Mir155 as the integration site to overcome acute graft versus host disease of gene-modified immune effector cells

Patent metadata

Field	Value
Publication number	US-2025032545-A1
Application number	US-202418785173-A
Country	US
Kind code	A1
Filing date	Jul 26, 2024
Priority date	Jul 26, 2023
Publication date	Jan 30, 2025
Grant date	—

Abstract

The present disclosure relates to CAR-T cells and uses thereof. Disclosed herein is a genetically modified T cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) polypeptide comprising a single-chain variable fragment (scFV) that specifically binds to a target molecule, wherein the nucleic acid sequence encoding the CAR polypeptide is integrated into an integration site located at a miR-155 host gene.

First claim

We claim:

1 . A genetically modified T cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) polypeptide comprising a single-chain variable fragment (scFV) that specifically binds to a target molecule, wherein the nucleic acid sequence encoding the CAR is integrated into an integration site located at a miR-155 host gene.

2 . The genetically modified T cell of claim 1 , wherein the integration site is at exon 1, exon 2, exon 3, or a transcriptional start site of the miR-155 host gene.

3 . The genetically modified T cell of claim 2 , wherein the integration site is at exon 3 of the miR-155 host gene.

4 . The genetically modified T cell of claim 1 , further comprising:
a ribonucleoprotein (RNP) complex comprising a CRISPR/Cas endonuclease (Cas9) system complexed with one or more guide RNAs targeting the miR-155 host gene or a fragment thereof; and an AAV vector comprising a polynucleotide sequence encoding the CAR polypeptide; wherein the polynucleotide sequence is flanked by a first homology arm and a second homology arm.

5 . The genetically modified T cell of claim 4 , wherein the first homology arm and the second homology arm comprise a sequence at least 80% identical to SEQ ID NO: 3 or 4, or a fragment thereof.

6 . The genetically modified T cell of claim 4 , wherein the CRISPR/Cas9 system comprises a first guide RNA and a second guide RNA.

7 . The genetically modified T cell of claim 6 , wherein the first guide RNA comprises a polynucleotide sequence at least 80% identical to SEQ ID NO: 1 or a fragment thereof.

8 . The genetically modified T cell of claim 6 , wherein the second guide RNA comprises a polynucleotide sequence at least 80% identical to SEQ ID NO: 2 or a fragment thereof.

9 . The genetically modified T cell of claim 1 , wherein the target molecule is a cancer-related protein.

10 . The genetically modified T cell of claim 9 , wherein the cancer-related protein is CD19, GD2, or CD33.

11 . The genetically modified T cell of claim 4 , wherein the AAV vector comprises a polynucleotide sequence at least 80% identical to SEQ ID NO: 6 or a fragment thereof.

12 . The genetically modified T cell of claim 1 , wherein the T cell is a primary T cell, a T cell line, a tumor infiltrating lymphocyte, an effector T cell, a memory T cell, a TEMRA, or a stem cell-like memory T cell.

13 . A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the genetically modified T cell of claim 1 .

14 . The method of claim 13 , wherein the T cell is cultured ex vivo for at least 2 days prior to administration to the subject.

15 . The method of claim 13 , wherein the T cell is cultured ex vivo for at least 7 days prior to administration to the subject.

16 . The method of claim 13 , wherein the T cell is cultured with IL-2, IL-7, or IL-15, or any combination thereof.

17 . The method of claim 13 , wherein the T cell is derived from the subject.

18 . The method of claim 13 , wherein administration of the genetically modified T cell prevents or treats acute graft-versus-host disease.

19 . A gene-editing system for engineering a CAR-T cell, said system comprising:
a ribonucleoprotein (RNP) complex comprising a CRISPR/Cas endonuclease (Cas9) system complexed with one or more guide RNAs targeting a miR-155 host gene or a fragment thereof; and an AAV vector comprising a polynucleotide sequence encoding a CAR polypeptide; wherein the polynucleotide sequence is flanked by a first homology arm and a second homology arm.

20 . A method of creating a genetically modified T cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) polypeptide integrated into an integration site located at a miR-155 host gene, said method comprising:
obtaining a T cell; and introducing into the T cell the gene-editing system of claim 19 , thereby creating the genetically modified T cell.

Assignees

• Ohio State Innovation Foundation
• Nationwide Childrens Hospital

Inventors

• Ranganathan Parvathi
• Naeimi Kararoudi Meisam
• Sezgin Yasemin
• Lee Dean

Classifications

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P37/06

  Immunosuppressants, e.g. drugs for graft rejection · CPC title

• A61K40/4224

  Molecules with a "CD" designation not provided for elsewhere · CPC title

• A61K40/31

  Chimeric antigen receptors [CAR] · CPC title