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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/44—Oxidoreductases (1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0066—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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
  • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
  • C12Y114/13017—Cholesterol 7-alpha-monooxygenase (1.14.13.17)

Definitions

• the present invention is directed to compositions and methods for modulating and/or altering cholesterol levels in an organism or altering cholesterol trafficking in an organism.
• the invention relates to RNA such as modified RNA in therapeutics.
• the RNA or modified RNA of the invention may encode peptides, polypeptides or multiple proteins.
• the RNA or modified RNA of the invention may also be used to produce polypeptides of interest.
• the modified RNA molecules of the invention may be mRNA and therefore be referred to as modified mR A.
• the polypeptides of interest may be used in therapeutics and/or clinical and research settings.
• High cholesterol is one of a number of risk factors for heart attack and stroke. Although poor diet and lack of excise are common causes of high
• cholesterolgenetic changes such as familiar hypercholesterolemia (FH), which is caused by deficiency in LDLR, can be causes of high cholesterol.
• FH hypercholesterolemia
• a number of cholesterol lowering drugs are currently on the market but they are not without risk or contraindications with certain conditions or other medications.
• Such drugs include statins, fibrates, niacin, bile acid sequestrants (resins), phytosterols, or other compounds that prevent absorption of fats, reduce absorption of cholesterol, or target genes in the cholesterol trafficking pathway.
• Nucleic acid based cholesterol lowering drugs include, for example an antisense oligonucleotide inhibitor which targets ApoB-100, mipomersen, which was approved in January 2013 for the treatment of homozygous familial
• hypercholesterolemia FH. In December of 2012, the FDA also approved lomitapide for the same condition.
• liver related problems associated with cholesterol targeting drugs particularly elevation in serum transaminases and accumulation of hepatic fat (or hepatic steatosis).
• the drug will carry a boxed warning about liver toxicity as well as requiring certification of prescribers and pharmacies, as well as documentation that the drug is being properly used with each new prescription.
• mipomersen was generally effective in lowering LDL cholesterol (more than half of patients in clinical trials had more than a 20% decrease in LDL levels and in the homozygous FH trial, it reduced LDL by 24.7%), a typical FH patient has an average LDL between 400-1000mg/dL.
• the present invention addresses both the problem of elevated LDL cholesterol levels and dysregulation of hepatic function by providing nucleic acid based compounds or polynucleotides which encode a polypeptide of interest (e.g., modified mRNA or mmRNA) and which have structural and/or chemical features that avoid one or more of the problems in the art.
• a polypeptide of interest e.g., modified mRNA or mmRNA
• compositions, methods and kits RNA using RNA such as modified RNA in the treatment, prevention or diagnosis of disease and disorders associated with cholesterol and/or cholesterol trafficking.
• the pathways associated with cholesterol trafficking are modulated by providing one or more polypeptides (including enzymes) which alter either the concentrations of cholesterol, its processing or transport.
• the transport of LDL cholesterol from plasma to liver cells is increased by providing the cell with either more receptor molecules or by minimizing the destruction of the LDL receptor.
• a cell with either more receptor molecules or by minimizing the destruction of the LDL receptor.
• polynucleotide, primary construct or mmRNA which encodes LDL receptor.
• a mutant form of LDL receptor is encoded by the polynucleotide, primary construct or mmRNA.
• Such mutant LDL receptors LDL-R or LDLR
• LDLR LDL receptors
• PCSK9 binding deficient LDLR would bring cholesterol into the hepatocyte.
• a modified mRNA may encode a LDLR mutant which may comprise at least one amino acid mutation in a region comprising amino acids 314-393 of LDLR such as, but not limited to, SEQ ID NO: 19.
• the region of amino acids comprises amino acids 316-339 of SEQ ID NO: 19.
• the modified mRNA may comprise at least one nucleoside modification such as, but not limited to, 1-methylpseudouridine.
• the modified mRNA may also comprise the nucleoside modification 5-methylcytosine.
• a modified mRNA may encode a LDLR mutant which may comprise at least one amino acid mutation in a region comprising amino acids 314-393 of LDLR such as, but not limited to, SEQ ID NO: 19.
• the region of amino acids comprises amino acids 316-339 of SEQ ID NO: 19.
• the hepatocyte is provided with one or more polynucleotides, primary constructs, or mrnRNA encoding and/or which
• CYP7A1 is the rate limiting enzyme for bile acid synthesis, and promotes removal of the incoming cholesterol. There are humans with CYP7A1 mutations that are associated with high plasma low-density lipoprotein (LDL) and hepatic cholesterol content, as well as deficient bile acid excretion.
• LDL low-density lipoprotein
• two polynucleotides, primary constructs, or mrnRNA are delivered resulting in lower plasma cholesterol and concomitant enhanced cholesterol disposal.
• the one or more polynucleotides, primary constructs or mrnRNA are modified in the 3'UTR to contain a microRNA binding site or seed.
• the CYP7A1 polynucleotide having a normal half life of approximately 30 minutes, may be made transcription-dependent by miR- destabilization (specifically the ubiquitous miR122a in hepatocytes).
• miR- destabilization specifically the ubiquitous miR122a in hepatocytes
• a miR122a binding site may be incorporated into the 3'UTR of the mrnRNA rendering the transcript less stable. This would allow, depending on the number of binding sites engineered into the construct, the titration of stability and therefore allow for control of expression of the encoded CYP7A1 enzyme.
• the polynucleotides, primary constructs primary constructs or mrnRNA encoding CYP7A1 may also be useful in creating mouse models useful in proof of concept studies and basic research. These studies would be analogous to producing dose dependent gene
• treatment regimes may be designed for rare diseases where patients present with CYP7A1 polymorphisms that are hyporesponsive to statins.
• studies of effective compositions of the present invention may be completed quickly and based on diet-based challenges.
• the compositions of the present invention are useful in the study and treatment of disease involving cholesterol related diseases, both rare and prevalent.
• compositions of the present invention may be administered along with other drug compounds.
• other drugs include specifically statins.
• statins include, but are not limited to, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, simvastatin, and combinations thereof.
• compositions comprising
• polynucleotides, primary constructs or mmRNA are useful in treating diseases such as rare non-alcoholic fatty liver disease.
• diseases such as rare non-alcoholic fatty liver disease.
• any therapeutic that drives hepatic cholesterol to its natural sink (out of the body through biles) would have superior treatment outcomes. Consequently, it is contemplated that administration of polynucleotides, primary constructs or mmRNA encoding LDLR would increase cholesterol in the hepatocyte but that coadministration of a second polynucleotides, primary construct, or mmRNA encoding CYP7A1 would continue to drive the cholesterol out through bile thereby avoiding the fatty liver symptoms currently seen with known therapeutics.
• a mix of mmRNA would be titrated along the cholesterol homeostasis pathway to promote mobilization of cholesterol out of the body.
• bile acid sequestrants or fat soluble vitamins may be co-administered.
• FIG. 1 is a schematic of a primary construct of the present invention.
• FIG. 2 is a schematic of a primary construct of the present invention.
• FIG. 3 shows the pathway of cholesterol trafficking in a liver cell.
• FIG. 4 is a flow cytometry plot of low density lipoprotein receptor (LDLR) modified mRNA.
• LDLR low density lipoprotein receptor
• FIG. 5 is a flow cytometry plot of LDLR modified mRNA.
• FIG. 6 is a graph of LDLR Expression.
• Figure 6A shows LDL Receptor Expression of cells compared to LDLR mRNA added.
• Figure 6B shows LDL Receptor Expression of cells post transfection.
• Figure 5C shows the saturation of BODIPY® labeled LRL.
• Figure 6D shows the binding affinity of BODIPY-LDL to cells.
• Figure 6E shows the total cholesterol content of each fraction.
• FIG. 7 is a bioanalyzer image of LDLR modified mRNA product. Lane 1, size markers in nucleotides; Lane 2, LDLR modified mRNA.
• FIG. 8 is a flow cytometry plot of 800 ng LDLR modified mRNA transfected in HEK293 cells.
• FIG. 9 is a flow cytometry plot of LDLR modified mRNA transfected in HEK293 cells.
• FIG. 10 shows the cholesterol level in serum.
• Figure 10A shows the absorbance profile of FPLC fractions from pooled LDLR knock out mice (Upper panel) and wild type mice (lower panel).
• Figure 10B shows the total cholesterol content of each fraction.
• FIG. 11 is a flow cytometry plot of variant LDLR modified mRNA transfected in HEK293 cells.
• FIG. 12 is a flow cytometry plot of variant LDLR modified mRNA transfected in HEK293 cells with or without PCSK9.
• FIG. 13 is a flow cytometry plot of transfected variant LDLR modified mRNA.
• Figure 13A shows contour plots of the binding of BODIPY-LDL to LDLR mRNA transfected cells.
• Figure 13B shows the half-maximal cell association of BODIPY-LDL.
• FIG. 14 shows the effect on half-life after transfection with LDLR mRNA.
• Figure 134 shows wild-type LDLR mRNA.
• Figure 14B shows a LDLR mRNA encoding a variant LDLR with 4 amino acid substitutions (N316A, E317A, D331A and Y336A).
• Figure 14C shows a LDLR mRNA encoding a variant LDLR with 1 amino acid substitution, Y336A.
• Figure 14D shows a LDLR mRNA encoding a variant LDLR with 1 amino acid substitution, E317A.
• Figure 14E shows a LDLR mRNA encoding a variant LDLR with 1 amino acid substitution, N316A.
• Figure 14F shows a LDLR mRNA encoding a variant LDLR with 1 amino acid substitution, L339D.
• Figure 14G shows a LDLR mRNA encoding a variant LDLR with 1 amino acid substitution, D331E.
• FIG. 15 shows the effect on cell surface LDLR when the amount of PCSK is varied.
• compositions including pharmaceutical compositions
• methods for the design, preparation, manufacture and/or formulation of polynucleotides encoding one or more polypeptides of interest are also provided.
• these polynucleotides are preferably modified as to avoid the deficiencies of other polypeptide-encoding molecules of the art. Hence these polynucleotides are referred to as modified mRNA or mmR A.
• polynucleotides, primary constructs and/or mmRNA encoding polypeptides of interest which have been designed to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretion efficiency (when applicable), accessibility to circulation, protein half-life and/or modulation of a cell's status, function and/or activity.
• the polynucleotides, primary constructs and/or mmRNA of the present invention are useful in altering cholesterol levels or cholesterol trafficking in an organism, particularly human patients.
• the pathways associated with cholesterol trafficking are modulated by providing one or more polypeptides (including enzymes) which alter either the concentrations of cholesterol, its processing or transport.
• the transport of LDL cholesterol from plasma to liver cells is increased by providing the cell with either more receptor molecules or by minimizing the destruction of the LDL receptor.
• a cell with either more receptor molecules or by minimizing the destruction of the LDL receptor.
• polynucleotide, primary construct or mmRNA which encodes LDL receptor.
• a mutant form of LDL receptor is encoded by the polynucleotide, primary construct or mmRNA.
• Such mutant LDL receptors would be deficient in some way in their binding of PCSK-9.
• the binding site of PCSK-9 has been previously localized to the EGF-A (or EGF-like repeat) domain of LDLR (see e.g., Kwon et al. Molecular Basis for LDL receptor recognition by PCSK9. PNAS. 2008 105(6), 1820-1825; the contents of which is herein incorporated by reference in its entirety). Accordingly, a PCSK9 binding deficient LDLR would bring cholesterol into the hepatocyte.
• a polynucleotide, primary construct or mmRNA may encode a mutant LDLR which is deficient in binding to PCSK-9.
• a polynucleotide, primary construct or mmRNA may encode a mutant LDLR which comprises at least one amino acid mutation in the PCSK-9 binding site.
• the mutant LDLR may comprise one, two, three, four, five, six, seven, eight, nine, ten or more than ten mutations.
• the mutant LDLR may comprise at least one amino acid mutation in the EGF-A domain (also known as the EGF-like repeat domain) of LDLR.
• the EGF-A domain is located in a region of LDLR comprising amino acids 314-393.
• the EGF- A domain is a region of SEQ ID NO: 19 comprising amino acids 314-393.
• a polynucleotide, primary construct or mmRNA may encode a mutant LDLR which comprises at least one amino acid mutation in the EGF-like 1 domain.
• the EGF-like 1 domain may be located in a region of LDLR comprising amino acids 314-353.
• the EGF-like 1 domain is a region of SEQ ID NO: 19 comprising amino acids 314-353.
• a polynucleotide, primary construct or mmRNA may encode a mutant LDLR which comprises at least one amino acid mutation in the EGF-like 2 domain.
• the EGF-like 2 domain may be located in a region of LDLR comprising amino acids 353-393.
• the EGF-like 2 domain is a region of SEQ ID NO: 19 comprising amino acids 353-393.
• a polynucleotide, primary construct or mmRNA may encode a PCSK-9 binding deficient mutant LDLR.
• the PCSK-9 binding deficient mutant LDLR may comprise at least one amino acid mutation in the region of SEQ ID NO: 19 comprising amino acids 314-393.
• at least one mutation may be located between amino acids 314-353.
• at least one mutation may be located between amino acids 315-340.
• at least one mutation may be located between amino acids 354-393.
• a polynucleotide, primary construct or mrnRNA may encode a PCSK-9 binding deficient mutant LDLR comprising at least one mutation in the PCSK-9 binding region.
• the mutation may be located in the region of SEQ ID NO: 19 comprising amino acids 314-393.
• the mutation may be located in the region of 314-353, 315-340 and 354- 393.
• the mutation may be at position 316, 317, 331, 336 or 339.
• the mutant LDLR may comprise a mutation at position 316, 317, 331 and 336.
• a polynucleotide, primary construct or mrnRNA may encode a PCSK-9 binding deficient mutant LDLR comprising at least one mutation at an amino acid position such as, but not limited to, 316, 317, 331, 336 and/or 339.
• the PCSK-9 binding deficient mutant LDLR may comprise at least one of the mutations N316A, E317A, D331A, D331E, Y336A and/or L339D where "N316 A” means Asparagine at position 316 is replaced with Alanine.
• the PCSK-9 binding deficient mutant LDLR may comprise the mutations N316A, E317A, D331 A and Y336A.
• a polynucleotide, primary construct or mrnRNA may encode a PCSK-9 binding deficient mutant LDLR comprising four mutations at amino acid positions such as, but not limited to, 316, 317, 331, 336 and/or 339.
• the PCSK-9 binding deficient mutant LDLR may comprise any four of the mutations such as N316A, E317A, D331A, D331E, Y336A and/or L339D where "N316 A” means Asparagine at position 316 is replaced with Alanine.
• the PCSK-9 binding deficient mutant LDLR may comprise the mutations N316A, E317A, D331 A and Y336A.
• the hepatocyte is provided with one or more polynucleotides, primary constructs, or mmRNA encoding and/or which
• CYP7A1 is the rate limiting enzyme for bile acid synthesis, and promotes removal of the incoming cholesterol. There are humans with CYP7A1 mutations that are associated with high plasma low-density lipoprotein (LDL) and hepatic cholesterol content, as well as deficient bile acid excretion.
• LDL low-density lipoprotein
• two polynucleotides, primary constructs, or mmRNA are delivered resulting in lower plasma cholesterol and concomitant enhanced cholesterol disposal.
• the one or more polynucleotides, primary constructs or mmRNA are modified in the 3'UTR to contain a microRNA binding site or seed.
• the CYP7A1 polynucleotide having a normal half life of approximately 30 minutes, may be made transcription-dependent by miR- destabilization (specifically the ubiquitous miR122a in hepatocytes).
• miR- destabilization specifically the ubiquitous miR122a in hepatocytes
• a miR122a binding site may be incorporated into the 3'UTR of the mmRNA rendering the transcript less stable. This would allow, depending on the number of binding sites engineered into the construct, the titration of stability and therefore allow for control of expression of the encoded CYP7A1 enzyme.
• the polynucleotides, primary constructs primary constructs or mmRNA encoding CYP7A1 may also be useful in creating mouse models useful in proof of concept studies and basic research. These studies would be analogous to producing dose dependent gene therapy.
• treatment regimes may be designed for rare diseases where patients present with CYP7A1 polymorphisms that are hyporesponsive to statins.
• studies of effective compositions of the present invention may be completed quickly and based on diet-based challenges.
• the compositions of the present invention are useful in the study and treatment of disease involving cholesterol related diseases, both rare and prevalent.
• compositions of the present invention may be administered along with other drug compounds.
• other drugs include specifically statins.
• statins include, but are not limited to, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, simvastatin, and combinations thereof.
• compositions comprising
• polynucleotides, primary constructs or mmRNA are useful in treating diseases such as rare non-alcoholic fatty liver disease.
• diseases such as rare non-alcoholic fatty liver disease.
• any therapeutic that drives hepatic cholesterol to its natural sink (out of the body through biles) would have superior treatment outcomes. Consequently, it is contemplated that administration of polynucleotides, primary constructs or mmRNA encoding LDLR would increase cholesterol in the hepatocyte but that coadministration of a second polynucleotides, primary construct, or mmRNA encoding CYP7A1 would continue to drive the cholesterol out through bile thereby avoiding the fatty liver symptoms currently seen with known therapeutics.
• a mix of mmRNA would be titrated along the cholesterol homeostasis pathway to promote mobilization of cholesterol out of the body.
• bile acid sequestrants or fat soluble vitamins may be co-administered.
• nucleic acid molecules specifically polynucleotides, primary constructs and/or mmRNA which encode one or more polypeptides of interest.
• nucleic acid in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides.
• nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (R As), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ - D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'- amino- a-LNA having a 2 '-amino functionalization) or hybrids thereof.
• R As ribonucleic acids
• DNAs deoxyribonucleic acids
• TAAs threose nucleic acids
• GNAs glycol nucleic acids
• PNAs peptide nucleic acids
• LNAs locked nucle
• the nucleic acid molecule is a messenger RNA (mRNA).
• mRNA messenger RNA
• the term "messenger RNA” (mRNA) refers to any polynucleotide, such as a synthetic polynucleotide, which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.
• the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly-A tail.
• the present invention expands the scope of functionality of traditional mRNA molecules by providing polynucleotides or primary RNA constructs which maintain a modular organization, but which comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the reprograrmming polynucleotides including, in some embodiments, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced.
• modified mRNA molecules of the present invention such as synthetic modified mRNA molecules, are termed "mmRNA.”
• mmRNA modified mRNA molecules of the present invention, such as synthetic modified mRNA molecules, are termed "mmRNA.”
• a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a
• polynucleotide primary construct or mmRNA without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides.
• the polynucleotide "ATCG” may be chemically modified to "AT-5meC-G".
• the same polynucleotide may be structurally modified from "ATCG” to "ATCCCG".
• the dinucleotide "CC” has been inserted, resulting in a structural modification to the polynucleotide.
• the mmRNA of the present invention are distinguished from wild type mRNA in their functional and/or structural design features which serve to, as evidenced herein, overcome existing problems of effective polypeptide production using nucleic acid-based therapeutics.
• Figure 1 shows a representative polynucleotide primary construct 100 of the present invention.
• primary construct or “primary mRNA construct” refers to a polynucleotide transcript which encodes one or more polypeptides of interest and which retains sufficient structural and/or chemical features to allow the polypeptide of interest encoded therein to be translated.
• Primary constructs may be polynucleotides of the invention. When structurally or chemically modified, the primary construct may be referred to as an mmRNA.
• the primary construct 100 here contains a first region of linked nucleotides 102 that is flanked by a first flanking region 104 and a second flaking region 106.
• the "first region” may be referred to as a "coding region” or “region encoding” or simply the "first region.”
• This first region may include, but is not limited to, the encoded polypeptide of interest.
