Patent Application: US-58799409-A

Abstract:
in its various embodiments , the invention provides , first , a composition comprising a vector for transfecting a cell . the vector comprises a first nucleic acid encoding an antisense agent having thereon an rna interference target for a transcript of a gene endogenous to the cell . the vector further comprises a second nucleic acid that encodes a cell - killing agent . the second nucleic acid further comprises a sequence of nucleotides transcribable into a non - coding region of a transcript of the second nucleic acid , such that the non - coding region becomes an rna interference target for the antisense agent . in the transfected cell , the vector operates to interfere with the expression of the cell - killing agent unless and until the vector senses certain endogenous gene signals , whereupon it releases the cell - killing agent . second , the invention provides a method of treating a disease in a patient by killing cells responsible for the disease , the method comprising administering the vector to the patient until the disease , or a symptom thereof , is ameliorated .

Description:
the eradication of any cancer in a patient is an elusive goal . the resistant nature of cancerous cells continues to thwart efforts to specifically “ aim ” a therapeutic agent at cancer cells and definitively eliminate them from the body . embodiments of the instant invention provide a highly selective , and largely autonomous system that is capable of detecting and destroying many cancer cell types under the control of a logic evaluator introduced into cancer cells for therapeutic purposes . rinaudo et al ., in “ a universal rnai - based logic evaluator that operates in mammalian cells ” nat . biotechnol . 25 : 795 - 801 ( 2007 ), proposed the use of short interfering rna (“ sirna ”) to suppress or promote gene expression in cells by means of a stochastic control system that operates automatically on intracellular inputs . conceptually , every ongoing process within a cell makes a “ statement ” about the state of the cell at any given time . to a logic evaluator , these statements are inputs susceptible to evaluation or “ interpretation ” as logical expressions . depending on the evaluator &# 39 ; s programming , a binary evaluator will find either “ truth ” or “ not truth ” in the set of inputs it interprets . rinaudo et al . demonstrated a biological version of a logic evaluator whose output is a translation product of messenger rna ( i . e ., a protein ). a simple version comprised a pair of identical mrnas and two distinct sirnas , each of which had its own rna interference target fused onto one member of the mrna pair . with both species of sirna absent from the system , protein was produced . also , if one but not the other sirna species was present , protein was produced . if both species were present , protein production was blocked because rna interference was triggered against both mrna species . rinaudo et al . incorporated this system into cells by transfecting the cells with genes that express mrnas bearing the sirna binding sites . various sirna transfections then caused the cells to make different logic statements . rinaudo et al . suggested that if the appearance of the sirnas in the cells could be made to be dependent on endogenous processes (“ inputs ”), the system would be able to sense the inputs and logically evaluate them to yield outputs that , in turn , could be exploited to control the state of the cell . differential gene expression profiles , catalogued in databases , reveal for many cancer cell types gene signatures that are so different from those of healthy cells as to be reliably diagnostic of the cancer . a “ gene signature ” is a gene ( usually , a plurality of genes ) that is overexpressed or underexpressed in a cell relative to a suitable control cell . such a gene may be referred to herein as “ biomarker ” of the disease in which it is differentially expressed . it may also be referred to as a “ signature gene ” to denote its membership in a set of genes that constitutes a signature . in some embodiments , the present invention provides a biomolecular logic circuit that selectively identifies a cancer cell by “ reading ” the cell &# 39 ; s signature from molecular signals it receives as inputs at its input ports . the “ reading ” or output from the logic circuit then automatically triggers an apoptotic or other destructive process in that cell . in this respect , the term “ biomarker ” may not refer to a single gene , but to a plurality of genes , all of which the logic circuit must detect as inputs to trigger a destructive output . in some embodiments , the system that effects the destructive process may be endogenous to the cell , but in preferred embodiments it is transferred into the cell along with the logic circuit . in preferred embodiments , the logic circuit identifies the cell from within . however , the entry means may comprise , along with the logic circuit , a means for identifying the cell from without , by binding at a binding site expressed uniquely on the surface of the target cell , for example . in various embodiments , the transfer is accomplished with a viral vector or other vector capable of transferring into the cell the genetic information necessary to the operation of the logic circuit . the vector , however , need not be capable of selecting only cancer cells for transfection , inasmuch as the transfected