WO2007050074A1 - Conditionally replicating viruses and methods for cancer virotherapy - Google Patents
Conditionally replicating viruses and methods for cancer virotherapy Download PDFInfo
- Publication number
- WO2007050074A1 WO2007050074A1 PCT/US2005/038918 US2005038918W WO2007050074A1 WO 2007050074 A1 WO2007050074 A1 WO 2007050074A1 US 2005038918 W US2005038918 W US 2005038918W WO 2007050074 A1 WO2007050074 A1 WO 2007050074A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sequence
- virus
- composition
- cells
- cancer
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10041—Use of virus, viral particle or viral elements as a vector
- C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2820/00—Vectors comprising a special origin of replication system
- C12N2820/002—Vectors comprising a special origin of replication system inducible or controllable
Definitions
- the present invention pertains to a translational control element placed in a vector to cause a selective replication translation of a viral vector.
- the present invention provides for methods and compositions for conditionally expressing a viral gene necessary for vector replication inside tumor cells, while leaving normal cells unaffected due to their inability to replicate the vector.
- gene therapy/virotherapy remains a promising strategy for the treatment of cancer.
- the existing approaches to gene therapy/virotherapy of cancer can be divided into five broad categories: 1) mutation compensation, 2) molecular chemotherapy, 3) genetic immunopotentiation, 4) genetic modulation of resistance/sensitivity and 5) oncolytic therapy or virotherapy.
- any of the above gene therapy/virotherapy approaches is fundamentally based on the ability of vector to deliver the therapeutic gene or replication-competent viral genome to target cells with a requisite level of efficiency.
- the present invention provides for a conditionally replicating recombinant vims vector, comprising a replicating recombinant vims vector genome, comprising: (a) a replicating recombinant vims vector genome nucleic acid transcription sequence; and (b) a promoter operatively linked to the transcription sequence; wherein the transcription sequence, when transcribed, produces a messenger RNA sequence that comprises an open reading frame encoding a viral protein necessary for replication, and a 5 '-untranslated region (5'-UTR) sequence; wherein the untranslated sequence inhibits translation of the viral protein sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E; and wherein the untranslated sequence allows translation of the viral protein sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E relative to normal cells.
- a replicating recombinant vims vector genome comprising: (a) a replica
- the untranslated sequence further comprises a hairpin secondary structure conformation having a stability measured as folded state free energy of ⁇ G ⁇ about -50 Kcal/Mol.
- control sequences operably linked to sequences include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed.
- promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
- the promoter is typically selected from promoters that are functional in mammalian cells, although promoters functional in other eukaryotic cells may be used.
- the promoter is typically derived from promoter sequences of viral or eukaryotic genes.
- it may be a promoter derived from the genome of the type of cell in which expression is to occur.
- eukaryotic promoters they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, b- actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase).
- the virus vector genome comprises an adenovirus.
- the adenovirus vector is a type 2 or type 5 virus vector.
- the El coding region is operatively associated with a promoter selected from the group consisting of liver-specific, skeletal muscle-specific, cardiac muscle-specific, smooth muscle-specific, diaphragm muscle-specific, prostate-specific, and/or brain-specific promoters.
- the El coding region is operatively associated with a cancer cell specific promoter.
- the El coding region is operatively associated with an inducible promoter.
- the recombinant virus vector genome is encapsidated within an virus capsid.
- the present invention provides for a cultured cell comprising the replicating recombinant virus vector of the present invention.
- the present invention provides for a method of introducing a nucleic acid sequence into a cell, comprising contacting a cell with a replicating recombinant virus vector according to the present invention under conditions sufficient for entry of the virus particle into the cell.
- the present invention provides for a method of administering a nucleotide sequence to a subject, comprising administering to a subject a replicating recombinant virus vector according to the present invention in a pharmaceutically acceptable carrier.
- the promoter is a cancer cell specific promoter.
- the vector may be conditionally regulated by means consisting of a tissue-specific promoter operably linked to an early gene (e.g., El, E2 and/or E4) and a mutation in an early gene (e.g., El, E2 and/or E4).
- tissue-specific promoters are derived from genes encoding proteins such as the prostate specific antigen (PSA), Carcinoembryonic antigen (CEA), secretory leukoprotease inhibitor (SLPI), alpha-fetoprotein (AFP), vascular endothelial growth factor, CXCR4 or survivin.
- the promoter is an inducible promoter. In another embodiment, the promoter is the CXCR4 promoter.
- the present invention provides for a method of treating cancer, comprising administering to a subject that has cancer a composition comprising a replicating recombinant vims vector according to the present invention in a pharmaceutically acceptable carrier; wherein the composition is administered in an therapeutically effective amount and under conditions sufficient for the subject to produce a therapeutic response against the cancer cell.
- the recombinant vims vector is administered by a route selected from the group consisting of oral, rectal, transmucosal, transdermal, inhalation, intravenous, subcutaneous, intradermal, intramuscular, and intraarticular administration.
- the recombinant vims vector is injected directly into a cancerous tissue.
- the present invention provides for compositions and methods for using adeno-associated vims for transduction of a target gene in a variety of tissues wherein the expression of the vims is under control of an untranslated sequence; wherein the untranslated sequence inhibits translation of the viral sequence in the absence of eukaryotic initiation factor eIF4E, and wherein the untranslated sequence allows translation of the viral vector sequence into a virus in the presence of eukaryotic initiation factor eIF4E.
- the present invention provides for cancer-specific transcriptional control using a promoter such as that from the human CXCR4 gene that can be used together with cancer-specific translational control utilizing a highly structured 5 '-untranslated region (5'-UTR) sequence in the context of a CRAd.
- a promoter such as that from the human CXCR4 gene that can be used together with cancer-specific translational control utilizing a highly structured 5 '-untranslated region (5'-UTR) sequence in the context of a CRAd.
- the invention provides for the use of dual-level cancer targeting as means to increase specificity of gene expression in cancers to overcome the problem of non-specific promoter activity (promoter leakage) in normal tissues.
- the invention has broad utility to advance cancer-specific CRAds as a novel class of highly specific oncolytic agents to therapeutic clinical trials.
- the present invention provides for dual-targeting of transgene expression to cancer cells using both transcriptional and translational control to substantially enhance Ad5 based cancer virotherapy.
- This combinatorial approach allows the creation of a highly cancer-specific virotherapy agent, which will be evaluated for the treatment of cancers such as HNSCC.
- Conditionally Replicative Adenoviruses represent new promising therapeutic agents applied to cancer treatment.
- cancer-specific replication of the CRAds result in viral-mediated oncolysis of infected tumor tissues and release of the virus progeny, capable of further propagating in surrounding tumor cells but not in those of normal tissues, which would be refractory to CRAd replication.
- TSP tumor-specific promoter
- the present invention shows that the CXCR4 gene promoter not only has a distinct cancer-specific activation profile, but also shows one of the highest activity levels among other TSPs in a panel of selected cancers compared to the ubiquitously utilized Cytomegalovirus (CMV) promoter, particularly in squamous cell carcinomas of the head (HNSCC).
- CMV Cytomegalovirus
- the background activity of cancer-specific promoters in non- cancer cells is reduced by introducing a second level of cancer targeting for the Ad5 El -genes expression in addition to transcriptional targeting.
- This can be achieved by placing ElA gene, which is essential for Ad5 replication function, under cancer-specific translational control via engineering a highly structured 5'- UTR sequence such as that from the Fibroblast Growth Factor 2 (FGF2) upstream of the ElA mRNA coding sequence.
- FGF2 Fibroblast Growth Factor 2
- FGF2 5'-UTR - heipes simplex virus thymidine kinase (HSV-Tk) mRNA chimera limits efficient protein translation to tumors but strongly inhibits or even blocks efficient protein translation in normal cells, where eIF4E is the rate-limiting initiation factor [2,3].
- the present invention provides for dual-targeting of transgene expression to cancer cells, namely that this approach augments the cancer targeting strategies currently used alone in Ad5 based cancer gene therapy/virotherapy.
- This combinational approach allows the creation of a highly cancer-specific virotherapy agent.
- this combinational approach is used for treatment of HNSCC as well as other cancers. Development of such a product also helps to overcome the problem of viral liver toxicity.
- the present invention provides for both the tumor specific promoter and the FGF2 5'-UTR sequence element combined within a single viral agent, such as an adenovirus.
- HNSCC head and neck region
- FGF2 5'-UTR sequence element a TSP in the context of CRAd.
- Squamous cell carcinoma of the head and neck region (HNSCC) is the sixth most frequent cancer worldwide, comprising almost 50% of all malignancies in some developing nations. In the United States, 30,000 new cases and 8,000 deaths are reported each year [63]. Survival rates vary depending on tobacco and alcohol consumption, age, gender, ethnic background, and geographic area. This variability reflects the multifactoral pathogenesis of the disease. Early detection and diagnosis has increased survival but the overall 5-year rate of 50% is among the lowest of the major cancers [64]. Thus, an effective alternative in the treatment of malignant diseases, including but not limited to HNSCC, is greatly needed.
- the present invention now provides for a combination of transcription and translation control to enhance cancer-specific replication of the CRAds, the major advantage of which is to achieve efficient tumor cell oncolysis and to mitigate tumor cell infection limitations.
- This ability to target both primary and metastatic lesions means that our approach has wide application in the treatment of many forms of cancer, beyond p53 mutation-positive cancers.
- virus vector The expression of a virus vector is translationally repressed in normal cells by placing a complex 5'-UTR sequence in front of the vector reading frame.
- the invention also provides formulations comprising the recombinant adenoviruses according to the invention that can be used to preserve the recombinant adenoviruses and to administer the recombinant adenoviruses to cells.
- the formulations are used to administer the recombinant adenoviruses to cells in vitro, in another variation the formulations are used to administer the recombinant adenoviruses to cells in vivo.
- the invention furthermore provides methods to administer the formulations according to the invention to cells, leading to infection of the cells with the recombinant adenoviruses of the invention.
- the methods are used to administer the formulations to cells in vitro, in another variation the methods are used to administer the formulations to cells in vivo.
- the invention also provides compositions of the recombinant adenoviruses according to the invention and cells in which the recombinant adenoviruses according to the invention induce accelerated cell lysis and/or a faster release of virus progeny, compared to recombinant adenoviruses lacking coding sequences for the restoring factor according to the invention.
- the cells are cancer cells and the cell lysis is oncolysis.
- the cells are human cells.
- the invention provides compositions of the recombinant adenoviruses according to the invention and tumors in which the recombinant adenoviruses according to the invention induce accelerated cell lysis and/or a faster release of virus progeny, compared to recombinant adenoviruses lacking coding sequence for the restoring factor according to the invention.
- the accelerated cell lysis and/or a faster release of villas progeny results m an accelerated lateral spread by the recombinant adenoviruses from infected cells to neighboring cells in the tumors, compared to recombinant adenoviruses lacking coding sequence for the restoring factor according to the invention.
- the tumors are growing in an animal body. I In a further variation, the animal body is a human body.
- the modified virus is administered by injection into or near the solid neoplasm.
- the modified vims is administered intravenously into the mammal.
- the modified vims is administered intraperitoneally into the mammal.
- the mammal is immunocompetent.
- the modified virus is encapsulated in a micelle or liposome.
- the modified vims is administered with an effective amount of an anti-antivirus antibody.
- approximately 1 to 10 15 plaque forming units of modified virus/kg body weight are administered.
- the modified virus is administered in a single dose.
- the modified virus is administered in more than one dose.
- the administration further comprises the administration of an effective amount of a chemotherapeutic agent.
