WO2006066268A2

WO2006066268A2 - Synthetic transcription regulators

Info

Publication number: WO2006066268A2
Application number: PCT/US2005/046351
Authority: WO
Inventors: Thomas E. Wagner; Xianxhang Yu
Original assignee: Wagner Thomas E; Xianxhang Yu
Priority date: 2004-12-17
Filing date: 2005-12-19
Publication date: 2006-06-22
Also published as: WO2006066268A3

Abstract

This invention relates to the regulation of transcription with novel compositions and methods. Compositions and methods providing for the re-addressing of target genes in order to regulate transcription are described.

Description

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SYNTHETIC TRANSCRIPTION REGULATORS

BACKGROUND

Field of Invention

This invention generally relates to the regulation of transcription. In particular, compositions and methods providing for the re-addressing of target genes in order to regulate transcription are described.

Background

The complicated patterns of differential gene expression observed in all species, and especially in higher species and man, are the result of exquisite regulation of gene expression. While every cell within a multi-cellular organism has the identical complement of genes, different genes are expressed by activating the production of their complementary mRNAs or suppressed by repression or blockage of the production of their complementary mRNAs. These patterns of gene expression/suppression result in generating any cell's specific function. For example, the cells of the rods and cones of the eye express specific protein gene products that allow these cells to act as light sensors and allow for the process of vision. White blood cells from the same organism and containing exactly the same genome and DNA sequence in their chromosomes do not express those genes coding for the photo-responsive proteins of the eye but do express those genes and produce those cognate proteins that act as the organism's immune response. In this manner, complex organisms like man are able to function.

One of the most intensely studied aspects of genetics and molecular biology is the mechanism by which genes are regulated. Recently these mechanisms have begun to be elucidated. In addition to containing a coding region which encodes its cognate protein, each gene contains sequences, which usually precede its coding region, for the sole purpose of regulating its expression. These regulatory non- coding regions of genes have historically been termed promoters, but more recently have been re-described as gene "addresses." These genetic addresses define the patterns of expression of each gene so that gene expression to be appropriately regulated for the whole organism's benefit. Within these genetic addresses, short stretches of gene-specific DNA sequences provide the basis for this regulation. These specific address sequences may bind to regulatory proteins that may then either turn on or suppress gene expression. Extensive literature regarding address sequence specific activators and suppressors of gene expression has been generated over the past few years.

Since many diseases and disorders are a result of dysregulation or misregulation of gene expression, there is a need in the art for compositions and methods that can regulate gene expression and compensate for the effects of overexpression or insufficient expression of a given gene. The present invention satisfies that need.

SUMMARY

The present invention provides a method of regulating expression of a gene of interest comprising: (i) binding a DNA binding molecule that comprises at least one DNA binding domain to a gene of interest, (ii) associating the DNA binding molecule with a nucleic acid molecule or PNA molecule that comprises a binding site for a protein that interacts directly with the transcription machinery of the gene of interest, (iii) binding of the protein that interacts with the transcription machinery of the gene of interest to the binding site on the nucleic acid or PNA molecule, and (iv) regulating transcription of the gene of interest. In a preferred embodiment of the provided method the gene of interest is Bcl2. In other embodiments of the present method, the DNA binding molecule may comprise a gal4 binding domain, a Zinc finger binding domain, or a zif-268. In other embodiments, the DNA binding molecule may comprise a nucleic acid capable of forming a three helix bundle.

In a preferred embodiment of the present method the DNA binding molecule is a peptide nucleic acid (PNA). Additionally, the nucleic acid molecule may comprise a single stranded nucleic acid molecule capable of forming a self complementary binding site, and the nucleic acid molecule binds in a self complimentary manner before binding to the protein that interacts with the transcription machinery of the gene of interest. In another embodiment of the present method, the nucleic acid molecule consists of at least 2 single stranded nucleic acid molecules that are capable of binding to each other.

In another embodiment of the present method, the DNA binding molecule and nucleic acid molecule or PNA molecule may form a Y structure or a U structure.

Another aspect of the present invention is a composition for regulating gene expression consisting essentially of: (i) a DNA binding molecule that comprises at least one DNA binding domain, and (ii) a nucleic acid molecule or PNA molecule that contains a binding site for a protein that directly interacts with the transcription machinery of a gene of interest. In a preferred embodiment of the provided composition, the gene of interest is Bcl2. Additionally, the DNA binding molecule may comprise a gal4 binding domain, a Zinc finger binding domain, or a zif-268. The DNA binding molecule may comprise a nucleic acid capable of forming a three helix bundle.

In a preferred embodiment of the present composition, the DNA binding molecule is a peptide nucleic acid (PNA). Additionally, the nucleic acid molecule may comprise a single stranded nucleic acid molecule capable of forming a self complementary binding site, and the nucleic acid molecule binds in a self complimentary manner before binding to the protein that interacts with the transcription machinery of the gene of interest.

In another embodiment of the present method, the nucleic acid molecule consists of at least 2 single stranded nucleic acid molecules that are capable of binding to each other.

In another embodiment of the present composition, the DNA binding molecule and nucleic acid molecule or PNA molecule may form a Y structure or a U structure.

Another aspect of the present invention is a method of making a composition for regulating gene expression comprising associating the DNA binding molecule with a nucleic acid molecule or PNA molecule that comprises a binding site for a protein that interacts directly with the transcription machinery of the gene of interest. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 - Schematic representation of gene readdressing using PNA molecules to bind the promoter for the gene of interest and the nucleic acid molecule.