logic circuit , in preferred embodiments , does not actuate in cells that do not bear the signature of interest . in one embodiment of the invention , the cell - destructive system comprises a nucleic acid that encodes an apoptotic protein . bak and bax are preferred , but the artisan will know of many other proteins whose expression is sufficient to cause apoptosis , so the present invention is not limited to these two apoptotic signals . the artisan can readily select any of a number of proteins encoded in commercially available nucleic acids incorporable into expression vectors by methods well - known in the art . in preferred embodiments , the nucleic acid that encodes the apoptotic signal further encodes an antisense rna specifically complementary to a portion of the messenger rna ( preferably a portion in a non - coding region ) that translates into the apoptotic protein selected for use . thus , although the apoptotic signal may be continually transcribed , a corresponding antisense signal is also transcribed . applicants do not wish to be bound by any theory of how embodiments of their invention work , but they believe that the transcribed antisense signal titrates away the apoptotic message by an rna interference mechanism . in a more preferred embodiment , the nucleic acid that encodes the aforementioned antisense transcript is engineered to further transcribe into the antisense transcript a sequence that the messenger rna of a cancer signature gene can interact with to set up an rna interference . the mrna from the cancer signature gene behaves , in effect , like an antisense molecule against the antisense transcript that heretofore acted as a “ governor ” on apoptosis . thus , an elevated cancer signature message erodes the antisense governor , and apoptosis proceeds apace . in preferred embodiments , the antisense transcript , although it can be interfered with by the messenger rna of the cancer signature gene , does not have significant interference potential vis a vis the cancer signature gene . to achieve this balance , the artisan introduces specific mismatches to reduce the degree of complementarity between the two transcripts , as the art teaches ( e . g ., v . patzel , drug discovery today , 12 : 139 - 148 , 2007 ). as noted above , the gene expression signature of a cell is usually not reliably represented by a single gene . it follows that a logic circuit that responds with a cell - disabling output to an input from a single gene may not be reliable , either . accordingly , in its most preferred embodiments , the logic circuit is constructed with input ports for two or more signature genes so that all such genes must be present and “ sensed ” by the logic circuit to trigger the events that result in apoptosis . any viral construct that can be made to reach the nucleus of a eukaryotic host cell without integrating into the genome of the cell is within the scope of the invention . as a further safeguard for normal cells that take up the logic circuit , a highly preferred embodiment of the invention comprises the use of a lentiviral vector . lentiviruses are “ rna viruses .” that is , their genomes are encoded in rna instead of dna . however , reverse transcriptase , an enzyme intrinsic to the virus ( that is , encoded in the viral genome , not “ co - opted ” from the host genome ), catalyzes the conversion of the rna into dna upon the virus &# 39 ; s entry into a cell . another intrinsic viral enzyme , integrase , then shuttles the dna into the nucleus of the cell and catalyzes its integration into the cellular genome . several well - known mutations of integrase , however , retain the shuttling function but lack the integrating function . in preferred embodiments , therefore , the logic circuit and the cell - destructive system come to reside in the cell &# 39 ; s nucleus where its nucleic acid codes are readily transcribed , but does not enjoy the same propensity for survival that nucleic acid codes integrated into the cell &# 39 ; s chromosomes would have . instead , it is susceptible to degradation within the nucleus . mutated integrases vary in the extent to which their chromosome integration function is disabled , but mutants that are most favorable in this respect are readily identified . persons of skill in the art know well how to select genes that the logic circuit can sense in particular cancer cell types . for example , highly expressed genes in a published and well - established gene signature for a particular cancer are preferred candidates . note , however , that gene signatures also comprise highly underexpressed genes , generally because the cell in question is producing a repressor signal . in such cases , the repressor mrna can serve in embodiments of the invention . accordingly , any gene that comprises a signature for a particular cancer cell type is within the scope of the invention . preferred genes are genes ( or repressor genes ) that are not highly expressed at relevant times ( e . g ., during a treatment period ) in any healthy cells of a treated subject . notwithstanding the foregoing , preferred embodiments of the invention comprise logic circuit designs that respond to gene signatures rather than individual genes . thus , in some embodiments , a logic circuit according to the invention may have an input port that “ senses ” a gene overexpressed in both healthy cells and diseased cells . such a logic circuit remains within the scope of the invention