• the polypeptide of interest may comprise at its 5 ' terminus one or more signal sequences encoded by a signal sequence region 103.
• the flanking region 104 may comprise a region of linked nucleotides comprising one or more complete or incomplete 5' UTRs sequences.
• the flanking region 104 may also comprise a 5' terminal cap 108.
• the second flanking region 106 may comprise a region of linked nucleotides comprising one or more complete or incomplete 3' UTRs.
• the flanking region 106 may also comprise a 3' tailing sequence 110.
• first operational region 105 Bridging the 5' terminus of the first region 102 and the first flanking region 104 is a first operational region 105.
• this operational region comprises a Start codon.
• the operational region may alternatively comprise any translation initiation sequence or signal including a Start codon.
• this operational region comprises a Stop codon.
• the operational region may alternatively comprise any translation initiation sequence or signal including a Stop codon. According to the present invention, multiple serial stop codons may also be used.
• Figure 2 shows a representative polynucleotide primary construct 130 of the present invention.
• Polynucleotide primary construct refers to a polynucleotide transcript which encodes one or more polypeptides of interest and which retains sufficient structural and/or chemical features to allow the polypeptide of interest encoded therein to be translated.
• Non-limiting examples of polypeptides of interest and polynucleotides encoding polypeptide of interest are described in Table 6 of U.S. Provisional Patent Application No 61/618,862, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Biologies; U.S.
• the polynucleotide primary construct 130 here contains a first region of linked nucleotides 132 that is flanked by a first flanking region 134 and a second flaking region 136.
• the "first region” may be referred to as a "coding region” or “region encoding” or simply the "first region.”
• This first region may include, but is not limited to, the encoded polypeptide of interest.
• the first region 132 may include, but is not limited to, the open reading frame encoding at least one polypeptide of interest.
• the open reading frame may be codon optimized in whole or in part.
• the flanking region 134 may comprise a region of linked nucleotides comprising one or more complete or incomplete 5' UTRs sequences which may be completely codon optimized or partially codon optimized.
• the flanking region 134 may include at least one nucleic acid sequence including, but not limited to, miR sequences, TERZAKTM sequences and translation control sequences.
• the flanking region 134 may also comprise a 5' terminal cap 138.
• the 5' terminal capping region 138 may include a naturally occurring cap, a synthetic cap or an optimized cap.
• Non- limiting examples of optimized caps include the caps taught by Rhoads in US Patent No. US7074596 and International Patent Publication No. WO2008157668, WO2009149253 and WO2013103659.
• the second flanking region 106 may comprise a region of linked nucleotides comprising one or more complete or incomplete 3' UTRs.
• the second flanking region 136 may be completely codon optimized or partially codon optimized.
• the flanking region 134 may include at least one nucleic acid sequence including, but not limited to, miR sequences and translation control sequences.
• polynucleotide primary construct may comprise a 3' tailing sequence 140.
• the 3' tailing sequence 140 may include a synthetic tailing region 142 and/or a chain terminating nucleoside 144.
• Non- liming examples of a synthetic tailing region include a polyA sequence, a polyC sequence, a polyA-G quartet.
• Non-limiting examples of chain terminating nucleosides include 2'-0 methyl, F and locked nucleic acids (LNA).
• first operational region 144 Bridging the 5' terminus of the first region 132 and the first flanking region 134 is a first operational region 144.
• this operational region comprises a Start codon.
• the operational region may alternatively comprise any translation initiation sequence or signal including a Start codon.
• this operational region comprises a Stop codon.
• the operational region may alternatively comprise any translation initiation sequence or signal including a Stop codon. According to the present invention, multiple serial stop codons may also be used.
• the shortest length of the first region of the primary construct of the present invention can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide.
• a dipeptide a tripeptide
• a tetrapeptide a pentapeptide
• hexapeptide a heptapeptide
• an octapeptide a nonapeptide, or a decapeptide.
• the length may be sufficient to encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids.
• the length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.
• the length of the first region encoding the polypeptide of interest of the present invention is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).
• the "first region” may be referred to as a "coding region” or "region encoding” or simply the "first region.”
• the polynucleotide, primary construct, or mmR A includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 1,500, from 500 to 2,000
• the first and second flanking regions may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).
• 15-1,000 nucleotides in length e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides.
• the tailing sequence may range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides).
• the length may be determined in units of or as a function of polyA Binding Protein binding.
• the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein.
• PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.
• the capping region may comprise a single cap or a series of nucleotides forming the cap.
• the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length.
• the cap is absent.
• the first and second operational regions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences.
• a primary construct or mmRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5 '-end binding proteins.
• the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed.
• the newly formed 5'- /3 '-linkage may be intramolecular or intermolecular.
• the 5 '-end and the 3 '-end of the nucleic acid contain chemically reactive groups that, when close together, form a new covalent linkage between the 5 '-end and the 3 '-end of the molecule.
• the 5 '-end may contain an NHS- ester reactive group and the 3 '-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3 '-end of a synthetic mR A molecule will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5 '-/3 '-amide bond.
• T4 RNA ligase may be used to enzymatically link a 5'- phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage.
• ⁇ g of a nucleic acid molecule is incubated at 37°C for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol.
• the ligation reaction may occur in the presence of a split oligonucleotide capable of base- pairing with both the 5'- and 3'- region in juxtaposition to assist the enzymatic ligation reaction.
• either the 5 '-or 3 '-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5 '-end of a nucleic acid molecule to the 3 '-end of a nucleic acid molecule.
• the ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).
• the ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C.
• multiple distinct polynucleotides, primary constructs or mmRNA may be linked together through the 3 '-end using nucleotides which are modified at the 3 '-terminus.
• Chemical conjugation may be used to control the stoichiometry of delivery into cells.
• the glyoxylate cycle enzymes, isocitrate lyase and malate synthase may be supplied into HepG2 cells at a 1 : 1 ratio to alter cellular fatty acid metabolism.
• This ratio may be controlled by chemically linking polynucleotides, primary constructs or mmR A using a 3'- azido terminated nucleotide on one polynucleotide, primary construct or mmRNA species and a C5-ethynyl or alkynyl-containing nucleotide on the opposite polynucleotide, primary construct or mmRNA species.
• the modified nucleotide is added post-transcriptionally using terminal transferase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol.
• the two polynucleotide, primary construct or mmRNA species may be combined in an aqueous solution, in the presence or absence of copper, to form a new covalent linkage via a click chemistry mechanism as described in the literature.
• more than two polynucleotides may be linked together using a functionalized linker molecule.
• a functionalized saccharide molecule may be chemically modified to contain multiple chemical reactive groups (SH-, NH 2 -, N 3 , etc%) to react with the cognate moiety on a 3 '-functionalized mRNA molecule (i.e., a 3'-maleimide ester, 3'-NHS-ester, alkynyl).
• the number of reactive groups on the modified saccharide can be controlled in a stoichiometric fashion to directly control the stoichiometric ratio of conjugated polynucleotide, primary construct or mmRNA.
• primary constructs or mmRNA of the present invention can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g.
• psoralene mitomycin C
• porphyrins TPPC4, texaphyrin, Sapphyrin
• polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
• artificial endonucleases e.g. EDTA
• alkylating agents phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.
• biotin e.g., aspirin, vitamin E, folic acid
• transport/absorption facilitators e.g., aspirin, vitamin E, folic acid
• synthetic ribonucleases proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.
• a specified cell type such as a cancer cell, endothelial cell, or bone cell
• hormones and hormone receptors non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.
• Conjugation may result in increased stability and/or half life and may be particularly useful in targeting the polynucleotides, primary constructs or mmRNA to specific sites in the cell, tissue or organism.
• the mmRNA or primary constructs may be administered with, or further encode one or more of RNAi agents, siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like.
• RNAi agents siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like.
• bifunctional polynucleotides e.g., bifunctional primary constructs or bifunctional mmRNA.
• bifunctional polynucleotides are those having or capable of at least two functions. These molecules may also by convention be referred to as multi-functional.
• the multiple functionalities of bifunctional polynucleotides may be encoded by the RNA (the function may not manifest until the encoded product is translated) or may be a property of the polynucleotide itself. It may be structural or chemical.
• Bifunctional modified polynucleotides may comprise a function that is covalently or electrostatically associated with the polynucleotides. Further, the two functions may be provided in the context of a complex of a mmRNA and another molecule.
• Bifunctional polynucleotides may encode peptides which are antiproliferative. These peptides may be linear, cyclic, constrained or random coil. They may function as aptamers, signaling molecules, ligands or mimics or mimetics thereof. Anti-proliferative peptides may, as translated, be from 3 to 50 amino acids in length. They may be 5-40, 10-30, or approximately 15 amino acids long. They may be single chain, multichain or branched and may form complexes, aggregates or any multi-unit structure once translated.
• Noncoding Polynucleotides and Primary Constructs As described herein, provided are polynucleotides and primary constructs having sequences that are partially or substantially not translatable, e.g., having a noncoding region. Such noncoding region may be the "first region" of the primary construct. Alternatively, the noncoding region may be a region other than the first region. Such molecules are generally not translated, but can exert an effect on protein production by one or more of binding to and sequestering one or more translational machinery components such as a ribosomal protein or a transfer RNA (tR A), thereby effectively reducing protein expression in the cell or modulating one or more pathways or cascades in a cell which in turn alters protein levels.
• translational machinery components such as a ribosomal protein or a transfer RNA (tR A)
• the polynucleotide or primary construct may contain or encode one or more long noncoding RNA (IncRNA, or lincRNA) or portion thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).
• RNA long noncoding RNA
• siRNA micro RNA
• siRNA small interfering RNA
• piRNA Piwi-interacting RNA
• the primary construct is designed to encode one or more polypeptides of interest or fragments thereof.
• a polypeptide of interest may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or variants of any of the aforementioned.
• the term "polypeptides of interest” refers to any polypeptides which are selected to be encoded in the primary construct of the present invention.
• polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds.
• polypeptides refers to proteins, polypeptides, and peptides of any size, structure, or function.
• polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long.
• polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
• a polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer.
• polypeptides may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides.
• polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
• polypeptide variant refers to molecules which differ in their amino acid sequence from a native or reference sequence.
• the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence.
• variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%>, more preferably at least about 90%> identical (homologous) to a native or reference sequence.
• variant mimics are provided.
• the term “variant mimic” is one which contains one or more amino acids which would mimic an activated sequence.
• glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine.
• variant mimics may result in deactivation or in an inactivated product containing the mimic, e.g., phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.
• homology as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
• homologs as it applies to polypeptide sequences means the corresponding sequence of other species having substantial identity to a second sequence of a second species.
• Analogs is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.
• compositions which are polypeptide based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives.
• derivative is used synonymously with the term “variant” but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule.
• mmR A encoding polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this invention.
• sequence tags or amino acids such as one or more lysines
• Sequence tags can be used for peptide purification or localization.
• Lysines can be used to increase peptide solubility or to allow for biotinylation.
• amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences.
• Certain amino acids e.g., C- terminal or N-terminal residues
• substitutional variants when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position.
• the substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
• conservative amino acid substitution refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity.
• conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.
• conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine.
• substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions.
• non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
• “Insertional variants” when referring to polypeptides are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. "Immediately adjacent" to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.
• deletional variants when referring to polypeptides are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
• Covalent derivatives when referring to polypeptides include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and/or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for
• polypeptides when referring to polypeptides are defined as distinct amino acid sequence-based components of a molecule.
• Features of the polypeptides encoded by the mrnPvNA of the present invention include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.
• manifestation refers to a polypeptide based component of a protein appearing on an outermost surface.
• conformational shape means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.
• fold refers to the resultant conformation of an amino acid sequence upon energy minimization.
• a fold may occur at the secondary or tertiary level of the folding process.
• secondary level folds include beta sheets and alpha helices.
• tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.
• turn as it relates to protein conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
• loop refers to a structural feature of a polypeptide which may serve to reverse the direction of the backbone of a peptide or polypeptide. Where the loop is found in a polypeptide and only alters the direction of the backbone, it may comprise four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997). Loops may be open or closed. Closed loops or "cyclic" loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the bridging moieties.
• Such bridging moieties may comprise a cysteine-cysteine bridge (Cys-Cys) typical in polypeptides having disulfide bridges or alternatively bridging moieties may be non-protein based such as the dibromozylyl agents used herein.
• Cys-Cys cysteine-cysteine bridge
• bridging moieties may be non-protein based such as the dibromozylyl agents used herein.
• domain refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein- protein interactions).
• sub-domains may be identified within domains or half- domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).
• site as it pertains to amino acid based embodiments is used synonymously with "amino acid residue” and "amino acid side chain.”
• a site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention.
• terminal refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions.
• the polypeptide based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C -terminus (terminated by an amino acid with a free carboxyl group (COOH)).
• Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini.
• the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.
• any of the features have been identified or defined as a desired component of a polypeptide to be encoded by the primary construct or mmR A of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.
• Modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis.
• the resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
• the polypeptides may comprise a consensus sequence which is discovered through rounds of experimentation.
• a "consensus" sequence is a single sequence which represents a collective population of sequences allowing for variability at one or more sites.
• protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest of this invention.
• any protein fragment meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical
• a reference protein 10 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length.
• any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%>, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the invention.
• a polypeptide to be utilized in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
• the polynucleotides, primary constructs or mmRNA of the present invention may be designed to encode polypeptides of interest such as pepetides and proteins.
• primary constructs or mmRNA may encode variant polypeptides which have a certain identity with a reference polypeptide sequence.
• a "reference polypeptide sequence” refers to a starting polypeptide sequence. Reference sequences may be wild type sequences or any sequence to which reference is made in the design of another sequence.
• a “reference polypeptide sequence” may be any enconding LDLR and/or CYP7al or variants thereof.
• identity refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.
• the polypeptide variant may have the same or a similar activity as the reference polypeptide.
• the variant may have an altered activity (e.g., increased or decreased) relative to a reference polypeptide.
• variants of a particular polynucleotide or polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
• Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI- BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.) Other tools are described herein, specifically in the definition of "Identity.”
• Default parameters in the BLAST algorithm include, for example, an expect threshold of 10, Word size of 28, Match/Mismatch Scores 1, -2, Gap costs Linear. Any filter can be applied as well as a selection for species specific repeats, e.g., Homo sapiens.
• the primary constructs or mmRNA disclosed herein may encode one or more validated or "in testing" proteins or peptides.
• one or more proteins or peptides currently being marketed or in development may be encoded by the polynucleotides, primary constructs or oncology-related mmRNA of the present invention. While not wishing to be bound by theory, it is believed that incorporation into the primary constructs or mmRNA of the invention will result in improved therapeutic efficacy due at least in part to the specificity, purity and selectivity of the construct designs.
• the polynucleotides, primary constructs and/or mmRNA may alter a biological and/or physiolocial process and/or compound such as, but not limited to, altering (e.g., slowing) the progression of a disease and/or disorder, reduce cholesterol and/or low-density lipoprotein (LDL) cholesterol, improve Crigler-Najjar syndrome, restore hepcidin and/or hemochromatosis type 2 function to regulate iron uptake, restore bile acid metabolism, reduce coronary heart disease risk for familial hypercholesterolemia and prevent hyperkeratotic plaques and corneal clouding which may heal hyperkeratotic plaques on the hands and/or feet.
• altering e.g., slowing
• LDL low-density lipoprotein
• the polynucleotides, primary constructs and/or mmRNA may be used to express a polypeptide in cells or tissues for the purpose of replacing the protein produced from a deleted or mutated gene.
• the polynucleotides, primary constructs or mmR A of the invention may be used to treat metabolic disorders related to rare liver diseases and/or disorders.
• UTRs Untranslated Regions
• UTRs Untranslated regions of a gene are transcribed but not translated.
• the 5'UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3 'UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
• the regulatory features of a UTR can be incorporated into the polynucleotides, primary constructs and/or mmRNA of the present invention to enhance the stability of the molecule.
• the specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
• Natural 5'UTRs bear features which play roles in for translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5'UTR also have been known to form secondary structures which are involved in elongation factor binding.
• mRNA such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII
• tissue-specific mRNA to improve expression in that tissue is possible - for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CDl lb, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD 18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A/B/C/D).
• non-UTR sequences may be incorporated into the 5' (or 3' UTR) UTRs.
• introns or portions of introns sequences may be incorporated into the flanking regions of the polynucleotides, primary constructs or mrnRNA of the invention. Incorporation of intronic sequences may increase protein production as well as mRNA levels.
• the 5 'UTR may selected for use in the present invention may be a structured UTR such as, but not limited to, 5 'UTRs to control translation.
• a structured 5 'UTR may be beneficial when using any of the terminal modifications described in copending U.S. Provisional Application No. 61/758,921 filed January 31, 2013, entitled Differential Targeting Using RNA Constructs; U.S. Provisional Application No. 61/781,139 filed March 14, 2013, entitled Differential Targeting Using RNA Constructs; U.S. Provisional Application No. 61/729,933, filed November 26, 2012 entitled Terminally Optimized RNAs; U.S.
• 3 'UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined.
• AREs 3' UTR AU rich elements
• polynucleotides, primary constructs or mmRNA of the invention When engineering specific polynucleotides, primary constructs or mmRNA, one or more copies of an ARE can be introduced to make polynucleotides, primary constructs or mmRNA of the invention less stable and thereby curtail translation and decrease production of the resultant protein.
• AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
• Transfection experiments can be conducted in relevant cell lines, using polynucleotides, primary constructs or mmRNA of the invention and protein production can be assayed at various time points post-transfection.
• cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, and 7 days post- transfection.
• microRNAs are 19-25 nucleotide long noncoding RNAs that bind to the 3 'UTR of nucleic acid molecules and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation.
• the polynucleotides, primary constructs or mmRNA of the invention may comprise one or more microRNA target sequences, microRNA seqences, microRNA binding sites, or microRNA seeds. Such sequences may correspond to any known microRNA such as those taught in US Publication US2005/0261218 and US Publication
• a microRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson- Crick complementarity to the miRNA target sequence.
• a microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.
• a microRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1.
• a microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked byan adenine (A) opposed to microRNA position 1.
• A an adenine
• the bases of the microRNA seed have complete complementarity with the target sequence.
• miR-122 a microRNA abundant in liver, can inhibit the expression of the gene of interest if one or multiple target sites of miR-122 are engineered into the 3'UTR of the polynucleotides, primary constructs or mmRNA.
• Introduction of one or multiple binding sites for different microRNA can be engineered to further decrease the longevity, stability, and protein translation of a polynucleotides, primary constructs or mmRNA.
• microRNA site refers to a microRNA target site or a microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that “binding” may follow traditional Watson-Crick hybridization rules or may reflect any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.
• microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues.
• miR-122 binding sites may be removed to improve protein expression in the liver. Regulation of expression in multiple tissues can be accomplished through introduction or removal or one or several microRNA binding sites.
• tissues where microRNA are known to regulate mRNA, and thereby protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-ld, miR-149), kidney (miR-192, miR- 194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).
• liver miR-122
• muscle miR-133, miR-206, miR-208
• endothelial cells miR-17-92, miR-126
• myeloid cells miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223,
• MicroRNA can also regulate complex biological processes such as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18: 171-176).
• angiogenesis miR-132
• mmRNA of the invention binding sites for microRNAs that are involved in such processes may be removed or introduced, in order to tailor the expression of the polynucleotides, primary constructs or mmRNA expression to biologically relevant cell types or to the context of relevant biological processes.
• polynucleotides, primary constructs or mmRNA can be engineered for more targeted expression in specific cell types or only under specific biological conditions.