Figure 2 - Schematic representation of gene readdressing using PNA molecules to bind the promoter for the gene of interest and the nucleic acid molecule.

Figure 3 - Proposed Synthesis of Protein - Peptide Oligo Complex. Synthesis may be used to create Y-Structure. Reacting a 5¹ or 3' Phosphate oligo with EDC and Imadazole, then adding Adipic hydrazide to yield the oligo hydrazide which will react with a C-terminal CHO peptide without interference from the possible free amino groups in a lysine residue is provided. These NH2 will form transient shift bases but the hydrazide will form a stable bond.

Figure 4 - Schematic representation of gene activation via re-addressing.

Figure 5 - Schematic showing nucleic acid molecule with a Silencer re- address sequence between DNA binding domains of the DNA binding molecule.

Figure 6 -Schematic representation showing a nucleic acid molecule capable of binding a Zinc finger domain and having the additional silencer re-address sequence.

Figure 7 - Model of the Y-Structure including two gal4 DNA binding domains and the associated readdressing sequence without the promoter for the target gene.

Figure 8 -Model of the Y-Structure as bound to a DNA helix representing the promoter for the target gene.

Figure 9 - Space filed molecular model of the U-structure. A Gal4 target DNA sequence is bound to two Gal4 peptide GGC binding domains that continue into an 8 base PNA going N-C. This is then bound to two oligos with the 5¹ end of one and the 3¹ end of the other complementary so that single stranded overhangs, each in the 5¹ to 3' direction hang off each end. These overhangs are hybridized to the PNA so that it is C-N PNA on 5¹ to 3¹ DNA. This places the re-address DNA high enough above the genomic DNA to function as a new address.

Figure 10 - Crystal structure of a re-address complex. Complex is formed by linking a synthetic peptide zinc finger with a C-terminal CHO instead of a COOH to a synthetic deoxy oligo with a 16 carbon linker with a terminal NH2 (commercially available from Invitrogen) to form a -CH2-NH-CH2- linkage after Na cyanoborohydride reduction.

Figure 11 - Representation of duplex - duplex hydrogen bonding of base pairs in the quadrahelix structure.

Figure 12 - 10 unit DNA PNA quadrahelix of GC CG. Model is energy minimized.

Figure 13 Space filed model of a double stranded DNA : double stranded PNA quadrahelix. (A) Side view. (B) Top view.

Figure 14 - Space filled molecular model of two gal4 DNA binding domains with associated PNA molecules on a DNA target.

Figure 15 - Schematic representation and description of the pM plasmid

Figure 16 - Schematic representation of the pFR-Luc plasmid

DETAILED DESCRIPTION OF THE INVENTION

Introduction

A dominant feature of gene regulatory proteins which bind to genetic addresses and either activate or suppress gene expression is their modular nature. See, for example, Alberts, et al., Molecular Biology of the Cell, 3^rd ed. (1994). These regulatory proteins all contain a specific sequence DNA binding domain and a transcription activator or suppressor domain that are distinct domains within the protein structure. No specific geometric or conformational relationship between the DNA sequence address binding domain and their activator or suppressor domain has been identified or shown necessary for the function of any of these proteins to date. The present invention takes advantage of this property by copying and optionally modifying the DNA binding domain of one regulatory protein, and associating it with a segment which acts as a binding site for a protein that will in turn directly interact with the transcription machinery of a target gene.

Thus, described herein are compositions and methods for regulating expression of a gene of interest. In particular, the inventors discovered that expression of a gene of interest can be activated or enhanced in an environment in which the gene of interest is not normally (or insufficiently) expressed. Likewise, the inventors discovered that expression of a gene of interest can be repressed in an environment in which the gene of interest is normally expressed.

Compositions

The present invention describes a composition comprising (i) a DNA binding molecule that comprise at least one DNA binding domain and (ii) a nucleic acid or PNA molecule that contains a binding site for a protein that directly interacts with the transcription machinery of a gene of interest. As disclosed herein, the DNA binding domain of the DNA binding molecule binds a target sequence on the gene of interest and the nucleic acid molecule or PNA molecule associates with the DNA binding molecule.

In another embodiment, the compositions of the present invention comprise a DNA binding molecule that is a protein that contains both (i) a DNA binding domain that binds to a particular region on a gene of interest (i.e., a target sequence), and (ii) a region that acts as a binding site for a nucleic acid domain.

DNA binding molecule

A DNA binding molecule as described in the present invention is a molecule that comprises at least one DNA binding domain which recognizes and specifically binds to a target DNA sequence, typically to a promoter region, on a gene of interest. The DNA binding molecule may be a protein, PNA, or a combination thereof.

Examples of DNA binding molecules suitable for use in the present invention include proteins with a zinc finger motif or a leucine zipper motif, or proteins with a helix-turn-helix motif. The DNA binding domain of the DNA binding molecule may also be derived from suitable regulatory proteins, i.e. either positive or negative regulators of transcription. For example, the DNA binding domain of the DNA binding molecule may comprise the appropriate DNA-binding domain from a λ repressor protein, e.g. λ Cro. Alternatively, suitable regions of protamine may be used. Protamines are positively charged proteins used to pack in DNA, for example, sperm cells. Thus, the DNA-binding domains may be modified or engineered to have unique binding affinity for a target sequence.

