WO2007005867A2

WO2007005867A2 - Compositions and methods for providing a graded response in a protein

Info

Publication number: WO2007005867A2
Application number: PCT/US2006/026038
Authority: WO
Inventors: Barbara J. Graves; Miles Pufall; Gregory M. Lee; Lawrence Mcintosh
Original assignee: University Of Utah Research Foundation; University Of British Columbia
Priority date: 2005-06-30
Filing date: 2006-06-30
Publication date: 2007-01-11
Also published as: WO2007005867A3

Abstract

The compositions and methods disclosed herein are of use for the graded control of proteins through modification of the post-translational state of said proteins. In particular, the methods provide for the subtle and gradual control of protein levels in direct contrast to the traditional theory of protein levels merely being 'on' at one consistent level or completely 'off.' The ability to modify protein levels may be useful in the production of a variety of cell types, including whole animals or plants, or for therapeutic or laboratory research purposes. Additionally, the methods of the present invention may be utilized to produce or inhibit particular proteins in patients suffering from diseases caused by the complete lack of a particular protein or an incorrect level of a protein.

Description

(RflHIBc&mONS AND METHODS FOR PROVIDING A GRADED RESPONSE IN A PROTEIN

Cross Reference to Related Applications This application claims priority from U.S. Provisional Patent Application No.

60/695,951 filed June 30, 2005.

Field of the Invention

The present invention relates to compositions and methods related to the control of proteins. In particular, the present invention provides a method of providing a graded response in the activity level of a protein via a modification that affects the post-translational state of said protein.

Background

Proteins are activated or repressed by post-translational modifications in response to extracellular cues. One such modification is phosphorylation, which often accumulates at multiple sites until a threshold level is reached and protein activity is altered. This outcome has been described as a sharp on/off switch without a transition in the level of protein expression between the two states. Additional mechanisms of protein regulation include conformational changes, the formation of complexes with additional molecules, and proteolysis, for example. The ability to regulate protein activity in a gradual (graded) manner would have a significant therapeutic effect as many diseases are caused by the complete absence or consistent over-abundance of a particular protein and the ability to increase or decrease protein levels in a slight manner would be highly desirable as a treatment for such diseases. Furthermore, the ability to genetically engineer a protein to enable it to produce a graded response as opposed to the traditional sharp on or off response would be of great therapeutic benefit.

SUMMARY OF THE INVENTION

The present invention thus provides compositions and methods for the production of a graded response in a protein by alteration of its post-translational state. In one embodiment, the alteration of the gene sequence for a particular protein causes a change in the post-translational state of the protein in a manner selected from the group consisting of phosphorylation, conformational changes, formation of complexes, acetylation, alkylation, biotinylation, deimination, deamidation, disulfide bridging, glutamylation, glycylation, glycosylation, isoprenylation, lipoylation, PEGylation, phosphopantetheinylation, phosphorylation, pP(li:ib9έlrliiϊiii^3i^Oylation, and ubiquitination. In a particular embodiment, the alteration causes a change in the phosphorylation of the post-translational state of the protein. Any protein susceptible to phosphatases can be controlled by the method of the present invention as long as it is capable of being phosphorylated and such phosphorylation provides a graded response from said protein. In a particular embodiment, the phosphorylation causes the structure of the protein to be altered affecting the function of the protein.

The protein of the present invention may be selected from the group consisting of, but not limited to, receptor proteins, enzymes, transcription factors and the like.

In one embodiment, a protein not susceptible to a graded response is engineered to respond in a gradual manner by alteration, such as by the inclusion of sequences capable of altering the post-translational state of the protein. In such a manner, proteins may be altered in order to provide a manner of controlling them in a gradual or graded manner.

In another embodiment, the protein is a transcription factor. In one such embodiment, alteration to the post-translational state of the protein may be desirable in order to control the expression level of a particular gene of interest. In a particular embodiment, the transcription factor is selected from the ETS family of regulatory transcription factors, for example Ets-1. For exemplary protein Ets-1 , phosphorylation provides a method of auto-inhibition and disallows DNA binding resulting in a direct correlation between the level of phosphorylation of Ets-1 and DNA binding by Ets-1, as well as transcription of genes containing ETS binding domains. In a third embodiment, a protein of the present invention is regulated by a conformational change in the post-translational state of the protein. In a particular embodiment, such conformational change includes alteration to one or more α- helices. In exemplary protein, Ets-1, there is an inhibitory module composed of 4 α- helices that appears to oppose the structural change. The proteins of the present invention may have one or more alterations to their post-translational state in order to obtain the desired graded response. For instance, in exemplary protein Ets-1 , there may be an alteration in the phosphorylation of the protein in its post-translational state, as well as to its conformation, such as by alteration to one or more of the 4 α-helices. The ^■dd^nh ϋ W Win-' of Hbfiy •! F-^;Wire alterations to the post-translational state of the protein may be useful in order to create the intended graded response in the protein. In yet another embodiment, the compositions and methods include the transformation of a host organism by introducing a nucleic acid molecule encoding a protein of the present invention. The transformation of the host organism may further include a nucleic acid encoding a protein capable of being regulated by the protein of the present invention. Such regulated protein may produce a therapeutic benefit to the host organism or it may merely be useful to have it produced in the host organism for any reason. The nucleic acids used in the present invention may further provide a control element. Such control element may be selected from the group consisting of, but not limited to, a constitutively active, inducible, tissue-specific or development stage- specific promoters, for example.

In another embodiment, a method for producing a particular gene product is provided whereby the gene product is produced upon exposure to a protein of the present invention. The gene product can be a product produced for the benefit of the host cell or organism (for example, gene therapy), or the product can be one that is produced for use outside the host cell or organism (for example, the product may be selected from, but not limited to, pharmaceuticals, hormones, protein products used in the manufacture of useful objects or devices, nutriceuticals, products used in chemical manufacture or synthesis, and the like).

