IP Library › Granted Patent US 12,398,193
Granted Patent B2
US 12,398,193 · App. 18/160,825 · Granted Aug 26, 2025

Targeted replacement of endogenous T cell receptors

Inventors: Theodore Lee Roth (San Francisco, CA); Eric Shifrut (San Francisco, CA); Alexander Marson (San Francisco, CA); Cristina Puig Saus (Los Angeles, CA); Antoni Ribas (Los Angeles, CA)
Assignee: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
C07K14/7051A61K40/11A61K40/32A61K40/4269C12N5/0637C12N9/22C12N15/113C12N15/85C12N15/907C12N2310/20
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,398,193
App. No.
18/160,825
Granted
Aug 26, 2025
Kind
B2
Abstract

Provided herein are methods and compositions for editing the genome of a human T cell. In some embodiments, a heterologous T cell receptor (TCR)-β chain and a heterologous TCR-α chain are inserted into exon 1 of a TCR subunit constant gene in the genome of the T cell.

Claims (40)

1. A primary human T cell comprising at least one nucleic acid sequence comprising at least one heterologous gene in at least one target region of one or both of:

an endogenous T cell receptor alpha subunit constant gene (TRAC),

and an endogenous T cell receptor beta subunit constant gene (TRBC),

wherein the T cell does not comprise a viral vector for introducing the at least one nucleic acid sequence to the T cell, and

wherein the at least one heterologous gene is at least 500 bp in size.

2. The T cell of claim 1 , wherein the at least one nucleic acid sequence is at least 1.5 kb in size.

3. The T cell of claim 1 , wherein the at least one nucleic acid sequence is in an exon of TRAC or in an exon of TRBC.

4. The T cell of claim 1 , wherein the target region is in exon 1, 2, or 3 of TRAC, or in exon 1, 2, or 3 of TRBC.

5. The T cell of claim 1 , wherein the at least one nucleic acid sequence is up to 1.5 kb in size.

6. The T cell of claim 1 , wherein the at least one heterologous gene comprises at least one of:

(a) a variable region of a heterologous T cell receptor alpha (TCR-α) chain; and

(b) a variable region of a heterologous T cell receptor beta (TCR-β) chain.

7. The T cell of claim 6 , wherein the at least one heterologous gene comprises at least one of:

(1) (a) variable region or (b) variable region and constant region of the heterologous TCR-α chain gene; and

(2) (a) variable region or (b) variable region and constant region of the heterologous TCR-β chain gene.

8. The T cell of claim 7 , wherein the T cell comprises each of:

(1) the a) variable region or b) variable region and constant region of the heterologous TCR-α chain gene; and

(2) the a) variable region or b) variable region and constant region of the heterologous TCR-β chain gene.

9. The T cell of claim 7 , wherein the at least one heterologous gene comprises:

(1) the (a) variable region or (b) variable region and constant region of the heterologous TCR-α chain gene; and

(2) the (a) variable region or (b) variable region and constant region of the heterologous TCR-β chain gene.

10. The T cell of claim 9 , wherein, upon expression, the heterologous TCR-α chain and the heterologous TCR-β form an antigen-specific T cell receptor (TCR).

11. The T cell of claim 10 , wherein the heterologous TCR-α chain gene and the heterologous TCR-β chain gene are operably linked by a linker sequence or a multicistronic element.

12. The T cell of claim 11 , wherein the linker sequence is a cleavable linker sequence.

13. The T cell of claim 9 , wherein the at least one heterologous gene encodes the heterologous TCR-α chain gene and the heterologous TCR-β chain gene, and wherein the at least one heterologous gene is in the endogenous TRAC.

14. The T cell of claim 1 , wherein the at least one heterologous gene is in an endogenous TRAC, wherein the at least one heterologous gene comprises:

(1) a) a variable region of the heterologous TCR-α chain or b) a variable region and constant region of the heterologous TCR-α chain; and

(2) a) a variable region of the heterologous TCR-β chain or b) a variable region and constant region of the heterologous TCR-β chain,

wherein the heterologous TCR-α chain and the heterologous TCR-β chain are operably linked by a cleavable linker sequence that is cleaved to generate the heterologous TCR-α chain and the heterologous TCR-chain, and wherein the heterologous TCR-α chain and the heterologous TCR-β form an antigen-specific T cell receptor (TCR) that recognizes the target antigen expressed by a cancer.