if , in operation , it distinguishes between healthy and diseased cells . embodiments of the invention also apply to treatment of a subject over time . for example , one currently significant problem is the recurrence of breast cancer after treatment and the transformation of treatable cancer into treatment - resistant cancer . this occurs because breast cancer cells are able to change in character over time . expression profiles of cells from the same tumor differ significantly at different stages of cancer progression . it is likely that a patient , at any given time , harbors cancer cells in several stages of cancer progression . if a therapy is effective at only one stage of cancer , the cancer will be able to recover and resume growth in a manner difficult to treat . thus , in some embodiments , the invention provides a method of treating a subject according to the gene signature that the subject &# 39 ; s cancer presents at any given time or stage of disease progression . such embodiments , moreover , need not be limited to logic circuit designs that are effective against one and only one cancer cell type at one and only one stage of disease progression . for example , a patient may present with metastatic cancer or primary tumors in multiple organs or multi - organ tumors secondary to metastasis , in which case several distinct signatures may need attention all at once . logic circuits equipped to sense and respond to multiple gene signatures are within the scope of the invention . in response to the need for detecting cells that are slated for destruction with a high degree of discrimination , a vector transferable into cancer ( and normal ) cells has been designed to ( 1 ) detect within cancer cells a cancer - specific genetic message and ( 2 ) release , in response to the detection of such message , a genetic signal that translates into a cell - killing protein . control over the production of the cell - killing protein is accomplished by means of rna interference . the design can be viewed as a logic circuit for controlling a cell - killing system . the logic circuit is capable of identifying multiple markers of a specific cancer such that cancerous cells are destroyed selectively , leaving other cells to grow normally . the vector is further designed to deliver the logic circuit and cell - killing system in such a way that its effective lifetime in the cell is brief . the integration of three major components of the vector is demonstrated in several in vitro experiments summarized herein . all three components have been independently verified in mammalian cells [ 1 - 3 ] and are integrated here with a novel approach to detecting endogenous mrna levels . in the demonstration , genetically engineered lentiviruses infect all cells with a genetic control system analyzable as a logic circuit . since the system , in operation , employs the phenomenon of rna interference , it is called an rnai logic circuit . biomarkers in the form of messenger rna , specific to mcf - 7 breast cancer cells , are first identified . one such marker is gata3 mrna . gata3 is a zinc finger protein important in tissue differentiation . to detect and respond to gata3 mrna , the rnai logic circuit delivered by the engineered lentiviruses results in constitutive ( or , optionally , inducible ) expression of gata3 sirna . the lentiviral vector further comprises a killer gene ( optionally under the control of an inducible promoter ) fused with a special target site for the gata3 sirna . in cells with low endogenous levels of gata3 mrna , the engineered gata3 sirna binds and degrades killer gene mrna ( fig1 a ). in cells with high endogenous gata3 mrna levels , gata3 mrna “ uses up ” or titrates away so much of the gata3 sirna that any remaining gata3 sirna is insufficient to interfere with killer gene expression , and hence cell death ensues ( fig1 b ). it is important to recognize that using a single marker is rarely sufficient for addressing a problem as complex as cancer . to more fully address the problem , a vector is designed ( in a non - limiting example ) that detects the simultaneous presence of three markers specific to mcf - 7 cells in a manner akin to a logic and gate ( fig1 c - d ): instead of equipping the vector to express a single sirna species , the vector is designed to express ( 1 ) three species of sirna and ( 2 ) a killer gene to which a binding site for each of the three is fused . one species is designed from tff1 . members of the trefoil family are characterized by having at least one copy of the trefoil motif , a 40 - amino acid domain that contains three conserved disulfides . they are stable secretory proteins expressed in gastrointestinal mucosa . the gene and two other related trefoil family member genes are found in a cluster on chromosome 21 . the other is npy1r , which encodes a receptor for neuropeptide y . to test functionality of the logic circuit in healthy cells and in cancer cells , the cells are transfected in vitro by means of a lentiviral transfer vector comprising ( 1 ) a killer gene sequence modified so that its transcription entails the insertion of an sirna target region and ( 2 ) a nucleic acid sequence from which the corresponding sirna is transcribed . these transiently transfected cells are then examined for changes in function , including cellular proliferation , cell cycle regulation , pro - apoptotic factor expression , and differential phosphorylation . cells are