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1. WO2020139843 - ENGINEERED HEPATITIS B CORE POLYPEPTIDE

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[ EN ]

WHAT IS CLAIMED IS:

1. A hepatitis B core protein (HBc) polypeptide comprising an amino acid sequence modification to enhance uptake and retention of encapsulated cargo.

2. The HBc polypeptide of claim 1 , wherein the amino acid modification comprises the addition of a cargo-loading domain at the carboxy terminus.

3. The HBc polypeptide of claim 2, wherein the cargo-loading domain is from 1 to

15 amino acids in length.

4. The HBc polypeptide of claim 2 or claim 3, wherein the cargo-loading domain comprises one cysteine residue.

5. The HBc polypeptide of claim 2 or claim 3, wherein the cargo-loading domain is selected from (SEQ ID NO:4) EGFGEGFGEGF; (SEQ ID NO:5) EGFGEGFGEGFC; (SEQ ID NO:6) IGIGC; (SEQ ID NO:7) IGIGIC; RRR; R; NIC; C; CRC; EEE.

6. The HBc polypeptide of any of claims 2-5, further comprising a C-terminal polyhistidine tag before or after the cargo-loading domain.

7. The HBc polypeptide of any of claims 1-6, comprising an amino acid substitution at residue A131 to a basic amino acid.

8. The HBc polypeptide of claim 7, wherein the substitution is A131 K.

9. The HBc polypeptide of any of claims 1-8, further comprising one or more amino acid substitutions selected from [D29C, R127C]; [T109C, V120C]; [Y132C, N136C]; [Y132C, A137C]; [R133C, N136C]; [R133C, A137C]; [P134C, P135C]; [P134C, N136C]; [P134C, A137C]; and [P135C, N136C],

10. The HBc polypeptide of claim 9, wherein the amino acid substitutions are [D29C,

R127C, P134C, N136C],

11. The HBc polypeptide of claim 7 or 8, further comprising amino acid substitutions [157V, L60S, G63R, D64E, L65V, M66T, T67D, L68F, A69G, T70D, T74N, L76M, E77Q, P79Q, S81A, S87N, T91A, V93I, F97I] or [T74N, L76M, E77Q, P79Q, S81A],

12. The HBc polypeptide of any one of Claims 1-11 , wherein the polypeptide further comprises at least one unnatural amino acid.

13. The HBc polypeptide of Claim 12, wherein the unnatural amino acid provides a reactant group for Click chemistry reactions.

14. The HBc polypeptide of Claim 13, wherein the unnatural amino acid is azidohomoalanine, homopropargylglycine, p-acetyl-L-phenylalanine or p-azido-L-phenylalanine.

15. The HBc polypeptide of Claim 14, further comprising one or more additional moieties conjugated to the unnatural amino acid.

16. A VLP comprised of HBc polypeptides according to any of claims 1-15.

17. The VLP of claim 16, wherein the VLP comprises encapsulated cargo.

18. The VLP of claim 17, wherein the cargo is ssRNA, dsRNA, ssDNA, dsDNA, protein, or small molecule drug.

19. The VLP of claim 17, wherein the cargo is a chemotherapeutic drug.

20. The VLP of claim 17, wherein the cargo is modified to enhance retention or uptake.

21. The VLP of claim 20, wherein the cargo is modified by conjugation to cholesterol.

22. The VLP of claim 20, wherein the cargo is modified to add a single sulfhydryl group.

23. A method of assembling a cargo loaded VLP, the method comprising adding cargo to a solution comprising a polypeptide according to any of claims 1-15, wherein the tonicity is less than 1 M NaCI to trigger VLP self-assembly.

treating the assembled VLP with an oxidizing agent to form disulfide bonds.

24. The method of claim 23, wherein the tonicity is less than 0.5 M NaCI.

25. A method of assembling a cargo loaded VLP, the method comprising adding cargo to a solution comprising a polypeptide according to any of claims 1-15, wherein the iconicity is greater than 1 M NaCI to trigger VLP self-assembly, and. in addition, the solution comprises at least 10% of an organic solvent.

26. The method of claim 25, wherein the organic solvent is DMSO.

27. The method of any of claims 23-26, wherein the cargo-loaded VLP is resuspended in normal saline or a comparable excipient.