- This invention is directed to a method for treating a cell proliferative disorder in a mammal, comprising administering to proliferating cells in a mammal in an effective amount of one or more viruses selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia vims and modified parapoxvirus orf vims under conditions which result in substantial lysis of the proliferating cells.
- viruses selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia vims and modified parapoxvirus orf vims under conditions which result in substantial lysis of the proliferating cells.
- the vims may be modified such that the virion is packaged in a liposome or micelle, or the proteins of the outer capsid have been mutated.
- the virus can be administered in a single dose or in multiple doses.
- the cell proliferative disorder may be a neoplasm. Both solid and hematopoietic neoplasms can be targeted.
- a method of treating a neoplasm in a human comprising administering to the neoplasm an effective amount of virus selected from the group consisting of modified adenovims, modified HSV, modified vaccinia vims and modified parapoxvims orf virus, to result in substantial oncolysis of the neoplastic cells.
- the virus may be administered by injection into or near a solid neoplasm.
- Also provided is a method of inhibiting metastasis of a neoplasm in a mammal comprising administering to the neoplastic cells in a mammal a virus selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia vims and modified parapoxvirus orf virus, in an amount sufficient to result in substantial lysis of the neoplasm.
- a vims selected from the group consisting of modified adenovims, modified HSV, modified vaccinia vims and modified parapoxvims orf virus
- a pharmaceutical composition comprising a virus selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia vims and modified parapoxvims orf vims, a chemotherapeutic agent and a pharmaceutically acceptable excipient.
- a pharmaceutical composition comprising a vims selected from the group consisting of modified adenovims, modified HSV, modified vaccinia vims and modified parapoxvims orf virus, and a pharmaceutically acceptable excipient.
- this invention includes a kit comprising a pharmaceutical composition comprising a vims selected from the group consisting of modified adenovims, modified HSV, modified vaccinia vims and modified parapoxvims orf vims, and a chemotherapeutic agent.
- a vims selected from the group consisting of modified adenovims, modified HSV, modified vaccinia vims and modified parapoxvims orf vims, and a chemotherapeutic agent.
- this invention provides a kit comprising a pharmaceutical composition comprising a vims selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia virus and modified parapoxvirus orf virus and an anti-antivirus antibody.
- Also provided is a method for treating a population of cells comprising a neoplasm in vitro comprising administering to the population of cells in vitro a vims selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia virus and modified parapoxviiiis orf virus in an amount sufficient to result in substantial lysis of the neoplasm.
- the invention is also directed to methods of treating a proliferative disorder in a mammal, by immunosuppressing, immunoinhibiting or otherwise rendering the mammal immunodeficient and, concurrently or subsequently, administering a virus selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia virus and modified parapoxvirus orf virus in an amount sufficient to result in substantial lysis of the neoplasm.
- modified virus is administered to proliferating cells in mammal.
- modified virus include adenovirus, HSV, parapoxvirus orf virus, or vaccinia vims, which infect humans.
- modified adenovims is used.
- the vims may be a recombinant vims from two or more types of vimses with differing pathogenic phenotypes such that it contains different antigenic determinants thereby reducing or preventing an immune response by a mammal previously exposed to a vims subtype.
- Such recombinant virions can be generated by co-infection of mammalian cells with different subtypes of vims with the resulting resorting and incorporation of different subtype coat proteins into the resulting virion capsids.
- the invention furthermore provides methods to construct the recombinant viruses according to the invention and to produce the formulations and compositions according to the invention.
- the invention furthermore contemplates the use of the recombinant viruses, methods and formulations according to the invention for the treatment of a disease which involves inappropriate cell survival, where it is preferred that the disease is a disease in a human being. In a particular embodiment of the invention the disease is cancer.
- the invention includes all combined uses of the recombinant viruses, formulations, methods and compositions of the invention together with other methods and means to kill a population of cells, including but not limited to irradiation, introduction of genes encoding toxic proteins, such as for example toxins or prodrug converting enzymes, and administration of chemical compounds, antibodies, receptor antagonists, and the like.
- FIG. 1 is a graphical depiction of conditionally replicative virus based therapy.
- the vector enters the target cell and expresses the effector gene to kill the tumor cells.
- replicative virus-based therapy after entry, the virus replicates primarily in the infected target cell and kills the cell by cytolysis as a consequence of lytic infection. Then, the released virus infects surrounding target cells. The achievement of this lateral spread is a key event for the effectiveness of replicative virus-based therapy.
- FIG. 2 depicts a graphical model of translation initiation.
- the initiation process is comprised of three steps: 1) formation of the 43S complex, composed of a 4OS ribosomal subunit and the initiation factors eIF-2, eIF-3, Met-tRNAi and GTP; 2) formation of the 48S complex containing mRNA; and 3) joining of the 60S subunit to form the complete 80S complex.
- FIG. 3 is a graphical depiction of construction of shuttle vectors are an intermediate step in the production of adenoviruses with single and/or dual-level expression control of the luciferase reporter gene or the ElA gene.
- Shuttle vectors in which transcription of the ElA or the luciferase genes are controlled by the cancer-specific CXCR4 gene promoter.
- FIG. 4 shows endogenous CXCR4 and eIF4E Expression in HNSCC Tumors
- FIG. 5 is a Western blot analysis of El A protein levels. Western blot analysis of
- ElA protein expression was performed using lysates from for each cell line used (MCF-IOA, and MCF-10A-4E) and infected (at an m.o.i. of 100 p.f.u./cell) with a wild-type Ad vector containing a deletion of the E3 gene (Ad-wt-dE3) or Ad vectors containing the gene for green fluorescent protein (Ad-CMV-GFP), ElA (Ad-CXCR4- ElA), or the 5'-UTR-modified ElA (Ad- CXCR4-UTR-E1A) .
- FIG 6. is an in vitro oncolysis assay.
- Two cell lines were used: a normal breast epithelial cell line expressing low levels of eIF4E (MCF-IOA), and the MCF- 10A-4E cell line transfected with a G418-selectable plasmid expression vector that constitiitively expresses high levels of eIF4E.
- the cell lines were infected with increasing multiplicities of infection (m.o.i.) from 0.1 to 100 of the indicated adenovirus, and the number of cells adherent after 10 days from infection were visualized by crystal violet staining of the viable cells attached to the wells.
- FIG 7. is an in vitro oncolysis assay. Two cancer cell lines (ZR-75-1 and MDA-
- MB-231) were infected with increasing multiplicities of infection (M.O.I.) from 0.1 to 100 of the indicated adenovirus, and the number of cells adherent after 10 days from infection were visualized by crystal violet staining of the viable cells attached to the wells.
- M.O.I. multiplicities of infection
- adenovirus vector is meant a vector derived from an adenovirus serotype, including without limitation, to any of the fifty distinct serotypes of human adenoviruses have been identified to date. These serotypes have been classified into six subgroups (A-F) based on sequence comparisons, each subgroup with different tropisms.
- an adenovirus vector is defined herein to include at least those sequences required in cis for replication and packaging ⁇ e.g., functional ITRs) of the virus.
- the ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
- adenovirus virion is meant a complete vims particle, such as a wild-type
- adenovirus particle comprising a linear, single-stranded adenovirus nucleic acid genome associated with a capsid protein coat.
- single-stranded viral nucleic acid molecules of either complementary sense, e.g., "sense” or “antisense” strands can be packaged into any one virion and both strands are equally infectious.
- Ad vector genome refers to the viral genomic DNA, in either its naturally occurring or modified form.
- a “rAd vector genome” is a recombinant Ad genome (i.e., vDNA) that comprises one or more heterologous nucleotide sequence(s).
- the Ad vector genome or rAd vector genome will typically comprise the Ad terminal repeat sequences and packaging signal.
- An “Ad particle” or “rAd particle” comprises an Ad vector genome or rAd vector genome, respectively, packaged within an Ad capsid.
- the Ad vector genome is most stable at sizes of about 28 kb to 38 kb (approximately 75% to 105% of the native genome size).
- "stuffer DNA” can be used to maintain the total size of the vector within the desired range by methods known in the art.
- Adenovirus is a double stranded DNA vims of about 3.6 kilobases. In humans, adenoviruses can replicate and cause disease in the eye and in the respiratory, gastrointestinal and urinary tracts.
- the term "adenovirus” as used herein is intended to encompass all adenoviruses, including the Mastadenovirus and Aviadenovirus genera. To date, at least forty-seven human serotypes of adenoviruses have been identified (see, e.g., Fields et al., Virology, volume 2, chapter 67 (3d ed., Lippincoft-Raven Publishers).
- the adenovirus is a serogroup C adenovirus, still more preferably the adenovirus is serotype 2 (Ad2) or serotype 5 (Ad5).
- Ad2 serotype 2
- Ad5 serotype 5
- the various regions of the adenovirus genome have been mapped and are understood by those skilled in the art (see, e.g., FIELDS et al., VIROLOGY, volume 2, chapters 67 and 68 (3d ed., Lippincoft-Raven Publishers).
- the genomic sequences of the various Ad serotypes, as well as the nucleotide sequence of the particular coding regions of the Ad genome, are known in the art and may be accessed, e.g., from GenBank and NCBI (See, e.g., GenBank Accession Nos. J0917, M73260, X73487, AF10S105, L19443, NC 003266 and NCBI Accession Nos. NC 001405, NC 001460, NC 002067, NC 00454).
- inventive adenovirus vectors may be modified or "targeted" as described in Douglas et al., (1996) Nature Biotechnology 14: 1574; U.S. Pat. No. 5,922,315 to Roy et al.; U.S. Pat. No. 5,770,442 to Wickham et al.; and/or U.S. Pat. No. 5,712,136 to Wickham et al.
- administering indicates that the virus is administered in a manner so that it contacts the proliferating cells or cells of the neoplasm (also referred to herein as “neoplastic cells”).
- carcinoma of esophagus lung cancer, cancer of rectum, nasopharyngeal carcinoma, carcinoma of stomach, pleural effusion, carcinoma of ovarium, ascites, and melanoma.
- cancer therapy refers to that vectors for infecting cancer cells, so as to destroy cancer cells.
- Such vectors may optionally include one or more therapeutic gene.
- the therapeutic genes include genes related to cell apoptosis, cell lysis, cell suicide, etc.
- conditionally regulated and “conditionally- replicative” refer to the expression of a viral gene or the replication of a virus or a vector, wherein the expression of replication is dependent (i.e., conditional) upon the presence or absence of specific factors in the target cell.
- cytokine refers to all small proteins with the properties of locally acting hormones. They serve to communicate between cells in a paracrine manner, and may also act in an autocrine manner on the same cell that produces the cytokine(s).
- Growth factors are types of cytokines that are anti- arthritic in that they maintain synthesis of the cartilaginous matrix. Growth factors include, but are not limited to, transforming growth factor (TGF), TGF-/3, TGF-/32 and TGF-/33, fibroblast growth factor (FGF), oFGF and /3FGF, insulin-like growth factor (IGF) IGF-I and IGF-2.
- TGF transforming growth factor
- FGF fibroblast growth factor
- IGF insulin-like growth factor
- IGF-2 insulin-like growth factor
- BMP bone morphogenetic proteins
- the phrase "delivering a gene” or “transferring a gene” refers to methods or systems for reliably inserting foreign DNA into host cells, such as into cells. Such methods can result in transient or long-term expression of nonintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episodes), or integration of transferred genetic material into the genomic DNA of recipients.
- Gene transfer provides a unique approach for the treatment of acquired and inherited diseases. A number of systems have been developed for gene transfer into mammalian cells. See, e.g., U.S. Pat. No. 5,399,346.
