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CN-121986170-A - Engineering regulatory elements

CN121986170ACN 121986170 ACN121986170 ACN 121986170ACN-121986170-A

Abstract

The present disclosure provides, inter alia, methods and compositions comprising engineered nucleic acids, such as engineered regulatory elements, that allow for improved transcriptional activity of operably linked polynucleotides in cells.

Inventors

  • J.J.Hua
  • N. Frankel
  • K.L.L.Zhu
  • A.B. Rogoff

Assignees

  • 森迪生物科学公司

Dates

Publication Date
20260505
Application Date
20241011
Priority Date
20231013

Claims (19)

  1. 1. An engineered regulatory element comprising a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs 1-108.
  2. 2. An engineered regulatory element comprising one or more TP63 Transcription Factor Binding Sites (TFBS), optionally wherein said one or more TP63 TFBS comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs 217-220 and their reverse complements.
  3. 3. The engineered regulatory element of claim 1 or 2, wherein any of the one or more TP63 TFBS comprises a TP63 TFBS half-site motif, wherein the TP63 TFBS half-site motif comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from SEQ ID NOs 221-233 and their reverse complements, optionally wherein the TP63 TFBS comprises two TP63 TFBS half-site motifs, optionally wherein the two TP63 TFBS half-site motifs are operably linked by a nucleic acid linker, optionally wherein the linker is between 1-10 base pairs, optionally wherein the two TP63 TFBS half-site motifs comprise (a) a first half-site motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from SEQ ID nos. and (at least 233%, at least 95% or at least 95% identical to a nucleotide sequence selected from SEQ ID nos. 221-80%, at least 95% or at least 95% complementary to at least 95% of the nucleotide sequence.
  4. 4. The engineered regulatory element of any one of claims 1 to 3 comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 TP63 TFBS, optionally wherein the engineered regulatory element comprises 1-500, 1-100, 1-50, 2-20, or 2-10 TP63 TFBS.
  5. 5. The engineered regulatory element of any one of claims 1 to 4, wherein the engineered regulatory element further comprises at least one additional non-TP 63 TFBS.
  6. 6. An engineered regulatory element comprising one or more TP63 Transcription Factor Binding Sites (TFBS) and comprising at least one additional non-TP 63 TFBS.
  7. 7. The engineered regulatory element of claim 5 or 6, wherein the at least one additional non-TP 63 TFBS is selected from BARX TFBS, NHLH1 TFBS, TP73 TFBS, HOXC10 TFBS, NFE2 TFBS, ATF4 TFBS, HES1 TFBS, FOS TFBS, JUN TFBS, and JUNB TFBS, optionally wherein: (a) The BARX TFBS comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 234, and/or (B) The NHLH1 TFBS comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 235, and/or (C) The TP73 TFBS comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 236, and/or (D) The HOXC10 TFBS comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 237, and/or (E) The NFE2 TFBS comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 238, and/or (F) The ATF4 TFBS comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 239, and/or (G) The HES1 TFBS comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID No. 240, and/or (H) The FOS TFBS comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 241, and/or (I) The JUN TFBS comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 242, and/or (J) The JUNB TFBS comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 243; Optionally wherein the at least one additional non-TP 63 TFBS comprises both BARX, 3, 4, 5, or more than 5 BARX TFBSs, optionally wherein the at least one additional non-TP 63 TFBS comprises about 2, 3, 4, 5, or more than 5 NHLH1 TFBSs, or NHLH1 TFBS or BARX TFBS and NHLH TFBS.
  8. 8. The engineered regulatory element of any one of the preceding claims, wherein the engineered regulatory element is operably linked to a core promoter, optionally wherein the core promoter comprises a sequence selected from the group consisting of minCMV minimal promoter, SV40 promoter, B2M promoter, SCP3 minimal promoter, YB-SCP3 minimal promoter, DPR containing SCP3 promoter, minP promoter, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, YB TATA, minTK, inducer molecule response promoter 、CMV、EFS、SFFV、SV40、MND、PGK、UbC、hEFlaV1、hCAGG、hEFlaV2、hACTb、heIF4A1、hGAPDH、hGRP78、hGRP94、hHSP70、hKINb、hUBIb and tandem repeats thereof, Optionally wherein the core promoter is selected from the group consisting of minCMV minimal promoter, SV40 promoter, B2M promoter, SCP3 minimal promoter, YB-SCP3 minimal promoter, and SCP3 promoter containing DPR.
  9. 9. An engineered regulatory element comprising a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs 109-216.
  10. 10. A heterologous construct, the heterologous construct comprising: (a) The engineered regulatory element of any one of claim 1 to 9, and (B) The payload of the heterologous payload is selected to be, Wherein the engineered regulatory element is operably linked to the heterologous payload.