• tissue-specific microRNA binding sites polynucleotides, primary constructs or mmRNA could be designed that would be optimal for protein expression in a tissue or in the context of a biological condition.
• Transfection experiments can be conducted in relevant cell lines, using engineered polynucleotides, primary constructs or mmRNA and protein production can be assayed at various time points post-transfection.
• cells can be transfected with different microRNA binding site-engineering polynucleotides, primary constructs or mmRNA and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, 72 hr and 7 days post- transfection.
• In vivo experiments can also be conducted using microRNA-binding site-engineered molecules to examine changes in tissue-specific expression of formulated polynucleotides, primary constructs or mmRNA.
• the polynucleotides, primary constructs and/or mmRNA may comprise at least one miR sequence or variant thereof.
• a non- exhaustive listing of miR sequences for use in polynucleotides, primary constructs and/or mmRNA is described in Table 9 of co-pending International Patent
• the polynucleotides, primary constructs and/or mmRNA may comprise at least one miR sequence that bind and inhibit the untranslated region of HMG-CoA reductase or PCSK9.
• miR sequences that bind and inhibit the untranslated region of HMG-CoA reductase or PCSK9 are described in International Patent Publication No. WO2013154766, the contents of which are herein incorporated by reference in its entirety.
• the polynucleotides, primary constructs and/or mmRNA may comprise a miR sequence that comprises miR-520d-5p, miR-224 or variants thereof (see e.g., International Patent Publication No.
• the polynucleotides, primary constructs and/or mmRNA may comprise a miR sequence that comprises or encodes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10 and/or SEQ ID NO: 11 of International Patent Publication No. WO2013154766, the contents of which are herein incorporated by reference in its entirety.
• the polynucleotides, primary constructs and/or mmRNA may comprise a miR sequence that comprises miR-224 or variants thereof (see e.g., International Patent Publication No.
• the polynucleotides, primary constructs and/or mmRNA may comprise a miR sequence that comprises miR-520d-5p and miR-224 or variants thereof (see e.g., International Patent Publication No. WO2013154766, the contents of which are herein incorporated by reference in its entirety).
• the 5' cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsibile for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
• CBP mRNA Cap Binding Protein
• the cap further assists the removal of 5' proximal introns removal during mRNA splicing.
• Endogenous mRNA molecules may be 5 '-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule.
• This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
• the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-0-methylated.
• 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.
• Modifications to the polynucleotides, primary constructs, and mmRNA of the present invention may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap. Additional modified guanosine nucleotides may be used such as a- methyl-phosphonate and seleno-phosphate nucleotides.
• Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and/or 5 '-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'-hydroxyl group of the sugar ring.
• Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as an mRNA molecule.
• Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non- enzymatically) or enzymatically synthesized and/linked to a nucleic acid molecule.
• the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'- triphosphate-5'-guanosine (m 7 G-3'mppp-G; which may equivaliently be designated 3' 0-Me-m7G(5')ppp(5')G).
• the 3'-0 atom of the other, unmodified, guanine becomes linked to the 5 '-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA).
• the N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA or mmRNA).
• mCAP which is similar to ARCA but has a 2'-0- methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'- guanosine, m 7 Gm-ppp-G).
• cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
• Polynucleotides, primary constructs and mmRNA of the invention may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5 '-cap structures.
• the phrase "more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects.
• Non- limiting examples of more authentic 5 'cap structures of the present invention are those which, among other things, have enhanced binding of cap binding proteins, increased half life, reduced susceptibility to 5' endonucleases and/or reduced 5'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5 'cap structure).
• recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5 '-terminal nucleotide of an mR A and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNA contains a 2'-0-methyl.
• Capl structure Such a structure is termed the Capl structure. This cap results in a higher
• Cap structures include 7mG(5')ppp(5')N,pN2p (cap 0),
• polynucleotides, primary constructs or mmRNA may be capped post-transcriptionally, and because this process is more efficient, nearly 100% of the polynucleotides, primary constructs or mmRNA may be capped. This is in contrast to -80% when a cap analog is linked to an mRNA in the course of an in vitro
• 5' terminal caps may include
• a 5' terminal cap may comprise a guanine analog.
• Useful guanine analogs include inosine, Nl- methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2- amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
• Additional viral sequences such as, but not limited to, the translation enhancer sequence of the barley yellow dwarf virus (BYDV-PAV) can be engineered and inserted in the 3' UTR of the polynucleotides, primary constructs or mmRNA of the invention and can stimulate the translation of the construct in vitro and in vivo.
• Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post- transfection.
• polynucleotides, primary constructs or mmRNA which may contain an internal ribosome entry site (IRES).
• IRES internal ribosome entry site
• An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
• polynucleotides, primary constructs or mmRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes ("multicistronic nucleic acid molecules").
• IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).
• picornaviruses e.g. FMDV
• CFFV pest viruses
• PV polio viruses
• ECMV encephalomyocarditis viruses
• FMDV foot-and-mouth disease viruses
• HCV hepatitis C viruses
• CSFV classical swine fever viruses
• MLV murine leukemia virus
• SIV simian immune deficiency viruses
• CrPV cricket paralysis viruses
• a long chain of adenine nucleotides may be added to a polynucleotide such as an mRNA molecules in order to increase stability.
• a polynucleotide such as an mRNA molecules
• the 3' end of the transcript may be cleaved to free a 3' hydroxyl.
• poly-A polymerase adds a chain of adenine nucleotides to the RNA.
• the process called polyadenylation, adds a poly-A tail that can be between 100 and 250 residues long.
• the length of a poly-A tail of the present invention is greater than 30 nucleotides in length.
• the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).
• the polynucleotide, primary construct, or mmRNA includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 2,500,
• the poly-A tail is designed relative to the length of the overall polynucleotides, primary constructs or mmRNA. This design may be based on the length of the coding region, the length of a particular feature or region (such as the first or flanking regions), or based on the length of the ultimate product expressed from the polynucleotides, primary constructs or mmRNA.
• the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotides, primary constructs or mmRNA or feature thereof.
• the poly-A tail may also be designed as a fraction of polynucleotides, primary constructs or mmRNA to which it belongs.
• the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail.
• engineered binding sites and conjugation of polynucleotides, primary constructs or mmRNA for Poly-A binding protein may enhance expression.
• polynucleotides, primary constructs or mmRNA may be linked together to the PABP (Poly-A binding protein) through the 3 '-end using modified nucleotides at the 3 '-terminus of the poly-A tail.
• Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-trans fection.
• the polynucleotide primary constructs of the present invention are designed to include a polyA-G quartet.
• the G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
• the G-quartet is incorporated at the end of the poly-A tail.
• the resultant mmRNA construct is assayed for stability, protein production and other parameters including half- life at various time points. It has been discovered that the polyA-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone.
• the polynucleotides, primary constructs or mmRNA of the present invention may be quantified in exosomes derived from one or more bodily fluid.
• bodily fluids include 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.
• exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
• the level or concentration of a polynucleotide, primary construct or mmRNA may be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker.
• the assay may be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
• immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods.
• Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
• Polynucleotides, primary constructs or mmRNA for use in accordance with the invention may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription (IVT) or enzymatic or chemical cleavage of a longer precursor, etc.
• IVT in vitro transcription
• Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M.J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference).
• the process of design and synthesis of the primary constructs of the invention generally includes the steps of gene construction, mRNA production (either with or without modifications) and purification.
• a target polynucleotide sequence encoding the polypeptide of interest is first selected for incorporation into a vector which will be amplified to produce a cDNA template.
• the target polynucleotide sequence and/or any flanking sequences may be codon optimized.
• the cDNA template is then used to produce mRNA through in vitro transcription (IVT). After production, the mRNA may undergo purification and clean-up processes.
• IVTT in vitro transcription
• the step of gene construction may include, but is not limited to gene synthesis, vector amplification, plasmid purification, plasmid linearization and cleanup, and cDNA template synthesis and clean-up.
• a primary construct is designed.
• a first region of linked nucleosides encoding the polypeptide of interest may be constructed using an open reading frame (ORF) of a selected nucleic acid (DNA or RNA) transcript.
• the ORF may comprise the wild type ORF, an isoform, variant or a fragment thereof.
• an "open reading frame” or “ORF” is meant to refer to a nucleic acid sequence (DNA or RNA) which is capable of encoding a polypeptide of interest. ORFs often begin with the start codon, ATG and end with a nonsense or termination codon or signal.
• the nucleotide sequence of the first region may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g.
• Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies) and/or DNA2.0 (Menlo Park CA).
• the ORF sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table 1.
• SECIS Selenocystein insertion element
• the modified mRNA nucleotide sequence may be codon optimized by methods known in the art and/or described herein. After a sequence has been codon optimized it may be further evaluated for regions containing restriction sites. At least one nucleotide within the restriction site regions may be replaced with another nucleotide in order to remove the restriction site from the sequence but the replacement of nucleotides does alter the amino acid sequence which is encoded by the codon optimized nucleotide sequence.
• flanking regions may be incorporated into the primary construct before and/or after optimization of the ORF. It is not required that a primary construct contain both a 5' and 3' flanking region. Examples of such features include, but are not limited to, untranslated regions (UTRs), Kozak sequences, an oligo(dT) sequence, and detectable tags and may include multiple cloning sites which may have Xbal recognition.
• a 5' UTR and/or a 3' UTR may be provided as flanking regions. Multiple 5 ' or 3' UTRs may be included in the flanking regions and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical modifications, before and/or after codon optimization. Combinations of features may be included in the first and second flanking regions and may be contained within other features.
• the ORF may be flanked by a 5' UTR which may contain a strong Kozak trans lational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.
• Tables 2 and 3 of co-pending U.S. Provisional Patent Application No 61/737,130 filed December 14, 2012 provide a listing of exemplary UTRs which may be utilized in the primary construct of the present invention as flanking regions.
• Variants of 5' or 3 'UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.
• any UTR from any gene may be incorporated into the respective first or second flanking region of the primary construct.
• multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide artificial UTRs which are not variants of wild type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5 ' or 3' UTR may be inverted, shortened, lengthened, made chimeric with one or more other 5' UTRs or 3' UTRs.
• the term "altered" as it relates to a UTR sequence means that the UTR has been changed in some way in relation to a reference sequence.
• a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an "altered" UTR (whether 3' or 5') comprise a variant UTR.
• a double, triple or quadruple UTR such as a 5 Or 3' UTR may be used.
• a "double" UTR is one in which two copies of the same UTR are encoded either in series or substantially in series.
• a double beta-globin 3' UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety.
• patterned UTRs are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.
• flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property.
• polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development.
• the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new chimeric primary transcript.
• a "family of proteins" is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
• the primary construct components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
• a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
• the optimized construct may be reconstituted and transformed into chemically competent E. coli, yeast, neurospora, maize, drosophila, etc. where high copy plasmid-like or chromosome structures occur by methods described herein.
• the primary constructs of the present invention may include at least two stop codons before the 3' untranslated region (UTR).
• the stop codon may be selected from TGA, TAA and TAG.
• the primary constructs of the present invention include the stop codon TGA and one additional stop codon.
• the addition stop codon may be TAA.
• the primary constructs of the present invention may include three stop codons before the 3' untranslated region (UTR).
• the vector containing the primary construct is then amplified and the plasmid isolated and purified using methods known in the art such as, but not limited to, a maxi prep using the Invitrogen PURELINKTM HiPure Maxiprep Kit (Carlsbad, CA).
• the plasmid may then be linearized using methods known in the art such as, but not limited to, the use of restriction enzymes and buffers.
• the linearization reaction may be purified using methods including, for example Invitrogen' s
• PURELINKTM PCR Micro Kit Carlsbad, CA
• HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC) and Invitrogen' s standard PURELINKTM PCR Kit (Carlsbad, CA).
• the purification method may be modified depending on the size of the linearization reaction which was conducted.
• the linearized plasmid is then used to generate cDNA for in vitro transcription (IVT) reactions.
• a cDNA template may be synthesized by having a linearized plasmid undergo polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• Table 4 of U.S. Provisional Patent Application No 61/737,130 filed December 14, 2012 provides a listing of primers and probes that may be usefully in the PCR reactions of the present invention. It should be understood that the listing is not exhaustive and that primer-probe design for any amplification is within the skill of those in the art. Probes may also contain chemically modified bases to increase base-pairing fidelity to the target molecule and base-pairing strength.
• the cDNA may be submitted for sequencing analysis before undergoing transcription.
• the process of mRNA or mmRNA production may include, but is not limited to, in vitro transcription, cDNA template removal and RNA clean-up, and mRNA capping and/or tailing reactions.
• the cDNA produced in the previous step may be transcribed using an in vitro transcription (IVT) system.
• the system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.
• NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein.
• the NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.
• the polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids.
• RNA polymerases or variants may be used in the design of the primary constructs of the present invention.
• RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence.
• the RNA polymerase may be modified to exhibit an increased ability to incorporate a 2 '-modified nucleotide triphosphate compared to an unmodified RNA polymerase (see
• Variants may be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and/or nucleic acid sequence and/or by using other methods known in the art.
• T7 RNA polymerase variants may be evolved using the continuous directed evolution system set out by Esvelt et al.
• T7 RNA polymerase may encode at least one mutation such as, but not limited to, lysine at position 93 substituted for threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A, H5
• T7 RNA polymerase variants may encode at least mutation as described in U.S. Pub. Nos. 20100120024 and 20070117112; herein incorporated by reference in their entireties.
• Variants of RNA polymerase may also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, deletional variants and/or covalent derivatives.
• the primary construct may be designed to be recognized by the wild type or variant RNA polymerases. In doing so, the primary construct may be modified to contain sites or regions of sequence changes from the wild type or parent primary construct.
• the primary construct may be designed to include at least one substitution and/or insertion upstream of an RNA polymerase binding or recognition site, downstream of the RNA polymerase binding or recognition site, upstream of the TATA box sequence, downstream of the TATA box sequence of the primary construct but upstream of the coding region of the primary construct, within the 5'UTR, before the 5'UTR and/or after the 5'UTR.
• the 5 'UTR of the primary construct may be replaced by the insertion of at least one region and/or string of nucleotides of the same base.
• the region and/or string of nucleotides may include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 nucleotides and the nucleotides may be natural and/or unnatural.
• the group of nucleotides may include 5-8 adenine, cytosine, thymine, a string of any of the other nucleotides disclosed herein and/or combinations thereof.
• the 5 'UTR of the primary construct may be replaced by the insertion of at least two regions and/or strings of nucleotides of two different bases such as, but not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein and/or combinations thereof.
• the 5 'UTR may be replaced by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine bases.
• the 5 'UTR may be replaced by inserting 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.
• the primary construct may include at least one substitution and/or insertion downstream of the transcription start site which may be recognized by an RNA polymerase.
• at least one substitution and/or insertion may occur downstream the transcription start site by substituting at least one nucleic acid in the region just downstream of the transcription start site (such as, but not limited to, +1 to +6). Changes to region of nucleotides just downstream of the transcription start site may affect initiation rates, increase apparent nucleotide triphosphate (NTP) reaction constant values, and increase the dissociation of short transcripts from the transcription complex curing initial transcription (Brieba et al, Biochemistry (2002) 41 : 5144-5149; herein incorporated by reference in its entirety).
• the modification, substitution and/or insertion of at least one nucleic acid may cause a silent mutation of the nucleic acid sequence or may cause a mutation in the amino acid sequence.
• the primary construct may include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or at least 13 guanine bases downstream of the transcription start site.
• the primary construct may include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 guanine bases in the region just downstream of the transcription start site.
• the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 adenine nucleotides.
• the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 cytosine bases.
• the guanine bases in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 thymine, and/or any of the nucleotides described herein.
• the primary construct may include at least one substitution and/or insertion upstream of the start codon.
• start codon is the first codon of the protein coding region whereas the transcription start site is the site where
• the primary construct may include, but is not limited to, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8
• nucleotide bases may be inserted or substituted at 1, at least 1 , at least 2, at least 3, at least 4 or at least 5 locations upstream of the start codon.
• the nucleotides inserted and/or substituted may be the same base (e.g., all A or all C or all T or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T or A, C and T) or at least four different bases.
• the guanine base upstream of the coding region in the primary construct may be substituted with adenine, cytosine, thymine, or any of the nucleotides described herein.
• the substitution of guanine bases in the primary construct may be designed so as to leave one guanine base in the region downstream of the transcription start site and before the start codon (see Esvelt et al. Nature (2011) 472(7344):499-503; herein incorporated by reference in its entirety).
• At least 5 nucleotides may be inserted at 1 location downstream of the transcription start site but upstream of the start codon and the at least 5 nucleotides may be the same base type.
• RNA clean-up may also include a purification method such as, but not limited to, AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, MA), HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) .
• AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, MA
• HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) .
• the primary construct or mmR A may also undergo capping and/or tailing reactions.
• a capping reaction may be performed by methods known in the art to add a 5' cap to the 5' end of the primary construct. Methods for capping include, but are not limited to, using a Vaccinia Capping enzyme (New England Biolabs, Ipswich, MA).
• a poly-A tailing reaction may be performed by methods known in the art, such as, but not limited to, 2' O-methyltransferase and by methods as described herein. If the primary construct generated from cDNA does not include a poly-T, it may be beneficial to perform the poly-A-tailing reaction before the primary construct is cleaned.
• Primary construct or mmRNA purification may include, but is not limited to, mRNA or mmRNA clean-up, quality assurance and quality control.
• mRNA or mmRNA clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
• AGENCOURT® beads Beckman Coulter Genomics, Danvers, MA
• poly-T beads poly-T beads
• LNATM oligo-T capture probes EXIQON® Inc, Vedbaek, Denmark
• HPLC based purification methods such as, but not limited to, strong anion exchange HPLC
• purified when used in relation to a polynucleotide such as a “purified mRNA or mmRNA” refers to one that is separated from at least one contaminant.
• a "contaminant” is any substance which makes another unfit, impure or inferior.
• polynucleotide e.g., DNA and RNA
• a form or setting different from that in which it is found in nature or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.
• a quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
• the mRNA or mmRNA may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.
• the mRNA or mmRNA may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).
• UV/Vis ultraviolet visible spectroscopy
• a non- limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA).
• the quantified mRNA or mmRNA may be analyzed in order to determine if the mRNA or mmRNA may be of proper size, check that no degradation of the mRNA or mmRNA has occurred.
• Degradation of the mRNA and/or mmRNA may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP- HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography- mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
• HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP- HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography- mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
• the primary constructs or mmRNA may also encode additional features which facilitate trafficking of the polypeptides to therapeutically relevant sites.
• One such feature which aids in protein trafficking is the signal sequence.
• a "signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-60 amino acids) in length which is incorporated at the 5' (or N-terminus) of the coding region or polypeptide encoded, respectively. Addition of these sequences result in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported.
• Protein signal sequences which may be incorporated for encoding by the polynucleotides, primary constructs or mmRNA of the invention include signal sequences from a- 1 -antitrypsin, G-CSF, Factor IX, Prolactin, Albumin, HMMSP38, ornithine carbamoyltransferase,
• Cytochrome C Oxidase subunit 8A Type III, bacterial, viral, secretion signals, Vrg-6, PhoA, OmpA, STI, STII, Amylase, Alpha Factor, Endoglucanase V, Secretion signal, fungal and fibronectin.
• SS secretion signal
• MLS mitochondrial leader signal.
• the primary constructs or mmRNA of the present invention may be designed to encode any of the signal sequences or fragments or variants thereof. These sequences may be included at the beginning of the polypeptide coding region, in the middle or at the terminus or alternatively into a flanking region.
