Homology-independent targeted DNA insertion in human t cells

HITI with CRISPR enrichment and GMP-grade reagents addresses inefficiencies in CAR integration in human T cells, enhancing cell yields and purity to achieve a therapeutically relevant dose range.

HK40134688APending Publication Date: 2026-07-10THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
View PDF 0 Cites 0 Cited by

Patent Information

Authority / Receiving Office
HK · HK
Patent Type
Applications
Current Assignee / Owner
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
Filing Date
2026-04-17
Publication Date
2026-07-10

AI Technical Summary

Technical Problem

Existing methods for site-directed CAR integration in primary human T cells using DNA delivery are inefficient, leading to low cell yields and require improvements for therapeutic relevance.

Method used

A method utilizing homology-independent targeted insertion (HITI) with CRISPR enrichment and GMP-grade reagents to enhance CAR T cell purity to approximately 80%, enabling higher cell yields.

Benefits of technology

The method achieves a therapeutically relevant dose range of CAR+ T cells by improving cell yields and purity through HITI and CRISPR enrichment.

✦ Generated by Eureka AI based on patent content.

Smart Images

  • Figure 00000000_0000_ABST
    Figure 00000000_0000_ABST
Patent Text Reader

Abstract

The present disclosure provides methods and compositions for site directed CAR integration in primary human T cells using DNA delivered via homology-independent targeted insertion (HITI). The methods provide higher cell yield compared to homology-directed repair (HDR) mediated gene insertion. Post-HITI CRISPR EnrichMENT (CEMENT) using GMP grade reagents enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of CAR+ T cells.
Need to check novelty before this filing date? Find Prior Art

Description

Abstract This disclosure provides methods and compositions for site-directed CAR integration in primary human T cells using DNA delivered via homology-independent targeted insertion (HITI). Compared to homology-directed repair (HDR)-mediated gene insertion, the method provides higher cell yields. CAR T cells are enriched to approximately 80% purity using CRISPR enrichment (cement) after HITI with GMP-grade reagents, thereby generating a therapeutically relevant dose range of CAR+ T cells.

Claims

WHAT IS CLAIMED IS:

1. A method for enriching for T cells that express a chimeric antigen receptor (CAR) inserted at a target genomic locus following homology-independent targeted insertion (HITI), comprising: transfecting a plurality of T cells with (a) a linear nucleic acid molecule comprising: (i) an expression cassette comprising a promoter operably linked to a sequence encoding a CAR and a sequence encoding a selectable marker; and (ii) a single protospacer adjacent motif (PAM) sequence that binds a guide RNA-CRISPR / Cas9 ribonucleoprotein (RNP), wherein the PAM is located 5’ or 3’ of the expression cassette of (i); and (b) a guide RNA-CRISPR / Cas9 ribonucleoprotein (RNP); wherein the nucleic acid molecule is inserted into a target site in the genome of the T cell via non-homologous end joining (NHEJ); and isolating T cells that express the selectable marker, wherein the number of CAR+ T cells is increased compared to T cells that do not express the selectable marker.

2. The method of claim 1, wherein the nucleic acid molecule does not comprise sequences that are homologous to genomic sequences at the target genomic locus.

3. The method of claim 1 or 2, wherein the single PAM is located 5’ of the expression cassette of (i).

4. The method of claim 1, wherein the nucleic acid molecule is inserted on the positive or negative strand of the target genomic locus.

5. The method of claim 1, wherein the nucleic acid molecule comprises a plasmid having a plasmid backbone of less than 500 bp.

6. The method of claim 1, wherein the plasmid comprises an insert size of at least 2000 bp.

7. The method of claim 6, wherein the plasmid comprises an insert size between 2000 and 5000 bp.

8. The method of claim 1, wherein the promoter is an EF-1Į promoter or EF-1Į short (EFS) promoter.

9. The method of claim 1, wherein the promoter comprises SEQ ID NO:

1.

10. The method of claim 1, wherein the T cells are activated T cells.

11. The method of claim 1, wherein the T cells are activated prior to transfection with the nucleic acid molecule.

12. The method of claim 1, wherein the T cells are incubated with an inhibitor of homology directed repair (HDR).

13. The method of claim 12, wherein the inhibitor of HDR is selected from the group consisting of an ATM / ATR kinase inhibitor, a Chk1 / Chk2 inhibitor, a BRCA1 inhibitor, a Rad51 inhibitor, and combinations thereof.

14. The method of claim 13, wherein the inhibitor of HDR is an ATM / ATR kinase inhibitor.

15. The method of claim 1, wherein the selectable marker is a protein expressed on the cell surface and expression of the selectable marker is detected by contacting an antibody to the selectable marker.

16. The method of claim 15, wherein the protein expressed on the cell surface is tEGFR or tNGFR.

17. The method of claim 1, wherein the selectable marker is a protein that confers resistance to a drug or compound.

18. The method of claim 17, wherein the selectable marker is DHFR-FS, and the T cells are cultured with methotrexate (MTX) for a period of time to reduce the number of cells that do not express DHFR-FS.

19. The method of claim 18, wherein the T cells are cultured with MTX beginning on day 1 after transfection until day 5 after transfection.

20. The method of claim 18, wherein the T cells are cultured with MTX beginning on day 1 after transfection until day 4 after transfection, followed by culturing the T cells without MTX for 3 to 7 days.

21. The method of claim 18, wherein the T cells are cultured with MTX beginning on day 5 after transfection until day 12 after transfection.

