miRNA switches for RNA-triggered control of RNA interference
Inventors
Green, Alexander Arthur • Zhou, Yu • Sheng, Peike • Xie, Mingyi
Assignees
Boston University • University of Florida Research Foundation Inc
Publication Number
US-12365900-B2
Publication Date
2025-07-22
Expiration Date
2043-12-21
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Provided herein are methods, compositions and systems comprising synthetic nucleic acid molecules that enable inducible or conditional pri-miRNA processing, preferably in mammalian cells in vivo. Provided herein are synthetic nucleic acid molecules referred to as Orthogonal RNA Interference induced by Trigger RNA (ORIENTR) that switches between an inactive form and an active form upon interaction with one or more specific RNA-trigger molecules, which can be e.g., a synthetic RNA-trigger, or a disease-specific RNA signals, such as disease-specific mRNA, miRNA, or other cellular RNA products with sequences that characterize a disease state of a cell. The interaction between the RNA-trigger molecules and the ORIENTR is preferably mediated by hybridization, which exposes, facilitates the formation, and/or allows the formation of a correctly folded pri-miRNA scaffold substrate that can be processed by proteins of the RNAi pathway (such as Dicer), leading to RNAi-mediated repression of a target gene. Also provided herein are methods of using such ORIENTR molecules for the treatment or prevention of a disease in a subject, as well as detecting the presence or absence of a target RNA in a biological sample or in vivo.
Core Innovation
The invention provides methods, compositions, and systems comprising synthetic nucleic acid molecules called Orthogonal RNA Interference induced by Trigger RNA (ORIENTR), which enable inducible or conditional pri-miRNA processing preferably in mammalian cells in vivo. These synthetic RNA molecules switch between inactive and active forms upon hybridization with one or more specific RNA-trigger molecules, which can be synthetic or disease-specific RNAs such as mRNAs or miRNAs characteristic of a disease state. The interaction exposes or facilitates formation of a correctly folded pri-miRNA scaffold substrate processed by RNAi pathway proteins like Drosha, leading to production of functional miRNAs that mediate RNA interference and target gene repression.
The problem being solved is the limitations of constitutively active, unregulated RNAi which competes with endogenous RNAi, causing reduced potency and off-target effects, as well as challenges in targeting essential genes due to tissue-wide knockdown toxicity. Current RNA switch technologies have been used to upregulate transgenes but have not been developed for downregulation of endogenous genes through RNAi in mammalian cells. Prior conditional RNAi systems focused on Dicer substrates, which pose challenging structural requirements and only work in vitro or in lysates, unable to function robustly in live cells. There is a need for methods to enable programmable, tunable, and conditional RNA interference with spatiotemporal control in living mammalian cells.
The core innovation is an engineered synthetic toehold-mediated RNA riboregulator (ORIENTR) comprising a sensing domain with a toehold and 3' sequestering arm that adopts an inactive secondary structure sequestering the 5' basal stem sequence and preventing formation of the pri-miRNA scaffold. Upon binding a specific RNA-trigger molecule, toehold-mediated strand displacement releases the sequestered 5' basal stem sequence, allowing it to base pair with the 3' basal stem sequence and form the basal stem in the reconfiguration domain. This refolding into an active pri-miRNA scaffold allows Drosha recognition and microprocessor processing of the output domain to generate mature miRNA, which induces target gene repression via RNA interference. The technology decouples RNA-trigger input sequence from miRNA output sequence, enabling programmable conditional RNAi responses in live mammalian cells.
Claims Coverage
The patent discloses twelve main inventive features covering the structure, configuration, and components of the ORIENTR nucleic acid construct, its conditional switching mechanism, and associated RNA triggers, as claimed in the independent claims.
ORIENTR nucleic acid construct with sensing, reconfiguration, and output domains
A nucleic acid construct comprising an ORIENTR molecule with a single-stranded 5′ toehold domain (T1), a partially double-stranded sequestering loop domain (SLD) with 3′ sequestering arm regions (S1, S2), a stem-loop, a 3′ flanking sequence (S2f), a 5′ basal stem sequence base paired to S1, and an output domain comprising a double-stranded RNAi hairpin with guide strand, loop, and passenger strand sequences, plus a 3′ basal stem capable of base pairing with 5′ basal stem to form a basal stem recognized by Drosha, and a spacer sequence containing a small hairpin sequence (SHS) forming a leak-reduction motif and a CNNC motif.
pri-miRNA hairpin output domain with an apical loop
The RNAi hairpin in the output domain is a pri-miRNA hairpin structure, and the loop sequence is an apical loop enabling processing by microprocessor.
