Patent Application: US-16356398-A

Abstract:
over the past decade , it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death . theoretically , the proteins encoded by these genes provide excellent tools for achieving tumor cell - specific expression . an approach to achieving specific expression of a desired protein in tumor cells is based on the selective expression of such oncoproteins . in outline , an endogenous cellular oncoprotein binds to exogenously introduced gene products , resulting in transcriptional activation of a desired gene . this approach is generally applicable to other diseases in which a particular protein is selectively expressed in disease - affected cells as compared to non - affected cells .

Description:
the present inventors have found that genetic alterations integral to disease development can be used to selectively target diseased cells for specific gene expression . as schematically depicted in fig1 an intracellular protein complex is created by binding a disease marker protein ( such as an oncoprotein ) to exogenously introduced nuclear proteins . the resultant complex then activates expression of a desired gene . because the rate of complex formation should be directly related to the cellular concentration of the disease marker protein , the strategy can potentially target not only novel oncoprotein , but any protein which is over - expressed as a direct or indirect result of the genetic alterations responsible for disease . disease marker proteins can be the products of endogenous cellular genes which are induced during disease . disease marker proteins can be normal proteins which are overexpressed during disease . disease marker proteins can be viral genes . diseases during which proteins are selectively expressed include , but are not limited to cancer , viral diseases , and autoimmune diseases . the strategy outlined in fig1 and 2 results in substantial selective expression for cells over - expressing a mutant oncoprotein relative to cells not expressing the mutant oncoprotein . this strategy is applicable to p53 as well as to other genes involved in neoplasia , such as rb , ras , neu , myc , erbb2 , bax2 , bcl2 , apc , mcc , and dcc , as well as to other genes involved in viral and autoimmune diseases . many oncogenic proteins , like mutant p53 , have intrinsic transactivation domains ( tad &# 39 ; s ), simplifying the approach . disease marker proteins normally present in the cytoplasm ( rather than the nucleus ) can also be used . they can be translocated to the nucleus through interaction with triggers containing nuclear localization signals ( finkel , 1993 ). powerful techniques for identifying polypeptides that interact with given proteins have been developed in the past few years , ( fields , 1994 ; gyuris , 1993 ; germino , 1993 ; finkel , 1993 ), facilitating the design of kbd &# 39 ; s for trigger and tad constructs . novel domains , such as the ones found in foreign ( e . g ., viral ) or mutant proteins represent the most attractive disease marker proteins for use in the present invention , but do not exhaust the possibilities . any gene which is selectively over - expressed in a tumor cell ( e . g ., telomerase , cyclins , myc ) or other diseased cell represents a potential target . the expression efficacy of the weapon should be directly related to the concentration of the disease marker protein in the diseased cells . selectivity will be limited by the concentration of the disease marker proteins in non - diseased cells . selective expression by a diseased cell is preferabley expression of at least 50 % more of a marker protein than a non - diseased cell expresses . more preferably , the ratio of expression in a diseased relative to a not - diseased cell is greater than 2 , 5 , 10 , or 50 . a set of genetic constructs , according to the present invention are genetically engineered , recombinant nucleic acids , which are used in a single diagnostic or therapeutic method . typically they are introduced into the same cells where they , and their gene products can interact . they may be packaged in a kit , in separate containers or mixed . instructions and ancillary reagents may also be provided . particularly desirable are reagents for facilitating transfection of cells with the dna constructs . such reagents are well known in the art . the constructs may be provided in liquid , frozen , or dehydrated form . the constructs may be on linear or circular molecules ; they may be on autonomously replicating molecules or on molecules without replication sequences . at a minimum , the set comprises two constructs , a weapon and a trigger . optionally , if desired , the set also comprises a transactivation construct . this is