• the primary constructs comprise at least a first region of linked nucleosides encoding at least one polypeptide of interest.
• the polypeptides of interest or “Targets" of the present invention are listed in Table 2 below. Shown in Table 2, in addition to the name and description of the gene encoding the polypeptide of interest are the ENSEMBL Transcript ID (ENST), the ENSEMBL Protein ID (ENSP) and when available the optimized sequence ID (OPT SEQ ID). For any particular gene there may exist one or more variants or isoforms. Where these exist, they are shown in the table as well. It will be appreciated by those of skill in the art that disclosed in the Table are potential flanking regions.
• each ENST transcript either to the 5' (upstream) or 3' (downstream) of the ORF or coding region.
• the coding region is definitively and specifically disclosed by teaching the ENSP sequence. Consequently, the sequences taught flanking that encoding the protein are considered flanking regions. It is also possible to further characterize the 5' and 3' flanking regions by utilizing one or more available databases or algorithms. Databases have annotated the features contained in the flanking regions of the ENST transcripts and these are available in the art.
• PCSK9 subtilisin/kexin type 9 15 PCSK9 subtilisin/kexin type 9 8 15 401598 25 proprotein convertase 30211
• the targets of the present invention may be any of the targets described in U.S. Provisional Patent Application No 61/618,862, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Biologies; U.S.
• PCT/US2013/030066 filed March 9, 2013, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins
• International Application No. PCT/US2013/030067 filed March 9, 2013, entitled Modified Polynucleotides for the Production of Nuclear Proteins
• the polypeptides of the present invention may include at least one protein cleavage signal containing at least one protein cleavage site.
• the protein cleavage site may be located at the N-terminus, the C-terminus, at any space between the N- and the C- termini such as, but not limited to, half-way between the N- and C-termini, between the N-terminus and the half way point, between the half way point and the C-terminus, and combinations thereof.
• the polypeptides of the present invention may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin or Factor Xa protein cleavage signal.
• Proprotein convertases are a family of nine proteinases, comprising seven basic amino acid-specific subtilisin-like serine proteinases related to yeast kexin, known as prohormone convertase 1/3 (PC 1/3), PC2, furin, PC4, PC5/6, paired basic amino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilases that cleave at non-basic residues, called subtilisin kexin isozyme 1 (SKI-1) and
• PCSK9 proproteinconvertase subtilisin kexin 9
• the primary constructs and mmRNA of the present invention may be engineered such that the primary construct or mmRNA contains at least one encoded protein cleavage signal.
• the encoded protein cleavage signal may be located before the start codon, after the start codon, before the coding region, within the coding region such as, but not limited to, half way in the coding region, between the start codon and the half way point, between the half way point and the stop codon, after the coding region, before the stop codon, between two stop codons, after the stop codon and combinations thereof.
• the primary constructs or mmRNA of the present invention may include at least one encoded protein cleavage signal containing at least one protein cleavage site.
• the encoded protein cleavage signal may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin and/or Factor Xa protein cleavage signal.
• a proprotein convertase or prohormone convertase
• thrombin or Factor Xa protein cleavage signal.
• Factor Xa protein cleavage signal may be used as Table 1 above or other known methods to determine the appropriate encoded protein cleavage signal to include in the primary constructs or mmRNA of the present invention. For example, starting with a signal sequence and considering the codons of Table 1 one can design a signal for the primary construct which can produce a protein signal in the resulting polypeptide.
• the polypeptides of the present invention include at least one protein cleavage signal and/or site.
• U.S. Pat. No. 7,374,930 and U.S. Pub. No. 20090227660 herein incorporated by reference in their entireties, use a furin cleavage site to cleave the N-terminal methionine of GLP-1 in the expression product from the Golgi apparatus of the cells.
• the polypeptides of the present invention include at least one protein cleavage signal and/or site with the proviso that the polypeptide is not GLP-1.
• the primary constructs or mmRNA of the present invention includes at least one encoded protein cleavage signal and/or site.
• the primary constructs or mmRNA of the present invention includes at least one encoded protein cleavage signal and/or site with the proviso that the primary construct or mmRNA does not encode GLP-1.
• the primary constructs or mmRNA of the present invention may include more than one coding region. Where multiple coding regions are present in the primary construct or mmRNA of the present invention, the multiple coding regions may be separated by encoded protein cleavage sites.
• the primary construct or mmRNA may be signed in an ordered pattern. On such pattern follows AXBY form where A and B are coding regions which may be the same or different coding regions and/or may encode the same or different polypeptides, and X and Y are encoded protein cleavage signals which may encode the same or different protein cleavage signals.
• a second such pattern follows the form AXYBZ where A and B are coding regions which may be the same or different coding regions and/or may encode the same or different polypeptides, and X, Y and Z are encoded protein cleavage signals which may encode the same or different protein cleavage signals.
• a third pattern follows the form ABXCY where A, B and C are coding regions which may be the same or different coding regions and/or may encode the same or different polypeptides, and X and Y are encoded protein cleavage signals which may encode the same or different protein cleavage signals.
• the polypeptides, primary constructs and mmR A can also contain sequences that encode protein cleavage sites so that the polypeptides, primary constructs and mmRNA can be released from a carrier region or a fusion partner by treatment with a specific protease for said protein cleavage site.
• modify refers to modification with respect to A, G, U or C ribonucleotides. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moieties.
• modification refers to a modification as compared to the canonical set of 20 amino acids, moiety
• the modifications may be various distinct modifications.
• the coding region, the flanking regions and/or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide
• a modified polynucleotide, primary construct, or mmRNA introduced to a cell may exhibit reduced degradation in the cell, as compared to an unmodified polynucleotide, primary construct, or mmRNA.
• the polynucleotides, primary constructs, and mmRNA can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g. to a linking phosphate / to a phosphodiester linkage / to the
• a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro).
• modifications e.g., one or more modifications
• RNAs ribonucleic acids
• DNAs deoxyribonucleic acids
• TAAs threose nucleic acids
• GAAs glycol nucleic acids
• PNAs peptide nucleic acids
• LNAs locked nucleic acids
• the polynucleotides, primary constructs, and mmRNA of the invention do not substantially induce an innate immune response of a cell into which the mRNA is introduced.
• Featues of an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc, and/or 3) termination or reduction in protein translation.
• the invention provides a modified nucleic acid molecule containing a degradation domain, which is capable of being acted on in a directed manner within a cell.
• the polynucleotides, primary constructs, and mmRNA can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.).
• the polynucleotides, primary constructs, or mmRNA may include one or more messenger RNAs (mRNAs) and one or more modified nucleoside or nucleotides (e.g., mmRNA molecules). Details for these polynucleotides, primary constructs, and mmRNA follow.
• the polynucleotides, primary constructs, and mmRNA of the invention includes a first region of linked nucleosides encoding a polypeptide of interest, a first flanking region located at the 5' terminus of the first region, and a second flanking region located at the 3' terminus of the first region.
• the polynucleotide, primary construct, or mmRNA (e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having any base, sugar, backbone, building block or other structure or formula, including but not limited to those of Formulas I through IX or any substructures thereof as described in International Application PCT/US12/58519 filed October 3, 2012 (Attorney Docket Number: M009.20), the contents of which are incorporated herein by reference in their entirety.
• Such structures include modifications to the sugar, nucleobase, internucleoside linkage, or combinations thereof.
• the nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil.
• the modified nucleobase is a modified uracil.
• nucleobases and nucleosides having a modified uracil include
• pseudouridine ( ⁇ ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio- 5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5 -hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm
• the modified nucleobase is a modified cytosine.
• exemplary nucleobases and nucleosides having a modified cytosine include 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl- cytidine (ac 4 C), 5-formyl-cytidine (f ⁇ C), N4-methyl-cytidine (m 4 C), 5-methyl- cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2- thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-
• the modified nucleobase is a modified adenine.
• exemplary nucleobases and nucleosides having a modified adenine include 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6- halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2- amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1- methyl-adenosine (rr ⁇ A), 2-methyl-adenine (m 2 A), N6-methyl-adenine (rr ⁇ A),
• the modified nucleobase is a modified guanine.
• exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m 1 !), wyosine (imG), methylwyosine (mimG), 4-demethyl- wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o 2 yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7- deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-
• the nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog.
• the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or
• the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4- d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2- thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8- thiol, 8-thioalkyl, 8-hydroxyl and other 8-sub
• each letter refers to the representative base and/or derivatives thereof, e.g., A includes adenine or adenine analogs, e.g., 7-deaza adenine).
• Modified nucleosides and nucleotides can be prepared according to the synthetic methods described in Ogata et al, J. Org. Chem. 74:2585-2588 (2009); Purmal et al, Nucl. Acids Res. 22(1): 72-78, (1994); Fukuhara et al, Biochemistry, 1(4): 563-568 (1962); and Xu et al, Tetrahedron, 48(9): 1729-1740 (1992), each of which are incorporated by reference in their entirety.
• polypeptides, primary constructs, and mmRNA of the invention may or may not be uniformly modified along the entire length of the molecule.
• nucleotide e.g. , purine or pyrimidine, or any one or more or all of A, G, U, C
• a given predetermined sequence region thereof e.g. one or more of the sequence regions represented in Figure 1).
• nucleotides X in a polynucleotide of the invention are modified, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
• internucleoside linkages may exist at various positions in the polynucleotide, primary construct, or mmRNA.
• nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide, primary construct, or mmRNA such that the function of the polynucleotide, primary construct, or mmRNA is not substantially decreased.
• a modification may also be a 5 ' or 3' terminal modification.
• the polynucleotide, primary construct, or mmRNA may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e.
• any one or more of A, G, U or C) or any intervening percentage e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 90% to 100%, and from 95% to 100%).
• any intervening percentage e.g.,
• the polynucleotide, primary construct, or mmR A includes a modified pyrimidine (e.g., a modified uracil/uridine/U or modified cytosine/cytidine/C).
• a modified pyrimidine e.g., a modified uracil/uridine/U or modified cytosine/cytidine/C.
• the uracil or uridine (generally: U) in the polynucleotide, primary construct, or mmRNA molecule may be replaced with from about 1%) to about 100% of a modified uracil or modified uridine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 20%
• the cytosine or cytidine (generally: C) in the polynucleotide, primary construct, or mmRNA molecule may be replaced with from about 1%) to about 100% of a modified cytosine or modified cytidine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%,
• the polynucleotide, primary construct, or mmRNA is translatable.
• polynucleotides, primary constructs, and mmRNA are optional, and are beneficial in some embodiments.
• a 5' untranslated region (UTR) and/or a 3'UTR are provided, wherein either or both may independently contain one or more different nucleotide modifications.
• nucleotide modifications may also be present in the translatable region.
• polynucleotides, primary constructs, and mmRNA containing a Kozak sequence are also be present in the translatable region.
• At least 25% of the cytosines are replaced by a compound of Formula (bl0)-(bl4) (e.g., at least about 30%>, at least about 35%, at least about 40%>, at least about 45%, at least about 50%>, at least about 55%, at least about 60%), at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).
• a compound of Formula (bl0)-(bl4) e.g., at least about 30%>, at least about 35%, at least about 40%>, at least about 45%, at least about 50%>, at least about 55%, at least about 60%
• At least 25% of the uracils are replaced by a compound of Formula (bl)-(b9) (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%), at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).
• a compound of Formula (bl)-(b9) e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%
• at least about 65%, at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
• At least 25% of the cytosines are replaced by a compound of Formula (bl0)-(bl4), and at least 25% of the uracils are replaced by a compound of Formula (bl)-(b9) (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%), at least about 65%, at least about 70%>, at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%, or about 100%).
• a compound of Formula (bl0)-(bl4) are replaced by a compound of Formula (bl)-(b9) (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%), at least about 65%, at least about 70%>, at least about 75%, at least about 80%>, at least about 85%, at least
• the present invention provides polynucleotides, primary constructs and mmRNA compositions and complexes in combination with one or more
• compositions may optionally comprise one or more additional active substances, e.g. therapeutically and/or prophylactically active substances.
• additional active substances e.g. therapeutically and/or prophylactically active substances.
• General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
• compositions are administered to humans, human patients or subjects.
• active ingredient generally refers to polynucleotides, primary constructs and mmRNA to be delivered as described herein.
• compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the
• compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
• Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
• Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
• a pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
• a "unit dose" is discrete amount of the
• composition comprising a predetermined amount of the active ingredient.
• the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one -third of such a dosage.
• compositions in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
• the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%>, between 1-30%, between 5-80%>, at least 80%> (w/w) active ingredient.
• the polynucleotide, primary construct, and mmRNA of the invention can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the polynucleotide, primary construct, or mmRNA); (4) alter the biodistribution (e.g., target the polynucleotide, primary construct, or mmRNA to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo.
• excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the polynucleotide, primary construct, or mmRNA); (4) alter the biodistribution (e.g., target the polynucleotide, primary construct, or mmRNA to specific tissues or cell types); (5) increase the translation of encoded protein in
• excipients of the present invention can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with polynucleotide, primary construct, or mmRNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
• the formulations of the invention can include one or more excipients, each in an amount that together increases the stability of the polynucleotide, primary construct, or mmRNA, increases cell transfection by the polynucleotide, primary construct, or mmRNA, increases the expression of polynucleotide, primary construct, or mmRNA encoded protein, and/or alters the release profile of polynucleotide, primary construct, or mmRNA encoded proteins.
• the primary construct and mmRNA of the present invention may be formulated using self-assembled nucleic acid nanoparticles.
• Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
• a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
• a "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
• the amount of the active ingredient may generally be equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage including, but not limited to, one-half or one-third of such a dosage.
• Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
• the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.
• the formulations described herein may contain at least one mmRNA.
• the formulations may contain 1, 2, 3, 4 or 5 mmRNA.
• the formulation may contain modified mRNA encoding proteins selected from categories such as, but not limited to, human proteins, veterinary proteins, bacterial proteins, biological proteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secreted proteins, plasma membrane proteins, cytoplasmic and cytoskeletal proteins, intrancellular membrane bound proteins, nuclear proteins, proteins associated with human disease and/or proteins associated with non-human diseases.
• the formulation contains at least three modified mRNA encoding proteins.
• the formulation contains at least five modified mRNA encoding proteins.
• compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
• a pharmaceutically acceptable excipient includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
• excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD,
• any conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the
• the particle size of the lipid nanoparticle may be increased and/or decreased.
• the change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the modified mRNA delivered to mammals.
• compositions include, but are not limited to, inert diluents, surface active agents and/or emulsifiers, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in the pharmaceutical formulations of the invention.
• the polynucleotides, primary constructs and/or mmRNA may be administered in or with, formulated in or delivered with
• nanostructures that can sequester molecules such as cholesterol.
• Non-limiting examples of these nanostructures and methods of making these nanostructures are described in US Patent Publication No. US20130195759, the contents of which are herein incorporated by reference in its entirety.
• Exemplary structures of these nanostructures are shown in Figure 1 of US Patent Publication No. US20130195759, the contents of which are herein incorporated by reference in its entirety, and may include a core and a shell surrounding the core.
• the present disclosure describes their formulation and use in delivering single stranded polynucleotides, primary constructs, or mmRNA.
• Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in an effective delivery of the polynucleotide, primary construct, or mmRNA, as judged by the production of an encoded protein, following the injection of a lipidoid formulation via localized and/or systemic routes of administration.
• Lipidoid complexes of polynucleotides, primary constructs, or mmRNA can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.
• nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle
• PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety).
• PEG poly(ethylene glycol)
• Formulations with the different lipidoids including, but not limited to penta[3-(l-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA- 5LAP; aka 98N12-5, see Murugaiah et al, Analytical Biochemistry, 401 :61 (2010)), CI 2-200 (including derivatives and variants), and MD1, can be tested for in vivo activity.
• TETA- 5LAP penta[3-(l-laurylaminopropionyl)]-triethylenetetramine hydrochloride
• CI 2-200 including derivatives and variants
• MD1 penta[3-(l-laurylaminopropionyl)]-triethylenetetramine hydrochloride
• the lipidoid referred to herein as "C 12-200" is disclosed by Love et al, Proc Natl Acad Sci U S A. 2010 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670; both of which are herein incorporated by reference in their entirety.
• the lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotide, primary construct, or mmRNA.
• formulations with certain lipidoids include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (CI 4 alkyl chain length).
• formulations with certain lipidoids include, but are not limited to, C12-200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.
• a polynucleotide, primary construct, or mmRNA formulated with a lipidoid for systemic intravenous administration can target the liver.
• an intravenous formulation using a CI 2-200 may have a molar ratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidyl
• choline/cholesterol/PEG-DMG with a weight ratio of 7 to 1 total lipid
• polynucleotide, primary construct, or mmRNA and a mean particle size of 80 nm may be effective to deliver polynucleotide, primary construct, or mmRNA to hepatocytes (see, Love et al, Proc Natl Acad Sci U S A. 2010 107: 1864-1869 herein incorporated by reference).
• an MD1 lipidoid-containing formulation may be used to effectively deliver polynucleotide, primary construct, or mmRNA to hepatocytes in vivo. The characteristics of optimized lipidoid
• formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879 herein incorporated by reference), use of a lipidoid-formulated polynucleotide, primary construct, or mmRNA to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited.
• lipidoid formulations to deliver siRNA in vivo to other non-hepatocyte cells such as myeloid cells and endothelium has been reported (see Akinc et al, Nat Biotechnol. 2008 26:561-569; Leuschner et al, Nat Biotechnol. 2011 29:1005-1010; Cho et al. Adv. Funct. Mater. 2009 19:3112-3118; 8 th International Judah Folkman Conference, Cambridge, MA October 8-9, 2010 herein incorporated by reference in its entirety).
• Effective delivery to myeloid cells, such as monocytes lipidoid formulations may have a similar component molar ratio.
• lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formulation of the polynucleotide, primary construct, or mmRNA for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc.
• the component molar ratio may include, but is not limited to, 50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG (see Leuschner et al., Nat Biotechnol 2011 29: 1005-1010; herein incorporated by reference in its entirety).
• lipidoid formulations for the localized delivery of nucleic acids to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, may not require all of the formulation components desired for systemic delivery, and as such may comprise only the lipidoid and the polynucleotide, primary construct, or mmRNA.
• Combinations of different lipidoids may be used to improve the efficacy of polynucleotide, primary construct, or mmRNA directed protein production as the lipidoids may be able to increase cell transfection by the polynucleotide, primary construct, or mmRNA; and/or increase the translation of encoded protein (see Whitehead et al., Mol. Ther. 2011, 19: 1688-1694, herein incorporated by reference in its entirety).
• Liposomes Liposomes, Lipoplexes, and Lipid Nanoparticles
• the polynucleotide, primary construct, and mmRNA of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.
• pharmaceutical compositions of polynucleotide, primary construct, or mmRNA include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations.
• Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.
• MLV multilamellar vesicle
• SUV small unicellular vesicle
• LUV large unilamellar vesicle
• Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
• Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
• liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients , the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
• compositions described herein may include, without limitation, liposomes such as those formed from l,2-dioleyloxy-N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), l,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l ,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).
• DOXIL® DiLa2 liposomes
• DiLa2 liposomes from Marina Biotech (Bothell, WA)
• DLin-DMA l,2-dil
• compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6: 1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Momssey et al, Nat Biotechnol. 2005 2: 1002-1007; Zimmermann et al, Nature. 2006 441 : 111-114; Heyes et al. J Contr Rel. 2005 107:276-287;
• liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for
• the original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method.
• the liposome formulations are composed of 3 to 4 lipid components in addition to the polynucleotide, primary construct, or mmRNA.
• a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%) l,2-dioleyloxy-A ,N-dimethylaminopropane (DODMA), as described by Jeffs et al.
• certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be l,2-distearloxy-N,N-dimethylaminopropane
• DSDMA DODMA
• DLin-DMA DODMA
• DLenDMA l,2-dilinolenyloxy-3-dimethylaminopropane
• the polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.
• the polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid-polycation complex.
• the formation of the lipid-polycation complex may be accomplished by methods known in the art and/or as described in U.S. Pub. No. 20120178702, herein incorporated by reference in its entirety.
• the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or
• polyarginine in another embodiment, the polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid-polycation complex which may further include a neutral lipid such as, but not limited to, cholesterol or dioleoyl
• DOPE phosphatidylethanolamine
• the liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size.
• the liposome formulation was composed of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and 1.4% PEG-c-DMA.
• changing the composition of the cationic lipid could more effectively deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated by reference in its entirety).
• the ratio of PEG in the LNP formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to CI 8 to alter the pharmacokinetics and/or biodistribution of the LNP formulations.
• LNP formulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol.
• the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG- DSG (1,2-Distearoyl-sn-glycerol,
• the cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.
• the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,
• the cationic lipid may be selected from, but not limited to, formula A described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259,
• the cationic lipid may be selected from, but not limited to, formula CLI-CLXXIX of International Publication No. WO2008103276, formula CLI-CLXXIX of US Patent No. 7,893,302, formula CLI-CLXXXXII of US Patent No. 7,404,969 and formula I-VI of US Patent
• the cationic lipid may be selected from (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N- dimemylhexacosa-17,20-dien-9-amine, (1 Z, 19Z)-N5N ⁇ dimethylpentacosa ⁇ l 6, 19- dien-8-amine, ( 13Z, 16Z)-N,N-dimethyldocosa- 13 Jl 6-dien-5 -amine, ( 12Z, 15Z)-NJN- dimethylhenicosa- 12, 15-dien-4-amine, ( 14Z, 17Z)-N,N-dimethyltricosa- 14,17-dien-6- amine, ( 15Z, 18Z)-N,N-dimethyltetracosa- 15,18-dien-7-amine, (
• the LNP formulations of the polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG 3% lipid molar ratio. In another embodiment, the LNP formulations of the polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG 1.5% lipid molar ratio.
• polynucleotides, primary constructs and/or mmRNA may include at least one of the PEGylated lipids described in International Publication No. 2012099755, herein incorporated by reference.
• the LNP formulation may contain PEG-DMG 2000 (l,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-2000).
• the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component.
• the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.
• the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol.
• the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40: 10:48 (see Geall et al, Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012; PMID: 22908294).
• the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or
• LNP formulations described herein may comprise a polycationic composition.
• the polycationic composition may be selected from formula 1-60 of US Patent Publication No. US20050222064; herein incorporated by reference in its entirety.
• the LNP formulations comprising a polycationic composition may be used for the delivery of the modified RNA described herein in vivo and/or in vitro.
• the LNP formulations described herein may be any suitable LNP formulations described herein.
• Non-limiting permeability enhancer molecules are described in US Patent Publication No. US20050222064; herein incorporated by reference in its entirety.
• the pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713)) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
• DiLa2 liposomes Marina Biotech, Bothell, WA
• SMARTICLES® Marina Biotech, Bothell, WA
• neutral DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine) based liposomes e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &
• Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP).
• Ionizable cationic lipids such as, but not limited to,
• the rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat.
• Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation.
• the ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.
• the internal ester linkage may be located on either side of the saturated carbon.
• reLNPs include,
• an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
• a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
• the polymer may encapsulate the nanospecies or partially encapsulate the nanospecies.
• Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier.
• Mucus is located on mucosal tissue such as, but not limted to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
• oral e.g., the buccal and esophageal membranes and tonsil tissue
• ophthalmic e.g., gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum)
• nasal, respiratory e.g., nasal, pharyngeal, tracheal and bron
• Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosla tissue within seconds or within a few hours. Large polymeric nanoparticles (200nm -500nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al.
• PEG polyethylene glycol
• the transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT).
• FRAP fluorescence recovery after photobleaching
• MPT high resolution multiple particle tracking
• the lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer.
• the polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes,
• polyacetylenes polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
• the polymeric material may be biodegradable and/or biocompatible.
• Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid- co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L- lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L- lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (
• polyurethanes derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate),
• nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer, and (poly(ethylene glycol))-(poly(propylene oxide))- (poly(ethylene glycol)) triblock copolymer (see US Publication 20120121718 and US Publication 20100003337; each of which is herein incorporated by reference in their entirety).
• the co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created.
• the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; herein incorporated by reference in its entirety).
• the vitamin of the polymer-vitamin conjugate may be vitamin E.
• the vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).
• the lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, mmR A, anionic protein (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example
• dimethyldioctadecyl-ammonium bromide sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin ⁇ 4 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase.
• nucleic acids polymers (e.g., heparin, polyethylene glycol and poloxamer)
• the surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle.
• the mucus penetrating lipid nanoparticles may comprise at least one mmRNA described herein.
• the mmRNA may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the paricle.
• the mmRNA may be covalently coupled to the lipid nanoparticle.
• Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.
• the polynucleotide, primary construct, or mmRNA is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine -based targeted and non- targeted delivery of nucleic acids acids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al.
• a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine -based targeted and non- targeted delivery of nucleic acids acids acids (Aleku et al. Cancer
• such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18:1357-1364; Song et al, Nat Biotechnol.
• One example of passive targeting of formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and MC3-based lipid nanoparticle formulations which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18: 1357-1364; herein incorporated by reference in its entirety).
• Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8: 197-206; Musacchio and Torchilin, Front Biosci. 2011 16: 1388-1412; Yu et al, Mol Membr Biol. 2010 27:286-298; Patil et al, Crit Rev Ther Drug Carrier Syst. 2008 25: 1-61; Benoit et al, Biomacromolecules.
• the polynucleotide, primary construct, or mmRNA is formulated as a solid lipid nanoparticle.
• a solid lipid nanoparticle may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers.
• the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al, ACS Nano, 2008, 2 (8), pp 1696-1702; herein incorporated by reference in its entirety).
• Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of polynucleotide, primary construct, or mmRNA directed protein production as these formulations may be able to increase cell transfection by the polynucleotide, primary construct, or mmRNA; and/or increase the translation of encoded protein.
• One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et al, Mol Ther. 2007 15:713- 720; herein incorporated by reference in its entirety).
• the liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the polynucleotide, primary construct, or mmRNA.
• the polynucleotides, primary constructs, and/or the mmRNA of the present invention can be formulated for controlled release and/or targeted delivery.
• controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
• the polynucleotides, primary constructs or the mmRNA may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
• the term "encapsulate” means to enclose, surround or encase. As it relates to the formulation of the compounds of the invention, encapsulation may be substantial, complete or partial.
• substantially encapsulated means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent.
• Partially encapsulation means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent.
• encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the invention using fluorescence and/or electron micrograph.
• At least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the delivery agent.
• the polynucleotides, primary constructs, or the mmR A may be encapsulated into a lipid nanoparticle or a rapidly eliminating lipid nanoparticle and the lipid nanoparticles or a rapidly eliminating lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art.
• the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer,
• surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).
• the lipid nanoparticle may be encapsulated into any polymer or hydrogel known in the art which may form a gel when injected into a subject.
• the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
• the polynucleotide, primary construct, or mmRNA formulation for controlled release and/or targeted delivery may also include at least one controlled release coating.
• Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer,
• polyvinylpyrrolidone hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and
• the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains.
• Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
• the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
• the polynucleotides, primary constructs, and/or the mmRNA of the present invention may be encapsulated in a therapeutic nanoparticle.
• Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645, US20100087337, US20100068285,
• therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, herein incorporated by reference in its entirety.
• the therapeutic nanoparticle of may be formulated for sustained release.
• sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years.
• the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the the polynucleotides, primary constructs, and mmRNA of the present invention (see International Pub No. 2010075072 and US Pub No. US20100216804 and
• the therapeutic nanoparticles may be formulated to be target specific.
• the thereapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO2011084518).
• the therapeutic nanoparticles may be formulated to be cancer specific.
• the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426,
• the nanoparticles of the present invention may comprise a polymeric matrix.
• the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters),
• polycyanoacrylates polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L- lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
• the diblock copolymer may include PEG in one embodiment
• polycarbonates polyanhydrides, polyhydroxyacids, polypropylfumerates,
• polycaprolactones polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L- lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
• the therapeutic nanoparticle comprises a diblock copolymer.
• the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, herein incorporated by reference in their entireties).
• the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, each of which is herein incorporated by reference in its entirety).
• the therapeutic nanoparticle may comprise at least one acrylic polymer.
• Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),
• the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.
• the therapeutic nanoparticles may comprise at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers and combinations thereof.
• the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains.
• Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L- lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
• the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
• the therapeutic nanoparticle may include a conjugation of at least one targeting ligand.
• the therapeutic nanoparticle may be formulated in an aqueous solution which may be used to target cancer (see International Pub No. WO2011084513 and US Pub No. US20110294717, each of which is herein incorporated by reference in their entirety).
• the polynucleotides, primary constructs, or mmRNA may be encapsulated in, linked to and/or associated with synthetic nanocarriers.
• the synthetic nanocarriers may be formulated using methods known in the art and/or described herein.
• the synthetic nanocarriers may be formulated by the methods described in International Pub Nos. WO2010005740, WO2010030763 and US Pub. Nos. US20110262491, US20100104645 and
• the synthetic nanocarrier formulations may be lyophilized by methods described in International Pub. No. WO2011072218 and US Pat No.
• the synthetic nanocarriers may contain reactive groups to release the polynucleotides, primary constructs and/or mmRNA described herein (see International Pub. No. WO20120952552 and US Pub No. US20120171229, each of which is herein incorporated by reference in their entirety).
• the synthetic nanocarriers may contain an immunostimulatory agent to enhance the immune response from delivery of the synthetic nanocarrier.
• the synthetic nanocarrier may comprise a Thl immunostimulatory agent which may enhance a Thl -based response of the immune system (see International Pub No. WO2010123569 and US Pub. No. US20110223201, each of which is herein incorporated by reference in its entirety).
• the synthetic nanocarriers may be formulated for targeted release.
• the synthetic nanocarrier is formulated to release the polynucleotides, primary constructs and/or mmRNA at a specified pH and/or after a desired time interval.
• the synthetic nanoparticle may be formulated to release the polynucleotides, primary constructs and/or mmRNA after 24 hours and/or at a pH of 4.5 (see International Pub. Nos. WO2010138193 and WO2010138194 and US Pub Nos. US20110020388 and US20110027217, each of which is herein incorporated by reference in their entireties).
• the synthetic nanocarriers may be formulated for controlled and/or sustained release of the polynucleotides, primary constructs and/or mmRNA described herein.
• the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Pub No. WO2010138192 and US Pub No.
• the polynucleotide, primary construct, and mmRNA of the invention can be formulated using natural and/or synthetic polymers.
• polymers which may be used for delivery include, but are not limited to, Dynamic POLYCONJUGATETM formulations from MIRUS® Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM (Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers.
• RONDELTM RNAi/Oligonucleotide Nanoparticle Delivery
• PHASERXTM pH responsive co-block polymers
• a non- limiting example of PLGA formulations include, but are not limited to, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
• PLGA injectable depots e.g., ELIGARD® which is formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
• the first of these delivery approaches uses dynamic polyconjugates and has been shown in vivo in mice to effectively deliver siRNA and silence endogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci U S A. 2007 104: 12982-12887).
• This particular approach is a multicomponent polymer system whose key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and ⁇ -acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (Rozema et al, Proc Natl Acad Sci U S A.
• asialoglycoprotein receptor-expressing hepatocytes to sinusoidal endothelium and Kupffer cells.
• Another polymer approach involves using trans ferrin-targeted cyclodextrin-containing polycation nanoparticles. These nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene product in transferrin receptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et al., Cancer Res.2005 65: 8984-8982) and siRNA formulated in these nanoparticles was well tolerated in non-human primates (Heidel et al., Proc Natl Acad Sci USA 2007 104:5715-21). Both of these delivery strategies incorporate rational approaches using both targeted delivery and endosomal escape mechanisms.
• the polymer formulation can permit the sustained or delayed release of the polynucleotide, primary construct, or mmRNA (e.g., following intramuscular or subcutaneous injection).
• the altered release profile for the polynucleotide, primary construct, or mmRNA can result in, for example, translation of an encoded protein over an extended period of time.
• the polymer formulation may also be used to increase the stability of the polynucleotide, primary construct, or mmRNA.
• Biodegradable polymers have been previously used to protect nucleic acids other than mmRNA from degradation and been shown to result in sustained release of payloads in vivo (Rozema et al, Proc Natl Acad Sci U S A. 2007 104:12982-12887; Sullivan et al, Expert Opin Drug Deliv. 2010 7: 1433-1446; Convertine et al,
• the pharmaceutical compositions may be sustained release formulations.
• the sustained release formulations may be for subcutaneous delivery.
• Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme
• surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).
• modified mRNA may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the modified mRNA in the PLGA microspheres while maintaining the integrity of the modified mRNA during the encapsulation process.
• EVAc are non-biodegradeable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications (e.g., extended release products Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a sustained release progesterone intrauterine deivce; transdermal delivery systems Testoderm, Duragesic and Selegiline; catheters).
• Poloxamer F-407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxyethylene- polyoxypropylene-polyoxyethylene having a low viscosity at temperatures less than 5°C and forms a solid gel at temperatures greater than 15°C.
• PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days.
• GELSITE® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic ineraction to provide a stabilizing effect.
• Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by, but not limited by, folate, transferrin, and N- acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 2011 12:2708- 2714; Rozema et al, Proc Natl Acad Sci U S A. 2007 104: 12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010 464: 1067-1070; each of which is herein incorporated by reference in its entirety).
• GalNAc N- acetylgalactosamine
• the polynucleotides, primary constructs and/or mmRNA of the invention may be formulated with or in a polymeric compound.
• the polymer may include at least one polymer such as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, linear biodegradable copolymer, poly[a-(4- aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cation-
• the polynucleotides, primary constructs and/or mmRNA of the invention may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No. 6,177,274 herein incorporated by reference in its entirety.
• the formulation may be used for transfecting cells in vitro or for in vivo delivery of the polynucleotides, primary constructs and/or mmRNA.
• the polynucleotides, primary constructs and/or mmRNA may be suspended in a solution or medium with a cationic polymer, in a dry pharmaceutical composition or in a solution that is capable of being dried as described in U.S. Pub. Nos. 20090042829 and 20090042825 each of which are herein incorporated by reference in their entireties.
• the polynucleotides, primary constructs or mmRNA of the invention may be formulated with a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, each of which is herein incorporated by reference in their entireties).
• the polynucleotides, primary constructs or mmRNA of the invention may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by reference in its entirety).
• a polyamine derivative may be used to deliver nucleic acids or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No. 20100260817 herein incorporated by reference in its entirety).
• a pharmaceutical composition may include the modified nucleic acids and mmRNA and the polyamine derivative described in U.S. Pub. No.
• the polynucleotides, primary constructs or mmRNA of the invention may be formulated with at least one acrylic polymer.
• Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid
• copolymers methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid),
• poly(methacrylic acid), polycyanoacrylates and combinations thereof are examples of poly(methacrylic acid), polycyanoacrylates and combinations thereof.
• the polynucleotides, primary constructs or mmRNA of the present invention may be formulated with at least one polymer described in International Publication Nos. WO2011115862, WO2012082574 and
• polynucleotides, primary constructs or mmRNA of the present invention may be formulated with a polymer of formula Z as described in WO2011115862, herein incorporated by reference in its entirety.
• polynucleotides, primary constructs or mmRNA may be formulated with a polymer of formula Z, Z' or Z" as described in WO2012082574 or
• WO2012068187 each of which are herein incorporated by reference in their entireties.
• the polymers formulated with the modified RNA of the present invention may be synthesized by the methods described in WO2012082574 or WO2012068187, each of which is herein incorporated by reference in their entireties.
• Formulations of polynucleotides, primary constructs or mmRNA of the invention may include at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers or combinations thereof.
• polynucleotides, primary constructs and/or mmRNA of the invention may be formulated in a pharmaceutical compound including a
• biodegradable cationic lipopolymer a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof.
• the biodegradable cationic lipopolymer may be made my methods known in the art and/or described in U.S. Pat. No. 6,696,038, U.S. App. Nos.
• the poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Pub. No. 20100004315, herein incorporated by reference in its entirety.
• the biodegradabale polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Pat. Nos.
• the linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,652,886.
• the PAGA polymer may be made using methods known in the art and/or as described in U.S. Pat. Nos. 6,217,912 herein incorporated by reference in its entirety.
• the PAGA polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines, polylactides and poly(lactide-co-glycolides).
• the biodegradable cross-linked cationic multi-block copolymers may be made my methods known in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 each of which is herein incorporated by reference in their entireties.
• the multi-block copolymers may be synthesized using linear
• polyethyleneimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines.
• the composition or pharmaceutical composition may be made by the methods known in the art, described herein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 each of which is herein incorporated by reference in their entireties.
• the polynucleotides, primary constructs, and mmRNA of the invention may be formulated with at least one degradable polyester which may contain polycationic side chains.
• Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
• the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
• the polymers described herein may be conjugated to a lipid-terminating PEG.
• PLGA may be conjugated to a lipid-terminating PEG forming PLGA-DSPE-PEG.
• PEG conjugates for use with the present invention are described in International Publication No. WO2008103276, herein incorporated by reference in its entirety.
• the polynucleotides, primary constructs and/or mmRNA described herein may be conjugated with another compound.
• conjugates are described in US Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties.
• the polynucleotides, primary constructs and/or mmRNA of the present invention may be conjugated with conjugates of formula 1-122 as described in US Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties.
• a gene delivery composition may include a nucleotide sequence and a poloxamer.
• the polynucleotide, primary construct and/or mmRNA of the present inveition may be used in a gene delivery composition with the poloxamer described in U.S. Pub. No. 20100004313.
• the polymer formulation of the present invention may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups.
• the polymer formulation may be contacted with a cationic lipopolymer using the methods described in U.S. Pub. No. 20090042829 herein incorporated by reference in its entirety.
• the cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine),
• DOGS diheptadecylamidoglycyl spermidine
• DDAB N,N-distearyl-N,N- dimethylammonium bromide
• DMRIE N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide
• DODAC N,N-dioleyl-N,N- dimethylammonium chloride
• the polynucleotide, primary construct, and mmRNA of the invention can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate.
• Components may be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-tuning of the nanoparticle so to delivery of the polynucleotide, primary construct and mmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller et al, Biomaterials. 2008 29: 1526-1532; DeKoker et al, Adv Drug Deliv Rev. 2011 63:748-761; Endres et al, Biomaterials. 2011 32:7721-7731; Su et al, Mol Pharm. 2011 Jun 6;8(3):774-87; herein incorporated by reference in its entirety).
• Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver polynucleotides, primary constructs and mmRNA in vivo.
• a lipid coated calcium phosphate nanoparticle which may also contain a targeting ligand such as anisamide, may be used to deliver the polynucleotide, primary construct and mmRNA of the present invention.
• a targeting ligand such as anisamide
• This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the siRNA.
• calcium phosphate with a PEG-polyanion block copolymer may be used to delivery polynucleotides, primary constructs and mmRNA (Kazikawa et al, J Contr Rel. 2004 97:345-356; Kazikawa et al, J Contr Rel. 2006 111 :368-370).
• a PEG-charge-conversional polymer (Pitella et al., Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle to deliver the polynucleotides, primary constructs and mmRNA of the present invention.