  11. 11. The heterologous construct of claim 10, wherein the heterologous payload comprises a polynucleotide, optionally wherein the polynucleotide comprises a nucleotide sequence encoding one or more polypeptides, optionally wherein the one or more polypeptides comprise a) at least one effector molecule or b) two or more separate polypeptides comprising a first effector molecule, a second effector molecule, optionally a third effector molecule, optionally a fourth effector molecule, optionally wherein the at least one effector molecule or each effector molecule is selected from one or more therapeutic classes, wherein the one or more therapeutic classes are selected from chimeric receptors, cytokines, chemokines, homing molecules, growth factors, polynucleotide molecules, coactivating molecules, tumor microenvironment modulators, receptors, ligands, transcription factors, antibodies, peptides, and enzymes, optionally wherein the chimeric receptor is a Chimeric Antigen Receptor (CAR).
  12. 12. The heterologous construct of claim 11 wherein the polynucleotide comprises E1-L1-E2, optionally wherein the polynucleotide comprises E1-L1-E2-L2-E3-L3-E4, wherein E1 is a nucleotide sequence encoding the first effector molecule, L1 is a first linker molecule, E2 is a nucleotide sequence encoding the second effector molecule, L2 is a second linker molecule, E3 is a nucleotide sequence encoding the third effector molecule, L3 is a third linker molecule, and E4 is a nucleotide sequence encoding the fourth effector molecule, optionally wherein L1, L2, L3, and L4 are independently selected from the group consisting of an Internal Ribosome Entry Site (IRES) and one or more nucleotide sequences encoding one or more 2A ribosome-hopping elements, optionally wherein the linker nucleotide sequence encodes one or more 2A ribosome-hopping elements, optionally wherein the one or more 2A ribosome-hopping elements include elements selected from the group consisting of P5326 a and P2A A, T a and P2A 392.
  13. 13. The heterologous construct of claim 10 or 11, wherein the one or more polypeptides comprise: a) The first effector molecule and the second effector molecule, and wherein: i) The first effector molecule and the second effector molecule are independently selected from a first CAR and a second CAR, or Ii) the first effector molecule and the second effector molecule are independently selected from a first CAR and a cytokine, or Iii) The first effector molecule and the second effector molecule are independently selected from the group consisting of a first cytokine and a second cytokine, and Optionally wherein the first CAR is an activated CAR (aCAR) and the second CAR is an Inhibitory CAR (iCAR), optionally wherein the first CAR and/or the second CAR is a divalent CAR, or B) The first effector molecule, the second effector molecule, and the third effector molecule, and wherein the first effector molecule, the second effector molecule, and the third effector molecule are independently selected from the group consisting of: i) A first CAR, a second CAR, and a cytokine, or Ii) a first CAR, a first cytokine and a second cytokine, or C) The first effector molecule, the second effector molecule, the third effector molecule, and the fourth effector molecule, and wherein the first effector molecule, the second effector molecule, the third effector molecule, and the fourth effector molecule are independently selected from the group consisting of a first CAR, a second CAR, a first cytokine, and a second cytokine.
  14. 14. A vector comprising the heterologous construct of any one of claims 10 to 13, optionally wherein the vector is a viral vector, optionally wherein the viral vector is a retroviral vector.
  15. 15. A dual expression vector comprising the heterologous construct of any one of claims 10 to 13 and a second construct comprising an additional payload, optionally wherein the dual expression vector is a retroviral vector.
  16. 16. An immune response cell comprising the heterologous construct of any one of claims 10 to 13, the vector of claim 14, or the dual expression vector of claim 15, optionally wherein the immune response cell is selected from the group consisting of a Natural Killer (NK) cell, a T cell, a cd8+ T cell, a cd4+ T cell, a gamma-delta T cell, a Cytotoxic T Lymphocyte (CTL), a regulatory T cell, a virus-specific T cell, a Natural Killer T (NKT) cell, a B cell, a macrophage, a tumor-infiltrating lymphocyte (TIL), a congenital lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a monocyte, a dendritic cell, a erythrocyte, a platelet cell, a human Embryonic Stem Cell (ESC), an ESC-derived cell, a pluripotent stem cell, a Mesenchymal Stromal Cell (MSC), an Induced Pluripotent Stem Cell (iPSC), and an iPSC-derived cell, optionally wherein the immune response cell expresses an activated immune receptor, optionally wherein the immune response cell comprises an antigen recognizing the antigen or the allogeneic receptor.
  17. 17. A pharmaceutical composition comprising the vector of claim 14, the dual expression vector of claim 15, or the immunoresponsive cell of claim 16, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.
  18. 18. A method of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of the vector of claim 14, the dual expression vector of claim 15, the immune response cell of claim 16, or the pharmaceutical composition of claim 17.
  19. 19. A kit for treating and/or preventing a disease or disorder, the kit comprising the immunoresponsive cell of claim 16 or the pharmaceutical composition of claim 17, optionally wherein the disease or disorder comprises a tumor, optionally wherein the kit further comprises written instructions for using the immunoresponsive cell or the pharmaceutical composition to treat and/or prevent the disease or disorder in a subject.

