JP2007500012A - System for external control of oncolytic virus replication - Google Patents

Abstract

  Compositions, methods and systems for external regulation of oncolytic virus replication are provided. The oncolytic virus includes a cell type specific transcriptional regulatory element (CT-TRE) and an inducible transactivator-regulated transcriptional regulatory element. The virus replicates selectively in cells where CT-TRE is allowed to function. In addition, the concentration or condition of the inducer is used to modulate the transactivator-regulated transcriptional regulatory element, thereby providing at least two control levels of oncolytic virus replication.

Description

  Gene therapy in which exogenous nucleotides are provided to cells forms an innovative and possibly instrumental principle to combat powerful diseases. This approach included not only cancer, but also cystic fibrosis, anemia, hemophilia, diabetes, Huntington's disease, AIDS, abnormally high serum cholesterol levels, certain immunodeficiencies, and many forms of cancer It has great potential for the treatment of many other diseases as well. Gene therapy generally requires a delivery carrier of exogenous sequences such as, for example, viral vectors or non-viral vectors. Various viral vectors have shown therapeutic efficacy against these diseases. For review, see, for example, Sandrin V et al., Curr Top Microbiol Immunol. 2003; 281: 137-78; Buning H., Gene Then 2003 Jul; 10 (14): 1142-51, and St George JA, Gene See Ther. 2003 Jul; 10 (14): 1135-41.

  In an alternative approach that can be applied to cancer therapy, specific attenuated replication-capable viral vectors have been developed in which selective replication in cancer cells selectively destroys those cells. For example, adenovirus constructs that have replication capabilities unique to various cells that selectively replicate (and thereby destroy) specific cell types are described in, for example, WO95 / 19434, WO98 / 39465, WO98 / 39467, WO98 / 39466, WO99 / 06576, WO98 / 39464, WO00 / 15820.

  Factors that make adenovirus a safe treatment are the following facts: (a) Adenovirus infection has minor clinical signs of disease; (b) Adenovirus is stable and well-explained; And has a characterized genome; (c) adenovirus generally does not incorporate its viral DNA into host DNA; (d) adenoviral infection results in transient gene expression; (e) adenovirus is Can infect dividing and non-dividing cells; (f) adenovirus can infect various human cell types; (g) adenovirus is physically stable; and (h A) Adenoviruses can withstand high infectious value production.

  A continuing concern regarding patient gene therapy is potential addiction. Thus, the ability to modulate viral infection and / or gene expression in vivo will be effective. Regulated gene expression for gene therapy purposes is described, for example, in Zoltick and Wilson (2001) Ann NY Acad Sci. 953: 53-63; Harrington et al. (2000) Adv Drug Deliv Rev. 44 (2-3 ): 167-84; and Clackson (2000) Gene Ther. 7 (2): 120-5. Vectors that are defective, unlike replication, commonly used for gene therapy, such as oncolytic vectors as exemplified herein, replicate in vivo to produce progeny that infect additional target cells. Thus, there will be great focus on being able to regulate the replication of oncolytic vectors in vivo.

SUMMARY OF THE INVENTION Viral vectors capable of replication and methods for their use are provided. The present invention relates to a composition comprising a replication competent virus having at least one viral gene under the transcriptional control of a regulated gene expression system, as exemplified herein by a replication competent adenovirus. I will provide a. A regulated gene expression system includes at least two levels of transcriptional regulation. At the first level of regulation, the transcriptional transactivator coding sequence is under transcriptional regulation of a cell type specific transcription response element (CT-TRE). At the second level of regulation, viral genes are regulated by the interaction of transcriptional response elements (referred to herein as response elements regulated by transcriptional transactivators) and transcriptional transactivators. Under control. A transcriptional activator of interest for carrying out the present invention is an inducer provided externally to the host cell of interest, usually a non-natural inducer, such as a chemical substance such as tetracycline, ecdysone, rapamycin, synthetic progesterone Functionally interacts with glucocorticoids, or analogs thereof. Similarly, the inducer may be a non-chemical inducer, i.e. ultrasound, heat, external radiation, hypoxia, etc.

  Transcriptional transactivator is produced only in cells where cell type specific TRE is active. The activity of the transcriptional transactivator depends on the presence / concentration or conditions of the particular inducer. Thus, the inducer or condition provides a means to modulate the function of the transcriptional transactivator. In the presence of the appropriate concentration of inducer and / or conditions, activation of a transcriptional transactivator activates transcription and an inhibitory transcriptional transactivator suppresses transcription. Expression of genes essential for viral replication is thus regulated, thereby regulating viral replication and transmission. Thus, oncolytic virus replication and transmission is regulated in vitro or in vivo by providing inducers and / or adjusting the concentration of inducers using the compositions and methods described herein. The

Detailed Description of Embodiments Regulated replication ability, including cell type specific regulatory transcriptional regulatory elements (CT-TRE) and transactivator regulated transcriptional regulatory elements that are inducible A viral vector is provided. Viruses are replicated selectively in cells that permit CT-TRE function and based on the presence or absence of inducers that regulate transactivator-regulated transcriptional regulatory elements.

  CT-TRE controls transcription of inducible transcriptional transactivator (TA) coding sequences. Transcriptional transactivators (a) require a functioning inducer and (b) control the transcription of viral genes. The inducer is an exogenous compound that is not normally present in the host cell of interest, including its analogs, such as tetracycline, ecdysone, rapamycin, synthetic progesterone, glucocorticoid, or the inducer is ultrasound, heat, external irradiation May be hypoxia, or some other treatment.

  Inhibitory transcriptional transactivators suppress transcription in the presence of inducers, and activated transcriptional transactivators activate transcription in the presence of inducers. In this way, the expression of viral genes essential for replication is regulated both by CT-TRE and transactivator-regulated transcriptional regulatory elements and indirectly by inducer concentrations or conditions. Thus, during treatment of a patient with oncolytic virus therapy, the replication and transmission of the virus will be regulated by adjusting the inducer concentration.

  Depending on the specific use of the viral vector, the inducer may act to suppress or enhance transcription. Transcriptional repressors provide a way to “stop” viral replication by delivering inducers to host cells. Transcription activators provide a way to induce viral replication by the addition of inducers.

  Preferably, the viral gene essential for replication is the early gene. In some embodiments, the viral vector of the invention is a viral gene under the transcriptional control of a transactivator-regulated transcriptional regulatory element and the same transactivator-regulated transcriptional regulatory element or first transactivator regulatory. At least one other gene, such as an additional viral gene or transgene, under the control of any of the second transactivator-regulated transcriptional regulatory elements that are substantially identical to the Including. Preferably, the first and second genes under the transcriptional control of the transactivator-regulated transcriptional regulatory element are both viral genes necessary for replication. By cell-specific transcription through the use of one or more transactivator-regulated transcriptional regulatory elements, the present invention can be used for cell-specific replication, resulting in selective cytolysis of target cells. Providing a viral vector, wherein viral replication is regulated by exposing target cells to exogenous inducers or conditions.

  The viral vectors of the present invention selectively replicate in CT-TRE functional cells, referred to herein as target cells. This replication preference is shown by comparing the level of replication (ie, titer) in cells where CT-TRE is active to that of cells where CT-TRE is not active (ie, non-target cells). Comparison of the virus titer following target cell infection with the titer following non-target cell (CT-TRE inactive) infection shows that the overall replication preference is enhanced by replication in the target cell, and It provides an important indicator that it decreases with replication in non-target cells. This provides a means to kill target cells so as to prevent irreversible infections. An additional level of replication control is provided by the presence or absence of inducers or conditions, or changes in their concentrations. The viral vectors of the present invention are exemplified herein by replication competent adenoviruses that exhibit target cell specific replication and whose regulation is further regulated by inducers.

General Technology The practice of the present invention, unless otherwise indicated, is within the skill of the art (including recombinant technology) in molecular biology, microbiology, cell biology, biochemistry and immunology. Use traditional techniques. Such techniques are described in the literature, for example "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" ( RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (DM Weir & CC Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (JM Miller & MP Calos, eds., 1987); "Current Protocols in Molecular Biology" (FM Ausubel et al., Eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., Eds., 1994) And “Current Protocols in Immunology” (JE Coligan et al., Eds., 1991).

Provisions "that there is a replication capability" of (interchangeably are used herein) the present invention or "oncolytic" vectors include, but are not limited to, naked DNA; encapsulated in viral coat Viral vectors packed into other viruses or virus-like forms (eg, herpes simplex virus and AAV); viral vectors encapsulated in liposomes; complexed with polylysine or other biocompatible polymers A viral vector complexed with a synthetic polycationic molecule; a viral vector conjugated with transferrin; and complexed with a compound such as PEG to immunize the molecule Viral vectors that have been “masked” and / or have increased half-life; or non-viral It may be in any of several forms including proteins, or viral vectors conjugated to any delivery carrier known to those skilled in the art. Such a vector represents one embodiment of the present invention. Preferably, the polynucleotide is DNA.

  As used herein, “DNA” does not include only bases A, T, C, and G, but also analogs thereof, or, for example, methylated nucleotides, internucleotide modifications, such as uncharged Includes all modified forms of these bases, such as linkages and thioates, the use of sugar analogs, and modified and / or alternative backbone structures such as polyamides. For the purposes of the present invention, viral vectors are capable of replication in target cells. Exemplary viruses capable of replication included within the scope of the present invention include, but are not limited to, adenovirus, vesicular stomatitis virus (VSV), herpes virus (eg, herpes simplex virus; HSV), reo Including viruses, paramyxoviruses, rhinoviruses, Newcastle disease viruses, polioviruses, West Nile viruses, Coxsackie viruses, measles viruses, and vaccinia viruses. All serotypes of virus capable of replication can be created for targeted and regulated expression using the techniques described herein.

  “Adenovirus vector” or “adenovirus vector” (used interchangeably herein) is a term well understood in the art and refers to all or part of the adenovirus genome. In general, a polynucleotide (as defined herein) is included. In the present specification, “adenovirus” represents the virus itself or a derivative thereof. The term covers all serotypes and subtypes, and natural and recombinant forms, except where otherwise indicated. For the purposes of the present invention, a regulatable adenoviral vector is provided. Cell type-specific transcriptional regulatory elements (CT-TRE) operably linked to inducible transactivator genes and transcriptional transactivator-regulated regulatory elements regulated by inducible transactivator Contains an adenoviral gene operably linked. The adenoviral vector is optionally a gene or transfer of a second adenovirus operably linked to a transactivator-regulated regulatory element or other type of transcriptional regulatory element that is not cell type specific. May contain genes.

  With regard to technologies specifically related to adenovirus, Feigner and Ringold (1989) Nature 337: 387-388; Berkner and Sharp (1983) Nucl. Acids Res. 11: 6003-6020; Graham (1984) EMBO J. 3: 2917-2922; Bett et al. (1993) J. Virology 67: 5911-5921; Bett et al. (1994) Proc. Natl. Acad. Sci. USA 91: 8802-8806. Publications describing various aspects of adenovirus biology and / or technology related to adenovirus include: Graham and Van de Eb (1973) Virology 52: 456-467; Takiff et al. (1981) Lancet ii: 832-834; Berkner and Sharp (1983) Nucleic Acid Research 6003-6020; Graham (1984) EMBO J 3: 2917-2922; Bett et al. (1993) J. Virology 67: 5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci. USA 91: 8802-8806 appear to act as gene transfer carriers Describes genetically modified replication-deficient adenoviruses (ie, gene therapy). In such vectors, the early adenoviral gene products, E1A and E1B, are generally deleted and provided in trans by packaging cell line 293 developed by Frank Graham (Graham et al. (1987 ) J. Gen. Birol. 36: 59-72 and Graham (1977) J. Genetic Virology 68: 937-940).

  The gene to be transduced is generally inserted into the deleted E1A and E1B regions of the adenoviral viral genome, Bett et al. (1990) supra. Adenoviral vectors for gene therapy are described in Stratford-Perricaudet (1990) Human Gene Therapy 1: 2-256; Rosenfeld (1991) Science 252: 431-434; Wang et al. (1991) Adv. Exp. Med. Biol. 309: 61-66; Jaffe et al. (1992) Nat Gent. 1: 372-378; Quantin et al. (1992) Proc Natl. Acad. Sci. USA 89: 2581-2584; Rosenfeld et al. 1992) Cell 68: 143-155; Stratford-Perricaudet et al. (1992) J. Clin. Invest. 90: 626-630; Le Gal La Salle et al. (1993) Science 259: 988-990; Mastrangeli et al (1993) J. Clin. Invest. 91: 225-234; Ragot et al. (1993) Nature 361: 647-650; Hayaski et al. (1994) J. Biol. Chem. 269: 23872-23875 It was done.

  By “transactivator”, “transactivator” or “transcription activator” is meant a polypeptide that facilitates transcription from a promoter. Inducible transactivators facilitate transcription by the presence (or absence) of a particular inducer or condition, or by changing its concentration. For example, a tet-regulated inducible transactivator may facilitate transcription from an inducible tetO promoter when the transactivator is not bound to a suitable inducer, such as tetracycline or an analog thereof. unknown. In the presence of tetracycline, such a transactivator is prevented from binding to its target, thereby preventing transcription. The reverse tet transactivator maintains the DNA binding specificity of the wild-type tet repressor, but is regulated in the reverse manner, ie it is a suitable inducer rather than a lack of inducer, eg tetracycline Alternatively, it binds to the tet operator sequence only in the presence of its analog. In general, transcriptional activators bind directly to transcription response elements, but in some cases, they then bind to transcription response elements or indirectly bind to other bound proteins.

  As used herein, a “transcriptional regulatory element” is also referred to as a “transcription response element” or “TRE” and regulates transcription of a polynucleotide sequence operably linked by RNA polymerase that forms RNA (ie, A polynucleotide sequence, preferably a DNA sequence. As used herein, TREs increase transcription of operably linked polynucleotide sequences in a host cell that allows the TRE to function. TREs include enhancer elements and / or promoter elements that are derived from or not derived from the same gene. As long as the desired transcriptional activity is obtained, the promoter and enhancer components of TRE can be in any direction and / or distance from the coding sequence of interest and may include the aforementioned multimers. As used herein, a TRE may lack a silencer element. TRE is cell type specific or, for example, a cancer cell derived from any of various tissues (prostate cancer cell, bladder cancer cell, colon cancer cell, liver cancer cell, kidney cancer cell, breast cancer cell, pancreatic cancer Cell specific) (cell status specific) or may be active in many cell types. As used herein, the term cell type specific transcriptional regulatory element (CT-TRE) refers to both cell type specific and cell status specific regulatory elements.

  A transcriptional regulatory element on which a transactivator acts is called a “transcriptional transactivator regulatory regulatory element”. Transcriptional transactivator-regulated regulatory elements regulate transcription of operably linked polynucleotide sequences and by inducible transactivators, for example to specific DNA binding sites in or near the promoter. Regulated by binding of inducible transactivator protein. Herein, a transcriptional transactivator-regulated regulatory element or TA-TRE alters the level of transcription in the presence of an inducer by downregulating or upregulating transcription.

