WO2005058368A1 - Methods and compositions for treatment of interferon-resistant tumors - Google Patents
Methods and compositions for treatment of interferon-resistant tumors Download PDFInfo
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- WO2005058368A1 WO2005058368A1 PCT/US2004/041307 US2004041307W WO2005058368A1 WO 2005058368 A1 WO2005058368 A1 WO 2005058368A1 US 2004041307 W US2004041307 W US 2004041307W WO 2005058368 A1 WO2005058368 A1 WO 2005058368A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
- A61K38/212—IFN-alpha
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/10—Drugs for disorders of the urinary system of the bladder
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
- C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- Interferon protein therapy is well established in the clinical environment as a treatment for a variety of cancers.
- Two widely used commercially available interferon species produced by recombinant DNA technology are interferon ⁇ -2b recombinant (Intron A®, Schering Corporation) and interferon ⁇ -2a recombinant (Roferon ® , Hoffman LaRoche, Inc.).
- Intron A is indicated for use in the treatment of malignant melanoma in combination with surgery, aggressive follicular Non-Hodgkin's Lymphoma in combination with anthracycline chemotherapy, intralesional treament of condylomata acuminata, hairy cell leukemia, and AIDS-Related Kaposi's Sarcoma.
- Roferon is indicated for use in the treatment of Philadelphia chromosome positive chronic myelogenous leukemia (CML) and AIDS-related Kaposi's sarcoma.
- CML chronic myelogenous leukemia
- the disease is typically characterized as a slowly progressing malignancy that originates from the surface lining of the urothelium. While most of these superficial cancers can be adequately managed with periodic transurethral resection (TUR) and surveillance, this is far from an optimal approach because 60 to 70 percent of superficial tumors recur after TUR, and up to 30 percent evolve into more aggressive, potentially lethal cancers. The high recurrence rate and the unpredictability of the progression patterns have led to the widespread use of intravesical therapy for local control of the disease. Immunotherapy with intravesically instilled Bacillus Calmette-Guerin (BCG) can usually delay disease progression in newly diagnosed patients.
- BCG Bacillus Calmette-Guerin
- Intravesical interferon- ⁇ 2b (IFN ⁇ 2b) recombinant (Intron A) is well tolerated as a monotherapy in superficial bladder cancer patients and is an approved indication for this agent in many countries. Intron A has demonstrated dose-related clinical efficacy as a salvage therapy in the scenario of BCG failure, although the durability of the response is limited and most patients relapse within the first year of treatment. Belldegrun et al, J. Urol, 159(6): 1793-801 (1998). Recently, Intron A has been combined with BCG in an attempt to enhance the cellular immune response to BCG and improve the response to therapy. O'Dormell et al, J. Urol, 7(5(5:1300-1304 (2001).
- Combination therapy has been effective in BCG refractory TCC, but many of these initial responders relapse with superficial disease and ultimately require cystectomy.
- Gene therapy employing recombinant vectors encoding interferon speicies provides a promising alternate approach to the treatment of refractory superficial bladder cancer. Local delivery can maximize transgene expression in the urothelium and minimize vector distribution to vital organs outside of the bladder.
- efficient transfer of a gene encoding a secreted protein (e.g., IFN ⁇ 2b) to both normal and malignant cells of the urothelium can generate a potent anti-tumor bystander effect coincident with sustained local protein concentrations in the urine.
- a secreted protein e.g., IFN ⁇ 2b
- the present invention provides methods for the treatment of interferon resistant tumors through the use of recombinant vectors encoding interferon species.
- interferon species provided by recombinant vectors possess properties not associated with the available recombinantly produced interferon proteins.
- the present invention further provides compositions useful in the treatment of interferon resistant tumors using recombinant vectors encoding interferons.
- BRIEF DESCRIPTION OF THE DRAWINGS [0008]
- Figure 1 provides a data of the effect of intravestical Ad-IFN- ⁇ 2 ⁇ l/Syn 3 or Ad-IFN- ⁇ 2b/Syn3 on tumor regression.
- To the right are a series of fluorescence micrographs providing a representative example of pre-treatment tumor images and those taken 21 days later from individual mice in each treatment group.
- Figure 2 provides a demonstration of prolonged IFN expression following Ad-IFN/Syn3 intravesical treatment.
- Panel A demonstrates that sustained levels of IFN are seen in the bladder homogenates of athymic mice treated once with Ad-IFN ⁇ 2b/Syn3 which was increased after two exposures. In contrast, similar treatment with 200,000 IU of Intron A protein itself did not show any such high or prolonged IFN levels. Arrows indicate when treatment 1 and 2 were given.
- Panel B provides data indicating that the same high levels of IFN in the urothelium and adjacent bladder tumor is seen by immunohistochemical analysis 2 days after two treatments with Ad-IFN ⁇ 2 ⁇ l/Syn 3 in athymic mice. Arrows show strong perinuclear IFN staining present in many cells.
