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 for production of proteins.

1 - 4 . (canceled) 
     
     
         5 . A method of producing an immunoglobulin protein, comprising delivering two modified mRNA constructs in vivo so as to cause intracellular translation of the mRNA constructs into heavy and light chains of an immunoglobulin protein, wherein the modified mRNA constructs are capable of evading an innate immune response of a cell into which they are introduced, and wherein the modified mRNA constructs are substantially devoid of cytidine or uracil nucleotides. 
     
     
         6 . The method of  claim 5 , wherein the modified mRNA constructs are substantially devoid of cytidine nucleotides. 
     
     
         7 . The method of  claim 5 , wherein the modified mRNA constructs are substantially devoid of uracil nucleotides. 
     
     
         8 . The method of  claim 6 , wherein the modified mRNA constructs comprise 5-methyl-cytidine. 
     
     
         9 . The method of  claim 7 , wherein the modified mRNA constructs comprise pseudouridine. 
     
     
         10 . The method of  claim 7 , wherein the modified mRNA constructs comprise 1-methyl-pseudouridine. 
     
     
         11 . A kit for immunoglobulin protein production, comprising a first isolated nucleic acid comprising i) a translatable region encoding the immunoglobulin protein and ii) a nucleic acid modification, wherein the first nucleic acid is capable of evading an innate immune response of a cell into which the first isolated nucleic acid is introduced, wherein the translatable region is substantially devoid of cytidine and uracil nucleotides, and packaging and instructions therefor. 
     
     
         12 . The kit of  claim 11 , wherein the immunoglobulin protein comprises a polypeptide selected from the group consisting of a full-length antibody, a heavy chain polypeptide, a light chain polypeptide, an Fab domain, or a single chain variable fragment (ScFv) polypeptide, and the first isolated nucleic acid comprises a messenger ribonucleic acid comprising 5-methyl-cytidine and pseudouridine. 
     
     
         13 . A mammalian cell generated by use of the kit of  claim 11 . 
     
     
         14 . An isolated immunoglobulin protein produced from a production cell comprising a first isolated nucleic acid comprising i) a translatable region encoding the immunoglobulin protein and ii) a nucleic acid modification, wherein the first nucleic acid is capable of evading an innate immune response of the cell, wherein the translatable region is substantially devoid of either cytidine or uracil nucleotides or the combination of cytidine and uracil nucleotides. 
     
     
         15 . A pharmaceutical preparation comprising an effective amount of a first nucleic acid comprising i) a translatable region encoding an immunoglobulin protein and ii) a nucleic acid modification, wherein the first nucleic acid exhibits reduced degradation by a cellular nuclease and is capable of evading an innate immune response of a cell into which the first nucleic acid is introduced, wherein the translatable region is substantially devoid of cytidine and uracil nucleotides. 
     
     
         16 . A method of producing a heterologous protein of interest in a cell, comprising the step
 providing a target cell capable of protein translation; and   introducing into the target cell a composition comprising a first isolated nucleic acid comprising a translatable region encoding the heterologous protein of interest and a nucleoside modification,   under conditions such that the protein of interest is produced in the cell.   
     
     
         17 . The method of  claim 16 , wherein the protein of interest is an immunoglobulin protein. 
     
     
         18 . A method of increasing the production of a recombinantly expressed protein of interest in a cell, comprising the step:
 providing a target cell comprising a heterologous nucleic acid encoding the protein of interest; and   introducing into the target cell a composition comprising a first isolated nucleic acid comprising a translatable region encoding a translation effector protein and a nucleoside modification under conditions such that the effector protein is produced in the cell,   thereby increasing the production of the recombinantly expressed protein in the cell.   
     
     
         19 . The method of  claim 18 , wherein the protein of interest is an immunoglobulin protein. 
     
     
         20 . A method for modulating the level of a protein of interest in a target cell, comprising the steps of:
 modulating the activity of at least one translation effector molecule in the target cell, wherein the modulation comprises introducing into the target cell a first isolated nucleic acid comprising a translatable region encoding the translation effector protein and a nucleoside modification; and   culturing the cell.   
     
     
         21 . A kit for protein production, comprising a first isolated nucleic acid encoding a translatable region encoding a protein, wherein the first nucleic acid comprises a nucleic acid modification, wherein the first nucleic acid displays decreased degradation in a cell into which the first isolated nucleic acid is introduced as compared to a nucleic acid not comprising a nucleic acid modification, and packaging and instructions therefor. 
     
     
         22 . A kit for protein production, comprising a first isolated nucleic acid encoding a translatable region encoding a protein, wherein the first nucleic acid comprises a nucleic acid modification, wherein the first nucleic acid is more stable in a cell into which the first isolated nucleic acid is introduced as compared to a nucleic acid not comprising a nucleic acid modification, and packaging and instructions therefor.