- DNA is meant a polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in double-stranded or single-stranded form, either relaxed and supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this te ⁇ n includes single- and double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
- linear DNA molecules e.g., restriction fragments
- viruses e.g., plasmids, and chromosomes.
- sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having the sequence homologous to the mRNA).
- the term captures molecules that include the four bases adenine, guanine, thymine, or cytosine, as well as molecules that include base analogues which are known in the art.
- eIF4E refers to the cap-binding sub-unit of the eIF4E complex. eIF4E binds the mRNA cap structure and forms eIF4Fcomplexes that recruit 4OS subunits to the mRNA.
- the eukaryotic initiation factor 4E (eIF4E) is a component of the cellular translational apparatus. Translation initiation on eukaryotic mRNA includes the recruitment of the 4OS ribosomal subunit to the 5' end of mRNA. This is mediated by eukaryotic translation initiation complex 4F (eIF4F) that is a heterotrimetic complex containing eIF4E, eIF4A, and eIF4G.
- eIF4A is an RNA-dependent RNA helicase which unwinds mRNA secondary structure and eIF4G is a large polypeptide containing binding sites for eIF4E, eIF4A, eIF3 and poly(A) binding protein.
- eIF4E facilitates the initiation of translation by directly binding to the mRNA 5' cap structure (m 7 GpppN).
- the sequence of DNA encoding human eIF4E has been determined [Reychlik, W. et al. (1987) Proc. Natl. Acad. USA 84:945-949].
- Yeast eIF4E and a fusion protein of mouse eIF4E have been expressed in E. coli [Edery, L, et al.
- “Expression control element”, or simply “control element” refers to DNA sequences, such as initiation signals, enhancers, promoters and silencers, which induce or control transcription of DNA sequences with which they are operably linked.
- Control elements of a gene may be located in introns, exons, coding regions, and 3' flanking sequences. Some control elements are "tissue specific”, i.e., and affect expression of the selected DNA sequence preferentially in specific cells (e.g., cells, of a specific tissue), while others are active in many or most cell types. Gene expression occurs preferentially in a specific cell if expression in this cell type is observably higher than expression in other cell types.
- a "gene” or “coding sequence” or a sequence which "encodes” a particular protein is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or In vivo when placed under the control of appropriate regulatory sequences. The boundaries of the gene are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
- a gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
- a transcription termination sequence will usually be located 3' to the gene sequence.
- HSV herpes simplex virus
- HSV gene gammal 34.5 encodes the gene product infected-cell protein 34.5 (ICP34.5) that can prevent the antiviral effects exerted by PKR.
- ICP34.5 has a unique mechanism of preventing PKLR activity by interacting with protein phosphatase 1 and redirecting it activity to dephosphorylate eIF-2alpha.29
- eIF-2alpha is phosphorylated and protein synthesis is turned off in cells infected with gammal 34.5 minus virus.
- the gammal 34.5 minus virus would be replication competent in cells with an activated Ras pathway in which the activity of ICP34.5 would be redundant.
- HSV is unable to replicate in cells which do not have an activated Ras-pathway. Thus, HSV can replicate in cells which have an activated Ras-pathway.
- heterologous as it relates to nucleic acid sequences such as gene sequences and control sequences, denotes sequences that are not normally joined together, and/or are not normally associated with a particular cell.
- a heterologous region of a nucleic acid construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature.
- a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature.
- heterologous coding sequence is a construct where the coding sequence itself is not found in nature ⁇ e.g., synthetic sequences having codons different from the native gene).
- a cell transformed with a construct which is not normally present in the cell would be considered heterologous for purposes of this invention. Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.
- heterologous nucleotide sequence or “heterologous nucleic acid sequence” will typically be a sequence that is not naturally-occurring in the virus.
- a heterologous nucleotide or nucleic acid sequence may refer to a viral sequence that is placed into a non-naturally occurring environment (e.g., by association with a promoter with which it is not naturally associated in the virus).
- homology refers to the percent of identity between two polynucleotide or two polypeptide moieties.
- the correspondence between the sequence from one moiety to another can be determined by techniques known in the ait. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single- stranded-specific nuclease(s), and size determination of the digested fragments.
- Two DNA, or two polypeptide sequences are "substantially homologous" to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids match over a defined length of the molecules, as determined using the methods above.
- infectious refers to that the vims can enter the cell by natural transduction mechanisms and express the transgene therein.
- an "infectious" vims is one that can enter the cell by other mechanisms and express the transgene therein.
- the vector can enter a target cell by expressing a ligand or binding protein for a cell-surface receptor in the adenovims capsid or by using an antibody(ies) directed against molecules on the cell-surface followed by internalization of the complex, as is described hereinbelow.
- “Initiator” refers to a short, weakly conserved element that encompasses the transcription start site and which is important for directing the synthesis of properly initiated transcripts.
- a "mammal suspected of having a proliferative disorder” means that the mammal may have a proliferative disorder or tumor or has been diagnosed with a proliferative disorder or tumor or has been previously diagnosed with a proliferative disorder or tumor, the tumor or substantially all of the tumor has been surgically removed and the mammal is suspected of harboring some residual tumor cells.
- mammalian subject any member of the class Mammalia including, without limitation, humans and non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rate and guinea pigs, and the like.
- the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
- naturally-occurring refers to the fact that an object can be found in nature.
- a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
- the term “recombinant” indicates that a polynucleotide construct (e.g., and adenovirus genome) has been generated, in part, by intentional modification by man.
- oncolytic techniques refers to all kinds of effective protocols that can induce the lysis or death of tumor cells including apoptosis and necrosis. These protocols include application of an oncolytic virus that lead to the lysis or death of cancer cells.
- oncolytic virus refers to a genetically engineered vims that may replicate immortally in cancer cells, so as to kill these cancer cells.
- Adenovirus dll520 is an example of oncolytic viruses.
- the oncolytic viruses referred to in this invention could be herpes simplex virus (HSV-I), adenovirus, newcastle disease virus (“NDV”), poliovirus, measles virus, vesicular stomatitis virus (“VSV”), etc.
- HSV-I herpes simplex virus
- NDV newcastle disease virus
- poliovirus measles virus
- VSV vesicular stomatitis virus
- the lytic viruses include other viruses, for example, baculovirus; members of the Herpesviridae such as HSVl, HSV2, VZV, HCMV, HHV8; parvoviruses such as B 19, AAV-2; members of the Togaviridae including alphaviruses such as equine encephalitis viruses & Sindbis and rubiviruses such as rubella; polyoma viruses such as SV40; arboviruses such as Getah arbovirus; diarrhoea viruses such as porcine epidemic diarrhoea virus; members of the Flaviviridae including flaviviruses such as yellow fever, dengue & encephalitis viruses, pestiviruses such as BVDV and unclassified viruses such as hepatitis C; members of the Bunyaviridae including phleboviruses, such as sandfly fever, nairoviruses, such as haemorrhagi
- the lyticvirus is an oncolytic vims selected from the group consisting of an adenovirus, a heipes simplex virus, a reovirus, a Newcastle disease vims, a poliovirus, a measles virus, or a vesicular stomatis vims). Additionally, the lytic vims may comprises a therapeutic gene (e.g.
- an apoptotic gene a gene for tumor necrosis, a gene for starving tumor cells to death, cytolytic gene, negative I-kappa- beta, caspase, gamma globulin, h-alpha-1 antitrypsin, or EIa of adenovirus).
- operably linked refers to the arrangement of various nucleic acid molecule elements relative to each such that the elements are functionally connected and are able to interact with each other.
- Such elements may include, without limitation, a promoter, an enhancer, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a gene of interest to be expressed (i.e., the transgene).
- the nucleic acid sequence elements when properly oriented or operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression of the transgene. By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element.
- the position of each element relative to other elements may be expressed in terms of the 5' terminus and the 3' terminus of each element, and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements.
- the terms "percentage of sequence identity” as used herein compares two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e. "gaps") as compared to a reference sequence for optimal alignment of the two sequences being compared.
- the percentage identity is calculated by determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window and multiplying the result by 100 to yield the percentage of sequence identity. Total identity is then determined as the average identity over all of the windows that cover the complete query sequence.
- computer software packages such as GAP, BESTFIT, BLASTA, FASTA and TFASTA can also be utilized to determine sequence identity.
- polypeptide encompasses both peptides and proteins, unless indicated otherwise.
- a “proliferative disorder” is any cellular disorder in which the cells proliferate more rapidly than normal tissue growth.
- a “proliferating cell” is a cell that is proliferating more rapidly than normal cells.
- the proliferative disorder includes but is not limited to neoplasms.
- a “neoplasm” is an abnormal tissue growth, generally forming a distinct mass, that grows by cellular proliferation more rapidly than normal tissue growth. Neoplasms show partial or total lack of structural organization and functional coordination with normal tissue. These can be broadly classified into three major types.
- Malignant neoplasms arising from epithelial structures are called carcinomas, malignant neoplasms that originate from connective tissues such as muscle, cartilage, fat or bone are called sarcomas and malignant tumors affecting hematopoetic structures (structures pertaining to the formation of blood cells) including components of the immune system, are called leukemias and lymphomas.
- a tumor is the neoplastic growth of the disease cancer.
- a neoplasm also referred to as a "tumor”
- Other proliferative disorders include, but are not limited to neurofibromatosis.
- promoter refers to a nucleic acid sequence that regulates, either directly or indirectly, the transcription of a corresponding nucleic acid coding sequence to which it is operably linked.
- the promoter may function alone to regulate transcription, or, in some cases, may act in concert with one or more other regulatory sequences such as an enhancer or silencer to regulate transcription of the transgene.
- promoter region is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.
- a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
- the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
- a transcription initiation site within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
- Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT” boxes.
- Prokaryotic promoters contain Shine-Dalgamo sequences in addition to the -10 and -35 consensus sequences.
- propagation refers to a productive viral infection wherein the viral genome is replicated and packaged to produce new virions, which typically can “spread” by infection of cells beyond the initially infected cell.
- a "propagation-defective" vims is impaired in its ability to produce a productive viral infection and spread beyond the initially infected cell.
- recombinant virus is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
- replication refers specifically to replication of the viral genome (i.e., making new copies of the virion DNA).
- replication-competent viruses refers to a virus capable of replication (i.e., a virus that yields progeny).
- replication deficient virus refers to a vims that preferentially inhibits cell proliferation or induces apoptosis in a predetermined cell population, which supports expression of a virus replication phenotype, and which is substantially unable to inhibit cell proliferation, induce apoptos function levels characteristic of non-replicating, non-transformed cells.
- replication phenotype refers to one or more of the following phenotypic characteristics of cells infected with a virus such as a replication deficient adenovirus: (1) substantial expression of late gene products, such as capsid proteins (e.g., adenoviral penton base polypeptide) or RNA transcripts initiated from viral late gene promoter(s), (2) replication of viral genomes or formation of replicative intermediates, (3) assembly of viral capsids or packaged virion particles, (4) appearance of cytopathic effect (CPE) in the infected cell, (5) completion of a viral lytic cycle, and (6) other phenotypic alterations which are typically contingent upon certain conditions for function in neoplastic cells.
- a replication phenotype comprises at least one of the listed phenotypic characteristics, preferably more than one of the phenotypic characteristics.
- substantially lysis means at least 10% of the proliferating cells are lysed, more preferably of at least 50% and most preferably of at least 75% of the cells are lysed.
- the percentage of lysis can be determined for tumor cells by measuring the reduction in the size of the tumor in the mammal or the lysis of the tumor cells in vitro.
