Periodic Reporting for period 1 - Holo-GT (Custom-designed gene editing of induced epidermal stem cells for gene therapy of genetic diseases of squamous epithelia)
Periodo di rendicontazione: 2021-09-01 al 2023-02-28
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).
During this reporting period, our focus was on WP1 and WP3.
In WP1, we conducted the collection of single-cell transcriptomic data from human primary keratinocyte cultures. Our objective was to identify transcription factors (TF) that have the potential to reprogram progenitors into stem cells (iHolo). We employed two bioinformatic approaches to infer the TF based on the gene regulatory network derived from the single-cell data. Subsequently, we validated the candidate TF using western blot, immunofluorescence, and digital real-time analysis. Moving forward, our plan is to assess whether modulating the predicted TF can indeed reprogram progenitors into stem cells.
For WP3, we pursued a structure-guided mutagenesis approach to engineer the RVRCpf1 nuclease. Utilizing the crystallographic structure as a starting point, we manually introduced amino acid mutations to enhance its selectivity towards a desired target. After assessing stability, folding, and DNA-protein interaction through bioinformatic analyses, we identified two specific mutations (M604Q and C608S) that significantly improved the specificity of the CRISPR system for the "new" PAM sequence 5'-TATG-3. In preliminary experiments conducted on the HEK293 cell line, this mutant demonstrated functionality and specificity.
In WP1, we conducted the collection of single-cell transcriptomic data from human primary keratinocyte cultures. Our objective was to identify transcription factors (TF) that have the potential to reprogram progenitors into stem cells (iHolo). We employed two bioinformatic approaches to infer the TF based on the gene regulatory network derived from the single-cell data. Subsequently, we validated the candidate TF using western blot, immunofluorescence, and digital real-time analysis. Moving forward, our plan is to assess whether modulating the predicted TF can indeed reprogram progenitors into stem cells.
For WP3, we pursued a structure-guided mutagenesis approach to engineer the RVRCpf1 nuclease. Utilizing the crystallographic structure as a starting point, we manually introduced amino acid mutations to enhance its selectivity towards a desired target. After assessing stability, folding, and DNA-protein interaction through bioinformatic analyses, we identified two specific mutations (M604Q and C608S) that significantly improved the specificity of the CRISPR system for the "new" PAM sequence 5'-TATG-3. In preliminary experiments conducted on the HEK293 cell line, this mutant demonstrated functionality and specificity.
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.