MODIFIED POLYNUCLEOTIDES FOR THE PRODUCTION OF PROTEINS ASSOCIATED WITH HUMAN DISEASE

• US 20140010861A1
• Filed: 03/09/2013
• Published: 01/09/2014
• Est. Priority Date: 04/02/2012
• Status: Abandoned Application

• Current Assignee: Moderna Therapeutics, Inc. (Moderna, Inc.)
• Original Assignee: Moderna Therapeutics, Inc. (Moderna, Inc.)
• 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
• Application Number:
  US13/791,910
• Publication Number:
  US 20140010861A1
• Time in Patent Office: Days
• US Class Current: 424/450
• 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 38/177 Receptors; Cell surface antigens; Cell surface determinants
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• CPC Class Codes: A61K 38/1891 Angiogenesic factors; Angiogenin
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• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61K 38/212 IFN-alpha
• CPC Class Codes: A61K 38/45 Transferases (2)
• 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 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 48/0033 the non-active part being non-polymeric
• CPC Class Codes: A61K 48/0041 the non-active part being 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 9/145 with organic compounds
• 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: A61P 1/00 : Drugs for disorders of the alimentary tract or the digestive system
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• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
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• CPC Class Codes: A61P 31/10 : Antimycotics
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• CPC Class Codes: A61P 31/14 : for RNA viruses
• CPC Class Codes: A61P 31/18 : for HIV
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 35/04 : specific for metastasis
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• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 : Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/04 : Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07H 21/02 : with ribosyl as saccharide radical
• CPC Class Codes: C07K 14/005 : from viruses
• CPC Class Codes: C07K 14/435 : from animals; from humans
• CPC Class Codes: C07K 14/43595 : from coelenteratae, e.g. medusae
• 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 14/4723 : Cationic antimicrobial peptides, e.g. defensins
• CPC Class Codes: C07K 14/4746 : p53
• 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 14/495 : Transforming growth factor [TGF]
• 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 14/5418 : IL-7
• CPC Class Codes: C07K 14/56 : IFN-alpha
• CPC Class Codes: C07K 14/61 : Growth hormone [GH], i.e. somatotropin
• 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 16/00 : Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2863 : against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2887 : against CD20
• CPC Class Codes: C07K 16/40 : against enzymes
• 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 15/85 : 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 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/0091 : oxidizing metal ions (1.16)
• CPC Class Codes: C12N 9/1051 : Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/1241 : Nucleotidyltransferases (2.7.7)
• 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 9/6435 : Plasmin (3.4.21.7), i.e. fibrinolysin
• CPC Class Codes: C12N 9/6437 : Coagulation factor VIIa (3.4.21.21)
• CPC Class Codes: C12N 9/644 : Coagulation factor IXa (3.4.21.22)
• CPC Class Codes: C12N 9/6443 : Coagulation factor XIa (3.4.21.27)
• 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 116/03001 : Ferroxidase (1.16.3.1), i.e. ceruloplasmin
• CPC Class Codes: C12Y 207/07012 : UDP-glucose--hexose-1-phosphate uridylyltransferase (2.7.7.12)
• CPC Class Codes: C12Y 304/21007 : Plasmin (3.4.21.7), i.e. fibrinolysin
• CPC Class Codes: C12Y 304/21022 : Coagulation factor IXa (3.4.21.22)
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• CPC Class Codes: C12Y 403/02001 : Argininosuccinate lyase (4.3.2.1)
• CPC Class Codes: C12Y 603/02019 : Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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 polynucleotide comprising;

(a) a first region of linked nucleosides, said first region encoding a polypeptide of interest, said polypeptide of interest selected from the group consisting of SEQ ID NOs 35608-71005;

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

terminus of said first region comprising;

(i) a sequence of linked nucleosides selected from the group consisting of the native 5′

UTR of any of the nucleic acids that encode any of SEQ ID NOs 35608-71005, SEQ ID NOs;

1-4 and functional variants thereof; 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 selected from the group consisting of the native 3′

UTR of any of the nucleic acids that encode any of SEQ ID NOs 35608-71005, SEQ ID NOs 5-21 and functional variants thereof; and

(ii′

) a 3′

tailing sequence of linked nucleosides.

