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EP-4735613-A1 - ARTIFICIAL EXPRESSION CONSTRUCTS FOR MODULATING GENE EXPRESSION IN CARDIOMYOCYTES

EP4735613A1EP 4735613 A1EP4735613 A1EP 4735613A1EP-4735613-A1

Abstract

Artificial expression constructs for modulating gene expression in cardiomyocytes within targeted anatomical regions are described. The artificial expression constructs can be used to express synthetic genes or modify gene expression in cardiomyocytes within targeted anatomical regions including the heart atria and/or heart ventricles.

Inventors

  • DAIGLE, Tanya
  • JOHANSEN, Nelson
  • LEIN, Edward Sebastian

Assignees

  • Allen Institute

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. An artificial expression construct comprising (i) an eHGT_1199h enhancer, (ii) a promoter, and (iii) a coding sequence.
  2. 2. An artificial expression construct comprising (i) an enhancer selected from eHGT_1199h, eHGT_1146h, eHGT_1147h, eHGT_1148h, eHGT_1149h, eHGT_1150h, eHGT_1151h, eHGT_1153h, eHGT_1154h, eHGT_1155h, eHGT_1156h, eHGT_1157h, eHGT_1161h, eHGT_1162h, eHGT_1163h, eHGT_1164h, eHGT_1198h, eHGT_1201h, eHGT_1202h, eHGT_1203h, eHGT_1204h, eHGT_1206h, and eHGT_1207h; (ii) a promoter; and (iii) a coding sequence.
  3. 3. The artificial expression construct of claim 2, wherein the coding sequence encodes an effector element or an expressible element.
  4. 4. The artificial expression construct of claim 3, wherein the effector element comprises a reporter protein or a functional molecule.
  5. 5. The artificial expression construct of claim 4, wherein the reporter protein comprises a fluorescent protein.
  6. 6. The artificial expression construct of claim 4, wherein the functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular trafficking protein, signaling molecule, calcium reporter, channelrhodopsin, CRISPR/Cas molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or a designer receptor exclusively activated by designer drug (DREADD).
  7. 7. The artificial expression construct of claim 3, wherein the expressible element comprises a non-functional molecule.
  8. 8. The artificial expression construct of claim 7, wherein the non-functional molecule comprises a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular trafficking protein, signaling molecule, calcium reporter, channelrhodopsin, CRISPR/Cas molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or DREADD.
  9. 9. The artificial expression construct of claim 2, wherein the artificial expression construct is associated with a capsid that comprises AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV1R6, AAV1 R7, AAVrh.74, and AAV-PHP.S.
  10. 10. The artificial expression construct of claim 2, wherein the artificial expression construct comprises or encodes a skipping element.
  11. 11. The artificial expression construct of claim 10, wherein the skipping element comprises a 2A peptide or an internal ribosome entry site (IRES).
  12. 12. The artificial expression construct of claim 11, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A.
  13. 13. The artificial expression construct of claim 2, wherein the artificial expression construct comprises or encodes a set of features selected from: eHGT_1199h, eHGT_1146h, eHGT_1147h, eHGT_1148h, eHGT_1149h, eHGT_1150h, eHGT_1151h, eHGT_1153h, eHGT_1154h, eHGT_1155h, eHGT_1156h, eHGT_1157h, eHGT_1161h, eHGT_1162h, eHGT_1163h, eHGT_1164h, eHGT_1198h, eHGT_1201 h, eHGT_1202h, eHGT_1203h, eHGT_1204h, eHGT_1206h, eHGT_1207h, AAV, scAAV, rAAV, pAAV, minBglobin, CMV, minCMV, minCMV*, minRho, minRho*, fluorescent protein, hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10, tag cassette, 10aa, nuclear localization protein, self-cleaving peptides, WPRE, WPRE3, hGHpA, and/or BGHpA.
