Modified nucleoside, nucleotide, and nucleic acid compositions

Abstract

The present disclosure provides, inter alia, formulation compositions comprising modified nucleic acid molecules which may encode a protein, a protein precursor, or a partially or fully processed form of the protein or a protein precursor. The formulation composition may further include a modified nucleic acid molecule and a delivery agent. The present invention further provides nucleic acids useful for encoding polypeptides capable of modulating a cell's function and/or activity.

What is claimed is: 
     
         1 . A method of producing a polypeptide of interest in a mammalian cell or tissue, the method comprising, contacting said mammalian cell or tissue with a formulation comprising a modified mRNA encoding the polypeptide of interest, wherein the formulation is selected from the group consisting of nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoid, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids, fibrin gel, fibrin hydrogel, fibrin glue, fibrin sealant, fibrinogen, thrombin, rapidly eliminated lipid nanoparticles (reLNPs) and combinations thereof. 
     
     
         2 . The method of  claim 1 , wherein the modified mRNA comprises a purified IVT transcript. 
     
     
         3 . The method of  claim 1 , wherein the formulation comprising the modified mRNA is a nanoparticle and wherein said nanoparticle comprises at least one lipid. 
     
     
         4 . The method of  claim 3 , wherein the lipid is selected from the group consisting of DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG and PEGylated lipids. 
     
     
         5 . The method of  claim 3 , wherein the lipid is cationic lipid. 
     
     
         6 . The method of  claim 5 , wherein the cationic lipid is selected from the group consisting of DLin-DMA, DLin-K-DMA, DLin-MC3-DMA, DLin-KC2-DMA and DODMA. 
     
     
         7 . The method of  claim 3 , wherein the lipid to modified mRNA weight ratio is between 10:1, and 30:1. 
     
     
         8 . The method of  claim 7 , wherein the mean size of the nanoparticle formulation comprising the modified mRNA is between 60-225 nm. 
     
     
         9 . The method of  claim 8 , wherein the PDI of the nanoparticle formulation comprising the modified mRNA is between 0.03 and 0.15. 
     
     
         10 . The method of  claim 3 , wherein the zeta potential of the lipid is from −10 to +10 at a pH of 7.4. 
     
     
         11 . The method of  claim 7 , wherein the nanoparticle formulation comprising the modified mRNA further comprises a fusogenic lipid, cholesterol, and a PEG lipid. 
     
     
         12 . The method of  claim 11 , wherein the nanoparticle formulation comprising the modified mRNA has a molar ratio of 50:10:38.5:1.5-3.0 (Cationic Lipid:fusogenic lipid:Cholesterol:PEG lipid). 
     
     
         13 . The method of  claim 12 , wherein the PEG lipid is selected from PEG-c-DOMG and PEG-DMG and the fusogenic lipid is DSPC. 
     
     
         14 . The method of  claim 1 , wherein contacting is through the use of a device selected from the group consisting of syringe pump, internal osmotic pump and external osmotic pump. 
     
     
         15 . The method of  claim 1 , wherein the formulation comprising the modified mRNA is a poly(lactic-co-glycolic acid) (PLGA) microsphere. 
     
     
         16 . The method of  claim 15 , wherein microspheres of the PLGA microsphere formulation comprising the modified mRNA are between 4 and 20 μm in size. 
     
     
         17 . The method of  claim 15 , wherein the PLGA microsphere formulation comprising the modified mRNA release less than 50% of the modified mRNA in a 48 hour time period. 
     
     
         18 . The method of  claim 15 , wherein the PLGA microsphere formulation comprising the modified mRNA is stable in serum. 
     
     
         19 . The method of  claim 18 , wherein the stability is determined relative to unformulated modified mRNA in 90% serum. 
     
     
         20 . The method of  claim 15 , wherein the loading weight percent is at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, or at least 0.4%. 
     
     
         21 . The method of  claim 15 , wherein the encapsulation efficiency of the modified mRNA in the PLGA microspheres is at least 50%. 
     
     
         22 . The method of  claim 15 , wherein the encapsulation efficiency of the modified mRNA in the PLGA microspheres is at least 70%. 
     
     
         23 . The method of  claim 15 , wherein the encapsulation efficiency of the modified mRNA in the PLGA microspheres is at least 90%. 
     
     
         24 . The method of  claim 15 , wherein the encapsulation efficiency of the modified mRNA in the PLGA microspheres is at least 97%. 
     
     
         25 . The method of  claim 11 , wherein contacting said mammalian cells or tissues occurs via a route of administration selected from the group consisting of intravenous, intramuscular, intravitreal, intrathecal, intratumoral, pulmonary, and subcutaneous. 
     