  “Enhancer” is a well-understood term in the art, and to the extent that it is stronger than the transcriptional activation caused by a promoter in the absence of an enhancer when operably linked to a gene. A nucleotide sequence derived from a gene that enhances the transcription of the operably linked gene, ie it increases transcription from the promoter. “Enhancer activity” is a well-understood term in the art, and refers to transcription of a gene operably linked to a promoter in the presence of a nucleotide sequence with “enhancer activity”. It means to increase to a level higher than the level of transcription provided by the promoter itself when operably linked to the gene in the absence of a nucleotide sequence with “activity”, ie increase transcription from the promoter.

  “Under transcriptional regulation” is a term well understood in the art, transcription in which transcription of a polynucleotide sequence, usually a DNA sequence, is operably linked so that it can operate. Depends on the elements that contribute to the regulation of either the promotion or suppression.

  The term “operably linked” relates to the orientation of the polynucleotide elements in a functional relationship. If the TRE facilitates transcription of the coding sequence, the TRE is operably linked to the coding region. Operatively linked means that the linked DNA sequences are generally contiguous and are contiguous and within the same reading frame where necessary to connect the two protein coding regions. However, enhancers generally function when separated from the promoter by several kilobases, and because the intron sequence is variable length, several polynucleotide elements are operably linked but are contiguous. Maybe not.

  The terms “inducer” and “induction condition” are used interchangeably, and are chemicals that facilitate binding of an inducible transactivator transcription response element to a specific DNA binding site in or near a promoter. Represents a condition. As used herein, when an inducer / condition acts on a transactivator transcription response element, transcription levels are altered or regulated by down-regulating or up-regulating transcription. Typical “inducers” and “induction conditions” include, but are not limited to chemicals or non-chemicals (eg, ultrasound, heat, external radiation, hypoxia, etc.). Exposure of the oncolytic vector of the present invention to such “inducers” or “induction conditions” results in transcription activation (induction) or repression (repression), respectively, resulting in increased or decreased viral replication. It is understood.

  A “host cell” includes an individual cell or cell culture that could or was the recipient of any virus and / or vector of the invention. A host cell contains the progeny of a single host cell, and the progeny is original (in terms of morphology or total DNA complement) due to natural, accidental or conscious mutations and / or changes. May not be exactly the same as the parent cell. Host cells include cells that have been transfected or infected with the viruses and / or vectors of the invention in vivo. Host cells are isolated from tissue or animal hosts for in vitro culture.

  As used herein, the terms “neoplastic cell”, “neoplasia”, “tumor”, “tumor cell”, “cancer”, and “cancer cell” (used interchangeably herein) are: Represents cells that exhibit relatively autonomous proliferation, as they exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. Neoplastic cells may be malignant or benign.

  With respect to viral vectors, eg, viral vectors of the present invention, a “heterologous” promoter or enhancer is one that is not present in the wild-type virus. Examples of heterologous promoters or enhancers are albumin promoters or enhancers, and other viral promoters and enhancers such as SV40.

  With respect to a virus (viral vector), the “endogenous” promoter, enhancer, or TRE is unique to or derived from the virus.

  The term “gene” is a polynucleotide that is well understood in the art and that encodes a polypeptide. In addition to the polypeptide coding region, a gene includes, but is not limited to, introns, transcribed but untranslated regions, and non-coding regions containing regulatory elements upstream and downstream of the coding region.

  With respect to a virus (viral vector), a “heterologous polynucleotide” or “transgene” is any gene that is not present in the wild-type virus. Preferably, the transgene will not be expressed or present in the target cell prior to introduction by the virus (viral vector). Examples of preferred transgenes are provided below.

  Regardless of the polynucleotide or polypeptide, the sequence “represented by” SEQ ID NO: means that the sequence is present as a sequence that is also adjacent to the sequence of SEQ ID NO.

  The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include single-stranded, double-stranded, or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, other natural, chemically, biologically Includes chemically modified, non-natural or derivatized nucleotide bases. The following are non-limiting examples of polynucleotides: gene or gene fragment, exon, intron, mRNA, tRNA, rRNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any Isolated DNA of any sequence, isolated RNA, nucleic acid probe, and primer of any sequence. Polynucleotides may include modified nucleotides, such as, for example, methylated nucleotides and nucleotide analogs, uracils, other sugars and linkage groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition include caps, substitution of one or more natural nucleotides with analogs, and polynucleotides as proteins, metal ions, labeling components, other polynucleotides, or solid supports It is the introduction of the means to attach to. Preferably, the polynucleotide is DNA. As used herein, “DNA” does not include only bases A, T, C, and G, but also analogs thereof or modified forms of these bases, such as methylated nucleotides, Includes any internucleotide modifications such as uncharged bonds and thioates, sugar analogs, and the use of modified and / or alternative backbone structures such as polyamides.

  A polynucleotide or polynucleotide region that has a percentage of “sequence identity” relative to other sequences (eg, 80%, 85%, 90%, or 95%), when aligned, in a comparison of two sequences , That proportion of bases means the same. This alignment and homology rate or sequence identity is determined by software programs known in the art, such as Current Protocols in Molecular Biology (FM Ausubel et al., Eds. 1987) Supplement 30, section 7.7.18, Table 7.7. It can be determined using the one described in .1. A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania), preferably using default parameters.

  The terms “polypeptide”, “peptide”, and “protein” are used interchangeably to denote a polymer of amino acids of any length. These terms likewise include proteins that are post-translationally modified through reactions that include glycosidation, acetylation, and phosphorylation.

  “Replication” and “propagation” are used interchangeably and represent the ability of a viral vector of the invention to reproduce or propagate. This term is well understood in the art. For the purposes of the present invention, replication is generally directed to viral replication with the production of viral proteins. Replication can be measured using assays standard in the art and described herein, such as a burst assay or plaque assay. “Replication” and “propagation” include, but are not limited to, viral gene expression, production of viral proteins, nucleic acids, or other components, packaging of viral components into complete viruses, and cell lysis. Includes any activity that is directly or indirectly related to the virus production process.

  A “gene essential for replication” is a gene whose transcription is required for a viral vector to be replicated in a cell.

  As used herein, a “target cell” is a cell that allows (ie, enables or induces) a cell type-specific transcriptional regulatory element (TRE) to function. In general, the target cell is a mammalian cell, preferably a human cell.

As used herein, “cytotoxicity” is a term well understood in the art that disrupts (ie, inhibits) one or more normal biochemical or biological functions of a cell. Status). These activities include, but are not limited to, metabolism, cell replication, DNA replication, transcription, translation, and molecular uptake. “Cytotoxicity” includes cell death and / or cell lysis. Assays showing cytotoxicity, such as dye exclusion, ³ H thymidine incorporation, and plaque assays are known in the art. As used herein, the term “selective cytotoxicity” is compared to the cytotoxicity afforded by the viral vectors of the present invention to cells that do not allow or only allow the same TRE to function. In some cases, it represents the cytotoxicity conferred by the viral vectors of the present invention to cells that allow the cell type specific TRE to function. Such cytotoxicity may be due to, for example, plaque assay, reduction or stabilization of the size of the tumor containing the target cell, or a marker characteristic of the tumor cell or a tissue specific marker, such as a cancer marker, such as a prostate specific antigen. It can be measured by reduction or stabilization of serum levels.

  As used herein, a “cytotoxic” gene is a gene whose expression alone or in cells associated with viral replication increases the degree and / or rate of cytotoxicity and / or cytolytic activity in the cell.

  A “therapeutic” gene is a gene whose expression in a cell is related to the desired result. In the context of cancer, this desirable result may be, for example, cytotoxicity, inhibition or slowing of cell proliferation, and / or cell death.

  A “biological sample” includes a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue culture or cells derived therefrom, and progeny thereof. The definition likewise includes samples that have been manipulated in any way after they are obtained, for example by treatment with reagents, solubilization or concentration of certain components, such as proteins or polynucleotides. The term “biological sample” includes medical samples and also includes cultured cells, cell supernatants, cell lysates, serum, plasma, body fluids, and tissue samples.

  An “individual” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, rodents, primates, farm animals, sport animals, and pets.

  An “effective amount” is an amount that is beneficial or sufficient to achieve the desired clinical result. An effective amount can be administered by one or more administrations. For the purposes of the present invention, an effective amount of viral vector is an amount sufficient to mitigate, ameliorate, stabilize, reverse, slow down or delay the progression of the disease state. is there.

  As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results, whether or not detectable, may be detected or reduced in symptoms, disease range, disease state stability Including prevention (ie, not worsening), prevention of disease transmission (ie, metastatic), slowing or slowing of disease progression, amelioration or alleviation of illness, and (partial or total) remission. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

  “Alleviating” the disease reduces the severity of the condition and / or its undesirable clinical signs and / or slows the progression over time compared to not administering the viral vectors of the invention. Means to do or extend.

Regulatable transcriptional transactivators and repressors The present invention utilizes transactivating proteins that function in mammalian cells that can serve the purpose of host cells for viral infection. Such transactivation proteins are synthetic, chimeric, and natural transcriptional transactivation proteins or protein domains from eukaryotic cells, including vertebrate cells, of viruses that can stimulate transcription from vertebrate promoters. Contains a trans-activating protein or any synthetic amino acid sequence. Such trans-activated proteins are either (1) native (wild-type) or (2) chimeric (natural protein DNA-binding domain and natural protein regulatory (activator or repressor) domain Or (3) synthetic with a novel DNA binding domain designed by structural modeling, phage display screening, or other methods, and (4) the form of the fusion protein It may be something that you might take.

  Types of transcription activation domains include acidic transcription activation domains, high proline transcription activation domains, high serine / threonine transcription activation domains, and high glutamine transcription activation domains. Examples of acidic transcriptional activation domains include the VP16 region of Saccharomyces cerevisiae Gal4 and amino acid residues 753-881 (Braselmann et al., 1993, Proc Natl Acad Sci USA. 90 (5): 1657- 1661). Examples of high proline activation domains include amino acid residues 399-499 of CTF / NF1 and amino acid residues 31-76 of AP2. Examples of high serine / threonine transcription activation domains include amino acid residues 11-427 of ITF and amino acid residues 2-451 of ITF2. Examples of high glutamine activation domains include Oct1 amino acid residues 175-269 and Sp1 amino acid residues 132-243. The amino acid sequences of each of the previously described regions and other useful transcriptional activation domains are disclosed by Seipel, K. et al. (EMBO J. (1992) 13: 4961-4968).

  Examples of transactivating proteins are the lymph specific transcription factor identified by Muller et al. (1988, Nature 336: 544-551), the fos protein (Lucibello et al., 1988, Oncogene 3: 43-52); jun protein (Bos et al., 1988, Cell 52: 705-712); Factor EF-C (Ostapchuk et al., 1989, Mol. Cell. Biol. 9: 2787-2797); HIV-1tat protein (Arya et al., 1985, Science 229: 69-73), papillomavirus E2 protein (Lambert et al., 1989, J. Virol. 63: 3151-3154), and adenovirus E1A protein (Flint and Shenk, 1989, Ann. As well as Rev. Genet.). In a preferred embodiment of the invention, the transactivating protein is herpes simplex virus VP16 (Sadowski et al., 1988, Nature 335: 563-564; Triezenberg et al., 1988, Genes and Dev. 2: 718-729 ). The human NF-kappaB activation domain (p65) was used in a number of systems as well (Vermeulen L et. Al., Biochem Pharmacol 64 (5-6): 963-70, 2002).

  Inducible transactivators include chimeric fusion proteins comprising (i) a functional part of a DNA binding protein and (ii) a functional part of a transcriptional activator protein as described above. DNA sequences encoding the DNA binding protein and the transactivation protein are combined to preserve each individual binding and transactivation property. Regions that are not required for DNA binding protein or transcriptional transactivation protein function will be less mutation sensitive than the mapping mutation assay, as well as other, more functionally related parts of the molecule. It can be identified by any method known in the art, including identification of regions lacking mutations. Suitable recombinant constructs are prepared using standard molecular biology techniques, including those described in Maniatis (1982, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY, Cold Spring Harbor Laboratory)). The

  The DNA binding protein portion can be from any vertebrate, nonvertebrate, fungal, plant, or bacterial source and can be natural, processed, or synthetic. Examples are Gal4 (Keegan et al., 1986, Science 231: 699-704), ADR1 (Hartshorne et al., 1986, Nature 320: 283-287), Swl (Stillman et al., 1988, EMBO J. 7 : 485-495) and generally as discussed by Johnson et al. (1989, Annual Rev. Biochem., 58: 799-839). The transactivator may be a repressor protein, such as a lexA repressor. In some embodiments, the chimeric transactivator protein results in bacterial DNA binding that provides specificity for the transactivator protein by binding to a site processed into a transcriptional transactivator regulatory regulatory element. It is derived from protein. DNA sequence homology to bacterial DNA binding sites is unlikely to occur frequently in the mammalian genome, and thus selectively controls expression of the gene of interest.

  Typical activator domains include, but are not limited to, VP16, NF-kappaB, TFE3, ITF1, Oct-1, Spl, Oct-2, NFY-A, ITF2, c-myc, and CTF (Seipel, et al., 1992, EMBO J 13: 4961-4968). Typical repressor domains include, but are not limited to, Kruppel (KRAB; Margolin et al., 1994, Proc Natl Acad Sci USA 91 (10): 4509-13), kox-1 (Deuschle et al ., 1995), even-skipped (Licht et al., 1994), LacR, engrailed (Li et al, 1997, J Biol Chem., 274 (12): 7803-15), hairy (HES; Fisher et al. , 1996, EMBO J 12 (13): 5075-82), Groucho (TLE; Fisher etal., 1996), RING1 (Satjin etal., 1997, Mol. Cell. Biol. 17 (7): 4105-4113), SSB16 and SSB24 (Sana et al., 1993), TUPI (Tzamarlas, Struhl, 1994), Nabl (Swirnoff et al., 1998), AREB (Ikeda et al., 1998, Mol. Cell. Biol. 18 (1) : 10-18), E4BP4 (Cowell & Hurst, 1996), HoxA7 (Schnabell et al, 1996), EBNA3 (Bourillot et al., 1998, J Gen Virol. 79 (Pt 2): 363-70.), And v-erbA (Busch et al., 1997, Mol Endocrinol. 11 (3): 379-89.). As a specific example, tTs (TET silencer) uses the repression domain of KRAB protein. See also PCT publication WO0052179 for additional examples of activator domains, repressor domains, and chimeric transcriptional transactivators.