The DNA-binding domain of the DNA binding molecule is chosen on the basis of its ability to bind to a selected target sequence on, or associated with, a gene of interest. The targeted sequence on the gene of interest is not necessarily a regulatory region within the gene of interest and therefore, the DNA binding domain may be engineered to bind to alterative sequences that are highly conserved in the gene of interest.

The use of the term "conserved" in the context of the gene of interest is intended to refer to a target sequence that is specific for that gene, i.e. not found in other unrelated genes. The target sequence will usually be greater than 6 nucleotides, preferably greater than 8 nucleotides, more preferably greater than 10 nucleotides, and most preferably 16 or more nucleotides. Suitable target sequences can be identified using conventional sequence analysis software programs, with comparisons to other gene sequences being accomplished based on the sequence information made available as part of the Human Genome Project. For example, the target sequence on the gene of interest can be analyzed using conventional computer programs to identify a "conserved" sequence that is specific for that gene. This sequence can then be used as the target and a suitable DNA binding domain of the DNA binding molecule can be designed based on this target sequence.

The DNA binding domain of the DNA binding molecule of the present invention may be selected by using conventional techniques. Once the conserved gene sequence on the gene of interest has been selected, this can be used as the target in an assay to make a suitable DNA binding molecule. Conventional DNA binding molecules may be adapted/modified using recombinant DNA techniques, to produce proteins that contain a DNA binding domain that binds specifically to the conserved sequence.

A particularly suitable technique is phage display, a review of which is given in Cannon et al., IVD Technology, 1996; November/December: 22-31. Phage display is an efficient way of producing large numbers of diverse proteins/peptides, and selecting those that bind to a particular target. Alternative techniques, for example, ribosome display, may also be used to select those molecules that bind to the conserved sequence.

In addition, Moore et al., Proc. Natl. Acad. ScL, 2001 ; 98(4): 1432-1436, and Moore et al., Proc. Natl. Acad. Sci., 2001 ; 98(4): 1437-1441 , show that polyzinc finger peptides can be adapted to produce "designer peptides" that have novel binding specificities. These publications show that it is possible to design peptides that bind to unique sites on a genome. Thus, in another embodiment of the present invention, the DNA binding domain of the DNA binding molecule is a multi-zinc finger peptide that binds to a unique target DNA sequence on the gene of interest. Preferably there are at least four, and more preferably at least six zinc fingers that make up the DNA binding domain.

The molecular interactions between several DNA binding domains and the sequences to which they bind have been extensively studied. Specifically, the zinc finger binding domains of a specific zinc finger DNA binding protein have been fully characterized and based upon this characterization zinc finger binding domains may be designed to selectively bind to any of the 16 triplet sequences GNN (US patent 6,140,081). In addition naturally existing DNA binding domains such as the yeast GaI 4 DNA binding domain that binds to the sequence GGC is known. These modular GNN sequence specific DNA binding domains are approximately 30 amino acids in length and may be chemically synthesized or produced biologically.

Because of the length and complexity of the human genome, a DNA sequence on a given gene must be at least 12 and preferably 18 base pairs in length to be unique within the human genome. Therefore, in order to build a DNA binding domain that can recognize any unique "address" it must recognize a DNA sequence of such length. This can be accomplished by, for example, stringing 6 modular GNN specific zinc finger domains together to produce a protein that recognizes an 18 base pair DNA sequence.

For GAL4 yeast transcription factor, two GGC recognition DNA binding modules constrained a precise distance from each other and in mirror image orientation to each other by a protein bridge to generate sequence uniqueness by targeting the sequence GGCNNNNNNNNNNNNNCGG. See Alberts, et. al, 1994. The lac repressor, a bacterial gene regulatory protein, utilizes a similar approach to create sequence recognition. Id.

Nucleic acid molecule and PNA molecule

A nucleic acid as described in the present invention contains a binding site for a protein such as a transcription regulator that directly interacts with the transcription machinery of a gene of interest. In addition, the nucleic acid molecule is associated with the DNA binding molecule described herein.

The nucleic acid of the present invention can be DNA or RNA. In one embodiment, the nucleic acid molecule is a promoter or enhancer sequence that can be used to enhance expression of a gene of interest. In another embodiment, the nucleic acid molecule is a promoter nucleic acid sequence that functions more effectively when associated with the appropriate transcription machinery than the promoter nucleic acid sequence endogenously associated with the gene of interest. In yet another embodiment, the nucleic acid molecule is a promoter nucleic acid sequence that contains a binding site for a transcription regulator which directly interacts with the transcription machinery of the gene of interest to either activate or repress gene transcription. Preferably, the transcription regulator which directly interacts with the transcription machinery of the gene of interest is an enhancer of expression.

A peptide nucleic acid ("PNA") molecule as described in the present invention is PNA that contains a binding site for a protein such as a transcription regulator that directly interacts with the transcription machinery of a gene of interest. In addition, the PNA molecule is associated with the DNA binding molecule described herein. Thus, in another embodiment of the present invention, the PNA molecule acts a promoter or enhancer sequence that can be used to enhance expression of a gene of interest. More specifically, one embodiment of the present invention discloses a DNA binding molecule that comprises a protein that contains the DNA binding domain and is associated with a PNA molecule that comprises a PNA clamp. The PNA clamp acts as a promoter sequence and provides a binding site for a protein that directly interacts with the transcription machinery of a gene of interest.

A PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997 7(4) 431-37). A PNA is able to be utilized in a number methods that traditionally have used RNA or DNA. In fact, PNA sequences often perform better in various techniques than the corresponding RNA or DNA sequences, and have utilities that are not inherent to RNA or DNA. A review of PNAs, including methods of making, characteristics of, and methods of using, is provided by Corey, Trends Biotechnol 1997 June;15(6):224-9.

PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al., Science 1991 Dec. 6;254(5037):1497-500; Hanvey et al., Science. 1992 Nov. 27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 January;4(1):5-23). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used.

PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et a/., Bioorg Med Chem. 1995 April;3(4):437-45). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs. As with peptide synthesis, the success of a particular PNA synthesis will depend on the properties of the chosen sequence. While in theory, PNAs can incorporate any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. In expectation of this difficulty, it is suggested that, in producing PNAs with adjacent purines, one should repeat the coupling of residues likely to be added inefficiently. This should be followed by the purification of PNAs by reverse-phase high-pressure liquid chromatography, providing yields and purity of product similar to those observed during the synthesis of peptides.

Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine. Alternatively, PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry. Several studies have made and utilized modifications of PNAs (for example, Norton et al., Bioorg Med Chem. 1995 April; 3(4):437-45; Petersen et al., J Pept Sci. 1995 May-June; 1(3): 175-83; Orum et al., Biotechniques. 1995 September; 19(3):472-80; Footer et al., Biochemistry. 1996 Aug. 20;35(33): 10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug. 11 ;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci USA. 1995 Jun. 6;92(12):5592-6; Boffa et al., Proc Natl Acad Sci USA. 1995 Mar. 14;92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 Aug. 15;88(4):1411-7; Armitage et al., Proc Natl Acad Sci USA. 1997 Nov. 11 ;94(23): 12320-5; Seeger et al., Biotechniques. 1997 September;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.

Also contemplated in the present invention is a PNA molecule that can act as both a DNA binding molecule and a PNA molecule, as described herein.

While this invention allows the selective targeting of any genetic address, it also offers a dramatic additional opportunity. The double stranded DNA helix utilized to selectively separate the DNA binding domains a precise distance from each other and so create sequence specific recognition and binding, like any other DNA sequence, may also serve as a target for regulatory protein binding. In any organism or cell ubiquitously expressed genetic regulatory proteins are naturally present. They act on their particular sequence specific genetic address targets of those genes they are intended to control. But, if the sequence of these specific address targets is included within the DNA helical portion of the zinc finger-DNA helix-zinc finger motif of the gene address targeting molecule described above, this sequence can act to bind its cognate regulatory protein. Due to the modular nature and lack of any specific geometric or conformational relationship between the DNA sequence address binding domain of these regulatory proteins and their activator or suppressor domain as described above, they act to regulate the gene targeted by gene address targeting molecule.

The present invention also describes numerous structural modes to provide for the nucleic acid or PNA molecule to properly form in order to bind a protein that directly interacts with the transcription machinery of a gene of interest. This protein may be an activator or repressor of transcription of the gene of interest. In one embodiment, a DNA binding molecule comprises at least two DNA binding domains that bind to separate binding sites on a promoter for the gene of interest. Each of the DNA binding domains on the DNA binding molecule preferably has a single stranded DNA or PNA linked to the DNA binding domain. Upon binding of each DNA binding domain to the promoter for the gene of interest, the single strand of PNA or DNA linked to one DNA binding domain would ligate to the single strand of PNA or DNA linked to the other DNA binding domain. An example of such a duplex structure is provided in Figure 7-8 and is termed a "Y-structure." In this regard, the ligated PNA or DNA becomes a double stranded PNA or DNA and is able to act as a binding site for a protein that directly interacts with the transcription machinery of a gene of interest.

One of skill in the art would appreciate that the DNA and PNA duplexes described herein must be anti-parallel. Consequently the single stranded DNA or PNA must be designed so as to allow for proper ligation. In certain instances, this may require linking the PNA or DNA strands in opposite orientation to each other on the DNA binding molecule. For instance, if a zinc finger DNA binding domain is used, one of the binding sites may require the PNA to have its "N terminus" linked to the end of the DNA binding domain, while the other zinc finger DNA binding domain will have a PNA linked by the "C terminus" to the DNA binding domain. Thus, the two oppositely connected PNA strands will be designed to ligate in the Y-structure. The same applies if a DNA strand instead of a PNA strand was linked to a DNA binding domain In certain situations one may be able to link all of the DNA or PNA strands in the same fashion and still have binding of the DNA or PNA portions in an anti-parallel fashion.

In addition, a skilled artisan may limit the flexibility of the single stranded portions of the Y-Structure by the additional of short oligonucleotides that ligate to these single stranded portions.

A different structural possibility is termed the "U-structure" for the purposes of this application. In this structure, two DNA binding domains have short PNA or DNA single strands linked to the N or C-terminal end of the DNA binding domains. Each DNA binding domain binds independently to DNA sequence on the target gene. Another double stranded molecule is then introduced which has short 5' and 3' single strand overhangs that may ligate to the single stranded DNA or PNA molecules linked to the DNA binding domain. This structure is provided in Figure 9 and places the readdressed binding site above the target sequence of the gene of interest positioned for the binding of an activator or repressor.

Alternatively, the PNA or DNA strands linked to the DNA binding domains of the DNA binding molecule may form an anti-parallel helix that may serve as the binding site for a protein that interacts directly with the transcription machinery of a gene of interest.

