Targeted gene integration of CRS inhibitor genes for improved immune cells therapy
The invention pertains to the field of adaptive cell immunotherapy. It provides with the genetic insertion of exogenous coding sequence(s) into genetically engineered immune cells to prevent cytokine release syndrome to arise during the course of cell therapy. These exogenous coding sequences are more particularly soluble human polypeptides placed under the transcriptional control of endogenous gene promoters that are sensitive to immune cells activation. Such method allows the production of safer immune primary cells of higher therapeutic potential.
1. A method for preparing engineered immune cells for cell immunotherapy, said method comprising:
providing a population of cells comprising T-cells;
introducing into a proportion of said T-cells by cleavage by at least one sequence-specific reagent selected from an RNA-guided endonuclease, a TAL-endonuclease, a zinc finger nuclease, a homing endonuclease, or any combination thereof that specifically targets PD-1 endogenous locus,
at least one nucleic acid comprising an exogenous polynucleotide sequence expressing a soluble form of GP130 consisting of the amino acid sequence of SEQ ID NO:61 such that the exogenous polynucleotide sequence is integrated into a human endogenous PD-1 gene locus and eliminates expression of the PD-1 protein, such that expression of the soluble form of GP130 is under control of the endogenous PD-1 promoter and
at least one nucleic acid encoding a CAR directed against a tumor antigen integrated into the genome of the T-cells under the control of a constitutive promoter,
wherein tumor cell engagement by the CAR induces the expression and secretion of the soluble form of GP130 from the PD-1 promoter, while expression of PD-1 protein is prevented by the integration of the exogenous polynucleotide.
2. The method of claim 1 , wherein said T cells are human primary T-cells.
3. The method of claim 1 , wherein the sequence-specific reagent is Cas9 or Cpf1.
4. The method of claim 1 , wherein the sequence-specific reagent is a TAL-endonuclease.
5. The method of claim 1 , wherein the sequence-specific reagent is a zinc finger nuclease.
6. The method of claim 1 , wherein the sequence-specific reagent is a homing endonuclease.
7. The method of claim 1 , wherein said exogenous polynucleotide sequence has the nucleotide sequence of SEQ ID NO:51.