Modified polynucleotides

• US 10,501,512 B2
• Filed: 03/09/2013
• Issued: 12/10/2019
• Est. Priority Date: 04/02/2012
• Status: Active Grant

• Current Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Original Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Inventors: De Fougerolles, Antonin, Guild, Justin
• Primary Examiner(s): Gonzalez, Antonio Galisteo
• Application Number:
  US14/390,097
• Publication Number:
  US 20150064235A1
• Time in Patent Office: 2,467 Days
• Field of Search: 425 691, 536 235
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/7115 Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/17 from animals; from humans enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/1767 from invertebrates
• CPC Class Codes: A61K 38/177 Receptors; Cell surface antigens; Cell surface determinants
• CPC Class Codes: A61K 38/1816 Erythropoietin [EPO]
• CPC Class Codes: A61K 38/1866 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: A61K 38/1891 Angiogenesic factors; Angiogenin
• CPC Class Codes: A61K 38/191 Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61K 38/212 IFN-alpha
• CPC Class Codes: A61K 38/215 IFN-beta
• CPC Class Codes: A61K 38/36 Blood coagulation or fibrinolysis factors
• CPC Class Codes: A61K 38/363 Fibrinogen
• CPC Class Codes: A61K 38/44 Oxidoreductases (1)
• CPC Class Codes: A61K 38/45 Transferases (2)
• CPC Class Codes: A61K 38/4833 Thrombin (3.4.21.5)
• CPC Class Codes: A61K 38/4846 Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 47/10 : Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• CPC Class Codes: A61K 47/34 : Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers A61K47/10 takes precedence
• CPC Class Codes: A61K 47/54 : the modifying agent being an organic compound
• CPC Class Codes: A61K 47/542 : Carboxylic acids, e.g. a fatty acid or an amino acid
• CPC Class Codes: A61K 47/543 : Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 48/00 : Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/0033 : the non-active part being non-polymeric
• CPC Class Codes: A61K 48/005 : characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 48/0066 : Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• CPC Class Codes: A61K 48/0075 : characterised by an aspect of the delivery route, e.g. oral, subcutaneous
• CPC Class Codes: A61K 9/0019 : Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/1271 : Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• CPC Class Codes: A61K 9/1272 : with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: A61K 9/1277 : Processes for preparing; Proliposomes
• CPC Class Codes: A61K 9/14 : Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles microspheres A61K9/16; microcapsules A61K9/50; nanocapsules, nanoparticles of the matrix type A61K9/51
• CPC Class Codes: A61K 9/145 : with organic compounds
• CPC Class Codes: A61K 9/5031 : obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
• CPC Class Codes: A61K 9/5123 : Organic compounds, e.g. fats, sugars
• CPC Class Codes: A61K 9/5146 : obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
• CPC Class Codes: A61K 9/5153 : Polyesters, e.g. poly(lactide-co-glycolide)
• CPC Class Codes: C07K 14/005 : from viruses
• CPC Class Codes: C07K 14/435 : from animals; from humans
• CPC Class Codes: C07K 14/47 : from mammals
• CPC Class Codes: C07K 14/4705 : stimulating, promoting or activating activity
• CPC Class Codes: C07K 14/4713 : Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
• CPC Class Codes: C07K 14/475 : Growth factors; Growth regulators
• CPC Class Codes: C07K 14/485 : Epidermal growth factor [EGF], i.e. urogastrone
• CPC Class Codes: C07K 14/505 : Erythropoietin [EPO]
• CPC Class Codes: C07K 14/515 : Angiogenesic factors; Angiogenin
• CPC Class Codes: C07K 14/525 : Tumour necrosis factor [TNF]
• CPC Class Codes: C07K 14/535 : Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/56 : IFN-alpha
• CPC Class Codes: C07K 14/565 : IFN-beta
• CPC Class Codes: C07K 14/62 : Insulins
• CPC Class Codes: C07K 14/705 : Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/745 : Blood coagulation or fibrinolysis factors
• CPC Class Codes: C07K 14/75 : Fibrinogen
• CPC Class Codes: C07K 16/00 : Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2887 : against CD20
• CPC Class Codes: C07K 16/32 : against translation products of oncogenes
• CPC Class Codes: C07K 19/00 : Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C12N 15/11 : DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/52 : Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/67 : General methods for enhancing the expression
• CPC Class Codes: C12N 15/85 : for animal cells
• CPC Class Codes: C12N 15/87 : Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/88 : using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 2840/00 : Vectors comprising a special translation-regulating system
• CPC Class Codes: C12N 2840/85 : mammalian
• CPC Class Codes: C12N 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/1051 : Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/16 : acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/2402 : hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12N 9/2445 : Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12N 9/644 : Coagulation factor IXa (3.4.21.22)
• CPC Class Codes: C12N 9/6451 : Coagulation factor XIIa (3.4.21.38)
• CPC Class Codes: C12N 9/88 : Lyases (4.)
• CPC Class Codes: C12N 9/93 : Ligases (6)
• CPC Class Codes: C12P 13/04 : Alpha- or beta- amino acids other amino acids C12P13/005
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/005 : Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 113/12007 : Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
• CPC Class Codes: C12Y 304/21005 : Thrombin (3.4.21.5)
• CPC Class Codes: C12Y 304/21022 : Coagulation factor IXa (3.4.21.22)
• CPC Class Codes: C12Y 603/02019 : Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme

Abstract

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

Claims

1. A composition comprising:

a plurality of lipid nanoparticles comprising a cationic lipid, a PEGylated lipid, cholesterol, and a phospholipid, wherein the mean particle size of the plurality of lipid nanoparticles is 80 nm to 160 nm and the lipid nanoparticles encapsulate a polynucleotide,

wherein the polynucleotide comprises;

(a) a first region of linked nucleosides, said first region encoding factor IX, wherein said first region of linked nucleosides consists of nucleotides selected from 1-methyl-pseudouridine, cytidine, adenosine, and guanosine;

(b) a first flanking region located at the 5′

-terminus of said first region comprising;

(i) a 5′

-untranslated region (5′

-UTR); and

(ii) 5′

terminal cap;

(c) a second flanking region located at the 3′

-terminus of said first region comprising;

(i′

) a 3′

-untranslated region (3′

-UTR); and

(ii′

) a 3′

-tailing sequence of linked nucleosides.

2. The composition of claim 1, wherein the 3′

-tailing sequence of linked nucleosides is selected from the group consisting of a poly-A tail of approximately 160 nucleotides and a polyA-G quartet.

3. The composition of claim 1, wherein the 5′

terminal cap is selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′

-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

4. A pharmaceutical composition comprising the composition of claim 1 and an excipient.

5. The pharmaceutical composition of claim 4, wherein the excipient is selected from a solvent, aqueous solvent, non-aqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, lipid, lipidoids liposome, lipid nanoparticle, core-shell nanoparticles, polymer, lipoplex peptide, protein, cell, hyaluronidase, and mixtures thereof.

6. The composition of claim 1, where the cationic lipid is selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA, and DLenDMA.

7. The composition of claim 1, wherein the 5′

-UTR and the 3′

-UTR are not derived from the same species.

8. The composition of claim 1, wherein at least one of the 5′

-UTR or the 3′

-UTR is not derived from beta-globin.

9. The composition of claim 1, wherein the nanoparticles comprise about 50 mol % cationic lipid, about 38.5% cholesterol, about 10% phospholipid and about 1.5% PEGylated lipid.

10. A method of producing factor IX in a mammalian cell, tissue or organism comprising administering to said cell, tissue or organism the pharmaceutical composition of claim 4.