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Processes using vlps with capsids resistant to hydrolases

a technology of hydrolase and capsids, which is applied in the field of viruslike particles, can solve the problems of high separation efficiency of recombinant proteins using these simple isolation processes, unique and complex isolation process, and limited application to simple systems

Inactive Publication Date: 2013-06-27
APSE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present patent provides a virus-like particle (VLP) that can encapsidate a heterologous cargo molecule and a packing sequence. The VLP can also contain a ribozyme enclosed by the capsid. The VLP can be used to deliver the cargo molecule and the ribozyme to specific cells or tissues. The VLP can also contain a linker to connect the cargo molecule and the ribozyme. The invention also provides a nucleic acid construct and a vector comprising the nucleic acid construct for delivering the cargo molecule and the ribozyme to cells or tissues. The technical effects of the invention include improved delivery of nucleic acid molecules to specific cells or tissues and the use of VLPs for gene therapy and gene silencing.

Problems solved by technology

However, these techniques are typically limited to applications to simple systems, and by the need to specify a different set of conditions for each protein and expression system.
Yet each target recombinant protein presents a unique set of binding interactions, thereby making its isolation process unique and complex.
The separation efficiency for recombinant proteins using these simple isolation processes is therefore low.
These methods are generally complex and high cost because of the large number of steps needed and the complexity of the reactions which predispose to technical difficulties, and the cost of the manufacturing systems.
In addition, the synthetic reagents involved are costly and so economy of scale is not easily obtained by simply increasing batch size.

Method used

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  • Processes using vlps with capsids resistant to hydrolases
  • Processes using vlps with capsids resistant to hydrolases
  • Processes using vlps with capsids resistant to hydrolases

Examples

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examples

[0108]The following non-limiting examples are included to illustrate various aspects of the present disclosure. It will be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the Applicants to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the instant disclosure, appreciate that many changes can be made in the specific examples described, while still obtaining like or similar results, without departing from the scope of the invention. Thus, the examples are exemplary only and should not be construed to limit the invention in any way. To the extent necessary to enable and describe the instant invention, all references cited are herein incorporated by reference.

example a

Propagation of MS2 Bacteriophage

[0109]MS2 bacteriophage (ATCC No. 15597-B1, from American Type Culture Collection, Rockville, Md.) and its E. Coli host (ATCC No. 15669) were obtained from ATCC and propagated using the procedure described by Strauss and Sinsheimer (1963) J. Mol. Biol. 7:43-54 J. Mol. Biol. 7:43-54. Results are plotted in FIG. 1. Optical Density (OD) at 600 nm and pH were followed during the reaction. ODi represents OD immediately after inoculation with host. Infection was done at 2.3 hours. Ln(OD / ODi) was plotted on the left axis (full diamonds) and pH was plotted on the right axis (open squares). This experiment was ended 5.3 hours after inoculation with host. Lysate obtained was centrifuged at 2,000 g and filtered through a 0.2 μm membrane to eliminate remaining bacteria and bacterial debris.

example b

Purification of MS2 Bacteriophage Using Proteinase K and Ultrafiltration

[0110]Purification of MS2 bacteriophage was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 2.

8 mL lysate obtained at end of Example A (sample in Lane 1, FIG. 2) was filtered through a 300 kDa membrane (Vivaspin 2, from Sartorius Stedim, Bohemia, N.Y.) and the filtrate was filtered through a 100 kDa membrane, from which 1 mL of retentate was obtained (sample in Lane 2, FIG. 2). This retentate was divided in two equal parts. To one half (control) 2064, mM CaCl2 aqueous solution at pH=7.5 was added. To the second half (Proteinase) 0.15 mg Proteinase K (Sigma Aldrich, St. Louis, Mo.) dissolved in 2064, 20 mM CaCl2 aqueous solution at pH=7.5 and was added. Both tubes were incubated at 37° C. and after 1 hour they were placed in an ice-water bath. Samples were then taken and analyzed: control sample in Lane 3, FIG. 2, and Pr...

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Abstract

Novel processes and compositions are described which use viral capsid proteins resistant to hydrolases to prepare virus-like particles to enclose and subsequently isolate and purify target cargo molecules of interest including nucleic acids such as siRNA's and shRNA's, and small peptides.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the priority of U.S. provisional application No. 61 / 578,706, filed Dec. 21, 2011, U.S. provisional application No. 61 / 607,900, filed Mar. 7, 2012, and U.S. provisional application No. 61 / 661,688, filed Jun. 19, 2012, the entire disclosures of which are hereby incorporated by reference.INCORPORATION OF SEQUENCE LISTING[0002]The entire contents of a paper copy of the “Sequence Listing” and a computer readable form of the sequence listing on diskette, containing the file named 450061_SequenceListing_ST25.txt, which is 77 kilobytes in size and was created on Dec. 20, 2012, are herein incorporated by reference.TECHNICAL FIELD[0003]The invention relates to virus-like particles, and in particular to methods and compositions using viral capsids as nanocontainers for producing, isolating and purifying heterologous nucleic acids and proteins.BACKGROUND OF THE INVENTION[0004]Virus-like particles (VLPs) are particles derived i...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/01C12N15/113A01N25/26
CPCC07K14/005C12N2310/14C12N2795/18123C12N2795/18142A01N25/26C07K14/01C12N15/113A01N25/28C12N2795/18122C12N2310/128C12N2310/123C12N7/00C12N2330/51C12N2310/16C12N15/111C12N2310/121A01N43/70A01N47/40A01N57/12A01N57/14A01N57/30C12N2310/531C12N15/63C12N7/04
Inventor ARHANCET, JUAN PEDRO HUMBERTOARHANCET, JUAN P.DELANEY, KIMBERLYHALL, KATHLEEN B.SUMMERS, NEENA
Owner APSE LLC
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