2. The isolated polynucleotide of claim 1 wherein the first region of linked nucleosides comprises at least an open reading frame of a nucleic acid sequence, wherein the nucleic acid sequence selected from the group consisting of SEQ ID NOs:

35554-35607 and 71006-251418.

3. The isolated polynucleotide 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 polynucleotide of any one of claims 1-3 which is purified.

5. The isolated polynucleotide of any one of claims 1-4, 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 polynucleotide of any preceding claim, 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 polynucleotide of claim 6, wherein at least one said modification is located in a nucleoside base and/or sugar portion.

8. The isolated polynucleotide of any one of claims 1-7, wherein said first region comprises n number of linked nucleosides having Formula (Ia):

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

) or 5-methyl-cytidine (m⁵C).

10. The isolated polynucleotide of any one of claims 8-9, 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 polynucleotide of claim 10, wherein each of R¹, R^1′

, and R^1″

, if present, is H.

12. The isolated polynucleotide 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 polynucleotide of claim 10, wherein each of R², R^2′

, and R^2″

, if present, is H.

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

16. isolated The polypeptide of claim 15, wherein said first region comprises n number of linked nucleosides having Formula (IIb) or (IIc), or a pharmaceutically acceptable salt thereof.

17. The isolated polynucleotide of claim 8, wherein said first region comprises n number of linked nucleosides having Formula (IId):

18. The isolated polypeptide of claim 17, wherein said first region comprises n number of linked nucleosides having Formula (IIe) or (IIf), or a pharmaceutically acceptable salt thereof.

19. The isolated polynucleotide of claim 8, wherein said first region comprises n number of linked nucleotides, each of said linked nucleotides independently having one of Formulas (IIg)-(IIj):

20. The isolated polynucleotide of claim 8, wherein said first region comprises n number of linked nucleosides having Formula (Ilk):

21. The isolated polynucleotide of claim 20, wherein said first region comprises n number of linked nucleosides having Formula (111):

22. The isolated polynucleotide of claim 20, wherein said first region comprises n number of linked nucleosides having Formula (IIm):

23. The isolated polynucleotide of any one of claims 11-22, 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.

24. The isolated polynucleotide of claim 8, wherein said first region comprises n number of linked nucleosides having Formula (IIn):

25. The isolated polynucleotide of any one of claims 8-24, wherein in said n number of B has, each B independently has a formula selected from Formula (b1)-(b5):

26. The isolated polynucleotide of any one of claims 8-25, wherein n number of B has Formula (b6)-(b9):

27. The isolated polynucleotide of claim 26, wherein R^12a, R^12b, R^12c, or R^Va is substituted with —

(CH₂)_s2(OCH₂CH₂)_s1(CH₂)_s3OR′

, wherein s1 is an integer from 1 to 10, each of s2 and s3, independently, is an integer from 0 to 10, and R′

is H or C_1-20 alkyl);

or —

NR^N1(CH₂)_s2(CH₂CH₂O)_s1(CH₂)_s3NR^N1, wherein s1 is an integer from 1 to 10, each of s2 and s3, independently, is an integer from 0 to 10, and each R^N1 is, independently, hydrogen or optionally substituted C_1-6 alkyl.

28. The isolated polynucleotide of any one of claims 8-27, wherein n number of B has Formula (b10)-(b14):

29. The isolated polynucleotide of any one of claims 7-27, wherein n number of B has Formula (b15)-(b17):

30. The polynucleotide of any one of claims 8-29, wherein n number of B has Formula (b18)-(b20):

31. The isolated polynucleotide of claim 30, wherein R^26a, R^26b, or R²⁹ is substituted with —

(CH₂)_s2(OCH₂CH₂)_s1(CH₂)_s3OR′

, wherein s1 is an integer from 1 to 10, each of s2 and s3, independently, is an integer from 0 to 10, and R′

is H or C_1-20 alkyl);

or —

NR^N1(CH₂)_s2(CH₂CH₂O)_s1(CH₂)_s3NR^N1, wherein s1 is an integer from 1 to 10, each of s2 and s3, independently, is an integer from 0 to 10, and each R^N1 is, independently, hydrogen or optionally substituted C_1-6 alkyl.

