# US 2013/0156818 A1 · Methods and compositions for controlling assembly of viral proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0156818 A1 - Document ID: US20130156818A1 - Status: Published application - Kind: A1 - Application number: 13/609,234 - Filed: 2012-09-10 - Published: 2013-06-20 - Earliest claimed: 2011-09-09 - Assignees: Biomed Realty, L.P. - Inventors: Miguel de los Rios; Stephanie de los Rios; Jacek Ostrowski; Kenneth J. Oh; Ilan Zipkin - Web page: https://patentgrep.com/patent/US20130156818A1 ## CPC - C07K 14/005: from viruses - A61K 9/5184: {Virus capsids or envelopes enclosing drugs (liposomes with additional exogenous lipids A61K9/127)} - A61K 31/713: Double-stranded nucleic acids or oligonucleotides - A61K 38/162: {from virus} - A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65) - A61P 9/00: Drugs for disorders of the cardiovascular system - A61P 25/00: Drugs for disorders of the nervous system - A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] - A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics - A61P 31/12: Antivirals - A61P 35/00: Antineoplastic agents - A61P 37/02: Immunomodulators - C07K 1/1072: {by covalent attachment of residues or functional groups} - C07K 1/1075: {by covalent attachment of amino acids or peptide residues} - C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids} - C07K 1/1133: {by redox-reactions involving cystein/cystin side chains} - C07K 1/1136: {by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents} - C12N 2730/10122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Provided herein are methods and compositions for controlling assembly of modified viral core proteins, for example, into a viral capsid or a nanocage. In some embodiments, the disclosed modified viral core proteins comprise at least one mutation or modification that can substantially prevent assembly of the viral core proteins until assembly is desired. In some embodiments, assembly of the viral core proteins may be triggered, for example, by contacting the viral core proteins with a reducing agent and/or by reducing the concentration of a denaturant. The viral core proteins may self-assemble to form a viral capsid or nanocage. ## Claims ### Claim 1 A method for assembling a modified Hepatitis B Virus (HBV) core protein into a capsid structure, the method comprising: providing a solution comprising a modified HBV core protein and a first concentration of a denaturing agent, wherein the spike region of the modified HBV core protein comprises a cysteine residue; and adding a reducing agent to the solution, thereby to form an assembled capsid structure. ### Claim 2 (depends on 1) The method of claim 1, wherein the modified HBV core protein comprises one or more modifications of as least one of amino acid sequence SEQ ID NO: 1 and SEQ ID NO: 2. ### Claim 3 (depends on 1) The method of claim 1, wherein the spike region comprises amino acids 74 to 84 of at least one of SEQ ID NO: 1 and SEQ ID NO: 2. ### Claim 4 (depends on 2) The method of claim 2, wherein the modified HBV core protein comprises a cysteine at amino acid position 77, 79 or 80 of at least one of SEQ ID NO: 1 and SEQ ID NO: 2. ### Claim 5 (depends on 2) The method of claim 2, wherein the modified HBV core protein comprises a cysteine at amino acid position 77 of at least one of SEQ ID NO: 1 and SEQ ID NO: 2. ### Claim 6 (depends on 1) The method of claim 1, wherein the reducing agent is at least one of beta-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP), glutathione (GSH), dithiothreitol (DTT), 2-mercaptoyethylamine (BMA) and free cysteine. ### Claim 7 (depends on 1) The method of claim 1, wherein the concentration of the reducing agent is from about 0.1 molar equivalent to about 100 molar equivalent. ### Claim 8 (depends on 1) The method of claim 1, further comprising diluting a first concentration of denaturing agent to a second concentration thereby to form an assembled capsid structure and wherein the denaturing agent is diluted at least one of prior to, during, and following the addition of the reducing agent. ### Claim 9 (depends on 8) The method of claim 8, wherein the first concentration of denaturing agent is diluted to the second concentration at least one of prior to, during, and after the addition of the reducing agent. ### Claim 10 (depends on 1) The method of claim 1, further comprising adding a negatively charged polymer to the solution. ### Claim 11 (depends on 1) The method of claim 1, wherein the pH of the solution is about pH 7.0 or lower. ### Claim 12 (depends on 1) The method of claim 1, further comprising adding a drug to the solution and wherein the drug is added at least one of prior to, during, or after the addition of the reducing agent. ### Claim 13 (depends on 1) The method of claim 1, wherein the drug binds to the amino acid tail portion of the modified HBV core protein. ### Claim 14 (depends on 1) The method of claim 1, wherein the drug is at least one of encapsulated in the capsid structure by diffusion following the addition of the reducing agent, and bound to the amino acid tail portion and encapsulated