Geminiviral vector for expression of rituximab

• US 10,125,373 B2
• Filed: 01/22/2014
• Issued: 11/13/2018
• Est. Priority Date: 01/22/2013
• Status: Active Grant

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Mason, Hugh, Arntzen, Charles, Rosenthal, Sun Hee, Winkle, Sean, Diamos, Andrew
• Primary Examiner(s): Worley, Cathy Kingdon
• Application Number:
  US14/762,128
• Publication Number:
  US 20150368660A1
• Time in Patent Office: 1,756 Days
• Field of Search: None
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 2317/13 isolated from plants
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C12N 15/8203 Virus mediated transformation
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8258 for the production of oral vaccines (antigens) or immunoglobulins
• CPC Class Codes: C12N 2750/12043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

A single vector or multiple separate vectors that contain two or more non-competing replicons for transient expression of the heavy and light chains of Rituximab in Nicotiana benthamiana leaves is described. The correct assembly of these subunit proteins into functional oligomeric structures to optimize the expression is also described. This system advances plant transient expression technology by eliminating the need for non-competing viruses, and thus, enhances the realistic commercial application of the multi-replicon single vector system for producing Rituximab in plant cells.

Claims

1. A vector comprising a long intergenic region (LIR) of a geminivirus genome, followed by two nucleic acid segments followed by a short intergenic region (SIR) of a geminivirus genome, followed by one or more nucleic acids encoding Rep and RepA proteins of a geminivirus genome, followed by a LIR, wherein the two nucleic acid segments comprise:

(a) a nucleic acid segment comprising at least one promoter, a 5′

UTR, a region encoding a heavy chain of Ritixumab, and a 3′

terminator; and

(b) a nucleic acid segment comprising at least one promoter, a 5′

UTR, a region encoding a light chain of Ritixumab, and a 3′

terminator;

wherein the two nucleic acid segments comprise a single replicon.

2. The vector of claim 1, further comprising a nucleic acid segment that comprises a gene-silencing inhibitor.

3. The vector of claim 1, wherein the 5′

UTR of the first and second nucleic acid segment comprise the same sequence and the 3′

terminator of the first and second nucleic acid segment comprise the same sequence.

4. The vector of claim 1, wherein the 5′

UTR of the first and second nucleic acid segment do not comprise the same sequence and the 3′

terminator of the first and second nucleic acid segment do not comprise the same sequence.

5. A method of producing Rituximab in a plant cell comprising:

(a) obtaining a vector of claim 1

(b) introducing the vector into a plant cell; and

(c) allowing expression of the replicon comprising the first and the second chain of Rituximab.

6. The method of claim 5, further comprising a full assembly of the first and the second chains of Rituximab.

7. The vector of claim 1, wherein the 3′

terminator comprises a tobacco extensin gene terminator region.