An example of a product produced for the benefit of the host organism would be to utilize the methods of the present invention to effect insertion of a promoter susceptible to activation by a protein of the present invention into a pancreatic cell linked to an insulin gene. The pancreatic cell would then produce insulin, thereby aiding a diabetic host.

Additionally, the methods and compositions of the present invention could be utilized to effect the production of pharmaceutically relevant gene products in the same manner. For example, a gene containing a promoter capable of being controlled by the proteins of the present invention, could be inserted into a host plant in order to produce a large amount of a pharmaceutically useful protein product in the host plant. Such protein products could then be isolated from the plant.

In another embodiment, the present invention is utilized to decrease gene expression in a particular cell. Such gene may be over-expressed in one or more

The decrease in gene expression by the protein of the present invention may only be successful in a minimal number of cells but such disruption may contribute to better health for an individual suffering from disease due to over-expression of such disease. In another embodiment, the present invention is utilized to increase gene expression in a particular cell. Such gene may be under-expressed in one or more cell types resulting in disease. The increase in gene expression may only be successful in a minimal number of cells but such increase may contribute to better health for an individual suffering from disease due to over-expression of such disease.

Diseases or indications capable of being treated by the present invention include, but are not limited to, Alzheimer's Disease, cancer, heart disease, muscular dystrophy, cystic fibrosis, inflammation, autoimmune disease, diabetes, obesity, macular degeneration, COPD, multiple sclerosis, depression, schizophrenia, infertility, hair loss, asthma, spinal cord injury, Parkinson's disease, Huntington's Disease, Spinal Muscular Atrophy, hemophilia, glaucoma, viral infections, fungal infections, bacterial infections, kidney disease, kwashiorkor, familial amyloidotic polyneuropathy, mitochondrial trifunctional protein deficiency, and age-related development of cataracts. The present invention can be utilized in both somatic and germ line cells to effect protein expression of any gene.

The transformed cells of the present invention may be used for any purpose. In a particular embodiment, such use is for research, development, diagnostic or therapeutic purposes. The compositions and methods of the present invention may be used to create transgenic plants and animals. They may further be used to create novel experimental systems, such as animal models, cell-based assays, in vitro assays, and the like. Additionally, they may be used to create animals or cells for the production of antibodies, vaccines, and the like. They may further be useful to create components of novel regulatable expression systems for use in animal or cell culture systems, synthetic or chimeric transcriptional regulators or other regulatory proteins, cells for autologous or heterologous transplantation, and the like. They may also be useful in generating cell lines for in-vitro ADME/Tox applications or high-

tagged cells or tissues may also be created for experimental, diagnostic or therapeutic purposes.

Methods of the present invention are applicable to all cell types and organisms. The present invention can apply to any of the following cells, although the methods of the invention are not limited to the cells or organisms herein listed: A single celled or multicellular organism; an oocyte; a gamete; a germline cell in culture or in the host organism; a somatic cell in culture or in the host organism; an insect cell, including an insect selected from the group consisting of Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Lepidoptera, or Orthoptera, including a fruit fly, a mosquito and a medfly; a plant cell, including a monocotyledon cell and a dicotyledon cell; a mammalian cell, including but not limited to a cell selected from the group consisting of mouse, rat, pig, sheep, cow, dog or cat cells; an avian cell, including, but not limited to a cell selected from the group consisting of chicken, turkey, duck or goose cells; or a fish cell, including , but not limited to zebrafish, trout or salmon cells. In a particular embodiment, such cell is a stem cell.

The methods of the present invention can be applied to whole organisms or in cultured cells or tissues or nuclei, including those cells, tissues or nuclei that can be used to regenerate an intact organism, or in gametes such as eggs or sperm in varying stages of their development. Because proteins are important regulators in essentially all cells or organisms, the proteins of the present invention may be used in any cells or organisms. The methods of the present invention can be applied to cells derived from any organism, including but not limited to insects, fungi, rodents, cows, sheep, goats, chickens, and other agriculturally important animals, as well as other mammals, including, but not limited to dogs, cats and humans. Additionally, the compositions and methods of the present invention may be used in plants. It is contemplated that the compositions and methods can be used in any variety of plant species, including monocots or dicots. In certain embodiments, the invention can be used in plants such as grasses, legumes, starchy staples, Brassica family members, herbs and spices, oil crops, ornamentals, woods and fibers, fruits, medicinal plants, poisonous plants, corn, cotton, castor bean and any other crop specie. In alternative embodiments, the invention can be used in plants such as sugar cane, wheat, rice, maize, potato, sugar beet, cassava, barley, soybean, sweet potato, oil palm fruit, tomato, sorghum, orange, grape, banana, apple, cabbage, watermelon, coconut, onion, cottonseed, rapeseed and yam. In ^■lέtlii^IMOdiii^^'fttlltl^^'Invention can be used in members of the Solanaceae specie, such as tobacco, tomato, potato and pepper. In other embodiments, the invention can be used in poisonous ornamentals, such as oleander, any yew specie and rhododendron. In a particular embodiment, the Brassica specie is Arabidopsis. In another embodiment, the plant species is tobacco.

