Mutations in connexin genes expressed in epidermal keratinocytes cause a variety of rare genodermatoses, ascribed to keratinocyte proliferation and/or differentiation defects that range in severity from increases in skin thickness, to life-threatening and fatal barrier break down. In particular, some pathological connexin 26 (Cx26) variants generate "leaky" or abnormally active hemichannels that are causally linked to keratitis-ichthyosis-deafness (KID) syndrome, a devastating disease for which there is no cure [1]. We previously showed that submicromolar concentrations of a human-derived monoclonal antibody (mAb), named abEC1.1, inhibit KID-related leaky Cx26 hemichannels in vitro [2,3]. Here, we performed abEC1.1 gene transfer experiments in vivo based on a recombinant AAV vector in a well characterized mouse model of KID syndrome. In this model, the inducible expression of the p.G45E variant in the epidermis of heterozygous transgenic Cx26G45E mice leads to skin abnormalities within few days of doxycycline administration [4, 5]. We developed an ad hoc technology based on ratiometric reflectance imaging in live animals and determined that a single systemic administration of the abEC1.1 AAVmAb significantly reduces the visible manifestation of epidermal pathology for up to 4 weeks. We also performed imaging of DAPI uptake by multiphoton intravital microscopy[6] and showed that treatment acted at the level of the mutant hemichannels expressed in epidermal keratinocytes after doxycycline induction. Finally, we examined treatment effects ex vivo, by histology and confocal immunofluorescence microscopy of mouse skin. We found that abEC1.1 AAVmAb recovered a normal epidermal thickness and keratinocytes size. It also caused partial recovery of the expression pattern of epidermal keratins and reduction of proliferation and apoptosis markers. Prolonged exposure to mAb had no effect on gap junction formation in liver and brain and did not affect locomotion activity, body weight, food or water consumption of treated mice up to 4 months after AAVmAb injection. These results in preclinical settings prove the efficacy of the AAVmAb-based treatment in the KID syndrome mouse model, support the clinical potential of our approach and pave the way to the development of innovative therapy to treat connexin-related human genodermatoses by mAbs targeting the extracellular domain of hemichannels.

UN APPROCCIO TERAPEUTICO PER LA SINDROME DA CHERATITE-ITTIOSI-SORDITÀ (KID) Stato dell'arte antecedente a questo studio: Le mutazioni della connessina26 (Cx26 o GJB2) che causano un' aumentata attività degli emicanali sono alla base di malattie della pelle che includono la sindrome cheratite-ittiosi-sordità (KID). Per malattie come la sindrome KID, non esiste una cura, ci sono opzioni limitate per la palliazione e l'impatto combinato di perdita dell'udito, deturpazione e cecità diminuisce notevolmente la qualità della vita delle persone colpite che sopravvivono all'infanzia. Valore aggiunto di questo studio: In questo studio, abbiamo dimostrato che la patologia epidermica può essere alleviata in un modello murino che replica la malattia della pelle associata alla sindrome KID umana mediante somministrazione sistemica di un anticorpo monoclonale. Abbiamo dimostrato che l'effetto terapeutico dell'anticorpo si esplica mediente il blocco degli emicanali della Cx26. Ulteriori implicazioni: Questi risultati (1) confermano l'ipotesi che l'aumento dell'attività degli emicanali contribuisca in modo significativo alla patologia epidermica nella sindrome KID e (2) mostrano il potenziale uso di specifici anticorpi monoclonali come nuovo intervento terapeutico per il trattamento di questa malattia. La disfunzione degli emicanali contribuisce all'insorgenza di numerose altre malattie, pertanto questi risultati potrebbero avere un ampio impatto per il trattamento di diversi stati patologici dipendenti dagli emicanali.

1. Avshalumova L. et al. International journal of dermatology 53, 192-205 (2014). 2. Xu, L. et al. "Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders". Front. in molecular neuroscience 10, 298 (2017). 3. Ziraldo, G. et al. "A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function". Front. in physiology 10, 392 (2019). 4. Mese, G. et al. "The Cx26-G45E mutation displays increased hemichannel activity in a mouse model of the lethal form of keratitis-ichthyosis-deafness syndrome". Molecular biology 22, 4776-4786 (2011). 5. Lilly, E. et al. "Connexin channels in congenital skin disorders". Seminars in cell & developmental biology 50, 4-12 (2016). 6. Nardin, C. et al. "Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing". Cancers 13 (2021).

Keratitis-ichthyosis-deafness (KID) syndrome

Sindrome da cheratite-ittiosi-sordità (KID)