desirable when the disease marker protein does not contain its own transactivation domain . the weapon , trigger , and transactivation constructs may also be provided individually , as can the fusion proteins they encode . &# 34 ; detectable markers &# 34 ; may be enzymes which catalyze reactions which produce a readily detectable product , typically a product with a color . detectable markers may also provide to the host cell a readily observable phenotype , such as morphology , growth habit , or drug resistance . toxic proteins can also be produced by the weapon construct , for example , cytotoxins such as ricin . alternatively , the protein produced by the weapon construct may be an enzyme which metabolizes a pro - drug , converting it to a drug which is cytotoxic or cytostatic . methods for introduction of the genetic constructs of the present invention into cells are those which are available in the art . these include , but are not limited to transfection with naked or encapsulated nucleic acids , cellular fusion , protoplast fusion , and viral infection . introduction of genetic constructs may be conducted in vitro or in vivo . cells containing the constructs can be re - introduced to a body , for example by infusion or inoculation . such methods can be used in the conventional manner . methods for detecting expression of the desired protein encoded on the weapon construct are any that are standardly used in the art for detection of marker proteins . detection of particular enzymes or proteins expressed by the weapon construct may be by assay , by formation of colored product , observation of cellular or colony morphology , immunodetection methods , etc ., as is convenient in a particular context for a desired protein . such methods are well known in the art . the following examples are provided for exemplification purposes only and are not intended to limit the scope of the invention which has been described in broad terms above . this example illustrates the general form of our strategy , using as the particular example of the gene on the &# 34 ; weapon &# 34 ; one which encodes a toxic product . the scheme is graphically depicted in fig1 . exogenous genes encoding &# 34 ; weapons &# 34 ; and &# 34 ; triggers &# 34 ; are delivered into cells . the &# 34 ; weapon &# 34 ; is encoded by a foreign , toxic gene whose expression is under the control of an inducible uas ( upstream activating sequence ). the gene will not be expressed unless a functional , uas - binding transcription factor is present in the cell . the &# 34 ; trigger &# 34 ; is encoded by a crippled transcription factor which can bind to the uas through its dna - binding domain ( dbd ) but cannot activate transcription because its transactivation domain ( tad ) has been replaced with an amino acid sequence , the killer binding domain ( kbd ) that interacts with the target oncoprotein . in cells expressing adequate levels of the oncoprotein ( termed the &# 34 ; killer &# 34 ;), a protein complex forms that can both bind to the uas and activate transcription of the enzyme through a recruited tad . the tad can either be naturally present within the oncoprotein or , if the oncoprotein lacks a tad , within another exogenously introduced protein that independently binds to the oncoprotein ( see fig1 ). the weapon will be expressed at high levels only in cells expressing high levels of the killer , resulting in cell death . this example demonstrates a test system used to validate the proposed strategy . we made the following choices ( fig2 ). for the killer , we chose the p53 gene , which has been shown to be genetically altered in numerous tumor types ( hollstein , 1991 ). immunohistochemical studies have shown that the resultant mutant p53 proteins are expressed at high levels in tumor cells but p53 is undetectable in most normal cells ( bartek , 1991 ). the p53 protein also contains a strong tad at its amino - terminus ( fields , 1990 ; raycroft , 1990 ). this tad is preserved , though other properties of p53 are disrupted , in most p53 mutants ( zambetti , 1993 ). for the trigger , we chose protein domains known to bind to the carboxyl - terminus of p53 , either from p53 itself ( iwabuchi , 1993 ; milner , 1991 ; jeffrey , 1995 ) or from the large t antigen of sv40 ( linzer , 1979 ; ruppert , 1993 ). these domains were fused in frame to the dbd of gal4 . for the weapon , we chose the e . coli gene encoding polynucleotide phosphorylase ( pnp ), which can convert the non - toxic pro - drug 6 - methylpurine deoxyribose ( mep - dr ) to the diffusible , toxic 6 - methylpurine ( mep ) ( sorscher , 1994 ). the pnp gene was placed under the control of a gal4 responsive promoter , uas g . plasmid construction . &# 34 ; trigger &# 34 ; plasmids contained the dna - binding domain ( dbd ) of gal4 ( aa 1 - 147 ; sadowski , 1989 ) fused in frame to a protein domain that could bind p53 ( p53bd ). in trigger ta , the p53bd consisted of amino acids ( aa ) 84 - 708 of the sv40 large t antigen ( from ptd1 , clontech , palo alto , calif . ), separated from the dbd by the 7 aa spacer pefglrp . in trigger p5 , the p53bd consisted of aa 305 - 393 of human p53 ( from pcep4 - p53wt , ( pietenpol , 1994 ), separated from the dbd by the 5 aa spacer pefpg . in both cases , the p53bd domains were inserted into a vector derived from psgvp ( finkel , 1993 ) containing the gal4 dbd . the &# 34 ; weapon &# 34 ; plasmid contained the bacterial deod gene , coding for pnp ( hershfield , 1991 ), amplified from escherichia coli genomic dna and modified to contain a consensus eukaryotic translation start sequence ( kozak , 1992 ). the deod gene was placed under control of a gal4 - responsive promoter obtained from pg5e1b - cat ( finkel , 1993 ) to create uas g - pnp . the gal4 - responsive uas g - βgal construct was identical to uas g - pnp except that the deod gene was replaced with the β - galactosidase gene from pcmv - β - gal ( clontech ). the &# 34 ; killer &# 34 ; plasmids were pcep4 - p53 ( wt or mutants r175h , r248w , or r273h ), each containing the complete p53 coding sequence under control of the cmv promoter ( pietenpol , 1994 ). the construction of these plasmids was generally performed by pcr amplification of the indicated templates . each of the components illustrated in fig1 and 2 can be independently optimized . we first tested the ability of various triggers to stimulate transcription of a uas g - controlled reporter gene in a p53 - dependent manner . two different human cell lines were used . the p53 - null h1299 cell line was derived from a lung cancer ( chen , 1993 ) and the 293 line was derived from human embryonic kidney cells transformed with adenovirus dna ( graham , 1977 ). the tad of the endogenous p53 in 293 cells is inactivated by the adenovirus encoded e1b protein ( yew , 1994 ). the 293 cells are more efficient recipients for pei - mediated gene transfer than h1299 cells . several trigger constructs were evaluated in these lines , and the most efficient chosen for further experiments ( fig2 b ). when h1299 cells were transfected with the ta trigger plus a uas g - controlled β - galactosidase ( β - gal ) reporter , only very low levels of β - gal activity could be detected ( fig3 ). however , when p53 was overexpressed in these cells as a result of p53 gene transfer , β - gal levels were increased by up to 30 - fold . three of the most common p53 mutants were tested in these assays . the r248w and r273h mutants were effective , while the r175h mutant was not . these results were consistent with the defective transactivation properties of the latter ( fields , 1990 ; raycroft , 1990 ). as expected , the wt p53 protein also showed activity in this system , though less than the r248w and r273h mutants , probably because of the greater protein stability of mutant forms of p53 ( zambetti , 1993 ). the p5 trigger also proved effective at conferring a p53 - dependent expression of the reporter , in both h1299 and 293 cells . the results with p5 confirm those of oren and co - workers , demonstrating the ability of the c - terminus of p53 to bind intact p53 and recruit its transactivation domain ( shaulian , 1993 ). cell culture and transfections . the 293 cell line ( graham , 1977 ) was obtained from atcc and cultured in dmem medium ( life technologies , gaithersburg , md .) supplemented with 10 % fetal bovine serum and antibiotics ( fbsa ). h1299 cells were obtained from a . fornace ( chen , 1993 ) and grown in ham &# 39 ; s f - 12 medium ( life technologies ) with fbsa . for transfections , cells were plated at a density of 5 - 8 × 10 4 cells per well of a 24 - well tissue culture plate . transfections were performed 36 - 60 h after plating , when cells were almost confluent , essentially as described in ( boussif , 1995 ). briefly , cells were washed once with optimem ( life technologies ) and 400 μl of optimem were added to each well . two μg of each plasmid in 3 mm tris , 0 . 2 mm edta were mixed with a sufficient amount of pbluescript ( stratagene , la jolla , calif .) and nacl ( 150 mm ) to constitute 6 μg plasmid dna in a total volume of 50 μl . a mixture of 12 μl polyethylenimine ( pei , 0 . 