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US-12624362-B2 - Mutant reverse tetracycline transactivators for expression of genes

US12624362B2US 12624362 B2US12624362 B2US 12624362B2US-12624362-B2

Abstract

Provided herein are mutant reverse tetracycline transactivator (rtTA) proteins and engineered nucleic acids that encode a mutant rtTA that are useful in regulating gene expression, inducing cellular reprogramming, tissue repair, tissue regeneration, organ regeneration, reversing aging, treating a disease, or any combination thereof. Also provided herein are recombinant viruses comprising the engineered nucleic acids and methods of regulating cellular reprogramming, tissue repair, tissue regeneration, or any combination thereof by administering an engineered nucleic acid or recombinant virus comprising the same in a cell, tissue or subject comprising administering a mutant rtTA and an inducible nucleic acid encoding a transgene.

Inventors

  • David A. Sinclair
  • Yuancheng Lu
  • Noah Justin Davidsohn

Assignees

  • PRESIDENT AND FELLOWS OF HARVARD COLLEGE

Dates

Publication Date
20260512
Application Date
20190927

Claims (20)

  1. 1 . An engineered nucleic acid comprising a promoter operably linked to a first nucleic acid comprising a sequence that is at least 80% identical to the sequence of SEQ ID NO: 12, wherein the first nucleic acid sequence encodes a mutant reverse tetracycline transactivator (rtTA) comprising an amino acid sequence at least 70% identical to the sequence of SEQ ID NO: 11, which comprises the following mutations relative to rtTA3 (SEQ ID NO: 11): (a) a G72V mutation; (b) a G12S or G12T mutation; (c) an F67S or F67T mutation; and (d) an R171K or R171H mutation.
  2. 2 . The engineered nucleic acid of claim 1 , wherein the promoter is a constitutive promoter selected from the group consisting of CP1, CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaMV 35S, Ubi, H1, and U6 promoters.
  3. 3 . The engineered nucleic acid of claim 1 , wherein the promoter is a tissue-specific promoter.
  4. 4 . The engineered nucleic acid of claim 1 further comprising a second nucleic acid sequence that encodes a tetracycline repressor.
  5. 5 . The engineered nucleic acid of claim 4 , wherein the promoter that is operably linked to the first nucleic acid is also operably linked to the second nucleic acid.
  6. 6 . The engineered nucleic acid of claim 4 further comprising a separator sequence encoding an internal ribosome entry site (IRES) or a 2A peptide between the first and the second nucleic acid.
  7. 7 . The engineered nucleic acid of claim 4 , wherein the tetracycline repressor is tetRKRAB.
  8. 8 . The engineered nucleic acid of claim 1 , wherein the engineered nucleic acid is a viral vector, wherein the viral vector is an adeno-associated virus (AAV), lentiviral, retroviral, adenoviral, or herpes viral vector.
  9. 9 . The engineered nucleic acid of claim 8 , wherein the viral vector comprises inverted terminal repeats (ITRs) flanking one or more nucleic acids.
  10. 10 . The engineered nucleic acid of claim 1 further comprising a WPRE3 sequence.
  11. 11 . The engineered nucleic acid of claim 1 , wherein the engineered nucleic acid comprises a sequence that is at least 70% identical to the sequence of SEQ ID NO: 17 or SEQ ID NO: 30.
  12. 12 . The engineered nucleic acid of claim 1 , wherein the engineered nucleic acid further comprises a first transgene sequence operably linked to an inducible promoter that comprises a tetracycline-responsive element (TRE).
  13. 13 . The engineered nucleic acid of claim 12 , wherein the TRE promoter is a TRE3G promoter.
  14. 14 . The engineered nucleic acid of claim 12 , wherein the TRE comprises at least one Tet-O sequence set forth as SEQ ID NO: 19.
  15. 15 . A recombinant virus comprising the engineered nucleic acid of claim 1 .
  16. 16 . A pharmaceutical composition comprising the recombinant virus of claim 15 .
  17. 17 . A pharmaceutical composition comprising the engineered nucleic acid of claim 1 and a pharmaceutically acceptable excipient.
  18. 18 . An in vitro or ex vivo cell comprising the engineered nucleic acid of claim 1 .
  19. 19 . The engineered nucleic acid of claim 1 , wherein the mutant rtTA comprises the following mutations: (a) G72V; (b) G12S; (c) F67S; and (d) R171K.
  20. 20 . The engineered nucleic acid of claim 1 , wherein the mutant rtTA comprises a sequence that is at least 70% identical to the sequence of SEQ ID NO: 13.

