Repressible control of gene expression
Inventors
WONG, Wilson Wai Chun • DING, Yage
Assignees
Publication Number
US-12180485-B2
Publication Date
2024-12-31
Expiration Date
2041-07-21
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
The technology described herein is directed to inducible and repressible polypeptides and polypeptide systems. In particular, described herein are split sequence-specific nucleases and split recombinases that are linked to drug-inducible or drug-repressible dimerization domains. In some embodiments, the polypeptides comprise sequestering domains and/or have their expression controlled by an inducible promoter. In multiple aspects described herein are polynucleotides, vectors, cells, and pharmaceutical compositions comprising said polypeptides or polypeptide systems. Also described herein are methods of using said polypeptide systems to modulate the expression of a target polypeptide or to treat a subject in need of a cell therapy.
Core Innovation
The invention is directed to repressible split-nuclease polypeptide systems, particularly involving sequence-specific nucleases such as Cas13. In these systems, the nuclease is divided into two inactive fragments, each fused to a component of a repressible dimerization domain. The fragments are designed to reassemble into a functional nuclease only under specific conditions: in the default (absence of repressor agent or signal), the two split halves interact and form an active nuclease; but in the presence of a repressor agent or signal, the dimerization interaction is disrupted and the fragments do not assemble, remaining inactive.
A key aspect of the invention is the use of a repressible protease, such as hepatitis C virus (HCV) NS3, paired with a peptide domain such as ANR or CP5-46-5D5E, as the repressible dimerization domains. The system allows specific binding between the protease and peptide in the absence of a small molecule inhibitor, with this interaction reversed in the presence of clinically approved protease inhibitors (for example, grazoprevir or danoprevir). Such switching provides tight and reversible control over the assembly and activation of the nuclease. The invention also provides specific split sites within Cas13 family endonucleases that enable the design of functional split fragments for this module.
The problem addressed is the need for programmable genetic circuits and regulatory elements that offer tunable, reversible, and safe control of gene expression for sophisticated cell therapies and research applications. Existing methods are limited by constitutive activity or lack specificity, reversibility, or compatibility with clinically approved molecules. This invention solves the problem by enabling precise, ligand- or drug-mediated repression of nuclease activity, thereby providing another layer of safety and dynamic regulation for engineered cellular therapies or genetic tools.
Claims Coverage
The claims cover a repressible split-nuclease system with specific structural and functional features as defined by the independent claim and its dependent claims.
Repressible split-nuclease system with protease–peptide domain dimerization
A system comprising two polypeptide fragments of a sequence-specific nuclease (such as Cas13), each fused to a member of a repressible dimerization domain. One member comprises a repressible protease (for example, HCV NS3), and the other a peptide domain (such as ANR or CP5-46-5D5E). In the absence of a specific repressor agent or signal, the dimerization domains interact, enabling protein complementation to form active nuclease; in the presence of the repressor agent (e.g., protease inhibitor), this interaction is disrupted and the system remains inactive.
Drug-responsive switching of nuclease activity via clinically approved inhibitors
The system's dimerization and resulting nuclease activity can be specifically inhibited by clinically approved small molecule protease inhibitors, including grazoprevir, danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, ritonavir, dasabuvir, and telaprevir. The protease specifically binds the peptide domain without inhibitor present and does not bind (or dissociates from) the peptide domain in the presence of these inhibitors.
Split Cas13 family endonucleases with defined functional split sites
The nuclease fragments are specifically Cas13, including Cas13d, Cas13a, or Cas13b, with split positions at specified amino acids: for Cas13d, splits at amino acid positions 88/89, 263/264, 384/385, 404/405, 507/508, 559/560, 565/566, 576/577, 655/656, or 903/904. For Cas13a and Cas13b, eligible split sites are also specified. These splits ensure functional assembly and repression as described.
Reversibility and kinetics of split-nuclease assembly
Upon removal of the repressor agent or signal, the system rapidly reassembles (lag time of 30 seconds or less), permitting prompt recovery of nuclease activity. Disassembly and loss of activity are similarly rapid upon repressor binding.
Optional incorporation of localization and sequestering domains
The system may further include at least one cytosolic sequestering domain (with a ligand-binding domain that allows for cytosol-to-nucleus transport control), nuclear export signal (NES), or nuclear localization signal (NLS) on either split polypeptide. This enables additional spatial or ligand-dependent regulation.
In summary, the patent claims are directed to a Cas13-based repressible split-nuclease system controlled by the interaction of a protease and peptide domain dimerization module, which is responsive to small molecule inhibitors. The claims further cover specific split-site arrangements within Cas13 family nucleases, options for fast reversibility, and the inclusion of localization/sequestering domains as supplementary control mechanisms.
Stated Advantages
Provides tunable and reversible repression of nuclease activity using clinically approved, non-toxic small molecules, enhancing safety and controllability in gene regulation.
Enables rapid switching between active and inactive states of the nuclease with minimal lag time (30 seconds or less), allowing precise temporal control.
Increases the versatility and applicability of gene regulatory circuits for cell therapy by allowing drug-mediated, tightly regulated modulation of gene expression.
Avoids constitutive nuclease activity, minimizing off-target effects and potential toxicity in engineered cells.
Documented Applications
Modulation of gene expression in engineered cells, including immune cells, through chemically controlled repression of RNA-targeting nucleases for cell therapy.
Implementation as a gene regulatory element or circuit component for programmable and safe therapeutic interventions, including control systems in T cell and CAR-T therapies.
Research use in reversible, ligand-responsive RNA knockdown or editing, enabling studies on gene function or temporal effects in mammalian systems.
Interested in licensing this patent?