- Panel C demonstrates that similar high levels of IFN are seen in the urothelium of outbred Balb-C mice 7 days after treatment with either Ad-IFN ⁇ 2 ⁇ l/Syn 3 or Ad-IFN ⁇ 2b/Syn3. No morphological changes in the urothelium were seen by histological examination or H&E stained sections.
- Figure 3 provides a comparison of Ad-IFN cytotoxicity to bladder cancer cell lines resistant to over 100,000 IU of Intron A.
- Panel A shows the negative IFN staining in control cells (10X Magnification) and marked perinuclear IFN staining in 253 J- BV cells treated with Ad-IFN ⁇ 2 ⁇ l (40X Magnification).
- Panel B is data from a flow cytometric analysis showing increase in the number of subdiploid cells at 48 hrs after treatment of a 50 MOI Ad-IFN ⁇ 2 ⁇ l in 253 J B-V cells. Red bars indicate the subdiploid population.
- Panel C illustrates an increase in the number of G2/M cells following Ad- IFN ⁇ 2 ⁇ l treatment to over 60% was observed.
- Panel D demonstrates the marked cytotoxicity to Ad-IFN ⁇ 2 ⁇ seen in the 253 J B-V cells at the same 50 MOI of Ad-IFN ⁇ 2 ⁇ l.
- a 50 MOI of Ad- ⁇ gal was also used for these studies as a control and no cytotoxicity was seen, nor were changes observed in these cells when treated with 100,000 IU of Intron A.
- FIG. 4 illustrates caspase dependent cell death occurs in bladder cancer cells resistant to IFN protein following Ad-IFN treatment including cells which show no IFN staining.
- Panel A provides KU7 or 253 J-BV cells treated with Ad-IFN- ⁇ 2 ⁇ l .
- Two by two optical channels merged images of triple staining of cells for IFN (red) and DNA (blue) upper panels and for active caspase-3 (green) and DNA (blue) lower panels are shown 36 h after treatment. Caspase activation and nuclear condensation is seen in cells expressing IFN (white arrows).
- FIG. 1 is a comparison of Ad-IFN and Intron A on bladder cancer' cells. Panel A illustrates the high and sustained production of IFN in the supernatant from B- V and KU7 following Ad-IFN treatment at two MOIs. Levels reached 10 million pg/ml (-2.5 million IU of IFN) in both cell lines.
- Panel B illustrates the percentage of subdiploid cells shown 48 and 72 hours after addition of 2.5 million IU/ml of Intron A or 50 MOI of Ad-IFN- ⁇ 2b.
- Panel C illustrates the lack of morphological changes or IFN staining in KU7 cells seen after Intron A treatment at the same concentration and time points as Panel B. In contrast, note the cells with increased size and strong perinuclear staining following treatment with Ad-IFN- ⁇ 2b along with several small apoptotic appearing cells (arrows) (Magnification 40X).
- the present invention provides a method of treating an interferon- resistant tumor by contacting the tumor with a recombinant vector encoding an interferon such that the cells of the tumor transduced by the vector express the interferon encoded by the vector.
- interferon-resistant tumor refers to a tumor that is ref actory to treatment by the administration of interferon protein in accordance with conventional interferon treatment protocols.
- a tumor is considered refractory to interferon treatment if the tumor fails to diminish in a course of therapy of intravesical administration of up to six weekly administrations of approximately 100 million units of Intron A for an exposure time of approximately 1 hour.
- a tumor is considered to be refractory to interferon treatment if the tumor fails to respond following a treatment regimen comprising the intravenous administration of approximately 20 MIU/m 2 of Intron A for 5 consecutive days per week for
- neoplastically transformed cells shall be considered tumors.
- the tumor is considered refractory to interferon treatment if the individual fails to respond to a treatment regimen comprising subcutaneous administration of
- tumors are considered refractory to treatment if the individual fails to respond to a treatment regimen consisting of a daily intramuscular or subcutaneous injection of approximately 9 MIU of Roferon for a treatment period of up to 5 to but as long as 18 months.
- a treatment regimen consisting of a daily intramuscular or subcutaneous injection of approximately 9 MIU of Roferon for a treatment period of up to 5 to but as long as 18 months.
- tumors may be identified as being refractory to administration of interferon protein as determined in vitro in accordance with conventional in vitro assay procedures.
- Tumor refers to a collection of neoplastic cells.
- neoplastic cell refers to a cell displaying an aberrant growth phenotype characterized by independence of normal cellular growth controls. As neoplastic cells are not necessarily replicating at any given time point, the term neoplastic cells comprise cells which may be actively replicating or in a temporary nonreplicative resting state (Gl or GO).
- neoplasms or tumors Localized populations of neoplastic cells are referred to as neoplasms or tumors (these terms are considered interchangeable).
- tumors as used herein may also refer to diffuse neoplastic diseases such as leukemia where no substantial localized tumor mass is present.
- Neoplasms or tumors may be malignant or benign. Malignant tumors are also referred to as cancers.
