RECOMBINATION ACTIVATING GENE (RAG) INDUCED V(D)J GENE TARGETING

• US 20200131540A1
• Filed: 04/18/2018
• Published: 04/30/2020
• Est. Priority Date: 04/20/2017
• Status: Active Grant

• Current Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Original Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Inventors: BARZEL, Adi, DOTAN, Iris, PUNDAK-MINTZ, Carmel, NATAF, Daniel, FRIED, Miriam, GRITSENKO, Natalia
• Granted Patent: US 12,060,572 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2239/28 Expressing multiple CARs, TCRs or antigens
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4612 B-cells
• CPC Class Codes: A61K 39/4622 Antigen presenting cells
• CPC Class Codes: A61K 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/4632 T-cell receptors [TCR]; antibody T-cell receptor constructs
• CPC Class Codes: A61K 39/464412 CD19 or B4
• CPC Class Codes: A61K 39/46447 Mucins, e.g. MUC-1
• CPC Class Codes: A61K 39/464488 NY-ESO
• CPC Class Codes: A61K 39/464499 Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
• CPC Class Codes: C07K 16/1027 Paramyxoviridae, e.g. respiratory syncytial virus
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 5/0635 : B lymphocytes

Abstract

The present invention relates to methods for targeted insertion of at least one nucleic acid sequence/s of interest into a target genomic locus of a mammalian cell. More specifically, the methods of the invention are based on using nucleic acid cassettes comprising the nucleic acid sequence/s of interest and at least one recognition signal sequence (RSS), for insertion of the nucleic acid sequence of interest into the target genomic locus that is mediated by RAG-catalyzed recombination. The invention further provides cassettes, vectors and vehicles and cells comprising said cassettes, compositions and uses thereof in immunotherapy.

Claims

1. A method for targeted insertion of at least one nucleic acid sequence of interest into a target genomic locus of a mammalian cell, said method comprises the step of contacting a mammalian cell expressing the recombination activating gene (RAG) complex with at least one nucleic acid cassette comprising said nucleic acid sequence/s of interest and at least one recognition signal sequence (RSS), or with a vector comprising the same, wherein the insertion of said nucleic acid sequence of interest into the target genomic locus is mediated by RAG-catalyzed recombination between at least one genomic RSS flanking the target genomic locus and at least one RSS comprised within said nucleic acid cassette.

2. The method according to claim 1, wherein said nucleic acid cassette is flanked on both the 5′

and 3′

ends thereof by RSS, optionally, said RSS are at least one of 12 RSS, 23 RSS and 22 RSS, optionally, said cassette further comprises at least one genetic element, said genetic element is at least one of;

an internal ribosome entry site (IRES), a 2A peptide coding sequence, a promoter or any functional fragments thereof, a splice donor (SD), a splice acceptor (SA), a degron, a 3 frame stop, a protein stabilizing sequence, a signal peptide, a stop codon, a polyadenylation site, a transcription enhancer, a switch region, an mRNA stabilizing sequence and a protein stabilizing sequence, optionally, said cassette comprises two or more nucleic acid sequences of interest separated by at least one genetic element, said genetic element is at least one of;

an IRES, a 2A peptide coding sequence, a promoter or any functional fragments thereof, a splice donor (SD) and a splice acceptor (SA).

6. The method according to claim 1, wherein said nucleic acid sequence of interest is a protein coding nucleic acid sequence or a non-coding nucleic acid sequence, optionally, wherein:

(a) said nucleic acid sequence of interest is a protein coding sequence, said protein is at least one of;

a therapeutic protein or peptide, a prophylactic protein, a tolerizing protein, an immunizing protein, a toxic protein, a suicide protein, a marker protein, an imaging protein and a fusion or chimeric protein;

or

(b) said nucleic acid sequence of interest encodes at least one small non-coding RNA molecule.