- the term “therapeutic” refers to the ability of a gene, product, protein, peptide, method and the like to alleviate at least one symptoms of a disorder, or the benefit realized from such alleviation.
- the term “prophylactic” refers to the ability of a gene, product, protein, peptide, method and the like to prevent or at least retard the onset of at least one symptom of a disorder, or the benefit realized from such action.
- the term “enhanced therapeutic benefit” refers to the therapeutic benefit realized when more than one gene of interest is introduced to a host at the same time; the enhanced therapeutic benefit is greater than the therapeutic benefit of each of the genes administered separately. The benefit can be either additive or synergistic.
- terapéuticaally effective amount refers to an amount of a selected DNA sequence that is sufficient to produce a therapeutic effect, e.g., inhibit metastatic tumor growth in a mammal.
- the virus is a lytic virus.
- a therapeutically effective amount of a lytic virus is an amount capable of producing substantial lysis of the target proliferating cells.
- therapeutically effective amount therefore includes, for example, an amount of such selected DNA sequence sufficient to prevent the growth of the patient's tumor, and preferably to reduce by at least 50%, and more preferably to reduce by at least 90%, the mass of a patient's tumor.
- the dosage ranges for the administration of the selected DNA sequence are those that produce the desired effect.
- the dosage will vary with the age, weight, condition, sex of the patient, type of tumor, and degree of tumor development.
- a person of ordinary skill in the art given the teachings of the present specification, may readily determine suitable dosage ranges.
- the dosage can be adjusted by the individual physician in the event of any contraindications. In any event, the effectiveness of treatment can be determined by monitoring the extent of tumor growth and remission by methods well known to those in the field.
- the term "UTR" refers to an untranslated region sequence of mRNA.
- a 5'-UTR is a non-coding nucleotide sequence which lies in a viral sequence directly 5', i.e. upstream, of the start codon of a coding gene.
- region stands for a certain range on nucleic acid (DNA or RNA).
- 5'-untranlated region of mRNA in the present specification stands for a region that, among the mRNA synthesized by the transcription from DNA, which is present at its 5'-side and does not code for a protein.
- Vaccinia virus refers to the virus of the orthopoxvirus genus that infects humans and produces localized lesions Vaccinia vims encodes two genes that play a role in the down regulation of PBCR activity through two entirely different mechanisms.
- E3L gene encodes two proteins of 20 and 25 kDa that are expressed early in infection and have dsRNA binding activity that can inhibit PKR activity.
- the term “vector” or “gene delivery vector” may refer to an viral particle that functions as a gene delivery vehicle, and which comprises vDNA (i.e., the vector genome) packaged within an viral capsid.
- the term “vector” may be used to refer to the vector genome/vDNA when used as a gene delivery vehicle in the absence of the virion capsid.
- vector is meant any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, vims, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
- the term includes cloning and expression vehicles, as well as viral vectors.
- a vector is a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment.
- a “replicon” is any genetic element (e.g., plasmid, chromosome, vims) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
- An "expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. .
- a coding sequence is "operably linked” and “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
- viral infection or "virus infection” as used herein refers to infection by one or more of adenovims, HSV, parapoxvirus orf vims, or vaccinia vims.
- the present invention provides a viral vector comprising (a) a viral genome and
- the present invention also provides an adenoviral vector comprising (a) an adenoviral genome and (b) a nucleic acid sequence coding a complex 5'-UTR operably linked to the viral genome, wherein the DNA sequence comprises a natural or synthetic hai ⁇ in conformation with a stability of at least about ⁇ G > an absolute 50 Kcal/mol.
- the novel strategy targets a general characteristic that distinguishes cancer cells from normal cells, i.e., elevated eIF4E expression.
- This property is exploited in the present invention to repress the expression of a viral vector translationally by placing a complex 5'-UTR in front of its open reading frame.
- cancer cells which have higher levels of eIF4E and hence increased helicase activity, are able to continue to translate this hybrid mRNA while normal cells are not.
- the present invention uses the 5'-UTR of basic fibroblast growth factor (FGF-2), an angiogenic factor previously found to be translationally regulated by eIF4E.
- FGF-2 basic fibroblast growth factor
- eIF4E angiogenic factor previously found to be translationally regulated by eIF4E.
- FGF-2 basic fibroblast growth factor
- VEGF vascular endothelial growth factor
- the DNA sequence comprises a natural or synthetic hai ⁇ in conformation with a stability of at least about ⁇ G > an absolute 50 Kcal/mol.
- the necessary conformation is achieved through a tight hai ⁇ in formation.
- a relatively long palindromic oligonucleotide sequence that is self-complementary is used. See A.E. Koromilas et al., "mRNAs containing extensive secondary structure in their 5' non-coding region translate efficiently in cells overexpressing initiation factor eIF4E," The EMBO Journal, vol. 11, pp. 4153-4158 (1992); and A. DeBenedetti et al, 1999.
- Expression of eIF4E is elevated in most solid tumors, causing translation of mRNAs that would normally be repressed by complex 5'-UTRs.
- the present methods and compositions provide a therapeutic for solid tumors.
- the viral vector system of the present invention can be used in lieu of an episome construct.
- the present compositions and methods allow for efficient delivery of the vector to a large number of tissues.
- translational regulation of a lytic virus or other toxin expression is used to treat wide variety of cancers.
- only a single injection of the vector is employed.
- repeated injections of the vector are used to ensure greater tissue coverage, and a longer treatment for a more effective therapeutic treatment.
- the complex 5'-UTR used as a promoter for translation in the present invention is such that by placing this complex 5'-UTR in front of the open reading frame of the virus or conditional vims, only cells that have higher levels of eIF4E and hence increased helicase activity, are able to continue to translate this hybrid mRNA.
- FGF-2 basic fibroblast growth factor
- eIF4E complex sequences
- eIF4E untranslated hairpin sequences on genes of the proto-oncogene c-myc, cyclin Dl, ornithine decarboxylase, or vascular endothelial growth factor ("VEGF”) (otherwise known as vascular permeability factor (“VPF”)) genes.
- VEGF vascular endothelial growth factor
- the UTR sequence comprise a natural or synthetic hairpin conformation with a stability of at least about ⁇ G > 50 Kcal/mol.
- the concentration of eIF4E required to allow translation of mRNAs that would normally be repressed by the present of the complex 5' UTRs is at least 1.5 times the concentration found in normal eukaryotic cells. In another embodiment, the concentration of eIF4E required to allow translation of mRNAs that would normally be repressed by the present of the complex 5' UTRs is at least 2, 2.2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, S, 9, or 10 times the concentration found in normal eukaryotic cells.
- the DNA sequence comprises the 5'-UTR is a natural or synthetic conformation with a stability of at least about ⁇ G > an absolute 50 Kcal/mol. In one embodiment, the DNA sequence comprises the 5'-UTR is a natural or synthetic conformation with a stability of at least about ⁇ G > an absolute 55 Kcal/mol.
- the UTR sequence comprises at least 30, 40, 50, 60, 70, 80,
- the UTR is an oligonucleotide sequence capable of forming a duplex.
- the UTR is a palindromic oligonucleotide sequence capable of forming a duplex.
- the UTR is an oligonucleotide sequence capable of forming a stable complex formation.
- the stable complex includes loop formations.
- the free energy " ⁇ G” is the free energy of an oligonucleotide, which is a measurement of an oligonucleotide duplex stability.
- the strength ( ⁇ G) of the resulting complexes is measured by thermal denaturation or duplex melting.
- the ⁇ G can either be expressed as a negative or a positive number depending upon whether you are looking at the stability as a measurement of free energy stored in the structure (negative) or free energy required to melt the duplex (positive).
- Energy must be released overall to form a base-paired structure, and a structure's stability is dete ⁇ nined by the amount of energy it releases.
- free energy stored in the structure is a negative value, then the complex formed is in the thermodynamically stable form.
- Predicted enthalpy, entropy and free energy of duplex formation are thermodynamic state functions, related by the Gibbs equation:
- T is the temperature in degrees K. hi practice
- the enthalpy and entropy are predicted via a thermodynamic model of duplex formation and used to calculate the free energy and melting temperature.
- the predicted free energy of an oligonucleotide that contains self-complementary sequences that can form intramolecular secondary structures is calculated as the most stable intramolecular structure of an oligonucleotide.
- "Secondary structure” refers to regions of a nucleic acid sequence that, when single stranded, have a tendency to form double-stranded hairpin structures or loops.
- Nucleic acids can be evaluated for their likely secondary structure by calculating the predicted ⁇ G of folding of each possible structure that could be formed in a particular strand of nucleic acid.
- One example is the MFOLD program.
- the ⁇ G as referred to in the specification and claims herein is given in absolute energy change value and is evident from the context by one skilled in the art.
- a folded state free energy a negative number
- the more negative the ⁇ G i.e., the lower the free energy
- the stability of a secondary structure is quantified as the amount of free energy released or used by forming base pairs or the input energy required to melt such secondary structure, which in the present case would have to be > 50 Kcal/Mol.
- the present description describes the required to melt the secondary structure since a structure having a positive free energy requires work to form a configuration arid hence would be unstable and not form the required structure.
- Negative free energies release stored work. When quantified as the amount of free energy released or used by forming base pairs, the more negative the free energy of a structure, the more likely is formation of that structure, because more stored energy is released.
- the stability of the oligonucleotides of the present invention can be described as "wherein the untranslated sequence further comprises a hairpin secondary structure conformation having a stability measured as folded state free energy of ⁇ G ⁇ about -50 Kcal/Mol" instead of in terms of absolute energy change.
- the protein eIF4E is the cap-binding subunit of the eIF4F complex, an ATP- dependent helicase that unwinds "excess" secondary structure in the 5' untranslated region (UTR) of mRNAs.
- the low-abundance of eIF4E/F is the limiting factor for the translation of some mRNAs, particularly those with long, G/C-rich 5'-UTRs with the potential to form a stable, secondary structure. .MJ. Clemens et al., "Translational control: the cancer connection," Int. J. Biochem. Cell Biol., vol. 31, pp. 1-23 (1999).
- UTR many of which encode factors required for cell proliferation, e.g., protooncogene c-myc, cyclin Dl, ornithine decarboxylase, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor ("VEGF,” otherwise known as vascular permeability factor, "VPF”).
- FGF-2 fibroblast growth factor-2
- VEGF vascular endothelial growth factor
- VPF vascular endothelial growth factor
- the present invention provides for a novel gene therapy for cancer, which unlike most prior approaches, does not require specific knowledge of the cancer cells, but instead targets a general characteristic that distinguishes cancer cells from normal cells, i.e., elevated eIF4E expression.
- the present invention provides conditionally replicative adenoviruses (CRAds) as therapeutic agents applied to cancer treatment.
- CRAds conditionally replicative adenoviruses
- cancer-specific replication of CRAds result in viral-mediated oncolysis of infected tumor tissues and release of the vims progeny, capable of further propagating in surrounding tumor cells but not in those of normal tissues, which would be refractory to CRAd replication.
- any of the above gene therapy/virotherapy approaches is fundamentally based on the ability of vector to deliver the therapeutic gene or replication-competent viral genome to target cells with a requisite level of efficiency.
- conditionally replicative adenoviruses represent a method to achieve efficient tumor cell oncolysis and mitigate tumor cell infection limitations.
- overall efficacy of Ad-based cancer gene therapy/virotherapy approaches remains limited by sub-optimal vector delivery efficiency in cancer tissues.
