Periodic Reporting for period 2 - Holo-GT (Custom-designed gene editing of induced epidermal stem cells for gene therapy of genetic diseases of squamous epithelia)
Período documentado: 2023-03-01 hasta 2024-08-31
Epidermolysis Bullosa (EB) is a severe genetic skin disease caused by mutations in the adhesion proteins responsible for maintaining the tight connection between the dermis and epidermis. The recessive form of EB, known as the junctional form, has been effectively treated using ex vivo gene therapy. However, more than 50% of EB cases are dominantly inherited and cannot be addressed through viral-mediated gene addition. In this project, we aim to develop a novel system to tackle the dominant form of EB by utilizing induced holoclones (iHolo) and allele-specific gene editing mediated by a newly developed Cas protein.
The Holo-GT project has the potential to bring about groundbreaking advancements for patients affected by dominant monogenic diseases, particularly for two key aspects:
1. Development of new allele-specific Cas proteins: This breakthrough will pave the way for innovative approaches in treating dominant genetic diseases affecting not only the skin but also other squamous epithelia.
2. Reprogramming of TA progenitors into holoclone-forming cells (iHolo): This technique may also find application in other pathologies, especially those caused by stem cells exhaustion.
The Holo-GT project is structured around four main objectives:
1. Identification of specific pathways that sustain holoclone-forming cells.
2. Generation of induced holoclone-forming cells (iHolo).
3. Development of a purpose engineered CRISPR/Cas nuclease capable of accurately distinguishing between wild type and mutant alleles.
4. Gene editing of holoclone-forming cells or iHolo obtained from patients with severe forms of dominant EB Simplex (EBS) or dominant Dystrophic EB (DDEB).
The Holo-GT project has the potential to bring about groundbreaking advancements for patients affected by dominant monogenic diseases, particularly for two key aspects:
1. Development of new allele-specific Cas proteins: This breakthrough will pave the way for innovative approaches in treating dominant genetic diseases affecting not only the skin but also other squamous epithelia.
2. Reprogramming of TA progenitors into holoclone-forming cells (iHolo): This technique may also find application in other pathologies, especially those caused by stem cells exhaustion.
The Holo-GT project is structured around four main objectives:
1. Identification of specific pathways that sustain holoclone-forming cells.
2. Generation of induced holoclone-forming cells (iHolo).
3. Development of a purpose engineered CRISPR/Cas nuclease capable of accurately distinguishing between wild type and mutant alleles.
4. Gene editing of holoclone-forming cells or iHolo obtained from patients with severe forms of dominant EB Simplex (EBS) or dominant Dystrophic EB (DDEB).
Thus far we have implemented activities on all four work packages designed for the project.
In WP1, we expanded previous research by incorporating single-cell data to better define the molecular signatures of holoclone-forming stem cells (KSC), transient amplifying progenitors (TAC), and differentiated cells. New KSC markers identified include H1B, NUSAP1, RRM2 and STMN1. Notably, H1B is a novel target gene of FOXM1, crucial for self-renewal and differentiation in epidermal stem cells. These markers help distinguish KSCs from TACs. In addition, we demonstrated that KSCs uniquely repair DNA damage mainly through homologous recombination. FOXM1, downstream of YAP, coordinates the response to genotoxic stress and sustains epithelial self-renewal.
In WP2, we used a new reference to predict genes involved in intra-lineage reprogramming, employing two bioinformatics tools. This approach identified 10 genes. We evaluated the expression of these genes at RNA and protein levels in keratinocyte cultures. To test the effects of these genes, we are setting up a single-cell CRISPR activation (sc-CRISPRa) screening. The experiments are ongoing.
In WP3, we are developing a "selection-based directed evolution" to develop Cas9 variants with specific PAM sequence recognition. We switched from luminescence-based screening to antibiotic selection and focused on mutations to improve Cas9 specificity and overcome the same technical concerns. We also created a user-friendly platform called “AlPaCas” to screen Cas proteins for allele-specific targeting. AlPaCas identifies SNV-derived PAMs and suggests Cas enzymes or mutational changes to enhance specificity towards specific PAM.
In WP4, we are developing He-RASE, a HEK293 cell line model mimicking a "biallelic genomic context" of a dominant disease for testing CRISPR/Cas systems identified by WP3. This model helps test new CRISPR tools due to the scarcity of primary EBS cells and aims to develop similar models for other dominant diseases, such as Dominant Dystrophic EB (DEB).
