Recombinant nucleic acid molecule of transcriptional circular rna and its application in protein expression

Abstract

The present disclosure relates to a recombinant nucleic acid molecule of the transcriptional circular RNA and its application in protein expression. Specifically, the present disclosure relates to a recombinant nucleic acid molecule of the transcriptional circular RNA, recombinant expression vector, pre-circularized RNA, circular RNA, recombinant host cell, pharmaceutical composition and protein preparing method. The transcription product of the recombinant nucleic acid molecule in this present disclosure is a circular RNA which containing specific IRES element. IRES element can increase the protein expression level of circular RNA in eukaryotic cells, achieve efficient and persistent expression of protein. It has important application value in many fields like: Preparation of mRNA infectious disease vaccines, therapeutic mRNA tumor vaccines, mRNA-based dendritic cell tumor vaccines, mRNA-based gene therapy, mRNA-based chimeric antigen receptor T cell therapy, and protein supplement therapy.

What is claimed is: 
     
         1 . A recombinant nucleic acid molecule comprising an IRES element; wherein, the IRES element includes any one of the following groups (i)-(iv):
 i. A nucleotide sequence comprising one or more sequences in the group consisting of any one of SEQ ID NO: 8-11;   ii. A nucleotide sequence comprising the reverse complement of the sequence shown in any one of SEQ ID NO: 8-11;   iii. A nucleotide sequence that its reverse complement sequence is capable of hybridizing with the nucleotide sequence shown in (i) or (ii), under high stringency hybridization conditions or very high stringency hybridization conditions;   iv. A sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (i) or (ii).   
     
     
         2 . The recombinant nucleic acid molecule of  claim 1 , wherein the recombinant nucleic acid molecule further comprises a 5′homology arm located upstream of the IRES element, and a 3′homology arm located downstream of the coding region and also complementary to the 5′ homology arm located upstream;
 preferably, 
 the 5′homology arm comprises a sequence shown in any one of the following (a1)-(a2): 
 (a1) The nucleotide sequence shown in any one of SEQ ID NO: 2-3; 
 (a2) A sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (a1); 
 The 3′homology arm includes the sequence shown in any one of (b1)-(b2) below: 
 (b1) The nucleotide sequence shown in any one of SEQ ID NO: 17-18; 
 (b2) A sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (b1); 
 preferably, the recombinant nucleic acid molecule further comprises a 5′spacer located between the 5′homology arm and the IRES element, and a 3′spacer located between the coding region and the 3′homology arm; 
 preferably, the 5′spacer contains the sequence shown in any one of the following (c1)-(c2): 
 (c1) The nucleotide sequence shown in any one of SEQ ID NO: 6-7; 
 (c2) A sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (c1); 
 the 3′spacer includes the sequence shown in any of the following (o1)-(o2): 
 (o1) The nucleotide sequence shown in any one of SEQ ID NO: 52-53; 
 (o2) A sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (o1). 
 
     
     
         3 . The recombinant nucleic acid molecule of  claim 1 , wherein the recombinant nucleic acid molecule further comprises a 3′intron and a second exon located between the 5′homology arm and the IRES element, and the first exon and 5′intron located between the coding region and the 3′homology arm;
 preferably, 
 The 3′intron is located upstream of the second exon, and the 5′spacer is included between the second exon and the IRES element; The first exon is located upstream of the 5′intron, and the 3′spacer is included between the first exon and the coding region; 
 preferably, the 3′intron comprises a sequence shown in any one of the following (d1)-(d2): 
 (d1) The nucleotide sequence shown in SEQ ID NO: 4; 
 (d2) A sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (d1); 
 The second exon includes the sequence shown in any one of (e1)-(e2) below: 
 (e1) The nucleotide sequence shown in SEQ ID NO: 5; 
 (e2) A sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the nucleotide sequence shown in (e1); 
 the first exon includes the sequence shown in any one of the following (f1)-(f2): 
 (f1) The nucleotide sequence shown in SEQ ID NO: 15; 
 (f2) A sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (f1); 
 the 5′intron includes the sequence shown in any one of (g1)-(g2): 
 (g1) The nucleotide sequence shown in SEQ ID NO: 16; 
 (g2) A sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (g1); 
 more preferably, the recombinant nucleic acid molecule further comprises a regulatory sequence for directing the recombinant nucleic acid molecule to transcribe the circular RNA. 
 