- human trials carried out to date have demonstrated relatively inefficient gene transfer to tumor cells in contexts whereby non-replicative adenoviruses have been employed using in vivo delivery schemas.
- the requirement for quantitative in vivo tumor transduction is the key issue that has to be addressed for further development of the cancer gene therapy/virotherapy field.
- augmenting the gene transfer efficacy of adenoviruses for cancer targets is an important tool for deriving their full benefit.
- transductional targeting alone does not allow sufficient cancer selectivity for adenoviruses, due to lack of known cell surface receptors (cell surface markers) highly specific for cancer cells that are not also present on normal cells.
- Another fact that has limited effective tumor cell transduction in the context of cancer gene therapy/virotherapy approaches is that the adenoviruses employed have been rendered replication-incompetent. In this case, tumor cell infection is a terminal event, whereby post-infection viral replication (and consequential amplification) does not occur.
- One conceptual approach to achieve an amplification effect is via selective replication of the delivered viral vector post-infection such that lateral spread of the progeny vector may occur. In this approach, a conditionally replication-competent virus replicates in transduced tumor cells and not in normal tissue.
- virus progeny from transduced tumor cells would then allow infection of the neighboring tumor cells (Fig. 1). Additionally, utilization of viruses, which replicate through out a lytic cycle, would result in viral-mediated oncolysis, a process that is of therapeutic utility.
- replicative viral systems have been utilized as novel anti-tumor therapies. Wild-type or attenuated viruses with the ability to replicate within specific tissues have been used in human clinical trials to achieve specific oncolysis of various neoplasms. Those viruses include: adenovirus [4], mumps virus [5,6], and West Nile virus [7].
- Adenoviruses possess unique attributes fundamental for development as conditionally replicating anti-tumor agents
- Ad vector as an anti-tumor therapy agent
- this virus possesses a lytic life cycle, which can be exploited for oncolysis.
- Bischoff et al. [12] have recently employed Conditionally Replicative Adenovirus (CRAd) defective in early gene regions to accomplish tumor-specific oncolysis.
- CRAd Conditionally Replicative Adenovirus
- dll520 (ONYX-015) is an E1B-55 kDa gene deleted CRAd that has been demonstrated to replicate in and selectively destroy tumor cells such as ovarian cancer, which lack p53 function [12].
- ONYX-015 both in vitro and in intraperitoneally treated murine models of ovarian cancer [13.14].
- ONYX- 015 has undergone extensive testing in the clinic, and has proven safe with promising signs of efficacy [4,15]. This experience thus established the concept that CRAd systems can accomplish a significant anti-tumor effect. A more detailed knowledge of the Ad replication cycle should make it feasible to develop advanced CRAds that achieve therapeutic oncolysis over-and-above that achieved with the first generation ONYX-015 CRAd system.
- CXCR4 gene a receptor for stromal derived factor- 1 (SDF-I), a member of the CXC-group of small chemo attractive cytokines (chemokines).
- SDF-I stromal derived factor- 1
- chemokines small chemo attractive cytokines
- a reporter gene driven by the CXCR4 promoter in the context of replication-deficient Ad vector shows the highest expression level among other TSPs in several HNSCC cell lines.
- the CXCR4 promoter is even more active than CMV promoter in the same Ad vector context (Fig. 3). Therefore, CXCR4 promoter activity should be high enough to drive efficient expression of El genes in the CRAd context.
- Improvement of the adenovirus vector based on targeting of ElA mRNA translation to cancer cells regulation of protein synthesis for restriction of transgene expression to cancer cells
- Protein synthesis is energetically the most expensive process in the cell, and not surprisingly, translation rates are tightly regulated [22]. In mammals, most of the regulation operates at the level of translation initiation, rather than elongation or termination [23,24].
- the initiation process is comprised of three steps: 1) formation of the 43 S complex, composed of a 4OS ribosomal subunit and the initiation factors eIF-2, eIF-3, Met-tRNA, and GTP; 2) formation of the 48S complex containing mRNA; and 3) joining of the 60S subunit to form the complete 80S complex. In most circumstances, the second step is rate limiting and hence, subject to regulation.
- This step is also a point of discrimination, since one particular mRNA is selected from the untranslated pool of messages and recruited to the ribosomes.
- This process is mediated by the eIF-4 group of factors, of which, eIF4E is the least abundant and rate limiting [25-27]
- eIF4E is the least abundant and rate limiting [25-27]
- the importance of protein synthesis in growth regulation and the role of eIF4E in this process was confirmed by the observation that overexpression of eIF4E causes malignant transformation of cells in culture [28,29].
- each protein ultimately depends on the relative abundance of its mRNA and its intrinsic translatability, i.e., the capacity of that particular mRNA to interact with components of the translation initiation machinery. This property of the translation initiation process establishes an order of priorities among the different mRNAs to be translated. Such a hierarchy in protein synthesis is extremely important for gene expression. In eukaryotes, the flow of information from genes to proteins is too slow to accommodate rapid changes in the environment. Eukaryotes compensate for this problem by maintaining a pool of mRNAs that are not immediately utilized.
- mRNAs may, for instance, encode growth factors which can be rapidly produced under conditions that require cells to re-enter rapid division [30,31].
- a theoretical treatment of mRNA competition for translation [32] identified two types of structurally different mRNAs: weak and strong. Weak mRNAs, about 5- 10% of the total, are translationally repressed in quiescent cells; weak and strong mRNAs are translated in rapidly proliferating cells [33,34] and in cancerous cells in particular, due to a change in the translation initiation capacity.
- the eIF4E specifically binds to the 7-methylguanosine-containing cap of the mRNA in the first step of mRNA recruitment for translation [35].
- the eIF4E is also a subunit of the eIF-4F complex, a helicase that unwinds the secondary structure at the 5'-UTR of mRNA. This latter function is critical during "scanning" for exposing and locating the translation start site [36-40].
- the low abundance of eIF4E creates a situation of competition among different mRNA species, such that mRNAs with long and highly structured 5'-UTRs (weak) are out competed for binding to ribosomes by the strong mRNAs [41].
- eIF4E activity a mechanism that is based largely on altered translational control (eIF4E activity) has evolved to respond quickly to the imperative of achieving adequate oxygenation [30]. Elevated eIF4E results in drastically increased synthesis of basic fibroblast growth factor (FGF2) [48,31] and vascular endothelial growth factor (VEGF) [49], both of which are encoded by mRNAs with long and complex 5'-UTRs. Indeed, there is a strong correlation between the expression of eIF4E and VEGF in tumor biopsies [50].
- FGF2 basic fibroblast growth factor
- VEGF vascular endothelial growth factor
- FGF2 and VEGF are the two most powerful mitogens for vascular endothelia and are essential for tumor vascularization [51].
- the up-regulation of eIF4E could also be a pre-requisite for establishing greater protein synthesis outputs, possibly a necessary development for cancer cells to sustain their rapid proliferation.
- eIF4E in cancer and as a target for gene therapy/virotherapy we now know that overexpression of eIF4E is ubiquitous in solid tumors and malignant cell lines [52-55]. Deregulation of eIF4E appears to be a pre-requisite for vascularization of the primary tumor [52]. Reduction of eIF4E with antisense RNA in a breast carcinoma line crippled its angiogenic and tumorigenic properties, along with the capacity to synthesize FG F2 [31]. It is important to emphasize that elevated eIF4E does not always correlate with rapid cell division. Even cells from the intestinal mucosa, which have rapid turnover rates, do not have high levels of eIF4E, although colon carcinomas do [53].
- TSPs tissue- or tumor-specific promoters
- the present invention provides for dual-level of cancer targeting to overcome any possible leakage of the CXCR4 promoter in normal tissues.
- the dual-level cancer targeting is further combined with infectivity enhancement/transductional re-targeting of CRAds to Ad3 receptor achieved by serotype chimerism technology of fiber modification developed previously [56- 58].
- the cassettes in the shuttle vectors are flanked by Ad5 sequences located right upstream and downstream of the El region (right and left arm homology sequences) in the Ad5 genome and used to insert the shuttle vector cassette in place of El by homologous recombination. Since the genomes of such recombinant Ad vectors derived from pVK400 contain the wild-type fiber gene, no additional homologous recombination steps were necessary to create replication defective recombinant Ad vectors expressing the reporter or ElA genes.
- FIG. 4 shows three tumor samples, Tl, T2, and T3 and T2, and the matched adjacent histologically normal mucosa, Ml, M2, and M3, obtained from surgical resection margins. All three tumor samples (T) overexpressed eIF4E; of the three margins (M) shown, two margins were negative for eIF4E, while one (Ml) expressed eIF4E at low but detectable levels. CXCR4 levels were high in all tumor samples and margins. Although CXCR4 was detected in both tumor and matched normal tissues, Western blot analysis showed strong increased expression of eIF4E in tumors compared with adjacent mucosa.
- Ad5 Ad-wt-dE3 in two different breast cancer cell lines.
- Each of the two CRAd agents tested used the CXCR4 tumor specific promoter to regulate expression of the adenovirus ElA.
- Ad-CXCR4-E1A a wild-type ElA transcript
- Ad-CXCR4-UTR-E1A a second construct expressed the 5'-UTR of FGF2 inserted upstream of the ElA open reading frame
- adenovirus type 5 (Ad5)-based conditionally replicative adenoviruses have a natural liver tropism
- any background activity of cancer-specific promoters in normal tissues (and subsequent liver toxicity) presents a particularly critical problem that warrants development of novel approaches to improve cancer specificity of CRAds.
- any subtype mixture of subtypes, or chimeric adenovirus can be used as the source of the viral genome for generation of an adenoviral vector in conjunction with the present invention.
- the genome of a human serotype adenovirus is used, such as a type 2 (Ad2) or type 5 (Ad5) adenoviral genome.
- Ad5 adenoviral genome is most preferred, and the present invention is described further herein with respect to the Ad5 serotype.
- the adenoviral genome used in conjunction with the present invention is replication deficient in addition to conditionally replicative in a cell with conditions with an overexpression of eIF4E. This would provide a second level of defense against undesirable replication in non-target cells.
- a deficiency in a gene, gene function, or gene or genomic region, as used herein, is defined as a deletion of sufficient genetic material of the viral genome to impair or obliterate the function of the gene whose nucleic acid sequence was deleted in whole or in part and to provide room in, or capacity of, the viral genome for the insertion of a nucleic acid sequence that is foreign to the viral genome.
- Such a deficiency can be in a gene or genome region essential or unessential for propagation of the adenoviral vector in a non-complementing cellular host.
- a deficiency in an adendviral genome region essential for such propagation e.g., early region 1 (El), early region 2A (E2A), early region 2B (E2B), early region 4 (E4), late region 1 (Ll), late region 2 (L2), late region 3 (L3), late region 4 (L4), and late region 5 (L5) renders an adenoviral vector based on that adenoviral genome replication deficient.
- the adenoviral vector of the present invention may be multiply replication deficient, i.e., it is deficient in at least two genome regions required for viral propagation in a non-complementing cellular host (i.e., viral replication in vitro.
- regions include the El, E2, E4, or L1-L5 regions.
- ElA early region IA
- ElB early region IB
- a deficiency in either or both of the ElA and/or ElB regions is considered as a single deficiency in the context of the present invention.
- such a vector can be deficient in one or more regions that are not required for viral propagation, e.g., the vectors can be additionally deficient in early region 3 (E3).
- the present invention is not limited to adenoviral vectors that are deficient in gene functions only in the early region of the genome. Also included are adenoviral vectors that are deficient in the early and late regions of the genome, as well as vectors in which essentially the entire genome has been removed, in which case it is preferred that at least either the viral ITRs and some of the promoters or the viral ITRs and a packaging signal are left intact.