Inclusion of RNA Pol III terminator sequence
The ORIENTR comprises a RNA Pol III terminator sequence located 3′ of the 3′ basal stem sequence or 3′ of the spacer sequence to enable proper transcription termination.
Single stranded 3′ basal stem sequence
The 3′ basal stem sequence is single stranded in the inactive ORIENTR configuration to enable conditional folding upon trigger binding.
Leak-reduction motif comprising SHS of at least 5 bp
The small hairpin sequence (SHS) within the spacer is at least 5 base pairs in length and partially base pairs with a 3′ portion of the 3′ basal stem sequence to form a leak-reduction motif lowering background miRNA biogenesis.
ORIENTR reconfigures upon RNA trigger binding
In the presence of an RNA trigger, the ORIENTR forms a complex where the trigger base pairs with the 5′ toehold domain and S1 and S2 regions of the 3′ sequestering arm, releasing the basal stem sequences to form an active pri-miRNA scaffold with Drosha-recognized basal stem structure enabling miRNA biogenesis.
Basal stem length and imperfection
The 5′ and 3′ basal stem sequences are at least 11 nucleotides long and form an imperfect duplex basal stem structure that can be recognized and bound by Drosha.
RNA trigger stability hairpin
The RNA trigger can include a stability hairpin at the 5′ end to enhance stability and/or localization.
RNA trigger length
The RNA trigger single-stranded region complementary to the ORIENTR sensing domain is between 9-50 nucleotides in length.
CRISPR RNA scaffold hairpin stability in trigger
The stability hairpin on the RNA trigger can be a CRISPR RNA (crRNA) hairpin, such as the rfxCas13d scaffold hairpin, and can be bound by dCas13d protein.
Synthetic sequences in sensing domains and triggers
One or more of the 5′ toehold domain (T1), S1, S2, and 3′ sequestering arm sequences can be synthetic sequences, with corresponding RNA triggers being synthetic sequences complementary to these regions.
Endogenous RNA-trigger sequences
The 5′ toehold domain (T1), S1, S2, and 3′ sequestering arm regions can form duplexes with endogenous RNA trigger sequences selected from tissue-specific, disease-specific, environmental, developmental, temporal, or cell-cycle specific RNA sequences.
The claims cover the design and structural components of the ORIENTR nucleic acid construct comprising sensing, reconfiguration, and output domains with specific nucleic acid elements (toehold, sequestering arm, basal stems, spacer with leak-reduction motif), the structural switch upon RNA-trigger binding forming a Drosha-recognized pri-miRNA scaffold, the RNA triggers with or without stabilizing hairpins, and the use of synthetic or endogenous RNA triggers to conditionally activate RNA interference.
Stated Advantages
Provides programmable and tunable inducible RNA interference in living mammalian cells with high specificity and sensitivity.
Reduces off-target effects and toxicity associated with constitutive RNAi by enabling spatial and temporal control of miRNA biogenesis.
Decouples RNA-trigger input sequence from miRNA output sequence, allowing modular design to target any desired nucleic acid.
Allows response to endogenous RNA triggers including disease- or tissue-specific RNAs for precise gene regulation.
Improves regulation dynamics and stability by using deactivated Cas13d proteins to facilitate RNA-RNA interactions and nuclear localization.
Offers advantages over prior Dicer substrate-based conditional RNAi systems which suffer from structural constraints and lack robust switching in living cells.
Documented Applications
Use in programmable cellular RNA switches to regulate RNA interference for therapeutic or diagnostic purposes in mammalian cells and organisms.
Treatment or prevention of diseases or disorders by conditional repression of target genes via RNAi activated by disease-specific or other endogenous RNA triggers.
Detection of the presence or absence of target RNA sequences in biological samples or in vivo through conditional miRNA production.
Application in cell-type-specific RNAi by responding to tissue-specific or cell-state-specific RNA triggers.
Potential use for targeting essential genes safely by conditional gene knockdown only in desired tissues or under specific conditions.
Use in synthetic biology to rewire cell behavior or gene expression circuits responsive to RNA expression profiles.
Interested in licensing this patent?