The aim of this proposal is to clarify the molecular mechanisms of a gene targeting approach called Small Fragment Homologous Replacement (SFHR). This approach can stably modify a genomic sequence by homologous replacement of a small DNA fragment and could correct mutations within disease genes. However, SFHR mechanisms are poorly understood and its practical application is limited by a low and variable frequency of correction.
We already published experimental evidences about the interconnection between SFHR, DNA methylation, chromatin remodelling, DNA repair and cell cycle pathways. We also published a selection of 18 specific genes within DNA repair and cell cycle pathways involved in the SFHR. Both the general pathways and the related specific genes are excellent targets for SFHR molecular mechanism study and manipulation.
This project will focus on the relationship between the 4 pathways listed above and SFHR, with particular regard to the enhancing of correction efficiency. This relationship will be studied in a reporter cellular system of mouse embryonic fibroblasts and in human Cystic Fibrosis (CF) cells of airway epithelium. Both primary differentiated and long-term stem-induced (by the culture reprogramming condition - CRC - approach) CF airway epithelial cells will be used. Drugs acting on each of the 4 pathways and single-gene targeting will be used to dissect the pathways and to manipulate SFHR efficiency. A possible enhancement of SFHR by the use of CRISPR/Cas 9 method will be evaluated.
These studies will contribute to a better comprehension of the molecular mechanisms of homologous genomic replacement after the uptake and intracellular processing of exogenous DNA. The selection of molecular targets to manipulate will provide suggestions for increasing gene repair efficiency. The application to both differentiated and stem-induced cells may highlight new perspective for SFHR therapeutic applications, in particular to CF.
The interaction of both the SFHR and the CRISPR/Cas 9 tool with the 4 cellular pathways studied and the related possible modulation of the correction efficiency and specificity of SFHR are almost unexplored fields.
Our results will allow an advancement of knowledge about gene targeting, focused on the enhancement of its correction ability. This will constitute a further step towards the possibility of correction of a mutation directly within the genome of patients. In particular about CF, the spin-offs are both the possibility of direct correction in the lung of patients, as well as the ex vivo approach as cellular therapy. In this case, the aim is to pick-up cells from the patient, reprogram them to stem cells (possibly easier to correct), to correct and re-administer them to the patient after correction, to complement the action of diseased cells.
Crucial effectors and gene-specific targets to be manipulated for the enhancement of SFHR efficiency, in particular in CF, will be revealed. This can allow the achievement of a higher and more specific correction for a practical SFHR and/or CRISPR/Cas 9 method application to both in vivo and ex vivo therapeutic approaches.
The most innovative aspects of our proposal are:
- the use of SFHR, a particularly innovative approach of gene targeting
- the evaluation of the interconnection between gene targeting and DNA methylation, chromatin remodelling, DNA repair and cell cycle pathways, a field almost unexplored till now
- the selection of specific genes to be manipulated for the enhancement of gene targeting
- the use of an approach suitable for ex vivo correction of mutated cells, suitable also for stem and stem-induced cells
- the combination of the cellular models used.
Due to their peculiarity and innovative features, the cellular models that will be used are briefly described below.
CELLULAR MODELS
MEF-mEGFP system
The first experimental system we will use was setup by us (32); it is a mouse embryonic fibroblast (MEF) cell line, stably integrating the enhanced green fluorescent protein (EGFP) either wild-type (wtEGFP) or nonsense mutated (mEGFP). In MEF-mEGFP, a small DNA fragment (SDF), homologous to the wtEGFP sequence, can correct the mutation and restore the fluorescence of the cells. Based on our previous results (32, 57), a clone of the MEF-mEGFP with inhibited expression of Trex1 gene (MEF-mEGFP-shRNA-Trex1-), chosen within the 18 genes selected as involved in SFHR, will be setup and used. A mock counterpart (MEF-mEGFP-shRNA-mock) will also be established. Due to the facility of its manipulation, this is a reporter system useful for a better comprehension of the molecular mechanisms of the SFHR in differentiated cells. The efficiency of SFHR is functionally quantified by FACS, which allows the detection of low frequency phenotypic changes.
Cystic Fibrosis (CF) nasal and lung airway primary epithelial cells
As a second cellular system we will use CF human nasal epithelial primary cells obtained by nasal brushing of CF patients, or healthy subjects as wild-type counterparts.
As a third cellular system we will use CF, and wild-type, human bronchial epithelial primary cells (obtained from lung transplants), provided by the facility of the Italian Cystic Fibrosis Foundation (Genetic Molecular Laboratory - Gaslini Institute - Genoa, Italy).
All the CF cells used will have a mutated genotype homozygous F508del / F508del.
These cells will be grown on transwells on air-liquid interface (ALI) conditions developing a high transepithelial resistance and differentiating. The efficiency of SFHR, after treatment with a SDF homologous to the wild type sequence of CFTR, will be evaluated on the overall cellular population by fluid reabsorption (61, 62) measurements and at DNA (by a cyclic clonal enrichment protocol (54)), RNA (by real-time PCR) and protein (by western blot and immunochemistry) level. Also a cloning strategy will be applied to measure the efficiency of SFHR on repaired clonal cellular populations.
CF nasal and lung stem-induced airway epithelial cells
The reprogramming of nasal and lung airway epithelial primary cells to airway epithelial stem cells will be obtained by the culture reprogramming condition (CRC) approach (63). Briefly, this approach consists in the treatment of differentiated cells with the Rho kinase (ROCK) inhibitor (Y-27632) in combination with irradiated Swiss 3T3 fibroblast feeder cells. This treatment induces in epithelial cells a stem-like phenotype including the expression of typical epithelial stem cell markers (Integrin-beta-1, CD44), lung epithelial stem cell markers (Integrin-alfa-6, NGFR/CD271, c-kit) and the ability to proliferate indefinitely in vitro, without the need of transduction of exogenous viral or cellular genes. The CRC reprogrammed cells retain a normal karyotype and remain non tumorigenic. Typically, the removal of the ROCK inhibitor induces the re-differentiation to airway epithelium.