- the vector comprises at least one expression cassette which includes (i.e., comprises) a nucleic acid sequence coding for a toxin.
- a nucleic acid sequence coding for a toxin is described in detail in U.S. Pat. No. 6,759,394.
- the nucleic acid sequence encoding a toxin further comprises a transcription-terminating region such as a polyadenylation sequence.
- Any suitable polyadenylation sequence can be used, including a synthetic optimized sequence, as well as the polyadenylation sequence of BGH (Bovine Growth Hormone), polyoma vims, TK (Thymidine Kinase), EBV (Epstein Barr Vims), and the papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus).
- BGH Bovine Growth Hormone
- polyoma vims polyoma vims
- TK Thymidine Kinase
- EBV Epstein Barr Vims
- papillomaviruses including human papillomaviruses and BPV (Bovine Papilloma Virus).
- TK Thimidine Kinase
- EBV Epstein Barr Vims
- papillomaviruses including human papillomaviruses and BPV (Bovine Papill
- the nucleic acid sequence encoding a toxin is operably linked to (i.e., under the transcriptional control of) one or more promoter and/or enhancer elements, for example, as part of a promoter variable expression cassette.
- promoter and/or enhancer elements for example, as part of a promoter variable expression cassette.
- Techniques for operably linking sequences together are well known in the art. Any suitable promoter or enhancer sequence can be used in conjunction with the present invention. Suitable promoters and enhancer sequences are generally known in the art.
- the present invention provides compositions and methods of producing a conditionally replicative adenoviral vector comprising (a) providing an adenoviral genome , (b) inserting a nucleic acid sequence DNA sequence comprising a promoter operatively linked to a transcription sequence; wherein the transcription sequence, when transcribed, produces a messenger RNA sequence that comprises a translatable sequence encoding a viral particle, and an untranslated sequence; wherein the untranslated sequence inhibits translation of the viral sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E;; and wherein the untranslated sequence allows translation of the toxin sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eEF4E relative to normal cells.
- the untranslated sequence further comprises a hairpin secondary structure conformation having a stability measured as folded state free energy of ⁇ G ⁇ about -50 Kcal/Mol.
- the present invention provides for a replication competent adenbvirus-free stock of the viral vector of the present invention.
- the present invention provides for a pharmaceutical composition comprising the viral vector of the present invention and a pharmaceutically acceptable earner, wherein the pharmaceutical composition does not contain replication-competent adenoviruses.
- the present invention provides for a host cell comprising the viral vector of the present invention.
- the present invention provides for a method of treating a tumor or cancer in a mammal comprising administering an anti-tumor or anticancer effective amount of the conditionally replicative viral vector of the present invention directly to the tumor or cancer of the mammal.
- the an anti-tumor effective amount of the viral vector is administered to a tumor in a mammal.
- the present invention provides for a method of treating a cell proliferation disease comprising administering to a subject in need of such treatment a therapeutically-effective amount of the compositions of the present invention.
- the administering is in an amount effective to inhibit cell growth
- the DNA sequence is administered by administering an expression vector encoding the DNA sequence to cells.
- the expression vector is delivered within a liposomal construct.
- the expression vector is delivered within a host cell.
- the expression vector is a viral vector.
- the untranslated sequence allows translation of the viral sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E at least 2-fold greater relative to normal cells.
- the vector further comprises a sequence encoding for a toxin.
- the encoded toxin is a conditional toxin.
- the encoded conditional toxin is a herpes thymidine kinase.
- the present invention provides for a method for selectively expressing a viral particle within a cell comprising administering to the cell a messenger RNA sequence that comprises a translatable sequence encoding a virus, and an untranslated sequence; wherein the untranslated sequence inhibits translation of the viral sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E and wherein the untranslated sequence allows translation of the viral sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E relative to normal cells.
- the untranslated sequence comprises an mRNA sequence with a secondary structure conformation having a stability measured as folded state free energy of ⁇ G ⁇ about -50 Kcal/Mol.
- the administering is in an amount effective to inhibit cell growth.
- the messenger RNA sequence is administered by administering an expression vector encoding the messenger RNA sequence.
- the viral vector is delivered within a host cell.
- the present invention provides for a method of treatment for cancer in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of a DNA sequence comprising a promoter operatively linked to a transcription sequence; wherein the transcription sequence, when transcribed, produces a messenger RNA sequence that comprises a translatable sequence encoding a virus, and an untranslated sequence; wherein the untranslated sequence inhibits translation of the viral sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E;; and wherein the untranslated sequence allows translation of the viral sequence under conditions that exist within rumor cells that overexpress eukaryotic initiation factor eIF4E; relative to normal cells.
- the untranslated sequence allows translation of the viral sequence under conditions that exist within tumor cells that overexpress eukaryotic initiation factor eIF4E at least 2-fold greater relative to normal cells.
- the untranslated sequence allows translation of the viral sequence within tumor cells in which the presence of eukaryotic initiation factor eIF4E allows the translation of the virus, the vims is translated to kill the tumor cells.
- the majority of non-tumor cells in the mammal are not killed due to the low levels of eukaryotic initiation factor eIF4E typically present in non-rumor cells.
- the cancer is a metastatic tumor.
- the cancer is a solid tumor.
- the metastatic tumor is associated with a mammalian cancer selected from the group consisting of bladder, breast, cervical, colon, lung, prostate, and head and neck.
- the present invention also provides a method of producing an adenoviral vector comprising (a) providing an adenoviral genome and (b) inserting into the adenoviral genome a nucleic acid sequence DNA sequence comprising a promoter operatively linked to the virus transcription sequence; wherein the transcription sequence, when transcribed, produces a messenger RNA sequence that comprises a translatable sequence encoding a viral particle, and an untranslated sequence; wherein the untranslated sequence inhibits translation of the viral sequence under conditions that exist within normal mammalian cells that do not overexpress eukaryotic initiation factor eIF4E and wherein the untranslated sequence allows translation of the viral sequence under conditions that exist within mammalian cells that overexpress eukaryotic initiation factor eIF4E relative to no ⁇ nal cells.
- the method provided by the present invention can include other steps or elements, such as the insertion of other nucleic acid sequences into, or deletion of such sequences from, the viral genome used to provide the viral vector.
- the various aspects of the present inventive method e.g., the viral genome, nucleic acid sequences coding for 5'- UTR sequence, etc. are as previously described herein with respect to the viral vector of the present invention.
- virus vector construction relies on the high level of recombination between adenoviral nucleic acid sequences in a cell.
- Two or three separate viral nucleic acid sequences e.g., DNA fragments
- regions of similarity or overlap
- the host cell's recombination machinery constructs a full-length viral vector genome by recombining the aforementioned sequences.
- a preferred method of constructing the present inventive adenoviral vector first involves constructing the necessary deletions or modifications (such as adding a spacer element to a deleted region) of a particular region of the adenoviral genome. Such modifications can be performed, for example, in a plasmid cassette using standard molecular biological techniques. The altered nucleic acid sequence (containing the deletion or modification) then is moved into a much larger plasmid that contains up to one-half of the virus genome to provide a base plasmid comprising the modified adenoviral genome. The next step is to insert an expression cassette into a desired region of the modified adenoviral genome.
- the expression cassette can be provided by standard methods known in the art, for example, by isolating the cassette from a plasmid.
- the isolated cassette then can be transfected with the plasmid DNA (containing the modified adenoviral genome) into a recipient cell.
- the plasmid is, optionally, linearized prior to transfection by digestion with a suitable restriction enzyme to facilitate the insertion of the expression cassette at a desired position in the adenoviral genome. Selection of a suitable restriction enzyme is well within the skill of the ordinary artisan.
- the two pieces of DNA recombine to form a plasmid comprising the modified adenoviral genome and the expression cassette. .
- the plasmid is isolated from the host cell and introduced into recipient cell that complements for the missing viral functions of the recombined viral genome to produce the adenoviral vector comprising the modified viral genome and the expression cassette.
- the vector can be further modified by alteration of the ITR and/or packaging signal.
- the open reading frame for a virus capable of inducing apoptosis is functionally linked to regulatory DNA sequences in such a manner that the virus is constitutively expressed in a cell into which the recombinant virus is introduced.
- the expression of the virus is driven by a constitutive or stable promoter.
- the present invention does not dictate the choice of the stable promoter.
- the type of promoter is chosen to accomplish a useful expression profile in the context of the recombinant virus.
- Non-limiting examples of useful promoters for this variation of the invention include the Cytomegalovirus (CMV) immediate early promoter, The Simian Virus 40 (SV40) immediate early promoter, and promoters from eukaryotic household genes.
- CMV Cytomegalovirus
- SV40 Simian Virus 40
- the open reading frame is functionally linked to one or more control sequences, i.e. regulatory DNA sequences, in such a manner that the virus is only expressed or is expressed in a cell into which the recombinant adenovirus is introduced under certain conditions that can be modulated by an external signal, where the term "external" means having its origin outside of the DNA fragment encompassing the open reading frame and the regulatory DNA sequences.
- the expression of the virus is driven by a so-called regulatable or inducible promoter.
- the external signal include, but are not limited to, the addition or deprivation of a chemical compound, a shift in temperature, a decreased oxygen concentration, irradiation, and the like.
- Non-limiting examples of this kind of promoter include the heat shock protein 70 promoter, the promoter of an acute phase protein gene, such as the serum amyloid A3 gene or the complement factor 3 gene, the early growth response protein 1 promoter, the multidrug resistance gene 1 promoter, and promoters comprising one or more hypoxia-responsive elements, and fragments thereof (Kohno et al., Biochem. Biophys. Res. Comm. 165(1989):1415-1421; Varley et al., Proc. Natl. Acad. Sci. USA 92(1995):5346-5350; Hallahan et al., Nature Med. 1(1995):786-791; Dachs et al., Nature Med.
- a special kind of regulatable promoter is a tissue- or cell type-specific promoter, where the external signal is provided by a protein that is only present in a particular type of cell or tissue.
- tissue- or cell-type specific promoters are the prostate specific antigen promoter, the alpha-fetoprotein promoter, the albumin promoter, the carcinoembryonic antigen promoter, the cytokeratin 18 gene promoter, the kallikrein 2 promoter, the tyrosinase promoter, the osteocalcin promoter, the PAX-5 promoter and the alpha-lactalbumin promoter (Kaneko et al., Cancer Res. 55(1995):5283-5287; Richards et al., Hum. Gene Ther. 6(1995):881- 893; Kozmik et al., Proc. Natl. Acad. Sci.
- regulatable promoter is a promoter that is responsive to an external signal that is provided by a protein that is not present in a particular type of cell or tissue.
- external signals that are absent in liver tissue are of interest in the context of in vivo administration of recombinant adenoviruses.
- promoters that are responsive to external signals that are absent in liver tissue are the cyclooxygenase-2 promoter and the midkine promoter (Adachi et al, Cancer Res. 60(2000):4305-4310; Yamamoto et al., MoI. Ther. 3(2001):385-394).
- regulatable promoter is a promoter that is responsive to an external signal that is provided by a protein that is only present during a certain stage of the cell cycle.
- a non-limiting example of this kind of promoter is the promoter of a gene that is responsive to E2F, such as for example the adenovirus E2 gene or the E2F-1 gene.
- Another, not mutually excluding, special kind of regulatable promoter is a so-called transactivation response element (TRE).
- the TRE is a first component of a transactivation system that comprises as a second component a transactivator protein, that is capable of binding with specificity to the TRE, thereby regulating the transcription of a gene linked to the TRE.