Description

RELATED APPLICATIONS This application is a national stage filing under 35 U.S.C. § 371 of international PCT application number PCT/US2019/053492, filed Sep. 27, 2019, which claims the benefit under 35 U.S.C. § 119 (e) of U.S. provisional application No. 62/738,894, filed Sep. 28, 2018, each of which is incorporated by reference herein in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (H082470300US01-SUBSEQ-FL.txt; Size: 101,807 bytes; and Date of Creation: Apr. 19, 2024) is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Inducible gene expression holds great promise for gene therapy and other biomedical applications. On-demand and tightly regulated gene expression can obviate toxicity associated with prolonged expression or toxicity associated with super physiological expression of exogenous genes. As an example, tetracycline-on (Tet-On) systems often use a reverse tetracycline transactivator (rtTA) to induce gene expression. Reverse tetracycline transactivators (rtTAs) comprise a mutant tetracycline repressor DNA binding protein (TetR) and a transactivation domain (e.g., Gossen et al., Science. 1995 Jun. 23;268 (5218): 1766-9). These transactivators can be activated in the presence of a tetracycline (e.g., doxycycline) and subsequently bind to promoters comprising a tetracycline-responsive element (TRE) to induce gene expression (Gossen et al., Science. 1995 Jun. 23: 268 (5218): 1766-9); Baron et al., Methods Enzymol. 2000; 327:401-21. A TRE comprises at least one Tet operator (Tet-O) sequence (e.g., multiple repeats of Tet-O sequences) and may be located upstream of a minimal promoter (e.g., minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter). The tendency of Tet-On systems to turn on gene expression (be “leaky”) even in the absence of a tetracycline, however, has hindered their use. Therefore, improved Tet-On systems with little to no leakiness and high tetracycline sensitivity are needed. SUMMARY OF THE INVENTION The present disclosure stems from the unexpected discovery that four mutations in the residues corresponding to positions G72, G12, F67, and R171 in rtTA3 (SEQ ID NO: 11) significantly improve the sensitivity and lower the leakiness of Tet-On systems in vivo. Provided herein, in some embodiments, are mutant rtTAs (e.g., rtTA4), engineered nucleic acids (e.g., expression vectors, including viral and non-viral vectors) encoding the same, recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) comprising the engineered nucleic acids (e.g., expression vectors), pharmaceutical compositions, and kits thereof. In some embodiments, the engineered nucleic acids encoding a mutant rtTA4 further encodes a transgene (e.g., a protein-encoding sequence, a gene-targeting nucleic acid, and/or a therapeutic sequence) operably linked to a tetracycline-responsive element (TRE) promoter. In some embodiments, the pharmaceutical compositions and kits further comprise a second vector (e.g., multiple second vectors) or a second recombinant virus (e.g., multiple second recombinant viruses) (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) that comprises a tetracycline-responsive element (TRE) promoter operably linked to a transgene. In some embodiments, the pharmaceutical compositions and kits further comprise multiple vectors or multiple recombinant viruses (e.g., lentiviruses, vaccinia viruses, alphaviruses, adenoviruses, retroviruses, herpes viruses, or adeno-associated viruses (AAV)) that comprise a tetracycline-responsive element (TRE) promoter operably linked to a transgene. Methods of promoting gene expression comprising administering (1) any of the engineered nucleic acids (e.g., expression vectors) described herein that encode a mutant rtTA (e.g., rtTA4) and (2) tetracycline to a cell, tissue, or subject in need thereof are also provided herein. In certain embodiments, the methods further comprise administering (3) a second nucleic acid (e.g., an engineered nucleic acid, including an expression vector) comprising a TRE promoter operably linked to a transgene. In certain embodiments, multiple second engineered nucleic acids (e.g., expression vectors) are administered. The mutant rtTAs (e.g., rtTA4s), engineered nucleic acids (e.g., expression vectors), recombinant viruses, pharmaceutical compositions, kits, and methods described herein are useful in regulating gene expression in vivo. Without being bound by a particular theory, the reduction in leakiness improves the toxicity profile of the rtTA4 Tet-On system compared to rtTA3 Tet-On system and allows for transient expression of transgenes. Aspects of the present disclosure provide mutant rtTAs (e.g., rtTA4). A mutant rtTA of the present disclosure comprises four mutations corresponding to positions G72, G12, F67, and R171 of rtTA