Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair

International audienceDifferent mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock

Interleukin-22 binding protein (IL-22BP) is constitutively expressed by a subset of conventional dendritic cells and is strongly induced by retinoic acid

Interleukin-22 (IL-22) is mainly produced at barrier surfaces by T cells and innate lymphoid cells and is crucial to maintain epithelial integrity. However, dysregulated IL-22 action leads to deleterious inflammation and is involved in diseases such as psoriasis, intestinal inflammation, and cancer. IL-22 binding protein (IL-22BP) is a soluble inhibitory IL-22 receptor and may represent a crucial regulator of IL-22. We show both in rats and mice that, in the steady state, the main source of IL-22BP is constituted by a subset of conventional dendritic cells (DCs) in lymphoid and non-lymphoid tissues. In mouse intestine, IL-22BP was specifically expressed in lamina propria CD103+CD11b+ DC. In humans, IL-22BP was expressed in immature monocyte-derived DC and strongly induced by retinoic acid but dramatically reduced upon maturation. Our data suggest that a subset of immature DCs may actively participate in the regulation of IL-22 activity in the gut by producing high levels of IL-22BP

Immunohistologic Analysis of Epithelial-Cell Populations in Oral Lichen-Planus

Previous studies have demonstrated heterogeneity within lesional lymphocytes in drug‐related oral lichen planus (D‐LP) and idiopathic lichen planus (I‐LP). This study examined the phenotype of Langerhans cells (LC) and keratinocytes in non‐erosive D‐LP and I‐LP. In I‐LP, keratinocytes expressed HLA‐DR antigens whilst LC co‐expressed CDIa, MHC Class II and CD4 antigens. The high levels of expression of MHC Class II antigens by LC were maintained during short term organ culture. In I‐LP, the epithelium contained occasional CD25 + dendritic cells (putative activated LC). These cell phenotypes are suggestive of cell activation and likely result from local production of gamma interferon. In D‐LP, expression of MHC Class II antigens on LC was reduced and no CD25+ cells were detected. The epithelium contained an increased number of CD45RA+ dendritic cells. While no differences between the production of interleukin‐1 and interleukin‐1 inhibitors by tissue samples could be detected, it is likely that the variations in epithelial cell phenotypes in I‐LP and D‐LP reflect altered cytokine production