Individual
ESR projects

Research project

Correction of mutations associated with primary immunodeficiencies using novel and engineered CRISPR tools

Rationale and Objectives

Primary immunodeficiencies are a group of highly heterogenous disorders, characterized by an increased susceptibility to infections, coupled with an higher risk of developing autoimmune diseases or cancer. Common variable immunodeficiency disorders (CVID) due to mutations in CTLA-4 have been particularly linked to the development of autoimmunity , . Dominant negative mutations in STAT3, which have been associated to Hyper-IgE syndrome (HIES), are also of interest in the gene editing field since its impaired function has been related to immunodeficiency as well as tumorigenesis. Alia Therapeutics has developed a discovery and engineering platform for the isolation of new CRISPR nucleases with high activity and specificity.

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DC10’s goal is to apply our gene editing technology to target monogenic primary immunodeficiencies to identify potential therapeutic approaches for the treatment of such disorders. Therefore, Alia’s editing technology will be applied to edit CTLA-4 or STAT3 dominant-negative mutated cells. The project will be split in four phases:

1)   Identify undescribed nucleases from our dataset which best fit the above-mentioned targets.

2)  Develop a tailored editing strategy for the two targets:a gene replacement strategy (knock-in) for CTLA-4 deficiency will be evaluated; and a classical allele-specific NHEJ-strategy for STAT3 mutant will be tested to downregulate its expression.

3) Validate in vitro the efficacy (editing, mRNA and protein downregulation or upregulation) and safety (genome wide off-targets, allele-specificity if needed) of the editing approaches.

4) Evaluate the functional recovery upon gene correction by exploiting available mouse models.

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Expected results

The project will lead to the identification of novel gene editing-based therapeutic strategies for relevant primary immune deficiencies. Additional relevant achievements include the discovery of novel gene editing tools (new CRISPR nucleases with defined characteristics which are desirable for clinical use) that can find additional applications beyond the currently investigated indications.

Planned secondments

IJC (Ballestar) to learn in vitro differentiation protocols of immune cells, m10-m12 (2 months); OSR (Di Micco) to learn in vivo differentiation assays, m13-15 (2 months); IRB (Geiger) to learn T cell biology, m16-17 (1 month); and GRL (Vento-Tormo) to perform and analyse sc-omics on corrected cells, m25-m28 (3 month).

PhD programme

PhD in Biomolecular Sciences, University of Trento, Italy