Development of a protocol for maintaining viability while shipping organoid-derived retinal tissue.

Retinal organoid technology enables generation of an inexhaustible supply of three-dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid-derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage-reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation-competent) hPSC-derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid-derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration

Cat LCA-CRX model, homozygous for an antimorphic mutation has a unique phenotype

PURPOSE: Mutations in the CRX transcription factor are associated with dominant retinopathies often with more severe macular changes. The CRX-mutant cat (Rdy-A182d2) is the only animal model with the equivalent of the critical retinal region for high-acuity vision, the macula. Heterozygous cats (CRXRdy/+) have a severe phenotype modeling Leber congenital amaurosis. This study reports the distinct ocular phenotype of homozygous cats (CRXRdy/Rdy). METHODS: Gene expression changes were assessed at both mRNA and protein levels. Changes in globe morphology and retinal structure were analyzed. RESULTS: CRXRdy/Rdy cats had high levels of mutant CRX mRNA and protein. The expression of photoreceptor target genes was severely impaired although there were variable effects on the expression of other transcription factors. The photoreceptor cells remained immature and failed to elaborate outer segments consistent with the lack of retinal function. The retinal layers displayed a progressive remodeling with cell loss but maintained overall retinal thickness due to gliosis. Rapid photoreceptor loss largely occurred in the macula-equivalent retinal region. The homozygous cats developed markedly increased ocular globe length. CONCLUSIONS: The phenotype of CRXRdy/Rdy cats was more severe compared to CRXRdy/+ cats by several metrics. TRANSLATIONAL RELEVANCE: The CRX-mutant cat is the only model for CRX-retinopathies with a macula-equivalent region. A prominent feature of the CRXRdy/Rdy cat phenotype not detectable in homozygous mouse models was the rapid degeneration of the macula-equivalent retinal region highlighting the value of this large animal model and its future importance in the testing of translational therapies aiming to restore vision

Re-assessing the diversity of negative strand RNA viruses in insects.

The spectrum of viruses in insects is important for subjects as diverse as public health, veterinary medicine, food production, and biodiversity conservation. The traditional interest in vector-borne diseases of humans and livestock has drawn the attention of virus studies to hematophagous insect species. However, these represent only a tiny fraction of the broad diversity of Hexapoda, the most speciose group of animals. Here, we systematically probed the diversity of negative strand RNA viruses in the largest and most representative collection of insect transcriptomes from samples representing all 34 extant orders of Hexapoda and 3 orders of Entognatha, as well as outgroups, altogether representing 1243 species. Based on profile hidden Markov models we detected 488 viral RNA-directed RNA polymerase (RdRp) sequences with similarity to negative strand RNA viruses. These were identified in members of 324 arthropod species. Selection for length, quality, and uniqueness left 234 sequences for analyses, showing similarity to genomes of viruses classified in Bunyavirales (n = 86), Articulavirales (n = 54), and several orders within Haploviricotina (n = 94). Coding-complete genomes or nearly-complete subgenomic assemblies were obtained in 61 cases. Based on phylogenetic topology and the availability of coding-complete genomes we estimate that at least 20 novel viral genera in seven families need to be defined, only two of them monospecific. Seven additional viral clades emerge when adding sequences from the present study to formerly monospecific lineages, potentially requiring up to seven additional genera. One long sequence may indicate a novel family. For segmented viruses, cophylogenies between genome segments were generally improved by the inclusion of viruses from the present study, suggesting that in silico misassembly of segmented genomes is rare or absent. Contrary to previous assessments, significant virus-host codivergence was identified in major phylogenetic lineages based on two different approaches of codivergence analysis in a hypotheses testing framework. In spite of these additions to the known spectrum of viruses in insects, we caution that basing taxonomic decisions on genome information alone is challenging due to technical uncertainties, such as the inability to prove integrity of complete genome assemblies of segmented viruses

Sudden acquired retinal degeneration syndrome (SARDS) â a review and proposed strategies toward a better understanding of pathogenesis, early diagnosis, and therapy

Sudden acquired retinal degeneration syndrome (SARDS) is one of the leading causes of currently incurable canine vision loss diagnosed by veterinary ophthalmologists. The disease is characterized by acute onset of blindness due to loss of photoreceptor function, extinguished electroretinogram with an initially normal appearing ocular fundus, and mydriatic pupils which are slowly responsive to bright white light, unresponsive to red, but responsive to blue light stimulation. In addition to blindness, the majority of affected dogs also show systemic abnormalities suggestive of hyperadrenocorticism, such as polyphagia with resulting obesity, polyuria, polydipsia, and a subclinical hepatopathy. The pathogenesis of SARDS is unknown, but neuroendocrine and autoimmune mechanisms have been suggested. Therapies that target these disease pathways have been proposed to reverse or prevent further vision loss in SARDSâ affected dogs, but these treatments are controversial. In November 2014, the American College of Veterinary Ophthalmologists' Vision for Animals Foundation organized and funded a Think Tank to review the current knowledge and recently proposed ideas about disease mechanisms and treatment of SARDS. These panel discussions resulted in recommendations for future research strategies toward a better understanding of pathogenesis, early diagnosis, and potential therapy for this condition.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/122446/1/vop12291.pd

Knowledge acquisition for the internationalization of the smaller firm: content and sources

Internationalization process research emphasizes accumulated experience and networks as sources of knowledge for internationalization. Our understanding, however, as to what this knowledge is in practice for smaller firms, the challenges they face in acquiring it, and how they address those challenges is limited. Integrating organizational learning concepts with our theoretical understanding of the small firm internationalization process, we develop a new framework for understanding knowledge acquisition processes, which are examined with a case study of 10 Scottish internationalizing firms. We find smaller firms may not have relevant experience or useful networks, and rely on sources rarely recognised before. Firms used recruitment, government advisors and consultants to acquire indirect experience. Recruitment is a source of market and technological knowledge and government advisors and consultants a source of internationalization knowledge. Accessing internal information is important for firms that have internationalized. Our integrated theoretical framework identifies knowledge content and sources that are critical for internationalization, but that may be absent

Patients and animal models of CNGβ1-deficient retinitis pigmentosa support gene augmentation approach.

Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel β 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGβ1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials

Effect of Gene Therapy on Visual Function in Leber's Congenital Amaurosis

Early-onset, severe retinal dystrophy caused by mutations in the gene encoding retinal pigment epithelium–specific 65-kD protein (RPE65) is associated with poor vision at birth and complete loss of vision in early adulthood. We administered to three young adult patients subretinal injections of recombinant adeno-associated virus vector 2/2 expressing RPE65 complementary DNA (cDNA) under the control of a human RPE65 promoter. There were no serious adverse events. There was no clinically significant change in visual acuity or in peripheral visual fields on Goldmann perimetry in any of the three patients. We detected no change in retinal responses on electroretinography. One patient had significant improvement in visual function on microperimetry and on dark-adapted perimetry. This patient also showed improvement in a subjective test of visual mobility. These findings provide support for further clinical studies of this experimental approach in other patients with mutant RPE65. (ClinicalTrials.gov number, NCT00643747.)Supported by grants from the U.K. Department of Health, the British Retinitis Pigmentosa Society, and the Special Trustees of Moorfields Eye Hospital, and by the Sir Jules Thorn Charitable Trust, the Wellcome Trust, the European Union (EVI-Genoret and Clinigene programs), the Medical Research Council, Foundation Fighting Blindness, Fight for Sight, the Ulverscroft Foundation, Fighting Blindness (Ireland), Moorfields Eye Hospital, and Institute of Ophthalmology Biomedical Research Centre for Ophthalmology, University College London