15. The T cell of claim 1 , wherein expression of the at least one heterologous gene is driven by an endogenous promoter.

16. The T cell of claim 1 , wherein expression of one or both of endogenous TRAC and endogenous TRBC is reduced in the cell relative to a control T cell, wherein the control T cell is a primary human T cell that lacks an insertion into the endogenous TRAC or TRBC.

17. The T cell of claim 1 , wherein the T cell is a CD8+ T cell or a CD4+ T cell.

18. A primary human T cell comprising at least one nucleic acid sequence comprising at least one heterologous gene non-virally inserted into at least one target region of one or both of:

an endogenous T cell receptor alpha subunit constant gene (TRAC),

and an endogenous T cell receptor beta subunit constant gene (TRBC),

wherein the at least one heterologous gene is at least 500 bp in size.

19. The T cell of claim 18 , wherein the T cell does not comprise a viral vector for introducing the at least one nucleic acid sequence to the T cell.

20. A population of T cells comprising a plurality of the primary human T cells of claim 1 .

21. The population of claim 20 , wherein the genome of at least 10% of the population comprises the at least one nucleic acid sequence.

22. A population of T cells comprising a plurality of the primary human T cells of claim 18 .

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 23, 2023
From: MARSON, ALEXANDER; ROTH, THEODORE LEE; SHIFRUT, ERIC; PUIG SAUS, CRISTINA; RIBAS, ANTONI
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 064044/0063 →
Continuity (7)
Continuation 17725478 · Apr 20, 2022
Continuation 17390673 · Jul 30, 2021
Continuation 17200301 · Mar 12, 2021
Continuation 16568116 · Sep 11, 2019
Continuation PCTUS2018058026 · Oct 29, 2018
Provisional Application 62578153 · Oct 27, 2017
Related Publication 20230310502A1 · Oct 5, 2023
References Cited (167)
US 6824978B1 · Cox, III et al. · 2004 [cited by applicant]
US 8133733B2 · Khan · 2012 [cited by applicant]
US 8586363B2 · Voytas et al. · 2013 [cited by applicant]
US 8795965B2 · Zhang · 2014 [cited by applicant]
US 9221886B2 · Liu et al. · 2015 [cited by applicant]
US 9512446B1 · Joung et al. · 2016 [cited by applicant]
US 9526784B2 · Liu et al. · 2016 [cited by applicant]
US 9758775B2 · Voytas et al. · 2017 [cited by applicant]
US 9855297B2 · Duchateau et al. · 2018 [cited by applicant]
US 9890393B2 · Duchateau et al. · 2018 [cited by applicant]
US 11033584B2 · Roth et al. · 2021 [cited by applicant]
US 11083753B1 · Roth et al. · 2021 [cited by applicant]
US 20020064802A1 · Raschke et al. · 2002 [cited by applicant]
US 20020160940A1 · Case et al. · 2002 [cited by applicant]
US 20030087817A1 · Cox, III et al. · 2003 [cited by applicant]
US 20030232410A1 · Liljedahl et al. · 2003 [cited by applicant]
US 20050136040A1 · Hart et al. · 2005 [cited by applicant]
US 20060182736A1 · Kim et al. · 2006 [cited by applicant]
US 20070254291A1 · Cui et al. · 2007 [cited by applicant]
US 20090082250A1 · Hart et al. · 2009 [cited by applicant]
US 20100093617A1 · Barrangou et al. · 2010 [cited by applicant]
US 20110145940A1 · Voytas et al. · 2011 [cited by applicant]
US 20110265198A1 · Gregory et al. · 2011 [cited by applicant]