- the tumor is an epithelial cancer, e.g., breast, lung, prostate, colorectal, kidney, stomach, bladder or ovarian, or any cancer of the gastrointestinal tract.
- epithelial cancer e.g., breast, lung, prostate, colorectal, kidney, stomach, bladder or ovarian, or any cancer of the gastrointestinal tract.
- a tumor may possess individual cells or cell subpopulations which are interferon-resistant and cells or cell subpopulations that are sensitive to interferon treatment.
- interferon-resistant tumors refers to tumors where a substantial population (i.e., at least 20%) of the cells which comprise the tumors are interferon-resistant as determined in accordance with the MTT assay described in Example 6 herein.
- treatment is intended to refer to the introduction of nucleic acid encoding an interferon to a patient for the purpose of exposing a tissue of interest comprising neoplastic cells to interferon.
- a "cancerous" tissue is intended to refer to a tissue in which one or more cells is classified as cancerous, malignant, tumorous, precancerous, transformed, or as an adenoma or carcinoma, or any other synonym commonly used in the art for these conditions.
- a treatment may be considered therapeutic if the treatment indicates an improvement in the physiological or psychological condition of the subject in which the neoplasm is present. Improvement in the condition may be measured by a variety of means including reduced tumor burden, reduction of rumor progression, slowing of tumor progression, increased survival, tumor regression, amelioration symptoms associated with tumor burden including but not limited to fatigue, anorexia, pain, depression, neutropenia and cognitive dysfunction.
- interferon refers collectively to type I and type 2 interferons including deletion, inse ⁇ ion, or substitution variants thereof, biologically active fragments, and allelic forms.
- interferon refers collectively to type 1 and type 2 interferons.
- Type 1 interferon includes interferons- ⁇ , - ⁇ and - ⁇ and their subtypes. Human interferon- ⁇ has at least 14 identified subtypes while interferon- ⁇ has 3 identified subtypes.
- preferred interferon-alphas include human interferon alpha subtypes including, but not limited to, ⁇ -1 (GenBank Accession Number NP 076918), ⁇ -lb (GenBank Accession
- Human interferon gamma has at least 5 identified subtypes, including interferon omega 1 (GenBank Accession Number NP 002168). Construction of DNA sequences encoding inteferons for expression may be accomplished by conventional recombinant DNA techniques based on the well-known amino acid sequences referenced above and as described in Goeddel et al, U.S. Patent No. 6,482,613, issued November 19, 2002, the teachings of which are herein incorporated by reference. [0019] "Biologically active" fragments of interferons may be identified as having any anti-rumor or anti-proliferative activity as measured by techniques well known in the art (see, for example, Openakker et al, supra; Mossman, J. Immunol. Methods, 65:55 (1983) and activate IFN responsive genes through IFN receptor mediated mechanisms.
- Soluble IFN- ⁇ and IFN- ⁇ proteins are generally identified as associating with the Type 1 IFN receptor complex (GenBank Accession Number NP 000865) and activate similar intracellular signaling pathways.
- IFN- ⁇ is generally identified as associating with the type II IFN receptor.
- Ligand-induced association of both types of IFN receptors results in the phosphorylation of the receptors by Janus kinases subsequently activating STATs (signal transducers and activators of transcription) proteins and additional phosphorylation events that lead to the formation of IFN-inducible transcription factors that bind to IFN response elements presented in IFN-inducible genes.
- interferons Polypeptides identified as activating the IFN pathways following association with Type 1 and/or Type 2 IFN receptors are considered interferons for purposes of the present invention.
- the term "recombinant vector” refers to viral and nonviral vectors comprising an interferon expression cassette that has been prepared using conventional recombinant DNA technology.
- interferon expression cassette is used herein to define a nucleotide sequence containing regulatory elements operably linked to the interferon coding sequence so as to result in the transcription and translation of an interferon in a cell.
- regulatory element refers to promoters, enhancers, transcription terminators, polyadenylation sites, and the like.
- the expression cassette may also contain other sequences aiding expression and/or secretion of the interferon gene.
- the regulatory elements may be arranged so as to allow, enhance or facilitate expression of the interferon only in a particular cell type.
- the expression cassette may be designed so that the interferon is under control of an inducible promoter, tissue specific or tumor specific promoter, or temporal promoter.
- the interferon is provided in an expression vector comprising the following elements linked sequentially at appropriate distances for functional expression: a promoter, an initiation site for transcription, a 3' untranslated region, a 5' mRNA leader sequence, a nucleic acid sequence encoding an interferon polypeptide, and a polyadenylation signal.
- operably linked refers to a linkage of polynucleotide elements in a functional relationship.
- a nucleic acid sequence is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
- a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
- Operably linked means that the nucleotide sequences being linked are typically contiguous.
- inducible promoter refers to promoters that facilitate transcription of the interferon transgene preferably (or solely) under certain conditions and/or in response to external chemical or other stimuli. Examples of inducible promoters are known in the scientific literature (.see, e.g., Yoshida et al, Biochem. Biophys. Res.