Description

Engineering regulatory elements Cross Reference to Related Applications The present application claims the benefit of U.S. provisional application No. 63/590,220, filed on 10/13 of 2023, the contents of which are hereby incorporated by reference in their entirety. Sequence listing The present application comprises a sequence listing, which is hereby incorporated by reference in its entirety. Electronic copies were created at 2024, 10/9, named 70012_seqlisting. Background Gene regulatory elements such as promoters and enhancers may have specific activities and may perform different functions in different environments (such as in different cell types). Such regulatory elements are generally suitable for use in therapies (such as cell and gene therapies) to treat diseases that benefit from expression of a particular gene and/or therapeutic payload. Thus, regulatory elements for regulating expression of a therapeutic payload may ultimately affect the overall functionality of an engineered cell or vector for gene therapy. However, the problem of transcription strength still exists in known regulatory elements that are commonly used to drive expression of payloads. By way of example only, engineered cell therapy applications may require low integrated transgene copy numbers for regulatory and safety reasons, however, known promoters may not be able to drive adequate therapeutic payload expression in an engineered cell population at the required copy numbers, particularly where the payload is large and/or polycistronic. Thus, there is a need to identify and develop regulatory elements that can effectively drive expression of payloads, particularly for use in therapy. Disclosure of Invention Provided herein are engineered regulatory elements comprising a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs 1-108. In some aspects, the nucleotide sequence is at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS 1-108. In some aspects, the nucleotide sequence is 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS 1-108. Also provided herein are engineered regulatory elements comprising one or more TP63 Transcription Factor Binding Sites (TFBS). In some aspects, the one or more TP63 TFBS comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from SEQ ID NOs 217-220 and their reverse complements. In some aspects, the one or more TP63 TFBS comprises a sequence that is at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS 217-220 and the reverse complement thereof. In some aspects, the one or more TP63 TFBS are selected from the group consisting of SEQ ID NOs 217-220 and their reverse complement. In some aspects, any of the one or more TP63 TFBS comprises a TP63 TFBS half-site motif, wherein the TP63 TFBS half-site motif comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs 221-233 and their reverse complements. In some aspects, the TP63 TFBS half-site motif comprises a sequence that is at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS 221-233 and the reverse complement thereof. In some aspects, the TP63 TFBS half-site motif is selected from the group consisting of SEQ ID NOS 221-233 and the reverse complement thereof. In some aspects, the TP63 TFBS comprises two TP63 TFBS half-site motifs. In some aspects, the two TP63 TFBS half-site motifs are operably linked by a nucleic acid linker, optionally wherein the linker is between 1 and 10 base pairs. In some aspects, the two TP63 TFBS half-site motifs comprise (a) a first half-site motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from SEQ ID NOs 221-233, and (b) a second half-site motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a reverse complement of a nucleotide sequence selected from SEQ ID NOs 221-233. In some aspects, the engineered regulatory element comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 TP63 TFBS. In some aspects, the engineered regulatory element comprises at least two TP63 TFBS. The engineered regulatory element of any one of claims 4 to 13 c