Disclosed herein are compositions and methods that regulate expression of two or more endogenous genes.

Description of the Invention

SUMMARY

The present disclosure overcomes the problems inherent in multigenic regulation, by simultaneously modulating (e.g., upregulating and/or downregulating) the expression of essentially some or all of the key enzymes within a specific metabolic pathway using a single transgene deliverable. The basis of our approach is to engineer a single customized zinc finger protein transcription factor (ZFP-TF) that will bind to and modulate expression of an entire set of endogenous genes (i.e., genes in their normal chromosomal context) that are specific for the target pathway.

In certain embodiments, a multi zinc finger protein is provided comprising two or more engineered zinc finger proteins, wherein the multi zinc finger protein modulates expression of two or more endogenous genes (e.g., three or more genes, five or more genes, eight or more genes, or even ten or more genes). Each zinc finger protein can comprise at least two zinc finger modules, for example a zinc finger module that binds to a 3 base pair subsite in target site of the endogenous gene. The zinc finger proteins can be linked together using linker molecules as described in the art. In certain embodiments, the multi zinc finger proteins further comprise at least one functional domain (e.g., activation and/or repression domain), for example a functional domain for each zinc finger protein. Any of the multi zinc finger proteins described herein can be included in a composition, for example a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

In certain aspects, the endogenous genes targeted by the multi zinc finger proteins are involved in a metabolic pathway, for example, synthesis of a product. In certain embodiments, the endogenous genes are plant genes, for example genes involved in tocopherol synthesis.

Any of the multi zinc finger proteins described herein may be encoded by one or more nucleic acid molecules.

In other embodiments, any of the compositions described in herein can be used in methods of modulating the level of a product in a eukaryotic cell, comprising contacting the eukaryotic cell with any of the compositions disclosed herein, under conditions such that levels of the product are modulated.

DETAILED DESCRIPTION

The practice of the disclosed methods employs, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, genetics, computational chemistry, cell culture, recombinant DNA and related fields as are within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Third Edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; and the series METHODS IN ENZYMOLOGY, Academic Press, San Diego.

The regulation of gene expression is one of the most fundamental processes in all of biology. Gene expression is normally controlled by the concerted action of transcription factors that bind to and regulate gene promoter activity at various chromosomal sites. These transcription factors typically bind to DNA elements located within regulatory regions of genes, and they can induce the activation or repression of gene expression. Transcription factors generally contain both (i) a DNA binding domain (e.g., to target the protein to a specific site in the genome) and (ii) a regulatory domain (e.g., signals whether the genes near this binding site should be turned on or turned off).

The capacity to engineer transcription factors that bind to and regulate the expression of endogenous genes, thereby tapping into the normal physiological mechanisms of gene control has been described, for example in WO/0183819 and WO/0119981 and the references cited therein. Briefly, this technology involves the engineering of artificial transcription factors (containing both DNA-binding and transcription-regulation domains) that can bind to any desired site in the genome. These transcription factors can be transiently or stably expressed within a plant or animal cell and thus, this strategy is an immensely powerful approach for controlling individual gene expression. To date, this technology has been applied to selectively target one gene per engineered transcription factor. The transition from single-gene to multi-gene regulation represents an enormous technical challenge but has incredibly exciting implications. Our approach uses designed transcription factors that have a zinc finger DNA binding domain (ZFPs), for example of the Cys.sub.2-His.sub.2 class. See, e.g., Tupler et al. (2001) Nature 409:832-3.

Design and selection studies have demonstrated the amazing versatility of this motif, and powerful strategies for the design of ZFPs (that contain, for example, 3, 4, or 6 fingers) that can recognize virtually any desired DNA sequence (of 9, 12 or 18 basepairs, respectively) have been developed. See, e.g., Jamieson et al. (1994) Biochemistry 33:5689-95; Rebar & Pabo (1994) Science 263:671-3; Rebar et al. (1996) Methods Enzymol 267:129-49; Desjarlais & Berg (1992) Proc Natl Acad Sci USA 89:7345-9; Greisman & Pabo (1997) Science 275: 657-61; Choo & Klug (1994) Proc Natl Acad Sci USA 91:11163-7.