- Tissue specific and tumor specific promoters are well known in the art and include promoters active preferentially in smooth muscle ( ⁇ -actin promoter), pancreas specific (Palmiter et al, Cell, 50:435 (1987)), liver specific (Rovet et al, J. Biol. Chem., 2.57:20765 (1992); Lemaigne et al , J. Biol Chem., 268: 19896 (1993); Nitsch et al , Mol. Cell. Biol, 13:4494 (1993)), stomach specific (Kovarik et al, J.
- temporally regulated promoters refers to promoters that drive transcription of the interferon transgene at a point later in the viral cycle relative to the promoter controlling expression of the response element and are used in conjunction with viral vector systems. Examples of such temporally regulated promoters include the adenovirus major late promoter (MLP), and other late promoters.
- MLP adenovirus major late promoter
- nonviral vector refers to an autonomously replicating, extrachromosomal circular DNA molecules, distinct from the normal genome and nonessential for cell survival under nonselective conditions capable of effecting the expression of an interferon coding sequence in the target cell. Plasmids autonomously replicate in bacteria to facilitate bacterial production, but it is not necessary that such plasmids containing the interferon coding sequence replicate in the target cell in order to achieve the therapeutic effect. Additional genes, such as those encoding drug resistance, can be included to allow selection or screening for the presence of the recombinant vector.
- Such additional genes can include, for example, genes encoding neomycin resistance, multi-drug resistance, thymidine kinase, ⁇ -galactosidase, dihydrofolate reductase (DHFR), and chloramphenicol acetyl transferase.
- a lipid encapsulated expression plasmid may incorporate modified surface cell receptor ligands to facilitate targeting.
- the liposomes either filled or decorated with a desired composition of the invention can delivered systemically, or can be directed to a tissue of interest, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions.
- ligands include antibodies, monoclonal antibodies, humanized antibodies, single chain antibodies, chimeric antibodies or functional fragments (Fv, Fab, Fab') thereof.
- nonviral vectors can be linked through a polylysine moiety to a targeting moiety as described in Wu et al. U.S. Patent No. 5,166,320, issued November 24, 1992, and Wu et al, U.S. Patent No.
- viral vector and "virus” are used interchangeably herein to refer to any of the obligate intracellular parasites having no protein-synthesizing or energy- generating mechanism.
- the viral genome may be RNA or DNA contained with a coated structure of protein of a lipid membrane.
- virus(es) and viral vector(s) are used interchangeably herein.
- viruses useful in the practice of the present invention include recombinantly modified enveloped or nonenveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesviridiae, poxviridae, or adenoviridiae.
- the viruses may be naturally occurring viruses or their viral genomes may be modified by recombinant DNA techniques to include expression of exogenous transgenes and may be engineered to be replication deficient, conditionally replicating or replication competent.
- Chimeric viral vectors which exploit advantageous elements of each of the parent vector properties (see, e.g., Feng, et al, Nature Biotechnology, 75:866-870 (1997)) may also be useful in the practice of the present invention.
- Minimal vector systems in which the viral backbone contains only the sequences need for packaging of the viral vector and may optionally include a transgene expression cassette may also be produced according to the practice of the present invention. Although it is generally favored to employ a virus from the species to be treated, in some instances, it may be advantageous to use vectors derived from different species that possess favorable pathogenic features. For example, equine herpes virus vectors for human gene therapy are described in PCT International Publication No. WO98/27216, published August 5, 1998. The vectors are described as useful for the treatment of humans as the equine virus is not pathogenic to humans.
- ovine adenoviral vectors may be used in human gene therapy as they are claimed to avoid the antibodies against the human adenoviral vectors.
- Such vectors are described in PCT International Publication No. WO 97/06826, published April 10, 1997.
- the term "replication deficient" refers to vectors that are highly attenuated for replication in a wild type mammalian cell.
- a producer cell line is generally created by co-transfection with a helper virus or genomically modified to complement the missing functions.
- HEK293 cells have been engineered to complement adenoviral El deletions allowing propagation of the El deleted replication deficient adenoviral vectors in 293 cells.
- replication competent viral vectors refers to a viral vector that is capable of infection, DNA replication, packaging and lysis of an infected cell.
- conditionally replicating viral vectors is used herein to refer to replication competent vectors that are designed to achieve selective expression in particular cell types. Such conditional replication may be achieved by operably linking tissue specific, tumor specific or cell type specific or other selectively induced regulatory control sequences to early genes (e.g., the El gene of adenoviral vectors).
- tissue specific, tumor specific or cell type specific or other selectively induced regulatory control sequences to early genes (e.g., the El gene of adenoviral vectors).
- Cell type specificity or cell type targeting may also be achieved in viral vectors derived from viruses having characteristically broad infectivities by the modification of the viral envelope proteins.
- cell targeting has been achieved with adenovirus vectors by selective modification of the viral genome knob and fiber coding sequences to achieve expression of modified knob and fiber domains having specific interaction with unique cell surface receptors.
