Invented by Yen Choo, Christopher Graeme Ullman, Nam-Hai Chua, Juan Pablo Sanchez, Gendaq Ltd, UK Research and Innovation, Rockefeller University, Sangamo Therapeutics Inc

Regulated gene expression is a process that allows cells to control the amount and timing of gene expression. This process is essential for the proper development and function of all living organisms, including plants. In recent years, the market for regulated gene expression in plants has been growing rapidly, driven by the increasing demand for sustainable agriculture and the need to improve crop yields. One of the main applications of regulated gene expression in plants is the development of genetically modified crops. By introducing genes that regulate the expression of other genes, scientists can create crops that are more resistant to pests, diseases, and environmental stressors. For example, a gene that regulates the expression of a plant’s immune system could be introduced into a crop to make it more resistant to fungal infections. Another application of regulated gene expression in plants is the development of crops that are more nutritious. By introducing genes that regulate the expression of specific nutrients, scientists can create crops that are richer in vitamins, minerals, and other essential nutrients. This could have a significant impact on global health, particularly in developing countries where malnutrition is a major problem. The market for regulated gene expression in plants is also being driven by the increasing demand for sustainable agriculture. As the world’s population continues to grow, there is a growing need for crops that can be grown in a more environmentally friendly way. Regulated gene expression can help to achieve this by reducing the need for pesticides and other harmful chemicals, as well as by improving the efficiency of crop production. Despite the potential benefits of regulated gene expression in plants, there are also concerns about the safety and ethical implications of genetically modified crops. Some people worry that these crops could have unintended consequences, such as the spread of modified genes to wild plant populations. Others worry about the impact of genetically modified crops on biodiversity and the environment. In conclusion, the market for regulated gene expression in plants is growing rapidly, driven by the increasing demand for sustainable agriculture and the need to improve crop yields. While there are concerns about the safety and ethical implications of genetically modified crops, the potential benefits of regulated gene expression are significant and could have a major impact on global health and food security. As such, it is important that scientists, policymakers, and the public continue to engage in a thoughtful and informed discussion about the future of this technology.

The Gendaq Ltd, UK Research and Innovation, Rockefeller University, Sangamo Therapeutics Inc invention works as follows

A method of controlling transcription in a plant cells is provided from a DNA sequence that comprises a target DNA operably connected to a codifying sequence. This method involves introducing a zinc finger polypeptide engineered in said plant cell. The polypeptide binds with the target DNA and modifies transcription of the codifying sequence.

Background for Regulated gene expression is in plants

Biotechnology has brought many agricultural benefits to the world. Biotechnology can be used to enhance various plant properties, such as resistance to diseases and pests, as well as the ability to produce improved seeds and fruits. There are many other applications for plant biotechnology. These include modification of traits that might be of agronomic or commercial interest, and the use and processing of plant-derived products. This could often be done by manipulating endogenous genetics that encode these traits. However, sophisticated methods to up- or down-regulate such endogenous gene are not yet available in many cases. Plants also have great potential as biological “factories”. There are many chemical products that plants can produce, including enzymes and pharmaceutical compounds. It is possible that high levels of gene products or compounds for such use could have adverse effects on the host plant. Therefore, better mechanisms to express such genes are needed.

Gene switches are of particular interest in controlling the timing and/or dose of gene expression in plants,” says Dr. Judith A. Sullivan. It is particularly desirable to develop gene switches that can direct gene expression towards any plant chromosome.

The present invention aims to overcome these problems by providing engineered, non-naturally occurring zinc finger proteins that confer specificity on gene regulatory for endogenous and transgenes. The present invention can be used in order to regulate any gene within a plant.

Accordingly, the present invention provides a method to regulate transcription in a plant cells which involves introducing an engineered zinc fingers polypeptide into said cell. This polypeptide binds with a target DNA and modifies transcription of a sequence that is operably linked to said DNA.

Previously, it was reported that engineered zinc fingers could be used to regulate genes within mammalian cells. (see, for example, Choo et.al., Nature 372; 642-645 (1994); Pomerantz and al. Science 267: 993-96 (1995); Liu. PNAS 94: 5525-5530 (1997); Beerli et al. PNAS 95: 146228-14633 (1998) Choo et al. The regulated gene was a gene that was integrated into a chromosome in the host mammalian cells. It is clear that mammalian and plants have very different biology and that each cell has evolved at a different level of structural, physiological and molecular biological levels. The inventors of the invention have demonstrated that an engineered zinc finger can be used to control a gene in a plant. The inventors also demonstrated that an engineered zinc fingers can regulate a gene in a plant chromosome. This is done by binding the engineered zinc fingers to a target sequence of DNA.