  The chimeric transactivator protein may further comprise a nuclear localization sequence so that the chimeric transactivator protein is selectively enriched in the cell nucleus. For example, the nucleic acid sequence encoding the nuclear localization signal of SV40 large T antigen, Pro-Lys-Lys-Lys-Arg-Lys-Val (Kalderon et al., 1984, Cell 39: 499-509) is transactivated Arranged identically to the factor protein coding sequence.

  A number of methods of controlling gene expression by inducers or conditions have been described, such as those induced by tetracycline, antiprogestins, and ecdysone.

  Inductive systems applicable to the present invention are disclosed in US Pat. Nos. 5,464,758, 5,589,362, PCT publications WO94 / 29442, WO96 / 01313, and WO96 / 40892, each of which is specifically incorporated herein by reference. Including tet (Tet ™) and reverse (RevTet ™) systems. For example, one of skill in the art will use Tet ™ and RevTet ™ systems (BDBiosciences) that utilize small molecules such as tetracycline (Tc) or analogs such as doxycycline to modulate (turn on or off) target transcription. Clontech) (Knott A et al., Biotechniques 32 (4): 796, 798, 800 (2002)). Both Tet ™ and RevTet ™ systems have been shown to regulate gene expression in vivo. Modified vectors based on parvovirus, adenovirus, retrovirus, and herpes simplex virus have been used to introduce TET technology in vitro. For example, Ho, DY et al. (1996) Mol. Brain Res. 41: 200-209; Hwang, JJ. Et al. (1996) J. Virol. 70: 8138-814; Hu, SX. Et al. (1997) Cancer Res. 57: 3339-3343; and Maxwell, IH et al. (1996) Gene Ther. 3: 28-36. Gossen et al. (1992) Proc Natl Acad Sci USA. 89 (12): 5547-51 describes the regulation of gene expression in mammalian cells by a tetracycline-sensitive promoter. Gossen et al. (1995) Science 268 (5218): 1766-9 describes transcriptional activation by tetracycline in mammalian cells. Urlinger ef al. (2000) Proc Natl Acad Sci USA 97 (14): 7963-8 discusses mutations in tetracycline-dependent transcriptional activators. Freundlieb et al. (1999) J Gene Med. 1 (1): 4-12 discusses a tetracycline-regulated activation / suppression system with a high capacity for gene transfer into mammalian cells.

  In other typical systems, transcription is activated by rapamycin (or an analog thereof) that joins two intracellular molecules, each of which is linked to a transactivator or DNA binding protein. When these components come together, transcription of the gene of interest is activated. Rapamycin mediates heterodimer formation between immunophilin FK506 binding protein (FKBP) and lipid kinase homolog FRAP (Standaert, RF et al., Nature 346, 671-674 (1990); Brown, EJ et al , Nature 369, 756-758 (1994); Rivera, VM ef al. Nat. Med. 2, 1028-1032 (1996); and Ho, SN et al., Nature 382, 822-826 (1996)). The rapamycin-based system (Ariad) has been shown to regulate target gene transcription in both cell cultures and animal models, and a high dose-dependent inducibility has been demonstrated after the addition of rapamycin.

  For example, Ye et al. Science 283 (5398): 88-91, 1999; Magari et a /. J Clin Invest. 100 (11): 2865-72,1997; Rivera et al. Nat Med. 2 (9): See 1028-32, 1996. Ariad has two major systems: a system based on homodimer formation and a system based on heterodimer formation (Rivera et al., 1996, Nature Med, 2 (9): 1028-1032; Ye et al., 2000, Science 283: 88-91; Sawyer TK et al., 2002, Mini Rev Med Chem. 2 (5): 475-88). The heterodimer formation system is based on the 93aa fragment of human FKBP12 (FK506 binding protein) and the large human PI3K homologue, FRAP (RAFT, mTOR). When a DNA binding protein called ZFHD1 (a fusion protein of two human DNA binding domains) is bound to FKBP and the human NF-kappa b p65 activation domain is FRAP (a rapamycin analog is used as an inducer) Instead, it is fused to a mutated version of FRAP). The addition of inducer molecules that bind to both FKBP12 and FRAP brings FKBP12 and FRAP together with the DNA binding protein and the activation domain. Gene transcription is thereby initiated.

  Other examples of regulatory gene expression systems or promoters are the metallothionein promoter system (Mulherkar et al. Biochem Biophys Res Commun. 177 (1): 90-6, 1991); the glucocorticoid promoter system (Ko ef al (1989) Gene 84 (2): 383-9), ecdysone-regulated gene switch (Saez ef al. Proc Natl Acad Sci USA. 97 (26): 14512-7), and macrolide-based transgene control System (Weber ef al. (2002) Nat Biotechnol. 20 (9): 901-7).

  Among the ecdysone systems, the Drosophila ecdysone system (Yao and Evans, 1996, Proc. Nat. Acad. Sci., 93: 3346), the Bombyx ecdysone system (Suhr et al., 1998) , Proc. Nat. Acad. Sci., 95: 7999). Steroid-based systems include a synthetic progesterone receptor system that utilizes RU-486 as an inducer (Wang et al., Biochim Biophys Acta, 1994, 1218 (3): 308-314; Delort and Capecchi, 1996 , Hum Gene Ther 7 (7): 809-820; and Osterwalder et al., 2001, Proc Natl Acad Sci 98 (22): 12596-601).

  A “functionally conserved” variant of a transcriptional transactivator, repressor, or response element therefor is a transcriptional transactivator that differs from the referenced transcriptional transactivator, repressor, or response element therefor A repressor, or a response element therefor, but maintains the ability to increase operably linked polynucleotide transcription, particularly cell type specific transcriptional activity. The differences may be due to altered linear sequences or conformations that occur, for example, due to single or multiple base mutations, additions, deletions, and / or base modifications. Differences may arise from sugar changes and / or linkages between bases as well.

  The oncolytic virus of the present invention can be used for a wide variety of purposes. The purpose depends on the target cell. Suitable target cells are characterized by the transcriptional activation of cell-specific transcription response elements in viral vectors. Regulation of transcriptional activation is the result of an interaction between the transcriptional activator that binds to the cis-regulatory element, the factor that binds to the basal transcription element, and the activity of the transcriptional mediator, coactivator, and the presence of inducers or conditions It is. The transactivator-regulated transcriptional regulatory element is operably linked to viral genes essential for growth so that replication competence can be achieved in the target cell and / or for the transgene. By transgene is intended any gene that is not in the wild-type virus, and often the transgene is not expressed in the target cell prior to introduction by the virus as well.

  Those skilled in the art will be aware that some changes in the bases in and around the transcription factor binding site are negatively affected by gene activation and cell specificity, while being associated with transcription factor binding. Recognize that no base change is expected to have such an impact. Certain mutations can increase transcriptional transactivator or repressor activity as well. Testing for the effect of changing bases can be performed in vitro or in vivo by any method known in the art, such as mobility shift assays or transfection of vectors containing these changes in target, non-target cells. Can be implemented. Moreover, those skilled in the art recognize that point mutations and deletions can be made to a transcriptional transactivator or repressor sequence, or a response element therefor, without altering the ability of the sequence to regulate transcription. To do.

  It is known by those skilled in the art that very low basal transcription levels may exist in non-target cell types. By transcriptional activation, transcription is at least about 2-fold; preferably at least about 5-fold; preferably at least about 10-fold; more preferably at least about 20-fold, 30-fold, or 40-fold; more preferably at least about 50-fold; 60 times, 70 times, 80 times, or 90 times; more preferably at least about 100 times; even more preferably at least about 200 times, even more preferably at least about 400 to about 500 times, even more preferably at least about 1000 times It is intended to increase above the basal level of the target cell.

  All of the previously described systems for control of gene expression find utility in practicing the present invention. Effective doses or effective induction conditions for inducers are readily determined from extrapolation of published results, from empirical assays, and from other methods known and routinely used by those skilled in the art . For example, an inducer is administered to an animal carrying a viral vector of the invention, and an effective virus titer is measured after administration of the inducer.

Transcriptional regulatory elements (TREs)
In the practice of the present invention, the gene sequence encoding the transactivator protein is desirably placed under the transcriptional regulation of a suitable cell type specific TRE. A “cell type specific TRE” functions selectively in a particular cell type relative to other cell types with different functions. A “cell type” is a reflection of the differentiation of a cell state that is irreversible and end-stage under normal physiological conditions. For example, the prostate specific antigen TRE functions in prostate cells but does not substantially function in other cell types such as hepatocytes, astrocytes, heart cells, lymphocytes and the like. In general, a cell type specific TRE is only active in one cell type. If a cell type-specific TRE is active in more than one cell type, its activity is limited to a limited number of cell types, ie not active in all cell types. The cell type specific TRE may be tumor cell specific. Such cell type specificity represents a relative increase in transcription in target host cells in which TRE is active compared to cells in which TRE is not active.

  As used herein, a TRE from a particular gene is represented by the gene from which it is derived, and a polynucleotide sequence that regulates the transcription of the operably linked polynucleotide sequence in the host cell expressing that gene. It is. Depending on the target cell type, various enhancers can be used to provide specific transcription. For lymphocytes, for B cells, one of skill in the art may use an Ig enhancer, and for T cells, one skilled in the art may use a T cell antigen receptor promoter. For different muscle cells, one skilled in the art may use promoters for different myosins. For endothelial cells, one skilled in the art may use different promoters for different selectins. For each cell type, there are specific proteins associated with the cell that allow target cell specific transcription.

  As used herein, a “cell state-specific transcriptional regulatory element” or cell state-specific TRE is induced under special physiological conditions that permit or induce the expression of a polynucleotide under its transcriptional regulation. Or a TRE that becomes active.

  An example of a cell state is the cell cycle. A typical gene whose expression is related to the cell cycle is the universally expressed growth regulatory gene, E2F-1, which shows a peak of transcriptional activity in S phase. Johnson et al. (1994) Genes Dev. 8: 1514-1525. The RB protein, as well as other members of the RB family, form a specific complex with E2F-1, thereby inhibiting its ability to activate transcription. Thus, the E2F-1-responsive promoter is downregulated by RB. Many tumor cells have RB function that can derepress the E2F-1 sensitive promoter and in turn cause deregulated cell division.

  A replication competent vector comprising a cell state showing a peak of transcriptional activity in S phase-specific TRE, such as the universally expressed growth regulatory gene “E2F-1-TRE”, is within the present invention. included. “E2F-1-TRE” refers to a host cell in which E2F-1-TRE is allowed to function, such as a cell expressing E2F-1 (preferably a mammalian cell, more preferably a human cell). A polynucleotide sequence, preferably a DNA sequence, that selectively increases transcription (of operably linked polynucleotide sequences). E2F-1-TRE is sensitive to cofactors related to transcription factors and / or E2F-1 producing cells and contains at least part of the E2F-1 promoter and / or enhancer. Hernandez-Alcoceba R et al. (Hum Gene Ther 2002 Sep 20; 13 (14): 1737-50); Tsukuda et al. (Cancer Res. 62: 3238-3447, 2002); Johnson et al., Cancer Cell. 2002 May; 1 (4): 325-37. (Onyx); Jakubczak JL et al., Cancer Res. 2003 Apr 1; 63 (7): 1490-9; US patent 09 / 392,822 published as 20010053352 And U.S. Pat. No. 10/081969, published as 20030104625. The sequence of the E2F promoter is known in the art and is described, for example, in Fueyo J et al., Nat Med. 1998 Jun; 4 (6): 685-90.

  A further example of a cellular state is a response to hypoxic conditions (eg, a hypoxic response element or HRE). Important mediators of hypoxic response are hypoxia-sensitive elements (HREs) within the regulatory region of several genes, including several genes that encode glycolytic enzymes, including vascular endothelial growth factor and enolase-1 Transcription complex HIF-1, or hypoxia-inducible factor-1 The mouse HRE sequence was confirmed and characterized. Firth et al. (1994) Proc. Natl. Acad. Sci. USA 91: 6496-6500. Hypoxia is an essential component of the tumor microenvironment that is expressed in most solid tumors regardless of their origin, configuration, or genetic changes, resulting in rapid growth of the tumor associated with its vascular supply. Hypoxia is a major contributor to cancer cell resistance to radiation therapy and chemotherapy, so select a highly malignant tumor clone to give the tumor a metastatic tendency and a new treatment that targets the hypoxic region of the tumor The development of a strategy for is important.

  Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that mediates the response to hypoxia by binding to the hypoxia response element (HRE) present in the target gene, thereby making the HIF / HRE system a tumor Is used to specifically target therapeutic gene expression for. Similarly, replication competent vectors containing hypoxia responsive elements or “HRE” are included within the invention. Hypoxia responsive elements function as HREs, such as cells that express hypoxia-inducible factor 1 (preferably mammalian cells, more preferably human cells) that interact with hypoxia sensitive elements (HRE) A polynucleotide sequence, preferably a DNA sequence, that selectively increases transcription (of operably linked polynucleotide sequences) in a host cell under conditions that permit it to do so. The sequence of the hypoxia responsive element is known in the art, for example, HRE from the mouse enolase-1 promoter is described in Jiang et al. (1997) Cancer Res. 57: 5328-533.

  Another typical gene whose expression is related to the cell cycle is telomerase. For example, published US patent application Ser. No. 10/081969, published as 20030104625, describing regulated expression of adenovirus using the human telomerase promoter; published US patent application Ser. No. WO00 / 46355; See U.S. Patent Application No. 10/206447 published as 10 / 023,969, US20030099616 (Geron); U.S. Patent Application No. 09 / 956,335 published as 20020028785 (University of St. Louis). The present invention provides replication-competent vectors that contain telomerase regulatory elements, eg, telomerase promoters. Numerous telomerase promoter sequences are known in the art, examples of which can be found in GenBank accession numbers AF128893.1 and AF121948.

  Other cell state specific transcription response elements include heat inducible (ie heat shock) promoters and radiation exposure responsive promoters including ionizing radiation and UV radiation. For example, the promoter region of the early growth response-1 (Egr-1) gene contains elements induced by ionizing radiation. Hallahan et al. (1995) Nat. Med. 1: 786-791; and Tsai-Morris et al. (1988) Nucl. Acids. Res. 16: 8835-8846. A heat-inducible promoter containing a heat-inducible element has been described. See, for example, Welsh (1990) in “Stress Proteins in Biology and Medicine”, Morimoto, Tisseres, and Georgopoulos, eds. Cold Spring Harbor Laboratory Press; and Perisic et al. (1989) Cell 59: 797-806.

  As a result, in some embodiments, the cell state specific transcription response element comprises one or more of ionizing radiation sensitive elements, such as 5 'flanking sequences of the Egr-1 gene, heat shock responsive elements, or heat inducible elements. Including.