Grasses include, but are not limited to, wheat, maize, rice, rye, triticale, oats, barley, sorghum, millets, sugar cane, lawn grasses and forage grasses. Forage grasses include, but are not limited to, Kentucky bluegrass, timothy grass, fescues, big bluestem, little bluestem and blue gamma. Legumes include, but are not limited to, beans like soybean, broad or Windsor bean, kidney bean, lima bean, pinto bean, navy bean, wax bean, green bean, butter bean and mung bean; peas like green pea, split pea, black-eyed pea, chick-pea, lentils and snow pea; peanuts; other legumes like carob, fenugreek, kudzu, indigo, licorice, mesquite, copaifera, rosewood, rosary pea, senna pods, tamarind, and tuba-root; and forage crops like alfalfa. Starchy staples include, but are not limited to, potatoes of any species including white potato, sweet potato, cassava, and yams. Brassica, include, but are not limited to, cabbage, broccoli, cauliflower, brussel sprouts, turnips, collards, kale and radishes. Oil crops include, but are not limited to, soybean, palm, rapeseed, sunflower, peanut, cottonseed, coconut, olive palm kernel. Woods and fibers include, but are not limited to, cotton, flax, and bamboo. Other crops include, but are not limited to, quinoa, amaranth, tarwi, tamarillo, oca, coffee, tea, and cacao.

Certain objects of the present invention are exemplified by the graded regulation of a transcription factor, Ets-1. The level of gene expression for genes containing ETS domains can be carefully and gradually controlled directly by the phosphorylation of Ets-1. This is in direct contrast to the traditional theory that phosphorylation merely provided an "on" and "off' switch. The elements necessary to carry out the methods of the present invention as herein disclosed can be adapted for application for any protein susceptible to gradual regulation by post-translational modification, as exemplified by phosphorylation of Ets-1. A graded response may be created in any protein by the methods of the present invention, whether by alteration to the endogenous gene sequences in order to create the desired result in the post-translational state of the protein or by engineering the desired alteration into the gene sequence. Certain embodiments of iftl^;|ϊrllint1rifliifSήiήIrefore provide a general method for providing gradual control of a protein.

Many alterations and variations of the invention exist as described herein.

Detailed Description of the Invention The present invention may be understood more readily by reference to the following detailed description of particular embodiments of the invention.

Particular advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Before the present compositions and/or methods are disclosed and described, it is to be understood that this invention is not limited to specific compounds or methods, as such may, of course, vary, unless it is otherwise indicated. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Definitions For the purposes of the present invention, the following terms shall have the following meanings:

As used herein, the term "graded" as in a "graded" response in a protein refers to a response which is gradual in direct contrast to a complete lack of response and/or one consistent level. In the case of a protein, a graded response means the protein may shift from its active to its inactive state, or vice versa, slowly or gradually instead of immediately shifting from one state to the other ("off or "on").

In the case of a protein that may have an affinity for another molecule, a graded response can gradually generate a greater or lesser affinity for the molecule over a period of time. As used herein, the term "targeted genetic recombination" refers to a process wherein recombination occurs within a DNA target locus present in a host cell or host organism. Recombination can involve either homologous or non-homologous DNA. The term "target" or "target locus" or "target region" refers herein to the gene or DNA segment selected for modification by the targeted genetic recombination 'rMM If tή^'i:pδliri?iivention. Ordinarily, the target is an endogenous gene, coding segment, control region, intron, exon or portion thereof, of the host organism. However, the target can be any part or parts of the host DNA.

As used herein, the terms "host cell" or "host organism" or, simply, "target host", refer to a cell or an organism that has been selected to be genetically transformed to carry one or more genes for expression of a function used in the methods of the present invention. A host can further be an organism or cell that has been transformed by the targeted genetic recombination or mutation methods of the present invention. For the purposes of the present invention, the term "gene" refers to a nucleic acid sequence that includes the translated sequences that encode a protein ("exons"), the untranslated intervening sequences ("introns"), the 5' and 3' untranslated region and any associated regulatory elements.

As used herein, the term "donor" or "donor construct" refers to the entire set of DNA segments to be introduced into the host cell or organism as a functional group. The term "donor DNA" as used herein refers to a DNA segment with sufficient homology to the region of the target locus to allow participation in homologous recombination at the intended sight.

For the purposes of the present invention, the term "marker" refers to a gene or sequence whose presence or absence conveys a detectable phenotype to the host cell or organism. Various types of markers include, but are not limited to, selection markers, screening markers and molecular markers. Selection markers are usually genes that can be expressed to convey a phenotype that makes an organism resistant or susceptible to a specific set of environmental conditions. Screening markers can also convey a phenotype that is a readily observable and distinguishable trait, such as Green Fluorescent Protein (GFP), GUS or beta- galactosidase. Molecular markers are, for example, sequence features that can be uniquely identified by oligonucleotide probing, for example RFLP (restriction fragment length polymorphism), or SSR markers (simple sequence repeat). For the purposes of the present invention, the term "sequence" means any series of nucleic acid bases or amino acid residues, and may or may not refer to a sequence that encodes or denotes a gene or a protein. Many of the genetic constructs used herein are described in terms of the relative positions of the various genetic elements to each other. ^■-

the term "adjacent" is used to indicate two elements that are next to one another without implying actual fusion of the two elements. Additionally, for the purposes of the present invention, "flanking" is used to indicate that the same, similar, or related sequences exist on either side of a given sequence. Segments described as "flanking" are not necessarily directly fused to the segment they flank, as there can be intervening, non-specified DNA between a given sequence and its flanking sequences. These and other terms used to describe relative position are used according to normal accepted usage in the field of genetics.

For the purposes of the present invention, the term "recombination" is used to indicate the process by which genetic material at a given locus is modified as a consequence of an interaction with other genetic material.

