Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
Inventors
Chakraborty, Tirtha • de Fougerolles, Antonin
Assignees
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Publication Number
US-9303079-B2
Publication Date
2016-04-05
Expiration Date
Abstract
The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.
Core Innovation
The invention relates to modified nucleosides, nucleoside building blocks, and polynucleotides, including modified mRNA (mmRNA) constructed from linked nucleosides. The subject matter includes modified mRNA encoding functional proteins, modified ribonucleoside and ribonucleotide building-block compounds, and structural formulas defining nucleobase and sugar modification options for uracil, cytosine, guanine, and adenine variants, together with pharmaceutically acceptable salt forms and stereoisomers.
The disclosure describes nucleoside chemistries including 5-methylcytosine, pseudouridine, and N1-methyl-pseudouridine, and states that N1-methyl-pseudouridine-containing mRNAs show higher expression and reduced cytokine stimulation compared with pseudouridine-containing controls. It also includes modified internucleoside linkages, coordinated selection of base, sugar, and linkage modifications, and polynucleotide/mmRNA synthesis concepts using repeated reaction cycles and stepwise coupling frameworks.
Additional subject matter concerns mmRNA architecture and formulation features, including stop codons, 3′ tailing sequence options, 5′UTR and 3′UTR origin constraints, Kozak sequence concepts, cap and poly-A tail design, tissue-specific regulatory elements, microRNA binding sites, and delivery or retention systems. The disclosure further includes intracellular delivery systems, cell-penetrating peptides or proteins, hyaluronidase and hyaluronan, lipid- and liposome-type delivery systems, and broader formulation concepts using selected excipients and lipids.
Claims Coverage
The consolidated claim coverage centers on one independent mRNA claim and related dependent refinements. The inventive features include a sequence-defined mRNA encoding SEQ ID NO: 4947 with at least 80% identity to SEQ ID NO: 31871, together with structural and formulation limitations such as two stop codons, defined 3′ tailing options, 5′UTR and 3′UTR origin constraints, and selected excipient and lipid materials.
mRNA encoding SEQ ID NO: 4947 with coding region identity
An mRNA encoding SEQ ID NO: 4947 wherein the mRNA comprises a coding region having at least 80% identity to SEQ ID NO: 31871.
mRNA with two stop codons
The mRNA comprises two stop codons.
Defined 3′ tailing sequence
The mRNA comprises a 3′ tailing sequence of linked nucleosides at the 3′ terminus, selected from an approximately 160-nucleotide poly-A tail or a poly A-G quartet.
5′UTR and 3′UTR from different species
The mRNA has a 5′UTR and a 3′UTR that are not derived from the same species.
Pharmaceutical composition with selected excipient classes
The pharmaceutical composition comprises a pharmaceutically acceptable excipient selected from solvents, aqueous and non-aqueous dispersion components, stabilizing or emulsifying agents, lipids/lipid nanoparticles, polymers, proteins/cells, hyaluronidase, and mixtures thereof.
Formulation with specified lipid materials
The formulation comprises a lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA, PEGylated lipids, and mixtures or combinations thereof.
The claim coverage is directed to an mRNA encoding SEQ ID NO: 4947 with a coding region identity requirement relative to SEQ ID NO: 31871, further narrowed by stop-codon, tailing, and UTR-origin features, and by pharmaceutical composition and formulation selections using specified excipient classes and lipid materials.
Stated Advantages
N1-methyl-pseudouridine-containing mRNAs show markedly higher luciferase/G-CSF expression compared with pseudouridine-containing controls.
N1-methyl-pseudouridine-containing mRNAs show reduced cytokine stimulation compared with pseudouridine-containing controls.
Most tested nucleoside modifications maintain expression or activity in in vivo and in vitro readouts.
Reduced innate immune activation, including reduced interferon/cytokine signatures via immune evasion and immunomodulation of modified polynucleotides/mmRNA.
Quantification of immune response reduction using interferon/cytokine signatures and Protein:Cytokine Ratio (PC ratio) thresholds measured using a PBMC assay.
Broad therapeutic applicability across multiple disease areas, including vaccines/adjuvants and targeting pathogens/diseased cells.
Documented Applications
In vivo mouse bioluminescence readouts and ELISA cytokine/protein readouts following administration of modified mRNA formulations.
PBMC innate immune assays measuring IFN-α and TNF-α stimulation after exposure to modified mRNA.
HeLa cell and rabbit reticulocyte translation comparisons for modified mRNA protein production.
Formulation and route studies using delivery vehicles including PBS, PLGA microspheres, fibrin sealant hydrogel, LNP/Lipoplex, and osmotic pumps, and administration routes including IM, SC, IV, and intranasal.
IVIS-based temporal expression kinetics and reported organ distribution for LNPs.
Protein production after formulation, including modified mRNA protein production after PLGA-encapsulated luciferase formulation.
Therapeutic applications directed to pathogens/diseased cells in broad disease areas spanning fungal, protozoal, parasitic, and prion infections.
Vaccines/adjuvants, including self-replicating/self-amplifying RNA, as part of the downstream therapeutic/application scope.
Use in contexts involving immune response reduction and immune evasion quantified by interferon/cytokine signatures and PC ratios in PBMC assays.
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