Each individual finger in a Cys.sub.2-His.sub.2 zinc finger protein contains an .alpha.-helix (FIG. 1a (see Original Patent)). The aminoterminal region of each .alpha.-helix contains four amino acid residue positions that are especially critical for making specific base pair contacts, and each finger contacts a 3-4 base pair region along the DNA (FIG. 1b (see Original Patent)). See, e.g., Jamieson et al. (1994) Biochemistry 33:5689-95. By varying the residues used at these key positions, the DNA-binding specificity of each individual finger can be altered to recognize the desired 3-4 basepair region. Therefore, the DNA binding domain of each engineered transcription factor contains a set of linked fingers that recognizes a specific site in the target gene promoter. However, DNA binding per se generally may not be sufficient to regulate transcription. In such instances, we attach appropriate transcription activation or repression domains to zinc finger proteins to produce artificial zinc finger protein transcription factors (ZFP-TFs) that will (by virtue of the specificity inherent in the DNA-binding domain) be able to turn on or turn off any endogenous gene (FIG. 1c (see Original Patent)).

A central step in designing these novel transcription factors involves creating zinc finger DNA binding units that are precisely targeted to the desired DNA sequence and thus will specifically regulate the genes of interest. Phage display libraries of zinc fingers can be used to select individual zinc fingers with desired DNA-binding specificities. See, e.g., Jameison et al (1994) Biochemistry 33:5689-95; Rebar & Pabo, supra; Greisman & Pabo, supra; Choo et al. (1994) Nature 372:642-5; Isalan et al. (1998) Biochemistry 37:12026-33; and Isalan & Choo (2000) J Mol Biol 295:471-7. Selection process is typically done using a library of "two-finger" modules--that can recognize any desired six-base pair site in duplex DNA. By linking together such two-finger units, four-finger or six-finger proteins that recognize twelve-base pair or eighteen-base pair target sites, respectively, can be rapidly assembled. Recognition sites of this size will typically be large enough such that they occur only once in the human genome, thus conferring specificity of gene targeting. Details of our sequence-specific zinc finger protein selection strategy are given in Example 8 and FIG. 2 (see Original Patent).

The disclosure herein relates to a novel approach in which multiple autonomous ZFP DNA binding domains are joined by linker peptides to create a single "multiZFP" that can selectively bind to each of the genes for which it contains the cognate ZFP module (FIG. 3 (see Original Patent)). In addition, a transcription regulatory domain can be added, for example to generate a functional multiZFP-TF that would simultaneously bind to and regulate each and all of the cognate target genes.

Currently, ZFP-TF approaches typically employ a single ZFP that recognizes a select 9-18 basepair sequence within the promoter of a target gene. Disclosed herein are compositions and methods involving a single ZFP-TF that simultaneously regulates several key genes, for example multiple genes in a biosynthetic pathway is engineered. Thus, a single multiZFP-TF as disclosed herein binds to several individual gene promoters, for example several genes within a synthesis pathway.

Further, the ZFPs described herein are preferably highly effective on each target gene to which they bind. As described herein, each ZFP-TF typically comprises two domains: a DNA binding domain, and a transcription regulatory domain (activator or repressor). Thus, binding function may be separate from regulatory function. The transcription regulatory function of that ZFP-TF is determined, in part, by the local chromatin environment and the presence of adjacent transcription factors and, accordingly, different regulatory domains may exhibit promoter context-dependent efficacy (e.g., one type of activation domain might be more effective on promoter A than on promoter B, while the converse may apply for a different activation domain). Thus, in the context of the present disclosure it is preferred that the ZFP-TF not only bind to all the selected target genes, but also retain the capacity to effectively modulate transcription from all of those genes.

This disclosure represents a significant improvement over current technologies by providing the ability to generate a single multiZFP-TF that modulates more than one target gene. For example, in the context of dietary supplements, administration of such multiZFPs (e.g., via insertion into the plant genome), will activate the major rate limiting genes in the .alpha.-tocopherol synthesis pathway and result in a dramatic increase in the level of .alpha.-tocopherol in the seed oil of the plant.

Table 1 (see Original Patent) illustrates some of the differences between the disclosure presented herein and other methods.

While the foregoing is applicable to genes in any organism, the disclosure is exemplified herein by showing production of tocopherol in Arabidopsis. The successful development of these systems will have a much larger impact on protein production and particularly agronomy in general. Thus, this technology could be broadly applied to increase the level of any high value product in any organism. Enhancing the level of these products in primary sources (e.g., as plants) will likely have a significant impact on the efficacy of downstream harvesting and extraction technologies. Furthermore, because the principles of gene regulation are conserved throughout eukarya, plant studies exemplified herein are directly applicable to the transfer of this technology to mammalian systems. Transgenic regulation of synthesis pathways in humans have great potential in medicine and healthcare. In addition, application of such a technology to animals could enhance the nutritional value of meat or milk products, with obvious economic rewards.
 

Claim 1 of 19 Claims

1. A protein comprising two or more engineered zinc finger domains, wherein: (i) each zinc finger domain comprises at least two fingers; (ii) each zinc finger domain binds a different target site; and (iii) the protein modulates expression of two or more endogenous genes.

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