22. The method of claim 18, wherein the frequency of CAR+ T cells is equal to or greater than 70%.

23. The method of claim 19, wherein the T cells T cells are cultured with MTX beginning on day 1 after transfection until day 5 after transfection, and are then cultured without MTX until day 12 after transfection, and the yield of CAR+ T cells at day 12 is similar to the yield of CAR+ T cells cultured with MTX beginning on day 5 after transfection until day 12 after transfection.

24. The method of claim 1, wherein the transfection comprises electroporation.

25. The method of claim 1, wherein the CAR binds to an antigen selected from the group consisting of Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan- A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA- 125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, Mesothelin, GPC3, MUC1 and CTAG1B.

26. The method of claim 1, wherein the nucleic acid molecule is integrated into the T cell receptor alpha constant (TRAC), beta-2-microglobulin (B2M), or adeno- associated virus integration site 1 (AAVS1) genomic locus.

27. The method of claim 1, wherein the T cell is a primary human T cell.

28. The method of claim 1, wherein the T cells are unstimulated T cells.

29. A method for treating a tumor in a subject, comprising administering to the subject an effective amount of a genetically modified T cell comprising:a nucleic acid molecule comprising: (i) an expression cassette comprising a promoter operably linked to a sequence encoding a chimeric antigen receptor (CAR) and a sequence encoding a selectable marker; and (ii) a single protospacer adjacent motif (PAM) sequence that binds a guide RNA-CRISPR / Cas9 ribonucleoprotein (RNP), wherein the PAM is located 5’ or 3’ of the expression cassette of (i); wherein the nucleic acid molecule is inserted into a target site in the genome of the T cell via non-homologous end joining (NHEJ), thereby treating the tumor.

30. The method of claim 29, wherein the nucleic acid molecule does not comprise sequences that are homologous to genomic sequences at the target genomic locus.

31. The method of claim 29 or 30, wherein the single PAM is located 5’ of the expression cassette of (i).

32. The method of claim 29, wherein the nucleic acid molecule comprises a plasmid having a plasmid backbone of less than 500 bp.

33. The method of claim 29, wherein the plasmid comprises an insert size between 2000 and 5000 bp.

34. The method of claim 29, wherein the promoter is an EF-1Į promoter or EF-1Į short (EFS) promoter.

35. The method of claim 29, wherein the promoter comprises SEQ ID NO:

1.

36. The method of claim 29, wherein the selectable marker is DHFR-FS, and the T cells are cultured with methotrexate (MTX) for a period of time to reduce the number of cells that do not express DHFR-FS.

37. The method of claim 36, wherein the T cells are cultured with MTX beginning on day 1 after transfection until day 4 after transfection, followed by culturing the T cells without MTX for 3 to 7 days.

38. The method of claim 36, wherein the T cells are cultured with MTX beginning on day 7 after transfection until day 14 after transfection.

39. The method of claim 29, wherein the selectable marker is a protein expressed on the cell surface and expression of the selectable marker is detected by contacting an antibody to the selectable marker.

40. The method of claim 39, wherein the protein expressed on the cell surface is tEGFR or tNGFR.

41. The method of claim 29, wherein the CAR binds to an antigen selected from the group consisting of Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan- A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA- 125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, Mesothelin, GPC3, MUC1 and CTAG1B.

42. The method of claim 29, wherein the nucleic acid molecule is integrated into the T cell receptor alpha constant (TRAC), beta-2-microglobulin (B2M), or adeno- associated virus integration site 1 (AAVS1) genomic locus.

43. The method of claim 29, wherein the T cells are activated prior to transfection with the nucleic acid molecule.

44. The method of claim 29, wherein 1 x 106to 1 x 109CAR+ T cells are administered to the subject.

45. The method of claim 29, wherein 0.1 x 106to 5 x 106CAR+ T cells / kg of the subject’s weight are administered to the subject.

46. The method of claim 29, wherein the CAR+ T cells are administered in one or more doses.

47. A genetically modified T cell comprising: a nucleic acid molecule comprising: (i) an expression cassette comprising a promoter operably linked to a sequence encoding a CAR and a sequence encoding DHFR-FS; and(ii) a single protospacer adjacent motif (PAM) sequence that binds a guide RNA-CRISPR / Cas9 ribonucleoprotein (RNP), wherein the PAM is located 5’ or 3’ of the expression cassette of (i).

48. The genetically modified T cell of claim 47, wherein the promoter is an EF-1Į promoter or EF-1Į short (EFS) promoter.

49. The genetically modified T cell of claim 48, wherein the promoter comprises SEQ ID NO:

1.

50. The genetically modified T cell of claim 47, wherein the CAR binds to an antigen selected from the group consisting of Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY- SEO-1, tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, Mesothelin, GPC3, MUC1 and CTAG1B.

51. The genetically modified T cell of claim 47, wherein the T cell further comprises a guide RNA and CRISPR / Cas9 nuclease.

52. The genetically modified T cell of claim 47, wherein the nucleic acid molecule is integrated into the T cell receptor alpha constant (TRAC), beta-2-microglobulin (B2M), or adeno-associated virus integration site 1 (AAVS1) genomic locus.

53. The genetically modified T cell of claim 47, wherein the T cell is a primary human T cell.

54. The genetically modified T cell of claim 47, wherein the T cell is an activated T cell.

55. The genetically modified T cell of claim 47, wherein the T cell is an unstimulated T cell.

56. A pharmaceutical composition comprising the genetically modified T cell of claim 47.