US 20120077270A1 · Hart et al. · 2012 [cited by applicant]
US 20120110685A1 · Bonas et al. · 2012 [cited by applicant]
US 20130236504A1 · Alexis et al. · 2013 [cited by applicant]
US 20140068797A1 · Doudna et al. · 2014 [cited by applicant]
US 20140189896A1 · Zhang et al. · 2014 [cited by applicant]
US 20140199767A1 · Barrangou et al. · 2014 [cited by applicant]
US 20140294773A1 · Brouns et al. · 2014 [cited by applicant]
US 20140301990A1 · Gregory et al. · 2014 [cited by applicant]
US 20140349402A1 · Cooper et al. · 2014 [cited by applicant]
US 20140357530A1 · Zhang et al. · 2014 [cited by applicant]
US 20150016954A1 · Thibodeau et al. · 2015 [cited by applicant]
US 20150071903A1 · Liu et al. · 2015 [cited by applicant]
US 20150110762A1 · Holmes et al. · 2015 [cited by applicant]
US 20150164954A1 · Bonini et al. · 2015 [cited by applicant]
US 20150259704A1 · Church et al. · 2015 [cited by applicant]
US 20150283265A1 · Peyman · 2015 [cited by applicant]
US 20150322457A1 · Kim et al. · 2015 [cited by applicant]
US 20150344912A1 · Kim et al. · 2015 [cited by applicant]
US 20160017366A1 · Chen et al. · 2016 [cited by applicant]
US 20160024524A1 · Joung et al. · 2016 [cited by applicant]
US 20160053274A1 · D'Halluin · 2016 [cited by applicant]
US 20160102322A1 · Ravinder et al. · 2016 [cited by applicant]
US 20160160210A1 · Mali et al. · 2016 [cited by applicant]
US 20160200779A1 · Liu et al. · 2016 [cited by applicant]
US 20160264999A1 · Rao et al. · 2016 [cited by applicant]
US 20160298134A1 · Chen et al. · 2016 [cited by applicant]
US 20160298135A1 · Chen et al. · 2016 [cited by applicant]
US 20160298138A1 · Chen et al. · 2016 [cited by applicant]
US 20160304893A1 · Daboussi et al. · 2016 [cited by applicant]
US 20170000743A1 · Ghoroghchian et al. · 2017 [cited by applicant]
US 20170016027A1 · Lee et al. · 2017 [cited by applicant]
US 20170044569A9 · Church et al. · 2017 [cited by applicant]
US 20170073705A1 · Chen et al. · 2017 [cited by applicant]
US 20170175128A1 · Welstead et al. · 2017 [cited by applicant]
US 20170191082A1 · Chen et al. · 2017 [cited by applicant]
US 20170211075A1 · Lee et al. · 2017 [cited by applicant]
US 20170290858A1 · Zhao et al. · 2017 [cited by applicant]
US 20170296676A1 · Stephan et al. · 2017 [cited by applicant]
US 20170335331A1 · Zhao et al. · 2017 [cited by applicant]
US 20180044700A1 · Doudna et al. · 2018 [cited by applicant]
US 20180161447A1 · Watson et al. · 2018 [cited by applicant]
US 20180312848A1 · Zhao et al. · 2018 [cited by applicant]
US 20190388469A1 · Marson et al. · 2019 [cited by applicant]
US 20200048606A1 · Marson et al. · 2020 [cited by applicant]
US 20200362355A1 · Roth et al. · 2020 [cited by applicant]
WO 0041566A1 · 2000 [cited by applicant]
WO 0183751A2 · 2001 [cited by applicant]
WO 0183751A3 · 2002 [cited by applicant]
WO 03033701A1 · 2003 [cited by applicant]
WO 03080809A2 · 2003 [cited by applicant]
WO 2004092194A2 · 2004 [cited by applicant]
WO 2004108883A2 · 2004 [cited by applicant]
WO 2005123962A2 · 2005 [cited by applicant]
WO 2007025097A2 · 2007 [cited by applicant]
WO 2008021207A2 · 2008 [cited by applicant]
WO 2011059836A2 · 2011 [cited by applicant]
WO 2011072246A2 · 2011 [cited by applicant]
WO 2011139336A1 · 2011 [cited by applicant]
