Compositions comprising circular polyribonucleotides and uses thereof

1. A method of expressing a polypeptide in a subject, comprising:

administering to the subject a covalently closed polyribonucleotide that: (a) comprises an internal ribosome entry site (IRES) element; (b) comprises an expression sequence that encodes the polypeptide and that lacks one or both of a 5′ cap and a poly-A sequence; and (c) comprises a termination element; and

translating the polypeptide from the expression sequence of the covalently closed polyribonucleotide in vivo over to of at least 7 days.

2. The method of claim 1 , wherein the polypeptide is a therapeutic polypeptide.

3. The method of claim 1 , wherein the covalently closed polyribonucleotide further comprises at least one element selected from:

(a) an encryptogen;

(b) a regulatory element; and

(c) a quasi-double-stranded secondary structure.

4. The method of claim 1 , wherein the internal ribosome entry site element comprises a sequence derived from picornavirus complementary DNA, encephalomyocarditis virus (EMCV) complementary DNA, poliovirus complementary DNA, or an Antennapedia gene from Drosophila melanogaster.

5. The method of claim 1 , wherein the termination element comprises a stop codon.

6. The method of claim 1 , wherein the covalently closed polyribonucleotide comprises two or more of the expression sequence encoding the polypeptide.

7. The method of claim 1 , wherein the covalently closed polyribonucleotide further comprises a second expression sequence encoding a second polypeptide.

8. The method of claim 1 , wherein the polypeptide comprises at least 150 amino acids.

9. The method of claim 1 , wherein the polypeptide is an antigen.

10. The method of claim 9 , wherein the antigen is a viral antigen, a bacterial antigen, or a tumor antigen.

11. The method of claim 1 , wherein the polypeptide is at least a functional portion of a viral envelope protein.

12. The method of claim 1 , wherein the polypeptide is selected from the group consisting of a hormone, a cytokine, a ligand, a receptor, an antibody, and an enzyme.

13. The method of claim 1 , wherein the polypeptide is a secreted protein.

14. The method of claim 1 , wherein the polypeptide is an epigenetic modifying agent.

15. The method of claim 1 , wherein the polypeptide is an epigenetic enzyme.

16. The method of claim 1 , wherein the polypeptide is a nuclease.

17. The method of claim 1 , wherein the polypeptide is a component of a CRISPR system.

18. The method of claim 1 , wherein the polypeptide is a nuclease and the covalently closed polyribonucleotide further comprises a guide RNA sequence.

19. The method of claim 1 , wherein the polypeptide is selected from the group consisting of a pore forming peptide, a cytotoxic peptide, and an anti-microbial peptide.

20. The method of claim 1 , wherein the covalently closed polyribonucleotide further comprises an encryptogen.

21. The method of claim 20 , wherein the encryptogen comprises a splicing element, a modified ribonucleotide, or a protein binding site.

22. The method of claim 20 , wherein the encryptogen comprises an immunoprotein binding site.

23. The method of claim 1 , wherein the covalently closed polyribonucleotide is formulated, for the administration, with:

a) a pharmaceutically acceptable excipient;

b) a polymeric carrier;

c) an exosome;

d) a lipid carrier; or

e) a lipid nanoparticle.

24. The method of claim 1 , wherein the covalently closed polyribonucleotide further comprises a miRNA target sequence.

25. The method of claim 1 , further comprising detecting expression of the expression sequence in a cell or a tissue of the subject before and/or after the administration.

26. The method of claim 1 , further comprising detecting the polypeptide in the subject over a time period of at least 7 days.

27. The method of claim 1 , further comprising detecting the polypeptide in the subject over a time period of at least 21 days.

28. The method of claim 1 , wherein the subject is a human.

29. The method of claim 1 , wherein the subject is a non-human mammal.

30. A method of delivering a therapeutic polypeptide to a human, comprising:

administering to the human a covalently closed polyribonucleotide that: (a) comprises an internal ribosome entry site (IRES) element; (b) comprises an expression sequence that encodes the therapeutic polypeptide and that lacks one or both of a 5′ cap and a poly-A sequence; and (c) comprises a termination element; and

translating the therapeutic polypeptide from the expression sequence of the covalently closed polyribonucleotide in vivo over a period of at least 7 days,

wherein the therapeutic polypeptide is selected from the group consisting of an antigen, at least a functional portion of a viral envelop protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a secreted protein, an epigenetic modifying agent, a nuclease, a component of a CRISPR system, a pore forming peptide, a cytotoxic peptide, and an anti-microbial peptide.