Genetic elements driving circular rna translation and methods of use

Abstract

Provided herein are recombinant circular RNA (circRNA) molecules comprising an internal ribosome entry site (IRES) operably linked to a protein-coding nucleic acid sequence. The IRES includes at least one RNA secondary structure element; and a sequence region that is complementary to an 18S ribosomal RNA (rRNA). Methods of producing a protein in a cell using the recombinant circRNA molecules are also provided.

1 .- 16 . (canceled) 
     
     
         17 . A polynucleotide comprising a protein-coding nucleic acid sequence and an internal ribosome entry site (IRES) sequence region operably linked to the protein-coding nucleic acid sequence, wherein the IRES sequence region comprises,
 (i) at least one RNA secondary structure element, and   (ii) a sequence that is complementary to an 18S ribosomal RNA (rRNA),   
       wherein the IRES sequence region has a minimum free energy (MFE) of less than −18.9 kJ/mol and a melting temperature of at least 35.0° C., and 
       wherein the at least one RNA secondary structure element is formed by the nucleotides corresponding to about positions 40-60 of the IRES in which the first nucleotide at the 5′ end of the IRES sequence is considered to be position 1. 
     
     
         18 . The polynucleotide of clam 1, wherein the at least one RNA secondary structure element is a stem-loop. 
     
     
         19 . The polynucleotide of clam 1, wherein the sequence that is complementary an 18S rRNA comprises a sequence of any one of SEQ ID NOs: 28977-28983. 
     
     
         20 . The polynucleotide of clam 1, wherein the at least one RNA secondary structure element is located 5′ to the sequence that is complementary to an 18S rRNA, or 3′ to the sequence that is complementary to an 18S rRNA. 
     
     
         21 . The polynucleotide of claim  1 , wherein the polynucleotide is a DNA sequence, or a linear RNA sequence. 
     
     
         22 . The polynucleotide of claim  5 , wherein the polynucleotide is a DNA sequence. 
     
     
         23 . The polynucleotide of claim  6 , wherein the DNA sequence is comprised in a viral vector, a viral like particle, or a non-viral vector. 
     
     
         24 . The polynucleotide of claim  7 , wherein the DNA sequence is comprised in a viral vector, and wherein the viral vector is an adeno-associated virus (AAV), an adenovirus vector (AV), a retrovirus vector, a lentivirus vector, a vaccinia, or a herpesvirus vector. 
     
     
         25 . The polynucleotide of claim  1 , wherein the IRES is about 200 to about 1,000 nucleotides in length. 
     
     
         26 . The polynucleotide of claim  1 , wherein the polynucleotide comprises two self-splicing intron fragments flanking the protein-coding nucleic acid and IRES sequence region, and wherein self-splicing the intron fragments makes a circular RNA. 
     
     
         27 . A polynucleotide comprising a protein-coding nucleic acid sequence and an internal ribosome entry site (IRES) sequence region operably linked to the protein-coding nucleic acid sequence, wherein the IRES sequence region comprises a nucleic acid sequence at least 90%, at least 95%, or 100% identical to any one of the sequences in the group consisting of SEQ ID NOs. 1-228, and SEQ ID NOs. 229-17201. 
     
     
         28 . The polynucleotide of claim  11 , wherein the IRES sequence region is operably linked to the IRES sequence region in a non-native configuration. 
     
     
         29 . The polynucleotide of claim  11 , wherein the IRES sequence region comprises any one of the nucleic acid sequences of SEQ ID NOs. 531, 2270, 2602, 3042, 3244 and 33948. 
     
     
         30 . The polynucleotide of claim  11 , wherein the IRES sequence region has a G-C content of at least 25%. 
     
     
         31 . The polynucleotide of claim  11 , wherein the polynucleotide comprises a spacer between the IRES sequence region and a start codon of the protein coding nucleic acid sequence region. 
     
     
         32 . The polynucleotide of claim  15 , wherein the length of the spacer is selected to increase translation of the protein-coding nucleic acid sequence region relative to translation of a circular RNA having no spacer or a spacer that differs from the selected spacer. 
     
     
         33 . The polynucleotide of claim  11 , wherein the IRES sequence region is configured to promote rolling circle translation. 
     
     
         34 . The polynucleotide of claim  11 , wherein the polynucleotide is a DNA sequence, or a linear RNA sequence. 
     
     
         35 . The polynucleotide of  claim 18 , wherein the polynucleotide is a DNA sequence, and wherein the DNA sequence is comprised in a viral vector, a viral like particle, or a non-viral vector. 
     
     
         36 . The polynucleotide of claim  11 , wherein the polynucleotide comprises two self-splicing intron fragments flanking the protein-coding nucleic acid and IRES sequence region, and wherein self-splicing the intron fragments makes a circular RNA. 
     
     
         37 . A method for producing a protein in a cell comprising introducing into the cell the polynucleotide of claim  1 , wherein the polynucleotide produces a circular RNA and wherein the protein coding nucleic acid sequence of the circular RNA is translated to produce the encoded protein in the cell.