Compositions comprising circular polyribonucleotides and uses thereof
This invention relates generally to pharmaceutical compositions and preparations of circular polyribonucleotides and uses thereof.
1. A pharmaceutical composition for expression of a chimeric antigen receptor (CAR) comprising: a lipid nanoparticle carrier; a covalently closed polyribonucleotide that (a) expresses the CAR for at least 7 days in a human after the pharmaceutical composition is administered to the human, and (b) comprises: (i) an internal ribosome entry site (IRES); (ii) an expression sequence that encodes the CAR, and lacks one or both of: a 5′ cap and a 3′ poly-A sequence; and (iii) a stop codon; and
a pharmaceutically acceptable excipient.
2. The pharmaceutical composition of claim 1 , wherein the expression sequence that encodes the CAR lacks both the 5′ cap and the 3′poly A sequence.
3. The pharmaceutical composition of claim 1 , wherein the IRES has at least about 5 nucleotides.
4. The pharmaceutical composition of claim 3 , wherein the IRES is derived from viral DNA selected from the group consisting of picornavirus complementary DNA, encephalomyocarditis virus (EMCV) complementary DNA, and poliovirus complementary DNA.
5. The pharmaceutical composition of claim 3 , wherein the IRES comprises a sequence having at least about 10 nucleotides and is derived from viral DNA selected from the group consisting of picornavirus complementary DNA, encephalomyocarditis virus (EMCV) complementary DNA, and poliovirus complementary DNA.
6. The pharmaceutical composition of claim 3 , wherein the IRES comprises a sequence having at least 10 nucleotides and is derived an Antennapedia gene from Drosophila melanogaster DNA.
7. The pharmaceutical composition of claim 3 , wherein the IRES is derived from viral DNA.
8. The pharmaceutical composition of claim 7 , wherein the IRES is an EMCV IRES or a CVB3 IRES.
9. The pharmaceutical composition of claim 1 , wherein the IRES is an EMCV IRES or a CVB3 IRES.
10. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide is detectable in vivo for at least 7 days after administration of the composition to the human.
11. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide is detectable in-vivo for at least 21 days after administration of the composition to the human.
12. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide comprises a second expression sequence.
13. The pharmaceutical composition of claim 1 , wherein expression sequence that encodes the CAR lacks a 3′ polyA sequence having greater than 30 nucleotides.
14. The pharmaceutical composition of claim 1 , wherein the expression sequence that encodes the CAR lacks a 3′ polyA sequence having between 50 and 250 nucleotides.
15. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises a miRNA target sequence.
16. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises a modified ribonucleotide.
17. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises a protein binding site or a miRNA binding site.
18. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises an untranslated region (UTR) or a spacer sequence.
19. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises a translation initiation sequence.
20. The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises a splicing element.
21. A pharmaceutical composition for expression of a fusion protein, comprising:
a covalently closed polyribonucleotide comprising: (i) an internal ribosome entry site (IRES); (ii) an expression sequence that encodes the fusion protein and lacks a 5′ cap or lacks a 3′ poly A sequence greater than 15 nucleotides in length, and (iii) a stop codon; a pharmaceutically acceptable excipient; and a pharmaceutical carrier to administer the covalently closed polyribonucleotide to a human subject; wherein the covalently closed polyribonucleotide expresses the fusion protein for at least 7 days upon the administration.
22. The pharmaceutical composition of claim 21 , wherein the carrier is a polymeric carrier, exosome, or lipid nanoparticle.
23. The pharmaceutical composition of claim 21 wherein the fusion protein is a chimeric antigen receptor.
24. The pharmaceutical composition of claim 21 , wherein the covalently closed polyribonucleotide is detectable in vivo for at least 7 days.
25. The pharmaceutical composition of claim 21 , wherein the expression sequence lacks a 3′ poly A sequence having from 30 to 750 nucleotides.
26. A pharmaceutical composition for expression of a chimeric antigen receptor (CAR) comprising: a covalently closed polyribonucleotide that (a) expresses the CAR for at least 7 days in a human after the pharmaceutical composition is delivered to the human, and (b) comprises: (i) an internal ribosome entry site (IRES); (ii) an expression sequence that encodes the CAR and lacks 3′ poly-A sequence having 100 to 500 nucleotides; and (iii) a stop codon; a lipid nanoparticle delivery carrier that includes the covalently closed polyribonucleotide for delivering the covalently closed polyribonucleotide to the human; and a pharmaceutically acceptable excipient.
27. The pharmaceutical composition of claim 26 , wherein the expression sequence that encodes the CAR also lacks a 5′ cap.
28. The pharmaceutical composition of claim 26 , wherein the IRES is derived from viral DNA.
29. A pharmaceutical composition for expression of a fusion protein comprising: a lipid nanoparticle carrier; a covalently closed polyribonucleotide that (a) expresses the fusion protein for at least 7 days in a human after the pharmaceutical composition is administered to the human, and (b) comprises: (i) an internal ribosome entry site (IRES) derived from viral DNA; (ii) an expression sequence that encodes the CAR and lacks 3′ poly-A sequence having 100 to 500 nucleotides; and (iii) a stop codon; and
a pharmaceutically acceptable excipient.
30. The pharmaceutical composition of claim 29 , wherein the IRES is synthesized using modified or non-modified nucleotides.