- modifications are described in Wickham et al, J. Virol, 77 f7 ⁇ :8221-8229 (1997) (incorporation of RGD peptides into adenoviral fiber proteins); Arnberg et al, Virology, 227:239-244 (1997) (modification of adenoviral fiber genes to achieve tropism to the eye and genital tract); Harris et al, TIG, 12 (10) A00-405 (1996); Stevenson et al, J.
- particular moieties may be conjugated to the viral surface to achieve targeting (see, e.g., Nilson et al, Gene Therapy, 3:280-286 (1996) (conjugation of EGF to retroviral proteins).
- Targeting of vectors may also be achieved in accordance with the teaching of Murphy, U.S. Patent No. 6,635, 476, issued October 21, 2003, the teachings of which are herein incorporated by reference.
- These targeting modifications may be introduced into the viral vectors of the present invention in addition to or in combination with other modifications to the viral genome. Targeting modifications may be used with replication deficient, replication competent or conditionally replicating viruses.
- cell type targeting with viral vectors may be achieved through the use of a pathway responsive promoter driving a repressor of viral replication.
- the term "pathway-responsive promoter” refers to DNA sequences that bind a certain protein and cause nearby genes to respond transcriptionally to the binding of the protein in normal cells. Such promoters may be generated by incorporating response elements which are sequences to which transcription factors bind. Such responses are generally inductive, though there are several cases where increasing protein levels decrease transcription. Pathway-responsive promoters may be naturally occurring or synthetic. Pathway-responsive promoters are typically constructed in reference to the pathway of a functional protein which is targeted.
- a naturally occurring p53 pathway-responsive promoter would include transcriptional control elements activated by the presence of functional p53 such as the p21 or bax promoter.
- synthetic promoters containing p53 binding sites upstream of a minimal promoter e.g. , the S V40 TATA box region
- Synthetic pathway-responsive promoters are generally constructed from one or more copies of a sequence that matches a consensus binding motif. Such consensus DNA binding motifs can readily be determined. Such consensus sequences are generally arranged as a direct or head-to-tail repeat separated by a few base pairs.
- the viral vector is an adenovirus.
- adenovirus is synonomous with the term “adenoviral vector” and refers to viruses of the genus adenoviridiae.
- adenoviridiae refers collectively to animal adenoviruses of the genus mastadenovirus including but no limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera.
- human adenoviruses includes the A-F sugenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (AdllA and Ad I IP), 12, 13,14,15,16,17,18,19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91.
- bovine adenoviruses includes, but is not limited to, bovine adenovirus types 1,2,3,4,7, and 10.
- canine adenoviruses includes, but is not limited to, canine types 1 (strains CLL, Glaxo, RI261, Utrect, Toronto 26-61) and 2.
- equine adenoviruses includes, but is not limited to, equine types 1 and 2.
- porcine adenoviruses includes, but is not limited to, porcine types 3 and 4.
- viral vector includes replication deficient, replication competent, and conditionally replicating viral vectors.
- More preferred are vectors derived from human adenovirus types 2 and 5. These vectors may incorporate particular modifications to enhance their therapeutic potential. For example, they may include deletions of Ela and Elb genes. Certain other regions may be enhanced or deleted to provide specific features.
- upregulation of the E3 region is described to reduce the immunogenicity associated with human adenoviral vectors administered to human subjects.
- the E4 region has been implicated as important to expression of transgenes from the CMV promoter, however the E4orf 6 protein has been described as leading to the degradation of p53 in target cells in the presence of Elb large protein.
- an adenoviral vector be employed containing specific deletions in the El A region so as to reduce the ability of the Ela gene product to bind to the p300 and Rb proteins while retaining the transactivating function of the Ela CR3 domain.
- the vectors of the present invention contain deletions in the Ela coding sequence to eliminate p300 and pl05-Rb binding sites in the 13S coding sequence.
- the p300 binding deletions are represented by deletions of amino acids from about 4 to about 25 or from about 36 to about 49.
- the Rb binding deletions are represented by elimination of amino acids from about 111-127, preferably from about 111-123. More preferred is a vector wherein the deletion in the Ela-p300 binding domain comprises a deletion of the codons for amino acids 4 to 25 of the adenoviral Ela gene product. More preferred is a vector wherein deletion in the Ela-Rb binding domain comprises a deletion of the codons for amino acids 111-123 of the adenoviral Ela gene product.
- pRb binding may be eliminated by the introduction of a mutation to eliminate amino acids 124- 127 of the El A 289R and 243R proteins.
- the vector comprises a deletion of amino acids 4-25 and 111- 123 of the Ela 13S gene product.
- the elimination of amino acids from approximately 219 to 289 of the Ela 289R protein and 173 to 243 of the El A 243R protein may be introduced.