For the purposes of the present invention, the term "homologous recombination" is used to indicate recombination occurring as a consequence of interaction between segments of genetic material that are homologous, or identical. In contrast, for purposes of the present invention, the term "non-homologous recombination" is used to indicate a recombination occurring as a consequence of interaction between segments of genetic material that are not homologous, or identical. Non-homologous end joining (NHEJ) is an example of non-homologous recombination. For the purposes of the present invention, the term "nutriceutical" shall refer to any substance that is a food or part of a food and provides medical or health benefits, including the prevention and treatment of disease. Exemplary "nutraceuticals" include isolated nutrients, dietary supplements, herbal products and the like. Moreover, for the purposes of the present invention, the term "a" or "an" entity refers to one or more than one of that entity; for example, "a protein" or "an nucleic acid molecule " refers to one or more of those compounds, or at least one compound. As such, the terms "a" or "an", "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising," "including," and "having" can be used interchangeably. Furthermore, a compound "selected from the group consisting of refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds. According to the present invention, an isolated or biologically pure compound is a compound that has been removed from its natural milieu. As such,

pure" do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis. Protein Regulation

The methods of the present invention can be used to create a graded response in any protein. If the post-translational modification is not known or is difficult to manipulate, a sequence creating the desired graded response in the protein can be engineered. An exemplary protein of the present invention is the transcription factor Ets-1 , which exhibits graded DNA binding activity due to different levels of phosphorylation in a particular region, the serine-rich region. Free Ets-1 exists in equilibrium between an active state that is poised to bind DNA and an inactive state. Accordingly, the protein's affinity for DNA is proportional to the balance of these two states. Ets-1 is regulated by an auto-inhibitory module composed of four α-helices

(HM , HI-2, H4, and H5) that flank the DNA binding ETS domain. In the native protein these helices appear to pack cooperatively on a surface of the ETS domain opposite the DNA binding interface, reducing the affinity of Ets-1 for DNA 10-fold compared to the minimal ETS domain. This inhibitory module appears to oppose the structural change that accompanies DNA binding, the most striking feature of which is the unfolding of inhibitory helix HM .

NMR spectroscopic studies indicates that helix HI-1 is labile, even in the absence of DNA, and may serve as a control point for modulating DNA binding affinity. These studies indicate the affinity for DNA decreases as the propensity of helix HM to unfold decreases. Further, it appears that phosphorylation shifts Ets-1 from a dynamic conformation poised to bind DNA to a well-folded, inhibited state. It appears in exemplary protein ETS-1, these phosphatases lie in an unstructured flexible region that functions as the allosteric effector of auto-inhibition. It appears that each phosphate has a distinct role in modulating the conformational equilibrium. It appears Ca²⁺-dependent phosphorylation of Ets-1 at multiple sites reinforces auto- inhibition by lowering the DNA affinity ~50-fold further to an overall inhibition of 500- to 1000-fold. Mulitple Ca²⁺-dependent phosphorylation sites within Ets-1 appear to act additively to produce graded DNA binding affinity. Therefore, in this exemplary embodiment of the present invention, Ets-1 indicates that variable phosphorylation is '^■'iiidlliS]S|lly.-d^li2)©<!!lFlslWISbh, rather it serves as a "rheostat" for cell signaling to fine- tune transcription at the level of DNA binding.

NMR data indicates the core hydrophobic packing of Ets-1 is dynamic in the active state, and that increasing inhibition attenuates these motions. The inhibitory module, DNA binding interface, and hydrophobic core may form a concerted unit that is linked as a dynamic hydrophobic network.

The dynamic character of exemplary protein, Ets-1, may be crucial for binding to DNA but it also appears to provide a novel opportunity for regulation by the methods of the present invention. Namely, signal-dependent phosphorylation appears to provide a mechanism of controlling protein levels in a graded manner. The use of a highly flexible segment in the graded regulation of exemplary protein, Ets-1 , to control the protein is but one example of a method of utilizing a post-translational modification to control a protein. Other proteins that contain flexible elements, like the SRR in Ets-1 , also often display post-translational modifications (e.g. MAPK Ets-1 phosphorylation; histone tails; SH2 domain targets, tether independent domains (e.g. PKA ), or require folding for activity (e.g. CREB/CBP ). The highly mobile SRR of Ets-1 appears to demonstrate an exemplary role for unstructured protein segments in integrating signals that direct variable regulation of protein activity. With the information disclosed herein, one skilled in the art would know how to manipulate the gene sequence of any protein in order to enable that protein to produce a graded response such as was demonstrated with Ets-1. Further, by manipulating the post-translational state of a protein, one skilled in the art may produce a graded response in any protein of interest either by modifying the endogenous sequences to create the desired change in the post-translational state of said protein or by genetically engineering the alteration in a gene sequence via genetic recombination. Targeted genetic recombination or mutation

The methods of the present invention may be utilized for targeted genetic recombination or mutation of any cell or organism. Minimum requirements include a method to introduce genetic material into a cell or organism (either stable or transient transformation), sequence information regarding the endogenous target region, and identification of the protein and its gene sequence in which a graded response is desired. According to some embodiments of the present invention, for example "Wriib'BlΘuέB&ϋfiib^'iilϊion, additional donor DNA may also be required.

According to another embodiment of the present invention, DNA encoding an identifiable marker may also be included with the DNA construct. Such markers may include a gene or sequence whose presence or absence conveys a detectable phenotype to the host cell or organism. Various types of markers include, but are not limited to, selection markers, screening markers and molecular markers. Selection markers are usually genes that can be expressed to convey a phenotype that makes an organism resistant or susceptible to a specific set of environmental conditions. Screening markers can also convey a phenotype that is a readily observable and distinguishable trait, such as Green Fluorescent Protein (GFP), beta- glucuronidase (GUS) or beta-galactosidase. Markers may also be negative or positive selectable markers. In a particular embodiment, such negative selectable marker is codA. Molecular markers are, for example, sequence features that can be uniquely identified by oligonucleotide probing, for example RFLP (restriction fragment length polymorphism), or SSR markers (simple sequence repeat).

