Cell-specific expression of modRNA
Inventors
Assignees
Icahn School of Medicine at Mount Sinai
Publication Number
US-12215336-B2
Publication Date
2025-02-04
Expiration Date
2037-09-18
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Abstract
Disclosed is an expression regulatory system for cell-specific transcription (expression) of a protein of interest, for example a cell cycle inducer that reactivates proliferation in adult or neonatal cardiomyocytes or insulin-producing beta cells. The expression regulatory system comprises a first nucleic acid that encodes a microRNA recognition element that specifically binds a target cell miR, and a translation suppressor protein; and a second nucleic acid that comprises a suppressor protein interaction motif that binds the translation suppressor protein, and a gene that encodes a protein of interest. When a cell of interest is co-transfected with the first and second nucleic acids of the system, the protein of interest expressed in a cell-specific fashion.
Core Innovation
The invention provides an expression regulatory system enabling cell-specific transcription and translation of a protein of interest using two nucleic acid constructs composed of modified messenger RNA (modRNA). One nucleic acid encodes a translation suppressor protein and includes a microRNA (miR) recognition element that binds selectively to a miR specific to the target cell, while the second nucleic acid encodes a protein of interest along with a suppressor protein interaction motif, such as a kink-turn motif (k-motif), recognized by the translation suppressor protein. By co-transfecting a cell with these two nucleic acids, expression of the protein of interest is restricted to cells where the suppressor protein translation is halted due to miR binding, overriding suppression.
The problem addressed is the lack of modRNA-based gene delivery methods that combine high levels of gene expression with specificity for a desired cell type, especially in vivo. Existing reagents show limited tissue targeting and efficacy, resulting in unwanted expression in non-target cells, which is a limitation for therapeutic gene delivery, such as inducing cardiomyocyte proliferation after myocardial infarction without affecting other cardiac or systemic cells.
A specific embodiment applies this system for cardiomyocyte-specific expression of cell cycle inducer genes like Lin28 and Pkm2, which reactivate cardiomyocyte proliferation crucial for cardiac regeneration after myocardial infarction or in heart failure. The system utilizes miR recognition elements like those for miR1 and miR208, predominantly expressed in cardiomyocytes, to ensure translation suppressor proteins are not produced in these cells, thus allowing selective expression of therapeutic proteins exclusively in cardiomyocytes.
Claims Coverage
The patent includes five independent claims focusing on methods for expressing proteins using a modRNA-based system with cell-specific miR recognition and cell cycle inducer protein expression targeting cardiomyocytes.
Dual modRNA system for cell-specific protein expression
A method involving contacting cells with a first ribonucleic acid encoding a translation suppressor protein and a miR recognition element that binds a cell-specific miR, and a second ribonucleic acid encoding a protein of interest and a suppressor protein interaction motif that binds the translation suppressor protein, enabling cell-specific protein expression.
Modified ribonucleic acid compositions
Use of modifications in the first and/or second ribonucleic acids selected from substitution of uridine with pseudouridine, substitution of cytidine with 5-methylcytidine, and 5′ end substitution with an anti-reverse cap analog to improve stability and reduce immunogenicity.
Cardiomyocyte-targeted stimulation of proliferation
Administering to a subject first and second modRNAs where the first includes miR recognition elements for miR-1 and miR-208 and encodes a translation suppressor protein, and the second includes a suppressor protein interaction motif and encodes a cell cycle inducer protein such as Lin28 or Pkm2, for cardiomyocyte-specific proliferation after myocardial infarction or heart failure.
Sequences defining modRNA constructs
Utilization of nucleic acids comprising one or both of the nucleotide sequences of SEQ ID NO: 2, 3, or 4 for the first modRNA and SEQ ID NO: 5, 6, 7, or 8 for the second modRNA defining the miR recognition elements, translation suppressor, suppressor interaction motif, and gene of interest.
The claims cover a modRNA-based system using paired nucleic acids encoding a translation suppressor with cell-specific miR recognition and a suppressor binding motif linked to a gene of interest, employing chemical modifications, with specific nucleotide sequences disclosed. They encompass methods for cardiomyocyte-specific expression of cell cycle inducer proteins to stimulate proliferation in therapeutic contexts.
Stated Advantages
Allows high-level, cell-specific gene expression by utilizing endogenous cell-specific miRs to override translation suppression selectively.
Provides transient and safe expression of therapeutic proteins, avoiding risks associated with long-term or systemic gene expression such as hypertrophy or tumorigenesis.
Enables cardiomyocyte-specific delivery and expression of cell cycle inducer proteins that reactivate proliferation, improving cardiac regeneration after myocardial infarction or heart failure.
Reduces off-target gene expression in non-target cells, minimizing unwanted proliferation and fibrosis.
The system uses modified mRNAs that reduce immunogenicity and enhance stability, making it suitable for in vivo gene delivery.
Documented Applications
Inducing or reactivating proliferation of cardiomyocytes following myocardial infarction or in heart failure by administering modRNAs encoding translation suppressor proteins with miR recognition elements and cell cycle inducer proteins like Lin28 and Pkm2.
Using the modRNA regulatory system for transient and cell-specific expression of therapeutic proteins in cardiomyocytes for cardiac repair and regeneration.
Application in lineage tracing of cardiomyocytes by co-expression of reporter genes using the cardiomyocyte-specific modRNA system.
Isolation and enrichment of transfected cardiomyocytes in vitro or ex vivo via expression of cell surface markers (inactive human CD25) included in the modRNA constructs to enable selection by affinity methods.
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