- a point mutation at a position corresponding to position 1131 of the human Ad5 genome i.e. , changing the cytosme to a guamne
- This mutation results in the elimination of amino acids 219 to 289 of the Ela 289R protein and 173 to 243 of the El A 243R protein.
- the practice of the present invention involves "contacting" the tumor with a recombinant vector encoding interferon. Contact of the tumor cells is accomplished by exposure of the tumor cells with the recombinant vector such that the cells of the tumor are transduced by the vector expressing the interferon encoded by the vector.
- the particular means by which the tumor is contacted by the vector will differ depending on the nature of the tumor to which the vector is being delivered.
- the compositions of the invention can be administered directly into a tissue by injection or into a blood vessel supplying the tissue of interest.
- compositions are administered through catheters or other devices to allow access to a remote tissue of interest, such as an internal organ.
- the compositions of the invention can also be administered in depot type devices, pumps, implants, or formulations to allow slow or sustained release of the compositions, hi further embodiments of the invention, the compositions of the invention are administered "locoregionally", i.e., intravesically, intralesionally, and/or topically.
- the compositions of the invention may be administered systemically by intravenous or intraarterial routes of administration.
- One vector contained the human interferon ⁇ 2b gene (Ad-IFN ⁇ 2b), and the other contained a chimeric human "hybrid" interferon gene comprised of the human ⁇ 2 N-terminus joined to the human ⁇ l C-terminus (Ad-IFN ⁇ 2 ⁇ l).
- Ad-IFN ⁇ 2b human interferon ⁇ 2b gene
- Ad-IFN ⁇ 2 ⁇ l chimeric human "hybrid" interferon gene comprised of the human ⁇ 2 N-terminus joined to the human ⁇ l C-terminus
- FIG. 1 The urothelium appeared similar in all treatment groups including a mild degree of hyperplasia which was related to the initial trypsinization of the urothelium required to promote initial tumor cell adhesion.
- a series of experiments were conducted using nontumor-bearing athymic mice with the same concentrations of Ad-IFN ⁇ 2b/Syn3 utilized for the foregoing efficacy studies, and compared a single intravesical treatment to two consecutive days of exposure for one hour.
- the IFN protein in the bladder tissue was assessed by harvesting the mouse bladders at various times after treatment and assaying for IFN protein present in tissue homogenates using an ELISA assay.
- bladders contained approximately 50,000 pg/mg of Intron A, which slowly decreased to 5,000 - 10,000 pg/mg by 7d as shown in Figure 2, Panel A of the attached drawings.
- tissue concentrations reached 100,000 pg/mg and remained higher over a 7d period compared to the single exposure.
- Some human bladder cancer cell lines are resistant to interferon induced cell death even after exposure to more than 100,000 IU/ml of either IFN ⁇ or IFN ⁇ proteins in vitro in accordance with the MTT assay described herein.
- Certain cell interferon resistant tumor cell lines include the KU7 cells (which were used in the foregoing efficacy studies) and 253 J B-V cells. When exposed to recombinant vectors encoding interferon, these interferon resistant tumor cell lines demonstrated sensitivity to Ad-IFNq treatment.
- the present invention further provides pharmaceutical formulations comprising the vectors of the present invention.
- the compositions of the invention will be formulated for administration by manners known in the art acceptable for administration to a mammalian subject, preferably a human.
- delivery systems may be formulated for intramuscular, intravenous, injectable depot type devices or topical administration.
- the expression plasmid containing the interferon gene may be encapsulated in liposomes. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
- liposome carriers The delivery of DNA sequences to target cells using liposome carriers is well known in the art.
- a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al, Ann. Rev. Biophys. Bioeng., 9:467 (1980), Szoka, et al, U.S. Patent No 4,394,448, issued July 19, 1983, as well as U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, inco ⁇ orated herein by reference.
- Liposomes useful in the practice of the present invention may be formed from one or more standard vesicle- forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
- vesicle forming lipids include DC-chol, DOGS, DOTMA, DOPE, DOSPA, DMRIE, DOPC, DOTAP, DORIE, DMRIE-HP, n-spermidine cholesterol carbamate and other cationic lipids as disclosed in U.S. Patent No. 5,650,096.
- the selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
- compositions of the invention will be formulated for administration by manners known in the art acceptable for administration to a mammalian subject, preferably a human.
- the compositions of the invention can be administered directly into a tissue by injection or into a blood vessel supplying the tissue of interest.
- compositions of the invention are administered "locoregionally,” i.e., intravesically, intralesionally, and/or topically.
- compositions of the invention are administered systemically by injection, inhalation, suppository, transdermal delivery, etc.
- the compositions are administered through catheters or other devices to allow access to a remote tissue of interest, such as an internal organ.
- the compositions of the invention can also be administered in topical formulations or polymer matrices, hydrogel matrices, polymer implants, or encapsulated formulations to allow slow or sustained release of the compositions.
- the delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, preferably an aqueous carrier.
- aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
- compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
- concentration of the compositions of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc. , in accordance with the particular mode of administration selected.
