Università degli studi di Modena e Reggio Emilia

Purpose This paper explores the feasibility of a new therapy for the treatment of hypospadias patients. Hypospadias is a very common congenital malformation of male genitals, with very high rate of recurrences after surgery. The field of regenerative medicine, which offers innovative solutions for many pathologies, still does not offer reliable solution for this pathology. Here, we propose quality, safety, and clinical feasibility assessment for an oral mucosa advanced therapy medicinal product (ATMP) grown on a biocompatible scaffold for a clinical study on urethral reconstruction of hypospadias patients. Methods Urethral and oral mucosal epithelia from donor biopsies were cultivated between two fibrin layers, under clinical-grade conditions for cell and tissue characterization and comparison, aimed at tissue engineering. In addition, single-clone analyses were performed to analyze gene expression profiles of the two epithelia by microarray technology. Results Oral mucosa appeared suitable for urethral reconstruction. The resulting ATMP was proven to maintain stem cells and regenerative potency. The preclinical safety studies were performed on human tissues to assess abnormalities and tumorigenicity, and confirmed the safety of the ATMP. Finally, the patient selection and the clinical protocol for the upcoming clinical trial were defined. Conclusions Against this backdrop, in this paper, we are proposing a new reproducible and reliable ATMP for the treatment of hypospadias.

The surface of the eye is a peculiar and extraordinary area, since the functions of all regions of the ocular surface system (OSS) are closely integrated forming a unique functional unit composed of different tissues and organs. Anatomically the ocular surface is composed principally by cornea and conjunctiva, but it also includes the lacrimal gland and the lacrimal drainage system for the tear film homeostasis, and the eyelids. Additional components as Meibomian glands, eyelashes and eyebrows complete the picture, contributing to the outermost lipid layer and to particle clearance (Fig. 1). Cornea and conjunc- tiva are generally covered by a complex liquid known as the tear film, providing a continuous moist environment connecting all areas at the front of the eye ball and protecting them from pathogens and small solid particles. This interconnected system provides the necessary conditions for the maintenance of functions needed for the vision process, as the final goal of eye function. The dysfunction of this apparatus could lead to several pathologies, such as corneal opacity or dry eye diseases (DED). Each component plays an important role for ocular surface maintenance, therefore some recent findings in stem cell biology aimed at the regeneration of different components of the ocular surface by tissue engineering, are examined in the following paragraphs.

PURPOSE: The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles. METHODS: Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC). RESULTS: The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression. CONCLUSIONS: Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.