Compositions and methods of generating novel amiRNA
Inventors
Militello, Giuseppe • Fellmann, Christof • Zuber, Johannes
Assignees
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Publication Number
US-12365902-B2
Publication Date
2025-07-22
Expiration Date
Abstract
Provided herein are Compositions and Methods of Generating Novel amiRNA's. Provided herein are methods for producing or generating one or more amiRNAs; also provided are constructs and compositions useful in the methods. The methods and constructs provided in this disclosure are highly efficient methods for production of a new generation of amiRNAs.
Core Innovation
The invention relates to generating novel artificial miRNAs (amiRNAs) with enhanced potency versus benchmark scaffolds miRE and miR155. It addresses the design of amiRNAs by modifying a selected primary miRNA at defined scaffold subregions that correspond to features used by Drosha and Dicer processing, including the Base region, the Basal Stem region, and the Loop region of the primary miRNA.
A base modifying step produces a single stranded region of at least 9 nucleotides and a GU dinucleotide in position −13 and −14 from a Drosha cleavage site to form a base modified construct. A basal stem step produces a double stranded RNA with a CHC motif and a bulge in position +8 from the Drosha cleavage site to form a basal stem modified construct.
A loop modifying step substitutes the endogenous loop region of the primary miRNA with the loop of a miRE, or another loop with the same number of nucleotides and the GUG motif in the same position, to form a loop modified construct. The modified base, basal stem, and loop constructs are operably linked with the primary miRNA into an amiRNA construct.
Claims Coverage
The independent claim describes a method with three principal structural modification modules applied to a selected primary miRNA, followed by operable linking to form an amiRNA construct. The dependent claims further constrain the structural parameters and add functional and processing comparisons, including PTEN mRNA downregulation and guide-versus-passenger strand enrichment with DROSHA/DICER processing.
Base modification with a GU dinucleotide near the Drosha cleavage site
Applying a base modifying step to produce a single stranded region of at least 9 nucleotides and a GU dinucleotide in position −13 and −14, respectively from a Drosha cleavage site to form a base modified construct.
Basal stem modification with CHC motif and a bulge at position +8
Applying a basal stem step to produce a double stranded RNA with a CHC motif and a bulge in position +8 from the Drosha cleavage site to form a basal stem modified construct.
Loop substitution with the miRE loop or a loop having the GUG motif
Applying a loop modifying step to substitute the endogenous loop region of the primary miRNA with the loop of a miRE, or another loop with same number of nucleotides and the GUG motif in the same position, to form a loop modified construct.
Operable linking of modified constructs with the primary miRNA into an amiRNA construct
Operably linking the base modified construct, the basal stem modified construct, and the loop modified construct with the primary miRNA into an amiRNA construct.
Modified basal stem length constraint
The modified basal stem has a total length of 9 to 13 base pairs, with an optimal length being 11 base pairs.
Loop sequence identity constraint
The loop region of the modified construct has at least 75% sequence identity to SEQ ID NOs: 3-9.
Stem sequence identity constraint
The stem modified construct has at least 75% sequence identity to SEQ ID NO: 1.
PTEN mRNA downregulation versus miRE and miR155
The method decreases PTEN mRNA expression using an amiRNA construct compared to miRE and miR155.
DROSHA and DICER processing with guide strand enrichment over Passenger strand
The method includes DROSHA and DICER processing of an amiRNA construct and detecting a guide strand at a magnitude of thousands more than the Passenger strand amount.
Overall, the claims cover amiRNA generation by base, basal stem, and loop modifications defined by Drosha-cleavage-relative nucleotide features, followed by operable linking into an amiRNA construct. Dependent claims add explicit structural constraints and functional or processing comparisons, including PTEN mRNA downregulation versus miRE and miR155 and DROSHA/DICER-associated guide-versus-passenger strand enrichment.
Stated Advantages
Enhanced potency of novel amiRNAs versus benchmark scaffolds miRE and miR155.
Greater silencing observed for novel amiRNAs in fluorescence-based potency assays.
Decreased PTEN mRNA expression compared to miRE and miR155.
High precision 5′ guide-strand processing.
Guide/passenger strand enrichment at thousands- to tens-of-thousands-fold magnitude.
Documented Applications
Delivery of the amiRNA construct using rAAV vectors for expression in hIPSC-derived NGN2 neurons, with a CAGG promoter-driven expression context mentioned in the document.
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