Modified polynucleotides for the production of proteins
Inventors
Bancel, Stephane • Chakraborty, Tirtha • de Fougerolles, Antonin • Elbashir, Sayda M. • John, Matthias • Roy, Atanu • Whoriskey, Susan • Wood, Kristy M. • Hatala, Paul • Schrum, Jason P. • Ejebe, Kenechi • Ellsworth, Jeff Lynn • Guild, Justin
Assignees
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Publication Number
US-9254311-B2
Publication Date
2016-02-09
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 disclosed subject matter relates to modified mRNA and polynucleotides encoding polypeptides, including mRNA architecture with a coding region flanked by 5' and 3' untranslated regions, together with a 5' terminal cap and a poly-A tail. The disclosed constructs are used for non-integrative polynucleotide delivery to achieve protein expression without genomic integration, and may include translation initiation and termination signals and optional signal sequences to support expression and, where applicable, protein localization and secretion.
The disclosure also relates to chemically modified nucleoside and nucleoside building block molecules used to form polynucleotides and mRNA, including modified nucleoside/mRNA-incorporated building block molecules as pharmaceutically acceptable salts and/or stereoisomers. The building blocks are described with variable positions including Y1, Y3, Y4, Y6, and an r parameter ranging from 0 to 5, and include nucleobase and sugar modifications, modified pyrimidine and nucleoside structures, modified ribose structures, and modified internucleoside linkages.
Advanced formats include cyclized mmRNA and concatemerized mmRNA in which intramolecular or intermolecular 5'-3' linkage is formed. The linkage can be implemented through chemical, enzymatic, or ribozyme routes, and mmRNA multimerization is described using 3' modified nucleotides and covalent linking, including covalent conjugation approaches such as click chemistry, together with mmRNA conjugation and combination with other molecules and RNAi-related agents.
Claims Coverage
The consolidated claim coverage centers on one independent mRNA claim and its dependent refinements, together with later dependent composition and method features. The inventive features comprise a specific mRNA encoding SEQ ID NO: 13267 with at least 80% identity to SEQ ID NO: 22033, plus structural mRNA elements, selected lipid composition features, and a mammalian administration-based production method.
mRNA encoding SEQ ID NO: 13267 with coding region identity
An mRNA encoding SEQ ID NO: 13267, wherein said mRNA comprises a coding region having at least 80% identity to SEQ ID NO: 22033.
3' tailing sequence of linked nucleosides
The mRNA includes a 3' tailing sequence selected as either an approximately 160-nucleotide poly-A tail or a polyA-G quartet.
5' terminal cap selected from specified cap types
The mRNA includes at least one 5' terminal cap selected from Cap0, Cap1, and various modified guanosines.
Cross-species UTR sourcing
The mRNA has a 5'UTR and a 3'UTR derived from different species.
Pharmaceutical composition with selected lipid excipient list
A pharmaceutical composition includes a selected lipid chosen from a specified list of lipids, including DLin variants, nucleoside-associated lipid names, reLNPs, PLGA, PEGylated lipids, and mixtures thereof.
Producing a target polypeptide in mammalian systems
A method of producing a target polypeptide in a mammalian cell, tissue, or organism by administering the pharmaceutical composition.
The claim coverage is anchored by the sequence-defined mRNA claim and is refined by a defined 3' tailing sequence, selected 5' cap types, different-species UTR sourcing, a pharmaceutical composition with selected lipids, and a method of producing a target polypeptide in mammalian systems by administering that composition.
Stated Advantages
Improved stability/half-life.
Improved translation efficiency.
Immune evasion by reducing or mitigating the innate immune response.
Support for protein localization and secretion where applicable.
Protein expression without genomic integration (non-integrative polynucleotide delivery).
Reduction of innate immune cytokines (IFN-β1 and TNF-β1) and innate immune transcript markers.
Higher protein expression is obtained with N1-methyl-pseudouridine-containing mRNA.
Low cytokine stimulation is associated with mRNA containing N1-methyl-pseudouridine.
Reduced cytokine induction is reported for selected chemistries, especially 2'-fluoro compounds and N1-methyl-pseudouridine-containing mRNA.
Loss of luciferase activity is reported for 2'-fluorouridine in the single-modification comparisons.
The disclosure frames delivery enhancement, increased tissue permeability, and measurable improvements including protein expression and a reduced innate immune response.
Documented Applications
In vitro and in vivo expression studies of modified mRNAs delivered by LNPs or lipoplexes, including expression and biodistribution assessment for proteins such as EPO, G-CSF, Factor IX, and IgG antibodies (Trastuzumab and Rituximab), using assays including ELISA, IHC, western blot, FACS, and bioluminescence imaging.
Assessment of immunological effects in PBMC and BJ fibroblasts, including reduction of innate immune cytokines (IFN-β1 and TNF-β1) and modulation of innate immune transcript markers in response to different modified mRNA chemistries.
Screening of alternative base/sugar chemical modifications across PBMC, HeLa, rabbit reticulocyte lysates, and in vivo mouse systems to evaluate protein expression versus cytokine induction tradeoffs.
Protein expression evaluation using modified mRNA encoding luciferase, including comparative readouts such as luciferase expression measurements.
Protein expression evaluation using modified mRNA encoding G-CSF.
Assessment of innate immune response, including cytokine and innate immune response gene measurements in BJ fibroblasts and PBMCs after mRNA exposure.
Delivery/formulation studies using multiple formulations and delivery formats including PLGA microspheres, saline, osmotic pumps, fibrin sealant, and lipid nanoparticle/cationic lipid formulations, with routes of administration and associated biodistribution signals described.
Cell delivery and expression of mRNA, polynucleotide/primary constructs, and mmRNA formulations.
Delivery using cell-penetrating peptides/proteins, liposomes, hyaluronidase with hyaluronan, cell-based carriers including RBCs and virosomes/VLPs, nanoparticle mimics, nanotubes including rosette nanotubes, and lipid nanoparticles/lipidoids and related lipid systems.
Producing a target polypeptide in a mammalian cell, tissue, or organism by administering a pharmaceutical composition.
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