US-20260124229-A1 - COMPOSITIONS COMPRISING CIRCULAR POLYRIBONUCLEOTIDES AND USES THEREOF

Abstract

This invention relates generally to pharmaceutical compositions and preparations of circular polyribonucleotides and uses thereof.

Inventors

• Avak Kahvejian
• Nicholas McCartney Plugis
• Alexandra Sophie DE BOER

Assignees

• FLAGSHIP PIONEERING INNOVATIONS VI, LLC

Dates

Publication Date

20260507

Application Date

20251222

Claims (20)

1. 1 . A pharmaceutical composition comprising: (a) a covalently closed polyribonucleotide comprising, in the following order: (i) an internal ribosome entry site (IRES) operably linked to an expression sequence encoding a chimeric antigen receptor (CAR); and (ii) the expression sequence encoding the CAR, wherein the covalently closed polyribonucleotide has been circularized by chemical ligation or ribozyme-mediated circularization; and (b) a pharmaceutical carrier.
2. 2 . The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises a spacer sequence.
3. 3 . The pharmaceutical composition of claim 2 , wherein the spacer comprises a nucleic acid sequence having low GC content.
4. 4 . The pharmaceutical composition of claim 2 , wherein the spacer is substantially free of a secondary structure.
5. 5 . The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide further comprises at least one microRNA binding site.
6. 6 . The pharmaceutical composition of claim 1 , wherein the covalently closed polyribonucleotide comprises at least one modified ribonucleotide.
7. 7 . The pharmaceutical composition of claim 6 , wherein the modified ribonucleotide is selected from pseudouridine and N6-methyladenosine (m6A).
8. 8 . The pharmaceutical composition of claim 1 , wherein the pharmaceutical carrier is a lipid carrier.
9. 9 . A linear polyribonucleotide comprising, in the following order from 5′ to 3′: (a) (i) a 5′ nucleotide comprising a first reactive group; (ii) an IRES operably linked to an expression sequence encoding a CAR; (iii) the expression sequence encoding the CAR; and (iv) a 3′ nucleotide comprising a second reactive group; or (b) (i) a first ribozyme; (ii) an IRES operably linked to an expression sequence encoding a CAR; (iii) the expression sequence encoding the CAR; and (iv) a second ribozyme.
10. 10 . The linear polyribonucleotide of claim 9 , wherein the first reactive group comprises an NHS-ester group and the second reactive group comprises a terminal amino group.
11. 11 . The linear polyribonucleotide of claim 9 , wherein the first and second ribozymes are permuted Group I intron-exons, Hepatitis Delta Virus ribozymes, or hairpin ribozymes.
12. 12 . The linear polyribonucleotide of claim 9 , wherein the linear polyribonucleotide further comprises a spacer sequence.
13. 13 . The linear polyribonucleotide of claim 12 , wherein the spacer comprises a nucleic acid sequence having low GC content.
14. 14 . The linear polyribonucleotide of claim 12 , wherein the spacer is substantially free of a secondary structure.
15. 15 . The linear polyribonucleotide of claim 9 , wherein the linear polyribonucleotide further comprises at least one microRNA binding site.
16. 16 . The linear polyribonucleotide of claim 9 , wherein the linear polyribonucleotide comprises at least one modified ribonucleotide.
17. 17 . The linear polyribonucleotide of claim 16 , wherein the modified ribonucleotide is selected from pseudouridine and N6-methyladenosine (m6A).
18. 18 . A method of producing a covalently closed polyribonucleotide comprising: (i) providing a linear polyribonucleotide of claim 9 ; and (ii) circularizing the linear polyribonucleotide to produce the covalently closed polyribonucleotide.
19. 19 . The method of claim 18 , wherein the first reactive group comprises an NHS-ester group and the second reactive group comprises a terminal amino group.
20. 20 . The method of claim 18 , wherein the first and second ribozyme ligate the 5′ end to the 3′ end of the linear polyribonucleotide.