  As used herein, the term “cell type specific” refers to a TRE sequence that is operably linked to a gene, ie, a gene that is essential for viral replication, or a transgene is operably linked, or Target cells such that transcription (and replication if the operably linked gene is essential for viral replication) proceeds selectively in the target cell or the transgene is expressed in the target cell Is intended to mean a specific function. This is caused by the presence of transcription factors in the target cells that activate transcription driven by operably linked transcriptional regulatory sequences, and not in non-target cells. It can also prevent transcription driven by transcriptional control sequences operatively linked and produced by the effects of transcriptional inhibitors that normally occur in non-target cells. As used herein, the term “cell type specific” is intended to include cell type specificity, tissue specificity, and specificity for a cancerous condition of a given target cell. In the latter case, specificity for normal cell cancer symptoms is by comparison with normal and non-cancer counterparts.

  In one embodiment, the present invention comprises an oncolytic viral vector in which CT-TRE is prostate cell specific. For example, TREs that function selectively in prostate cells and can be used in the present invention for targeted virus replication against prostate neoplasms are not limited to this glandular (from the human gene hKLK2-TRE) TREs derived from the kallikrein-1 gene, prostate specific antigen gene (PSA-TRE), and probasin gene (PB-TRE). All three of these genes are selectively expressed in prostate cells and expression is inducible by androgens. In general, expression of genes sensitive to androgen induction requires the presence of androgen receptor (AR).

  Human glandular kallikrein (hKLK2 encoding hK2 protein) is expressed exclusively in the prostate, and its expression is regulated by androgens mainly by transcriptional activation. The levels of hK2 found in the sera of patients with various tumors and prostate cancer are substantially different from those of PSA, indicating that the hK2 antigen may be an important marker for prostate cancer.

  “Human glandular kallikrein transcriptional regulatory elements” or “hKLK2-TRE” are included in the replication competent vectors of the present invention. “HKLK2-TRE” is operably linked to, for example, a cell that expresses an androgen receptor (preferably a mammalian cell, more preferably a human cell), or a host cell that allows hKLK2-TRE to function. A polynucleotide sequence, preferably a DNA base sequence, that increases transcription of the polynucleotide sequence. Thus, hKLK2-TRE is sensitive to androgen receptor binding and includes at least one portion of the hKLK2 promoter and / or hKLK2 enhancer (ie, ARE or androgen receptor binding site). hKLK2-TREs are further described in US patent application Ser. No. 09 / 875,228.

  The hKLK2 5 ′ promoter activity has been previously described and up to -2256 regions related to the transcription initiation region have been previously disclosed (SEQ ID NO: 3). Schedlich et al. (1987) DNA 6: 429-437. The hKLK2 promoter is androgen-responsive, and in a plasmid construct in which the promoter alone controls the expression of the reporter gene, the expression of the reporter gene is increased approximately 10-fold by the presence of androgen. hKLK2 enhancer activity is found within a polynucleotide sequence of about nt-12,014 to nt-2257 relative to the transcription start point (shown in SEQ ID NO: 3) and the hKLK2 promoter and reporter to enable this sequence to operate. When linked to a gene, transcription of the operably linked sequence in prostate cells is enhanced by the presence of androgen to a level that exceeds the transcription level in the absence of androgen by about 30-fold to about 100-fold. This induction is generally orientation-independent and position-independent. Enhancer activity is similarly in the following regions (all relative to the transcription start site): about nt-3993 to nt-3643 (nt8021 to 8371 of SEQ ID NO: 3), about nt-4814 to nt-3643 (nt7200 to SEQ ID NO: 3) 8371), about nt-5155 to nt-3387 (nt6859 to nt8627 of SEQ ID NO: 3), and about nt-6038 to nt-2394 (nt5976 to 9620 of SEQ ID NO: 3).

  Thus, the hKLK2 enhancer is operably linked to the hKLK2 promoter or heterologous promoter to form the hKLK2 transcriptional regulatory element (hKLK2-TRE). HKLK2-TRE can then be operably linked to a heterologous polynucleotide to confer KLK2-TRE specific transcriptional control on the linked gene and increase its expression.

  In another example, a “probasin (PB) transcriptional regulatory element” or “PB-TRE” is included in the replication competent vector of the present invention. “PB-TRE” is operably linked in a host cell that allows PB-TRE to function, such as a cell that expresses an androgen receptor (preferably a mammalian cell, more preferably a human cell). A polynucleotide sequence, preferably a DNA sequence, that selectively increases transcription of the polynucleotide sequence. Thus, APB-TRE is sensitive to androgen receptor binding and contains at least a portion of the PB promoter and / or PB enhancer (ie, ARE or androgen receptor binding site). PB-TREs are further described in US Pat. No. 6,436,394.

  For example, the specificity of PB-TRE activity for prostate cells expressing androgen receptor (AR) was demonstrated as follows. A chimeric PB-TRE-luc plasmid was constructed by inserting the PB 5 'flanking DNA region (nt-426 to nt + 28) (SEQ ID NO: 9) containing the promoter sequence within the sequence upstream of the firefly luciferase gene. To do. Cation-mediated, transient transfection of LNCaP cells (PSA-producing and AR-producing prostate cancer cells) and PC-3 cells (PSA-deficient and AR-deficient prostate cancer cells) was performed. The results showed that LNCaP cells transfected with PB-TREluc were about 400 times more active than untransfected cells, indicating that PR-TRE was intact. Furthermore, the overall luciferase activity recovered in the cell extract of transfected LNCaP cells was approximately 30-40 times higher than that measured in the cell extract of transfected PC-3 cells. Thus, the above results indicate that PB-TRE expression is selectively functioning in PSA-producing, AR-producing prostate cancer cells compared to PSA-deficient, AR-deficient prostate cancer cells, and that PB-TRE is It shows that it can mediate expression specific to cells producing the receptor. The rat probasin (PB) gene encodes the nuclear and secreted protein, probasin. It is expressed only on the dorsal side of the prostate. As shown in (SEQ ID NO: 4), PB-TRE was represented by a sequence of about 0.5 kb fragment upstream of the probasin coding sequence, about nt-426 to nt + 28 relative to the transcription start site. This minimal promoter sequence from the PB gene appears to provide sufficient information for the direct development and hormonally regulated expression of operably linked heterologous genes specific to the prostate of transgenic mice It is.

  In other examples, a “prostate specific antigen (PSA) transcriptional regulatory element” or “PSA-TRE” or “PSE-TRE” is included in the replication competent vector of the invention. “PSA-TRE” or “PSE-TRE” operates in a host cell that allows PSA-TRE to function, such as a cell that expresses an androgen receptor (preferably a mammalian cell, more preferably a human cell). A polynucleotide sequence, preferably a DNA sequence, that selectively increases transcription of the ligated polynucleotide sequences. Thus, PSE-TRE is responsive to androgen receptor binding and includes at least a portion of the PSA promoter and / or PSA enhancer (ie, ARE or androgen receptor binding site). PSE-TREs are further described in US Pat. Nos. 5,648,478, 6,057,299, and 6,136,792.

  The region of the PSA gene used to provide androgen-dependent cell specificity, particularly in prostate cells, spans approximately 6.0 kb. Schuur et al. (1996) J. Biol. Chem. 271: 7043-7051. The enhancer region of about 1.5 kb in human is located between nt-5322 and nt-3739 with respect to the transcription start site of the PSA gene. The PSA promoter consists of a sequence of about nt-540 to nt + 8 relative to the transcription start site. These two proximal genetic elements are fully functional and result in a minimal prostate-specific enhancer / promoter (PSE) TRE. Other parts consisting of the approximately 6.0 kb region of the PSA gene can be used in the present invention so long as the description of the required function is maintained.

  The PSE-TRE represented by (SEQ ID NO: 1) is the same as that given by GenBank accession number U37672. The mutant PSA-TRE nucleotide sequence is shown in (SEQ ID NO: 2). This is the PSA-TRE contained within CN706 clone 35.190.13. CN706 is an adenovirus vector in which the E1A gene in Ad5 is under the transcriptional control of PSA-TRE. CN706 has demonstrated selective cytotoxicity against PSA-expressing cells in vitro and in vivo. Rodriauez et al. (1997).

  The region utilized to provide androgen-dependent cell specificity, particularly in prostate cells, spans an approximately 1.5 kb enhancer region and a 0.5 kb promoter region. The enhancer region in humans is between nt-5322 and nt-3739 with respect to the transcription start site of the prostate specific antigen (PSA) gene. The promoter consists of nt-540 to nt + 8. The proximal two genetic elements give rise to a fully functional, minimal prostate-specific enhancer promoter (PSE). The enhancer includes three regions that bind to prostate-specific DNA binding protein, one of which includes the assumed androgen response element. The promoter region contains typical TATA and CAAT boxes, as well as a potential second androgen response element.

  CEA is a 180,000 dalton glycoprotein tumor-associated antigen present in neoplasms of the gastrointestinal tract, such as colon cancer, gastric (stomach) and pancreatic cancer, endoderm-derived neoplasia, and other adenocarcinomas such as breast and lung cancer It is. In yet another example, a “carcinoembryonic antigen (CEA) transcriptional regulatory element” or “CEA-TRE” is included in the replication competent vector of the invention. CEA-TRE is a polynucleotide sequence operably linked in a host cell that allows CEA-TRE to function, such as a cell that expresses CEA (preferably a mammalian cell, more preferably a human cell). A polynucleotide sequence, preferably a DNA sequence, that selectively increases transcription. CEA-TRE is responsive to transcription factors and / or cofactors associated with CEA producing cells and includes at least a portion of the CEA promoter and / or enhancer. The 5 ′ upstream flanking sequence of the CEA gene has been shown to confer cell specific activity. The first approximately 424 nucleotide CEA promoter region upstream of the translation start site within the 5 'flanking region of the gene confers cell-specific activity in CEA-producing cells when provided with higher promoter activity than non-producing HeLa cells It was shown that.

  In addition, a cell-specific enhancer region was found. It appears that the entire 5 ′ CEA flanking region (including promoter, putative silencer, and enhancer elements) is contained within about 14.5 kb upstream from the transcription start region. When linked to a multimerizing promoter, two upstream regions, -13.6 to -10.7 kb, or -6.1 to -4.0 kb, provide selective expression at high levels of reporter constructs in CEA-producing cells . The promoter region is located at nt-90 to nt + 69 relative to the transcription start site with the nt-41 to nt-18 region essential for expression. WO95 / 14100 describes a series of 5 ′ flanking CEA fragments that confer cell specific activity, such as about nt-299 to about nt + 69; about nt-90 to nt + 69; nt-14,500 to nt-10,600; nt− 13,600 to nt-10,600 and nt-6100 to nt-3800 will be described. Furthermore, cell-specific transcriptional activity is conferred on the operably linked gene by the CEA fragment from nt-402 to nt + 69 shown in (SEQ ID NO: 7). The CEA-TREs used in the present invention are derived from mammalian cells including human cells without being limited thereto. Thus, any CEA-TREs can be used in the present invention as long as a description of the desired function required is shown. Cloning and characterization of CEA sequences is described in the literature (eg, US Patent Application No. 10 / 045,116 published as 200030026792).

  In a further example, an “alpha fetoprotein (AFP) transcriptional regulatory element” or “AFP-TRE” is included in the replication competent vector of the present invention. AFP is an oncofetal protein. Serum concentrations of AFP are increased in patients with liver cancer with high levels of AFP found in patients with advanced disease. Patient serum AFP levels appear to be regulated by AFP expression in hepatocellular carcinoma but not in the surrounding normal liver. Thus, the AFP gene appears to be involved in liver cancer cell specific expression. Cell-specific TREs from the AFP gene were identified. “AFP-TRE” refers to a operably linked poly- mer in a host cell that allows AFP-TRE to function, such as a cell expressing AFP (preferably a mammalian cell, more preferably a human cell). A polynucleotide sequence, preferably a DNA sequence, that selectively increases transcription (of the nucleotide sequence). AFP-TRE is responsive to transcription factors and / or cofactors associated with AFP producing cells and includes at least a portion of an AFP promoter and / or enhancer. AFP-TREs are further described in US patent application Ser. No. 09 / 898,883, which is expressly incorporated herein by reference. The entire 5 ′ AFP flanking region (including promoter, putative silencer, and enhancer molecules) is contained within about 5 kb upstream from the transcription start region (SEQ ID NO: 5).

  The human AFP enhancer region is located at about nt-3954 to nt-3335 relative to the transcription start site of the AFP gene. The human AFP promoter contains a region from nt-174 to nt + 29. These two proximal genetic elements result in a fully functional AFP-TRE. Ido et al. (1995) describe a 259 bp promoter fragment (nt-230 to nt + 29) specific for HCC. Cancer Res. 55: 3105-3109. The AFP enhancer contains two regions, defined as A and B, located between nt-3954 and nt-3335 relative to the transcription start site. The promoter region contains typical TATA and CAAT boxes. Preferably, AFP-TRE contains at least one enhancer region. More preferably, the AFP-TRE contains both enhancer regions.

  Suitable target cells for oncolytic viral vectors containing AFP-TREs are all cell types that allow AFP-TRE to function. Without being limited thereto, cells that express or produce AFP are preferred, including tumor cells that express AFP. Examples of such cells are hepatocellular carcinoma cells, gonadal and other germ cell tumors (especially endoderm tumors), brain tumor cells, ovarian tumor cells, pancreatic acinar cell carcinoma, first gallbladder tumor, intrauterine Membrane adenocarcinoma cells and any of the metastases previously (which may occur in the lung, adrenal gland, bone marrow, and / or spleen). In some cases, certain pancreatic and gastric cancer to liver metastases produce AFP. Hepatocellular carcinoma cells and any of their metastatic properties are particularly preferred. AFP production can be accomplished using standard assays in the art, such as RIA, ELISA or Western blot (immunoassay) to measure AFP protein production, or Northern blot to measure AFP mRNA production levels. Can be measured using. Alternatively, such cells can be identified and / or characterized by their ability to transcriptionally activate AFP-TRE (ie, to allow AFP-TRE to function).

  In yet a further example, urothelial cell specific transcriptional regulatory elements are included in the replication competent vectors of the invention. A urothelial cell-specific transcriptional regulatory element is a polynucleotide sequence, preferably a DNA sequence, that selectively increases transcription (of operably linked polynucleotide sequences) in a urothelial host cell. For example, urothelial cell-specific TRE is operably linked in cells that allow UP-TRE to function, such as cells expressing uroplakin (preferably mammalian cells, more preferably human cells). It may be derived from the 5 ′ flanking region of the uroplakin gene that increases TRE selectively (ie, UP-TRE). In some of these embodiments, the urothelial cell specific TRE is derived from the 5 'flanking region of the UPIa gene. In other embodiments, the urothelial cell specific TRE is derived from the 5 'flanking region of the UPIb gene. In yet other embodiments, the urothelial cell specific TRE is derived from the 5 'flanking region of the UPII gene. UP-TRE may contain a urothelial cell specific promoter and a heterologous enhancer. In other embodiments, the urothelial cell specific TRE comprises a urothelial cell specific promoter. In other embodiments, the urothelial cell specific TRE comprises a urothelial cell specific enhancer and a heterologous promoter. In other embodiments, the urothelial cell specific TRE comprises a urothelial cell specific promoter and a urothelial cell specific enhancer. UP-TREs are further described in PCT Publication WO01 / 72994.

