Micro-RNA scaffolds and non-naturally occurring micro-RNAs
Inventors
Kelley, Melissa • Birmingham, Amanda • Karpilow, Jon • Khvorova, Anastasia • Sullivan, Kevin
Assignees
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Publication Number
US-9284554-B2
Publication Date
2016-03-15
Expiration Date
Abstract
The present disclosure provides a non-naturally occurring miRNA having a stem-loop structure comprising a scaffold derived from a first endogenous miRNA (e.g., miR-196a-2 or miR-204), a mature strand derived from a second endogenous miRNA, and a star strand sequence that is at least partially complementary to the mature strand sequence. The present disclosure also provides a non-naturally occurring miRNA having a stem-loop structure comprising a scaffold derived from an endogenous miRNA (e.g., miR-196a-2 or miR-204), a mature strand designed to be at least partially complementary to a target RNA, and a star strand sequence that is at least partially complementary to the mature strand sequence. The methods and compositions of the disclosure may be used to mediate gene silencing via the RNAi pathway.
Core Innovation
The invention provides non-naturally occurring miRNAs that include a stem-loop structure with a miRNA stem-loop scaffold derived from an endogenous miRNA, combined with a non-endogenous mature-strand sequence and a star strand that is complementary to the mature strand. In particular, it discloses non-naturally occurring miR-196a-2 miRNAs in which the stem of the stem-loop structure incorporates a miRNA-196-a-2 scaffold, and a mature strand-star strand duplex is formed by the designed mature and star sequences.
The core design controls processing in the cell to yield a mature miRNA having the sequence of the mature strand derived from a mature endogenous miRNA distinct from the endogenous mature strand sequence of miR-196a-2. The duplex is constrained by specified rules for nucleotide and mismatch/wobble-pair patterns, including position-specific requirements for the mature strand and its complementary star strand, and a mismatch at position 12 with additional restrictions on mismatches or wobble pairs at other positions.
The disclosure further includes compositions and expression concepts for mediating RNAi gene silencing, including vector- and promoter-based expression, and scaffold engineering concepts for generating high-performance scaffolds. It provides design rationale for shMIMICs, as well as targeting algorithms and criteria that govern mature-strand nucleotide preferences and star-strand structural features intended to maintain processing and functionality.
Claims Coverage
Independent claim clm-00001 covers delivery and intracellular processing of a non-naturally occurring miR-196a-2 stem-loop miRNA to yield a specified mature strand sequence, with major inventive control over the stem-loop scaffold and the mature–star duplex mismatch/wobble pattern. The dependent claims refine the delivery modality and further constrain the miRNA design using explicit identifiers and/or additional sequence and structure constraints.
Delivery and intracellular processing of a non-naturally occurring miR-196a-2 stem-loop miRNA to yield a mature strand
Introducing in a cell a non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid with a stem-loop structure whose stem incorporates a miRNA-196-a-2 scaffold and a mature strand-star strand duplex, where the miRNA is processed in the cell to yield a mature miRNA having the mature strand sequence.
Stem-loop scaffold incorporates a miRNA-196-a-2 scaffold and forms a mature strand-star strand duplex
Employing a stem-loop structure with a stem that incorporates a miRNA-196-a-2 scaffold, together with a mature strand and a star strand forming a mature strand-star strand duplex.
Mature strand derived from a distinct mature endogenous miRNA with position-specific nucleotide and duplex mismatch constraints
Defining the mature strand as having a sequence derived from a mature endogenous miRNA whose sequence is distinct from the endogenous mature strand of miR-196a-2, with positional constraints including U at position 1 and the opposite nucleotide in the star strand as G, and a duplex containing a mismatch at position 12 of the mature strand under specified restrictions on additional mismatches or wobble pairs at other positions.
Constrained mature strand length and controlled mismatch/wobble pattern positions within the duplex
Providing that the mature strand is about 19–25 nucleotides in length, and specifying which positions other than 1 and 12 may contain mismatches or wobble pairs via two alternative constraint sets, either no additional mismatches or wobble pairs, or mismatches or wobble pairs limited to particular positions within the duplex.
Processing yields the mature miRNA sequence of the mature strand
Stating that the non-naturally occurring miR-196a-2 miRNA is processed in the cell to yield a mature miRNA having the sequence of the mature strand.
Across the independent claim, the core inventive coverage is delivery of a non-naturally occurring miR-196a-2 stem-loop miRNA that uses a miRNA-196-a-2 scaffold and a designed mature/star duplex, together with explicit positional mismatch/wobble and length constraints so that cellular processing yields a mature miRNA sequence derived from a distinct mature endogenous miRNA.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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