Description

SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 22, 2025, is named 51509-0050014_Sequence_Listing_12_22_25.xml and is 181,409 bytes in size. BACKGROUND Certain circular polyribonucleotides are ubiquitously present in human tissues and cells, including tissues and cells of healthy individuals. SUMMARY In one aspect, the invention includes a pharmaceutical composition comprising a circular polyribonucleotide that comprises at least one structural element selected from a) an encryptogen; b) a stagger element; c) a regulatory element; d) a replication element; f) quasi-double-stranded secondary structure; and g) expression sequence; and at least one functional characteristic selected from: a) greater translation efficiency than a linear counterpart; b) a stoichiometric translation efficiency of multiple translation products; c) less immunogenicity than a counterpart lacking an encryptogen; d) increased half-life over a linear counterpart; and e) persistence during cell division. In some embodiments, the circular polyribonucleotide is translation competent. In one such embodiment, the quasi-helical structure comprises at least one double-stranded RNA segment with at least one non-double-stranded segment. In another such embodiment, the quasi-helical structure comprises a first sequence and a second sequence linked with a repetitive sequence, e.g., an A-rich sequence. In some embodiments, the circular polyribonucleotide comprises an encryptogen. In some embodiments, the encryptogen comprises at least one modified ribonucleotide, e.g., pseudo-uridine, N(6)methyladenosine (m6A). In some embodiments, the encryptogen comprises a protein binding site, e.g., ribonucleotide binding protein. In some embodiments, the encryptogen comprises an immunoprotein binding site, e.g., to evade immune Reponses, e.g., CTL responses. In some embodiments, the circular polyribonucleotide comprises at least one modified ribonucleotide. In some embodiments, the circular polyribonucleotide has at least 2× less immunogenicity than a counterpart lacking the encryptogen, e.g., as assessed by expression or signaling or activation of at least one of RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, IFN-beta. In some embodiments, the circular polyribonucleotide further comprises a riboswitch. In some embodiments, the circular polyribonucleotide further comprises an aptazyme. In some embodiments, the circular polyribonucleotide comprises a translation initiation sequence, e.g., GUG, CUG start codon, e.g., expression under stress conditions. In some embodiments, the circular polyribonucleotide comprises at least one expression sequence, e.g., encoding a polypeptide. In one such embodiments, the expression sequence encodes a peptide or polynucleotide. In some embodiments, the circular polyribonucleotide comprises a plurality of expression sequences, either the same or different. In some embodiments, the circular polyribonucleotide comprises a stagger element, e.g., 2A. In some embodiments, the circular polyribonucleotide comprises a regulatory nucleic acid, e.g., a non-coding RNA. In some embodiments, the circular polyribonucleotide comprises a regulatory element, e.g., that alters expression of an expression sequence. In some embodiments, the circular polyribonucleotide has a size in the range of about 20 bases to about 20 kb. In some embodiments, the circular polyribonucleotide is synthesized through circularization of a linear polynucleotide. In some embodiments, the circular polyribonucleotide is substantially resistant to degradation, e.g., exonuclease. In some embodiments, the circular polyribonucleotide lacks at least one of: a) a 5′-UTR; b) a 3′-UTR; c) a poly-A sequence; d) a 5′-cap; e) a termination element; f) an internal ribosomal entry site; g) degradation susceptibility by exonucleases and h) binding to a cap-binding protein. In one aspect, the invention includes a method of producing the composition comprising a circular polyribonucleotide described herein. In one aspect, the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a circular polyribonucleotide that comprises one or more expression sequences, wherein the circular polyribonucleotide is competent for rolling circle translation. In some embodiments, each of the one or more expression sequences is separated from a succeeding expression sequence by a stagger element in the circular polyribonucleotide, wherein rolling circle translation of the one or more expression sequences generates at least two polypeptide molecules, e.g., the stagger elements stalls or halts the ribosome such that the elongating polypeptide falls off the ribosome. In some embodiments, the stagger element prevents generation of a single polypeptide (a) from two rounds of transla