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-9353368-B2
Publication Date
2016-05-31
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 t 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 disclosed subject matter concerns non-naturally occurring miRNAs built from miRNA scaffolds, including miR-196a-2 scaffolds and other scaffolds such as miR-204 and miR-26b. A non-naturally occurring miR-196a-2 miRNA includes a nucleic acid having a stem-loop structure comprising a miR-196a-2 scaffolding and a mature strand-star strand duplex.
The mature strand sequence is derived from a mature endogenous miRNA but is distinct from the sequence of the endogenous mature strand of miR-196a-2. The duplex defines specific nucleotide pairing and mismatch rules, including that position 1 of the mature strand is U and the opposite nucleotide in the star strand is G, and that the duplex contains a mismatch at position 12 of the mature strand.
Additional constraints limit where other mismatches or wobble pairs occur within the duplex, either disallowing additional mismatches or wobble pairs at any position other than positions 1 and 12, or allowing mismatches or wobble pairs only at one or more of positions 5 and 18 to 23 while requiring no mismatches or wobble pairs at positions 2 to 4 and 6 to 17. The mature strand is defined as 19 to 25 nucleotides in length.
Claims Coverage
The partial claim set includes three independent claims. Across these claims, the coverage centers on a non-naturally occurring miR-196a-2 miRNA with a defined stem-loop scaffold and mature/paired duplex mismatch pattern, applied in a recombinant expression vector and a pharmaceutical composition, with additional refinements regarding reporter constructs and carrier/context.
Non-naturally occurring miR-196a-2 miRNA scaffolded stem-loop with restricted mature-star duplex mismatch rules
A non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure with a miR-196a-2 scaffolding and a mature strand-star strand duplex, where the mature strand sequence is derived from a mature endogenous miRNA but is distinct from the endogenous mature strand of miR-196a-2; where position 1 of the mature strand is U and the opposite nucleotide in the star strand is G; where the duplex contains a mismatch at position 12; and where either no additional mismatches or wobble pairs occur at any position other than positions 1 and 12, or mismatches or wobble pairs occur only at one or more of positions 5, 18, 19, 20, 21, 22 or 23 with no mismatches or wobble pairs at positions 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16 and 17; wherein the mature strand is 19 to 25 nucleotides in length.
Recombinant expression vector encoding the non-naturally occurring miR-196a-2 miRNA
A recombinant expression vector comprising a nucleotide sequence that encodes a non-naturally occurring miR-196a-2 miRNA having a nucleic acid stem-loop structure incorporating a miR-196a-2 scaffold and a mature strand-star strand duplex, with the same mature-strand derivation and distinction from endogenous miR-196a-2, position 1 U/opposite G, mismatch at position 12, and the defined constraints on additional mismatches or wobble pairs plus no mismatches or wobble pairs at the specified other positions, and where the mature strand is 19 to 25 nucleotides in length.
Pharmaceutical composition including the non-naturally occurring miR-196a-2 miRNA with specified duplex mismatch pattern and pharmaceutically acceptable carrier
A pharmaceutical composition comprising a non-naturally occurring miR-196a-2 miRNA with a stem-loop nucleic acid incorporating a miR-196a-2 scaffold and a mature strand-star strand duplex, where the mature strand is derived from a mature endogenous miRNA but is distinct from the endogenous mature strand of miR-196a-2; where position 1 of the mature strand is U and the opposite nucleotide in the star strand is G; where the duplex contains a mismatch at position 12; where either no additional mismatches or wobble pairs occur at any position other than positions 1 and 12, or mismatches or wobble pairs occur at one or more of positions 5, 18, 19, 20, 21, 22, or 23 with no mismatches or wobble pairs at positions 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16 and 17; where the mature strand is 19 to 25 nucleotides in length; and at least one pharmaceutically acceptable carrier.
Overall, the claims protect a miR-196a-2 scaffolded non-naturally occurring miRNA with a specific mature-strand/U and star-strand/G relationship at position 1, a mismatch at position 12, and tightly limited additional mismatch or wobble positions, with the mature strand length constrained to 19 to 25 nucleotides. That miRNA is then covered both as part of a recombinant expression vector and as part of a pharmaceutical composition including a pharmaceutically acceptable carrier.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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