  The protein product of the MUC1 gene (mucin or MUC1 protein; episialin; polymorphic epithelial mucin or PEM; EMA; DF3 antigen; NPGP; PAS-O; or CA15.3 antigen) is known as stomach, pancreas, lung, trachea It is usually expressed primarily on the apical surface of epithelial cells lining the glands and ducts of the kidney, uterus, salivary gland, and mammary gland. However, mucin is overexpressed in 75-90% of human breast cancer. Mucin protein expression is associated with the degree of breast cancer differentiation. This overexpression appears to be controlled at the transcriptional level. Overexpression of the MUC1 gene in human breast cancer cells MCF-7 and ZR-75-1 appears to be regulated at the transcriptional level. “MUC1-TRE” is a operably linked poly- mer in host cells that allow MUC1-TRE to function, such as cells that express MUC1 (preferably mammalian cells, more preferably human cells). A polynucleotide sequence, preferably a DNA sequence, that selectively increases the transcription of the nucleotide sequence). MUC1-TRE is responsive to transcription factors and / or cofactors associated with MUC1-producing cells and includes at least a portion of the MUC1 promoter and / or enhancer. MUC1-TREs are further described in US Pat. No. 6,432,700. In a further example, a “mucin gene (MUC) transcriptional regulatory element” or “MUC1-TRE” is included in the replication competent vector of the present invention. The regulatory sequence of the MUC1 gene was cloned, containing about 0.9 kb upstream of the transcription start site containing TRE that appears to be involved in cell-specific transcription as set forth in SEQ ID NO: 8.

  The MUC1-TREs used in the present invention are derived from mammalian cells including, but not limited to, human cells. Preferably, MUC7-TRE is human. In one embodiment, the MUC1-TRE may comprise the entire 0.9 kb 5 ′ flanking sequence of the MUC1 gene. In other embodiments, the MUC1-TREs comprise the following sequences (related to the transcription start site of the MUC1 gene): from about nt-725 to about nt + 31 (operably linked to a promoter), nt- 743 to about nt + 33, nt-750 to about nt + 33, and nt-598 to about nt + 485.

  The c-erbB2 / neu gene (HER-2 / neu or HER) is a transforming gene that encodes a 185 kD epidermal growth factor receptor-related transmembrane glycoprotein. In humans, the c-erbB2 / neu protein is expressed during fetal development, but in adults, the protein is slightly detectable (by immunohistochemistry) in the epithelium of many normal tissues. Amplification and / or overexpression of the c-erbB2 / neu gene has been implicated in many human cancers including breast cancer, ovarian cancer, uterine cancer, prostate cancer, gastric cancer and lung cancer.

  In still further examples, “HER-2 / neu transcriptional regulatory elements” or “HER-2 / neu-TRE” are included in the replication competent vectors of the present invention.

  Additional tumor and / or cell type specific TREs are known in the art and included in the oncolytic vectors of the present invention include the following: aromatase, mammary gland specific promoter, mammaglobin, plasminogen activator Urokinase (uPA), and its receptor genes (related to breast cancer, colon cancer, and liver cancer; TREs that regulate uPA and uPAR transcription are provided in SEQ ID NO: 6: and Riccio et al. Nucleic Acids Res. 13 : 2759-2771, 1985; Cannio et al., Nucleic Acids Res. 19: 2303-2308, 1991; further described in human α-lactalbumin (related to breast tissue); BCSG1, BRCA1, and BRCA2 (Related to breast cancer); human papillomavirus (HPV) cell type-dependent regulatory elements (related to cervical cancer); BLCA4 (related to bladder cancer); uroplakin (related to bladder) Shi NCA (related to gastric cancer); hypoxanthine phosphoribosyltransferase (HPRT; related to glioma); AVP, human alveolar surfactant protein B gene, and puromycin N-acetyltransferase ( Related to lung cancer);

  Tyrosinase, gplOO; melanoma specific TREs, eg human MART promoter (hMART), mouse tyrosinase gene enhancer and promoter (mEP), mouse tyrosinase gene enhancer and promoter (mEEP) containing TRE, human tyrosinase promoter containing TRE (HTYR) and melanocyte-specific factor (MSF); HER2 / neu, urokinase, and CA125 (related to ovarian cancer); SL3-3 and T cell antigen receptors (related to T cell type lymphoma) ); Prostatic acid phosphatase (associated with prostate cancer); EBV-specific TRE (associated with EBV expressing tumors), eg LMP1 promoter; LMP2A promoter, LMP2B promoter; or Cp promoter (eg Sjoblom, A et al, J. Virol. (1998) 72, (2), 1365-1376; Franken et al, J. Virol (1995) 69 (12) 8011-8019.; Laux et al, J. Gen. Virol, 1989, 70, 3079-3084; and and Fuentes-Panana et al, J. Virol., (1999) 73, 826-833 and V01555); liver-specific TRE (Related to liver cancer), for example the CRGL2 promoter; and the WT1 promoter and enhancer (Hosen N. Leukemia. 2004 Jan 22) involved in leukemia-specific gene expression.

  In addition, tumor-specific promoters include artificial hTERT-cHSF1 / HSE promoters (as described, for example, in Wang J et al. FEES Lett. Jul 10; 546 (2-3): 315-20, 2003), or the complete May be obtained by chimeric constructs using different promoter elements as illustrated by synthetic constructs (Li X et al Nat Biotechnol. 1999 Mar; 17 (3): 241-5). Descriptions of these cell-specific TREs can be found in various scientific publications, and a large number of promoters, enhancers, and regulatory sequences associated with these TREs are available at http: //www.ncbi.nlm It can be found in the GenBank database available on the Internet at .nih.gov / PubMed /. Thus, many related sequences are available for practicing the present invention and may not be described in detail herein.

  The TREs listed above are provided as non-limiting examples of TREs that function in the invention. Additional cell specific TREs are known in the art, such as methods for identifying and testing candidate TRE cell specificity. TRE may lack a silencer. The presence of a silencer (ie, a negative regulatory element) may help stop transcription (and thus replication) in non-permissive cells (ie, cells in a normal cellular state). Thus, the presence of the silencer may provide high inducibility or cell specific replication by more effectively preventing non-target cell oncolytic virus vector replication. Alternatively, silencer deficiency may help achieve replication in the target cell, thereby providing high cell type specific replication due to more effective replication in the target cell. TREs can contain multimers as well. For example, a TRE can include a tandem series of at least 2, at least 3, at least 4, or at least 5 TREs. These multimers may also contain heterologous promoter and / or enhancer sequences.

  In one exemplary embodiment, substantially the same two TREs control transcription of viral genes, such as adenovirus E1A and E1B genes. However, any of a number of combinations of genes are known to be used with these at least two TREs. In the adenovirus example, other preferred embodiments include those comprising substantially the same TREs that carry out the expression of E1A, E1B, and E4. Such a construct may further comprise a transgene that may be under the control of substantially the same TRE. Preparation of these and other aspects is provided below and in the examples.

  Transcriptional activation can be measured in a number of ways known in the art (and described in more detail below), but is under the control of TRE (ie, TRE operatively Generally measured by detection and / or quantification of the mRNA or protein product of the coding sequence (linked to). TRE activity can be measured as follows. A TRE polynucleotide sequence or a set of such sequences can be generated using methods known in the art, such as chemical synthesis, site-directed mutagenesis, PCR, and / or recombinant methods. it can. The sequences tested are: promoter (as long as the promoter element is not in TRE), and, without limitation, chloramphenicol acetyltransferase (CAT), β-galactosidase (encoded by the lacZ gene) , Luciferase (encoded by the luc gene), alkaline phosphatase, green fluorescent protein, and horseradish peroxidase, and can be inserted into a vector containing a suitable reporter gene encoding a reporter protein. Such vectors and assays are readily available from commercial sources, among others. The plasmid constructed in this way is controlled by the assumed TRE using transfection methods known in the art, such as calcium phosphate precipitation, electroporation, liposomes (lipofection), and DEAE dextran, for example. In order to test the expression of the reporter gene, it is transfected into a suitable host cell.

  After introduction of the TRE-reporter gene construct into the host cell under appropriate conditions, TRE activity is detected and / or detected in the reporter gene-derived mRNA or protein product, including the presence or absence of a suitable inducer or condition. Or it can be measured quantitatively. The reporter gene protein can be detected directly (eg, immunochemically) or through its enzymatic activity, if any, using an appropriate substrate. In general, TRE reporter gene constructs are introduced into various cell types to measure the cell-specific activity of TRE. The amount of TRE activity is measured in each cell type and compared to that of a reporter gene construct without TRE. A TRE is cell-specific when it functions selectively in a particular type of cell over different types of cells.

  In the present specification, “a cell that allows TRE to function” or a cell in which TRE function is “sufficiently preserved” or “functionally preserved”, or “cell in which TRE functions” , When operably linked to a promoter (if not included in the TRE), the TRE is at least 2-fold, preferably at least about 5-fold, preferably at least about 10-fold, more preferably at least about 20-fold, More preferably at least about 50 times, more preferably at least about 100 times, even more preferably at least about 200 times, even more preferably at least about 400-500 times, even more preferably at least about 1000 times the basal level of the target cell Will enhance transcription above. The basal level is generally the level of activity in non-target cells, if any, or the level of activity (if any) of the reporter construct that lacks the TRE of interest when tested in the target cell type. Methods for measuring expression levels (whether relative or absolute) are known in the art and are described herein. Similarly, when utilizing the regulatable oncolytic virus system of the present invention, an inducible transactivator in the presence of an effective amount of inducer or under induction conditions sufficient to induce gene expression. Is at least 2-fold, preferably at least about 5-fold, preferably at least about 10-fold, more preferably at least about 20-fold, more preferably when compared to expression of the same promoter and gene in the absence of inducer It enhances gene expression by at least about 50-fold, more preferably at least about 100-fold, even more preferably at least about 200-fold, even more preferably at least about 400-500-fold, even more preferably at least about 1000-fold.

  “Functionally conserved” variants of TRE, unlike other TREs, still conserve the ability to increase transcription of operably linked polynucleotides, especially with cell type-specific transcriptional activity TRE. TRE differences may arise due to linear sequence or conformational differences, such as single or multiple base mutations, additions, deletions, and / or modifications. Differences arise from changes in linkage between sugars and / or TRE bases as well.

  Certain point mutations within the TREs sequence have been shown to reduce transcription factor binding and gene activation. Those skilled in the art will recognize that some changes in the base in and near the transcription factor binding region are likely to negatively affect gene activation and cell specificity, while bases not involved in transcription factor binding. Recognize that changes in are unlikely to have such an impact. Certain mutations can increase TRE activity as well. Testing the effects of base changes can be done by any method known in the art, such as mobility shift assays, or transfection of vectors containing these changes in TRE functional and non-TRE functional cells, By in vitro or in vivo. Furthermore, those skilled in the art recognize that point mutations and deletions are made to TRE sequences without altering the ability of the sequences to regulate transcription.

Viral vectors capable of system replication (oncolysis) are provided for external control of oncolytic virus replication . The vector includes a cell type specific transcriptional regulatory element (CT-TRE) and an inducible transactivator-regulated transcriptional regulatory element. CT-TRE controls transcription of inducible transactivator (TA) coding sequences. Inducible transactivators (a) require an inducer or condition to function and (b) control transcription of viral genes essential for replication. Expression of viral genes essential for replication is regulated by CT-TRE, and transactivator-regulated transcriptional regulatory elements, and indirectly by inducer concentration or conditions.

  Depending on the specific use of the viral vector, the inducer or condition acts to suppress or enhance transcription. Transcription repressors provide a means of viral replication that is “stopped” by delivering an inducer to the host cell. Transcriptional activators provide a means to induce viral replication by the addition of inducers when the virus is otherwise inactive.

  All oncolytic viruses in which control of viral genes essential for replication is achieved using the methods of the present invention are included within the scope of the present invention. Typical replication-competent (oncolytic) viruses include, but are not limited to, adenovirus, vesicular stomatitis virus (VSV), herpes virus (eg, herpes simplex virus; HSV), reovirus, para Includes myxovirus, rhinovirus, Newcastle disease virus, poliovirus, West Nile virus, Coxsackie virus, measles virus, and vaccinia virus.

  In embodiments where two substantially the same TREs are used, the present invention does not require that the TREs are derived from the same gene. As long as the TRE sequences are substantially the same and are shown for the required function, TREs can be from different genes.

  In some embodiments, an oncolytic vector of the invention comprises a first viral gene under transactivator-regulated transcriptional regulation and at least one other under the control of another heterologous TRE that is different from the first TRE. Includes genes, such as viral genes or transgenes, where heterologous TREs function in the same cell but do not have the same polynucleotide sequence (ie, have different polynucleotide sequences). Preferably, at least two of the heterologous TREs within the oncolytic vector are cell specific or inducible to the same cell, inducer or condition. In one approach, the viral gene is one that promotes cell death, more preferably essential for viral replication. Preferably, at least one of the viral genes required for cell replication is an early gene and a gene under the regulation of heterologous TRE transcription is required for replication. By providing cell-specific transcription through the use of multiple heterologous TREs, the present invention provides an oncolytic vector that can achieve cell-specific cytotoxic effects by selective replication.

  The novel system of the present invention for the regulated expression of oncolytic viruses is exemplified herein by a regulatable adenoviral vector. In this exemplary embodiment, the gene that is regulated by the transactivator-regulated transcriptional regulatory element may be an early or late adenovirus gene and / or a transgene. By providing regulated transcription, one skilled in the art will provide an adenovirus that can be used as a vehicle for the introduction of the ability of a gene into a host target cell, unlike other non-target cell types. Can do. In addition to either genotype or expression type modifications due to the presence of adenovirus, the transgene serves to modify the genotype or expression type of the target cell. As representative of the oncolytic vector of the present invention with adenovirus capable of replication, adenovirus propagation can be used for its cytotoxic effect.

  It has been demonstrated that adenoviral vectors containing at least two different heterologous TREs are more stable than the previously described adenoviral vectors and provide high cell specificity for replication. See, for example, US Pat. No. 6,432,700. Therefore, an adenoviral vector was constructed in which each of the E1A and E1B genes is under the transcriptional regulation of two different heterologous TREs. However, it is understood that any of a number of combinations of genes can be used with any combination of at least two TREs.

  There are many different serotypes of adenovirus, such as Ad2, Ad5, Ad3, Ad35, and Ad40, which differ slightly or to a significant degree. In particular, adenovirus serotypes differ with respect to host cell tropism. For the purposes of the present invention, Ad5 is exemplified, but any or all of the adenovirus serotypes are included within the scope of the present invention.