- the open reading frame is functionally linked to regulatory DNA sequences in such a manner that the virus is only expressed in a cell into which the recombinant virus is introduced during the late phase of virus replication.
- Expression of the virus confined to the late phase of virus replication is of particular interest in the context of a CRAd. Since the replication will only occur in cells in which certain conditions exist that are exploited by the CRAd to allow the replication, expression of the virus will also be confined to the cells in which the certain conditions exist. This variation of the invention will thus add to the specificity of the CRAd.
- it is preferred that expression of the virus is driven by the adenovirus major late promoter (MLP).
- MLP adenovirus major late promoter
- the expression cassette for the open reading frame comprises the in cis acting sequences required to confer full transcriptional activity of the MLP during the late phase of adenovirus replication as defined by Mondesart et al. (Nucleic Acids Res. 19(1991):3221-3228), included by reference herein.
- a useful expression cassette for this aspect of the invention was disclosed in U.S. Pat. No. 5,518,913, included by reference herein.
- the open reading frame is functionally linked to the endogenous MLP.
- the present invention provides a method of treating a tumor or cancer in a host comprising administering an anti-cancer or anti-tumor effective amount of the viral vector of the present invention to a host in need thereof.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising the viral vector of the present invention and a earner, especially a pharmaceutically acceptable (e.g., a physiologically or pharmacologically acceptable) earner (e.g., excipient or diluent).
- a pharmaceutically acceptable e.g., a physiologically or pharmacologically acceptable
- earner e.g., excipient or diluent.
- suitable formulations of the pharmaceutical composition of the present invention The following formulations and methods are merely exemplary and are in no way limiting. However, oral, injectable and aerosol formulations are preferred.
- Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effectn ⁇ e amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
- Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
- Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
- a flavor usually sucrose and acacia or tragacanth
- pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
- the vectors of the present invention can be made into aerosol formulations to be administered via inhalation.
- aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also can be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.
- Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the fo ⁇ nulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
- the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
- Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
- the vectors employed in the present invention can be made into suppositoiies by mixing with a vaiiety of bases such as emulsifying bases or watersoluble bases.
- bases such as emulsifying bases or watersoluble bases.
- Formulations suitable for vaginal admini stration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such earners as are known in the art to be appropriate.
- the dose administered to an animal, particularly a human, in the context of the present invention will vary with the particular viral vector, the composition containing the viral vector, the method of administration, and the particular site and organism being treated.
- the dose should be sufficient to effect a desirable response, e.g., therapeutic or prophylactic response, within a desirable time frame.
- the dose and dosage regimen will depend upon the nature of the cancer (primary or metastatic) and its population, the characteristics of the particular immunotoxin, e.g., its therapeutic index, the patient, the patient's history and other factors.
- the amount of virus administered will typically be in the range of about 10 10 to about 10 1 ' viral particles per patient.
- the schedule will be continued to optimize effectiveness while balanced against negative effects of treatment. See Remington's Pharmaceutical Science, 17th Ed. (1990) Mark Publishing Co., Easton, Penn.; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press; which are incorporated herein by reference.
- the present method of treating a tumor or cancer in a host further can comprise the administration (i.e., pre-administration, co-administration, and/or post- administration) of other treatments and/or agents to modify (e.g., enhance) the effectiveness thereof.
- the replication deficient viral vectors of the present invention also have utility in vitro. For example, they can be used to study viral gene function and assembly, the production of TNF, or the expression of other foreign nucleic acid sequences in a suitable target cell.
- One of ordinary skill can identify a suitable target cell by selecting one that can be transfected by the viral vector, resulting in expression of the thereby inserted viral nucleic acid sequence complement.
- a suitable target cell is selected that has receptors for attachment and penetration of virus into a cell.
- Such cells include, but are not limited to, those originally isolated from any mamma.
- the target cell is contacted with viral vector of the present invention, thereby effecting transfection or infection, respectively.
- Expression, toxicity, and other parameters relating to the insertion and activity of the nucleic acid sequence, or other foreign nucleic acid sequences, in the target cell then is measured using conventional methods well known in the art.
- cells explanted or removed from a patient having a disease that is suitably treated by gene therapy in the context of the present invention usefully are manipulated in vitro.
- cells cultured in vitro from such an individual are placed in contact with viral vector of the present invention under suitable conditions to effect transfection, which are readily determined by one of ordinary skill in the art.
- Such contact suitably results in transfection of the vector into the cultured cells, where the transfected cells are selected using a suitable marker and selective culturing conditions.
- the cells of the individual are tested for compatibility with, expression in, and toxicity of the vector of the present invention, thereby providing information as to the appropriateness and efficacy of treatment of the individual with the vector system so tested.
- explanted and transfected cells in addition to serving to test the potential efficacy/toxicity of a given gene therapy regime, also can be returned to an in vivo position within the body of the individual.
- Such cells so returned to the individual can be returned unaltered and unadorned except for the in vitro transfection thereof, or encased by or embedded in a matrix that keeps them separate from other tissues and cells of the individual's body.
- a matrix can be any suitable biocompatible material, including collagen, cellulose, and the like.
- the transfection can be implemented in vivo by administration means as detailed hereinabove.
- the viral vector, and methods involving the same, provided by the present invention can comprise any combination or permutation of the elements described herein.
- nucleic acid sequence many modifications and variations of the present illustrative nucleic acid sequence are possible.
- the degeneracy of the genetic code allows for the substitution of nucleotides throughout coding regions, as well as in the translational stop signal, without alteration of the encoded polypeptide coding sequence.
- substitutable sequences can be deduced from the known amino acid or nucleic acid sequence of a given gene and can be constructed by conventional synthetic or site-specific mutagenesis procedures. Synthetic DNA methods can be carried out in substantial accordance with the procedures of Itakura et al, Science, 198, 1056-1063 (1977), and Crea et al., Proc. Natl. Acad. Sci. USA, 75, 5765-5769 (1978).
- the vectors described also can comprise genes used to treat other similar or different diseases and/or afflictions including, but not limited to, other chronic lung diseases, such as emphysema, asthma, adult respiratory distress syndrome, and chronic bronchitis, as well as coronary heart disease, and other afflictions suitably treated or prevented by gene therapy, vaccination, and the like. Accordingly, any gene or nucleic acid sequence can be inserted into the viral vector.
- the viral vector can be modified in other ways without departing from the scope and spirit of the present invention.
- the coat protein of the present inventive viral vector can be manipulated to alter the binding specificity or recognition of the virus for a viral receptor on a potential host cell.
- Such manipulations can include deletion of regions of the fiber, penton, or hexon, insertions of various native or non-native ligands into portions of the coat protein, and the like.
- Manipulation of the coat protein can broaden the range of cells infected by a viral vector or enable targeting of a viral vector to a specific cell type.
- the vector can comprise a chimeric coat protein (e.g., a fiber, hexon or penton protein), which differs from the wild-type (i.e., native) coat protein by the introduction of a nonnative amino acid sequence, preferably at or near the carboxyl terminus.
- a nonnative amino acid sequence is inserted into or in place of an internal coat protein sequence.
- the resultant chimeric viral coat protein is able to direct entry into cells of the viral vector comprising the coat protein that is more efficient than entry into cells of viral vector that is identical except for comprising a wild-type viral coat protein rather than the chimeric viral coat protein.
- the chimeric virus coat protein desirably binds a novel endogenous binding site present on the cell surface.
- a result of this increased efficiency of entry is that the viral virus can bind to and enter numerous cell types which a virus comprising wild-type coat protein typically cannot enter or can enter with only a low efficiency.
- the viral vector of the present invention can comprise a chimeric virus coat protein that is not selective for a specific type of eukaryotic cell.
- Such chimeric coat protein differs from the wild-type coat protein by an insertion of a normative amino acid sequence into or in place of an internal coat protein sequence.
- the virus coat efficiently binds to a broader range of eukaryotic cells than a wild-type vims coat, such as described in International Patent Application WO 97/20051.
- Specificity of binding of virus to a given cell also can be adjusted by use of virus comprising a short-shafted viral fiber gene, as discussed in U.S. Pat. No. 5,962,311.
- Use of virus comprising a short-shafted viral fiber gene reduces the level or efficiency of viral fiber binding to its cell-surface receptor and increases viral penton base binding to its cell-surface receptor, thereby increasing the specificity of binding of the virus to a given cell.
- use of virus comprising a short-shafted fiber enables targeting of the virus to a desired cell- surface receptor by the introduction of a nonnative amino acid sequence either into the penton base or the fiber knob.
- the ability of a viral vector to recognize a potential host cell can be modulated without genetic manipulation of the coat protein.
- complexing vims with a bispecific molecule comprising a penton base-binding domain and a domain that selectively binds a particular cell surface binding site enables one of ordinary skill in the art to target the vector to a particular cell type.
- Suitable modifications for viral vector include those modifications described in U.S. Pat. Nos. 5,559,099; 5,731,190; 5,712,136; 5,770,442; 5,846,782; 5,926,31 1; and 5,965,541 and International Patent Applications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO 98/07877, and WO 98/54346.
- the vims may be modified by incorporation of mutated coat proteins into the virion outer capsid.
- the vims is a vims modified to reduce or eliminate an immune reaction to the vims.
- Such modified virus are termed "immunoprotected vims".
- Such modifications could include packaging of the virus in a liposome, a micelle or other vehicle to mask the virus from the mammals immune system.
- Neoplasms that are particularly susceptible to treatment by the methods of the invention include breast cancer, central nervous system cancer (e.g., neuroblastoma and glioblastoma), peripheral nervous system cancer, lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, lymphoma and leukemia.
- central nervous system cancer e.g., neuroblastoma and glioblastoma
- peripheral nervous system cancer e.g., central nervous system cancer
- lung cancer e.g., prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, lymphoma and leukemia.
- the virus is administered to a proliferating cell or neoplasm in a manner so that it contacts the proliferating cells or cells of the neoplasm or neoplastic cells.
- the route by which the virus is administered, as well as the formulation, carrier or vehicle, will depend on the location as well as the type of the neoplasm.
- a wide variety of administration routes can be employed.
- the virus can be administered by injection directly to the neoplasm, or a hematopoietic neoplasm, for example, the vims can be administered intravenously or intravascularly.
- the virus is administered in a manner such that it can be transported systemically through the body of the mammal and thereby reach the neoplasm (e.g., intrathecally, intravenously or intramuscularly) .
- the vims can be administered directly to a single solid neoplasm, where it then is carried systemically through the body to metastases.
- the vims can also be administered subcutaneously, intraperitoneally, topically (e.g., for melanoma), orally (e.g., for oral or esophageal neoplasm), rectally (e.g., for colorectal neoplasm), vaginally (e.g., for cervical or vaginal neoplasm), nasally or by inhalation spray (e.g., for lung neoplasm).
- Vims can be administered systemically to mammals which are immune compromised or which have not developed immunity to the virus epitopes.
- virus administered systemically i.e. by intraveneous injection, will contact the proliferating cells resulting in lysis of the cells.
- Immunocompetent mammals previously exposed to a particular virus may have developed humoral and/or cellular immunity to that virus. Nevertheless, it is contemplated that direct injection of the virus into a solid tumor in immunocompetent mammals will result in the lysis of the neoplastic cells.
- the mammals may produce an immune response to the vims.
- an immune response may be avoided if the vims is of a subtype to which the mammal has not developed immunity, or the virus has been modified as previously described herein such that it is immunoprotected, for example, by protease digestion of the outer capsid or packaging in a micelle.
