Compositions comprising circular polyribonucleotides and uses thereof

1. A pharmaceutical composition for persistent expression of a therapeutic polypeptide, comprising:

a pharmaceutically acceptable excipient; and

a covalently closed polyribonucleotide that (a) persistently expresses the therapeutic polypeptide for at least 7 days in a mammalian subject after the pharmaceutical composition is administered to the mammalian subject, and (b) comprises: (i) an internal ribosome entry site (IRES) element; (ii) an expression sequence that encodes the therapeutic polypeptide, and lacks either a 5′ cap or a 3′ poly-A sequence, and (iii) a termination element.

2. The pharmaceutical composition 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.

3. The pharmaceutical composition 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.

4. The pharmaceutical composition of claim 1 , wherein the termination element comprises a stop codon.

5. The pharmaceutical composition of claim 1 , wherein the expression sequence lacks both the 5′ cap and the 3′ poly-A sequence.

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

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

8. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide comprises at least 400 nucleotides.

9. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide comprises at least 150 amino acids.

10. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide is an antigen.

11. The pharmaceutical composition of claim 10 , wherein the antigen is a viral antigen, a bacterial antigen, or a tumor antigen.

12. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide is at least a functional portion of a viral envelope protein.

13. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide is selected from the group consisting of a hormone, a cytokine, a ligand, a receptor, an antibody, and an enzyme.

14. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide is a secreted protein.

15. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide is an epigenetic modifying agent.

16. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide is an epigenetic enzyme.

17. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide is a nuclease.

18. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide is a component of a CRISPR system.

19. The pharmaceutical composition of claim 1 , wherein the therapeutic polypeptide is a nuclease and the covalently closed polyribonucleotide further comprises a guide RNA sequence.

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

21. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises an encryptogen.

22. The pharmaceutical composition of claim 21 , wherein the encryptogen comprises a splicing element, a modified ribonucleotide, a protein binding site, or an immunoprotein binding site.

23. The pharmaceutical composition of claim 1 , further comprising a polymeric carrier.

24. The pharmaceutical composition of claim 1 , further comprising an exosome.

25. The pharmaceutical composition of claim 1 , further comprising a lipid carrier.

26. The pharmaceutical composition of claim 1 , further comprising a lipid nanoparticle.

27. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition does not include a carrier.

28. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises a miRNA target sequence.

29. A pharmaceutical composition for persistent expression of a therapeutic polypeptide, comprising:

a pharmaceutically acceptable excipient; and

a covalently closed polyribonucleotide that (a) persistently expresses the therapeutic polypeptide for at least 7 days in a mammalian subject after the pharmaceutical composition is administered to the mammalian subject, and (b) comprises: (i) an internal ribosome entry site (IRES) element; (ii) an expression sequence that encodes the therapeutic polypeptide, and lacks a 5′ cap and a 3′ poly-A sequence, and (iii) a termination element,

wherein the therapeutic polypeptide is selected from the group consisting of an antigen, at least a functional portion of a viral envelope 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.

30. A pharmaceutical composition comprising a covalently closed RNA encoding a therapeutic polypeptide, wherein the pharmaceutical composition is produced by a method comprising:

(a) providing a non-coding RNA;

(b) engineering the non-coding RNA (i) to produce a translation-competent RNA and (ii) to add a coding sequence for a therapeutic polypeptide operably linked to a promoter;

(c) covalently joining a first portion and a second portion of the translation-competent RNA produced in step (b) to produce a translation-competent, covalently closed RNA encoding the therapeutic polypeptide;

(d) purifying the translation-competent, covalently closed RNA encoding the therapeutic polypeptide produced in step (c); and

(e) formulating the purified translation-competent, covalently closed RNA encoding the therapeutic polypeptide produced in step (d) with a pharmaceutical excipient to produce a pharmaceutical composition;

wherein the pharmaceutical composition, when administered to a mammalian subject, expresses the therapeutic polypeptide in vivo for at least 7 days.