Guide RNA combinations and methods of use
Inventors
Schlabach, Michael R. • Hohmann, Anja Fides
Assignees
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Publication Number
US-12188045-B2
Publication Date
2025-01-07
Expiration Date
Abstract
The present disclosure provides gRNA compositions, CRISPR/Cas systems comprising the same, and methods of their use in the modification of immune effector cells. Methods of treating a cell proliferative disorder, such as a cancer, using the modified immune effector cells described herein are also provided.
Core Innovation
The invention relates to CRISPR/Cas systems for immune effector cells. It provides modified immune effector cells comprising a SOCS1-targeting gRNA molecule and a PTPN2-targeting gRNA molecule together with a Cas endonuclease. The SOCS1-targeting gRNA molecule comprises a targeting domain sequence encoded by any one of SEQ ID NOs: 1-3.
The disclosure covers immune effector cells including T cells, tumor infiltrating lymphocytes (TILs), NK cells, and NKT cells. By targeting SOCS1 and PTPN2 in the same modified cell, the disclosure describes enhanced immune effector functions. The modified immune effector cells are engineered using CRISPR/Cas systems that target SOCS1 and PTPN2.
The disclosure further addresses gRNA and Cas formats and configuration, including end modifications on gRNA molecules, guide formats such as sgRNA or dual gRNA (crRNA+tracrRNA), and delivery formats including vectors, Cas-gRNA ribonucleoprotein complexes, and Cas9 mRNA. It also describes therapeutic use contexts, including combinations with immune checkpoint inhibitors such as PD-1/PD-L1/CTLA4, and kit-related content.
Claims Coverage
The independent claim set centers on one inventive cell concept: a modified immune effector cell containing a SOCS1-targeting gRNA, a PTPN2-targeting gRNA, and a Cas endonuclease, with dependent refinements to gRNA structure, Cas format, delivery format, and cell type.
Engineered immune effector cell with dual SOCS1/PTPN2 gRNAs and Cas endonuclease
A modified immune effector cell comprising a SOCS1-targeting gRNA molecule and a PTPN2-targeting gRNA molecule, together with a Cas endonuclease.
SOCS1 targeting domain sequence limited to SEQ ID NOs: 1-3
The SOCS1-targeting gRNA molecule comprises a targeting domain sequence encoded by any one of SEQ ID NOs: 1-3.
End-modified SOCS1- and PTPN2-targeting gRNAs
The SOCS1-targeting gRNA and the PTPN2-targeting gRNA include modifications at or near their 5′ ends and/or at or near their 3′ ends.
Cas-delivered dual gRNAs as ribonucleoproteins
The SOCS1-targeting gRNA molecule and the PTPN2-targeting gRNA molecule are complexed with Cas endonuclease as ribonucleoproteins (RNPs).
Cas endonuclease provided by vector or mRNA polynucleotide
A vector encodes the Cas endonuclease or an mRNA polynucleotide encodes the Cas endonuclease.
Wild-type Cas endonuclease for double-stranded DNA breaks
The Cas endonuclease is a wild-type Cas endonuclease with two enzymatically active domains that induce double-stranded DNA breaks.
Human T cell with engineered chimeric antigen receptor
The modified immune effector cell includes a human T cell that has an engineered chimeric antigen receptor.
Overall, the claims cover modified immune effector cells combining dual SOCS1- and PTPN2-targeting gRNAs with a Cas endonuclease, further refined by gRNA end modifications, Cas delivery or format, specification to a wild-type double-strand break-inducing Cas, and restriction to human T cells with an engineered chimeric antigen receptor.
Stated Advantages
The modified immune effector cells are described as having enhanced immune effector functions.
Documented Applications
Therapeutic use contexts, including combinations with immune checkpoint inhibitors such as PD-1/PD-L1/CTLA4.
Gene therapy.
Antiviral, anti-pathogenic, and anti-cancer uses.
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