More than 2000 different sequence variations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described. Mutation-specific personalized therapies of cystic fibrosis (CF) is currently in clinical use for the most frequent CFTR pathogenic variant, the F508del, and some other pathogenic variants. However, CFTR function and relation with clinical status remain poorly understood for the large group of remaining and often rare, "orphan" mutations. A new emerging approach for precision medicine in CF is the so-called theratyping. It has the primary goal to identify, at cellular level, which CFTR pathogenic variants respond to certain biochemical therapeutic modulators, allowing a quick clinical translation of their use. On the other hand, the genetic correction of the defect is the final goal. Within the various approaches of gene therapy, the editing of CRTR gene by the Small Fragment Homologous Replacement (SFHR) revealed particularly promising. This proposal has two main objectives related to CF therapy: the theratyping and the gene editing by SFHR approach. From nasal epithelia of CF patients, by the "culture reprogramming condition" (CRC), we setup patient-specific airway epithelial stem cells (CF-CRC-AESC) and corresponding airway organoids. For both objectives, we will use the CF-CRC-AESC and airway organoids with different CFTR mutated genotypes. For theratyping, the pharmacological response will be assessed for: a) therapeutic agents already in clinical use in CF, that will be tested on rare (till now unexplored) CFTR mutated genotypes; b) an experimental drug (not in clinical use and until know unexplored in CF). The molecular mechanism and the efficacy of the CFTR gene editing by SFHR approach will be studied, on F508del homozygous CF-CRC-AESC, following the manipulation of 4 main cellular pathways: DNA methylation, chromatin remodeling, DNA repair and cell cycle control.
INNOVATION
The most innovative aspects of this proposal are:
- the use of the innovative CF-CRC-AESC and airway organoids as preclinical patient-specific model of CF; they represent a powerful tool to have patient-specific (and so genotype-specific) 2D cellular and 3D organoid CF airway epithelial models for therayping, functional characterization of CFTR variants and gene editing;
- the application of theratyping as unique step needed to extend the personalized therapeutic options already clinically available for CF to rare (previously untested) CFTR mutated genotypes;
- the test of an unexplored amplificatory and ionocyte-inducing possible therapeutic strategy;
- the application of an enhanced SFHR as an innovative approach of gene targeting;
- the evaluation of the interconnection between gene targeting and DNA methylation, chromatin remodelling, DNA repair and cell cycle pathways, an almost unexplored field.
The results of our studies will contribute to the enhancement of personalized treatment of CF by the advancement of knowledge in both theratyping and CFTR gene targeting. A better comprehension of the biochemical modulation of CFTR function and of molecular mechanisms of recognition and cellular processing of exogenous DNA will arise. The theratyping of each CF patient (with specific CFTR mutated genotype and genetic background), challenging CF-CRC-AESC cultures and organoids with both already known and experimental therapeutics, is of great translational impact, with particular relevance for CF patients with rare CFTR variants. At short-term, patients showing an in vitro response could benefit from the treatment as soon as the theratyping will be an approved approach also in Europe. The advancement in SFHR gene editing will constitute a further step towards the possibility of correction of a CFTR pathogenic variant directly within the genome of CF patients. At long-term, the spin-offs are both the possibility of direct correction in the lung, as well as the ex vivo cellular therapy.
The preliminary results of our group related to this project have been published by us in 6 recent papers quoted in the reference section with the following numbers: (31,33,35-38).
ANTICIPATED OUTPUT
At the end of this project, taking into account the already available and newly collected samples, at least 51 CF patients, with 35 different genotypes, 33 o which rare, will be characterized for their response to the already clinically approved and experimental therapies. A comparison between the efficacy of treatments on F508del homozygous patients will be available. An advancement in the knowledge of molecular and cellular targets involved in the mechanism of gene editing by SFHR and its applicability to CF gene therapy will be available. The best framework of the 4 main cellular pathways studied, allowing the highest level of correction, will be defined.
RELEVANCE
The possibility to obtain large amounts of CF-CRC-AESC from CF patients¿ upper airways, represents a powerful tool toward the introduction of more effective patient-specific biochemical and genetic therapeutic options, as well as to predict response to specific therapies for patients with rare pathogenic variants. The possibility to test cell response, virtually from each CF patient, to pharmacologic and genetic approaches may be of great translational impact, in the direction of theratyping and gene therapy.