assayed by means of dna microarrays , which allows a wide - scale comparison of the expression levels of multiple metabolic indicators , including genes differentially expressed during tumor progression ( 33 , 41 ). especially pertinent indicators are assayed with q - pcr including caseins a and g ( csna , csng ), fatty acid bp4 ( fabp4 ), carbonic anhydrases 3 and 6 ( car3 , car6 ), whey acidic protein ( wap ), lipoprotein lipase ( lpl ), zinc finger protein 503 ( zfp503 ), adipsin ( and ), thyroid spot14 homologue ( thrsp ), lactalbumin a ( lalba ), sterol carrier protein 2 ( scp2 ), acyl - coa synthetase l4 ( acs14 ), cdc - like kinase 1 ( clk1 ), catenin d1 ( ctnnd1 ), and tumor differential expression 1 ( tde1 ). the effects of the circuit on phosphorylation - dependent cellular signaling pathways are assayed using a tailored elisa scaffold treated with whole - cell lysates ( sigma - aldrich ). cell cycle progression and distribution throughout an experimental population is monitored with bromodeoxyuridine incorporation and dna content assays [ 42 ]). cellular membrane integrity is visualized by a fluorescence - enhanced cytotoxicity assay ( cytotox - one ™, promega , a homogeneous , fluorometric method for estimating the number of non - viable cells present in multiwell plates ). finally , proliferation rates of the four infected and uninfected cell lines are determined by cell counting . cancer cells exhibit genetic expression profiles that deviate from their normal progenitor tissues . for example , a comparison of genetic expression profiles of various cancerous and noncancerous cell types in the 6000 cancer - related arrays in the ncbi gene expression omnibus ( a database repository of high throughput gene expression data and hybridization arrays , chips , and microarrays well - known to artisans ), revealed 160 that are specific to mcf - 7 breast adenocarcinoma cells . mcf - 7 cells represent the most common type of breast cancer and have been characterized extensively in the literature [ 33 - 36 ]. under normal conditions mcf - 7 cells overexpress gata3 , tff1 and npy1r mrna relative to healthy cells ( fig3 a - c ). the precision and recall in distinguishing mcf - 7 cells using each individual marker and the combination of all three markers was compared . a tradeoff always exists between optimizing for true positives and excluding false negatives . receiver operating characteristic ( roc ) curves demonstrated this tradeoff for all cases and also showed the vast improvement obtained in both precision and recall when all three markers were used simultaneously ( fig3 d ). genetic combinatorial logic is not only a viable way to assess and control cell function , but is also a highly flexible technique ideally suited for the dynamic process of cancer progression . from normal to premalignant to invasive stages , the state of cancer cells ( and biomarker levels expressed by those cells ) evolves over time . the state of the cell at any stage implies a particular logic circuit for treatment . a logic circuit , embodied in one or more lentiviral transfer vectors , is assembled from a library of logic gates , themselves embodied in nucleic acids that encrypt genetic information . separate lentiviral constructs are then combined into a single treatment to simultaneously treat cells in different stages of cancer . an exemplary combination is represented in fig2 a , which depicts an rnai logic circuit that combines two ands with an or operation . the circuit comprises two output mrna species encoding the same output protein but different noncoding regions (“ different ” because each has its own sirna target sequence ). the output protein will be ‘ on ’ ( or ‘ true ’) as long as at least one of the two mrna species is translated . fig2 b summarizes experimental results with mammalian cells “ programmed ” to respond ( with a fluorescent signal ) only when input combinations satisfy the logic function “( a and b and c ) or (( d and e ).” for this experiment , the presence or absence of endogenous biomarkers was simulated by directly adding sirna as appropriate . the circuit output protein is zsyellow . numbers represent median facs values for zsyellow fluorescence . a three - input rnai - based and gate as described above and specifically exemplified below reliably and selectively differentiates between cancerous and noncancerous cells and , when the and gate is “ open ” ( i . e ., passes its signal along in the system ), kills the cells in which the gate operates , while producing no significant deleterious effects in healthy cells as defined by a series of cell function assays ( see above ). three candidate mrna biomarkers found to be overexpressed in mcf - 7 cells are detected by engineered sirna interactions . relative to the use of each individual biomarker separately , simultaneous detection of these biomarkers using an rnai - based and gate provides higher precision and recall for distinguishing mcf - 7 cells from other cell types . this rnai - based and gate , combined with an apoptosis - inducing mechanism based on regulated expression of bax and bak proteins , eliminates mcf - 7 cells within a heterogeneous cell population . the construction of a particular logic circuit operable in mcf - 7 cells first requires identifying , among phenotypically and genomically different sublines [ 35 ], the phenotype and genotype of the specific subline to be modified with the logic circuit . using