9. The method according to claim 6, wherein said nucleic acid sequence of interest encodes at least one of a receptor and an antibody or any fragment/s or chimera/s thereof, optionally, wherein at least one of:

(a) said nucleic acid sequence of interest encodes at least one receptor, said receptor is any one of;

a T cell receptor (TCR), chimeric antigen receptor (CAR) and a B cell receptor (BCR);

(b) said nucleic acid sequence of interest encodes at least one antibody, said antibody is any one of;

full length antibody, antibody fragment, single-chain variable fragment (scFv), bi-specific antibody, tri-specific antibody, Bi-specific T-cell engagers (BiTE) and variable new antigen receptor antibody (V-NAR); and

(c) said nucleic acid cassette comprises a nucleic acid sequence encoding a secreted antibody specific for an autoantigen and a BCR specific for a foreign antigen.

13. The method according to claim 1, wherein said target genomic locus is at least one of Immunoglobulin heavy chain locus, Immunoglobulin κ

chain locus, Immunoglobulin λ

chain locus, TCRβ

chain locus, TCRα

chain locus, TCRγ

chain and the TCRδ

chain locus.

14. The method according to claim 1, wherein said cassette is comprised within a nucleic acid vector, said vector is any one of a viral vector, a non-viral vector and a naked DNA vector, optionally wherein:

(a) said vector is a viral vector, said viral vector is any one of recombinant adeno associated vectors (rAAV), single stranded AAV (ssAAV), self-complementary rAAV (scAAV), Simian vacuolating virus 40 (SV40) vector, Adeno virus vector, helper-dependent Adeno viral vector, retroviral vector and lentiviral vector;

or

(b) said vector is a non-viral vector, said vector is any one of plasmid, minicircle and linear DNA;

or

(c) said vector is a naked DNA vector, said vector is any one of plasmid, minicircle and linear DNA.

18. The method according to claim 1, wherein said mammalian cells is any one of a naturally RAG expressing cell, stably RAG expressing cell, inducibly RAG expressing cell and a cell transfected with RAG expression vector/s, optionally, said cell is any one of a lymphocyte progenitor, ex vivo differentiating lymphocytes, hematopoietic stem and progenitor cells (HSPCs) and Induced pluripotent stem cells (iPSCs), optionally, said lymphocyte progenitor is any one of a T cell progenitor, a B cell progenitor and an NK cell progenitor.

22. The method according to claim 1, for targeted insertion of at least one nucleic acid sequence of interest into a target genomic locus of a cell in a mammalian subject, the method comprising administering to said subject an effective amount of at least one nucleic acid cassette comprising said nucleic acid sequence of interest and at least one RSS, or of a vector comprising said cassette, wherein the insertion of said nucleic acid sequence of interest into the target genomic locus is mediated by RAG-catalyzed recombination between at least one genomic RSS flanking the target genomic locus and at least one RSS comprised within said nucleic acid cassette, optionally, said cassette further comprises at least one genetic element, said genetic element is at least one of:

an IRES, a 2A peptide coding sequence, a promoter or any functional fragments thereof, a splice donor (SD), a splice acceptor (SA), a degron, a 3 frame stop, a protein stabilizing sequence, a signal peptide, a stop codon, a polyadenylation site, a splice donor, a transcription enhancer, a switch region, an mRNA stabilizing sequence and a protein stabilizing sequence.

24. The method according to claim 22, wherein said nucleic acid sequence of interest is a protein coding nucleic acid sequence or a non-coding nucleic acid sequence, wherein optionally, said nucleic acid sequence of interest encodes at least one of a receptor and an antibody or any fragment/s or chimera/s thereof, optionally, said target genomic locus is at least one of Immunoglobulin heavy chain locus, Immunoglobulin κ

chain locus, Immunoglobulin λ

chain locus, TCRβ

chain locus, TCRα

chain locus, TCRγ

chain and the TCRδ

chain locus.

27. The method according to claim 22, wherein at least one of:

(a) said cassette is comprised within a nucleic acid vector, said vector is any one of a viral vector, a non-viral vector and a naked DNA vector;

(b) said cell is any one of Thymocytes, HSPCs and mobilized HSPCs; and

(c) said subject is a subject suffering from an immune-related disorder.