9 mg / ml ) and 38 μl nacl ( 150 mm ) was then added to the dna . after a 10 - 30 min incubation at room temperature , the dna - pei mix was added to each well . two to four hours later , the liquid in the wells was aspirated and replaced with culture medium . β - gal assays . cells used for β - gal assays were harvested 24 - 48 hours after transfection . cells were washed once with hank &# 39 ; s balanced saline solution ( hbss ) and subjected to three cycles of freezing and thawing in 100 μl of phosphate buffered saline ( pbs ). cell lysates were transferred to microfuge tubes containing 11μ of 10 mm dithiothreitol ( dti ) in pbs , pelleted for 10 min at 4 ° c . and stored at - 20 ° c . until assayed for β - gal activity . in a typical assay , 50 μl of cell lysate were added to 450 μl of reaction buffer ( 60 mm na 2 hpo 4 , 40 mm nah 2 po 4 , 10 mm kcl , 1 mm mgso , ph 7 . 0 , 0 . 27 % β - mercaptoethanol ). following a 5 min pre - incubation at 28 ° c ., reactions were initiated by addition of 100 μl of a 4 mg / ml solution of ortho - nitrophenyl - galactoside ( onpg ) in reaction buffer ( kern , 1992 ). reactions were stopped by addition of 250 μl of 1m na 2 co 3 and absorbances at 420 nm measured . this example demonstrates that the observed cytotoxicity in the system is dependent on the presence of the oncogene p53 . we sought to determine whether this p53 - induced expression of a uas g - controlled gene could be translated into differential killing of p53 - overexpressing cells . we chose h1299 cells for these experiments , as the absence of all endogenous p53 in this line simplified interpretation . h1299 cells were transfected with both a trigger ( p5 ) and a weapon ( uas g - pnp ), with or without a killer ( p53 - r273h ). various amounts of mep - dr were added to the culture medium one day after transfection and cell number and viability assessed five days later . a dramatic difference in mep - dr sensitivity was observed between cells transfected with or without the killer ( fig4 and 5 ). at drug concentrations of 3 - 10 μm , most cells without p53 remained healthy and continued to proliferate . in contrast , after co - transfection with the killer , only a relatively small number of cells remained ( fig4 a ). cells transfected with the killer and weapon , but without the trigger , also remained healthy . these and additional controls ( see below ) demonstrated that only the combination of weapon , trigger , and killer resulted in substantial cell death at low drug concentrations . cellular viability assays . one day after transfection , mep - dr was added to the culture medium to final concentrations of 0 , 3 , or 10 μm . five days later , cells were harvested by trypsinization and suspended in a final volume of 600 μl . a 50 μl aliquot of cells was mixed with an equal volume of 1 % np40 , 6 . 7 % formaldehyde , 20 μg / ml hoechst 33258 in pbs for counting and assessing nuclear morphology . over 90 % of cells with a normal nuclear morphology were found to exclude dye ( wotring , 1985 ) in control experiments . for colony - forming assays , aliquots of 400 , 40 , or 4 μl were plated in 25 cm 2 tissue culture flasks in 5 ml of the appropriate medium . after 6 - 8 days , colonies were stained with crystal violet and counted . this example demonstrates that colony forming ability is another useful assay for drug toxicity . in these experiments and others , we noted that the number of morphologically intact cells was an inadequate measure of drug toxicity . when cells were harvested and replated after drug treatment , many of the apparently intact , dye - excluding cells proved incapable of growing ( compare fig4 a and b at 3 μm mep - dr ). accordingly , there was a striking difference in colony - forming ability between cells expressing killer , trigger , and weapon versus those expressing only trigger and weapon following exposure to drug . representative examples of the colonies formed after mep - dr treatment of such cells are shown in fig5 and the results quantitated in fig4 b . at 3 or 10 μm mep - dr , there was over a 1000 - fold greater effect in the p53 - expressing cells compared to the cells without p53 . it is also important to note that the pei protocol transduced less than 3 % of the h1299 cells , as assessed by β - gal staining . the fact that virtually all h1299 cells were incapable of forming colonies following appropriate transfection and mep - dr treatment attests to the powerful bystander effect of this system . the principles , preferred embodiments and modes of operation of the present invention have been described in the foregoing specification . the invention which is intended to be protected herein , however , is not to be construed as limited to the particular forms disclosed , since they are to be regarded as illustrative rather than restrictive . variations and changes may be made by those skilled in the art without departing from the spirit of the invention . 1 . stillman , b . 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