JP-2022531577-A5 -

Dates

Publication Date

20230511

Application Date

20200430

Description

A kit is also provided, comprising: one or more activators capable of inducing gene disruption at a target site within the CD247 locus; a polynucleotide comprising a nucleic acid sequence encoding a chimeric receptor or a portion thereof, wherein the transgene encoding the chimeric receptor or an antigen-binding fragment or chain is targeted for integration at or near the target site via homology-directed repair (HDR); and instructions for carrying out any of the methods provided herein. [Invention 1001] Genetically engineered T cells comprising a modified CD247 locus, wherein the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric receptor that includes an intracellular region comprising a CD3 zeta (CD3ζ) signaling domain. [Invention 1002] The genetically engineered T cell according to the present invention 1001, wherein the nucleic acid sequence includes a transgene sequence encoding a portion of a chimeric receptor, and the transgene sequence is optionally incorporated into the endogenous CD247 locus of the T cell via homology-directed repair (HDR). [Invention 1003] Genetically engineered T cells according to Invention 1001 or Invention 1002, wherein the entire CD3ζ signaling domain or a fragment of the CD3ζ signaling domain is encoded by an open reading frame or a partial sequence of the endogenous CD247 locus. [Invention 1004] A genetically engineered T cell according to any of invention 1001 to 1003, wherein the nucleic acid sequence encoding the chimeric receptor comprises an in-frame fusion of (i) a transgene sequence encoding a portion of the chimeric receptor and (ii) an open reading frame or a portion thereof of the endogenous CD247 locus. [Invention 1005] Genetically engineered T cells comprising a modified CD247 locus, wherein the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric receptor comprising an intracellular region comprising a CD3 zeta (CD3ζ) signaling domain, and the nucleic acid sequence comprises an in-frame fusion of (i) a transgene sequence encoding a portion of the chimeric receptor and (ii) an open reading frame or a sub-sequence of an endogenous CD247 locus encoding a CD3ζ signaling domain. [Invention 1006] A genetically engineered T cell according to any of invention 1002 to 1005, wherein the transgene sequence is in-frame with one or more exons of the open reading frame or a sub-frame of the endogenous CD247 locus. [Invention 1007] A genetically engineered T cell according to any of invention 1002 to 1006, wherein the transgene sequence does not contain a sequence encoding the 3'UTR and/or does not contain an intron. [Invention 1008] A genetically modified T cell according to any of invention 1002 to 1007, wherein the transgene sequence either encodes a fragment of the CD3ζ signaling domain or does not encode the CD3ζ signaling domain or a fragment thereof. [Invention 1009] A genetically engineered T cell according to any of invention 1003 to 1008, wherein the open reading frame or a subsequence thereof comprises at least one intron and at least one exon of the endogenous CD247 locus and/or encodes the 3'UTR of the endogenous CD247 locus. [Invention 1010] Genetically modified T cells according to any of invention 1002 to 1009, wherein the transgene sequence is located downstream of exon 1 and upstream of exon 8 of the open reading frame of the endogenous CD247 locus; optionally, downstream of exon 1 and upstream of exon 3 of the open reading frame of the endogenous CD247 locus. [Invention 1011] Genetically engineered T cells according to any of the inventions 1001 to 1010, wherein at least a fragment of the CD3ζ signaling domain of the encoded chimeric receptor, optionally the entire CD3ζ signaling domain, is encoded by the open reading frame or a partial sequence of the endogenous CD247 locus, and optionally the CD3ζ signaling domain is encoded by a sequence of nucleotides comprising at least a portion of exon 2 and exons 3-8 of the open reading frame of the endogenous CD247 locus; or a sequence of nucleotides that does not include exon 1, does not include the full length of exon 1, and/or does not include the full length of exon 2. [Invention 1012] A genetically engineered T cell according to any of invention 1001 to 1011, wherein the encoded chimeric receptor can be transmitted via the CD3ζ signaling domain. [Invention 1013] Genetically modified T cells according to any of invention 1001 to 1012, wherein the encoded CD3ζ signaling domain comprises a sequence selected from any one of SEQ ID NO:13 to 15, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with any one of SEQ ID NO:13 to 15, or a fragment thereof. [Invention 1014] A genetically modified T cell according to any of the invention 1001 to 1013, wherein the chimeric receptor is a chimeric antigen receptor (CAR). [Invention 1015] A genetically modified T cell according to any of inventi