  The adenoviral genes that are of interest to the invention are divided into two groups, early genes and late genes, the latter expression being controlled by the major late promoter. For early genes, E1A, E1B, E2, E3, and E4 are present. The E1A gene is expressed immediately after viral infection (0-2h) and before all other viral genes. The E1A protein acts as a trans-acting, positive-acting transcriptional regulator and is required for expression of other early viral genes and major late genes adjacent to the promoter. Regardless of the name, promoter flanking genes performed by the main late promoter were expressed during the early hours after Ad5 infection. In the absence of a functional E1A gene, the viral infection does not proceed because the gene product required for viral DNA replication is not produced.

  The E1B protein functions in trans and is required for transport of late mRNA from the nucleus to the cytoplasm. A defect in E1B expression results in poor expression of late viral proteins and the inability to block host cell protein synthesis.

There are a number of transcripts in the E4 gene. Open reading frame (ORF) 3 and ORF6 of the E4 transcription unit increase the accumulation of the major late transcription unit mRNA by binding the 55 kDa protein from E1B to the heterodimer of E2F-1 and DP-1 . In the absence of functional proteins from ORF3 and ORF6, plaques are produced with lower efficiency than that of 10 ⁶ wild-type viruses.

  The major late genes associated with the invention are, for example, the L1, L2, and L3 genes that encode the proteins of the virions of AD5 virus.

  The region of the adenovirus that is deleted, usually at least 500 nt, more usually at least about 1000 nt, AD5 genomic nucleotides, 300-3600 to E1, especially 342-3523; 27000-31000, especially 28133-30818, or 27865 Includes ~ 30995 in E3. The deletion is at least sufficient for insertion of the desired construct and allows packaging. In some embodiments, the E3 sequence is included in the regulatable replication competent adenovirus of the invention and is further described in US Patent Application No. 6,495,130. In other embodiments, an in-sequence ribosome entry region (IRES) is included in the regulatable adenovirus of the invention as regulatable as further described in US patent application Ser. No. 09 / 814,351 published as 20030148520. be written.

  As illustrated by utilizing adenoviral vectors with cell-specific response elements including promoter and enhancer constructs that are specific for prostate cells, the ability of various genes is introduced into prostate cells and Expresses a specific antigen. Of particular interest is the opportunity to introduce cytotoxic effects controlled by a particularly active transcription initiation region in prostate cells. Other cell types with specific active transcription factors that are involved in conditions where modulation is desired include leukocytes, especially lymphocytes, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, neuronal cells, and keratinocytes . Since adenoviruses generally result in transient expression (about 6-8 weeks), one skilled in the art will provide transient capacity to cells, for example, in situations where the desired result only requires a limited reaction period. Can do. In other cases, different oncolytic vectors are preferred depending on the time and level of expression desired.

  In order to further increase the specificity of the control mediated by the inducer, it is likewise desirable to control the expression of two viral genes, namely the expression of the E1A and E1B adenoviral genes. In this manner, specificity can be increased, i.e., oncolytic virus replication in non-target cells where CT-TRE is not active can be further reduced. Both E1A and E1B are controlled by the inducer-responsive promoter element illustrated in FIG. 1, which describes a prostate-specific oncolytic virus vector with tetracycline-regulated replication control, where the tetracycline-responsive element The promoter (TRE) provides E1A gene expression, and the prostate specific antigen (PSA) promoter provides reverse tet-responsive transactivator (rtTA) expression.

  In this approach, the expression of the E1A and E1B genes is linked by an IRES between the E1A and E1B genes. In this viral construct, the in-sequence E1B promoter element is removed and replaced with an IRES element. Thus, both E1A and E1B expression is under the control of an inducible factor responsive promoter element. The 2A peptide sequence from the foot-and-mouth disease virus (FMDV) as an IRES variant has been shown to provide efficient bicistronic expression of both E1A and the transgene (Purler S et al., Gene Ther. 2001 Jun ; 8 (11): 864-73) could be used instead of the IRES sequence.

  Preferred adenoviral embodiments include those comprising at least two different heterologous TREs that carry out the expression of E1A, E1B, and E4. Such a construct may further comprise a transgene that may be under the control of the TRE, where the TRE may be CT-TRE. See, for example, US Pat. Nos. 6,436,394 and 6,432,700.

  Therefore, the present invention provides an oncolytic virus comprising a viral gene under transcriptional control of a transactivator-regulated transcriptional regulatory element and an inducible transactivator under transcriptional control of TRE, preferably CT-TRE. Provide vector. In some embodiments, the first transactivator-regulated transcriptional regulatory element controls expression of the first viral gene and the second transactivator-regulated transcriptional regulatory element regulates expression of the second viral gene. Adjusted. The genes controlled under these TREs are preferably viral genes essential for growth. Alternatively, the genes regulated under these TREs may be the first viral genes essential for growth, where the second gene is a transgene. It will be appreciated that there may be additional TREs in the viral vector, and these additional TREs may be substantially identical to the first and / or second TREs. The present invention includes the use of 3 or more, 4 or more TREs.

The use of a viral vector that is capable in a transgene- specific target cell allows growth of the virus in the target cell resulting in the death of that host cell, and of the virus against other host cells. To further enhance the efficacy of the treatment, the oncolytic vector of the present invention may contain one or more transgenes that provide a therapeutic effect.

  Thus, the viral vectors of the present invention can further comprise a heterologous polynucleotide (transgene) encoding a therapeutic gene product under the control of a transactivator-regulated transcriptional regulatory element. Alternatively, a viral vector may contain a heterologous transgene that encodes a therapeutic gene product under the control of a constitutive or inducible promoter. Numerous examples of constitutive or inducible promoters are known in the art and are routinely utilized for transgene expression in the context of viral vectors. In this way, the ability of various genes can be introduced into target cells. For example, in certain instances, it may be desirable to increase the degree of therapeutic efficacy by increasing cytotoxic activity. This allows cell-specific replicative cytotoxic activity so that cells can metabolize 5-fluorocytosine (5-FC) to the chemotherapeutic drugs 5-fluorouracil (5-FU) and carboxypeptidase G2 (CPG2). In combination with the expression of one or more metabolic enzymes such as HSV-tk, nitroreductase, cytochrome P450 or cytosine deaminase (cd). This type of transgene may be used to give a bystander effect as well.

  Additional transgenes that may be introduced into the viral vectors of the present invention have other potential but essential proteins for growth, such as structural proteins, transcription factors, polymerases, etc. Artificial cytoplasmic properties of viruses or other pathogenic proteins, cytotoxic proteins such as diphtheria chains, lysine, abrin, etc., nucleases (eg RNaseA) or proteases (eg trypsin, papain, proteinase K, carboxypeptidase etc.) Genes encoding mutants, chemokines such as MCP3 alpha or MIP-1, pore-forming proteins from viruses, bacteria, or mammalian cells, fusgenic genes, chemotherapy sensitizing genes and radiosensitizing genes Directed to the encoding mRNA Includes factors, antisense, or ribozymes that can initiate apoptosis. Other genes of interest include cytokines, antigens, transmembrane proteins, such as IL-1, -2, -6, -12, GM-CSF, G-CSF, M-CSF, IFN-α, -β,- γ, TNF-α, -β, TGF-α, -β, NGF, MDA-7 (melanoma differentiation-related gene-7, mda-7 / interleukin-24) and the like. Further examples are pro-apoptotic genes such as Fas, Bax, caspases, TRAIL, Fas ligand; fusion genes that can result in or facilitate cell fusion, such as V22, VSV, etc .; tumor suppressor genes such as p53, RB, p16, p17, W9, etc .; including genes involved in the cell cycle and genes encoding anti-angiogenic proteins such as endostatin, angiostatin, etc.

  Other opportunities for modification of specific genes include T cells, such as tumor infiltrating lymphocytes (TILs), which promote proliferation, increase cytotoxicity, reduce response to growth inhibitors, lymphokine It can be modified to enhance expression or the like. Those skilled in the art may also wish to increase target cell susceptibility by preparing for the expression of certain surface membrane proteins, such as B7, SV40T antigen variants, and the like.

  In some embodiments, the adenovirus death protein (ADP) encoded within the E3 region is maintained (ie, included) in an adenovirus vector. The ADP gene under the control of the major late promoter (MLP) appears to encode a protein (ADP) that is important for the rapid processing of host cell lysis. Tollefson et al., (1996) J. Virol. 70 (4), 2296; Tollefson et al., (1992) J. Virol. 66 (6): 3633. Thus, adenoviral vectors containing ADP genes have made adenoviral vectors more powerful and have allowed for more effective treatment and / or lower dosage requirements.

  As a result, in one embodiment, the present invention provides an adenoviral vector in which an adenoviral gene is under transcriptional regulation of a first transactivator-regulated transcriptional regulatory element, and a second transactivator-regulated transcriptional regulatory element. A polynucleotide sequence encoding ADP that is under the control of, and preferably an adenoviral gene is essential for replication. The DNA sequence encoding ADP and the amino acid sequence of ADP are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively. In short, the ADP coding sequence is preferably obtained from Ad2 using techniques known in the art such as PCR (since this is a more fully characterized strain of ADP). Preferably, a Y leader (which is an important sequence for the correct expression of late genes) is similarly obtained and linked to the ADP coding sequence. The ADP coding sequence (which may involve a Y leader) can then be introduced into the adenovirus genome, eg, the E3 region (where the ADP coding sequence is driven by MLP). The ADP coding sequence could also be inserted at other locations in the adenovirus genome, such as the E4 region. Alternatively, the ADP coding sequence could be operably linked to different types of TREs including but not limited to other viral TREs.

  It is understood that the present invention does not exclude oncolytic vectors containing additional genes under the control of transactivator-regulated transcriptional regulatory elements. Therefore, the present invention provides a viral vector comprising a third gene that is under the transcriptional control of a third TRE. The third TRE may be substantially identical to the first and / or second TA-TREs, with all three TREs functional in the same cell. Preferably, the third gene is involved in cytotoxicity (whether directly and / or indirectly), more preferably is involved in and / or enhances cell death.

Delivery of oncolytic vectors into cells Oncolytic vectors include, but are not limited to, naked polynucleotide (usually DNA) constructs; polynucleotide constructs complexed with agents that facilitate entry into cells, such as Cationic liposomes or other compounds such as polylysine; packaged within infectious adenoviral particles (which may make it more immunogenic than adenoviral vectors); other particulate viruses such as HSV or AAV, Packaged in the form of: complexed with an agent to enhance or weaken the immune response; an agent that facilitates in vivo transfection, such as DOTMA ™, DOTAP ™, and It can be used in various forms including polyamines and complexes formed with them.

  If the oncolytic vector is packaged into a virus, the virus itself can be chosen to further facilitate targeting. For example, for adenoviral vectors, adenoviral fibers mediate initial contact with cellular receptors that helped direct. See, for example, Arnberg et al. (1997) Virol. 227: 239-244. If a particular subgenus of the adenovirus serotype is directed against the target cell type and / or exhibits a low affinity for the target cell type, such subgenus (or subgeneras) are cytotoxic and / or It could be used to further increase the cell specificity of cell lysis. Adenovirus fibers, hexons, or other surface proteins can be modified to increase the specificity of uptake by target cells.

  The modified oncolytic vector can be delivered to the target cell by a variety of methods depending on whether the cell is in culture, ex vivo or in vivo. In situations where in vivo delivery is desired, delivery can be accomplished in a variety of ways using liposomes, direct injection, subcutaneous injection, intramuscular injection, catheters, intravenous inhalation, topical application, intravenous infusion, etc. Can do. Due to the high efficiency of transfection of various oncolytic vectors, one skilled in the art can achieve a large number of modified cells. In the case of neoplasms where toxicity occurs, they can be released locally to affect cells that may not have been transfected with toxicity. In this manner, one skilled in the art can specifically remove neoplastic cells without significant impact on normal cells. Furthermore, the expression of viral proteins plays a role in activating the immune system against target cells. Finally, propagation of a viral vector capable of replication in the host cell results in cell death. The means of delivery will depend primarily on the particular vector (including morphology) and the type and location of the target cell (ie, whether the cell is in vitro or in vivo).

In the example of a packaged virus, such as an adenovirus, the adenovirus can be administered in a suitable physiologically acceptable carrier at a dose of about 10 ⁴ to 10 ¹¹ . The multiplicity of infection is generally in the range of about 0.001-100. Depending on the host's immune response capabilities, the virus may be administered one or more times. If necessary, the immune response can be reduced by utilizing various immunosuppressive agents or treatments to reduce circulating antibody levels to allow repeated administration without a strong immune response.

  If administered as a polynucleotide construct (ie, not packaged as a virus), about 0.01 microgram to 1000 micrograms of viral vector can be administered. Oncolytic vectors can be administered one or more times or as multiple simultaneous injections. Of the replication competent viruses utilized, such as herpes simplex virus (HSV), reovirus, vesicular stomatitis virus (VSV), Newcastle disease virus, vaccinia virus, West Nile virus, Coxsackie virus, poliovirus, and measles virus Depending on the type, the amount of virus administered can be determined based on standard knowledge about that particular virus (eg, readily available from published literature) and can be determined empirically. it can.

  In some embodiments, the packaged viral vector forms a complex with the hydrophilic polymer to produce a masked virus. The hydrophilic polymer binds (covalently or non-covalently) to the viral capsid protein, in the case of adenovirus, in particular to hexon and fiber proteins. In a preferred embodiment, the viral vector of the invention yields a masked viral vector by forming a complex with the masking agent. (See, eg, US Patent Application No. 10 / 139,089, published as 20030152553). In the adenovirus vector embodiment of the invention, the masked virus is (a) masking the adenovirus surface against circulating adenovirus neutralizing antibodies or opsonins, and (b) non-both by the body (ie macrophages, etc.). Advantageously, the systemic circulation time of adenovirus particles is increased by reducing the specific clearance mechanism. In vivo, systemic delivery of masked virus is less immunogenic and results in a longer circulation of increased biodistributed virus particles due to decreased clearance by the liver and spleen.

Host cells and target cells The present invention provides host cells and target cells that also include (ie, have been transformed by) the viral vectors described herein. A host cell includes both prokaryotic and eukaryotic host cells, as long as there are sequences necessary for the maintenance of the host, eg, an appropriate origin of replication. For convenience, selectable markers are provided as well. Prokaryotic hosts include bacterial cells such as E. coli and mycobacteria. Eukaryotic host cells are yeast, insect, avian, amphibian, plant and mammalian host cells. Numerous host cells are known in the art and need not be described in detail herein.