As one of skill in the art will appreciate, PNAs bind tightly to single stranded and double stranded DNA. When binding to double stranded DNA, however, the third strand introduces non-Watson-Crick base pairings, such as Hookstein pairs. As such, a PNA may act as a DNA binding molecule. One piece of PNA may act as a "bridge" linking a target sequence in a gene of interest with a nucleic acid molecule, such as another double stranded piece of DNA that contains a binding site for a protein that directly interacts with the transcription machinery of the gene of interest. In one embodiment, this second piece of DNA contains the binding site for an activator or repressor or repressor of transcription. See, for example, Figures 1 and 2. Similarly, the use of two PNA molecules to recognize a single site on a DNA helix may also be used. See below, discussion of quadrahelices and Figures 11-13.

There is no reason that the nucleic acid or PNA molecule, as defined herein, containing the "new address" (maybe a binding sequence for a gene silencer which is ubiquitously expressed in all cells or the target cells for turning off a gene) need be between the two DNA binding domains. As described above, these are two separate concepts in this composition. One is the concept of spacing two GNN binding domains, or other DNA binding domains, at a prescribed distance from each other by a DNA double helix stretch. A separate concept is the binding (in any way) a short oligonucleotide containing a "new address" to the promoter region of a gene of interest.

While one may covalently link the DNA binding molecule and nucleic acid molecule or PNA molecule to each other, as we have noted in this description, non- covalent binding may also be employed in order for two molecules to be "associated." As noted in the structures described above, binding between these two molecules (DNA binding molecule and nucleic acid or PNA molecule) can occur through base pairings, or other means, such as electrostatic, Van der Waals interactions,, ionic bonds, or hydrophobic forces. Interactions between DNA or PNA and a DNA binding molecule may similarly utilize any of the above mentioned means. As used throughout the application, the terms "associated," "associates," or "associating" when describing the relationship between two molecules includes both covalent and non-covalent means.

An additional aspect of this invention will take a known protein transcription factor which binds to a gene of interest (maybe even a factor that turns on that gene too much in cancer cells), delete its transcriptional regulatory module and replace it with a three zinc finger module which can recognize a specific GNNGNNGNN sequence. This novel protein will bind to the gene of interest but will not activate or enhance gene transcription since the protein's transcriptional regulatory region has been deleted. When one adds to these cells a double stranded oligonucleotide that acts as a nucleic acid molecule containing two important regions, first the GNNGNNGNN sequence that will cause it to bind to the added three zinc finger module of our novel protein and second a sequence which will bind a ubiquitously expressed gene suppressor in the cell, the suppressor can bind to the nucleic acid domain and repress transcription. See, for example, Figure 5. A skilled artisan would understand that standard recombinant DNA technology can be used to create a sequence encoding such a novel protein.

The concept of positioning a nucleic acid molecule at an appropriate position in a gene's promoter will re-address the gene. The means of doing this is novel, whether 1) the DNA binding molecule has a DNA binding domain to recognize the promoter of the gene of interest and the DNA binding molecule also binds to the nucleic acid molecule (or PNA molecule) or 2) the DNA binding molecule is covalently bound to the nucleic acid molecule (or PNA molecule). See Figures.

In another embodiment of the present invention, the protein binding site on nucleic acid molecule or PNA molecule (e.g., transcription factor binding site) is adjacent to the TATA box overriding the genomic promoter signals and effectively controlling the gene of interest. The TATA box is close to the start site of transcription on the gene of interest. Since transcription factors binding far from the TATA box probably regulate transcription by interacting with other transcription factors in the genomic DNA "context," one embodiment of the present invention is to override these complex signals by directly affecting the basic transcriptional machinery at the TATA box position.

Thus, since the effective distance between the activator or repressor and the transcription machinery may be limited, one embodiment of the present invention is to target the portion of the gene of interest that is close to the TATA box. In turn, this reduces the percentage of the genome where the protein which directly interacts with the transcription machinery of the gene of interest can function. For example, if the present invention could only function within 50 bases of the TATA box, then the amount of sequence within which the binding site must be unique is approximately 30,000 (number of genes) X 50 bases = 1 ,500,000 bases. Compared to the 3 billion bases of the human genome, this reduction in bases may allow for a six base sequence that is unique within this number of bases. As described above, the present invention has an additional method to engineer and gene of interest. By targeting the invention at two different sites with, for example, two zinc finger GNN DNA binding domains a fixed distance from each other by a DNA bridge, additional specificity may be generated. Therefore, to target a given gene one may analyze the promoter sequence, identify two GNN triplets within ~50 bases of the TATA box, count the bases between these triplets and build a targeting molecule accordingly.

Another method of recognizing sites at different distances along the DNA helix is to utilize DNA binding domains that bind DNA sites at the appropriate distances from each other. These domains often occur as homodimers, such as with the Gal4 and similar DNA binding molecules which may be used in this approach to re- address genes. By placing a PNA/DNA molecule of appropriate design onto the end of each DNA binding domain and allowing the domains to form homodimers, DNA binding domains recognize sites at the appropriate positions on the DNA and can position the PNA/DNA molecules to ligate and act as the nucleic acid or PNA molecule. There are several gene regulatory proteins which use the identical mode of DNA sequence recognition (CGG palindromic sequences with differing space between them, but otherwise identical). These proteins all have a DNA binding domain which is virtually identical ~32 amino acids with 6 Cys and two Zn or Cd to form a CGG binding cluster. They differ only in the spacing between these clusters and therefore the spacing between the GGCs in their DNA binding sequence, e.g., GaW, 11 bases; PPR1 , 6 bases, and PUT3, 10 bases. See, for example, DNA sequence preferences of Gal4 and PPR1 : How a subset of Zn2Cys6 binuclear cluster proteins recognize DNA. Stanley D. Laing, Ronen Marorstein, Stephen C. Harrison and Mark Ptashne, Molecular and Cellular Biology, July 1996, Vol. 16. No. 7 pp. 3773-3780, and How do Zn2Cys6 proteins distinguish between similar upstream activation sites? Vashee, S., Xu, H., Johnston, S.A., and Kodadek, T. The Journal of Biological Chemistry, Vol. 268, No. 33, pp. 24699-24706 (1993).