- enhancing agents that facilitate the transfer of the nucleic acid encoding interferon to the target cell.
- delivery enhancing agents include detergents, alcohols, glycols, surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetates.
- Alcohols include for example the aliphatic alcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol.
- Glycols include glycerine, propyleneglycol, polyethyleneglycol and other low molecular weight glycols such as glycerol and thioglycerol.
- Acetates such as acetic acid, gluconic acid, and sodium acetate are further examples of delivery-enhancing agents.
- Hypertonic salt solutions like 1M NaCl are also examples of delivery-enhancing agents.
- Bile salts such as taurocholate, sodium tauro-deoxycholate, deoxycholate, chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid and other astringents such as silver nitrate may be used.
- Heparin-antagonists like quaternary amines such as protamine sulfate may also be used.
- Anionic, cationic, zwitterionic, and nonionic detergents may also be employed to enhance gene transfer.
- Exemplary detergents include but are not limited to taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium chloride, Zwittergent 3-14 detergent, CHAPS (3-[(3-Cholamidopropyl) dimethylammoniol]-l-propanesulfonate hydrate), Big CHAP, Deoxy Big CHAP, Triton-X-100 detergent, C12E8, Octyl-B-D- Glucopyranoside, PLURONIC- F68 detergent, Tween 20 detergent, and TWEEN 80 detergent (CalBiochem Biochemicals).
- Additional enhancing agents useful in the practice of the present invention include, but are not limited to, the compounds of the Formulas II, III, IV, and V and their pharmaceutically acceptable salts: [0056]
- Initial efforts to transduce the urothelium using adenovirus vectors had limited success, due in part to the presence of an anti-adherence barrier that protects against infections.
- An excipient, Syn3 has been identified that dramatically enhances adenovirus transduction of the urothelium.
- Connor et al Gene Then, 5:41-8 (2001); Yamashixa et al, Cancer Gene Therapy, 9:687-691 (2002).
- the present invention further provides a method of treating a mammalian organism containing a tumor by administering to the mammalian organism a pharmaceutical formulation comprising a recombinant vector encoding an interferon.
- mammalian organism includes, but is not limited to, humans, pigs, horses, cattle, dogs, and cats.
- the vector is a recombinant adenoviral vector
- adenoviral vector preferably one employs an adenoviral vector endogenous to the mammalian type being treated.
- virus preferably one employs an adenoviral vector endogenous to the mammalian type being treated.
- it may be advantageous to use vectors derived from different species which possess favorable pathogenic features. For example, it is reported (PCT International Publication No. WO 97/06826, published April 10, 1997) that bovine adenoviral vectors may be used in human gene therapy to minimize the immune response characteristic of human adenoviral vectors.
- immunosuppressive agents include, but are not limited to, cyclosporine, azathioprine, methotrexate, cyclophosphamide, lymphocyte immune globulin, antibodies against the CD3 complex, adrenocorticosteroids, sulfasalzaine, FK-506, methoxsalen, and thalidomide.
- transient elimination of antiviral antibodies may be accomplished in accordance with the teaching of La Face et al, U.S. Patent No. 6,464,976, issued October 15, 2002, the teaching of which is herein inco ⁇ orated by reference.
- the determination of the optimal dosage regimen for treatment of the disease will be based on a variety of factors which are within the discretion of the attending health care provider, such as the progression of the disease at the time of treatment, age, weight, sex, the type of vector being employed, whether it is being formulated with a delivery enhancing agent, the frequency of administration, etc.
- recombinant adenoviral vectors have been demonstrated to be safe and effective in human beings in the dosage range between lxlO 5 and lxlO 12 viral particles per dose in a multiple dosing regimen over a period of several weeks. Consequently, administration of recombinant adenoviral vectors encoding interferon may be used in such dosage ranges.
- course of treatment comprising a dose of from lxl0 10 /ml to lxl0 12 /ml (most preferably approximately lxl0 ⁇ /ml) recombinant adenoviral particles encoding interferon ⁇ 2b in a volume of approximately 100 ml is instilled intravesically for a period of approximately one hour.
- An alternate course of treatment comprises a dose of from lxl0 10 /ml to lxl0 12 /ml (most preferably approximately lxl0 ⁇ /ml) recombinant adenoviral particles encoding interferon ⁇ 2b in a volume of approximately 100 ml is instilled intravesically for a period of approximately one hour followed by a second substantially equivalent dose within 7 days, 5 days, 4 days, 3 days, 2 days or on consecutive days following the first dose.
- Each course of treatment is repeatable, depending on the course of disease progression.
- a dosage regimen comprising approximately 1 x 10 10 - lx 10 12 particles of a replication deficient recombinant adenoviral vector expressing an intracellular interferon species is administered intratumorally or via the hepatic artery for a period of five to seven consecutive days. This dosage regimen may be repeated over a course of therapy of approximately three to six weeks.