The genetic constructs of the present invention may be any genetic sequence capable of producing a protein capable of a graded response. As such, one skilled in the art will be able to create such constructs with knowledge of the gene sequence of the protein product of interest and either knowledge of the portions of the sequence responsible for post-translational activity of the protein OR knowledge of the response desired in the protein in order to genetically engineer additional sequences to create the desired graded response.

The efficiency with which endogenous homologous recombination occurs in the cells of a given host varies from one class of cell or organism to another. However the use of an efficient selection method or a sensitive screening method can compensate for a low rate of recombination. Therefore, the basic tools for practicing the invention are available to those of ordinary skill in the art for a wide range and diversity of cells or organisms such that the successful application of such tools to any given host cell or organism is readily predictable. The compositions and methods of the present invention can be designed to introduce a targeted mutation or genetic recombination into any host cell, organism or protein. The flexibility of the present invention allows for genetic manipulation in order to create genetic models of disease or to investigate gene function.

The compositions and methods of the present invention can also be used to /qffι8§^jt^|G[e.te|)di;f;e©tj^,f^combination or mutation in a mammalian cell.

In another embodiment, homologous recombination can be used as follows. First, a site for integration is selected within the host cell. Sequences homologous to those located upstream and downstream from the integration site are then included in a genetic construct, flanking the selected gene to be integrated into the genome. Flanking, in this context, simply means that target homologous sequences are located both upstream (5') and downstream (3') of the selected gene. The construct is then introduced into the cell, thus permitting recombination between the cellular sequences and the construct. As a practical matter, the genetic construct will normally act as far more than a vehicle to insert the gene into the genome. For example, it is important to be able to select for recombinants and, therefore, it is common to include within the construct a selectable marker gene. The marker permits selection of cells that have integrated the construct into their genomic DNA. In addition, homologous recombination may be used to "knock-out" (delete) or interrupt a particular gene. Thus, another approach for inhibiting gene expression involves the use of homologous recombination, or "knock-out technology". This is accomplished by including a mutated or vastly deleted form of the heterologous gene between the flanking regions within the construct. Thus, it is possible, in a single recombi national event, to (i) "knock out" an endogenous gene, (ii) provide a selectable marker for identifying such an event and (iii) introduce a transgene for expression.

The frequency of homologous recombination in any given cell is influenced by a number of factors. Different cells or organisms vary with respect to the amount of homologous recombination that occurs in their cells and the relative proportion of homologous recombination that occurs is also species-variable. The length of the region of homology between donor and target affects the frequency of homologous recombination events, the longer the region of homology, the greater the frequency. The length of the region of homology needed to observe homologous recombination is also species specific. However, differences in the frequency of homologous recombination events can be offset by the sensitivity of selection for the recombinations that do occur. It will be appreciated that absolute limits for the length of the donor- target homology or for the degree of donor-target homology cannot be fixed but depend on the number of potential events that can be scored and the sensitivity of the selection for homologous recombination events. Where it is possible to screen 10⁹ events, for

a selection that can identify 1 recombination in 10⁹ cells will yield useful results. Where the organism is larger, or has a longer generation time, such that only 100 individuals can be scored in a single test, the recombination frequency must be higher and selection sensitivity is less critical. It will be understood by those skilled in the art that the invention is operative independent of the method used to transform the organism. Further, the fact that the invention is applicable to such disparate organisms as plants and insects demonstrates the widespread applicability of the invention to living organisms generally. Nucleic Acid Delivery Transformation can be carried out by a variety of known techniques which depend on the particular requirements of each cell or organism. Such techniques have been worked out for a number of organisms and cells, and can be adapted without undue experimentation to all other cells. Stable transformation involves DNA entry into cells and into the cell nucleus. For single-celled organisms and organisms that can be regenerated from single-cells (which includes all plants and some mammals), transformation can be carried out in in vitro culture, followed by selection for transformants and regeneration of the transformants. Methods often used for transferring DNA or RNA into cells include forming DNA or RNA complexes with cationic lipids, liposomes or other carrier materials, micro-injection, particle gun bombardment, electroporation, and incorporating transforming DNA or RNA into virus vectors. Other techniques are well known in the art.

Examples of some Delivery Systems useful in practicing the present invention

Liposomal formulations: In certain broad embodiments of the invention, the oligo- or polynucleotides and/or expression vectors containing the gene products for the proteins of the present invention and, where appropriate, donor DNA, may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are cationic lipid-nucleic acid complexes, such as lipofectamine-nucleic acid complexes. Lipids suitable for use according to the

;|ip obtained from commercial sources. Liposomes used according to the present invention can be made by different methods and such methods are known in the art. The size of the liposomes varies depending on the method of synthesis. Microinjection: Direct microinjection of DNA into various cells, including egg or embryo cells, has also been employed effectively for transforming many species. In the mouse, the existence of pluripotent embryonic stem (ES) cells that are culturable in vitro has been exploited to generate transformed mice. The ES cells can be transformed in culture, then micro-injected into mouse blastocysts, where they integrate into the developing embryo and ultimately generate germline chimeras. By interbreeding heterozygous siblings, homozygous animals carrying the desired gene can be obtained.

Adenoviruses: Human adenoviruses are double-stranded DNA tumor viruses with genome sizes of approximate 36 Kb. As a model system for eukaryotic gene expression, adenoviruses have been widely studied and well characterized, which makes them an attractive system for development of adenovirus as a gene transfer system. This group of viruses is easy to grow and manipulate, and they exhibit a broad host range in vitro and in vivo. In lytically infected cells, adenoviruses are capable of shutting off host protein synthesis, directing cellular machineries to synthesize large quantities of viral proteins, and producing copious amounts of virus.