Another embodiment of this invention includes the binding of multiple DNA binding domains and nucleic acid and/or PNA molecules upstream of the start site in the gene of interest. These multiple compositions may provide multiple binding sites for proteins, such as regulatory proteins, which will further concentrate and localize the required regulatory proteins {e.g., activators or repressors of transcription) to the gene of interest.

Protein that interacts with the transcription machinery of the gene of interest

In addition to associating with the DNA binding molecule of the present invention, the nucleic acid or PNA molecule described herein attracts a protein necessary for the regulation of expression (e.g., a regulatory protein) of the gene of interest. Protein binding to the nucleic acid or PNA molecule allows the regulatory protein to be brought into close enough proximity of the transcription machinery of the gene of interest so as to effect activation or repression of transcription of the gene of interest.

The nucleic acid or PNA molecule may therefore contain a binding site for an activator of transcription. Activators of transcription are well known in the art. For example, nuclear protein Oct-1 is well characterized as an activator of gene transcription. This factor is specific for an octamer motif having the consensus sequence ATGCAAAT, which is a common regulatory domain of immunoglobulin (Ig) genes. An alternative activator is the herpes simplex virus vision protein 16 (VP16), the amino acid sequence of which is disclosed in Triezenberg et al., Genes Dev., 1988; 2: 718-729. Alternatively, Gal4 may be used or lac may be used. In this regard, an activator of transcription may bind the nucleic acid molecule or PNA molecule and by virtue of its proximity to the transcription machinery of a gene of interest, the activator is able to activate transcription of the gene.

A person of skill in the art will appreciate that the nucleic acid molecule or PNA molecule may serve as a binding site not only for activators, but many types of regulatory proteins and transcription factors. These proteins may include repressors of transcription when there is a need to repress the target gene. In addition, if a molecule is required to interact with the transcription machinery, readdressing of the target gene will allow one to place the protein of interest in close proximity to the transcription machinery.

Gene of interest The compositions of the present invention allows for selective re-addressing of any gene. In other words, the gene of interest as described herein refers to the gene that is desired to be regulated (either activated, enhanced or repressed) by the compositions described herein. The gene of interest may be any gene such as an oncogene, or other genes that are can lead to oncogenesis, such as the over- expression genes encoding growth factors and/or hormones. The gene of interest may also be viral DNA that is incorporated into a host genome. It is desirable to repress certain integrated viral genes, as these may be implicated in pathogenesis.

The present invention also provides for methods and compositions that repress Bcl-2 expression in tumor cells. By building a composition comprising a DNA binding molecule and a nucleic acid or PNA molecule, the Bcl2 gene may be re-addressed for repression or silencing, thereby selectively reducing or eliminating the Bcl2 protein from tumor cells.

The under-expression of genes can also result in disease, due to a lack of an endogenous product. It is therefore desirable to enhance or activate expression of these genes to correct the deficiency. For example, the gene of interest may encode a product used in metabolism, and so the correct expression of the gene is necessary to maintain healthy metabolic function.

Methods

The present invention includes a method of making a composition for regulating gene, comprising associating the DNA binding molecule with a nucleic acid molecule or PNA molecule that comprises a binding site for a protein that interacts directly with the transcription machinery of the gene of interest.

As disclosed herein, methods for making the compositions according to the present invention, will be apparent to those skilled in the art.

The DNA binding molecule and nucleic acid or PNA molecule will be functional when they become associated with each other, the target sequence on gene of interest, and the protein which interacts directly with the transcription machinery of the gene of interest and which is endogenous to a cell containing the gene of interest. The present invention also provides for a method of treatment comprising introducing into a cell (i) a first vector encoding a DNA binding molecule that comprises at least one DNA binding domain, and (ii) a second vector comprising a nucleic acid molecule, wherein the nucleic acid molecule is a nucleic acid sequence that comprises a binding site for a protein that directly interacts with the transcription machinery of a gene of interest. The first and second vectors, however, may be administered in a reverse order. Likewise, a kit comprising (i) a first vector encoding a DNA binding molecule that comprises at least one DNA binding domain, and (ii) a second vector comprising a nucleic acid molecule, wherein the nucleic acid molecule is a nucleic acid sequence that comprises a binding site for a protein that directly interacts with the transcription machinery of a gene of interest, are also contemplated in the present invention.

In another embodiment, the present invention discloses a method of treatment comprising introducing into a cell a composition comprising a DNA binding molecule that comprises at least one DNA binding domain, associated with a PNA molecule, wherein the PNA molecule comprises a binding site for a protein that directly interacts with the transcription machinery of a gene of interest.