  Suitable target cells for the viral vectors of the invention are all eukaryotic cell types that allow the function of TREs and transactivator-regulated transcriptional regulatory elements, preferably mammals, more preferably humans, even more preferably Includes neoplastic cells. Suitable target cells also include all cells that produce proteins and other factors necessary for the expression of genes under the control of TREs, such factors necessary for the expression being produced naturally or recombinantly. The For example, if the TRE (s) used is prostate cell specific, the cell is preferably a prostate cell. Depending on whether the promoter used is androgen-inducible, the prostate used by the cells may be producing the androgen receptor. If an androgen-inducible promoter is used, an androgen receptor-producing cell, such as HLF, HLE, and 3T3, as well as non-androgen receptor-producing cells can be used if an androgen receptor-encoding expression vector is introduced into the cell along with the adenovirus. AR-producing prostate cancer cells, PC3 and DU145 can be used. If the oncolytic vector contains a TRE from the AFP gene, suitable host cells include all cell types that produce AFP, such as, but not limited to, Hep3B, HepG2, HuH7, HuH1 / C12. A given TRE activity of a given cell can be assessed by measuring the expression level of a reporter gene linked so that it can be operated using standard assays. Comparison of expression between cells suspected of functioning TRE and control cells indicates the presence or absence of increased transcription.

  Comparisons between or within various TREs can be assessed by measuring and comparing expression levels within a single target cell line. The absolute transcriptional activity of TRE is known to depend on several factors, such as the nature of the target cell, the mode of delivery, and the form of TRE, and the coding sequence that selectively activates transcription . In order to correct the size of the various plasmids used, the activity can be expressed as relative activity per mole of transfected plasmid. Alternatively, transcription levels (ie, mRNA) can be measured by standard Northern assays and hybridization techniques. The level of transfection (ie, transfection efficiency) is measured by co-transfecting plasmids encoding different reporter genes under the control of different TREs, such as the CMV immediate early promoter. This assay can similarly indicate negative regulatory regions, ie silencers.

Compositions The invention also includes compositions, including pharmaceutical compositions comprising the viral vectors described herein. Such compositions are useful for in vivo administration, for example when measuring the degree of transduction and / or cell killing effectiveness within an individual. Preferably, these compositions further comprise a pharmaceutically acceptable excipient. These compositions comprising an effective amount of a viral vector of the invention in a pharmaceutically acceptable excipient may be in unit dosage form, sterile injectable solution or suspension, sterile non-injectable solution orally Suitable for systemic administration to individuals such as solutions or suspensions, oil-in-water or water-in-oil emulsions. Formulations for parenteral and non-parenteral drug delivery are known in the art and described in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing (1990). Compositions also include lyophilized and / or reduced forms of viral vectors (including those packaged as viruses such as adenovirus) of the invention.

  Other compositions are used to aid in the detection methods described herein. For these compositions, the viral vector is usually suspended in a solution such as a suitable solvent or buffer system. Such solvent systems are well known in the art.

Kit The present invention also includes a kit comprising the oncolytic vector of the present invention. These kits can be used for diagnostic and / or monitoring purposes, preferably in monitoring. Procedures using these kits can be performed by a medical laboratory, laboratory, practitioner, or private individual. The kit included in the present invention makes it possible to detect the presence of target cells in a suitable biological sample, such as a biopsy specimen.

  The kit of the invention comprises the oncolytic vector described herein in a suitable package. The kit contains additional components useful for the procedure including, but not limited to, buffers, developer reagents, labels, reacting surfaces, detection means, control samples, instructions, and explanatory information. May offer.

Preparation of the viral vector of the present invention The viral vector of the present invention can be prepared using recombinant techniques that are standard in the art. In general, TREs are inserted into the adenovirus and transactivator genes of interest, preferably one or more early genes (or late genes can be used). TREs may be prepared using oligonucleotide synthesis or recombinant methods (eg, PCR and / or restriction enzymes) if the sequence is known. Restriction regions that are convenient within the native DNA sequence or induced by methods such as PCR or site-directed mutagenesis also provide insertion regions for TREs. Thus, convenient restriction regions for annealing (ie, inserting) TREs can be engineered into the 5 ′ and 3 ′ ends of TRE using standard recombination methods such as PCR.

  The polynucleotides used to make the oncolytic viral vectors of the invention can be obtained using standard methods in the art, such as chemically synthesized recombinant methods, and / or obtained from biological sources. It is done.

  In the case of adenovirus, the vector provides for one plasmid to provide the right hand region of adenovirus and the other plasmid to provide the left hand region, and the two plasmids to be at least about 500 nt of homologous recombination. It can generally be prepared by utilizing two plasmids that share the middle region of In this way, each plasmid is followed by a suitable transcription factor for co-transfection in a competent host providing gene complement as needed or for initiation of transcription from the PSE for adenovirus propagation. Can be operated independently as desired.

  For convenience, plasmids are commercially available that provide the necessary parts of adenovirus. Plasmid pXC.1 (McKinnon (1982) Gene 19: 33-42) contains the wild type left hand end of Ad5. pBHG10 has a deletion of E3 and provides the right hand end of Ad5. The deletion of E3 provides room for the virus to insert a 2 kb minimal PSE without the deletion of the wild type enhancer promoter. The gene for E3 is located on the opposite strand from E4 (r chain).

  For early gene manipulation, the transcription start site of Ad5E1A is nt560, and the ATG start site of the E1A protein is nt610 of the viral genome. This region can be used for the insertion of cell specific elements such as PSE. Conveniently, restriction sites are introduced by using the polymerase chain reaction (PCR) and the primers utilized are limited to the Ad5 genome or comprise a portion of a plasmid carrying Ad5 genomic DNA. For example, if pBR322 is the backbone, the primer may use the EcoRI site of the pBR322 backbone and the Xpal site of nt1339 of Ad5. By performing a two-step PCR where the overlapping primer in the center of the region introduces a sequence change that results in a unique restriction site, one skilled in the art can provide for insertion of a cell-specific response element at that site.

  Similar strategies are similarly used for the insertion of cell-specific response elements that regulate E1B. The Ad5 E1B promoter consists of a single high affinity recognition region for the Spl and TATA boxes. This region ranges from 1636 to 1701 nt. By inserting a cell-specific response element into this region, one skilled in the art can provide cell-specific transcription of the E1B gene. By using the left hand region modified by a cell-specific response element that regulates E1A as a template to introduce a cell-specific response element to regulate E1B, the resulting adenovirus can be expressed as E1A and E1B. Will depend on cell-specific transcription factors to express both.

  For example, we inserted a 12 bp 5 'AgeI site into the E1A start codon by oligo-directed mutagenesis and ligated PCR (Ad5 nucleotide 547). In addition, an EagI region was created upstream of the E1B start region by inserting a G residue at 1682 nt of Ad5 by oligonucleotide directed mutagenesis. To simplify the insertion of TRE into the EagI region, the in-sequence EagI region of CN95 was removed by cleavage with EagI, treated with mung bean nuclease, and religated to the CN114 construct. In this way, we have created adenoviral vectors that contain the unique AgeI and EagI regions within the proximal upstream region in E1A and E1B, respectively. Using these unique regions, one skilled in the art can insert a TRE that has engineered the AgeI or EagI region, thereby simplifying the recombinant adenoviral vector construct. Thus, the present invention includes adenoviral vectors comprising a unique AgeI site 5 'of the E1A initiation codon and a 5' unique EagI site of E1B.

  Similarly, TRE can be inserted upstream of the E2 gene. The E2 early promoter mapped within Ad5 from 27050-27150 consists of extensive and narrow transcription initiation sites, the latter consisting of E2 transcripts, two non-standard TATA boxes, two E2F transcription factor binding sites, and ATF transcription Approximately 5% of the factor binding site (see Swaminathan et al., Curr. Topics in Microbiol. And Immunol. (1995) 199 part 3: 177-194 for a detailed study of the E2 promoter architecture) Occupy.

  The E2 late promoter overlaps with the coding sequence of the gene encoded by the counter strand and is therefore less susceptible to genetic manipulation. However, the E2 early promoter overlaps with the sequence encoding the 33 kD protein of the counter strand only at a few base pairs. In particular, the SpeI restriction region (position 27082 of Ad5) is part of the stop codon of the 33 kD protein described above, and the extensive E2 early transcription initiation region and TATA binding protein region, upstream transcription factor binding site, E2F And conveniently separated from ATF. Thus, insertion of a TRE with a SpeI end at the SpeI site in the plus strand should disrupt the intrasequence E2 early promoter of Ad5, allowing TRE-regulated expression of the E2 transcript.

  For E4, those skilled in the art must use the right hand portion of the adenovirus genome. The E4 transcription initiation region is primarily nt35609, the TATA box is nt35638, and the first AUG / CUG of ORF1 is nt35532. irtanen et al. (1984) J. Virol. 51: 822-831. Using any of the strategies described above for other genes, a heterologous TRE may be introduced upstream of the transcription start site. For mutant constructs of the E4 region, co-transfection and homologous recombination are performed in W162 cells that provide E4 proteins to complement defects in the synthesis of these proteins (Weinberg et al. (1983) Proc. Natl. Acad. Sci. USA 80: 5383-5386).

  Methods for packaging adenoviral polynucleotides into adenoviral particles are known in the art and described in the examples.

Methods of Use of the Oncolytic Vectors of the Invention The oncolytic vectors can be used for a wide variety of purposes that vary depending on the desired or intended results. Accordingly, the present invention includes methods using the oncolytic viral vectors described above. In one embodiment, the method of using an oncolytic vector comprises introducing the vector into a cell, preferably into a eukaryotic cell, more preferably into a mammalian cell, in vitro or in vivo. In one preferred embodiment, the oncolytic vector of the invention is administered in vivo for the treatment of cancer.

  The purpose of introducing transient expression is for unwanted growth apart from tumors such as psoriatic lesions, restenosis, wound healing, tissue regeneration, high immune response, resistance to infection, factor production, high growth Presence or absence of transgene-introduced virus by comparing cellular activity before, during, and after modification with viral, etc., investigation of metabolic or other physiological pathways, modification with viruses, etc. Comparison of cellular activity in The vector can be used to remove a cell mixture of a particular cell group, which cell group is a target cell. By having oncolytic viruses that are selectively proliferative in the target cells, only those cells are killed by the growth of the oncolytic virus. By combining the virus with a cell mixture, for example in culture or in vivo, oncolytic viruses can only grow in target cells and are regulated by the presence of inducers. Thus, cells other than the target cell are not affected by the oncolytic virus, while the target cell is killed. Growth of oncolytic virus by propagation in the target cells ensures that the mixture is substantially cleared of the target cells. Once the target cells are destroyed, the oncolytic virus can no longer grow, but is maintained in culture to constantly monitor the mixture for recurrence of the target cells, such as mutant or neoplastic cells. The presence or concentration of inducers and / or conditions provides further control over viral replication.

  By identifying genes that are specifically expressed by the target host cell, based on the nature of the cells, their maturity level, or their conditions, the target cell-specific response element can determine the ability of the gene to such cells. The ability of that gene is not present in other cells, even if transfected with an oncolytic virus.

  In one embodiment, a method of using an oncolytic virus vector comprises introducing an oncolytic virus vector into a target cell, preferably a neoplastic cell. In other embodiments, a method of using an oncolytic viral vector comprises introducing an oncolytic viral vector into prostate cells. In other embodiments, a method of using an oncolytic viral vector comprises introducing an oncolytic viral vector into liver cells. In other embodiments, a method of using an oncolytic viral vector comprises introducing an oncolytic viral vector into breast cancer cells. In other embodiments, the method of using an oncolytic viral vector comprises introducing an oncolytic viral vector into colon cancer cells.

In one embodiment, the method comprises contacting a cell with an oncolytic viral vector described herein such that the oncolytic viral vector enters, ie, transforms the cell, and the function of TRE Providing cell-selective cytotoxicity in cells that permissive. Cytotoxicity can be measured using standard assays in the art, such as dye exclusion, ³ H thymidine incorporation, and / or lysis.

  In other embodiments, the method comprises tumor lysis specific for cells that are cell type specific and that permit the function of a transactivator-regulated transcriptional regulatory element, preferably eukaryotic cells, more preferably mammalian cells. Provides virus growth. These methods involve combining an oncolytic virus vector with mammalian cells that permit the function of TREs, whereby the oncolytic virus is propagated.

  Another embodiment provides a method of killing cells that allow TRE to function (ie, target cells), comprising combining a cell mixture with an oncolytic virus vector of the invention. The cell mixture is generally a mixture of cancer cells in which TREs are functional and normal cells, and may be an in vivo mixture or an in vitro mixture.

  The invention also includes methods for detecting cells in a biological sample that function as CT-TRE and / or transactivator-regulated transcriptional regulatory elements. These methods are particularly useful for monitoring the clinical and / or physiological symptoms of an individual (ie, a mammal) regardless of the experimental or clinical environment. For these methods, cells of a biological sample are contacted with an oncolytic virus vector to detect oncolytic virus vector replication. Suitable biological samples are those that may or may be suspected of having target cells present. In general, in mammals, suitable medical samples are those suspected of having target cancer cells. Such cells can be obtained, for example, by needle biopsy or other surgical procedure. The contacted cells are treated, eg, selective enrichment and / or solubilization, to create assay conditions. In these methods, target cells are detected using in vitro assays that detect proliferation, which are standard in the art. Examples of such standard assays include, but are not limited to, burst assays (which measure virus yield) and plaque assays (which measure infectious particles per cell). Proliferation can also be detected by measuring specific oncolytic virus DNA replication, which is also a standard assay.

  The present invention is similar to the method of modifying the genotype of a target cell comprising contacting the target cell with an oncolytic virus vector as described herein, wherein the oncolytic virus vector enters the cell. To provide.

The present invention comprises contacting a tumor cell with the oncolytic virus vector of the present invention such that the oncolytic virus vector enters the tumor cell and exhibits selective cytotoxicity against the tumor cell. Further provided is a method for inhibiting the growth of tumor cells. Tumor cell growth is not limited to this: measuring tumor size, measuring whether tumor cells are proliferating using a ³ H thymidine incorporation assay, or counting tumor cells. And can be evaluated by any means known in the art. “Suppressing” tumor cell growth means part or all of the following conditions: slowing, slowing and stopping tumor growth, and tumor shrinkage. “Inhibiting” tumor growth indicates a suppressed growth state when compared to growth without contact, ie, transfection with, the oncolytic viral vectors described herein. .

  The present invention also provides a method for reducing the level of an individual's tumor cell marker comprising providing the individual with an oncolytic viral vector of the present invention, wherein said oncolytic viral vector comprises a tumor cell It is selectively cytotoxic in cells producing the marker. Tumor cell markers include, but are not limited to, PSA, CEA, and hK2. Methods for measuring levels of tumor cell markers are known to those of skill in the art and are not limited to immunological assays, such as enzyme-linked immunosorbent assays using antibodies specific for tumor cell markers. Including the law (ELISA). In general, a biological sample is obtained from the individual to be tested and a suitable assay, such as an ELISA, is performed on the biological sample.