32. The isolated polynucleotide of any one of claims 8-31, wherein n number of B has Formula (b21):

33. The isolated polynucleotide of any one of claims 8-32, wherein n number of B has Formula (b22):

34. The isolated polynucleotide of any one of claims 8-33, wherein n number of B has Formula (b23):

35. The isolated polynucleotide of any one of claims 8-34, wherein n number of B has Formula (b24):

36. The isolated polynucleotide of any one of claims 1-35, further comprising a targeting moiety, wherein said targeting moiety is covalently bound to said polynucleotide.

37. The isolated polynucleotide of claim 36, 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.

38. A pharmaceutical composition comprising the isolated polynucleotide of any one of claims 1-37.

39. A pharmaceutical composition comprising the isolated polynucleotide of any one of claims 1-37 and a pharmaceutically acceptable excipient.

40. The pharmaceutical composition of claim 39, 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, lipoplexe peptide, protein, cell, hyaluronidase, and mixtures thereof.

41. The pharmaceutical composition of claim 40, 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.

42. A method of producing a polypeptide of interest in a mammalian cell, tissue or organism comprising administering to said cell, tissue or organism the isolated polynucleotide of any one of claims 1-37 or the pharmaceutical composition of any of claims 38-41.

43. The method of claim 42, wherein the isolated polynucleotide is formulated.

44. The method of claim 43, 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.

45. The method of claim 44, wherein the isolated polynucleotide is administered at a total daily dose of between 1 ug and 150 ug.

46. The method of claim 45, wheriein administration is by injection.

47. The method of claim 46, wherein administration is intradermal or subcutaneous or intramuscular or intravitreal.

48. The method of claim 45, 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.

49. The method of claim 45, 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.

50. The method of claim 49, 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.

51. The method of claim 44, 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 polnucleotide ratio (wt/wt) of between 10-20.

52. A method for producing an increased level of a polypeptide of interest selected from the group consisting of SEQ ID NOs 35608-71005 in a mammalian cell, tissue or organism, comprising administering to said cell, tissue or organism a total daily dose of the isolated polynucleotide of any one of claims 4-37 or the pharmaceutical composition of any one of claims 38-41 in two or more equal or unequal split doses.

53. The method of claim 52, wherein the level of the polypeptide produced in response to said administration is greater than the levels produced by administering the same total daily dose of the isolated polynucleotide or pharmaceutical composition as a single administration.

54. The method of claim 52, wherein the mammalian organism is a human patient in need of an increased level of the polypeptide of interest.

55. The method of claim 54, wherein the increased level of the polypeptide of interest is detectable in a bodily fluid of said patient.

56. The method of claim 55, wherein the bodily fluid is selected from the group consisting of peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper'"'"'s fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood.

57. The method of claim 56, wherein the bodily fluid is serum and the polpeptide per unit drug (PUD) is greater than 1.

58. The method of claim 57, wherein the dose splitting factor (DSF) is greater than 4.

59. The method of claim 55, wherein administration is transdermal.

60. The method of claim 59, wherein transdermal administration comprises utilization of one or more members selected from the group consisting of a patch, cream, ointment, mechanical device, needle, sponge, depot and fabric.

61. The method of claim 59, wherein administration is according to a dosing regimen which occurs over the course of hours, days, weeks, months, or years.

62. The method of claim 52, wherein said two or more split doses comprise a first dose of the polynucleotide or pharmaceutical composition administered at a time T1 followed by a second dose of the polynucleotide or pharmaceutical composition administered at a time T2, wherein said time T1 and said time T2 are separated by no more than 1 minute and wherein said first dose and said second dose are administered in amounts that result in higher levels of the polypeptide of interest in said subject than if the amounts of polynucleotide or pharmaceutical composition were administered together in a single unit dose.

63. The method of claim 62, further comprising administering a plurality of doses of said polynucleotide or pharmaceutical composition, Nx at times Tn, wherein x and n are independently selected from 3 to about 1000 and where the time between Tn and Tn+1 is separated by increments of no more than 10 seconds.

64. The method of claim 63, wherein administration occurs by direct injection.

65. The method of claim 64, wherein direct injection is selected from the group consisting of intravenous, intradermal, subcutaneous, intramuscular and intravitreal.