In WP1, we expanded previous research by incorporating single-cell data to better define the molecular signatures of holoclone-forming stem cells (KSC), transient amplifying progenitors (TAC), and differentiated cells. New KSC markers identified include H1B, NUSAP1, RRM2 and STMN1. Notably, H1B is a novel target gene of FOXM1, crucial for self-renewal and differentiation in epidermal stem cells. These markers help distinguish KSCs from TACs. In addition, we demonstrated that KSCs uniquely repair DNA damage mainly through homologous recombination. FOXM1, downstream of YAP, coordinates the response to genotoxic stress and sustains epithelial self-renewal.
In WP2, we used a new reference to predict genes involved in intra-lineage reprogramming, employing two bioinformatics tools. This approach identified 10 genes. We evaluated the expression of these genes at RNA and protein levels in keratinocyte cultures. To test the effects of these genes, we are setting up a single-cell CRISPR activation (sc-CRISPRa) screening. The experiments are ongoing.
In WP3, we are developing a "selection-based directed evolution" to develop Cas9 variants with specific PAM sequence recognition. We switched from luminescence-based screening to antibiotic selection and focused on mutations to improve Cas9 specificity and overcome the same technical concerns. We also created a user-friendly platform called “AlPaCas” to screen Cas proteins for allele-specific targeting. AlPaCas identifies SNV-derived PAMs and suggests Cas enzymes or mutational changes to enhance specificity towards specific PAM.
In WP4, we are developing He-RASE, a HEK293 cell line model mimicking a "biallelic genomic context" of a dominant disease for testing CRISPR/Cas systems identified by WP3. This model helps test new CRISPR tools due to the scarcity of primary EBS cells and aims to develop similar models for other dominant diseases, such as Dominant Dystrophic EB (DEB).
Human epithelial stem cells are used for clinical applications since 1980 but the mechanism driving self-renewal is still not completely characterized. With this project we aim at defining the pathways required for generation of clinical-grade stem cells from transient amplifying progenitors. To reach this objective we will take advantage of emerging technologies based on single cell analysis and CRISPR modifications.
Concurrently, our project aims at developing a new technology to treat dominant genetic diseases that cannot be tackled by gene addition strategy, using dominant forms of Epidermolysis Bullosa as a model system. We will apply the innovative “allele-specific intervention” to selectively disrupt EB simplex (EBS) and Dystrophic EB (DEB) dominant mutations through the use of custom designed CRISPR/Cas here described. Within HoloGT project, we aim at establishing a safe and efficacious protocol for ex vivo gene editing of EBS and dominant DEB (DDEB). At the end of the project, our scope is to develop a potential therapeutic approach for the treatment of EBS and DDEB.
The data produced in HoloGT will improve our knowledge on epidermal stem cell, shed light on molecular mechanism underlying EB dominant diseases, define safe and efficaceous gene editing approach, essential for future clinical application. Although the activities foreseen by the project are limited to basic and preclinical research, the proof of principle of feasibility of our approach will represent a proof of concept for its application in clinical setting.
The in vitro EB model used to test the feasibility of this approach will be instrumental for tackling other genetic diseases affecting the skin other squamous epithelia.
Concurrently, our project aims at developing a new technology to treat dominant genetic diseases that cannot be tackled by gene addition strategy, using dominant forms of Epidermolysis Bullosa as a model system. We will apply the innovative “allele-specific intervention” to selectively disrupt EB simplex (EBS) and Dystrophic EB (DEB) dominant mutations through the use of custom designed CRISPR/Cas here described. Within HoloGT project, we aim at establishing a safe and efficacious protocol for ex vivo gene editing of EBS and dominant DEB (DDEB). At the end of the project, our scope is to develop a potential therapeutic approach for the treatment of EBS and DDEB.
The data produced in HoloGT will improve our knowledge on epidermal stem cell, shed light on molecular mechanism underlying EB dominant diseases, define safe and efficaceous gene editing approach, essential for future clinical application. Although the activities foreseen by the project are limited to basic and preclinical research, the proof of principle of feasibility of our approach will represent a proof of concept for its application in clinical setting.
The in vitro EB model used to test the feasibility of this approach will be instrumental for tackling other genetic diseases affecting the skin other squamous epithelia.