     
         26 . The method of  claim 25 , wherein the polypeptide of interest is detectable in the serum for up to 72 hours after contacting at levels higher than the levels prior to contacting. 
     
     
         27 . The method of  claim 26 , wherein the polypeptide of interest is detectable in the serum of female subjects at levels greater than in the serum of male subjects. 
     
     
         28 . The method of  claim 1 , wherein the formulation further comprises a second modified mRNA. 
     
     
         29 . The method of  claim 28 , wherein the formulation further comprises a third modified mRNA. 
     
     
         30 . The method of  claim 1 , wherein the formulation comprising the modified mRNA comprises a rapidly eliminated lipid nanoparticle. 
     
     
         31 . The method of  claim 30 , wherein the rapidly eliminated lipid nanoparticle comprises an reLNP lipid, fusogenic lipid, cholesterol and a PEG lipid at a molar ratio of 50:10:38.5:1.5(reLNP Lipid:Fusogenic lipid:Cholesterol:PEG lipid). 
     
     
         32 . The method of  claim 31 , wherein the fusogenic lipid is DSPC and the PEG lipid is PEG-c-DOMG. 
     
     
         33 . The method of  claim 31  wherein the reLNP lipid is selected from the group consisting of DLin-DMA with an internal ester, DLin-DMA with a terminal ester, DLin-MC3-DMA-with an internal ester, and DLin-MC3-DMA with a terminal ester. 
     
     
         34 . The method of  claim 30 , wherein in the total lipid to modified mRNA weight ratio is between 10:1 and 30:1. 
     
     
         35 . The method of  claim 1 , wherein contacting occurs via injection using a split dosing schedule. 
     
     
         36 . The method of  claim 35 , wherein the injection is made to the tissue selected from the group consisting of intradermal space, epidermis, subcutaneous tissue, and muscle. 
     
     
         37 . The method of  claim 1 , wherein the formulation comprising the modified mRNA comprises a fibrin sealant. 
     
     
         38 . The method of  claim 1 , wherein the formulation comprising the modified mRNA comprises a lipidoid and wherein the lipid is selected from the group consisting of C12-200 and 98N12-5. 
     
     
         39 . The method of  claim 1 , wherein the formulation comprising the modified mRNA is a polymer and said polymer is coated, covered, surrounded, enclosed or comprises a layer of hydrogel or surgical sealant. 
     
     
         40 . The method of  claim 39 , wherein the polymer is selected from the group consisting of PLGA, ethylene vinyl acetate, poloxamer and GELSITE®. 
     
     
         41 . The method of  claim 40 , further comprising an additional layer of polymer, hydrogel or surgical sealant. 
     
     
         42 . The method of  claim 2 , wherein the modified mRNA comprises at least one 5′ terminal cap selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′ fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. 
     
     
         43 . The method of  claim 42 , wherein the 5′ terminal cap is Cap1. 
     
     
         44 . The method of  claim 43 , wherein the modified mRNA comprises at least two modifications. 
     
     
         45 . The method of  claim 44 , wherein the at least two modifications are independently selected from the group consisting of 5-methylcytidine, pseudouridine, and 1-methyl-pseudouridine. 
     
     
         46 . A method of producing a polypeptide of interest in a mammalian cell or tissue, the method comprising, contacting said mammalian cell or tissue with a buffer formulation comprising a modified mRNA encoding the polypeptide of interest. 
     
     
         47 . The method of  claim 46 , wherein the buffer formulation is selected from the group consisting of saline, phosphate buffered saline, and Ringer's lactate. 
     
     
         48 . The method of  claim 46 , wherein the buffer formulation comprises a calcium concentration of between 1-10 mM. 
     
     
         49 . The method of  claim 46 , wherein the modified mRNA comprises a purified IVT transcript. 
     
     
         50 . The method of  claim 46 , wherein contacting said mammalian cells or tissues occurs via a route of administration selected from the group consisting of intravenous, intramuscular, intravitreal, intrathecal, intratumoral, pulmonary, and subcutaneous. 
     
     
         51 . The method of  claim 25  or  50 , wherein said polypeptide of interest is produced in said cell or tissue in a location systemic from the location of contacting. 
     
     
         52 . The method of  claim 51  wherein the route of administration is either via intramuscular or subcutaneous. 
     
     
         53 . The method of  claim 3 , wherein the lipid nanoparticle formulation is further formulated in a sealant. 
     
     
         54 . The method of  claim 53 , wherein said sealant is a fibrin sealant.