  14. 14. The artificial expression construct of claim 2, wherein the artificial expression construct comprises or encodes a set of features selected from: eHGT_1199h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1146h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1147h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1148h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1149h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1150h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1151 h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1153h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1154h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1155h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1156h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1157h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1161 h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1162h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1163h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1164h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1198h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1201 h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1202h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1203h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1204h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1206h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1207h-minBglobin-[coding sequence]-[post-regulatory elements]; eHGT_1199h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1146h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1147h-minBglobin-[coding sequence]-WPRE3-bGHpA; eHGT_1148h-minBglobin-[coding sequence]-WPRE3-bGHpA; eHGT_1149h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1150h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1151 h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1153h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1154h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1155h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1156h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1157h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1161 h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1162h-minBglobin-[coding sequence]-WPRE3-bGHpA; eHGT_1163h-minBglobin-[coding sequence]-WPRE3-bGHpA; eHGT_1164h-minBglobin-[coding sequence]-WPRE3-bGHpA; eHGT_1198h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1201 h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1202h-minBglobin-[coding sequence]- WPRE3-bGHpA; eHGT_1203h-minBglobin-[coding sequence]-WPRE3-bGHpA; eHGT_1204h-minBglobin-[coding sequence]-WPRE3-bGHpA; eHGT_1206h-minBglobin-[coding sequence]-WPRE3-bGHpA; or eHGT_1207h-minBglobin-[coding sequence]- WPRE3-bGHpA.
  15. 15. A vector comprising an artificial expression construct of claim 2.
  16. 16. The vector of claim 15, wherein the vector comprises a viral vector.
  17. 17. The vector of claim 16, wherein the viral vector comprises a recombinant adeno-associated viral (AAV) vector.
  18. 18. An adeno-associated viral (AAV) vector comprising at least one coding sequence, wherein the coding sequence is under transcriptional control of a promoter and an enhancer selected from eHGT_1199h, eHGT_1146h, eHGT_1147h, eHGT_1148h, eHGT_1149h, eHGT_1150h, eHGT_1151h, eHGT_1153h, eHGT_1154h, eHGT_1155h, eHGT_1156h, eHGT_1157h, eHGT_1161h, eHGT_1162h, eHGT_1163h, eHGT_1164h, eHGT_1198h, eHGT_1201 h, eHGT_1202h, eHGT_1203h, eHGT_1204h, eHGT_1206h, and eHGT_1207h.
  19. 19. The AAV vector of claim 18, wherein the coding sequence encodes an effector element or an expressible element.
  20. 20. The AAV vector of claim 19, wherein the effector element comprises a reporter protein or a functional molecule.

