Gene-edited natural killer cells
Inventors
SLUCH, Valentin • Rezania, Alireza • Sagert, Jason • SWAIN, Danielle
Assignees
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Publication Number
US-12344655-B2
Publication Date
2025-07-01
Expiration Date
Abstract
The present invention relates to, inter alia, an engineered cell (e.g., iPSC, IPS-derived NK, or NK cell) comprising a disrupted B2M gene and an inserted polynucleotide encoding one or more of SERPINB9, a fusion of IL15 and IL15Rα, and/or HLA-E. The engineered cell can further comprise a disrupted CIITA gene and an inserted polynucleotide encoding a CAR, wherein the CAR can be an anti-BCMA CAR or an anti-CD30 CAR. The engineered cell may further comprise a disrupted ADAM17 gene, a disrupted FAS gene, a disrupted CISH gene, and/or a disrupted REGNASE-1 gene. Methods for producing the engineered cells are also provided, and therapeutic uses of the engineered cells are also described. Guide RNA sequences targeting described target sequences are also described.
Core Innovation
The invention provides an in vitro method of generating a genetically modified cell by delivering CRISPR associated endonuclease components. The method includes a first CRISPR associated endonuclease and a first guide RNA targeting a target site in the CIITA gene locus, and a first vector is delivered such that a nucleic acid having left and right sequence homology with genomic regions flanking the CIITA target site is inserted after CIITA cleavage. Insertion disrupts the CIITA gene locus at the target site.
The inserted nucleic acid encodes HLA class I histocompatibility antigen, alpha chain E (HLA-E) and specifies that the encoded HLA-E forms an HLA-E trimer. The HLA-E trimer comprises a beta-2-microglobulin (B2M) signal peptide fused to an HLA-G presentation peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide. This construct is integrated via homology arms into the cleaved CIITA gene locus, combining CIITA disruption with expression of a defined HLA-E trimer design.
The claim coverage further refines the genetically modified cell generation by adding additional CRISPR mediated gene disruptions and by modifying the HLA-E coding sequence. Examples include targeting B2M with a vector encoding SERPINB9 and introducing mutations to disrupt CISH via a guide RNA and CRISPR associated endonuclease. The HLA-E encoding polynucleotide can include a coding sequence for a chimeric antigen receptor (CAR), and the junction between CAR and HLA-E can be specified as a self-cleaving peptide such as P2A, E2A, F2A, or T2A.
Claims Coverage
The partial content includes one independent claim describing an in vitro method for generating a genetically modified cell. The dependent claims refine the core features by specifying construct components and adding additional CRISPR disruptions.
In vitro CRISPR cleavage and CIITA locus insertion of an HLA-E trimer construct
An in vitro method comprising delivering a first CRISPR associated endonuclease, a first guide RNA targeting a target site in the CIITA gene locus, and a first vector with a nucleic acid containing left and right genomic homology regions flanking the CIITA target site, where the nucleic acid encodes HLA-E as an HLA-E trimer with a B2M signal peptide fused to an HLA-G presentation peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide; wherein CIITA is cleaved at the target site and the nucleic acid is inserted into the CIITA gene locus thereby disrupting CIITA.
Additional CRISPR-mediated disruption of a gene locus via targeted insertion or mutation
A method delivering an additional CRISPR associated endonuclease and an additional guide RNA targeting a target site in a gene locus, wherein the locus is cleaved and an insertion or deletion mutation is introduced to disrupt the gene, including examples directed to B2M and CISH.
CAR incorporated into the HLA-E coding sequence with defined self-cleaving peptide junction
The polynucleotide encoding HLA-E includes a coding sequence for a chimeric antigen receptor (CAR), and the junction between CAR and HLA-E is defined using a self-cleaving peptide selected from P2A, E2A, F2A, or T2A.
Narrowed CRISPR nuclease and guide RNA target sequence selections
The first CRISPR associated endonuclease is a Cas9 nuclease, and the first gRNA includes a spacer sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 13-17.
The dominant inventive concept is in vitro CRISPR cleavage at the CIITA gene locus with insertion of a defined HLA-E trimer construct flanked by CIITA homology regions to disrupt CIITA. Refinements include additional CRISPR disruptions at other loci, incorporation of a CAR into the HLA-E coding sequence with a specified self-cleaving peptide junction, and narrowed CRISPR nuclease and guide RNA spacer selections.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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