The present invention also provides a therapeutic method of administering to an individual an effective amount of an oncolytic virus vector described herein. For example, treatment with an oncolytic viral vector wherein at least one cell type specific TRE is specific for prostate cells (eg, PSE-TRE, PB-TRE, and / or hKLK2-JRE) is described above. It is indicated for individuals with diseases related to the prostate such as hyperplasia and cancer. In this example, it is equally indicated for individuals deemed to be at high risk for prostate-related disease, such as those who had an excised disease and those who had a family history of prostate-related disease. An assay for the validity of the administration of the oncolytic virus vector of the present invention relies, inter alia, on assessable clinical parameters, such as serological indicators, and histological examination of tissue biopsies. In general, a pharmaceutical composition comprising an oncolytic virus vector is administered. The pharmaceutical composition has been described previously.
The following examples are provided as a means of illustration and not as a means of limitation.

Example 1
Prostate-specific oncolytic adenoviruses whose replication is regulated using a Tet-On system that controls the expression of at least one adenoviral gene specific for prostate tissue-specific tetracycline-regulated oncolytic virus replication is provided. In the case of a Tet-On regulated gene expression system that controls oncolytic viral replication, the expression of the early adenovirus E1A gene region is due to deletion of the adenovirus promoter element in the sequence and insertion of a tetracycline responsive element. Placed under the control of a chimeric promoter element, a tetracycline responsive element (Figure 1). For the production of a modified reverse tetracycline transactivator (rtTA (2) s-M2) that binds to and induces expression in the presence of tetracycline and its derivatives (eg doxycycline) The expression cassette is placed in the E3 region of the virus under the control of the tissue / tumor specific promoter (CT-TRE) exemplified herein by the human prostate specific antigen (PSA) promoter.

  In this manner, expression of rtTA transactivator (TA-TRE) is restricted to tissues or tumor tissues where the PSA promoter is active, such as prostate-derived tissue. With the addition of tetracycline (or a derivative thereof), the rtTA transactivator binds to the tetracycline responsive element and switches on E1A transcription and thereby expression. From the expression of the E1A protein, the adenovirus replication process continues, resulting in the final death of the host cell and the release of further adenovirus progeny. In contrast, in the absence of inducer, or in viruses that have entered non-target cells, rtTA cannot bind to the tetracycline responsive element, the E1A protein is not expressed, and viral replication does not continue. The E1A gene region is described in this example, but any essential ORF of adenovirus can be regulated in this manner, i.e., the tetracycline responsive element contains an E1a, E1b, E2, or E4 region. Can be controlled. In addition, multiple ORFs may be placed under tetracycline regulatory control, ie E1 and E4, E1 and E2, to increase control of the system.

1A. Cloning and characterization of a PSE-driven expression minimal prostate specific enhancer (PSE) of E1A by Ad5 is described in Schuur et al., 1996. Plasmid CN71 contains minimal PSE (-5322 to 3875 bp related to the transcription start site of the PSA gene) and -532 to +11 of the PSA promoter. CN71 was cut with HindIII, which removes the XhoI / PSA promoter. A short promoter from -230 to +7 was amplified by PCR. The PCR product was cut with XhoI / HindIII, and XhoI / HindIII cut CN71 was returned and ligated to produce CN105. Plasmid pUHrt62-1 (2) (a generous gift from W. Hillen) was cut with EcoRI, site blunted with Klenow fragment, and cut with XhoI to remove the CMV promoter from the construct. The PSA promoter fragment from the above is then placed 5 ′ of the rtTA2 (s) -M2 transgene (within pUHrt62-1 (2)) instead of CMV, and pUH-PSA-rt62-1 (2) was produced. The PSA-rtTA2 (s) -M2 fragment (incorporating SV40 late polyA) was then released from pUH-PSA-rt62-1 (2) using XhoI / HindIII digestion and pABS4 (Microbix , Toronto) was ligated / inserted into pABS4-PSA-rtTA, a shuttle plasmid containing the kanamycin resistance gene. By digesting pABS4-PSA-rtTA with PacI, the fragment containing the Kan ^R gene and the PSA-rtTA2 (s) -M2 expression cassette were isolated and similarly cut, processed in the E3 region of Ad5 BHG11-PSA-rtTA-Kan ^R was prepared by ligation into BHG11 (Microbix) containing a unique PacI region. BHG11-PSA-rtTA-Kan ^R was digested with SwaI and the Kan ^R gene was removed by ligating the vector (BHG11-PSA-rtTA).

1B. Attenuated Ad5 with a tetracycline-responsive promoter that drives E1A and carries the in-sequence Ad5 E1A promoter and enhancer
In the absence of a functional E1A gene, the viral infection does not proceed because the gene product required for viral DNA replication is not produced (Nevins (1989) Adv. Virus Res. 31: 35-81). The transcription start site of Ad5E1A is nt560, and the ATG start site of the E1A protein is nt610 of the viral genome.

  pXC.1 was purchased from Microbix Biosystems (Tronto). pXC.1 contains 5 sequences of adenovirus from bp 22-5790. The AgeI region was introduced at 5 ′ 12 bp into the E1A start codon (Ad5, 547 nucleotides) by oligo site-directed mutagenesis and ligated PCR. Plasmid pXC.1 was PCR amplified using primers containing extra A to introduce the AgeI region. This created a segment from the EcoRI region of the pBR322 skeleton to Ad5 nt560. The second segment of pXC.1 from Ad nucleotide 541 to the XbaI site of Ad nucleotide 1339 was amplified using a primer containing extra T to introduce an AgeI site. A mixture of these two PCR amplified DNA segments was mixed and amplified with primers 3 and 4 to create a DNA segment from the EcoRI site to the XbaI site of pXC.1. This DNA segment includes the leftmost 1317 bases of the adenovirus sequence and contains the AgeI site of Ad nucleotide 547. This DNA segment was used to replace the corresponding segment of pXC.1, creating CN95. Similarly, a tetracycline responsive element with AgeI ends was PCR amplified from plasmid pTRE2 (purchased from BD Biosciences Clontech) to generate pXC-TRE-E1a.

1C. Virus-CG1974 produced by homologous recombination
An Ad5 recombinant virus containing a PSA promoter driving the rtTA (2) s-M2 gene in the E3 region and a tetracycline responsive element driving E1a in the E1 region was constructed as described above. Viruses were produced by homologous recombination with 293 cells of low passage in a human kidney cell line expressing Ad E1A and E1B proteins. This was achieved by co-transfection of pXC-TRE-E1a and BHG11-PSA-rtTA. The genomic integrity of the resulting recombinant virus construct was confirmed using HindIII digestion and designated CG1974 (FIG. 1).

Example 2
In a further example, an oncolytic vector is “armed” with a therapeutic transgene to enhance efficacy, such as granulocyte macrophage colony stimulating factor (GMCSF) or thymidine kinase (TK). The expression of these therapeutic transgenes is placed under the control of a regulatory gene expression system. In this manner, the inducer used within the regulatory gene expression system switches on both viral replication and therapeutic gene expression simultaneously. An example of such a vector is given in FIG. 2, which illustrates a prostate-specific oncolytic virus vector armed with GMCSF with tetracycline-regulated replication and expression control. In this example, a tetracycline responsive element (TRE) drives E1 and GMCSF expression. IRES allows for two coding sequences to be expressed from one promoter, and the prostate specific antigen (PSA) promoter drives the expression of reverse tet-responsive transactivator (rtTA). Here, expression of both E1 and GMCSF, which are switched on by the addition of Tet to the system, makes the vector function as described above. In this example, an in-sequence ribosome entry site (IRES) or 2A sequence can express two coding regions using one promoter.

Example 3
A number of different tetracycline-regulated gene control systems for use in the manner described above. For example, the tetracycline-regulated transcription silencer (tTS) system (Freundlieb et al 1999) in combination with the previously described Tet-On system is used to more closely control gene expression (for use in bladder cancer) (See FIG. 3 illustrating an oncolytic viral vector with dual tetracycline-regulated replication control). In this example, the tetracycline responsive element (TRE) drives E1 gene expression and the human uroplakin II (hUPII) promoter is a reverse tet-responsive transactivator (rtTA) and a tet-regulated transcription silencer (tTS) Drive expression. In addition to the rtTA transactivator, the use of tTS repressor molecules reduces the “off” state basal or “leaky” gene expression and increases the degree of gene regulation. For example, the system of FIG. 3 expresses both a Tet-On transactivator and a tTs repressor from the human uroplakin II promoter using an in-sequence ribosome entry site (IRES). Expression of rtTA and tTS in this manner is specific to bladder tissue cell types. The expression of the essential E1 gene is again under TRE control. In the absence of Tet, tTS molecules bind to TRE elements and actively suppress E1 gene expression. Since adenoviruses have multiple transcriptional enhancer elements present in their genome, this active repression by tTS molecules is required. Despite the absence of a promoter in its sequence, it can cause “leaky” expression of the E1 promoter leading to a limited amount of virus formation in the absence of the inducer. With the addition of tetracycline, structural changes in the tTS molecule prevent binding to TRE and active repression of expression from TRE. The rtTA and Tet-On transactivator can induce the expression of E1 by the ability to bind to TRE obtained so that replication proceeds.

  An alternative system is shown in FIG. 4, which represents an example of an oncolytic viral vector with rapamycin (ARIAD system) regulated replication control for use in pan-cancer applications. This system includes a chimeric promoter that contains 8 copies of the recognition site upstream of ZFHD1 of the minimal IL-2 promoter that drives E1 gene expression, and an activation domain (in the activation region of the p65 subunit of NF-KB). The human E2F promoter (E2F), which drives the expression of the fused FRAP rapamycin binding domain (FRB), and the “DNA binding domain” (encoding a chimeric DNA binding molecule with ZFHD1 fused to 3 copies of FKBP) Dependent.

  A regulatable replication competent virus can be provided as a carrier specific to a particular host cell so that the virus can selectively replicate in a particular target cell and the replication of the virus can be regulated Is clear from the above description.

  All publications and patent applications mentioned in this specification are herein incorporated as if each individual publication or patent application was specifically and individually incorporated.

  It will be apparent to those skilled in the art that the present invention has been fully described herein and that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Schematic illustration of the genome of a prostate-specific adenoviral vector with tetracycline-regulated replication control. Schematic illustration of the genome of a prostate-specific adenoviral vector armed with GM-CSF with tetracycline-regulated replication and transgene control. Briefly illustrates the genome of an adenoviral vector with dual tetracycline-regulated replication control. Brief Description of the Drawings Figure 1 schematically illustrates the genome of an adenoviral vector with ARIAD system regulatory control for use in pan-cancer applications.

Claims (27)

below:
An inducible transcriptional transactivator coding sequence under transcriptional control of a cell type specific transcriptional response element (CT-TRE); and an adenovirus under transcriptional control of the transcriptional response element regulated by the transcriptional transactivator gene;
A replication competent adenoviral vector, wherein the transcriptional transactivator is functionally responsive to an exogenous inducer.   2. The adenovirus vector according to claim 1, wherein the adenovirus gene is an essential gene for adenovirus replication.   3. The adenovirus vector according to claim 2, wherein the gene essential for replication is an adenovirus early gene.   4. The adenovirus vector according to claim 3, wherein the adenovirus early gene is E1A.   4. The adenovirus vector according to claim 3, wherein the adenovirus early gene is E1B.   The adenovirus vector according to claim 2, wherein the gene essential for adenovirus replication is the E4 gene of adenovirus.   3. The adenovirus vector according to claim 2, wherein the gene essential for adenovirus replication is an adenovirus late gene.   2. The adenoviral vector according to claim 1, wherein the transcriptional transactivator is repressed by the inducer.   2. The adenovirus of claim 1, wherein the transcriptional transactivator is activated by the inducer.   2. The adenovirus of claim 1, wherein the inducer is selected from the group consisting of tetracycline, doxycycline and analogs thereof, FKBP homodimer and heterodimer, ecdysone, rapamycin and analogs thereof. below:
An inducible transcriptional transactivator coding sequence under transcriptional control of a cell type specific transcriptional response element (CT-TRE); and an adenovirus under transcriptional control of the transcriptional response element regulated by the transcriptional transactivator A gene and a second gene under transcriptional control of a second transcription response element;
A replication-competent adenoviral vector, wherein the transcriptional transactivator is activated by an exogenous inducer.   12. The adenovirus vector according to claim 11, wherein the second gene is an adenovirus gene.   13. The adenovirus vector according to claim 12, wherein the adenovirus gene is an essential gene for adenovirus replication.   14. The adenovirus vector according to claim 13, wherein the gene essential for adenovirus replication is an adenovirus early gene.   15. The adenovirus vector according to claim 14, wherein the adenovirus early gene is E1A.   15. The adenovirus vector according to claim 14, wherein the adenovirus early gene is E1B.   13. The adenoviral vector according to claim 12, wherein the first and second genes are essential for adenoviral replication.   18. The adenoviral vector according to claim 17, wherein the first and second genes are adenoviral early genes.   19. The adenovirus vector according to claim 18, wherein the first gene is E1A and the second gene is E1B.   18. The adenoviral vector according to claim 17, wherein the expression of the first or second gene is regulated by IRES.   12. The adenovirus vector according to claim 11, wherein the second gene is a transgene.   The adenoviral vector according to claim 21, wherein the transgene is a cytotoxic gene.   An isolated host cell comprising the adenovirus vector of claim 1.   A composition comprising the adenoviral vector of claim 1 and a pharmaceutically acceptable excipient. below:
An inducible transcriptional transactivator coding sequence under transcriptional control of a cell type specific transcriptional response element (CT-TRE); and an adenovirus under transcriptional control of the transcriptional response element regulated by the transcriptional transactivator gene;
A method of propagating an adenoviral vector comprising: wherein said transcriptional transactivator is activated by an exogenous inducer and is replication competent, comprising the following steps:
Introducing the adenoviral vector into a cell allowing cell-specific TRE to function; and administering the inducer, wherein the adenoviral vector is propagated;
A method for propagating the adenoviral vector comprising: below:
An inducible transcriptional transactivator coding sequence under transcriptional control of a cell type specific transcriptional response element (CT-TRE); and an adenovirus under transcriptional control of the transcriptional response element regulated by the transcriptional transactivator gene;
Wherein the transcriptional transactivator is replication-inhibited by an exogenous inducer and has the following steps:
Introducing the adenoviral vector into a cell allowing cell-specific TRE to function, wherein the adenoviral vector is propagated; and administering the inducer, wherein the adenovirus Transmission is inhibited;
Including said method. below:
An inducible transcriptional transactivator coding sequence under the transcriptional control of a cell type specific transcriptional response element (CT-TRE); and an adenovirus under the transcriptional control of the transcriptional response element regulated by the transcriptional transactivator gene;
A method for selective cell lysis of target tumor cells, wherein the transcriptional transactivator is activated by an exogenous inducer and comprises the following steps:
Introducing the adenoviral vector into a cell allowing cell-specific TRE to function; and administering the inducer, wherein the adenoviral vector is propagated and lysing the target prostate cell cause;
Including said method.

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