in the capsid structure following addition of the reducing agent. ### Claim 15 (depends on 14) The method of claim 14, wherein the drug is selected from the group consisting of a nucleic acid, a peptide, a protein, and a small molecule. ### Claim 16 A method for assembling a modified Hepatitis B Virus (HBV) core protein into a capsid structure, the method comprising: providing a solution comprising a modified HBV core protein and a first concentration of a denaturing agent; and diluting the first concentration of denaturing agent to a second concentration, thereby to form an assembled capsid structure. ### Claim 17 (depends on 16) The method of claim 16, wherein the denaturing agent is at least one of urea, guanidinium hydrochloride (GuHCl), guanidinium thiocyanate (GITC), methanol, ethanol, trifluoroethanol (TFE), acetonitrile, and lithium perchloride. ### Claim 18 (depends on 16) The method of claim 16 wherein the first concentration of denaturing agent is from about 2 M to about 8 M. ### Claim 19 (depends on 16) The method of claim 16, wherein the second concentration of denaturing agent following the dilution step is from about 0.25 M to about 4 M. ### Claim 20 (depends on 16) The method of claim 16, further comprising adding a negatively-charged polymer to the solution. ### Claim 21 (depends on 16) The method of claim 16, wherein the pH of the solution is about pH 7.0 or lower. ### Claim 22 (depends on 16) The method of claim 16, further comprising adding a drug to the solution prior to the dilution step. ### Claim 23 (depends on 22) The method of claim 22, wherein the drug binds to the amino acid tail portion of the HBV core protein. ### Claim 24 (depends on 23) The method of claim 23, wherein the drug is at least one of bound to the amino acid tail portion and encapsulated in the capsid structure following the dilution step, and encapsulated in the capsid structure by diffusion following the dilution step. ### Claim 25 (depends on 22) The method of claim 22, wherein the drug is selected from the group consisting of a nucleic acid, a peptide, a protein, and a small molecule. ### Claim 26 (depends on 16) The method of claim 16, wherein the HBV core protein is a modified HBV core protein comprising one or more modifications in at least one of amino acid sequence SEQ ID NO: 1 and SEQ ID NO: 2. ### Claim 27 (depends on 26) The method of claim 26, wherein the modified HBV core protein is a C terminal truncation mutant. ### Claim 28 (depends on 27) The method of claim 27, wherein one to four arginine-rich repeats are truncated from the carboxyl-terminus of the modified HBV core protein. ### Claim 29 (depends on 27) The method of claim 27, wherein the C-terminal truncation mutant is selected at least one of: SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; and SEQ ID NO: 6. ### Claim 30 (depends on 26) The method of claim 26, wherein the modified HBV core protein further comprises a carboxyl-terminal amino acid tail portion. ### Claim 31 (depends on 30) The method of claim 30, wherein the amino acid tail portion is at least one of a wild-type tail portion and a synthetic tail portion. ### Claim 32 (depends on 30) The method of claim 30, wherein the amino acid tail portion is positively charged. ### Claim 33 (depends on 32) The method of claim 32, wherein the positively-charged amino acid tail portion is at least one of a lysine-rich tail portion and an arginine-rich tail portion. ### Claim 34 (depends on 33) The method of claim 33, wherein the lysine-rich tail portion comprises at least one of from about 4 to about 30 lysines, and a lysine domain of from about 4 to about 30 lysines. ### Claim 35 (depends on 34) The method of claim 34, wherein the lysine domain is about 9 lysines. ### Claim 36 (depends on 26) The method of claim 26, further comprising at least one of measuring the formation of the assembled capsid structure and purifying the assembled capsid structure. ### Claim 37 (depends on 1) The viral capsid produced by the method of claim 1. ### Claim 38 (depends on 16) The viral capsid produced by the method of claim 16. ### Claim 39 A method for assembling a Hepatitis B Virus (HBV) core protein into a capsid structure, the method comprising: providing a solution comprising a modified HBV core protein and a first concentration of denaturing agent wherein the spike region of the modified HBV core protein comprises a cysteine residue; adding a drug to the solution; adding a reducing agent to the solution wherein the drug is added to the solution prior to the addition of the reducing agent: and diluting the first concentration of the denaturing agent to a second concentration after the addition of the reducing agent wherein the drug is added to the solution prior to diluting the denaturing agent, thereby to form an assembled capsid structure wherein the drug is encapsulated in the capsid structure. ### Claim 40 A method of treatment, comprising: administering to a patient in need of treatment a modified Hepatitis B Virus (HBV) core protein viral capsid bearing an appropriate therapeutic and associated with a pharmaceutically acceptable carrier.