- the vims may be administered to immunocompetent mammals immunized against the virus in conjunction with the administration of anti-antivirus antibodies.
- anti-antivirus antibodies may be administered prior to, at the same time or shortly after the administration of the virus.
- an effective amount of the anti-antivirus antibodies are administered in sufficient time to reduce or eliminate an immune response by the mammal to the administered vims.
- the immunocompetency of the mammal against the vims may be suppressed either by the prior or co-administration of pharmaceuticals known in the art to suppress the immune system in general or alternatively the administration of such immunoinhibitors as anti-antivirus antibodies.
- the humoral immunity of the mammal against the virus may also be temporarily reduced or suppressed by plasmapheresis of the mammals blood to remove the anti-vims antibodies.
- the anti-virus antibodies removed by this process correspond to the virus selected for administration to the patient.
- Such immunoinhibitors also include anti- antivirus antibodies, which are antibodies directed against anti-vims antibodies.
- Such anti-antivirus antibodies may be administered prior to, at the same time or shortly after the administration of the vims.
- an effective amount of the anti-antivirus antibodies are administered in sufficient time to reduce or eliminate an immune response by the mammal to the administered vims.
- a vims selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia vims and modified parapoxvirus orf vims is administered to proliferating cells in the individual mammal.
- a course of this therapy is administered one or more times.
- particular immune constituents that may interfere with subsequent administrations of vims are removed from the patient. These immune constituents include B cells, T cells, antibodies, and the like.
- Removal of either the B cell or T cell population can be accomplished by several methods.
- the blood may be filtered and heme-dialysis may be performed.
- Another method is the filtration of the blood coupled with extra corporeal compounds that can remove the cell populations, for example, with immobilized antibodies that recognize specific receptors on the cell population which is to be remove.
- Yet another method for removal of a cell population is by immune suppression. This can be done by first line radiation therapy or by cyclic steroids such as cyclosporin. Selective removal of anti-vims antibodies can also prevent the patient's immune system from removing therapeutically administered vims. Preventing antibody interaction with the administered virus may also assist systemic treatment strategies.
- Rhoads, R.E. Cap recognition and the entry of mRNA into the protein synthesis initiation cycle. TIBS. 13: 52-56, (1988).
- Kevil, C De Benedetti, A., Payne, K.D., Coe, L.L., Laroux, S., and Alexander, S., Translational regulation of Vascular Permeability Factor by eukaryotic initiation factor 4E: Implications for tumor angiogenesis. International J. Cancer. 65:785- 790, (1996).
- INGN 201 Ad-p53, Ad5CMV- ⁇ 53, Adenoviral p53, INGN 101, p53 gene therapy-Introgen, RPR/INGN 201. BioDrugs. 17:216-222, (2003).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- General Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2005/038918 WO2007050074A1 (en) | 2005-10-28 | 2005-10-28 | Conditionally replicating viruses and methods for cancer virotherapy |
CA002627638A CA2627638A1 (en) | 2005-10-28 | 2005-10-28 | Conditionally replicating viruses and methods for cancer virotherapy |
JP2008537668A JP2009513133A (en) | 2005-10-28 | 2005-10-28 | Conditionally replicating viruses and methods for viral therapy of cancer |
AU2005337612A AU2005337612A1 (en) | 2005-10-28 | 2005-10-28 | Conditionally replicating viruses and methods for cancer virotherapy |
EP05824940A EP1948792A4 (en) | 2005-10-28 | 2005-10-28 | Conditionally replicating viruses and methods for cancer virotherapy |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2005/038918 WO2007050074A1 (en) | 2005-10-28 | 2005-10-28 | Conditionally replicating viruses and methods for cancer virotherapy |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2007050074A1 true WO2007050074A1 (en) | 2007-05-03 |
Family
ID=37968085
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2005/038918 WO2007050074A1 (en) | 2005-10-28 | 2005-10-28 | Conditionally replicating viruses and methods for cancer virotherapy |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1948792A4 (en) |
JP (1) | JP2009513133A (en) |
AU (1) | AU2005337612A1 (en) |
CA (1) | CA2627638A1 (en) |
WO (1) | WO2007050074A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016029833A1 (en) * | 2014-08-26 | 2016-03-03 | 广州威溶特医药科技有限公司 | Use of alphavirus in preparation of antitumor drugs |
CN114729353A (en) * | 2019-06-21 | 2022-07-08 | 科纳勒生物公司 | Expression of engineered tumor-selective proteins |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000063406A2 (en) * | 1999-04-21 | 2000-10-26 | Genzyme Corporation | Adenoviral vectors having nucleic acids encoding immunomodulatory molecules |
US20030017138A1 (en) * | 1998-07-08 | 2003-01-23 | Menzo Havenga | Chimeric adenoviruses |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6759394B2 (en) * | 2001-07-26 | 2004-07-06 | Board Of Supervisors Of La. State Un. & Agricultural And Mechanical College | Cancer gene therapy based on translational control of a suicide gene |
-
2005
- 2005-10-28 EP EP05824940A patent/EP1948792A4/en not_active Withdrawn
- 2005-10-28 AU AU2005337612A patent/AU2005337612A1/en not_active Abandoned
- 2005-10-28 CA CA002627638A patent/CA2627638A1/en not_active Abandoned
- 2005-10-28 JP JP2008537668A patent/JP2009513133A/en active Pending
- 2005-10-28 WO PCT/US2005/038918 patent/WO2007050074A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030017138A1 (en) * | 1998-07-08 | 2003-01-23 | Menzo Havenga | Chimeric adenoviruses |
WO2000063406A2 (en) * | 1999-04-21 | 2000-10-26 | Genzyme Corporation | Adenoviral vectors having nucleic acids encoding immunomodulatory molecules |
Non-Patent Citations (5)
Title |
---|
GEMMILL ET AL.: "Synergistic Growth by Iressa and Rapamycin is modulated by VHL mutation in renal cell carcinoma", BRIT. J. CAN., vol. 92, June 2005 (2005-06-01), pages 2266 - 2277, XP003001047 * |
HAVIV ET AL.: "Transcriptional Targeting in renal Cancer cell lines via the human CRCR4 promoter", MOLECULAR CANCER THERAPEUTICS, vol. 3, no. 6, June 2004 (2004-06-01), pages 687 - 691, XP003001046 * |
REIN ET AL.: "Evaluations of tissue-specific promoters in carcinomas of the cervix uteri", J. OF GENE MED., vol. 6, September 2004 (2004-09-01), pages 1281 - 1289, XP002395588 * |
See also references of EP1948792A4 * |
STOFF-KHALI ET AL.: "Preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system", BREAST CANCER RES., vol. 7, no. 6, November 2005 (2005-11-01), pages R1141 - R1152, XP003001048 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016029833A1 (en) * | 2014-08-26 | 2016-03-03 | 广州威溶特医药科技有限公司 | Use of alphavirus in preparation of antitumor drugs |
AU2015309403B2 (en) * | 2014-08-26 | 2017-03-16 | Guangzhou Virotech Pharmaceutical Co., Ltd. | Use of alphavirus in preparation of antitumor drugs |
EP3187588A4 (en) * | 2014-08-26 | 2018-01-10 | Guangzhou Virotech Pharmaceutical Co. Ltd. | Use of alphavirus in preparation of antitumor drugs |
RU2693938C2 (en) * | 2014-08-26 | 2019-07-08 | Гуанчжоу Виротек Фармасьютикал Ко., Лтд. | Use of alphavirus in preparation of antitumor drugs |
US10517909B2 (en) | 2014-08-26 | 2019-12-31 | Guangzhou Virotech Pharmaceutical Co., Ltd | Use of alphavirus in preparation of antitumor drugs |
US11235011B2 (en) | 2014-08-26 | 2022-02-01 | Guangzhou Virotech Pharmaceutical Co., Ltd. | Use of alphavirus in preparation of antitumor drugs |
CN114729353A (en) * | 2019-06-21 | 2022-07-08 | 科纳勒生物公司 | Expression of engineered tumor-selective proteins |
Also Published As
Publication number | Publication date |
---|---|
EP1948792A4 (en) | 2010-08-04 |
AU2005337612A1 (en) | 2007-05-03 |
AU2005337612A8 (en) | 2008-07-03 |
EP1948792A1 (en) | 2008-07-30 |
JP2009513133A (en) | 2009-04-02 |
CA2627638A1 (en) | 2007-05-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10016470B2 (en) | Oncolytic adenoviruses for cancer treatment | |
US7048920B2 (en) | Recombinant oncolytic adenovirus for human melanoma | |
US10538744B2 (en) | Use of adenovirus and nucleic acids coding therefor | |
Zhang et al. | An armed oncolytic adenovirus system, ZD55-gene, demonstrating potent antitumoral efficacy | |
CN109576231B (en) | Isolated recombinant oncolytic adenoviruses, pharmaceutical compositions and their use in medicaments for the treatment of tumors and/or cancers | |
US20090270485A1 (en) | Cell specific replication-competent viral vectors comprising a self processing peptide cleavage site | |
US8142770B2 (en) | Drug comprising as the active ingredient proliferative vector containing survivin promoter | |
KR20130015270A (en) | Tumor-specific promoter and oncolytic virus vector comprising the same | |
Kim et al. | Antitumoral effects of recombinant adenovirus YKL-1001, conditionally replicating in α-fetoprotein-producing human liver cancer cells | |
WO2012163119A1 (en) | Construction and application of mutant type b human adenovirus ad11 having enhanced oncolytic power | |
US20060275262A1 (en) | Conditionally replicating viruses and methods for cancer virotherapy | |
AU2002346084B2 (en) | Viral mutants that selectively replicate in targeted human cancer cells | |
Gao et al. | RETRACTED: Selective Targeting of Checkpoint Kinase 1 in Tumor Cells with a Novel Potent Oncolytic Adenovirus | |
WO2006125381A1 (en) | Tumor targeting gene-virus zd55-il-24, construction method and application thereof | |
EP1948792A1 (en) | Conditionally replicating viruses and methods for cancer virotherapy | |
WO1999044423A1 (en) | Amplification of gene transfer and gene therapy by controlled replication | |
RU2814581C1 (en) | GENETIC VECTOR Ad6/3-hTERT-GMCSF CONTAINING GENOMIC SEQUENCES OF RECOMBINANT ADENOVIRUS SEROTYPE 6, HUMAN TELOMERASE PROMOTER, HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE, AS WELL AS FIBER PROTEIN GENE WITH INSERTION OF FIBER KNOB DOMAIN OF ADENOVIRUS SEROTYPE 3 WITH INCREASED TRANSDUCTION INTO TUMOR CELLS | |
CN113774031B (en) | Replication type human adenovirus and application thereof | |
Tong | Oncolytic viral therapy for human cancer: challenges revisited | |
TWI391486B (en) | A novel promoter and viral vector containing the same | |
AU2021371806A1 (en) | Mesenchymal stem cells that enable tumor targeting improvement and virus mass-production | |
Alemany | Conditionally replicating adenoviruses for cancer treatment | |
Abbas | UNDERSTANDING THE RELATIONSHIP BETWEEN ONCOLYTIC AD5 DELETED E1b55KDA LYTIC INFECTION AND P53 IN MAMMALIAN CELLS | |
AU2003252891A1 (en) | Adenovirus vectors containing cell status-specific response elements and methods of use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2005337612 Country of ref document: AU |
|
ENP | Entry into the national phase |
Ref document number: 2627638 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 567568 Country of ref document: NZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005824940 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2008537668 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2005337612 Country of ref document: AU Date of ref document: 20051028 Kind code of ref document: A |