• the PEG- charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a polycation at acidic pH, thus enhancing endosomal escape.
• core-shell nanoparticles have additionally focused on a high- throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al, Proc Natl Acad Sci U S A. 2011 108: 12996-13001).
• the complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle.
• the core-shell nanoparticles may efficiently deliver siRNA to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.
• a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containg PEG may be used to delivery of the polynucleotide, primary construct and mmRNA of the present invention.
• a luciferease-expressing tumor it was determined that the lipid-polymer-lipid hybrid nanoparticle significantly suppressed luciferase expression, as compared to a conventional lipoplex (Shi et al, Angew Chem Int Ed. 2011 50:7027-7031).
• the polynucleotide, primary construct, and mmRNA of the invention can be formulated with peptides and/or proteins in order to increase transfection of cells by the polynucleotide, primary construct, or mmRNA.
• peptides such as, but not limited to, cell penetrating peptides and proteins and peptides that enable intracellular delivery may be used to deliver pharmaceutical formulations.
• pharmaceutical formulations of the present invention includes a cell-penetrating peptide sequence attached to polycations that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cell- penetrating peptides (see, e.g., Caron et al, Mol. Ther. 3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton FL, 2002); El-Andaloussi et al, Curr. Pharm. Des. 11(28):3597-611 (2003); and
• compositions can also be formulated to include a cell penetrating agent, e.g., liposomes, which enhance delivery of the compositions to the intracellular space
• a cell penetrating agent e.g., liposomes
• polynucleotides, primary constructs, and mmRNA of the invention may be complexed to peptides and/or proteins such as, but not limited to, peptides and/or proteins from Aileron Therapeutics (Cambridge, MA) and Permeon Biologies (Cambridge, MA) in order to enable intracellular delivery (Cronican et al, ACS Chem. Biol.
• the cell-penetrating polypeptide may comprise a first domain and a second domain.
• the first domain may comprise a supercharged polypeptide.
• the second domain may comprise a protein-binding partner.
• protein-binding partner includes, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides.
• the cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein- binding partner.
• the cell-penetrating polypeptide may be capable of being secreted from a cell where the polynucleotide, primary construct, or mmRNA may be introduced.
• Formulations of the including peptides or proteins may be used to increase cell transfection by the polynucleotide, primary construct, or mmRNA, alter the biodistribution of the polynucleotide, primary construct, or mmRNA (e.g., by targeting specific tissues or cell types), and/or increase the translation of encoded protein.
• the polynucleotide, primary construct, and mmRNA of the invention can be transfected ex vivo into cells, which are subsequently transplanted into a subject.
• the pharmaceutical compositions may include red blood cells to deliver modified RNA to liver and myeloid cells, virosomes to deliver modified RNA in virus-like particles (VLPs), and electroporated cells such as, but not limited to, from MAXCYTE® (Gaithersburg, MD) and from ERYTECH® (Lyon, France) to deliver modified RNA.
• red blood cells, viral particles and electroporated cells to deliver payloads other than mmRNA have been documented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133; Fang et al., Expert Opin Biol Ther. 2012 12:385-389; Hu et al, Proc Natl Acad Sci U S A. 2011 108: 10980- 10985; Lund et al, Pharm Res. 2010 27:400-420; Huckriede et al, J Liposome Res. 2007;17:39-47; Cusi, Hum Vaccin. 2006 2: 1-7; de Jonge et al, Gene Ther. 2006 13:400-411; all of which are herein incorporated by reference in its entirety).
• polynucleotides, primary constructs and mmRNA may be delivered in synthetic VLPs synthesized by the methods described in International Pub No.
• Cell-based formulations of the polynucleotide, primary construct, and mmRNA of the invention may be used to ensure cell transfection (e.g., in the cellular carrier), alter the biodistribution of the polynucleotide, primary construct, or mmRNA (e.g., by targeting the cell carrier to specific tissues or cell types), and/or increase the translation of encoded protein.
• nucleic acid A variety of methods are known in the art and suitable for introduction of nucleic acid into a cell, including viral and non- viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to,
• microprojectile mediated transfer nanoparticles
• cationic polymer mediated transfer PEE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like
• cell fusion cell fusion
• Sonoporation or cellular sonication
• sound e.g., ultrasonic frequencies
• Sonoporation methods are known to those in the art and are used to deliver nucleic acids in vivo (Yoon and Park, Expert Opin Drug Deliv. 2010 7:321- 330; Postema and Gilja, Curr Pharm Biotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 2007 14:465-475; all herein incorporated by reference in their entirety).
• Electroporation techniques are also well known in the art and are used to deliver nucleic acids in vivo and clinically (Andre et al., Curr Gene Ther. 2010 10:267-280; Chiarella et al, Curr Gene Ther. 2010 10:281-286; Hojman, Curr Gene Ther. 2010 10: 128-138; all herein incorporated by reference in their entirety).
• polynucleotides, primary constructs or mmRNA may be delivered by electroporation as described in Example 26.
• the intramuscular or subcutaneous localized injection of polynucleotide, primary construct, or mmRNA of the invention can include hyaluronidase, which catalyzes the hydrolysis of hyaluronan.
• hyaluronidase catalyzes the hydrolysis of hyaluronan.
• hyaluronidase By catalyzing the hydrolysis of hyaluronan, a constituent of the interstitial barrier, hyaluronidase lowers the viscosity of hyaluronan, thereby increasing tissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440; herein incorporated by reference in its entirety). It is useful to speed their dispersion and systemic distribution of encoded proteins produced by transfected cells.
• the hyaluronidase can be used to increase the number of cells exposed to a polynucleotide, primary construct, or mmRNA of the invention administered intramus
• the polynucleotide, primary construct or mmRNA of the invention may be encapsulated within and/or absorbed to a nanoparticle mimic.
• a nanoparticle mimic can mimic the delivery function organisms or particles such as, but not limited to, pathogens, viruses, bacteria, fungus, parasites, prions and cells.
• the polynucleotide, primary construct or mmRNA of the invention may be encapsulated in a non-viron particle which can mimic the delivery function of a virus (see International Pub. No. WO2012006376 herein incorporated by reference in its entirety).
• the polynucleotides, primary constructs or mmRNA of the invention can be attached or otherwise bound to at least one nanotube such as, but not limited to, rosette nanotubes, rosette nanotubes having twin bases with a linker, carbon nanotubes and/or single-walled carbon nanotubes,
• the polynucleotides, primary constructs or mmRNA may be bound to the nanotubes through forces such as, but not limited to, steric, ionic, covalent and/or other forces.
• the nanotube can release one or more polynucleotides, primary constructs or mmRNA into cells.
• the size and/or the surface structure of at least one nanotube may be altered so as to govern the interaction of the nanotubes within the body and/or to attach or bind to the polynucleotides, primary constructs or mmRNA disclosed herein.
• the building block and/or the functional groups attached to the building block of the at least one nanotube may be altered to adjust the dimensions and/or properties of the nanotube.
• the length of the nanotubes may be altered to hinder the nanotubes from passing through the holes in the walls of normal blood vessels but still small enough to pass through the larger holes in the blood vessels of tumor tissue.
• At least one nanotube may also be coated with delivery enhancing compounds including polymers, such as, but not limited to, polyethylene glycol.
• delivery enhancing compounds including polymers, such as, but not limited to, polyethylene glycol.
• at least one nanotube and/or the polynucleotides, primary constructs or mmRNA may be mixed with pharmaceutically acceptable excipients and/or delivery vehicles.
• the polynucleotides, primary constructs or mmRNA are attached and/or otherwise bound to at least one rosette nanotube.
• the rosette nanotubes may be formed by a process known in the art and/or by the process described in International Publication No. WO2012094304, herein incorporated by reference in its entirety.
• At least one polynucleotide, primary construct and/or mmRNA may be attached and/or otherwise bound to at least one rosette nanotube by a process as described in International Publication No.
• rosette nanotubes or modules forming rosette nanotubes are mixed in aqueous media with at least one polynucleotide, primary construct and/or mmRNA under conditions which may cause at least one polynucleotide, primary construct or mmRNA to attach or otherwise bind to the rosette nanotubes.
• the polynucleotides, primary constructs, and mmRNA of the invention include conjugates, such as a polynucleotide, primary construct, or mmRNA covalently linked to a carrier or targeting group, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting group and therapeutic protein or peptide).
• conjugates such as a polynucleotide, primary construct, or mmRNA covalently linked to a carrier or targeting group, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting group and therapeutic protein or peptide).
• the conjugates of the invention include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid.
• the ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g. an aptamer).
• polyamino acids examples include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co- glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N- isopropylacrylamide polymers, or polyphosphazine.
• polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine,
• pseudopeptide-polyamine peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
• the conjugate of the present invention may function as a carrier for the polynucleotides, primary constructs and/or mmRNA of the present invention.
• the conjugate may comprise a cationic polymer such as, but not limited to, polyamine, polylysine, polyalkylenimine, and polyethylenimine which may be grafted to with poly(ethylene glycol).
• the conjugate may be similar to the polymeric conjugate and the method of synthesizing the polymeric conjugate described in U.S. Pat. No. 6,586,524 herein incorporated by reference in its entirety.
• the conjugates can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
• a cell or tissue targeting agent e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
• a targeting group can be a 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.
• Targeting groups can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
• Targeting groups may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N- acetyl-gulucosamine multivalent mannose, multivalent fucose, or aptamers.
• the ligand can be, for example, a lipopolysaccharide, or an activator of p38 MAP kinase.
• the targeting group can be any ligand that is capable of targeting a specific receptor. Examples include, without limitation, folate, GalNAc, galactose, mannose, mannose-6P, apatamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII,
• the targeting group is an aptamer.
• the aptamer can be unmodified or have any combination of
• compositions of the present invention may include chemical modifications such as, but not limited to, modifications similar to locked nucleic acids.
• LNA locked nucleic acid
• Some embodiments featured in the invention include polynucleotides, primary constructs or mrnRNA with phosphorothioate backbones and
• oligonucleosides with other modified backbones and in particular ⁇ CH 2 ⁇ NH ⁇ CH 2 ⁇ , ⁇ CH2 ⁇ N(CH3) ⁇ 0 ⁇ CH2 ⁇ [known as a methylene (methylimino) or MMI backbone], -CH 2 ⁇ 0-N(CH3) ⁇ CH 2 ⁇ , -CH 2 --N(CH3) ⁇ N(CH 3 ) ⁇ CH 2 -- and -N(CH 3 )-CH 2 - CH 2 ⁇ [wherein the native phosphodiester backbone is represented as— O— P(0) 2 — O— CH2— ] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240.
• the native phosphodiester backbone is represented as— O— P(0) 2 — O— CH2— ] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbone
• polynucletotides featured herein have morpholino backbone structures of the above- referenced U.S. Pat. No. 5,034,506.
• Modifications at the 2' position may also aid in delivery.
• modifications at the 2' position are not located in a polypeptide-coding sequence, i.e., not in a translatable region.
• Modifications at the 2' position may be located in a 5'UTR, a 3'UTPv and/or a tailing region.
• Modifications at the 2' position can include one of the following at the 2' position: H (i.e., 2'-deoxy); F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C10 alkyl or C2 to C10 alkenyl and alkynyl.
• Exemplary suitable modifications include 0[(CH 2 )nO] m CH3,
• the polynucleotides, primary constructs or mmRNA include one of the following at the 2' position: Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ONO2, NO2, N 3 , NH2, heterocycloalkyl, heterocycloalkaryl,
• the modification includes a 2'-methoxyethoxy (2'-0 ⁇ CH2CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl) or 2'- MOE) (Martin et al, Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
• 2'-dimethylaminooxyethoxy i.e., a 0(CH 2 ) 2 ON(CFI 3 )2 group, also known as 2'-DMAOE, as described in examples herein below
• 2'-dimethylaminoethoxyethoxy also known in the art as 2'-0- dimethylaminoethoxyethyl or 2*-DMAEOE
• 2*-0 ⁇ CH2 ⁇ 0 ⁇ CH2 ⁇ N(CH 2 )2 also described in examples herein below.
• the polynucleotide, primary construct, or mmRNA is covalently conjugated to a cell penetrating polypeptide.
• the cell- penetrating peptide may also include a signal sequence.
• the conjugates of the invention can be designed to have increased stability; increased cell transfection; and/or altered the biodistribution (e.g., targeted to specific tissues or cell types). Self-Assembled Nucleic Acid Nanoparticles
• Self-assembled nanoparticles have a well-defined size which may be precisely controlled as the nucleic acid strands may be easily reprogrammable.
• the optimal particle size for a cancer-targeting nanodelivery carrier is 20- 100 nm as a diameter greater than 20 nm avoids renal clearance and enhances delivery to certain tumors through enhanced permeability and retention effect.
• oligonucleotide nanoparticles are prepared using programmable self-assembly of short DNA fragments and therapeutic siRNAs. These nanoparticles are molecularly identical with controllable particle size and target ligand location and density. The DNA fragments and siRNAs self-assembled into a one-step reaction to generate
• compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
• a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
• Remington's The Science and Practice of Pharmacy 21 st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in
• a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
• an excipient is approved for use in humans and for veterinary use.
• an excipient is approved by United States Food and Drug Administration.
• an excipient is pharmaceutical grade.
• an excipient meets the standards of the United States
• compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
• Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
• Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross- linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), micro crystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM ® ), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
• crospovidone cross- linked poly(vinyl-pyrrolidone)
• Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM ® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
• natural emulsifiers e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
• colloidal clays e.g. bentonite [aluminum si
• stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol
• carbomers e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer
• carrageenan cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan
• polyoxyethylene esters e.g. polyoxyethylene monostearate [MYRJ ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL ®
• sucrose fatty acid esters e.g. CREMOPHOR ®
• polyoxyethylene ethers e.g.
• polyoxyethylene lauryl ether [BRIJ ® 30]), polyvinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC ® F 68, POLOXAMER ® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
• Exemplary binding agents include, but are not limited to, starch (e.g.
• cornstarch and starch paste cornstarch and starch paste
• gelatin e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g.
• acacia sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum ® ), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol;
• inorganic calcium salts silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
• Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
• Exemplary antioxidants antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
• antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite.
• Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
• Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,
• antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
• Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
• Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
• preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS ® , PHENONIP ® , methylparaben, GERM ALL ® 115,
• GERMABEN ® II NEOLONE TM , KATHON TM , and/or EUXYL ® .
• Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,
• Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
• Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana
• oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
• Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
• the present disclosure encompasses the delivery of polynucleotides, primary constructs or mmRNA for any of therapeutic, pharmaceutical, diagnostic or imaging by any appropriate route taking into consideration likely advances in the sciences of drug delivery. Delivery may be naked or formulated.
• the polynucleotides, primary constructs or mmRNA of the present invention may be delivered to a cell naked.
• naked refers to delivering polynucleotides, primary constructs or mmRNA free from agents which promote transfection.
• the polynucleotides, primary constructs or mmRNA delivered to the cell may contain no modifications.
• polynucleotides, primary constructs or mmRNA may be delivered to the cell using routes of administration known in the art and described herein.
• the polynucleotides, primary constructs or mmRNA of the present invention may be formulated, using the methods described herein.
• the formulations may contain polynucleotides, primary constructs or mmRNA which may be modified and/or unmodified.
• the formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot.
• the formulated polynucleotides, primary constructs or mmRNA may be delivered to the cell using routes of administration known in the art and described herein.
• compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.
• the polynucleotides, primary constructs or mmRNA of the present invention may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, ( into the base of the penis), intravaginal administration, intrauter
• compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier .
• Non-limiting routes of administration for the polynucleotides, primary constructs or mmRNA of the present invention are described below.
• Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
• liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
• oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
• adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
• compositions are mixed with solubilizing agents such as CREMOPHOR ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or
• Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents.
• Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol.
• the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P., and isotonic sodium chloride solution.
• Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil can be employed including synthetic mono- or diglycerides.
• Fatty acids such as oleic acid can be used in the preparation of injectables.
• Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
• the rate of drug release can be controlled.
• biodegradable polymers include poly(orthoesters) and poly(anhydrides).
• Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
• compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
• suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
• an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g.
• the dosage form may comprise buffering agents.
• solution retarding agents e.g. paraffin
• absorption accelerators e.g. quaternary ammonium compounds
• wetting agents e.g. cetyl alcohol and glycerol monostearate
• absorbents e.g. kaolin and bentonite clay
• lubricants e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate
• the dosage form may comprise buffering agents.
• compositions containing the polynucleotides, primary constructs or mmRNA of the invention may be formulated for administration topically.
• the skin may be an ideal target site for delivery as it is readily accessible. Gene expression may be restricted not only to the skin, potentially avoiding nonspecific toxicity, but also to specific layers and cell types within the skin.
• the site of cutaneous expression of the delivered compositions will depend on the route of nucleic acid delivery.
• Three routes are commonly considered to deliver polynucleotides, primary constructs or mmRNA to the skin: (i) topical application (e.g. for local/regional treatment and/or cosmetic applications); (ii) intradermal injection (e.g. for local/regional treatment and/or cosmetic applications); and (iii) systemic delivery (e.g. for treatment of dermatologic diseases that affect both cutaneous and extracutaneous regions), polynucleotides, primary constructs or mmRNA can be delivered to the skin by several different approaches known in the art.
• the invention provides for a variety of dressings (e.g., wound dressings) or bandages (e.g., adhesive bandages) for conveniently and/or effectively carrying out methods of the present invention.
• dressing or bandages may comprise sufficient amounts of pharmaceutical compositions and/or polynucleotides, primary constructs or mmRNA described herein to allow a user to perform multiple treatments of a subject(s).
• the invention provides for the polynucleotides, primary constructs or mmRNA compositions to be delivered in more than one injection.
• tissue such as skin
• a device and/or solution which may increase permeability.
• the tissue may be subjected to an abrasion device to increase the permeability of the skin (see U.S. Patent Publication No. 20080275468, herein incorporated by reference in its entirety).
• the tissue may be subjected to an ultrasound enhancement device.
• An ultrasound enhancement device may include, but is not limited to, the devices described in U.S. Publication No. 20040236268 and U.S. Patent Nos. 6,491,657 and 6,234,990; each of which is herein incorporated by reference in their entireties.
• a device may be used to increase permeability of tissue before delivering formulations of the polynucleotides, primary constructs and mmRNA described herein.
• the permeability of skin may be measured by methods known in the art and/or described in U.S. Patent No. 6,190,315, herein incorporated by reference in its entirety.
• formulation may be delivered by the drug delivery methods described in U.S. Patent No. 6,190,315, herein incorporated by reference in its entirety.
• tissue may be treated with a eutectic mixture of local anesthetics (EMLA) cream before, during and/or after the tissue may be subjected to a device which may increase permeability.
• EMLA local anesthetics
• enhancers may be applied to the tissue before, during, and/or after the tissue has been treated to increase permeability.
• Enhancers include, but are not limited to, transport enhancers, physical enhancers, and cavitation enhancers. Non-limiting examples of enhancers are described in U.S. Patent No. 6,190,315, herein incorporated by reference in its entirety.
• a device may be used to increase permeability of tissue before delivering formulations of polynucleotides, primary constructs and/or mmR A described herein, which may further contain a substance that invokes an immune response.
• a formulation containing a substance to invoke an immune response may be delivered by the methods described in U.S. Publication Nos. 20040171980 and 20040236268; each of which is herein incorporated by reference in their entirety.
• Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches.
• an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.
• the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body.
• dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium.
• rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.
• Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
• Topically-administrable formulations may, for example, comprise from about 0.1% to about 10% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
• Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
• the composition is formulated in depots for extended release.
• a specific organ or tissue a "target tissue" is targeted for administration.
