Engineered nucleic acids and methods of use thereof

Abstract

Provided are compositions and methods for delivering biological moieties such as modified nucleic acids into cells to modulate protein expression. Such compositions and methods include the use of modified messenger RNAs, and are useful to treat or prevent diseases, disorders or conditions, or to improve a subject's heath or wellbeing.

What is claimed is: 
     
         1 . A pharmaceutical formulation comprising:
 i) an effective amount of a synthetic messenger ribonucleic acid (mRNA) encoding a granulocyte colony-stimulating factor (G-CSF) polypeptide; and   ii) a pharmaceutically acceptable carrier, wherein i) the mRNA comprises pseudouridine, 5′methyl-cytidine or a combination thereof, or ii) wherein the mRNA does not comprise a substantial amount of a nucleotide or nucleotides selected from the group consisting of uridine, cytidine, and a combination of uridine and cytidine, and wherein the formulation is suitable for repeated intravenous administration to a mammalian subject in need thereof.   
     
     
         2 . The pharmaceutical formulation of  claim 1 , wherein the G-CSF polypeptide comprises:
 a) the amino acid sequence of SEQ ID NOs: 13 or 14;   b) an amino acid sequence at least 95% identical to SEQ ID NOs: 13 or 14;   c) a fragment of SEQ ID NOs: 13 or 14;   d) the amino acid sequence encoded by the nucleic acid of SEQ ID NO: 2 or SEQ ID NO: 17; or   e) the amino acid sequence encoded by a nucleic acid at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 17.   
     
     
         3 . The pharmaceutical formulation of  claim 1 , further comprising a lipid-based transfection reagent. 
     
     
         4 . The pharmaceutical formulation of  claim 1 , wherein the mRNA lacks at least one destabilizing element. 
     
     
         5 . A kit comprising the pharmaceutical formulation of  claim 1  and AMD3100 (1,1′-[1,4-phenylene-bis(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane), in one or more containers, and instructions for use thereof. 
     
     
         6 . A pharmaceutical formulation consisting essentially of:
 i) an effective amount of a synthetic messenger ribonucleic acid (mRNA) encoding a granulocyte colony-stimulating factor (G-CSF) polypeptide;   ii) a cell penetration agent; and   iii) a pharmaceutically acceptable carrier, wherein i) the mRNA comprises pseudouridine, 5′methyl-cytidine or a combination thereof, or ii) wherein the mRNA does not comprise a substantial amount of a nucleotide or nucleotides selected from the group consisting of uridine, cytidine, and a combination of uridine and cytidine, and wherein the formulation is suitable for repeated intravenous administration to a mammalian subject in need thereof.   
     
     
         7 . A method to obtain progenitor and/or stem cells from a mammalian subject, comprising the steps of: (a) administering to the subject a composition comprising a synthetic messenger ribonucleic acid (mRNA) encoding a granulocyte colony-stimulating factor (G-CSF) polypeptide in an amount effective to mobilize the progenitor and/or stem cells into the peripheral blood of the subject; followed by (b) harvesting the progenitor and/or stem cells. 
     
     
         8 . The method of  claim 7  wherein the mRNA or a pharmaceutically acceptable salt thereof is administered to the subject in the dosage range of about 1.0 μg/kg-100 mg/kg of body weight. 
     
     
         9 . The method of  claim 7 , wherein the subject is human. 
     
     
         10 . The method of  claim 7 , wherein the composition is administered to the subject by an intravenous route. 
     
     
         11 . The method of  claim 7 , further comprising the step of administering an effective amount of AMD3100 (1,1′-[1,4-phenyl ene-bi s(methyl ene)]-bi s-1,4,8, 11-tetraazacyclotetradecane) to the subject after administering the composition. 
     
     
         12 . The method of  claim 11 , wherein the composition is administered at least twice but fewer than ten times prior to administering the AMD3100. 
     
     
         13 . The method of  claim 7 , wherein the composition further comprises a synthetic messenger ribonucleic acid (mRNA) encoding i) a macrophage inflammatory protein (MIP) polypeptide or ii) an antibody that prevents EGF binding by EGFR. 
     
     
         14 . An improved regimen for mobilizing hematopoietic stem and progenitor cells in a mammalian subject in need thereof, the regimen comprising an amount of a synthetic messenger ribonucleic acid (mRNA) encoding a G-CSF polypeptide effective to enhance mobilization of hematopoietic stem cells and progenitor cells (HSPC), and an effective amount of AMD3100 (1,1′[1,4-phenylene-bis(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane). 
     
     
         15 . A pharmaceutical formulation comprising:
 i) an effective amount of a synthetic messenger ribonucleic acid (mRNA) encoding an insulin polypeptide; and   ii) a pharmaceutically acceptable carrier, wherein i) the mRNA comprises pseudouridine, 5′methyl-cytidine or a combination thereof, or ii) wherein the mRNA does not comprise a substantial amount of a nucleotide or nucleotides selected from the group consisting of uridine, cytidine, and a combination of uridine and cytidine, and wherein the formulation is suitable for repeated intravenous administration or intramuscular administration to a mammalian subject in need thereof.   
     
     
         16 . The pharmaceutical formulation of  claim 15 , wherein the insulin polypeptide comprises:
 a) the amino acid sequence of SEQ ID NOs: 15 or 16;   b) an amino acid sequence at least 95% identical to SEQ ID NOs: 15 or 16;   c) a fragment of SEQ ID NOs: 15 or 16;   d) the amino acid sequence encoded by the nucleic acid of SEQ ID NOs: 3 or 4, or SEQ ID NOs: 21or 23; or e) the amino acid sequence encoded by a nucleic acid at least 95% identical to SEQ ID NOs: 3 or 4, or SEQ ID NOs: 21 or 23.   
     
     
         17 . The pharmaceutical formulation of  claim 15 , further comprising a lipid-based transfection reagent. 
     
     
         18 . A method of regulating carbohydrate and lipid metabolism in a mammalian subject in need thereof, comprising the step of: (a) administering to the subject a composition comprising a synthetic messenger ribonucleic acid (mRNA) encoding an insulin polypeptide in an amount effective to regulate carbohydrate and lipid metabolism in the subject.