Particular advantages of an adenovirus system for delivering DNA encoding foreign proteins to a cell include (i) the ability to substitute relatively large pieces of viral DNA with foreign DNA; (ii) the structural stability of recombinant adenoviruses; (iii) the safety of adenoviral administration to humans; and (iv) lack of any known association of adenoviral infection with cancer or malignancies; (v) the ability to obtain high titers of recombinant virus; and (vi) the high infectivity of adenovirus.

In general, adenovirus gene transfer systems are based upon recombinant, engineered adenovirus which is rendered replication-incompetent by deletion of a portion of its genome, such as E1 , and yet still retains its competency for infection. Sequences encoding relatively large foreign proteins can be expressed when additional deletions are made in the adenovirus genome. For example, adenoviruses deleted in both the E1 and E3 regions are capable of carrying up to 10 kB of foreign DNA and can be grown to high titers in 293 cells. " IJ S CBIiherMrøailitøeltBs as Expression Constructs: Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from, for example, vaccinia virus, adeno-associated virus (AAV), and herpes viruses may be employed. Defective hepatitis B viruses, may be used for transformation of host cells. In vitro studies show that the virus can retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome. Potentially large portions of the viral genome can be replaced with foreign genetic material. The hepatotropism and persistence (integration) are particularly attractive properties for liver-directed gene transfer. The chloramphenicol acetyltransferase (CAT) gene has been successfully introduced into duck hepatitis B virus genome in the place of the viral polymerase, surface, and pre-surface coding sequences. The defective virus was cotransfected with wild-type virus into an avian hepatoma cell line, and culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was suvsequently detected. Non-viral Methods: Several non-viral methods are contemplated by the present invention for the transfer into a host cell of DNA constructs. These include calcium phosphate precipitation, lipofectamine-DNA complexes, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use. In one embodiment of the invention, the expression construct may simply consist of naked recombinant DNA. Transfer of the construct may be performed by any of the DNA transfer methods mentioned above which physically or chemically permeabilize the cell membrane. For example, polyomavirus DNA in the form of CaPO₄ precipitates was successfully injected into liver and spleen of adult and newborn mice which then demonstrated active viral replication and acute infection. In addition, direct intraperitoneal injection of CaPO₄ precipitated plasmid expression vectors results in expression of the transfected genes.

Transformation of Plants: Transformed plants are obtained by a process of transforming whole plants, or by transforming single cells or tissue samples in culture and regenerating whole plants from the transformed cells. When germ cells or seeds are transformed there is no need to regenerate whole plants, since the transformed plants can be grown directly from seed. A transgenic plant can be produced by any means known in the art, including but not limited to Agrobacterium tumefaciens-medlated DNA transfer, preferably with a disarmed T-DNA vector,

transfer, and particle bombardment. Techniques are well-known to the art for the introduction of DNA into monocots as well as dicots, as are the techniques for culturing such plant tissues and regenerating those tissues. Regeneration of whole transformed plants from transformed cells or tissue has been accomplished in most plant genera, both monocots and dicots, including all agronomically important crops. Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, the technique of site-specific mutagenesis is well known in the art. As will be appreciated, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site- directed mutagenesis include vectors such as the M 13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining a single- stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed

the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Screening for Mutations

Methods for genetic screening to accurately detect mutations in genomic DNA, cDNA or RNA samples may be employed, depending on the specific situation. A number of different methods have been used to detect point mutations, including denaturing gradient gel electrophoresis ("DGGE"), restriction enzyme polymorphism analysis, chemical and enzymatic cleavage methods, and others. The more common procedures currently in use include direct sequencing of target regions amplified by PCR™ and single-strand conformation polymorphism analysis ("SSCP"). SSCP relies upon the differing mobilities of single-stranded nucleic acid molecules of different sequence on gel electrophoresis. Techniques for SSCP analysis are well known in the art.

Another method of screening for point mutations is based on RNase cleavage of base pair mismatches in RNA/DNA and RNA/RNA heteroduplexes. As used herein, the term "mismatch" is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single and multiple base point mutations.

Examples

It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should appreciate, in light of the present disclosure, that many changes can be made in the specific embodiments disclosed herein which will still obtain a like or similar result without departing from the spirit and scope of the invention. In vivo phosphorylation of Ets-1 was accomplished in this example by mutating phosphoacceptor serines to alanines singly and in pairs, and the DNA binding affinity was then measured for both the phosphorylated and unmodified forms. A minimal fragment, ΔN244, (SEQ ID 1) which displays the DNA binding properties of full-length Ets-1 was utilized. It is a form of Ets-1 homogenously phosphorylated by CamKII, ΔN244^5P, contains five specific phosphoserines in the SRR (residues 244-300), and appears to undergo the same structural transition upon DNA binding as unmodified ΔN244 and other Ets-1 species. Mutating three (251 , 282, and 285) of the five phosphoacceptor serines, but not two others (270 and 273), in a manner that disrupted the phosphoacceptor sites, significantly reduced inhibition. Furthermore, mutation of one, two, or all three critical sites yielded a graded reduction in phosphorylation-dependent inhibition with each phosphate contributing additively to the change in free energy of binding. This appears to indicate that both the number and context of sites, and not simply a build up of charge, affects inhibition. Thus, in contrast to proteins such as Sid and NFAT1 for which a threshold level of phosphorylation serves as a binary switch ("on" or "off), multiple sites within Ets-1 appear to regulate DNA binding in a graded fashion across a wide range of affinities. Example 2:

The phosphorylated SRR appeared to be predominantly unstructured and highly flexible. Based on main chain ¹H and ¹³C chemical shifts, NMR spectra revealed no predominant secondary structure within this region and the amide hydrogen exchange (HX) rates were comparable to those of a random coil polypeptide. NMR relaxation experiments appeared to demonstrate a high degree of backbone conformational mobility on the nsec-psec timescale. The dynamic, phosphorylated SRR may function to impart variable regulation of DNA binding by making transient interactions with the auto-inhibitory module, ETS domain, or other portions of the protein. Example 3:

Despite the flexibility SRR appears to demonstrate, it also appears to have a profound impact on the structure, stability, and dynamics of the DNA binding domain and the inhibitory helices. Three Ets-1 fragments with increasing levels of inhibition were compared to further investigate this matter: partially activated ΔN301 ; ΔN280, a ilJiWiaEfca'gibSiπOeMpiitulating unmodified, autoinhibited Ets-1 binding; and ΔN244^5P (K_D~1CT¹¹,10-¹⁰, and 10^"8 M, respectively).

