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New Prime Editing and non-viral delivery strategies for Gene Therapy

Periodic Reporting for period 1 - EdiGenT (New Prime Editing and non-viral delivery strategies for Gene Therapy)

Reporting period: 2022-10-01 to 2023-09-30

Genome editing technologies based on CRISPR/Cas systems allow targeted genomic modification with unprecedented precision and have emerged as powerful alternatives to conventional gene therapy approaches for various human diseases, with a series of clinical trials in progress. However, some crucial challenges remain to be addressed to enhance efficiency and safety and decrease the costs of treatments.
Current viral-based delivery systems are associated with a high risk of toxicity and immunogenicity and remain highly expensive. We will develop a new generation of non-viral delivery systems for gene editing tools based on the use of modified nanoparticles with human-derived protein moieties that will allow targeting the tissue and cells of interest in vivo with minimal adverse effects.

Prime editors have raised exciting possibilities for double-strand break-free genome editing. However, a major limitation of current prime editors is highly variable efficiency both from one target to another and between cell types. We will design and evaluate novel prime editor tools in order to both increase activity per se and overcome cell-specific limitations.
We will test our approach on the hematopoietic system to treat Sickle Cell Disease, avoiding the challenges and risks of hematopoietic stem cell manipulation associated with current gene therapy approaches, and thus providing a treatment much simpler, safer, and cost-effective to implement.

Our technological breakthroughs address two key obstacles in cell and gene therapy: gene editing efficiency and systemic delivery. The novel prime editors and targeted nanoparticles that we will engineer will be combined to make unprecedented off-the-shelf, recombinant biologics for gene therapy. The versatility of the design of these novel recombinant biologics makes them suitable for the treatment of a vast majority of genetic diseases
1) Development of a new generation of gene correction tools.
We have started to develop a strategy to improve the efficiency of prime editing, replacing the reverse transcriptase with a DNA-dependent polymerase, we named this approach POLYPRIME. Transgenic zebrafish models for testing POLYPRIME in vivo have been generated.

2) Establishment of targeted non-viral in vivo delivery systems for CRISPR/Cas9-based ribonucleoprotein complexes using functionalized nanoparticles.
We have evaluated the efficiency of the current prime editing system to correct the SCD mutation achieving modest frequency of gene corrected cells and set up a model of mobilization of human hematopoietic stem/progenitor cells in the blood of immunodeficient mice using healthy donor samples. Transgenic zebrafish models for testing the delivery of RNPs in vivo have been generated.

3) Assessment of efficacy and safety of the breakthrough approaches in cellular and humanized animal models.
Transgenic zebrafish models for testing the safety and toxicity of the delivery of RNPs in vivo have been generated. Fully automated workflows for the evaluation of toxicity of the approaches in zebrafish embryos have been developed.
The project is still in its early stage, but awareness on the project has been achieved through participation of the Principal investigators of the consortium to several scientific events. The first high-impact scientific publication will be prepared after the IPR protection process has started with the patent office.