The present invention also provides for a method of treatment comprising introducing into a cell (i) a DNA binding molecule that comprises at least one DNA binding domain bound to a single stranded DNA or PNA molecule, and (ii) a vector comprising a nucleic acid molecule, wherein the nucleic acid molecule is a nucleic acid sequence that comprises a binding site for a protein that directly interacts with the transcription machinery of a gene of interest and which binds to the single stranded DNA or PNA molecule bound the DNA binding molecule. The DNA binding molecule and vector, however, may be administered in a reverse order. Likewise, a kit comprising (i) a DNA binding molecule that comprises at least one DNA binding domain bound to a single stranded DNA or PNA molecule, and (ii) a vector comprising a nucleic acid molecule, wherein the nucleic acid molecule is a nucleic acid sequence that comprises a binding site for a protein that directly interacts with the transcription machinery of a gene of interest and which binds to the single stranded DNA or PNA molecule bound the DNA binding molecule, are also contemplated in the present invention. The compositions of the present invention may be introduced into a cell of any animal, and humans in particular. The appropriate dosage can be selected according to various factors that are known to those skilled in the art.

Pharmaceutically acceptable carriers

Compositions of the present invention may be used in the manufacture of a pharmaceutical composition to treat a disease. The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

The vectors, DNA binding molecules and/or nucleic acid or PNA molecules of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the inventive molecules, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). Thus, the pharmaceutical composition of the present invention may be formulated for administration by inhalation or insufflation (either through the mouth or the nose), parenteral injection (e.g., intravenous, intramuscular, or subcutaneous), oral administration in solid, liquid, or aerosol form, buccal, rectal, vaginal, nasal, ocular, local (powders, ointments or drops), intracistemal, intraperitoneal, or topical administration, and the like.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they maybe presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents {e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

For administration by inhalation, the composition for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compositions described herein may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the composition may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compositions of the present invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions can be administered in a therapeutically effective amount by any suitable route. In particular, oral, transdermal, parenteral or mucosal delivery may be appropriate. In general, a therapeutically effective amount of the compositions described herein largely will depend on particular patient characteristics, route of administration, and the nature of the disorder being treated. General guidance can be found, for example, in the publications of the International Conference on Harmonisation and in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528 (Mack Publishing Company 1990).

Ddetermining a therapeutically effective amount will also depend on such factors as toxicity and efficacy of the medicament, including half-life of the composition. Toxicity may be determined using methods well known in the art and found in the foregoing references. Efficacy may be determined utilizing the same guidance in conjunction with the methods described below in the Examples. A therapeutically effective amount will be an amount that is deemed by the clinician to be toxicologically tolerable, yet efficacious.

The following examples are given to illustrate the present invention. It should be understood, however, that the spirit and scope of the invention is not to be limited to the specific conditions or details described in these examples but should only be limited by the scope of the claims that follow. All references identified herein, including U.S. patents, are hereby expressly incorporated by reference.

Examples

Example 1

Production of pGal4-zif268 plasmid

A gene to encode the fusion protein Gal4-zif268(three zinc finger region) is produced using the following procedure. A commercially available plasmid (Stratagene) pM (see Figure 15) is used as starting material. Using BamH1 and Xba1 restriction site containing PCR primers we will PCR amplify the three zinc finger domain from zif268 (see enclosed description of this gene from ATCC) which encodes the binding domain which can recognize and bind to the sequence 5'- GCGTGGGCG-3¹. These PCR generated sequences will be digested with BamH1 and Xba1 and then ligated between the BamH1 and Xba1 sites at the cloning site in pM adjacent to the gene coding for the binding domain of Gal4 (contains both the CCG binding region and the dimerization region encoding for a protein which will dimerize and bind to the Gal4 DNA binding site but which will not activate gene expression since it lacks a transcription activation domain). The resulting plasmid pGal4-zif268 will encode the Gal4-zif268 fusion protein.

Example 2

pGal4-zif268 is stably transfected into a commercially available (Stratgene) HeLa cell line stably transfected with the plasmid pFR_Luc. See Figure 16. The pFR_Luc sequences within this cell line contain a Gal4 binding site adjacent to a TATA box followed by the luciferase gene. No luciferase gene expression is observed from this cell line because it contains no other promoter sequences upstream of the luciferase gene. Once this cells line is stably transfected with pGal4-zif268, it will now express the Gal-zif268 fusion protein which can bind just upstream of the TATA box prior to the luciferase gene. No luciferase activity should be expected from these pGal4-zif268 transfected cells either, since still no gene activation domain is present in this complex adjacent to the TATA box to organize and activate the transcription machinery for the luciferase gene.

Example 3

Transfection of the pGal4-zif268 and pFR_Luc (See Figure 16) containing HeLa cells with a double stranded oligo nucleotide having the sequence GCGTGGGCG at its 5' end and having the DNA binding site for the activating transcription factor fos on its 3' end should transactivate the luciferase gene. It will do so by providing this double stranded oligonucleotide which will bind to the zif268 domain of the Gal4-zif268 protein already bound to the Gal4 binding site adjacent to the TATA box preceding the luciferase gene and link it to the activating transcription factor fos which can organize and activate the transcription machinery to express the luciferase gene. See Figure 4.

Example 4

The crystal structure of a complex to re-address genes shown in Figure 10 is synthesized by linking a synthetic peptide zinc finger with a C-terminal CHO instead of a COOH to a synthetic deoxy oligo with a 16 carbon linker with a terminal NH2 (commercially available from Invitrogen) to form a -CH2-NH-CH2- linkage after Na cyanoborohydride reduction.