WO 2013134349A1 · 2013 [cited by applicant]
WO 2013176772A1 · 2013 [cited by applicant]
WO 2014089290A1 · 2014 [cited by applicant]
WO 2014093661A2 · 2014 [cited by applicant]
WO 2014144761A2 · 2014 [cited by applicant]
WO 2014153470A2 · 2014 [cited by applicant]
WO 2014161821A1 · 2014 [cited by applicant]
WO 2015035136A2 · 2015 [cited by applicant]
WO 2015048690A1 · 2015 [cited by applicant]
WO 2015057980A1 · 2015 [cited by applicant]
WO 2015073867A1 · 2015 [cited by applicant]
WO 2015086795A1 · 2015 [cited by applicant]
WO 2015089419A2 · 2015 [cited by applicant]
WO 2015089462A1 · 2015 [cited by applicant]
WO 2015089486A2 · 2015 [cited by applicant]
WO 2015115903A1 · 2015 [cited by applicant]
WO 2015117021A1 · 2015 [cited by applicant]
WO 2015089419A9 · 2015 [cited by applicant]
WO 2016036754A1 · 2016 [cited by applicant]
WO 2016049251A1 · 2016 [cited by applicant]
WO 2016057951A2 · 2016 [cited by applicant]
WO 2016069282A1 · 2016 [cited by applicant]
WO 2016097751A1 · 2016 [cited by applicant]
WO 2016118697A1 · 2016 [cited by applicant]
WO 2016123578A1 · 2016 [cited by applicant]
WO 2016118697A9 · 2016 [cited by applicant]
WO 2016135559A2 · 2016 [cited by applicant]
WO 2016172359A2 · 2016 [cited by applicant]
WO 2016196388A1 · 2016 [cited by applicant]
WO 2016205680A1 · 2016 [cited by applicant]
WO 2016205703A1 · 2016 [cited by applicant]
WO 2017004509A1 · 2017 [cited by applicant]
WO 2017011519A1 · 2017 [cited by applicant]
WO 2017035659A1 · 2017 [cited by applicant]
WO 2017044661A1 · 2017 [cited by applicant]
WO 2017053729A1 · 2017 [cited by applicant]
WO 2017058751A1 · 2017 [cited by applicant]
WO 2017062451A1 · 2017 [cited by applicant]
WO 2017070056A1 · 2017 [cited by applicant]
WO 2017070169A1 · 2017 [cited by applicant]
WO 2017070429A1 · 2017 [cited by applicant]
WO 2017079673A1 · 2017 [cited by applicant]
WO 2017091630A1 · 2017 [cited by applicant]
WO 2017106528A2 · 2017 [cited by applicant]
WO 2017115128A2 · 2017 [cited by applicant]
WO 2017156484A1 · 2017 [cited by applicant]
WO 2017177137A1 · 2017 [cited by applicant]
WO 2017180989A2 · 2017 [cited by applicant]
WO 2017181110A1 · 2017 [cited by applicant]
WO 2017186550A1 · 2017 [cited by applicant]
WO 2017189336A1 · 2017 [cited by applicant]
WO 2017210334A1 · 2017 [cited by applicant]
WO 2017220527A1 · 2017 [cited by applicant]
WO 2018035387A1 · 2018 [cited by applicant]
WO 2018039084A1 · 2018 [cited by applicant]
WO 2018068135A1 · 2018 [cited by applicant]
WO 2018073391A1 · 2018 [cited by applicant]
WO 2018073393A2 · 2018 [cited by applicant]
WO 2018094291A1 · 2018 [cited by applicant]
WO 2019089610A1 · 2019 [cited by applicant]
Sharpe et al. (2015, Disease, Models and Mechanisms, vol. 8, pp. 337-350) (Year: 2015). [cited by examiner]
Baldanzi et al., “SAP-Mediated Inhibition of Diacylglycerol Kinase a Regulates TCR-Induced Diacylglycerol Signaling,” The Journal of Immunology, vol. 187, No. 11, Dec. 1, 2011, pp. 5941-5951. [cited by applicant]
Chira et al., “Progresses Towards Safe and Efficient Gene Therapy Vectors,” Oncotarget, vol. 6, No. 31, 2015, pp. 30675-30703. [cited by applicant]
Cornu et al., “T Refining Strategies to Translate Genome Editing to the Clinic,” Nat. Med. 23, 2017, pp. 415-423. [cited by applicant]