3-5. -5. (canceled)

7-8. -8. (canceled)

10-12. -12. (canceled)

15-17. -17. (canceled)

19-21. -21. (canceled)

23. (canceled)

25-26. -26. (canceled)

28-29. -29. (canceled)

30. A method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathologic disorder in a mammalian subject, said method comprising the step of administering to said subject an effective amount of at least one of:

(a) nucleic acid cassette;

(b) a vector comprising said nucleic acid cassette; and

(c) a cell transduced or transfected with said nucleic acid cassette;

wherein said cassette comprises at least one nucleic acid sequence of interest and at least one RSS, wherein the cassette is for targeted insertion of at least one nucleic acid sequence of interest into a target genomic locus, and wherein the insertion of said nucleic acid sequence of interest into the target genomic locus is mediated by RAG-catalyzed recombination between at least one genomic RSS flanking the target genomic locus and at least one RSS comprised within said nucleic acid cassette.

31. The method according to claim 30, wherein said subject undergoes a preceding treatment of at least one of HSPC mobilization, immune ablation and steroid administration.

32. The method according to claim 30, wherein said nucleic acid sequence of interest is a protein coding nucleic acid sequence or a non-coding nucleic acid sequence, optionally, said nucleic acid sequence of interest encodes at least one of a receptor and an antibody or any fragment/s or chimera/s thereof.

34. The method according to claim 30, wherein said target genomic locus is at least one of Immunoglobulin heavy chain locus, Immunoglobulin κ

chain locus, Immunoglobulin λ

chain locus, TCRβ

chain locus, TCRα

chain locus, TCRγ

chain and the TCRδ

chain locus.

35. The method according to claim 30, wherein said subject is administered with a nucleic acid vector comprising said cassette, said vector is any one of a viral vector, a non-viral vector and a naked DNA vector, optionally, said nucleic acid vector is administered by at least one of systemic injection, intrathymic injection, bone marrow injection, splenic injection and lymph node injection.

37. The method according to claim 30, wherein said subject is administered with at least one cell transduced or transfected with said nucleic acid cassette or any vector or vehicle comprising said cassette, optionally, said cells are of an autologous or allogeneic source.

39. The method according to claim 30, wherein said subject is a subject suffering from an immune-related disorder.

33. (canceled)

36. (canceled)

38. (canceled)

40. A nucleic acid cassette or any composition thereof, wherein said nucleic acid cassette comprises at least one nucleic acid sequence of interest and at least one RSS.

41. The cassette according to claim 40, wherein said cassette is for targeted insertion of at least one nucleic acid sequence of interest into a target genomic locus in a mammalian cell, wherein the insertion of said nucleic acid sequence of interest into the target genomic locus is mediated by RAG-catalyzed recombination between at least one genomic RSS flanking the target genomic locus and at least one RSS comprised within said nucleic acid cassette, optionally, at least one of:

(a) said cassette comprises two or more nucleic acid sequences of interest separated by at least one genetic element, said genetic element is at least one of;

an IRES, a 2A peptide coding sequence, a promoter or any functional fragments thereof, a splice donor (SD), a splice acceptor (SA);

(b) said nucleic acid sequence of interest encodes at least one of a receptor and an antibody or any fragment/s or chimera/s thereof;

(c) said target genomic locus is at least one of Immunoglobulin heavy chain locus, Immunoglobulin κ

chain locus, Immunoglobulin λ

chain locus, TCRβ

chain locus, TCRα

chain locus, TCRγ

chain and the TCRδ

chain locus; and

(d) said cassette is comprised within a nucleic acid vector, said vector is any one of a viral vector, a non-viral vector and a naked DNA vector.

46. The pharmaceutical composition according to claim 40, comprising at least one nucleic acid cassette, or any vector or cell comprising said cassette, wherein said cassette comprises at least one nucleic acid sequence of interest and at least one RSS, said cassette is for targeted insertion of said at least one nucleic acid sequence of interest into a target genomic locus, wherein the insertion of said nucleic acid sequence of interest into the target genomic locus is mediated by RAG-catalyzed recombination between at least one genomic RSS flanking the target genomic locus and at least one RSS comprised within said nucleic acid cassette, optionally said composition further comprise at least one of pharmaceutically acceptable carrier/s, diluent/s, excipient/s and additive/s.

42-45. -45. (canceled)

47-50. -50. (canceled)