• the polynucleotides, primary constructs or mmR A are spatially retained within or proximal to a target tissue.
• compositions to a target tissue of a mammalian subject by contacting the target tissue (which contains one or more target cells) with the composition under conditions such that the composition, in particular the nucleic acid component(s) of the composition, is substantially retained in the target tissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the composition is retained in the target tissue.
• retention is determined by measuring the amount of the nucleic acid present in the composition that enters one or more target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the nucleic acids administered to the subject are present.
• intracellularly at a period of time following administration.
• intramuscular injection to a mammalian subject is performed using an aqueous composition containing a ribonucleic acid and a transfection reagent, and retention of the composition is determined by measuring the amount of the ribonucleic acid present in the muscle cells.
• aspects of the invention are directed to methods of providing a composition to a target tissue of a mammalian subject, by contacting the target tissue (containing one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue.
• the composition contains an effective amount of a polynucleotide, primary construct or mmRNA such that the polypeptide of interest is produced in at least one target cell.
• the compositions generally contain a cell penetration agent, although "naked" nucleic acid (such as nucleic acids without a cell penetration agent or other agent) is also contemplated, and a pharmaceutically acceptable carrier.
• the amount of a protein produced by cells in a tissue is desirably increased.
• this increase in protein production is spatially restricted to cells within the target tissue.
• a composition is provided that contains polynucleotides, primary constructs or mmRNA characterized in that a unit quantity of composition has been determined to produce the polypeptide of interest in a substantial percentage of cells contained within a predetermined volume of the target tissue.
• the composition includes a plurality of different polynucleotides, primary constructs or mmRNA, where one or more than one of the polynucleotides, primary constructs or mmRNA encodes a polypeptide of interest.
• the composition also contains a cell penetration agent to assist in the intracellular delivery of the composition. A determination is made of the dose of the composition required to produce the polypeptide of interest in a substantial percentage of cells contained within the predetermined volume of the target tissue (generally, without inducing significant production of the polypeptide of interest in tissue adjacent to the predetermined volume, or distally to the target tissue).
• the determined dose is introduced directly into the tissue of the mammalian subject.
• the invention provides for the polynucleotides, primary constructs or mmRNA to be delivered in more than one injection or by split dose injections.
• the invention may be retained near target tissue using a small disposable drug reservoir or patch pump.
• patch pumps include those manufactured and/or sold by BD® (Franklin Lakes, NJ), Insulet Corporation (Bedford, MA), SteadyMed Therapeutics (San Francisco, CA),
• a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity.
• a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm.
• Such compositions are suitably in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
• Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm.
• Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
• Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50%> to 99.9% (w/w) of the composition, and active ingredient may constitute 0.1% to 20% (w/w) of the composition. A propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
• additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
• compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension.
• Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device.
• Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate.
• Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.
• Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition.
• Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 ⁇ to 500 ⁇ . Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
• Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
• a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration.
• Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, 0.1% to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
• formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
• Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
• a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration.
• Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0%) (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient.
• Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein.
• Other ophthalmically- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this invention.
• polynucleotides, primary constructs or mmRNA described herein can be used in a number of different scenarios in which delivery of a substance (the "payload") to a biological target is desired, for example delivery of detectable substances for detection of the target, or delivery of a therapeutic agent.
• Detection methods can include, but are not limited to, both imaging in vitro and in vivo imaging methods, e.g., immunohistochemistry, bioluminescence imaging (BLI), Magnetic Resonance Imaging (MRI), positron emission tomography (PET), electron microscopy, X-ray computed tomography, Raman imaging, optical coherence tomography, absorption imaging, thermal imaging, fluorescence reflectance imaging, fluorescence microscopy, fluorescence molecular tomographic imaging, nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging, photoacoustic imaging, lab assays, or in any situation where tagging/staining/imaging is required.
• imaging in vitro and in vivo imaging methods e.g., immunohistochemistry, bioluminescence imaging (BLI), Magnetic Resonance Imaging (MRI), positron emission tomography (PET), electron microscopy, X-ray computed tomography, Raman imaging, optical coherence tomography, absorption imaging, thermal imaging
• the polynucleotides, primary constructs or mmRNA can be designed to include both a linker and a payload in any useful orientation.
• a linker having two ends is used to attach one end to the payload and the other end to the nucleobase, such as at the C-7 or C-8 positions of the deaza-adenosine or deaza- guanosine or to the N-3 or C-5 positions of cytosine or uracil.
• the polynucleotide of the invention can include more than one payload (e.g., a label and a transcription inhibitor), as well as a cleavable linker.
• the modified nucleotide is a modified 7-deaza-adenosine triphosphate, where one end of a cleavable linker is attached to the C7 position of 7-deaza-adenine, the other end of the linker is attached to an inhibitor (e.g., to the C5 position of the nucleobase on a cytidine), and a label (e.g., Cy5) is attached to the center of the linker (see, e.g., compound 1 of A*pCp C5 Parg Capless in Fig. 5 and columns 9 and 10 of U.S. Pat. No. 7,994,304, incorporated herein by reference).
• an inhibitor e.g., to the C5 position of the nucleobase on a cytidine
• a label e.g., Cy5
• the resulting polynucleotide Upon incorporation of the modified 7-deaza-adenosine triphosphate to an encoding region, the resulting polynucleotide will have a cleavable linker attached to a label and an inhibitor (e.g., a polymerase inhibitor).
• an inhibitor e.g., a polymerase inhibitor
• the linker e.g., with reductive conditions to reduce a linker having a cleavable disulfide moiety
• the label and inhibitor are released. Additional linkers and payloads (e.g., therapeutic agents, detectable labels, and cell penetrating payloads) are described herein.
• the polynucleotides, primary constructs or mmRNA described herein can be used in induced pluripotent stem cells (iPS cells), which can directly track cells that are transfected compared to total cells in the cluster.
• iPS cells induced pluripotent stem cells
• a drug that may be attached to the polynucleotides, primary constructs or mmRNA via a linker and may be fluorescently labeled can be used to track the drug in vivo, e.g. intracellularly.
• Other examples include, but are not limited to, the use of a polynucleotide, primary construct or mmRNA in reversible drug delivery into cells.
• the polynucleotides, primary constructs or mmRNA described herein can be used in intracellular targeting of a payload, e.g. , detectable or therapeutic agent, to specific organelle.
• exemplary intracellular targets can include, but are not limited to, the nuclear localization for advanced mRNA processing, or a nuclear localization sequence (NLS) linked to the mRNA containing an inhibitor.
• NLS nuclear localization sequence
• polynucleotides, primary constructs or mmRNA described herein can be used to deliver therapeutic agents to cells or tissues, e.g. , in living animals.
• the polynucleotides, primary constructs or mmRNA attached to the therapeutic agent through a linker can facilitate member permeation allowing the therapeutic agent to travel into a cell to reach an intracellular target.
• the payload may be a therapeutic agent such as a cytotoxin, radioactive ion, chemotherapeutic, or other therapeutic agent.
• a cytotoxin or cytotoxic agent includes any agent that may be detrimental to cells.
• Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No.
• Radioactive ions include, but are not limited to iodine ⁇ e.g., iodine 125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium.
• Other therapeutic agents include, but are not limited to,
• antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine
• alkylating agents e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g.,
• dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
• anti-mitotic agents e.g., vincristine, vinblastine, taxol
• the payload may be a detectable agent, such as various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials (e.g., 18 F, 67 Ga, 81m Kr, 82 Rb, m In, 123 I, 133 Xe, 201 T1, 125 1, 35 S, 14 C, 3 H, or 99m Tc (e.g., as pertechnetate (technetate(VII), TcO-f)), and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticle
• fluorescent materials e.g.
• optically-detectable labels include for example, without limitation, 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives (e.g., acridine and acridine
• isothiocyanate 5-(2'-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4- amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-l- naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives (e.g., coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), and 7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes; cyanosine; 4 ',6- diaminidino-2-phenylindole (DAPI); 5' 5"-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyan
• orthocresolphthalein nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o- phthaldialdehyde; pyrene and derivatives(e.g., pyrene, pyrene butyrate, and succinimidyl 1 -pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRONTM Brilliant Red 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of
• sulforhodamine 101 (Texas Red), ⁇ , ⁇ , ⁇ ', ⁇ 'tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolic acid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5); cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.
• TAMRA ⁇ , ⁇ , ⁇ ', ⁇ 'tetramethyl-6-carboxyrhodamine
• TRITC tetramethyl rhodamine isothiocyanate
• riboflavin riboflavin
• rosolic acid terbium
• the detectable agent may be a non-detectable precursor that becomes detectable upon activation (e.g., fluorogenic tetrazine- fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenic agents (e.g., fluorogenic tetrazine- fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenic agents (e.g., fluorogenic tetrazine- fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY T
• ELISAs immunosorbent assays
• EIA enzyme immunoassays
• RIA radioimmunoassays
• polynucleotides, primary constructs or mmRNA may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents.
• combination with it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure.
• Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be
• the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
• the polynucleotides, primary constructs and/or mmRNA may be used in combination with a pharmaceutical agent for the treatment of cancer or to control hyperproliferative cells.
• metastasized tumor is described using a pharmaceutical composition including a DNA plasmid encoding for interleukin-12 with a lipopolymer and also administering at least one anticancer agent or chemotherapeutic.
• the polynucleotides, primary constructs and/or mmRNA of the present invention that encodes antiproliferative molecules may be in a pharmaceutical composition with a lipopolymer (see e.g., U.S. Pub. No. 20110218231, herein incorporated by reference in its entirety, claiming a pharmaceutical composition comprising a DNA plasmid encoding an antiproliferative molecule and a lipopolymer) which may be administered with at least one chemotherapeutic or anticancer agent.
• the present invention provides methods comprising administering polynucleotides, primary constructs and/or mmRNA and their encoded proteins or complexes in accordance with the invention to a subject in need thereof, nucleic acids, proteins or complexes, or pharmaceutical, imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and/or condition (e.g., a disease, disorder, and/or condition relating to working memory deficits).
• a disease, disorder, and/or condition e.g., a disease, disorder, and/or condition relating to working memory deficits.
• the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
• compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
• compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect.
• the desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
• the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
• a split dose is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose.
• a "single unit dose” is a dose of any therapeutic administed in one dose/at one time/single route/single point of contact, i.e., single administration event.
• a "total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.
• the mmRNA of the present invention are administed to a subject in split doses.
• the mmRNA may be formulated in buffer only or in a formulation described herein.
• a pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).
• injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous.
• Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
• liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art including, but not limited to,
• compositions may be mixed with solubilizing agents such as CREMOPHOR ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
• solubilizing agents such as CREMOPHOR ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
• Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents.
• Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol.
• the acceptable vehicles and solvents that may be employed include, but are not limited to, are water, Ringer's solution, U.S. P., and isotonic sodium chloride solution.
• Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil can be employed including synthetic mono- or diglycerides.
• Fatty acids such as oleic acid can be used in the preparation of injectables.
• Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
• delayed absorption of a parenterally administered polynucleotide, primary construct or mmRNA may be accomplished by dissolving or suspending the polynucleotide, primary construct or mmRNA in an oil vehicle.
• injectable depot forms are made by forming microencapsule matrices of the polynucleotide, primary construct or mmRNA in biodegradable polymers such as polylactide-polyglycolide.
• the rate of polynucleotide, primary construct or mmRNA release can be controlled.
• biodegradable polymers examples include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the polynucleotide, primary construct or mmRNA in liposomes or microemulsions which are compatible with body tissues.
• Formulations described herein as being useful for pulmonary delivery may also be use for intranasal delivery of a pharmaceutical composition.
• Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 ⁇ to 500 ⁇ .
• Such a formulation may be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
• Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
• a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration.
• Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
• formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
• Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
• Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active
• embedding compositions which can be used include polymeric substances and waxes.
• Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• compositions described herein can be characterized by one or more of bioavailability, therapeutic window and/or volume of distribution.
• the polynucleotides, primary constructs or mmRNA when formulated into a composition with a delivery agent as described herein, can exhibit an increase in bioavailability as compared to a composition lacking a delivery agent as described herein.
• bioavailability refers to the systemic availability of a given amount of polynucleotides, primary constructs or mmRNA administered to a mammal. Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration (Cmax) of the unchanged form of a compound following administration of the compound to a mammal.
• AUC is a determination of the area under the curve plotting the serum or plasma concentration of a compound along the ordinate (Y-axis) against time along the abscissa (X-axis).
• the AUC for a particular compound can be calculated using methods known to those of ordinary skill in the art and as described in G. S. Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, New York, Inc., 1996, herein incorporated by reference.
• the Cmax value is the maximum concentration of the compound achieved in the serum or plasma of a mammal following administration of the compound to the mammal.
• the Cmax value of a particular compound can be measured using methods known to those of ordinary skill in the art.
• phrases "increasing bioavailability” or “improving the pharmacokinetics,” as used herein mean that the systemic availability of a first polynucleotide, primary construct or mmRNA, measured as AUC, C max, Or Lmin in a mammal is greater, when co-administered with a delivery agent as described herein, than when such co-administration does not take place.
• the bioavailability of the polynucleotide, primary construct or mmRNA can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%o, at least about 60%>, at least about 65%, at least about 70%>, at least about 75%, at least about 80%>, at least about 85%, at least about 90%>, at least about 95%, or about 100%.
• the polynucleotides, primary constructs or mmRNA when formulated into a composition with a delivery agent as described herein, can exhibit an increase in the therapeutic window of the administered polynucleotide, primary construct or mmRNA composition as compared to the therapeutic window of the administered polynucleotide, primary construct or mmRNA composition lacking a delivery agent as described herein.
• therapeutic window refers to the range of plasma concentrations, or the range of levels of therapeutically active substance at the site of action, with a high probability of eliciting a therapeutic effect.
• the therapeutic window of the polynucleotide, primary construct or mmRNA when co-administered with a delivery agent as described herein can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%), at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%), at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
• the polynucleotides, primary constructs or mmR A when formulated into a composition with a delivery agent as described herein, can exhibit an improved volume of distribution (Vdist), e.g., reduced or targeted, relative to a composition lacking a delivery agent as described herein.
• the volume of distribution (Vdist) relates the amount of the drug in the body to the concentration of the drug in the blood or plasma.
• volume of distribution refers to the fluid volume that would be required to contain the total amount of the drug in the body at the same concentration as in the blood or plasma: Vdist equals the amount of drug in the body/concentration of drug in blood or plasma.
• the volume of distribution would be 1 liter.
• the volume of distribution reflects the extent to which the drug is present in the extravascular tissue.
• a large volume of distribution reflects the tendency of a compound to bind to the tissue components compared with plasma protein binding.
• Vdist can be used to determine a loading dose to achieve a steady state concentration.
• the volume of distribution of the polynucleotide, primary construct or mmRNA when co-administered with a delivery agent as described herein can decrease at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%), at least about 35%, at least about 40%>, at least about 45%, at least about 50%>, at least about 55%, at least about 60%>, at least about 65%, at least about 70%.
• the biological effect of the modified mRNA delivered to the animals may be categorized by analyzing the protein expression in the animals.
• the reprogrammed protein expression may be determined from analyzing a biological sample collected from a mammal administered the modified mRNA of the present invention.
• the expression protein encoded by the modified mRNA administered to the mammal of at least 50 pg/ml may be preferred.
• a protein expression of 50-200 pg/ml for the protein encoded by the modified mRNA delivered to the mammal may be seen as a therapeutically effective amount of protein in the mammal.
• Mass spectrometry is an analytical technique that can provide structural and molecular mass/concentration information on molecules after their conversion to ions.
• the molecules are first ionized to acquire positive or negative charges and then they travel through the mass analyzer to arrive at different areas of the detector according to their mass/charge (m/z) ratio.
• Mass spectrometry is performed using a mass spectrometer which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis.
• ionization of the sample may be performed by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization.
• ESI electrospray ionization
• APCI atmospheric pressure chemical ionization
• FAB fast atom bombardment
• LIMS liquid secondary ionization
• MALDI matrix assisted laser desorption/ionization
• field ionization field desorption
• thermospray/plasmaspray ionization and particle beam ionization.
• the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio (i.e., m/z).
• Suitable analyzers for determining mass-to-charge ratios include quadropole analyzers, ion traps analyzers, and time-of-flight analyzers.
• the ions may be detected using several detection modes. For example, selected ions may be detected (i.e., using a selective ion monitoring mode (SIM)), or alternatively, ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).
• SIM selective ion monitoring mode
• MRM multiple reaction monitoring
• SRM selected reaction monitoring
• LC-MS/MRM Liquid chromatography-multiple reaction monitoring
• MRM multiple reaction monitoring
• a biological sample which may contain at least one protein encoded by at least one modified mR A of the present invention may be analyzed by the method of MRM-MS.
• the quantification of the biological sample may further include, but is not limited to, isotopically labeled peptides or proteins as internal standards.
• the biological sample once obtained from the subject, may be subjected to enzyme digestion.
• digest means to break apart into shorter peptides.
• the phrase "treating a sample to digest proteins” means manipulating a sample in such a way as to break down proteins in a sample.
• enzymes include, but are not limited to, trypsin, endoproteinase Glu-C and chymotrypsin.
• a biological sample which may contain at least one protein encoded by at least one modified mRNA of the present invention may be digested using enzymes.
• a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed for protein using electrospray ionization.
• Electrospray ionization (ESI) mass spectrometry (ESIMS) uses electrical energy to aid in the transfer of ions from the solution to the gaseous phase before they are analyzed by mass spectrometry.
• Samples may be analyzed using methods known in the art (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12).
• the ionic species contained in solution may be transferred into the gas phase by dispersing a fine spray of charge droplets, evaporating the solvent and ejecting the ions from the charged droplets to generate a mist of highly charged droplets.
• the mist of highly charged droplets may be analyzed using at least 1, at least 2, at least 3 or at least 4 mass analyzers such as, but not limited to, a quadropole mass analyzer.
• the mass spectrometry method may include a purification step.
• the first quadrapole may be set to select a single m/z ratio so it may filter out other molecular ions having a different m/z ratio which may eliminate complicated and time-consuming sample purification procedures prior to MS analysis.
• a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed for protein in a tandem ESIMS system (e.g., MS/MS).
• the droplets may be analyzed using a product scan (or daughter scan) a precursor scan (parent scan) a neutral loss or a multiple reaction monitoring.
• a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MALDIMS).
• MALDI matrix-assisted laser desorption/ionization
• MALDIMS matrix-assisted laser desorption/ionization mass spectrometry
• MALDI provides for the nondestructive vaporization and ionization of both large and small molecules, such as proteins.
• the analyte is first co-crystallized with a large molar excess of a matrix compound, which may also include, but is not limited to, an ultraviolet absorbing weak organic acid.
• Non-limiting examples of matrices used in MALDI are a-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4- hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.
• Laser radiation of the analyte- matrix mixture may result in the vaporization of the matrix and the analyte.
• the laser induced desorption provides high ion yields of the intact analyte and allows for measurement of compounds with high accuracy.
• Samples may be analyzed using methods known in the art (e.g., Lewis, Wei and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894).
• mass analyzers used in the MALDI analysis may include a linear time-of-flight (TOF), a TOF reflectron or a Fourier transform mass analyzer.
• the analyte -matrix mixture may be formed using the dried-droplet method.
• a biologic sample is mixed with a matrix to create a saturated matrix solution where the matrix-to-sample ratio is approximately 5000: 1.
• An aliquot (approximately 0.5-2.0 uL) of the saturated matrix solution is then allowed to dry to form the analyte-matrix mixture.
• the analyte-matrix mixture may be formed using the thin-layer method.
