Scholarship 21/09900-0 - Proteína 9 associada à CRISPR, Repetições palindrômicas curtas agrupad - BV FAPESP
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Multiplex CRISPR/Cas9 genome editing using ribonucleoprotein complexes for the generation of off-the-shelf allogeneic CAR-T cells

Grant number: 21/09900-0
Support Opportunities:Scholarships abroad - Research Internship - Master's degree
Start date: February 01, 2022
End date: July 31, 2022
Field of knowledge:Health Sciences - Medicine - Medical Clinics
Principal Investigator:Lucas Eduardo Botelho de Souza
Grantee:Sarah Caroline Gomes de Lima
Supervisor: Krishanu Saha
Host Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil
Institution abroad: University of Wisconsin-Madison (UW-Madison), United States  
Associated to the scholarship:20/02043-2 - Development of CRISPR/Cas9-based genome editing for the generation of allogeneic CAR-T cells, BP.MS

Abstract

The use of autologous T cells expressing chimeric antigen receptors (CAR) has revolutionized the therapy of B-cell neoplasms. However, the use of allogeneic CAR-T cells has numerous advantages over this autologous approach, such as the immediate availability of cryopreserved batches for infusion, standardization of the final cell product, time for multiple genetic modifications in cells, and redosing. This strategy also allows scaling-up the production of CAR-T cells resulting in cost reduction, which currently represents a challenge for low-mid income countries. Thus, one of the next frontiers in the field is to develop an allogeneic, 'off-the-shelf' therapy. This can be achieved by knocking out the T cell receptor (TCR) from CAR-T cells using genome-editing tools to prevent the development of graft-versus-host disease. Also, depletion of the human leukocyte antigen I (HLA-I) on CAR-T cells would reduce the chances of CAR-T cell rejection by the host immune system. During the development of the FAPESP project 2020/02043-2, we successfully established a platform based on lentiviral transduction and plasmid electroporation to express CAR and to knockout TCR or HLA-I using the CRISPR/Cas9 system. This platform allowed us to identify gRNA sequences with the highest editing efficiencies. However, this specific plasmid-delivery system does not easily allow simultaneous editing of TCR and HLA-I. In addition, plasmid delivery is considerably toxic to T cells and expression of Cas9 using this system is longer and associated with more off-target mutations when compared to Cas9 ribonucleoprotein complexes (RNPs). In this project, our aim is to refine our platform through the establishment of a workflow for the production of allogeneic CAR-T cells deficient in the expression of endogenous TCR and HLA-I by multiplex CRISPR/Cas9 gene editing using RNP complexes. RNP delivery is quickly overtaking plasmids as an optimal approach since they are immediately active, less toxic to T cells, display less off-target effects and easily allow the editing of multiple genes at once. To be successful, we established a strategic collaboration with Prof. Krishanu Saha, who has extensive experience with synthetic biology and genome editing across many cell types, including T cells. We are confident that this funding opportunity will contribute to the nucleation of an international network for the development of safer allogenic advanced cell therapies, which are potentially accessible to a larger number of patients in comparison to the current autologous approach. In addition, the gene-editing platform established in this collaboration could be easily applied to modulate pathways limiting the therapeutic efficacy of CAR-T cells and to correct genetic deficiencies underlying currently incurable diseases.

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