Engineered hepatitis b core polypeptide

Abstract

Genetically modified HBc polypeptides are provided.

1 . A hepatitis B core protein (HBc) polypeptide comprising the addition of a cargo-loading domain from 1 to 15 amino acids in length at the carboxy terminus to enhance uptake and retention of encapsulated cargo. 
     
     
         2 - 3 . (canceled) 
     
     
         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 1 , wherein the cargo-loading domain is selected from RRR; R; IIIC; C; CRC; EEE. 
     
     
         6 . The HBc polypeptide of any of  claim 1 , further comprising a C-terminal poly-histidine tag before or after the cargo-loading domain. 
     
     
         7 . The HBc polypeptide of  claim 1 , comprising an amino acid substitution at residue A131 to a basic amino acid. 
     
     
         8 . The HBc polypeptide of  claim 7 , wherein the substitution is A131K. 
     
     
         9 . The HBc polypeptide of  claim 1 , 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 1 , further comprising amino acid substitutions [T74N, L76M, E77Q, P79Q, S81A]. 
     
     
         12 . The HBc polypeptide of any  claim 1 , 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  claim 1 . 
     
     
         17 . The VLP of  claim 16 , wherein the VLP comprises encapsulated cargo. 
     
     
         18 - 19 . (canceled) 
     
     
         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  claim 1 , wherein the ionicity is less than 1 M NaCl to trigger VLP self-assembly,
 treating the assembled VLP with an oxidizing agent to form disulfide bonds.   
     
     
         24 . (canceled) 
     
     
         25 . A method of assembling a cargo loaded VLP, the method comprising adding cargo to a solution comprising a polypeptide according to  claim 1 , wherein the iconicity is greater than 1 M NaCl to trigger VLP self-assembly, and, in addition, the solution comprises at least 10% of an organic solvent. 
     
     
         26 - 27 . (canceled)