     
     
         4 . A recombinant expression vector, wherein the recombinant expression vector comprises the recombinant nucleic acid molecule of  claim 1 . 
     
     
         5 . A pre-circularized RNA formed by transcribing the recombinant nucleic acid molecule of  claim 1 ;
 preferably, the pre-circularized RNA includes 5′homology arms, 3′introns, second exons, 5′spacers, IRES elements, coding regions, 3′spacers, first exons, 5′introns and 3′homology arm.   
     
     
         6 . A circular RNA formed by circularization of the recombinant nucleic acid molecule of  claim 1 ;
 optionally, the circular RNA includes a second exon, a 5′spacer, an IRES element, a coding region, a 3′spacer and a first exon that are sequentially connected.   
     
     
         7 . The circular RNA of  claim 6 , wherein the circular RNA expresses a target polypeptide. 
     
     
         8 . The circular RNA of  claim 7 , wherein the target polypeptide is the receptor binding domain (RBD) of the S protein of the novel coronavirus; preferably, the RBD protein is selected from any one of the following (h1)-(h4):
 (h1) A polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 32 and having RBD protein activity;   (h2) The amino acid sequence shown in SEQ ID NO: 32 has been substituted, repeated, deleted or added one or more amino acids, and has RBD protein activity polypeptide;   (h3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (h1) or (h2);   (h4) Encoded by a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 31, And a polypeptide with RBD protein activity;   preferably, the circular RNA contains at least 90%, optionally at least 95%, preferably at least 97% of the nucleotide sequence shown in SEQ ID NO: 33, more preferred are sequences with at least 98%, and most preferred at least 99% sequence identity.   
     
     
         9 . The circular RNA of  claim 7 , wherein the target polypeptide is selected from the group consisting of programmed cell death protein 1 (PD-1), programmed cell death ligand-1 (programmed cell death ligand-1, PD-L1) or Cytotoxic T-Lymphocyte Associated Protein-4 (CTLA-4) monoclonal antibody;
 preferably, the PD-1 monoclonal antibody comprises any one of the following (j1)-(j6):   (j1) A light chain comprising the amino acid sequence shown in SEQ ID NO: 38;   (j2) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 41;   (j3) Contains a polypeptide with light chain protein activity after substitution, repetition, deletion or addition of one or more amino acids with the amino acid sequence shown in SEQ ID NO: 38;   (j4) Contains a polypeptide with heavy chain protein activity after substitution, repetition, deletion or addition of one or more amino acids with the amino acid sequence shown in SEQ ID NO: 41;   (j5) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in any one of (j1)-(j4);   (j6) Encoded by a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 37, and a polypeptide having light chain protein activity; or, having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 90% of the nucleotide sequence shown in SEQ ID NO: 40 A polypeptide that is encoded by a sequence with 99% sequence identity and has heavy chain protein activity;   preferably, the circular RNA contains at least 90%, optionally at least 95%, preferably at least 97% of the nucleotide sequence shown in SEQ ID NO: 39 or 42, more preferably at least 98%, most preferably at least 99% sequence identity.   
     
     
         10 . The circular RNA of  claim 7 , wherein the target polypeptide is a chimeric antigen receptor; optionally, the target polypeptide is a CD16 protein of a chimeric antigen receptor, and the CD16 protein is selected from the following any one of (k1)-(k4):
 (k1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 50 and having CD16 protein activity;   (k2) The amino acid sequence shown in SEQ ID NO: 50 has been substituted, repeated, deleted or added one or more amino acids, and has CD16 protein activity;   (k3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (k1) or (k2);   (k4) Encoded by a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 49, And a polypeptide with CD16 protein activity;   preferably, the circular RNA contains at least 90%, optionally at least 95%, preferably at least 97% of the nucleotide sequence shown in SEQ ID NO: 51, More preferred are sequences with at least 98%, and most preferred at least 99% sequence identity.   
     
