Previous studies have tested gene replacement therapy in RPE65-deficient dogs using recombinant adeno-associated virus 2/2 (rAAV2/2), -2/1 or -2/5 mediated delivery of the RPE65 gene. They all documented restoration of dark-and light-adapted electroretinography responses and improved psychophysical outcomes. Use of a specific RPE65 promoter and a rAAV vector that targets transgene expression specifically to the RPE may, however, provide a safer setting for the long-term therapeutic expression of RPE65. Subretinal injection of rAAV2 pseudotyped with serotype 4 (rAAV2/4) specifically targets the RPE. The purpose of our study was to evaluate a rAAV2/4 vector carrying a human RPE65cDNA driven by a human RPE65 promoter, for the ability to restore vision in RPE65 À/À purebred Briard dogs and to assess the safety of gene transfer with respect to retinal morphology and function. rAAV2/4 and rAAV2/2 vectors containing similar human RPE65 promoter and cDNA cassettes were generated and administered subretinally in eight affected dogs, ages 8-30 months (n ¼ 6 with rAAV2/4, n ¼ 2 with rAAV2/2). Although fluorescein angiography and optical coherence tomography examinations displayed retinal abnormalities in treated retinas, electrophysiological analysis demonstrated that restoration of rod and cone photoreceptor function started as soon as 15 days post-injection, reaching maximal function at 3 months postinjection, and remaining stable thereafter in all animals treated at 8-11 months of age. As assessed by the ability of these animals to avoid obstacles in both dim and normal light, functional vision was restored in the treated eye, whereas the untreated contralateral eye served as an internal control. The dog treated at a later age (30 months) did not recover retinal function or vision, suggesting that there might be a therapeutic window for the successful treatment of RPE65 À/À dogs by gene replacement therapy.

Use of the in-practice CARD test allows identification of type A- and type B-positive cats, but weak reactions of type AB blood with the anti-A monoclonal antibody raise concerns. The modified GEL and TUBE tests appear to be reliable clinical laboratory methods for feline blood typing.

Use of the CARD test allows for rapid identification of DEA 1.1 but may produce weak reactions with blood from DEA 1.2-positive dogs. The GEL test is a reliable and rapid clinical laboratory method for identification of DEA 1.1. The MSU test requires Coombs' reagent for identification of DEA 1.1 and 1.2.

Prof. Hamel is credited as one of the discoverers of the RPE65 gene, a cause of a rare form of genetic blindness, which laid the groundwork for the understanding of this gene and the subsequent gene therapy studies that have led to a potential gene-based treatment for these patients. Prof. Hamel was a consummate scientist, clinician, and champion for those with inherited retinal disease and was dedicated to their care and to the discovery of potential treatments.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) technology enables rapid and precise genome editing at any desired genomic position in almost all cells and organisms. In this study, we analyzed the impact of different repair templates on the frequency of homology-directed repair (HDR) and non-homologous end joining (NHEJ). We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to quantify HDR and NHEJ events following transfection with Cas9, eight different guide RNAs, and a 1,000 bp donor template generated either as circular plasmid, as linearized plasmid with long 3′ or 5′ backbone overhang, or as PCR product. The sequence to be corrected was either centrally located (RS55), with a shorter 5′ homologous region (RS37), or with a shorter 3′ homologous region (RS73). Guide RNAs targeting the transcriptionally active strand (T5, T7) showed significantly higher NHEJ frequencies compared with guide RNAs targeting the transcriptionally inactive strand. HDR activity was highest when using the linearized plasmid with the short 5′ backbone overhang and the RS37 design. The results demonstrate the importance of the design of the guide RNA and template DNA on the frequency of DNA repair events and, ultimately, on the outcome of treatment approaches using HDR.