quantitative pcr ( q - pcr ), total rna extracted from mcf - 7 , mcf - 10a , 293 - ft and cce cell lines are analyzed and gata3 , npy1r and tff1 mrna levels are quantified in order to verify mcf - 7 overexpression of these three biomarkers . lentiviruses are selected for circuit delivery into the cells since they infect all cell types , both dividing and non - dividing . it is to be stressed , however , that delivery vectors other than lentiviruses or even viruses , are within the scope of the invention . for ease of observation , an experimental rnai circuit comprising green fluorescent protein instead of an apoptotic protein or other killer is constructed . the circuit is used to verify the functionality of such circuits in cce cells ( fig4 a ) and in mcf7 and mcf - 10a cells . cells are infected with hef1a - tetrkrab - ires2 - puro . cells successfully infected with the construct are resistant to the antibiotic puromycin and thus may be isolated . they are then infected with poliii teto : sirna egfp and ubc : egfp . to determine the efficacy of the rnai system , the cells are grown with 1 mm anydrotetracycline (“ atc ”) and gfp fluorescence is quantified periodically for 72 hours using facs . successful repression of gfp by sirna results in gradual reduction in fluorescence ( similar to fig4 a ). any of a wide variety of killer proteins are within the scope of the invention . extensive literature on apoptosis suggests that overexpression of one or both of the proteins bax and bak is sufficient to induce apoptosis [ 3 , 29 - 32 ]. to demonstrate that bax and bak are sufficient to induce apoptosis in the embodiment detailed herein , a lentivirus was constructed from two plasmids engineered to express bax or bak under control of doxycycline ( dox ) inducible tre promoter . these constructs also contain ubiquitin ( ubc )- driven constitutive expression of blasticidin ( bla ) expression . fig4 b shows experimental results with ainv stem cells where dox induction activates bax and bak expression , resulting in apoptosis . mcf - 7 and mcf - 10a cells are also infected with these constructs , selected for bla resistance , dox added to induce expression of bax and bak , and monitored for cell death over the next 48 hours . uninduced infected cells are used as negative controls . apoptotic cell death is verified using the annexin v - pe apoptosis detection kit ( biovision ). each biomarker site is first verified individually by co - infecting with a corresponding poliii : sirna construct and a ubc : gfp fusion with the intended rnai target site upstream or downstream of gfp . reduction of fluorescence in cells where a given biomarker is not overexpressed indicates that sirna for this biomarker successfully binds and silences gfp expression . for gata3 , three different shrna sequences are used ( fig5 ). in an experiment with cce and mcf - 7 cells , gata3mut02 sirna expression was induced from the poliii / teto promoter . simultaneously , gfp mrna fused to the target site for gata3mut02 was transcribed constitutively from the ubc promoter . as expected , after induction mcf - 7 cells continued to fluoresce ( indicating gata3 presence and sirna titration ) whereas cce cells did not ( fig4 c ). constructs that individually detect the gata3 , nfy1r and tff1 biomarkers are made first , and then combined into one construct to implement the and gate ( fig1 b ). as above , reduction of fluorescence indicates that sirna successfully binds one of the target sites and silences gfp expression , in this case indicating that at least one of the biomarkers is not overexpressed . to confirm that the and gate does not result in “ high ” output in cells other than mcf - 7 , the experiment is also conducted in mcf - 10a , 293ft , and cce cells . as the final step , the and gate is coupled to the killing mechanism by replacing gfp with bak or bak . the full circuit within mcf - 7 cells results in apoptosis , while mcf - 10a , 293ft , and cce cells remain unaffected by the circuit . to eliminate the tendency of the virus from inserting a signal into host cells that promotes cell cycling ( insertional mutagenesis ) while retaining the virus &# 39 ; s ability to replicate so that it can be grown up in packaging cell lines , achieve reverse transcription after infection and express a nuclear import signal , a specific type i lentiviral mutant integrase is used . the integrase was made using a known d64v mutation of the pol gene ( encoding the integrase protein ) [ 2 ]. as expected , initially high gfp expression ultimately decreased in infections with hef1a : egfp lentivirus created with the mutant integrase , whereas wild - type integrase maintained high gfp expression throughout ( fig6 ). the sirna design includes specific mismatches with endogenous mrna sequences that have been shown to essentially eliminate rnai activity of the sirna [ 38 ]. sequence similarity thereto . there are no such mismatches in the portion of the sirna that targets kill messages , so the rnai activity of the sirna in this respect is robust . western blots and immunocytochemical staining for gata3 , npy1r , and tff1 protein and proteins with sequence similarity thereto confirm this . 1 . k . rinaudo , l . bleris , r . maddamsetti , s . subramanian , r . weiss_ , and y . benenson_ . a universal rnai - based logic evaluator that operates in mammalian cells . nature biotechnology , 25 ( 7 ), july 2007 . 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