EP18871537.9, Extended European Search Report, Oct. 6, 2021, 14 pages. [cited by applicant]
Eyquem et al., “Targeting a CAR to the TRAC Locus with CRISPR/Cas9 Enhances Tumour Rejection,” Nature, vol. 543, No. 7643, 2017, pp. 113-117. [cited by applicant]
Foley et al., “HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer,” Molecular Therapy Oncolytics, vol. 5, Jun. 16, 2017, pp. 105-115. [cited by applicant]
Glusman et al., “Comparative Genomics of the Human and Mouse T Cell Receptor Loci,” Immunity, vol. 15, No. 3, Sep. 2001, pp. 337-349. [cited by applicant]
Hornung et al., “Intracellular DNA Recognition,” Nat. Rev. Immunol. 10, 2010, pp. 123-130. [cited by applicant]
Kaneko et al., “Simple Knockout by Electroporation of Engineered Endonucleases into Intact Rat Embryos,” Scientific Reports vol. 4, No. 6382, Oct. 1, 2014, 5 pages. [cited by applicant]
Liang et al., “Enhanced CRISPR/Cas9-Mediated Precise Genome Editing by Improved Design and Delivery of gRNA, Cas9 Nuclease, and Donor DNA,” Journal of Biotechnology, vol. 241, Jan. 10, 2017, pp. 136-146. [cited by applicant]
Mandal et al., “Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells Using CRISPR/Cas9,” Cell Stem Cell, vol. 15, No. 5, Nov. 6, 2014, 15 pages. [cited by applicant]
Okamoto et al., “A Promising Vector for TCR Gene Therapy: Differential Effect of siRNA, 2A Peptide, and Disulfide Bond on the Introduced TCR Expression,” Molecular Therapy—Nucleic Acids, vol. 1, No. 12, Dec. 18, 2012, p… [cited by applicant]
PCT/US2018/058026, “International Search Report and Written Opinion,” Jan. 17, 2019, 13 pages. [cited by applicant]
Rosenberg, “Adoptive Cell Transfer as Personalized Immunotherapy for Human Cancer,” Science vol. 80, No. 348, 2015, pp. 62-68. [cited by applicant]
Roth et al., “Reprogramming Human T Cell Function and Specificity with Non-Viral Genome Targeting,” bioRxiv preprint doi: https://doi.org/10.: 10:/183418, Dec. 7, 2017, pp. 1-75. [cited by applicant]
Roth et al., “Reprogramming Human T Cell Function and Specificity with Non-Viral Genome Targeting,” Nature, vol. 559, No. 7714, Jul. 19, 2018, pp. 405-409. [cited by applicant]
Sadelain et al., “Therapeutic T Cell Engineering,” Nature 545, 2017, pp. 423-431. [cited by applicant]
Schumann et al., “Generation of Knock-In Primary Human T Cells Using Cas9 Ribonucleoproteins,” Proceedings of the National Academy of Sciences, vol. 112, No. 33, 2015, pp. 10437-10442. [cited by applicant]
Verhoeven et al., “Lentiviral Vector Gene Transfer into Human T Cells,” Methods Mol. Biol. vol. 506, 2009, pp. 97-114. [cited by applicant]
Zhao et al., “High-Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation,” Mol. Ther., vol. 13, No. 1, Jan. 2006, pp. 151-159. [cited by applicant]
U.S. Appl. No. 16/568,116, “Corrected Notice of Allowability,” Apr. 1, 2021, 5 pages. [cited by applicant]
U.S. Appl. No. 16/568,116, “Non-Final Office Action,” Jun. 22, 2020, 8 pages. [cited by applicant]
U.S. Appl. No. 16/568,116, “Notice of Allowance,” Feb. 5, 2021, 10 pages. [cited by applicant]
PCT/US2018/058026, “International Preliminary Report on Patentability,” May 7, 2020, 11 pages. [cited by applicant]
U.S. Appl. No. 17/200,301, Notice of Allowance, May 27, 2021, 6 pages. [cited by applicant]