The zinc fingers of this invention can be used in order to regulate or down-regulate any plant gene. A zinc finger can be designed with a transactivating area to induce an endogenous gene. This allows for the target gene to be induction directly and without any external regulation. The only way to insert a transgene into another part of the genome was through the use of a separate promoter. The present invention’s zinc fingers can be used to down-regulate any plant gene. This was previously possible only using antisense, co-suppression and ribozymes, which can all be somewhat unpredictable. The zinc finger method of down-regulation is extremely potent. It allows for the targeted targeting of a specific gene family without affecting other members.

Engineered” is a term that refers to the process of engineering. The term “engineered” means that the zinc fingers have been created or modified in vitro. This means that the zinc finger has been generated or modified in vitro. You can also create engineering zinc fingers for the invention de novo by using rational design strategies.

Introduced into” is a term that refers to a plant, a part of he plant or cellular cell. “Induced into” refers to a procedure that is applied to a plant, part of a plant, or cell so that the zinc finger protein is present. Microinjection, bombardment and agrobacterium conversion are all suitable methods.

The term “target DNA sequence” is used. Any nucleic acid sequence that a zinc finger can bind to. This is, however, a sequence of DNA within a plant’s chromosome to which an engineered zinc fingers is capable of binding. The target DNA sequence will usually be associated with a target genes (see below). Binding the engineered zinc finger will allow for the up-or down-regulation of that gene. One embodiment of the target DNA sequence is part an endogenous genome sequence. Another embodiment introduces the target DNA sequence and the coding sequence into the cell. A target DNA sequence can be part of a transcription regulatory region, such as an enhancer or promoter. The most preferred embodiment of the target DNA is a sequence from a plant gene that is known to be a promoter.

The term “target gene” is a generic term that refers to a specific gene. A gene or cell in which one might wish to alter the expression of a target gene. A target gene may be an endogenous gene (i.e. One that is found in the genome of the plant, or cell, or one that is heterologous (i.e.

Heterologous to the cells” is a term that means: The sequence is not present in the cell’s genome but has been introduced to it. An heterologous sequence may include a modified sequence that is added to any chromosome or is not integrated into one. It can also be introduced through homologous recombination so that it is in the same place as the native allele.

In a preferred embodiment, the zinc fingers polypeptide is fused with a biological effectordomain. “Biological effector domain” is a term that refers to a polypeptide with a biological function. Any polypeptide with a biological function, which includes enzymes, transcriptional regulatory domains, proteins and additional sequences such as nuclear localization sequences.

Preferably, the zinc finger polypeptide has been fused to either a transcriptional activator or transcriptional repressor domain.

In another embodiment of the invention, the plant cell is part or can be regenerated to a plant. The target sequence is part a regulatory sequence to the nucleotide sequence in interest.

The present invention also provides a plant cell that contains a polynucleotide encoded an engineered zinc fingers polypeptide, and a target sequence to where the zinc finger protein binds.

The present invention also includes a transgenic plant that encodes an engineered zinc finger protein and a target sequence to where the zinc finger protein binds.

The present invention also provides a transgenic plants that contain a polynucleotide encoded an engineered zinc finger polypeptide as well as a target sequence within a plant’s chromosome.

The present invention also provides a transgenic plants that contain a polynucleotide encoded an engineered zinc finger polypeptide as well as a target sequence within the sequence of an endogenous gene to the plant.

Unless otherwise stated, technical and scientific terms used in this document have the same meanings as those commonly used in art (e.g. in cell culture, molecular gene therapy, nucleic acid chemical chemistry, hybridization techniques, and biochemistry). Standard methods are used to molecular, biochemical, and genetic methods (see Sambrook et. al. Molecular Cloning:A Laboratory Manual 2d Edition. (1989) Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y., Ausubel et al. Short Protocols in Molecular Biology (1999), 4th Ed, John Wiley & Sons, Inc., chemical methods, pharmaceutical formulations, and delivery and treatment for patients.

A. Zinc Fingers.

A zinc finger chimera (transcription factor) is composed of a DNA binding domain. It can bind to specific DNA sequences and one or more additional domains. A nuclear localization domain usually attaches to the zinc finger domain in order to direct the chimera towards the nucleus. The chimera usually includes an effector domain, which can be either a transactivation domain or a repression domain to regulate gene expression. Choo and Klug (1995). Curr. Opin. Biotech. 6:431-436. Choo and Klug (1997). Rebar and Pabo (1994). Science 263:671-673. Jamieson et.al. (1994) Biochem. 33:5689-5695. Other domains may be added to the zinc finger chimera, which could also be beneficial in the context of this invention. The zinc finger domain can include DNA-modifying domains, such as endonucleases or methylases, which give the zinc finger chimera the ability regulate the expression of any gene of interest, or modify any particular DNA. Wu et al. (1995) Proc. Natl. Acad. Sci. USA 92.344-348; Nahon & Raveh (1998); Smith, et al. (1999); and Carroll et.al. (1999). Within the context of this invention, zinc finger proteins of Cys2-His2 are preferred.

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