Description

ARTIFICIAL EXPRESSION CONSTRUCTS FOR MODULATING GENE EXPRESSION IN CARDIOMYOCYTES CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Patent Application No. 63/510,838 filed on June 28, 2023, which is incorporated herein by reference in its entirety as if fully set forth herein. REFERENCE TO SEQUENCE LISTING [0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 3946465.xml. The text file is 136,808 bytes, was created on June 17, 2024, and is being submitted electronically via Patent Center. FIELD OF THE DISCLOSURE [0003] The current disclosure provides artificial expression constructs for modulating gene expression in cardiomyocytes within targeted anatomical regions of the heart. The artificial expression constructs can be used to express synthetic genes or modify gene expression in cardiomyocytes within the heart atria and/or heart ventricles. BACKGROUND OF THE DISCLOSURE [0004] Current treatments for cardiovascular disease focus on managing symptoms and preventing complications, such as in the use of interventional procedures, implantable devices, and medications. Although considerable progress has been made in the prevention of cardiovascular diseases caused by environmental factors, such as nicotine, hypercholesterolemia, or diabetes, there is still a need for improvement in the treatment of cardiovascular disease, including in the treatment of inherited cardiomyopathies. Gene therapy has emerged as a promising treatment; however, assessment of cardiac gene therapy clinical trials indicates that efficiency of gene delivery to specific cardiac cell types (e.g., cardiomyocytes) and to specific cardiac anatomical regions remains an obstacle. SUMMARY OF THE DISCLOSURE [0005] The current disclosure provides artificial expression constructs that drive gene expression in cardiomyocytes within targeted anatomical regions of the heart. Targeted anatomical regions of the heart include: only the atria, which include the left and right atria; only the ventricles, which include the left and right ventricles; or the atria and the ventricles. [0006] Particular embodiments of the artificial expression constructs utilize the following enhancers to drive gene expression in cardiomyocytes within targeted anatomical regions of the heart as follows (enhancer I targeted anatomical region): eHGT_1153h / atria; eHGT_1157h, eHGT_1161h, and eHGT_1162h / ventricles; and eHGT_1146h, eHGT_1147h, eHGT_1148h, eHGT_1149h, eHGT_1150h, eHGT_1151h, eHGT_1154h, eHGT_1155h, eHGT_1156h, eHGT_1163h, eHGT_1164h, eHGT_1198h, eHGT_1199h, eHGT_1201h, eHGT_1202h, eHGT_1203h, eHGT_1204h, eHGT_1206h, and eHGT_1207h I atria and ventricles. [0007] Particular embodiments provide artificial expression constructs including the features of vectors described herein including vectors: HCT 27, HCT 31 , HOT 35, HOT 36, HOT 20, HOT 21 , HOT 22, HCT 23, HCT 24, HCT 25, HCT 28, HCT 29, HCT 30, HCT 37, HCT 38, HCT60, HCT61 , HCT62, HCT63, HCT64, HCT65, HCT66, and HCT67. BRIEF DESCRIPTION OF THE FIGURES [0008] FIGs. 1A, 1 B: Enhancer eHGT_1199h drives robust expression of SYFP2 in cardiomyocytes. Viral vector HCT61 was packaged with PHP.eB capsid and delivered to mice by retro-orbital administration. SYFP2+ cell bodies were found throughout the heart tissue and exhibit elongated morphology that lacks obvious polarity, as expected for cardiomyocytes. Black box with dashed lines in FIG. 1A indicates the region magnified in FIG. 1B. [0009] FIGs. 2A, 2B: Enhancer eHGT_1164h drives robust expression of SYFP2 in cardiomyocytes. Viral vector HCT38 was packaged with PHP.eB capsid and delivered to mice by retro-orbital administration. SYFP2+ cell bodies were found throughout the heart tissue and exhibit elongated morphology that lacks obvious polarity, as expected for cardiomyocytes. Black box with dashed lines in FIG. 2A indicates the region magnified in FIG. 2B. [0010] FIG. 3. Sequences supporting the disclosure including eHGT_1146h (SEQ ID NO: 1), eHGT_1147h (SEQ ID NO: 2), eHGT_1148h (SEQ ID NO: 3), eHGT_1149h (SEQ ID NO: 4), eHGT_1150h (SEQ ID NO: 5), eHGT_1151h (SEQ ID NO: 6), eHGT_1153h (SEQ ID NO: 7), eHGT_1154h (SEQ ID NO: 8), eHGT_1155h (SEQ ID NO: 9), eHGT_1156h (SEQ ID NO: 10), eHGT_1157h (SEQ ID NO: 11), eHGT_1161h (SEQ ID NO: 12), eHGT_1162h (SEQ ID NO: 13), eHGT_1163h (SEQ ID NO: 14), eHGT_1164h (SEQ ID NO: 15), eHGT_1198h (SEQ ID NO: 16), eHGT_1199h (SEQ ID NO: 17), eHGT_1201h (SEQ ID NO: 18), eHGT_1202h (SEQ ID NO: 19), eHGT_1203h (SEQ ID NO: 20), eHGT_1204h (SEQ ID NO: 21), eHGT_1206h (SEQ ID NO: 22), eHGT_1207h (SEQ ID NO: 23), Beta-Globin Minimal Promoter (pBGmin/minBGIobin/minBGprom) (SEQ ID NO: 24), minCMV Promoter (SEQ ID NO: 25), Mutated minCMV Promoter (Sacl RE site removed) (SEQ ID NO: 26), minRho Promoter (SEQ ID