• a matrix homogeneous film is first formed and then the sample is then applied and may be absorbed by the matrix to form the analyte-matrix mixture.
• the analyte-matrix mixture may be formed using the thick-layer method.
• a matrix homogeneous film is formed with a nitro-cellulose matrix additive. Once the uniform nitro-cellulose matrix layer is obtained the sample is applied and absorbed into the matrix to form the analyte-matrix mixture.
• the analyte-matrix mixture may be formed using the sandwich method.
• a thin layer of matrix crystals is prepared as in the thin-layer method followed by the addition of droplets of aqueous trifluoroacetic acid, the sample and matrix. The sample is then absorbed into the matrix to form the analyte- matrix mixture.
• the polynucleotides, primary constructs and mmRNA of the present invention may be used to alter the phenotype of cells.
• the polynucleotides, primary constructs and mmRNA of the invention may encode peptides, polypeptides or multiple proteins to produce polypeptides of interest.
• the polypeptides of interest may be used in therapeutics and/or clinical and research settings.
• the polypeptides of interest may include reprogramming factors, differentiation factors and de-differentiation factors.
• polynucleotides, primary constructs or mmRNA of the present invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine, therapy and preventative treatments.
• a polynucleotide, primary construct or mmRNA described herein can be administered to a subject, wherein the polynucleotide, primary construct or mmRNA is translated in vivo to produce a therapeutic or prophylactic polypeptide in the subject.
• compositions, methods, kits, and reagents for diagnosis, treatment or prevention of a disease or condition in humans and other mammals are provided.
• the active therapeutic agents of the invention include polynucleotides, primary constructs or mmR A, cells containing the polynucleotides, primary constructs or mmR A or polypeptides translated from the polynucleotides, primary constructs or mmRNA.
• combination therapeutics containing one or more polynucleotide, primary construct or mmRNA containing translatable regions that encode for a protein or proteins.
• a recombinant polypeptide in a cell population using the polynucleotide, primary construct or mmRNA described herein.
• Such translation can be in vivo, ex vivo, in culture, or in vitro.
• the cell population is contacted with an effective amount of a composition containing a nucleic acid that has at least one nucleoside modification, and a translatable region encoding the recombinant polypeptide.
• the population is contacted under conditions such that the nucleic acid is localized into one or more cells of the cell population and the recombinant polypeptide is translated in the cell from the nucleic acid.
• an "effective amount" of the composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the nucleic acid (e.g., size, and extent of modified nucleosides), and other determinants.
• an effective amount of the composition provides efficient protein production in the cell, preferably more efficient than a composition containing a corresponding unmodified nucleic acid. Increased efficiency may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the nucleic acid), increased protein translation from the nucleic acid, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified nucleic acid), or reduced innate immune response of the host cell.
• aspects of the invention are directed to methods of inducing in vivo translation of a recombinant polypeptide in a mammalian subject in need thereof.
• an effective amount of a composition containing a nucleic acid that has at least one structural or chemical modification and a translatable region encoding the recombinant polypeptide is administered to the subject using the delivery methods described herein.
• the nucleic acid is provided in an amount and under other conditions such that the nucleic acid is localized into a cell of the subject and the recombinant polypeptide is translated in the cell from the nucleic acid.
• the cell in which the nucleic acid is localized, or the tissue in which the cell is present, may be targeted with one or more than one rounds of nucleic acid administration.
• the administered polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that provide a functional activity which is substantially absent in the cell, tissue or organism in which the recombinant polypeptide is translated.
• the missing functional activity may be enzymatic, structural, or gene regulatory in nature.
• the administered polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that increases (e.g.,
• the administered polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that replace a polypeptide (or multiple polypeptides) that is substantially absent in the cell in which the recombinant polypeptide is translated. Such absence may be due to genetic mutation of the encoding gene or regulatory pathway thereof.
• the recombinant polypeptide increases the level of an endogenous protein in the cell to a desirable level; such an increase may bring the level of the endogenous protein from a subnormal level to a normal level or from a normal level to a super-normal level.
• the recombinant polypeptide functions to antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell.
• the activity of the endogenous protein is deleterious to the subject; for example, due to mutation of the endogenous protein resulting in altered activity or localization.
• the recombinant polypeptide antagonizes, directly or indirectly, the activity of a biological moiety present in, on the surface of, or secreted from the cell.
• antagonized biological moieties include lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), a nucleic acid, a
• carbohydrate a protein toxin such as shiga and tetanus toxins, or a small molecule toxin such as botulinum, cholera, and diphtheria toxins.
• antagonized biological molecule may be an endogenous protein that exhibits an undesirable activity, such as a cytotoxic or cytostatic activity.
• the recombinant proteins described herein may be engineered for localization within the cell, potentially within a specific compartment such as the nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
• compositions containing polynucleotide, primary construct, or mmRNA can be formulated for administration intramuscularly, transarterially,
• composition may be formulated for extended release.
• the subject to whom the therapeutic agent may be administered suffers from or may be at risk of developing a disease, disorder, or deleterious condition.
• GWAS genome-wide association studies
• the polynucleotide, primary construct, or mmRNA of the present invention may be used to treat familial hypercholesterolemia (FH).
• FH familial hypercholesterolemia
• the term "familial hypercholesterolemia” or “FH” refers to an autosomal dominant genetic disorder characterized by elevated levels of low density lipoprotein (LDL)-associated cholesterol in the plasma. Compared with LDL cholesterol levels in normal patients (e.g., ⁇ 130mg/dL), levels in heterozygous and homozygous FH patients often rise to 350-550 mg/dL and to > 600 mg/dL, respectively.
• LDL low density lipoprotein
• Elevation in LDL cholesterol at these levels in patients or subjects with FH leads to cholesterol deposition within tisssues and may have an increased risk for cardiovascular disease at a young age.
• high levels of LDL in the blood of these individuals may be the result of mutations in the gene encoding the LDL receptor.
• LDL is produced within the circulation by lipolytic catabolism of triglyeride-rich very low density lipoproteins or VLDL. Following lipid transfer and esterification reactions,it is believed, and is no means limiting, that the LDL receptor binds LDL in the circulation and facilitates endocytosis of LDL into the hepatic cell surface that the receptor is expressed on.
• LDL levels remain elevated in the circulation during to their prolonged retention in the bloodstream and promote the development of atherosclerosis.
• Inactivating mutations in the LDLR gene are responsible for the majority of FH cases with LDLR expression in heterogygous and homozygous patients generally -50% and -10-15% of normal, respectively.
• Individuals with FH may be heterozygous or homozygous for FH-related gene mutations.
• Heterozygous FH is one of the most common genetic disorders with a prevelance of -1/500 in the general population; while homozygous forms of the disease are more rare with a prevelance of -1/1,000,000. Symptoms in homozygous individuals can be more severe.
• Diagnosis is possible during childhood or young adulthood by methods known in the art including, but not limited to, a physical exam that reveals xanthomas (fatty skin growths). Earlier diagnosis of FH may be made through an analysis of family history and genetics. (Sjouke, B. et al, Familial hypercholesterolemia: present and future management. Curr Cardiol Rep. 2011 Dec;13(6):527-36; Avis, H.J. et al., A systematic review and meta-analysis of statin therapy in children with familial hypercholesterolemia. Arterioscler Thromb Vase Biol. 2007 Aug;27(8): 1803-10; each of which are herein incorporated by reference in their entireties).
• statins can reduce serum cholesterol levels, either directly or indirectly, through induction of LDLR expression in the liver. While effective for many heterozygous FH patients, these approaches can be problematic. At least 25-30% of patients taking these drugs fail to achieve their desired LDL cholesterol goals. These agents can be even less effective in treating homozygous FH primarily due to the low residual levels of functional LDLRs in the liver of these patients. Most of these patients are non-responsive to statins and in severe forms of disease, treatment is limited to LDL apheresis and liver transplantation. Current treatments are not always successful in lowering LDL- Cholesterol levels to target; therefore, new treatments are urgently needed.
• patients with FH may be administered a composition comprising at least one polynucleotide, primary construct or mmRNA of the present invention.
• the polynucleotide, primary construct or mmRNA may encode a peptide, protein or fragment thereof such as, but not limited to, low density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase
• LDLR low density lipoprotein receptor
• APOB apolipoprotein B
• PCSK9 subtilisin/kexin type 9
• FH may be treated by administering a composition of the present invention comprising at least one polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof of LDLR. In another embodiment, FH may be treated by administering a composition of the present invention comprising at least one polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof.
• FH may be treated by administering a composition of the present invention comprising at least one polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof of APOB. In another embodiment, FH may be treated by administering a composition of the present invention comprising at least one polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof. In one embodiment, FH may be treated by administering a composition of the present invention comprising at least one polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof of PCSK9.
• FH may be treated by administering a composition of the present invention comprising at least one polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof.
• a composition of the present invention comprising at least one polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof.
• it may be useful to optimize the expression of a specific polypeptide in a cell line or collection of cell lines of potential interest, particularly a polypeptide of interest such as a protein variant of a reference protein having a known activity.
• a method of optimizing expression of a polypeptide of interest in a target cell by providing a plurality of target cell types, and independently contacting with each of the plurality of target cell types a modified mR A encoding a polypeptide.
• culture conditions may be altered to increase protein production efficiency. Subsequently, the presence and/or level of the polypeptide of interest in the plurality of target cell types is detected and/or quantitated, allowing for the optimization of a polypeptide of interest's expression by selection of an efficient target cell and cell culture conditions relating thereto. Such methods may be useful when the polypeptide of interest contains one or more post-translational modifications or has substantial tertiary structure, which often complicate efficient protein production.
• Methods and compositions described herein may be used to produce proteins which are capable of attenuating or blocking the endogenous agonist biological response and/or antagonizing a receptor or signaling molecule in a mammalian subject.
• IL-12 and IL-23 receptor signaling may be enhanced in chronic autoimmune disorders such as multiple sclerosis and
• a nucleic acid encodes an antagonist for chemokine receptors.
• Chemokine receptors CXCR-4 and CCR-5 are required for HIV enry into host cells (Arenzana-Seisdedos F et al, (1996) Nature. Oct 3; 383 (6599):400).
• the polynucleotides, primary constructs or mmRNA described herein can be used to express a ligand or ligand receptor on the surface of a cell (e.g., a homing moiety).
• a ligand or ligand receptor moiety attached to a cell surface can permit the cell to have a desired biological interaction with a tissue or an agent in vivo.
• a ligand can be an antibody, an antibody fragment, an aptamer, a peptide, a vitamin, a carbohydrate, a protein or polypeptide, a receptor, e.g., cell-surface receptor, an adhesion molecule, a glycoprotein, a sugar residue, a therapeutic agent, a drug, a glycosaminoglycan, or any combination thereof.
• a ligand can be an antibody that recognizes a cancer-cell specific antigen, rendering the cell capable of preferentially interacting with tumor cells to permit tumor-specific localization of a modified cell.
• a ligand can confer the ability of a cell composition to accumulate in a tissue to be treated, since a preferred ligand may be capable of interacting with a target molecule on the external face of a tissue to be treated. Ligands having limited cross-reactivity to other tissues are generally preferred.
• a ligand can act as a homing moiety which permits the cell to target to a specific tissue or interact with a specific ligand.
• homing moieties can include, but are not limited to, any member of a specific binding pair, antibodies, monoclonal antibodies, or derivatives or analogs thereof, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems
• (SCFV)2 fragments diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments; and other homing moieties include for example, aptamers, receptors, and fusion proteins.
• the homing moiety may be a surface-bound antibody, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site.
• multiple antibodies are expressed on the surface of a cell, and each antibody can have a different specificity for a desired target. Such approaches can increase the avidity and specificity of homing interactions.
• an estrogen receptor ligand such as tamoxifen
• tamoxifen can target cells to estrogen-dependent breast cancer cells that have an increased number of estrogen receptors on the cell surface.
• ligand/receptor interactions include CCRI (e.g., for treatment of inflamed joint tissues or brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8 (e.g., targeting to lymph node tissue), CCR6, CCR9,CCR10 (e.g., to target to intestinal tissue), CCR4, CCRIO (e.g., for targeting to skin), CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., for treatment of inflammation and inflammatory disorders, bone marrow), Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4/VCAM-1 (e.g., targeting to endothelium).
• any receptor involved in targeting e.g., cancer metastasis
• any receptor involved in targeting can be harnessed for use in the methods and compositions described herein.
• kits for conveniently and/or effectively carrying out methods of the present invention.
• kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments, and contact cells and/or a population of cells at least once.
• kits comprising the molecules (polynucleotides, primary constructs or mmRNA) of the invention.
• the kit comprises one or more functional antibodies or function fragments thereof.
• Kits and devices useful in combination with the polynucleotides, primary constructs or mmRNA) of the invention include those disclosed in co-pending U.S. Provisional Patent Application No 61/737,130 filed December 14, 2012, the contents of which are incorporated herein by reference in their entirety.
• substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges.
• the term "Ci-6 alkyl” is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C 4 alkyl, C5 alkyl, and C 6 alkyl.
• Administered in combination means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial ⁇ e.g. , a synergistic) effect is achieved.
• animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal ⁇ e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically-engineered animal, or a clone.
• association means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.
• An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the "associated" entities remain physically associated.
• bifunctional refers to any substance, molecule or moiety which is capable of or maintains at least two functions. The functions may effect the same outcome or a different outcome. The structure that produces the function may be the same or different.
• bifunctional modified R As of the present invention may encode a cytotoxic peptide (a first function) while those nucleosides which comprise the encoding R A are, in and of themselves, cytotoxic (second function).
• delivery of the bifunctional modified RNA to a cancer cell would produce not only a peptide or protein molecule which may ameliorate or treat the cancer but would also deliver a cytotoxic payload of nucleosides to the cell should degradation, instead of translation of the modified RNA, occur.
• Biocompatible As used herein, the term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.
• Biodegradable As used herein, the term “biodegradable” means capable of being broken down into innocuous products by the action of living things.
• Biologically active refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
• a polynucleotide, primary construct or mmRNA of the present invention may be considered biologically active if even a portion of the polynucleotide, primary construct or mmRNA is biologically active or mimics an activity considered biologically relevant.
• Cancer stem cells are cells that can undergo self-renewal and/or abnormal proliferation and differentiation to form a tumor.
• stereomer as used herein means stereoisomers that are not mirror images of one another and are non-superimposable on one another.
• an effective amount of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied.
• an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of cancer, as compared to the response obtained without
• enantiomer means each individual optically active form of a compound of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.
• the term "isomer,” as used herein, means any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention can have one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis/trans isomers).
• the chemical structures depicted herein, and therefore the compounds of the invention encompass all of the corresponding stereoisomers, that is, both the stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates.
• Enantiomeric and stereoisomeric mixtures of compounds of the invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid
• Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
• stereoisomer refers to all possible different isomeric as well as conformational forms which a compound may possess (e.g., a compound of any formula described herein), in particular all possible
• Compound As used herein, the term “compound,” is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
• the compounds described herein can be asymmetric ⁇ e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.
• Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton.
• Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
• Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds.
• “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei.
• isotopes of hydrogen include tritium and deuterium.
• the compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
• Committed means, when referring to a cell, when the cell is far enough into the differentiation pathway where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell type instead of into a different cell type or reverting to a lesser differentiated cell type.
• conserved refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
• two or more sequences are said to be “completely conserved” if they are 100% identical to one another.
• two or more sequences are said to be "highly conserved” if they are at least 70%> identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another.
• two or more sequences are said to be "highly conserved” if they are about 70%> identical, about 80%> identical, about 90%> identical, about 95%, about 98%, or about 99% identical to one another.
• two or more sequences are said to be "conserved” if they are at least 30% identical, at least 40%> identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be "conserved” if they are about 30%) identical, about 40%> identical, about 50%> identical, about 60%> identical, about 70%) identical, about 80%> identical, about 90%> identical, about 95% identical, about 98%) identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of an oligonucleotide or polypeptide or may apply to a portion, region or feature thereof.
• Controlled Release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
• Cyclic or Cyclized As used herein, the term “cyclic” refers to the presence of a continuous loop. Cyclic molecules need not be circular, only joined to form an unbroken chain of subunits. Cyclic molecules such as the engineered RNA or mRNA of the present invention may be single units or multimers or comprise one or more components of a complex or higher order structure.
• Cytostatic refers to inhibiting, reducing, suppressing the growth, division, or multiplication of a cell ⁇ e.g., a mammalian cell ⁇ e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof.
• Cytotoxic refers to killing or causing injurious, toxic, or deadly effect on a cell ⁇ e.g., a mammalian cell ⁇ e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof.
• Delivery refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.
• Delivery Agent refers to any substance which facilitates, at least in part, the in vivo delivery of a polynucleotide, primary construct or mmRNA to targeted cells.
• Delivery agent refers to any substance which facilitates, at least in part, the in vivo delivery of a polynucleotide, primary construct or mmRNA to targeted cells.
• Destabilized As used herein, the term “destable,” “destabilize,” or
• stabilizing region means a region or molecule that is less stable than a starting, wild-type or native form of the same region or molecule.
• Detectable label refers to one or more markers, signals, or moieties which are attached, incorporated or associated with another entity that is readily detected by methods known in the art including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like. Detectable labels may be located at any position in the peptides or proteins disclosed herein. They may be within the amino acids, the peptides, or proteins, or located at the N- or C- termini.
• developmental potential or “developmental potency” refers to the total of all developmental cell fates or cell types that can be achieved by a cell upon differentiation.
• developmental potential altering factor refers to a protein or R A which can alter the
• Digest As used herein, the term “digest” means to break apart into smaller pieces or components. When referring to polypeptides or proteins, digestion results in the production of peptides.
• Differentiated cell As used herein, the term “differentiated cell” refers to any somatic cell that is not, in its native form, pluripotent. Differentiated cell also encompasses cells that are partially differentiated.
• Differentiation factor refers to a developmental potential altering factor such as a protein, RNA or small molecule that can induce a cell to differentiate to a desired cell-type.
• Differentiate refers to the process where an uncommitted or less committed cell acquires the features of a committed cell.
• distal As used herein, the term “distal” means situated away from the center or away from a point or region of interest.
• DSF Dose splitting factor
• Embryonic stem cell As used herein, the term “embryonic stem cell” refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst.
• Encapsulate As used herein, the term “encapsulate” means to enclose, surround or encase.
• Encoded protein cleavage signal refers to the nucleotide sequence which encodes a protein cleavage signal.
• Engineered As used herein, embodiments of the invention are "engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.
• Exosome is a vesicle secreted by mammalian cells or a complex involved in R A degradation.
• expression refers to one or more of the following events: (1) production of an RNA template from a DNA sequence ⁇ e.g., by transcription); (2) processing of an RNA transcript ⁇ e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
• Feature refers to a characteristic, a property, or a distinctive element.
• formulation includes at least a
• polynucleotide, primary construct or mmRNA and a delivery agent are examples of delivery agents.
• fragment refers to a portion.
• fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.
• a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
• Homology refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules ⁇ e.g. DNA molecules and/or R A molecules) and/or between polypeptide molecules.
• polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.
• homologous polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids.
• homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids.
• two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.
• Identity refers to the overall relatedness between polymeric molecules, e.g., between oligonucleotide molecules ⁇ e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
• Calculation of the percent identity of two polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes ⁇ e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
• the length of a sequence aligned for comparison purposes is at least 30%>, at least 40%>, at least 50%>, at least 60%>, at least 70%>, at least 80%), at least 90%>, at least 95%, or 100%> of the length of the reference sequence.
• the nucleotides at corresponding nucleotide positions are then compared.
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
• the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.

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