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 . 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. 
     
     
         2 . The kit of  claim 1 , 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. 
     
     
         3 . The kit of  claim 1 , wherein the first isolated nucleic acid comprises a nucleotide sequence selected from the group consisting of:
 a) the nucleotide sequence of SEQ ID NOs: 4 and/or 6 [rituximab];   b) a nucleotide sequence at least 95% identical to the nucleotide sequence of a);   c) a nucleotide sequence encoding the polypeptide of SEQ ID NOs: 5 and/or 7 [rituximab];   d) a nucleotide sequence at least 95% identical to the nucleotide sequence of c);   e) a nucleotide sequence encoding a polypeptide at least 95% identical to SEQ ID NOs: 5 and/or 7 [rituximab];   f) a nucleotide sequence at least 95% identical to the nucleotide sequence of e);   g) a nucleotide sequence comprising a fragment of any one of a)-f); and   h) a codon-optimized variant of the nucleotide sequence of any one of a)-g).   
     
     
         4 . The kit of  claim 3 , wherein the immunoglobulin protein immunospecifically binds to a CD20 polypeptide. 
     
     
         5 . The kit of  claim 3 , wherein the immunoglobulin protein specifically induces antibody-dependent cellular cytotoxicity of CD20+ cells when contacted therewith. 
     
     
         6 . The kit of  claim 3 , wherein the immunoglobulin protein is produced for use in the treatment of a leukemia, a lymphoma, an organ transplant rejection, or an autoimmune disease or disorder. 
     
     
         7 . A mammalian cell generated by use of the kit of  claim 1 . 
     
     
         8 . The kit of  claim 1 , wherein the first isolated nucleic acid comprises a nucleotide sequence selected from the group consisting of:
 a) the nucleotide sequence of SEQ ID NOs: 8 and/or 10;   b) a nucleotide sequence at least 95% identical to the nucleotide sequence of a);   c) a nucleotide sequence encoding the polypeptide of SEQ ID NOs: 9 and/or 11;   d) a nucleotide sequence at least 95% identical to the nucleotide sequence of c);   e) a nucleotide sequence encoding a polypeptide at least 95% identical to SEQ ID NOs: 9 and/or 11;   f) a nucleotide sequence at least 95% identical to the nucleotide sequence of e); and   g) a nucleotide sequence comprising a fragment at least 30 nucleotides in length of any one of a)-f).   
     
     
         9 . The kit of  claim 8 , wherein the immunoglobulin protein immunospecifically binds to a HER-2/neu receptor polypeptide. 
     
     
         10 . The kit of  claim 8 , wherein the immunoglobulin protein specifically induces antibody-dependent cellular cytotoxicity, apoptosis, cell cycle arrest or a combination thereof, of HER2/neu+ cells when contacted therewith. 
     
     
         11 . The kit of  claim 8 , wherein the immunoglobulin protein is produced for use in the treatment of a HER2/neu+ breast cancer. 
     
     
         12 . 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. 
     
     
         13 . The protein of  claim 12 , wherein the production cell is a cell isolated from a human subject. 
     
     
         14 . A pharmaceutical preparation comprising an effective amount of the protein of  claim 13 . 
     
     
         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 . The pharmaceutical preparation of  claim 15 , wherein the first nucleic acid exhibits reduced degradation by a cellular nuclease. 
     
     
         17 . A method of producing a heterologous protein of interest in a cell, comprising the step:
 i) providing a target cell capable of protein translation; and   ii) 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.   
     
     
         18 . The method of  claim 17 , further comprising the step of substantially purifying the protein of interest from the cell. 
     
     
         19 . The method of  claim 17 , wherein the protein of interest is a secreted protein. 
     
     
         20 . The method of  claim 17 , wherein the protein of interest is an immunoglobulin protein. 
     
     
         21 . The method of  claim 17 , wherein the protein of interest is an intracellular protein. 
     
     
         22 . A method of increasing the production of a recombinantly expressed protein of interest in a cell, comprising the step:
 i) providing a target cell comprising a heterologous nucleic acid encoding the protein of interest; and   ii) 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.   
     
     
         23 . The method of  claim 22 , wherein the protein of interest is an immunoglobulin protein. The method of  claim 22 , wherein the protein of interest is a secreted protein. 
     
     
         24 . The method of  claim 22 , wherein the protein if interest is an intracellular protein. 
     
     
         25 . The method of  claim 22 , wherein the target cell is a mammalian cell. 
     
     
         26 . The method of  claim 22 , wherein the target cell is a yeast cell. 
     
     
         27 . The method of  claim 22 , wherein the target cell is a bacterial cell, an insect cell, or a plant cell. 
     
     
         28 . The method of  claim 22 , wherein the translation effector protein is ceramide transfer protein (CERT). 
     
     
         29 . 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   ii) culturing the cell.   
     
     
         30 . 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. 
     
     
         31 . 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 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.