0.01, t = 2.69, p = 0.02). Although the overall rate of adoption and euthanasia did not differ, the numbers adopted and euthanized decreased significantly for both dogs and cats, reflecting the significantly decreased intake. We also found significant variability between shelters. During the pandemic, several shelters sought to expand their foster care networks through operational changes (n = 6) and statements made to the public (n = 7). However, the proportion of dogs and cats housed in foster care did not differ between March-June 2019 and 2020 in our sample. Our findings offer preliminary insights regarding the impact of a worldwide pandemic on the functioning of animal shelters.

Nrf2 overexpression rescues the RPE in mouse models of retinitis pigmentosa.

Nrf2, a transcription factor that regulates the response to oxidative stress, has been shown to rescue cone photoreceptors and slow vision loss in mouse models of retinal degeneration (rd). The retinal pigment epithelium (RPE) is damaged in these models, but whether it also could be rescued by Nrf2 has not been previously examined. We used an adeno-associated virus (AAV) with an RPE-specific (Best1) promoter to overexpress Nrf2 in the RPE of rd mice. Control rd mice showed disruption of the regular array of the RPE, as well as loss of RPE cells. Cones were lost in circumscribed regions within the cone photoreceptor layer. Overexpression of Nrf2 specifically in the RPE was sufficient to rescue the RPE, as well as the disruptions in the cone photoreceptor layer. Electron microscopy showed compromised apical microvilli in control rd mice but showed preserved microvilli in Best1-Nrf2-treated mice. The rd mice treated with Best1-Nrf2 had slightly better visual acuity. Transcriptome profiling showed that Nrf2 upregulates multiple oxidative defense pathways, reversing declines seen in the glutathione pathway in control rd mice. In summary, Nrf2 overexpression in the RPE preserves RPE morphology and survival in rd mice, and it is a potential therapeutic for diseases involving RPE degeneration, including age-related macular degeneration (AMD).