Long poly(a) plasmids and methods for introduction of long poly(a) sequences into the plasmid

Abstract

Disclosed herein are polynucleotides having a plurality of thymine nucleotides and an endonuclease recognition site inserted therein, methods of engineering the polynucleotides having a plurality of thymine nucleotides and an endonuclease recognition site inserted therein, and methods of enhancing transcription, translation, and increasing stability of a polynucleotide.

1 . (canceled) 
     
     
         2 . An isolated polynucleotide encoding a polypeptide, the polynucleotide comprising:
 (a) a first nucleic acid encoding the polypeptide; and   (b) a second nucleic acid comprising a plurality of thymine nucleotides of at least 130 consecutive thymine nucleotides, and an endonuclease cleavage site located within the plurality of thymine nucleotides or within 40 consecutive nucleotides from the 5′ terminal thymine of the plurality of thymine nucleotides, and   (c) a third nucleic acid comprising an endonuclease recognition site, wherein the endonuclease recognition site is within 55 consecutive nucleotides of the endonuclease cleavage site, wherein the second nucleic acid is covalently linked to the third nucleic acid at one end of the third nucleic acid.   
     
     
         3 . The isolated polynucleotide of  claim 2 , wherein the polypeptide comprises a nuclease, a chimeric antigen receptor (CAR), an antigen for a cancer vaccine, an antigen for an infectious disease, or a protein for a replacement protein therapy. 
     
     
         4 . The isolated polynucleotide of  claim 3 , wherein the polypeptide comprises an antigen for an infectious disease vaccine. 
     
     
         5 . The isolated polynucleotide of  claim 2 , wherein the first nucleic acid is codon optimized for expression in a human cell. 
     
     
         6 . The isolated polynucleotide of  claim 2 , further comprising a promoter operably linked to the first nucleic acid. 
     
     
         7 . The isolated polynucleotide of  claim 2 , wherein the endonuclease recognition site is a Type II restriction endonuclease recognition site. 
     
     
         8 . The isolated polynucleotide of  claim 7 , wherein the endonuclease recognition site is a Type II restriction endonuclease recognition site is a BsaI recognition site or a StuI recognition site. 
     
     
         9 . The isolated polynucleotide of  claim 2 , further comprising a telomere. 
     
     
         10 . The isolated polynucleotide of  claim 2  prepared by a method comprising:
 (i) obtaining a linear plasmid comprising a telomere, and a double-stranded nucleic acid comprising at least 130 consecutive thymine nucleotides; and 
 (ii) inserting the double-stranded nucleic acid into the vector, thereby preparing the isolated polynucleotide. 
 
     
     
         11 . A linear vector comprising the isolated polynucleotide of  claim 2 . 
     
     
         12 . A method for preparing a therapeutic polynucleotide, comprising:
 contacting the isolated polynucleotide of  claim 2  with a polymerase and a plurality of nucleotides, thereby obtaining the therapeutic polynucleotide.   
     
     
         13 . The method of  claim 12 , further comprising preparing the isolated polynucleotide comprising:
 (i) obtaining a linear plasmid comprising a telomere, and a double-stranded nucleic acid comprising at least 130 consecutive thymine nucleotides; and   (iii) inserting the double-stranded nucleic acid into the vector, thereby preparing the isolated polynucleotide.   
     
     
         14 . The method of  claim 12 , wherein the therapeutic polynucleotide is RNA. 
     
     
         15 . A pharmaceutical composition comprising a therapeutic polynucleotide prepared according to the method of  claim 12 , and a pharmaceutically acceptable vehicle. 
     
     
         16 . A method for treating, ameliorating or inhibiting a disorder in a subject, comprising: administering a therapeutic polynucleotide to the subject, wherein the therapeutic polynucleotide is prepared according to the method of  claim 12 . 
     
     
         17 . The method of  claim 16 , further comprising preparing the therapeutic polynucleotide. 
     
     
         18 . The method of  claim 16 , wherein the polypeptide is a nuclease, a chimeric antigen receptor (CAR), an antigen for a cancer vaccine, an antigen for an infectious disease, or a protein for a replacement protein therapy. 
     
     
         19 . The method of  claim 18 , wherein the polypeptide comprises an antigen for an infectious disease. 
     
     
         20 . The method of  claim 16 , wherein the disorder comprises an infectious disorder. 
     
     
         21 . The method of  claim 16 , wherein the subject is human.