The aim of this proposal is to clarify the molecular mechanisms of the Small Fragment Homologous Replacement (SFHR) gene targeting approach. SFHR can stably modify a genomic sequence by homologous replacement of a small DNA fragment. This approach can potentially correct mutations within disease genes. The practical application of SFHR is limited by a low and variable frequency of correction, mainly depending on the poor knowledge of underlying mechanisms.
We already published the results 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. These general pathways and the related specific genes appears to be excellent targets for SFHR molecular mechanism study and manipulation, aimed to the enhancement of correction efficiency.
This proposal is the continuation of the previous funded project and will focus on the relationship between the 4 pathways listed above and SFHR. These studies will be performed in a reporter cellular system of mouse embryonic fibroblasts and in human Cystic Fibrosis (CF) cells of airway epithelium. Long-term stem-induced CF airway epithelial cells, obtained by the culture reprogramming condition approach, will be used. Also the differentiated counterparts of stem-induced CF cells will be used. Drugs acting on each of the 4 pathways, single-gene targeting and the CRISPR/Cas 9 method will be used to dissect the pathways, clarify SFHR mechanisms and enhance the correction efficiency.
These studies will allow a better comprehension of the molecular mechanisms of homologous genomic replacement. The selected molecular targets will provide suggestions for increasing gene repair efficiency. New perspectives for SFHR therapeutic applications to both differentiated and stem-induced cells will arise, in particular for the mutation-specific treatment of CF at DNA level.
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, 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 peculiarity and innovative features of the cellular models used (described below).
CELLULAR MODELS
MEF-mEGFP system
The first experimental system we have been using 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). A small DNA fragment (SDF), homologous to the wtEGFP sequence, can correct the nonsense mutation and restore the fluorescence of the cells. Based on our previous results (32, 57), an already setup clone of the MEF-mEGFP with inhibited expression of Trex1 gene (MEF-mEGFP-shRNA-Trex1-), chosen within the 18 genes selected by us as involved in SFHR, will be used. A mock counterpart will also be used. The efficiency of SFHR is functionally quantified by FACS. This is a reporter system, easy to manipulate, useful for a better comprehension of the molecular mechanisms of the SFHR in differentiated cells.
Cystic Fibrosis (CF) nasal and lung airway primary epithelial cells
As a second cellular system we have been using 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 have been using 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).
These CF cells we have been using have several CFTR mutated genotypes, including the homozygous F508del / F508del.
These cells are 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, is evaluated on the overall cellular population as described in Experimental plan. 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-beta1, CD44), lung epithelial stem cell markers (Integrin-alfa6, 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. The 11 CRC reprogrammed CF cell lines so far obtained showed both a CFTR gene sequence and a CFTR gene expression perfectly matched with those of the corresponding CF patient they are derived from (also after several cell culture passages).