Achromatopsia is a hereditary form of day blindness caused by cone photoreceptor dysfunction. Affected patients suffer from congenital color blindness, photosensitivity, and low visual acuity. Mutations in the CNGA3 gene are a major cause of achromatopsia, and a sheep model of this disease was recently characterized by our group. Here, we report that unilateral subretinal delivery of an adeno-associated virus serotype 5 (AAV5) vector carrying either the mouse or the human intact CNGA3 gene under the control of the red/green opsin promoter results in long-term recovery of visual function in CNGA3-mutant sheep. Treated animals demonstrated shorter maze passage times and a reduced number of collisions with obstacles compared with their pretreatment status, with values close to those of unaffected sheep. This effect was abolished when the treated eye was patched. Electroretinography (ERG) showed marked improvement in cone function. Retinal expression of the transfected human and mouse CNGA3 genes at the mRNA level was shown by polymerase chain reaction (PCR), and cone-specific expression of CNGA3 protein was demonstrated by immunohistochemisrty. The rescue effect has so far been maintained for over 3 years in the first-treated animals, with no obvious ocular or systemic side effects. The results support future application of subretinal AAV5-mediated gene-augmentation therapy in CNGA3 achromatopsia patients.

SummaryAge-related macular degeneration is caused by dysfunction and loss of retinal pigment epithelium (RPE) cells, and their transplantation may rescue visual functions and delay disease progression. Human embryonic stem cells (hESCs) may be an unlimited source of RPE cells for allotransplantation. We analyzed the immunomodulatory properties of hESC-derived RPE (hESC-RPE) cells, and showed that they inhibited T cell responses. Co-culture experiments showed that RPE cells inhibited interfon-γ secretion and proliferation of activated T cells. Furthermore, hESC-RPE cells enhanced T cell apoptosis and secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). In addition, RPE cells altered the expression of T cell activation markers, CD69 and CD25. RPE cells transplanted into RCS rats without immunosuppression survived, provided retinal rescue, and enhanced IL-10 blood levels. Our data suggest that hESC-RPE cells have immunosuppressive properties. Further studies will determine if these properties are sufficient to alleviate the need for immunosuppression therapy after their clinical allotransplantation.

Injuries to ligaments are common, painful and debilitating, causing joint instability and impaired protective proprioception sensation around the joint. Healing of torn ligaments usually fails to take place, and surgical replacement or reconstruction is required. Previously, we showed that in vivo application of the recombinant human amelogenin protein (rHAM +) resulted in enhanced healing of the tooth‐supporting tissues. The aim of this study was to evaluate whether amelogenin might also enhance repair of skeletal ligaments. The rat knee medial collateral ligament (MCL) was chosen to prove the concept. Full thickness tear was created and various concentrations of rHAM +, dissolved in propylene glycol alginate (PGA) carrier, were applied to the transected MCL. 12 weeks after transection, the mechanical properties, structure and composition of transected ligaments treated with 0.5 μg/μl rHAM + were similar to the normal un‐transected ligaments, and were much stronger, stiffer and organized than control ligaments, treated with PGA only. Furthermore, the proprioceptive free nerve endings, in the 0.5 μg/μl rHAM + treated group, were parallel to the collagen fibres similar to their arrangement in normal ligament, while in the control ligaments the free nerve endings were entrapped in the scar tissue at different directions, not parallel to the axis of the force. Four days after transection, treatment with 0.5 μg/μl rHAM + increased the amount of cells expressing mesenchymal stem cell markers at the injured site. In conclusion application of rHAM + dose dependently induced mechanical, structural and sensory healing of torn skeletal ligament. Initially the process involved recruitment and proliferation of cells expressing mesenchymal stem cell markers.

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Purpose Usher syndrome (USH) is the most common syndromic inherited retinal disease, causing retinitis pigmentosa and sensorineural hearing loss. We reported previously that a nonsense mutation in the centrosome-associated protein CEP250 gene (encoding C-Nap1) causes atypical USH in patients of Iranian Jewish origin. To better characterize CEP250 , we aimed to generate and study a knockout (KO) mouse model for Cep250 . Methods Mice heterozygous for a “knockout-first” Cep250 construct were generated and bred with Cre recombinase mice to generate the null allele and produce homozygous Cep250 KO mice. Retinal function was evaluated by full-field electroretinography (ffERG) at variable ages, and retinal structure changes were examined using histological analysis. Hearing thresholds were detected using auditory brainstem response (ABR) at the age of 20 months. Results The Cep250 KO mouse model was generated by activating a construct harboring a deletion of exons 6 and 7. At 6 months, the ffERG was normal, but it decreased gradually with age. For both photopic and scotopic ffERG responses, very low amplitudes were evident at 20 months. Histological analysis confirmed late-onset retinal degeneration. ABR tests illustrated that hearing threshold significantly increased at the age of 20 months. Conclusions Although most USH animal models have normal retinal function and structure, the Cep250 KO mouse model shows both retinal degeneration and hearing loss with a relatively late age of onset. This model may shed more light on CEP250 -associated retinal and hearing deficits and represents an efficient platform for the development of treatment modalities for USH. Translational Relevance Our study demonstrates better understanding of Cep250 -associated retinal and hearing disease in a mouse model and may help in developing more efficient gene therapy modalities.

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