Alternative nucleic acid molecules containing reduced uracil content and uses thereof
Inventors
Hoge, Stephen G. • ISSA, William Joseph • Miracco, Edward John • Nelson, Jennifer • Reynders, John • Stanton, Matthew
Assignees
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Publication Number
US-10072057-B2
Publication Date
2018-09-11
Expiration Date
Abstract
The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them. In some aspects, the disclosure provides mRNA wherein the uracil content has been modified and which may be particularly effective for use in therapeutic compositions, because they may benefit from both high expression levels and limited induction of the innate immune response. In some aspects, the disclosure provides methods for the production of pharmaceutical compositions including mRNA without reverse phase chromatography.
Core Innovation
The invention relates to therapeutic pharmaceutical compositions comprising an mRNA encoding a polypeptide, with a 5-cap structure, a 5-UTR, an open reading frame (ORF), a 3-UTR, and a poly-A region. A defining feature is that at least 95% of uracils in the ORF are 5-methoxyuracils, and the uracil content in the ORF is between the theoretical minimum and 150% of the theoretical minimum.
In a comparative arrangement, the level of expression in a mammalian cell of the encoded polypeptide from the mRNA is increased relative to a reference mRNA comprising a reference ORF encoding the polypeptide, wherein at least 95% of uracils in the rORF are 5-methoxyuracils and wherein the uracil content in the rORF is from about 190% to about 200% of the theoretical minimum. The description further addresses uracil distribution constraints, including uracil clusters and a 20-nucleotide sliding window, and ties these constraints to reduced innate immune response including TLR7 and cytokine or IFN-b2 effects.
The invention also addresses mRNA quality and composition constraints intended to reduce innate immune activation, including minimizing RNA impurities such as aberrant transcription products and short RNAs, and references double-stranded RNA and RNA-dependent RNA polymerase contexts. It further discusses modified 5 cap structures with defined cap-analog variations, and states that more authentic caps can improve translational competency and stability.
Claims Coverage
The document includes one independent claim directed to a pharmaceutical composition with defined mRNA structural features and uracil-content constraints, and explicitly provides a comparative increased expression requirement versus a reference mRNA with higher uracil content.
Uracil-constrained therapeutic mRNA with increased mammalian cell expression
A pharmaceutical composition comprising an mRNA encoding a polypeptide and a pharmaceutically acceptable excipient, wherein the mRNA comprises a 5-cap structure, a 5-UTR, an ORF encoding the polypeptide, a 3-UTR, and a poly-A region, where at least 95% of uracils in the ORF are 5-methoxyuracils and the uracil content in the ORF is between the theoretical minimum and 150% of the theoretical minimum, and wherein the level of expression in a mammalian cell of the encoded polypeptide from the mRNA is increased relative to a reference mRNA comprising a reference ORF encoding the polypeptide, wherein at least 95% of uracils in the rORF are 5-methoxyuracils and wherein the uracil content in the rORF is from about 190% to about 200% of the theoretical minimum.
Claim coverage centers on an mRNA-based pharmaceutical composition with defined structural elements and a core uracil-content design specifying that at least 95% of ORF uracils are 5-methoxyuracils and that ORF uracil content is between the theoretical minimum and 150% of the theoretical minimum, resulting in increased mammalian cell expression relative to a reference mRNA having a higher specified uracil content.
Stated Advantages
Increased level of expression in a mammalian cell of the encoded polypeptide from the mRNA relative to a reference mRNA.
Reduced innate immune induction, including reduced TLR7 and cytokine/IFN-b2 responses.
More authentic 5 caps are described as improving translational competency and stability.
Documented Applications
Therapeutic mRNA use in pharmaceutical mRNA compositions for mammalian cell expression.
Reporter expression and protein expression assessment using luciferase and mCherry reporter mRNAs in mammalian cells, including measurements of luciferase/mCherry signals and ELISA-based measurement of secreted hEPO and GCSF.
Assessment of innate immune activation by measuring IFN-b2 levels after administration/expression in mammalian cell contexts.
In vivo expression of hEPO in CD-1 mice and cynomolgus monkeys (non-human primates), including reporting of hEPO Cmax and AUC.
Limiting innate immune induction in mammalian cells, including TLR7 and cytokine/IFN-b2-related responses.
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