Compositions and methods for synthesizing 5'-Capped RNAs
Inventors
Hogrefe, Richard I. • Lebedev, Alexandre • McCaffrey, Anton P. • Shin, Dongwon
Assignees
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Publication Number
US-11578095-B2
Publication Date
2023-02-14
Expiration Date
Abstract
Provided herein are methods and compositions for synthesizing 5′Capped RNAs wherein the initiating capped oligonucleotide primers have the general form m7Gppp[N2′Ome]n[N]m wherein m7G is N7-methylated guanosine or any guanosine analog, N is any natural, modified or unnatural nucleoside, “n” can be any integer from 0 to 4 and “m” can be an integer from 1 to 9.
Core Innovation
The invention is directed to an initiating capped oligonucleotide primer for synthesizing 5′-capped RNAs. The primer includes an m7Gppp[N2′Ome]n[N]m motif, where m7G is an N7-methylated guanosine or analog, and N2′-Ome is an N2′-O-methyl.
The disclosed primer is used as an initiating capped oligonucleotide primer in promoter-controlled in vitro transcription using a DNA template, including T7 RNA polymerase. Co-transcriptional initiation is described to produce 5′ cap structures including Cap 0, Cap 1, Cap 2, and TMG-cap-like 5′ structures.
The document further describes downstream embodiments of RNA molecules produced using the initiating capped oligonucleotide primer, including isolated cells containing the RNA and modified cells in which a protein or peptide is produced by translation of the RNA. Pharmaceutical compositions including the RNA and a pharmaceutically acceptable carrier are also described.
Claims Coverage
The independent claim covers a selected set of initiating capped oligonucleotide primer salt forms having an m7G 3′Ome cap structure and defined downstream nucleotide/block compositions, including variants containing pppA, pppApG, and ppp(N6-methyladenine) with 2′Ome phosphate. Dependent claims extend the scope to an RNA molecule and to isolated or modified cells, and to pharmaceutical compositions with a pharmaceutically acceptable carrier.
Initiating capped oligonucleotide primer salt forms with m7G 3′Ome cap structure
An initiating capped oligonucleotide primer selected from salt forms having an m7G 3′Ome motif and defined downstream compositions including pppA, pppApG, and ppp(N6-methyladenine), with 2′Ome and terminal 2′Ome phosphate as recited.
RNA molecule comprising the capped primer-defined structure
An RNA molecule that includes the initiating capped oligonucleotide primer defined as in the prior claim set.
Isolated cell containing the RNA molecule
An isolated cell that contains an RNA molecule as defined in the corresponding claim dependency.
Modified cell producing a translated protein or peptide
A modified cell that includes a protein or peptide produced by translating an RNA molecule as defined in the corresponding claim dependency.
Pharmaceutical composition containing the RNA molecule and pharmaceutically acceptable carrier
A pharmaceutical composition containing an RNA molecule as defined in the corresponding claim dependency together with a pharmaceutically acceptable carrier.
Overall claim coverage is centered on a defined initiating capped oligonucleotide primer salt-form selection with an m7G 3′Ome cap motif and specific downstream compositions, and then extends the resulting RNA into cell and pharmaceutical composition embodiments, including isolated cells, modified cells with translated proteins or peptides, and pharmaceutical compositions with pharmaceutically acceptable carriers.
Stated Advantages
Improved capping efficiency.
Improved initiation fidelity.
Reduced bidirectional initiation and uncapped products.
Eliminate extra enzymatic cap/2′-O-methylation steps.
Documented Applications
Producing 5′-capped RNAs via co-transcriptional initiation using a DNA template and promoter-controlled in vitro transcription.
Providing RNA molecules used for downstream embodiments including isolated cells containing the RNA and modified cells producing a protein or peptide translated from the RNA.
Pharmaceutical compositions comprising the RNA molecule together with a pharmaceutically acceptable carrier.
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