Compositions comprising circular polyribonucleotides and uses thereof

1. A method of providing a subject with persistent in vivo expression of a polypeptide, comprising:

administering to the subject a circular polyribonucleotide that: (a) comprises an expression sequence encoding the polypeptide, (b) comprises an internal ribosome entry site (IRES) and a termination element, and (c) lacks a poly-A sequence, a free 3′ end, and an RNA polymerase recognition motif; and

translating a biologically effective amount of the polypeptide from the expression sequence of the circular polyribonucleotide in vivo in a cell of the subject over a period of at least 7 days.

2. The method of claim 1 , wherein the circular polyribonucleotide further comprises a stagger element at a 3′ end of the expression sequence.

3. The method of claim 2 , wherein the stagger element encodes a sequence with a C-terminal consensus sequence that is D(V/I)ExNPGP, where x=any amino acid.

4. The method of claim 1 , wherein the expression sequence encodes a secreted protein.

5. The method of claim 1 , wherein the expression sequence encodes an epigenetic modifying agent.

6. The method of claim 1 , wherein the circular polyribonucleotide further comprises at least one element selected from:

(a) an encryptogen;

(b) a regulatory element; and

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

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

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

9. The method of claim 1 , wherein the expression sequence encodes an enzyme.

10. The method of claim 1 , wherein expression sequence encodes a therapeutic protein.

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

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

13. The method of claim 12 , wherein the antigen is a viral antigen, a bacterial antigen, or a tumor antigen.

14. The method of claim 1 , wherein the expression sequence encodes an anti-microbial peptide.

15. The method of or claim 1 , wherein the circular polyribonucleotide is formulated with an exosome.

16. The method of claim 15 , wherein the circular polyribonucleotide further comprises a miRNA target sequence.

17. The method of claim 1 , wherein the circular polyribonucleotide is formulated with a lipid carrier.

18. The method of claim 17 , wherein the circular polyribonucleotide further comprises a miRNA target sequence.

19. The method of claim 1 , wherein the expression sequence encodes an epigenetic enzyme.

20. The method of claim 1 , wherein the expression sequence encodes a nuclease.

21. The method of claim 1 , wherein the expression sequence encodes a component of a CRISPR system.

22. The method of claim 1 , wherein the expression sequence encodes a pore forming peptide.

23. The method of claim 1 , wherein the expression sequence encodes a cytotoxic peptide.

24. The method of claim 1 , wherein the circular polyribonucleotide is formulated with a lipid nanoparticle.

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

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

27. The method of claim 1 , further comprising detecting the biologically effective amount of the polypeptide in the subject over a time period of at least 14 days.

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

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

30. The method of claim 1 , wherein the polypeptide comprises at least 200 amino acids.

31. The method of any claim 1 , wherein the polypeptide comprises at least 250 amino acids.

32. The method of claim 1 , wherein the circular polyribonucleotide comprises at least 400 nucleotides.

33. The method of claim 1 , wherein the circular polyribonucleotide comprises at least 500 nucleotides.

34. The method of claim 1 , wherein the circular polyribonucleotide comprises at least about 1,000 nucleotides.

35. The method of claim 1 , wherein the circular polyribonucleotide further comprises two or more of the expression sequence encoding the polypeptide.

36. The method of claim 1 , wherein the circular polyribonucleotide further comprises a second expression sequence encoding a second polypeptide.