     
         11 . The circular RNA of  claim 7 , wherein the target polypeptide is a recombinant humanized protein, optionally, the recombinant humanized protein is a recombinant human erythropoietin (EPO) protein, and the EPO protein Selected from any of the following (l1)-(l4):
 (l1) A polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 35 and having EPO protein activity;   (l2) The amino acid sequence shown in SEQ ID NO: 35 has been substituted, repeated, deleted or added with one or more amino acids, and has EPO protein activity;   (l3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (l1) or (l2);   (l4) Encoded by a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the nucleotide sequence shown in SEQ ID NO: 34, And a polypeptide with EPO protein activity;   preferably, the circular RNA contains at least 90%, optionally at least 95%, preferably at least 97% of the nucleotide sequence shown in SEQ ID NO: 36, more preferred are sequences with at least 98%, and most preferred at least 99% sequence identity.   
     
     
         12 . The circular RNA of  claim 7 , wherein the target polypeptide is a cytokine; preferably, the cytokine is an IL-15 protein, and the IL-15 protein is selected from the following (m1)-(m4) Any one of:
 (m1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 44 and having IL-15 protein activity;   (m2) The amino acid sequence shown in SEQ ID NO: 44 has been substituted, repeated, deleted or added with one or more amino acids, and has IL-15 protein activity;   (m3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (m1) or (m2);   (m4) Encoded by a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 43, And a polypeptide with IL-15 protein activity;   preferably, the circular RNA contains at least 90%, optionally at least 95%, preferably at least 97% of the nucleotide sequence shown in SEQ ID NO: 45, more preferred are sequences with at least 98%, and most preferred at least 99% sequence identity.   
     
     
         13 . The circular RNA of  claim 7 , wherein the target polypeptide is a tumor-associated antigen or a tumor-specific antigen, optionally, the tumor-specific antigen is a PAP protein, and the PAP protein is selected from the following (n1 Any one of)-(n4):
 (n1) A polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 47 and having PAP protein activity;   (n2) The amino acid sequence shown in SEQ ID NO: 47 has been substituted, repeated, deleted or added one or more amino acids, and has PAP protein activity polypeptide;   (n3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (n1) or (n2);   (n4) Encoded by a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 46, and has PAP protein activity polypeptide;   preferably, the circular RNA contains at least 90%, optionally at least 95%, preferably at least 97% of the nucleotide sequence shown in SEQ ID NO: 48, more preferred are sequences with at least 98%, and most preferred at least 99% sequence identity.   
     
     
         14 . A recombinant host cell, wherein the recombinant host cell comprises the recombinant nucleic acid molecule of  claim 1 . 
     
     
         15 . A recombinant nucleic acid molecule of  claim 1  for use in the production of a protein. 
     
     
         16 . A pharmaceutical composition, comprising
 the recombinant nucleic acid molecule of  claim 1 .   
     
     
         17 . A method for preparing a protein, comprising expressing a target protein using the recombinant host cell of  claim 14 . 
     
     
         18 . A method for preventing or treating diseases in a subject, comprising the steps of administering the recombinant nucleic acid molecule of  claim 1  to the subject. 
     
     
         19 . The recombinant nucleic acid molecule of  claim 1 , wherein the recombinant nucleic acid molecule further includes a coding region encoding a target polypeptide, and the IRES element can increase the expression level of the target polypeptide; preferably, the IRES element can increase the expression level of the target polypeptide in eukaryotic cells. 
     
     
         20 . The recombinant nucleic acid molecule of  claim 1 , wherein the IRES element is selected from any one of the following (q1)-(q7):
 (q1) A nucleotide sequence comprising the sequence shown in SEQ ID NO: 8;   (q2) A nucleotide sequence comprising the sequence shown in SEQ ID NO: 9;   (q3) A nucleotide sequence comprising the sequence shown in SEQ ID NO: 10;   (q4) A nucleotide sequence comprising the sequence shown in SEQ ID NO: 11;   (q5) A nucleotide sequence comprising the sequence shown in SEQ ID NO: 12;   (q6) A nucleotide sequence comprising the sequence shown in SEQ ID NO: 13;   (q7) A nucleotide sequence comprising the sequence shown in SEQ ID NO: 14.