Modified polynucleotides for the production of proteins

• US 9,827,332 B2
• Filed: 11/13/2015
• Issued: 11/28/2017
• Est. Priority Date: 04/02/2012
• Status: Active Grant

• Current Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Original Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Inventors: Bancel, Stephane, Chakraborty, Tirtha, de Fougerolles, Antonin, Elbashir, Sayda M., Ellsworth, Jeff Lynn, Ejebe, Kenechi, Guild, Justin, John, Matthias, Hatala, Paul, Roy, Atanu, Schrum, Jason P., Whoriskey, Susan, Wood, Kristy M.
• Primary Examiner(s): Galisteo Gonzalez, Antonio
• Application Number:
  US14/940,512
• Publication Number:
  US 20160151516A1
• Time in Patent Office: 746 Days
• Field of Search: None
• 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. An isolated mRNA comprising;

(a) a first region of linked nucleosides, said first region having at least 80% identity to SEQ ID NO;

18020 encoding a polypeptide of interest, said polypeptide of interest having the sequence of SEQ ID NO;

9254;

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

terminus of said first region comprising;

(i) a sequence of linked nucleosides having the sequence of the native 5′

UTR of the nucleic acid that encodes SEQ ID NO;

9254, or having the sequence of SEQ ID NOs;

1-4; and

(ii) at least one 5′

terminal cap;

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

terminus of said first region comprising;

(i′

) a sequence of linked nucleosides having the sequence of the native 3′

UTR of the nucleic acid that encodes SEQ ID NO;

9254, or having the sequence of SEQ ID NOs;

5-21; and

(ii′

) a 3′

tailing sequence of linked nucleosides.

2. The isolated mRNA of claim 1, wherein the first region of linked nucleosides comprises at least an open reading frame of a nucleic acid sequence having the sequence of SEQ ID NO:

18020.

3. The isolated mRNA 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.

4. The isolated mRNA of claim 1 which is purified.

5. The isolated mRNA of claim 1, wherein the at least one 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.

6. The isolated mRNA of claim 1, wherein at least one of said linked nucleosides comprises at least one modification as compared to the chemical structure of an A, G, U or C ribonucleotide.

7. The isolated mRNA of claim 6, wherein at least one said modification is located in a nucleoside base and/or sugar portion.

8. The isolated mRNA of claim 1, wherein said first region comprises n number of linked nucleosides having Formula (Ia):

9. The isolated mRNA of claim 8, wherein B is not pseudouridine (ψ

) or 5-methyl-cytidine (m⁵C).

10. The isolated mRNA of claim 8, wherein

U is O or C(R^U)_nu, wherein nu is an integer from 1 to 2 and each R^U is, independently, H, halo, or optionally substituted alkyl;

each of R¹, R^1′

, R^1″

, R², R^2′

, and R^2″

, if present, is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, or optionally substituted aminoalkyl;

each of R³ and R⁴ is, independently, H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxyalkoxy;

each of Y¹, Y², and Y³, is, independently, O, S, —

NR^N1—

, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R^N1 is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

each Y⁴ is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, or optionally substituted amino;

each Y⁵ is, independently, O or optionally substituted alkylene; and

n is an integer from 10 to 10,000.

11. The isolated mRNA of claim 10, wherein each of R¹, R^1′

, and R^1″

, if present, is H.

12. The isolated mRNA of claim 11, wherein each of R², R^2′

, and R^2″

, if present, is, independently, H, halo, hydroxy, optionally substituted alkoxy, or optionally substituted alkoxyalkoxy.

13. The isolated mRNA of claim 10, wherein each of R², R^2′

, and R^2″

, if present, is H.

14. The isolated mRNA of claim 13, wherein each of R¹, R^1′

, and R^1″

, if present, is, independently, H, halo, hydroxy, optionally substituted alkoxy, or optionally substituted alkoxyalkoxy.

15. The isolated mRNA of claim 8, wherein said first region comprises n number of linked nucleotides having Formula (IIa):

16. The isolated mRNA of claim 8, wherein said first region comprises n number of linked nucleosides having Formula (IIk):

17. The isolated mRNA claim 11, wherein

U is O or C(R^U)_nu, wherein nu is an integer from 1 to 2 and each R^U is, independently, H, halo, or optionally substituted alkyl;

each of R¹ and R² is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, or optionally substituted aminoalkyl;

each of R³ and R⁴ is, independently, H or optionally substituted alkyl;

each of Y¹, Y², and y³, is, independently, O, S, —

NR^N1—

, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R^N1 is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

each Y⁴ is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, or optionally substituted amino;

each Y⁵ is, independently, O or optionally substituted alkylene; and

n is an integer from 10 to 10,000.

18. The mRNA of claim 8, wherein n number of B has Formula (b18)-(b20):

19. The isolated polynucicotidc mRNA of claim 1, further comprising a targeting moiety, wherein said targeting moiety is covalently bound to said polynucleotide.

20. The isolated mRNA of claim 19, wherein said targeting moiety is an antibody, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic, or an aptamer.

21. A pharmaceutical composition comprising the isolated mRNA of claim 1 and a pharmaceutically acceptable excipient.

22. The pharmaceutical composition of claim 21, 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.

23. The pharmaceutical composition of claim 22, where the pharmaceutical composition comprises a lipid and wherein said lipid is selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA and PEGylated lipids and mixtures thereof.

24. A method of producing a polypeptide of interest in a mammalian cell, tissue or organism comprising administering to said cell, tissue or organism the pharmaceutical composition of claim 21.

25. The method of claim 24, wherein the isolated polynucleotide is formulated.

26. The method of claim 25, wherein the formulation comprises a lipid which is selected from one of 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.

27. The method of claim 26, wherein the isolated polynucleotide is administered at a total daily dose of between 1 ug and 150 ug.

28. The method of claim 27, wherein administration is by injection.

29. The method of claim 27, wherein administration is intradermal or subcutaneous or intramuscular or intravitreal.

30. The method of claim 27, wherein levels of the polypeptide of interest in the serum of the mammal are at least 50 pg/mL at least two hours after administration.

31. The method of claim 27, wherein the levels of the polypeptide of interest in the serum of the mammal remain above 50 pg/mL for at least 72 hours after administration.

32. The method of claim 31, wherein the levels of the polypeptide of interest in the serum of the mammal remain above 60 pg/mL for at least 72 hours after administration.

33. The method of claim 26, wherein the resulting polynucleotide formulation has a mean particle size of 80 nm-160 nm, a PDI of between 0.02 and 0.20 and a lipid to polynucleotide ratio (wt/wt) of between 10-20.