Micro-RNA scaffolds, non-naturally occurring micro-RNAs, and methods for optimizing non-naturally occurring micro-RNAs
Inventors
Kelley, Melissa • Birmingham, Amanda • Karpilow, Jon • Khvorova, Anastasia • Sullivan, Kevin
Assignees
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Publication Number
US-8841267-B2
Publication Date
2014-09-23
Expiration Date
Abstract
The present disclosure provides non-naturally occurring miR-196a-2 miRNAs and non-naturally occurring miR-204 miRNAs. The non-naturally occurring miRNAs of the disclosure have mature strand sequences distinct from their endogenous counterparts. The disclosure also provides methods of selecting mature strand sequences that function optimally in non-naturally occurring miR-196a-2 miRNAs. The methods and compositions of the disclosure may be used to mediate gene silencing via the RNAi pathway.
Core Innovation
The invention relates to RNA interference using a non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure. The stem-loop structure incorporates a miR-196a-2 scaffold and a mature strand and star strand duplex, and the mature strand sequence is distinct from the sequence of the endogenous mature strand of any miRNA.
The mature strand is designed for complementarity to a target RNA, either fully or with defined wobble and mismatch constraints. The star strand is complementary to the mature strand under a defined rule at position 1, where position 1 of the mature strand is U and the opposite nucleotide in the star strand is G, and the duplex contains a mismatch at position 12 with further restricted mismatches or wobble pairs at specified positions.
The mature strand of the duplex is between about 19 and 25 nucleotides in length, and positions of the mature strand are numbered relative to the 5′ end. The documented platform also provides scaffold screening and scaffold modification using restriction sites to enable cloning while tuning mature versus star activity.
Claims Coverage
The partial content includes three independent claims for the miRNA itself, a recombinant expression vector, and a pharmaceutical composition. Each independent claim centers on a non-naturally occurring miR-196a-2 stem-loop with a miR-196a-2 scaffold and a specified mature strand–star strand duplex that follows defined complementarity and mismatch or wobble rules to a target RNA, with a mature-strand length constraint of about 19 to 25 nucleotides.
Non-naturally occurring miR-196a-2 miRNA with defined mature–star duplex complementarity rules
A non-naturally occurring miR-196a-2 miRNA nucleic acid having a stem-loop structure incorporating a miR-196a-2 scaffold and a mature strand and star strand duplex, where the mature strand sequence is distinct from the endogenous mature strand of any miRNA and is either 100% complementary to a portion of a target RNA or 100% complementary except that the target RNA forms a wobble base pair with position 1 of the mature strand; the star strand is complementary with position 1 defined as U in the mature strand and G in the star strand; the duplex contains a mismatch at position 12 and further allows mismatches or wobble pairs only at specified positions while excluding mismatches or wobble pairs at other specified positions; and the mature strand is between about 19 and 25 nucleotides in length.
Recombinant expression vector encoding the non-naturally occurring miR-196a-2 miRNA with defined duplex mismatch/wobble rules
A recombinant expression vector comprising a nucleotide sequence encoding a non-naturally occurring miR-196a-2 miRNA whose stem-loop structure incorporates a miR-196a-2 scaffold and a mature strand–star strand duplex with mature-strand complementarity to a target RNA under the same specified full or exceptional complementarity rules, position 1 U in the mature strand and G in the star strand, mismatch at position 12, and restricted allowed mismatch or wobble positions with excluded mismatches or wobble in other specified positions, wherein the mature strand is between about 19 and 25 nucleotides in length.
Pharmaceutical composition comprising the non-naturally occurring miR-196a-2 miRNA with defined duplex mismatch/wobble rules and pharmaceutically acceptable carrier
A pharmaceutical composition comprising a non-naturally occurring miR-196a-2 miRNA with a stem-loop structure incorporating a miR-196a-2 scaffold and a mature strand–star strand duplex, where the mature strand sequence is distinct from endogenous miRNA mature strands and is either 100% complementary to a portion of a target RNA or 100% complementary except for a wobble base pair at position 1, with the star strand complementarity defined at position 1 as U in the mature strand and G in the star strand; the duplex contains a mismatch at position 12 and allows mismatches or wobble pairs only at specified positions while excluding mismatches or wobble pairs at other specified positions; the mature strand is between about 19 and 25 nucleotides in length; and at least one pharmaceutically acceptable carrier.
Across the independent claims, the central inventive structure is a non-naturally occurring miR-196a-2 stem-loop with a miR-196a-2 scaffold and a mature strand–star strand duplex engineered for tightly specified target-RNA complementarity with defined wobble or mismatch constraints, including a mismatch at position 12 and allowed or disallowed wobble or mismatch positions, together with a mature-strand length between about 19 and 25 nucleotides. The claims further differ by including the miRNA alone, the miRNA encoded in a recombinant expression vector, or the miRNA formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier.
Stated Advantages
The miR-196a-2 algorithm-designed targeting outperforms an siRNA design algorithm for multiple genes in the documented comparisons.
Improved functionality is linked to low GC in the seed region.
Documented Applications
Scaffold screening using reporter knockdown, including reporter gene targeting such as GFP.
Targeting through GAPDH target walks to assess dependence on secondary structure.
Evaluation of gene targeting performance for multiple genes including CDC2, CD28, CD69, LAT, MAPK1, EGFR, and Zap70.
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