Optimized plant expression systems
Inventors
Mason, Hugh • Pardhe, Mary • Diamos, Andrew
Assignees
National Institutes of Health NIH • Arizona State University Downtown Phoenix campus
Publication Number
US-12297443-B2
Publication Date
2025-05-13
Expiration Date
2039-03-04
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Abstract
Improved plant transient expression systems using optimized geminiviral vectors that efficiently produce heteromultimeric proteins are described herein. Examples of high yields are shown herein, including two, three, or four fluorescent proteins coexpressed simultaneously. Various antibodies were produced using the optimized vectors with special focus given to the creation and production of a chimeric broadly neutralizing anti-flavivirus antibody. The variable regions of this murine antibody, 2A10G6, were codon optimized and fused to a human IgG1. Analysis of the chimeric antibody showed that it was efficiently expressed in plants, can be purified to near homogeneity by a simple one-step purification process, retains its ability to recognize the Zika virus envelope protein, and induce an immune response against Zika virus. Two other monoclonal antibodies were produced at similar levels. This technology is versatile tool for the production of a wide spectrum of pharmaceutical multi-protein complexes in a fast, powerful, and cost-effective way.
Core Innovation
The invention describes improved plant transient expression systems using optimized geminiviral vectors based on bean yellow dwarf virus (BeYDV) that efficiently produce heteromultimeric proteins by coexpressing multiple genes in a single vector. These optimized vectors enable the simultaneous high-level coexpression of two, three, or four proteins, such as fluorescent proteins, in a noncompetitive manner with near-equal expression levels. The system incorporates optimized 5′ untranslated regions (UTRs) and 3′ UTRs to enhance gene expression, allowing yields of 3-5 g of recombinant protein per kilogram of leaf fresh weight.
The problem addressed arises from the toxicity to plants caused by expression of heteromultimeric therapeutic proteins, such as antibodies, using existing plant expression vectors. This toxicity limits production. Additionally, previous vectors showed lower yields and issues with replicon size constraints and configuration for multi-gene expression. The optimized BeYDV vector system overcomes these issues by enabling high copy number replication in plant nuclei without causing toxicity, using multiple long and short intergenic regions (LIRs and SIRs) to facilitate replication of multiple expression cassettes either as single large replicons or multiple smaller ones.
The vectors feature optimized genetic elements, including a 35S promoter with duplicated enhancer region, various efficient 5′ UTRs (such as from Nicotiana benthamiana psaK gene, tobacco mosaic virus, tobacco etch virus, alfalfa mosaic virus), and optimized 3′ UTR terminators (including tobacco extension terminator with intron removed, ACT3 gene 3′ UTR, tobacco Rb7 matrix attachment region, pea rbcS 3′ UTR, and soybean vspB 3′ UTR). These optimizations enhance transcription and translation, reduce cell death, and improve expression levels. Using these vectors, pharmaceutical multi-protein complexes, including chimeric antibodies, can be produced rapidly, powerfully, and cost-effectively.
Claims Coverage
The claims disclose multiple independent claims directed to geminiviral vectors comprising optimized genetic elements for plant transient expression systems.
Optimization of untranslated regions for enhanced expression
The vector comprises at least one transgene encoding a protein, wherein the transgene includes an optimized 5′ untranslated region (5′ UTR) featuring a 35S promoter with duplicated enhancer from cauliflower mosaic virus and a selected 5′ UTR from Nicotinana benthamiana psaK gene, tobacco mosaic virus, tobacco etch virus, or alfalfa mosaic virus; and an optimized 3′ untranslated region (3′ UTR) comprising a tobacco extension terminator with intron removed, the 3′ UTR from the ACT3 gene of N. benthamiana, and the tobacco Rb7 matrix attachment region.
Geminiviral vector structure enabling replication and gene expression
The geminiviral vector includes a first long intergenic region (LIR), a first nucleotide sequence encoding at least one transgene flanked by optimized UTRs, a short intergenic region (SIR), a second LIR, and a second nucleotide sequence encoding Rep/RepA proteins of BeYDV positioned between the SIR and second LIR, facilitating replication of the transgene sequence in plant cells.
Sequence identity requirements for optimized elements and vector backbones
The optimized 5′ UTRs and 3′ UTRs have at least 99% sequence identity to specific nucleotide regions of SEQ ID NOS: 16, 17, or 18. The vector backbones include pBYKEHM-Bsa, pBYKEAM-BAGFPas6H, or pBY11HA-GFP, each with specified sequence identity to defined regions of SEQ ID NOS: 16-18.
Overall, the independent claims cover geminiviral vectors tailored for optimized plant expression comprising specific promoter-enhancer-UTR combinations, replicon configurations with LIRs and SIRs, Rep/RepA coding sequences, and defined sequence identities that enable efficient and high-level coexpression of heteromultimeric proteins in plants.
Stated Advantages
The vectors can produce high yields of recombinant proteins (up to 3-5 g/kg leaf fresh weight) efficiently and rapidly in plants.
The system produces heteromultimeric proteins with noncompetitive expression of multiple protein subunits, allowing simultaneous coexpression of two to four (or more) proteins at near-equal levels.
Optimized vectors reduce plant cell toxicity commonly caused by expression of pharmaceutical heteromultimeric proteins, enabling higher protein accumulation without necrosis or significant cell death.
The optimized plant expression vectors facilitate simple purification procedures, including a one-step purification resulting in near-homogeneous protein preparations.
The system is versatile and cost-effective, avoiding expensive cell culture requirements and sterile conditions, with scalability benefits, particularly useful for developing countries.
The BeYDV-based vectors maintain stability and high fidelity of DNA, allowing insertion of large genetic information without compromising expression.
Documented Applications
Production of heteromultimeric proteins such as antibodies, including chimeric broadly neutralizing anti-flavivirus antibodies like 2A10G6 targeting flavivirus envelope proteins.
Simultaneous coexpression of multiple fluorescent proteins (green, red, cyan, yellow) in plants for protein expression studies or reporter applications.
Production of monoclonal antibodies for therapeutic uses, including antibodies neutralizing Ebola virus, Herpes Simplex Virus, and Zika virus.
Production of pharmaceutical multi-protein complexes, including multi-subunit vaccines, immunotherapies, and multi-component virus-like particles.
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