66. The method of claim 64, wherein said first dose is administered proximal to said second or plurality of doses.

67. The method of claim 64, wherein said first dose is administered distal to said second or plurality of doses.

68. The method of claim 64, wherein the distance between the site of injection of said first dose and the site of injection of any second or plurality of doses is from about 1 mm to about 10 cm.

69. The method of claim 64, wherein injection is made at a depth of from 0.1 mm to about 1 cm.

70. The method of claim 65, wherein direct injection is achieved by using one or more devices selected from multineedle injection systems, catheter or lumen systems, and ultrasound, electrical or radiation based systems.

71. The method of claim 63, wherein the amount polynucleotide or pharmaceutical composition administered in any dose is substantially equal.

72. The method of claim 63, wherein time T1 and time T2 are separated by no more than 30 seconds.

73. The method of claim 63, wherein time T1 and time T2 are separated by no more than 10 seconds.

74. The method of claim 63, wherein the first dose, the second dose and any of a plurality of doses are administered at substantially the same time.

75. The method of claim 63, wherein the single unit dose is between about 10 mg/kg and about 500 mg/kg.

76. The method of claim 63, wherein the single unit dose is between about 1.0 mg/kg and about 10 mg/kg.

77. The method of claim 63, wherein the single unit dose is between about 0.001 mg/kg and about 1.0 mg/kg.

84. The method of any of claims 78-83, wherein the polynucleotide is selected from the polynucleotide of any of claims 1-37.

86. A lipid nanopartical formulation of claim 85, wherein the polynucleotide is selected from the polynucleotide of any of claims 1-37.

88. A reLNP formulation of claim 87, wherein the polynucleotide is selected from the polynucleotide of any of claims 1-36.

90. A sustained release formulation of claim 89, wherein the polynucleotide is selected from the polynucleotide of any of claims 1-36.

78. A method of preparing a lipid nanopartical formulation of a polynucleotide encoding a polypeptide of interest comprising rapidly injecting a first ethanolic solution into a second aqueous solution wherein,

(a) said first ethanolic solution comprises a mixture of lipid:

DSPC;

Cholesterol;

PEG-c-DOMG to yield a molar ratio of 50;

10;

38.5;

1.5 and having a final lipid concentration of approximately 25 mM, and

(b) said second aqueous solution comprises a sodium citrate buffered solution of the polynucleotide encoding the polypeptide of interest having a concentration of 1-2 mg/mL and a pH of approximately 3,

wherein the rapid injection results in a suspension containing 33% ethanol and a total lipid to polynucleotide weight ratio of at least 10;

1.

79. The method of claim 78, wherein the rapid injection is performed either manually (MI) or by the aid of a syringe pump (SP).

80. The method of claim 79, further comprising dialyzing the resultant suspension against phosphate buffered saline (PBS) at pH 7.4.

81. The method of claim 80, wherein dialysis is performed more than once.

82. The method of claim 81, further comprising filtering the dialyzed suspension through a 0.2 μ

m sterile filter.

83. The method of any of claims 78-82, wherein the lipid is selected from the group consisting of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA and PEGylated lipids.

85. A lipid nanopartical formulation of a polynucleotide encoding a polypeptide of interest produced by the method of any of claims 78-83 and having a particle size of 80 nm-160 nm, a PDI of between 0.02 and 0.20 and a lipid to polnucleotide ratio (wt/wt) of between 10-30.

87. A reLNP formulation of a polynucleotide said polynucleotide encoding a polypeptide of interest.

89. A sustained release formulation of a polynucleotide said polynucleotide encoding a polypeptide of interest.

91. A polynucleotide encoding a fusion protein said fusion protein comprising a first polypeptide and a second polypeptide.

92. The polynucleotide of claim 91 where the first polypeptide is selected from the group consisting of Fc receptor, Fab fragment, Fab′

fragment, F(ab′

)₂ fragment, Fv fragment, IgA domain, IgD domain, IgE domain, IgD domain, IgM domain, IgV domain, IgC1 domain, IgC2 domain and IgI domain and the second polypeptide is a polypeptide of interest.