Comparison of ¹H-¹⁵N HSQC spectra detected significant changes in backbone amide chemical shifts for ~25 residues common to the three species, which may be indicative of structural perturbations. The labile inhibitory helix HI-1 and helices H1 and H3 of the DNA binding domain appeared to be the most widely affected. The perturbed residues, many of which were non-polar, formed a hydrophobic network connecting the inhibitory elements and the DNA binding interface. Mutation of L429 to alanine within this network appeared to reduce autoinhibition and impaired phosphorylation reinforcement, which appeared to indicate that the integrity of this hydrophobic network is required for regulation.

A striking, linear pattern of change was observed from ΔN301 to ΔN280 to ΔN244^5P in the amide ¹H^N and ¹⁵N chemical shifts of almost all of the affected residues. This progressive, co-linear pattern appears to indicate the signature of an allosterically regulated molecule that is in conformational equilibrium between at least two states with the intermediate chemical shifts representing a population- weighted average of these states. Based on the correlation between the linear pattern of amide chemical shifts with DNA binding affinity, it appears that free Ets-1 exists in equilibrium between an active state (represented most closely by ΔN301) that is poised to bind DNA and an inactive state (represented by ΔN244^5P).

According to this allosteric model, Ets-1 DNA binding affinity appears to reflect the balance of these two states.

A comparison of the backbone amide chemical shifts of phosphorylated ΔN244 with single or double serine-to-alanine mutations also appeared to reveal a co-linear shifting pattern between ΔN280 and ΔN244^5P in at least 16 of the 25 residues within the aforementioned hydrophobic network. The position of the chemical shifts for each species, phosphorylated at the remaining CamKII sites, roughly appeared to correlate with affinity. This finding appears to emphasize the context dependence of each phosphate and appears to suggest that each has a distinct role in modulating the conformational equilibrium. These NMR studies indicate that differential phosphorylation regulates DNA binding by fine-tuning the balance between the active and inactive states of Ets-1. Example 4: ^{' 1}LiK¹Ei SDHiiid€JHiK'ilkpfefilhrients helped characterize the dynamics of the active and inactive states. They appeared to indicate the backbone amides within inhibitory helices HI-1 and HI-2, as well as the DNA binding helix H3 of the active ΔN301 exhibited limited protection from exchange, indicating that these helices sample locally unfolded conformations even in the absence of DNA. In contrast, the same amides appeared to exhibit elevated HX protection within ΔN280, and further in ΔN244^5P, suggestive of a progressively less dynamic, more stably folded species. A dynamic active state and more stable inactive state appear to be consistent with the affinity for DNA binding decreasing as the propensity of helix HI-1 to unfold decreases.

Example 5:

A dynamic connection between the autoinhibitory module and the DNA binding interface appeared to be demonstrated by NMR backbone amide and sidechain methyl relaxation dispersion measurements. The contribution of msec- Δsec timescale motions to linewidth broadening, denoted R_ex, are indicative of conformational switching. The backbone amides of ΔN301 appeared to exhibit mobility on this timescale (exchange lifetimes, Δ_ex, ~0.3 msec) for residues that shift co-linearly between ΔN301 , ΔN280, and ΔN244^5P. Strikingly, these motions were dampened in ΔN280, and further in ΔN244^5P. ¹³C-methyl relaxation dispersion of Leu, He, and VaI sidechains within the hydrophobic network of ΔN301 appeared to indicate motions on the same timescale (Δ_ex ~ 0.3 msec), that again were progressively reduced in ΔN280 and ΔN244^5P. These data indicate that the core hydrophobic packing of Ets-1 is dynamic in the active state, and that increasing inhibition attenuates these motions. Localized collective motions on a similar time scale within both the backbone and sidechains appear to support the proposal that the inhibitory module, DNA binding interface, and hydrophobic core form a concerted unit that is linked as a dynamic, hydrophobic network. Furthermore, the HX and R_ex data appear to be consistent with a conformational equilibrium within this concerted unit between at least two states, one stable and inactive, another more dynamic and active.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in

be applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

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1. A method of producing a graded response in a protein comprising;

altering the gene sequence for said protein in such a manner as to effect the post- translational activity of said protein.

2. The method of claim 1 , wherein said alteration in gene sequence affects said protein in a manner selected from the group consisting of phosphorylation, conformational changes, formation of complexes, acetylation, alkylation, biotinylation, deimination, deamidation, disulfide bridging, glutamylation, glycylation, glycosylation, isoprenylation, lipoylation, PEGylation, phosphopantetheinylation, phosphorylation, proteolysis, sulfation, SUMOylation, and ubiquitination.

3. The method of claim 1 , wherein said protein is selected from the group consisting of receptor proteins, enzymes and transcription factors.

4. The method of claim 3, wherein said protein is Ets-1.

5. The method of claim 4, wherein said alteration of the gene sequence effects phosphorylation of the serine rich region of said Ets-1.