Example 5

Synthesis of the Y Structure re-addressing molecule involves reacting a 5¹ or 3' Phosphate oligo with EDC and Imadazole, and then adding Adipic hydrazide to yield the oligo hydrazide which will react with a C-terminal CHO peptide without interference from the possible free amino groups in a lysine residue. These NH2 should form transient shift bases but the hydrazide will form a stable bond.. See Figure 3 enclosed diagrams of synthesis and final product.

Example 6

See Figure 9 providing the U Structure. A Gal4 target DNA sequence bound to two Gal4 peptide GGC binding modules that continue into an 8 base PNA going N-C is provided. This is then bound to two oligos with the 5¹ end of one and the 3¹ end of the other complementary so that single stranded overhangs, each in the 5' to 3¹ direction hang off each end. These overhangs then are hybridized to the PNA so that it is C-N PNA on 5' to 3¹ DNA. This places the re-address DNA high enough above the genomic DNA to function as a new address.

Example 7

See Figure 12 showing a 10 unit DNA PNA quadrahelix of GC CG. This structure is energy minimized and without any steric hindrance. Also, see Figure 13.

Example 8 A GNN and NNG pair where N may be any base is selected within the regulatory region or address of a target gene. The exact base pair length between these common triplets is noted so a target is defined as shown here: GGTXXXXXXXXXXXXXXXXXXXXACG. Such a target would be unique within the human genome. Two peptide zinc finger DNA binding domains to recognize the triplets GNN (GGT) and NNG (ACG) are designed or mimicked from such natural occurring domains and synthesized with a specific chemical linkage group attached to the terminus of each peptide to allow attachment of a single strand of DNA or a modified polydeoxyribonucleotide resistant to enzymatic degradation but able to bind transcription factors. These two GNN binding peptides are attached to complementary strands of DNA or a modified polydeoxyribonucleotide resistant to enzymatic degradation but able to bind transcription factors. When the two complementary strands are allowed to hybridize into a DNA duplex, this rigid helix will specifically separate them by the exact distance necessary to make a single molecular structure with two GNN binding domains juxtaposed so as to recognize and bind to the GGTXXXXXXXXXXXXXXXXXXXXACG target specifically.

The double stranded DNA helix utilized to selectively separate the DNA binding domains acts as the Nucleic acid molecule and is chosen to selectively bind a regulator of interest to control target gene expression.

Claims

In the Claims:

1 ) A method of regulating expression of a gene of interest comprising:

(i) binding a DNA binding molecule that comprises at least one DNA binding domain to a gene of interest, and

(ii) associating the DNA binding molecule with a nucleic acid molecule or PNA molecule that comprises a binding site for a protein that interacts directly with the transcription machinery of the gene of interest, and

(iii) binding of the protein that interacts with the transcription machinery of the gene of interest to the binding site on the nucleic acid or PNA molecule, and

(iv) regulating transcription of the gene of interest.

2) A composition for regulating gene expression consisting essentially of::

(i) a DNA binding molecule that comprises at least one DNA binding domain, and

(ii) a nucleic acid molecule or PNA molecule that contains a binding site for a protein that directly interacts with the transcription machinery of a gene of interest.

3) The composition of claim 2, wherein the DNA binding molecule comprises a gal4 DNA binding domain.

4) The composition of claim 2, wherein the DNA binding molecule comprises a Zinc finger DNA binding domain.

5) The composition of claim 4, wherein the DNA binding molecule comprises a zif-268 DNA binding domain.

6) The composition of claim 2, wherein the DNA binding molecule comprises a nucleic acid capable of forming a three helix bundle. 7) The composition of claim 2, wherein the DNA binding molecule is a peptide nucleic acid (PNA).

8) The composition of claim 2, wherein the nucleic acid molecule is a single stranded nucleic acid molecule capable of forming a self complementary binding site.

9) The composition of claim 2, wherein the nucleic acid molecule or PNA molecule consists of at least 2 single stranded nucleic acid molecules that are capable of binding to each other.

10) The composition of claim 9, wherein the PNA molecule forms a Y structure.

11) The composition of claim 9, wherein the nucleic acid molecule forms a U structure.

12) The method of claim 1 , wherein the gene of interest is Bcl2.

13) The method of claim 1 , wherein the DNA binding molecule comprises a gal4 binding domain

14) The method of claim 1 , wherein the DNA binding molecule comprises a Zinc finger binding domain.

15) The method of claim 1 , wherein the DNA binding molecule comprises a zif-268.

16) The method of claim 1 , wherein the DNA binding molecule comprises a nucleic acid capable of forming a three helix bundle.

17) The method of claim 1 , wherein the DNA binding molecule is a peptide nucleic acid (PNA).

18) The method of claim 1 , wherein the nucleic acid molecule comprises a single stranded nucleic acid molecule capable of forming a self complementary binding site, and the nucleic acid molecule binds in a self complimentary manner before binding to the protein that interacts with the transcription machinery of the gene of interest.

19) The method of claim 1 , wherein the nucleic acid molecule consists of at least 2 single stranded nucleic acid molecules that are capable of binding to each other.

20) The method of claim 19, wherein the DNA binding molecule and nucleic acid molecule or PNA molecule forms a Y structure.

21) The method of claim 19, wherein the DNA binding molecule and nucleic acid molecule forms a U structure.

22) A method of making a composition of claim 2, comprising:

associating the DNA binding molecule with a nucleic acid molecule or PNA molecule that comprises a binding site for a protein that interacts directly with the transcription machinery of the gene of interest.