System and method for cell type-specific translation of RNA molecules in eukaryotes
Inventors
BOHLEN, HERIBERT • Hoffmann, Bernd
Assignees
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Publication Number
US-12180470-B2
Publication Date
2024-12-31
Expiration Date
Abstract
The present technology comprises methods and RNA constructs for the targeted translation of a polypeptide of interest in a eukaryotic target cell, and to the use thereof in therapeutic clinical applications.
Core Innovation
The invention relates to a cell-type-specific RNA translation system in which translation of a polypeptide of interest is prevented in a non-target eukaryotic cell and takes place in a eukaryotic target cell. This is achieved by introducing an RNA construct having an antisense sequence, a blocking element, a stabilizing element, a translation initiator element, and a coding sequence. The antisense sequence base-pairs with an mRNA expressed by the target cell to induce processing that activates the translation initiator element.
In the non-target eukaryotic cell, translation is prevented by base pairing of the blocking element with the translation initiator element, which blocks the translation initiator. In the target cell, interaction of the antisense sequence with the endogenous, cell-specific target mRNA removes or suppresses the blocking, thereby enabling translation initiation that is described as cap-independent. The translation initiator element functions independently of 5' cap in the construct and is active only in target cells.
To preserve downstream translation, the RNA construct includes a stabilizing element configured to prevent degradation in the 3' direction from the stabilizing element by exonucleases. The stabilizing element comprises binding partners and/or secondary structures configured to restrict complete degradation from the stabilizing element. The overall architecture includes optional 5' and 3' regulatory regions, including 5' cap, 5' untranslated region, 3' untranslated region, poly(A), and linkers, arranged in a 5' to 3' direction relative to the antisense, blocking, stabilizing, translation initiator, and coding sequence.
Claims Coverage
The provided material includes two independent claims. The claim set covers targeted translation in a eukaryotic target cell and a corresponding RNA construct configured to enable cap-independent, target-cell selective translation while maintaining inhibition in non-target cells via a blocking element and 3' exonuclease-resistant stabilizing element.
Targeted translation using an RNA construct architecture
A method for targeted translation of a polypeptide of interest in a eukaryotic target cell by introducing an RNA construct with, in 5' to 3' order, an antisense sequence complementary to an mRNA of a gene expressed by the target cell, a blocking element that interacts with a translation initiator element, a stabilizing element that prevents 3' exonuclease degradation, the translation initiator element, and a coding sequence, such that translation is prevented in a non-target eukaryotic cell by base pairing of the blocking element with the translation initiator element and occurs in the target cell due to processing induced by antisense-mRNA interaction that activates the translation initiator element.
Cap-independent, target-cell active RNA construct with antisense processing and blocking inhibition
An RNA construct for targeted translation in a eukaryotic target cell having, in 5' to 3' direction, an antisense sequence complementary to an mRNA of a gene expressed by the target cell, a blocking element complementary to or inhibiting at least a portion of the translation initiator element, a stabilizing element preventing 3' exonuclease degradation from the stabilizing element, a translation initiator element that functions independently of 5' cap and is active only in target cells, and a coding sequence, wherein translation is prevented in a non-target eukaryotic cell by base pairing of the blocking element with the translation initiator element and translation takes place in a eukaryotic target cell due to processing induced by antisense-mRNA interaction that activates the translation initiator element.
Across the independent claims, the central coverage is the RNA construct-and-processing logic: antisense sequence-mRNA interaction in the target cell removes inhibition by a blocking element that otherwise suppresses a translation initiator element, while a stabilizing element prevents 3' exonuclease degradation to maintain the translation region. The translation initiator element is described as functioning independently of 5' cap and being active only in target cells.
Stated Advantages
Prevents translation in a non-target eukaryotic cell while enabling translation of the encoded polypeptide in a eukaryotic target cell.
Provides cap-independent translation initiation in target cells.
Maintains RNA integrity by preventing degradation of RNA in the 3' direction from the stabilizing element by exonucleases.
Documented Applications
Therapeutic clinical applications and gene editing-related applications are described in the partial content.
KRT13-targeted constructs with IRES-controlled eGFP expression in keratinocytes are described, with strong expression in WT but minimal expression in keratin 13 knockout (KO) cells and quantitative evaluation by flow cytometry.
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