6. The method of claim 1 , wherein said graded response in a protein results in an alteration in the rate of transcription of a gene regulated by said protein.

7. The method of claim 1, wherein said protein is useful in the inhibition of transcription of a particular gene product.

8. The method of claim 1 , wherein said protein is useful in promoting transcription of a particular gene product.

9. The method of claim 1 , wherein said protein is useful in the treatment of a disease selected from the group consisting of Alzheimer's Disease, cancer, heart disease, muscular dystrophy, cystic fibrosis, inflammation, autoimmune disease, liffiSISijiobifeifyjLilHi'dOlar degeneration, COPD, multiple sclerosis, depression, schizophrenia, infertility, hair loss, asthma, spinal cord injury, Parkinson's disease, Huntington's Disease, Spinal Muscular Atrophy, hemophilia, glaucoma, viral infections, fungal infections, bacterial infections, kidney disease, kwashiorkor, familial amyloidotic polyneuropathy, mitochondrial trifunctional protein deficiency, and age-related development of cataracts.

10. The method of claim 1 , wherein said protein is within a single celled organism, a cell from a multicellular organism, a gamete cell or an oocyte.

11. The method of claim 1 , further comprising administering a second nucleic acid wherein expression of said second nucleic acid is regulated by said protein.

12. The method of claim 11 , wherein said second nucleic acid encodes a protein of therapeutic benefit to said patient.

13. A method for treating disease comprising administering a protein of Claim 1 to a patient in need of such treatment.

14. A method for treating disease comprising administering a nucleic acid encoding a protein of Claim 1 to a patient in need of such treatment.

15. The method of claim 14, further comprising administering a second nucleic acid wherein expression of said second nucleic acid is regulated by said protein.

16. The method of claim 15, wherein said second nucleic acid encodes a protein of therapeutic benefit to said patient.

17. A method of treating a disease, comprising introducing into a patient a nucleic acid encoding a protein capable of a graded response, such that a level of said protein effective to alleviate the symptoms of said disease is expressed in the patient. IMMMIe1τ(f£iidi$ αf claim 17, wherein said disease is Alzheimer's Disease, cancer, heart disease, muscular dystrophy, cystic fibrosis, inflammation, autoimmune disease, diabetes, obesity, macular degeneration, COPD, multiple sclerosis, depression, schizophrenia, infertility, hair loss, asthma, spinal cord injury, Parkinson's disease, Huntington's Disease, Spinal Muscular Atrophy, hemophilia, glaucoma, viral infections, fungal infections, bacterial infections, kidney disease, kwashiorkor, familial amyloidotic polyneuropathy, mitochondrial trifunctional protein deficiency, and age-related development of cataracts.

19. A composition comprising: a nucleic acid sequence including an alteration to said sequence that affects the post-translational state of the protein encoded by said sequence in order to create a graded response in said protein.

20. The composition of claim 19, wherein said alteration in gene sequence affects said protein in a manner selected from the group consisting of phosphorylation, conformational changes, formation of complexes, acetylation, alkylation, biotinylation, deimination, deamidation, disulfide bridging, glutamylation, glycylation, glycosylation, isoprenylation, lipoylation, PEGylation, phosphopantetheinylation, phosphorylation, proteolysis, sulfation, SUMOylation, and ubiquitination.

21. The composition of claim 19, wherein said protein is selected from the group consisting of receptor proteins, transcription factors, enzymes and transcription factors.

22. A method of producing a protein capable of a graded response comprising; altering the nucleic acid sequence encoding said protein in such a way as to affect the post-translational state of said protein in order to create said graded response; introducing into a host cell said nucleic acid sequence; inducing expression of said protein within the host cell; and identifying a recombinant host cell in which the selected host DNA sequence exhibits said nucleic acid sequence. ^'SSSOTIMe-fiiffidSoiROfeiirn 22, wherein said alteration in gene sequence affects said protein in a manner selected from the group consisting of phosphorylation, conformational changes, formation of complexes, acetylation, alkylation, biotinylation, deimination, deamidation, disulfide bridging, glutamylation, glycylation, glycosylation, isoprenylation, lipoylation, PEGylation, phosphopantetheinylation, phosphorylation, proteolysis, sulfation, SUMOylation, and ubiquitination.

24. The method of claim 22, wherein said protein is selected from the group consisting of receptor proteins, transcription factors, enzymes and transcription factors.

25. The method of claim 22, wherein said protein is Ets-1.

26. The method of claim 23, wherein said alteration of the gene sequence effects phosphorylation of the serine rich region of said Ets-1.

27. The method of claim 22, wherein said graded response in a protein results in an alteration in the rate of transcription of a gene regulated by said protein.

28. The method of claim 22, wherein said protein is useful in the inhibition of transcription of a particular gene product.

29. The method of claim 22, wherein said protein is useful in promoting transcription of a particular gene product.

30. The method of claim 22, wherein said protein is useful in the treatment of a disease selected from the group consisting of Alzheimer's Disease, cancer, heart disease, muscular dystrophy, cystic fibrosis, inflammation, autoimmune disease, diabetes, obesity, macular degeneration, COPD, multiple sclerosis, depression, schizophrenia, infertility, hair loss, asthma, spinal cord injury, Parkinson's disease, Huntington's Disease, Spinal Muscular Atrophy, hemophilia, glaucoma, viral infections, fungal infections, bacterial infections.

22, wherein said protein is within a single celled organism, a cell from a multicellular organism, a gamete cell or an oocyte.

32. The method of claim 22, wherein said protein is useful for a purpose selected from the group consisting of research, development, diagnostic or therapeutic purposes.

33. A method for treating disease comprising administering a protein of Claim 22 to a patient in need of such treatment.