US2015252081A1PendingUtilityA1

Methods and compositions for controlling assembly of viral proteins

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Assignee: BIOMED REALTY L PPriority: Sep 9, 2011Filed: Oct 17, 2014Published: Sep 10, 2015
Est. expirySep 9, 2031(~5.1 yrs left)· nominal 20-yr term from priority
Inventors:Miguel De Los RiosStephanie De Los RiosJacek OstrowskiKenneth J. OhIlan Zipkin
A61P 37/02A61P 9/00A61P 35/00A61P 31/00A61P 31/12A61P 29/00A61P 25/00C07K 14/005C07K 1/1136C07K 1/1072C07K 1/1133C12N 2730/10122A61K 31/713A61K 9/5184A61K 38/162A61K 47/64C07K 1/1077C07K 1/1075A61K 47/48246
45
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Claims

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

exact text as granted — not AI-modified
What is claimed is: 
     
         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.   
     
     
         2 . The method of  claim 1 , wherein the modified HBV core protein comprises one or more modifications selected from the group consisting of amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2. 
     
     
         3 . The method of  claim 1 , wherein the spike region comprises amino acids 74 to 84 of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         4 . The method of  claim 2 , wherein the modified HBV core protein comprises a cysteine at amino acid position 77, 79 or 80 of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         5 . The method of  claim 2 , wherein the modified HBV core protein comprises a cysteine at amino acid position 77 of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         6 . 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. 
     
     
         7 . The method of  claim 1 , wherein the concentration of the reducing agent is from about 0.1 molar equivalent to about 100 molar equivalent. 
     
     
         8 .- 15 . (canceled) 
     
     
         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.   
     
     
         17 . 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. 
     
     
         18 . The method of  claim 16 , wherein the first concentration of denaturing agent is from about 2 M to about 8 M. 
     
     
         19 . 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. 
     
     
         20 . The method of  claim 16 , further comprising adding a negatively-charged polymer to the solution. 
     
     
         21 . The method of  claim 16 , wherein the pH of the solution is about pH 7.0 or lower. 
     
     
         22 . The method of  claim 16 , further comprising adding a drug to the solution prior to the dilution step. 
     
     
         23 . The method of  claim 22 , wherein the drug binds to the amino acid tail portion of the HBV core protein. 
     
     
         24 . 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. 
     
     
         25 . 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. 
     
     
         26 .- 36 . (canceled) 
     
     
         37 . The viral capsid produced by the method of  claim 1 . 
     
     
         38 . The viral capsid produced by the method of  claim 16 . 
     
     
         39 . 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 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 addition of the reducing agent; and   diluting the first concentration of the denaturing agent to a second concentration after 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.   
     
     
         40 . (canceled)

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