- a particularly preferred dosage regimen for the treatment of hepatocellular carcinoma in a human subject suffering therefrom would be to provide intrahepatic arterial administration of from approximately 1 x 10 10 - lx 10 12 particles of a replication deficient recombinant adenoviral vector expressing inteferon- ⁇ 2b under control of the AFP promoter for approximately five consecutive days. Most preferably, this dosage regimen is carried out in parallel with other chemotherapeutic regimens. [0061] In the situation where the vector is a replication competent vector, the dosage regimen may be reduced.
- a replication competent adenoviral vector may be constructed wherein the replication is substantially restricted to hepatocellular carcinoma cells by using the AFP promoter (for example) to drive expression of El proteins in lieu of the native El promoter.
- AFP promoter for example
- Such vector would preferentially replicate in and express interferon in tumor cells and possess the desirable ability to spread to surrounding cells expanding the therapeutic effect and allowing for a reduced dosage or shorter duration of treatment.
- the compositions and methods of the present invention may be practiced alone or in combination with conventional chemotherapeutic agents or treatment regimens.
- chemotherapeutic agents include inhibitors of purine synthesis (e.g., pentostatin, 6-mercaptopurine, 6-thioguanine, methotrexate) orpyrimidine synthesis (e.g., Pala, azarbine), the conversion of ribonucleotides to deoxyribonucleotides (e.g., hydroxyurea), inhibitors of dTMP synthesis (5-fluorouracil), DNA damaging agents (e.g., radiation, bleomycines, etoposide, teniposide, dactinomycine, daunorubicin, doxorubicin, mitoxantrone, alkylating agents, mitomycin, cisplatin, procarbazine) as well as inhibitors of microtubule function (e.g., vinca alkaloids and colchicine).
- purine synthesis e.g., pentostatin, 6-mercaptopurine, 6-thioguanine, methot
- Chemotherapeutic treatment regimens refers primarily to nonchemical procedures designed to ablate neoplastic cells such as radiation therapy. These chemotherapeutic agents may be administered seperately or may be included with the formulations of the present invention for co-administration. The present invention may also be practiced in combination with conventional immunotherapeutic treatment regiments such as BCG in the case of superficial bladder cancer.
- Example 1 Cell lines. Vectors, and Reagents: [0064] The bladder cancer cell lines, KU7/GFP clone 6 and 253 J-B-V, were used for these studies. KU7/GFP clone 6 is stably transfected with the green-flourescent protein and was used for all in vivo studies. These cell lines are described in Watanabe et al, Cancer Gene Tlierapy, 7:1575-1580 (2000). The cells were grown in modified minimum essential medium supplemented with 10% FCS and incubated at 37 C in 5% CO 2 and 95% air.
- Example 2 Superficial tumor formation, treatment and imaging: [0068] The methods for growing superficial KU7-GFP human bladder tumors in athymic mice and their imaging were conducted as previously been described in Izawa et al, Clin.Cancer Res, 5:1258-1270 (2002) and Zhou et al, Cancer Gene Therapy, °:681-686 (2002). Briefly, two weeks after the tumor cells were instilled, the bladders were imaged for the presence of GFP-containing tumor.
- mice then received an intravesical instillation of lOO ⁇ l of either Ad-IFN- ⁇ 2 ⁇ l/Syn3, Ad-IFN- ⁇ 2b/Syn3, Ad-IFN- ⁇ 2 ⁇ l, Ad- ⁇ -gal/Syn3, Syn3 or the IFN- ⁇ 2 ⁇ l protein for 1 hour.
- a purse-string suture was tied around the urethra to ensure retention during the procedure.
- Virus, Syn3 and interferon protein concentrations were lxlO 11 P/ml, lmg/ml and 2xl0 5 IU/lOO ⁇ l, respectively.
- Example 3 Homogenate gene expression studies: [0070] Female athymic mice were anesthetized, catheterized and received either a single 100 ⁇ l intravesical administration of Intron A (2 x 10 6 IU/ml) or 1-2 daily treatments with Ad-IFN/Syn3 (1 x 10 11 P/ml:l mg/ml) for 1 hour as described above. At various intervals the bladders were harvested and frozen. The bladders were later thawed and transferred into lysis buffer (Promega). Samples were homogenized for 20 sec (Fast Prep, Q- BlOgene). The homogenates were then assayed for human interferon ⁇ protein concentration using an ELISA (Endogen). Interferon protein concentration was expressed as pg IFN/mg bladder tissue.
- Example 4 Immunochemical analysis: [0071] For in vivo studies interferon immunohistochemistry was performed in substantial accordance with the teaching of Izawa et al. above. A positive reaction was indicated by a brown staining. Similarly, interferon staining for cells in culture was performed in substantial accordance with the teaching of Xu et al, Oncogene, :807-812 (1989), except that the primary antibody used was a 1 :500 dilution of rabbit polyclonal antibody against human interferon ⁇ (Hu-IFN- ⁇ , PBL).
- Example 5 Statistical analysis: [0072] Statistical analysis was performed with Image-Pro® Plus version 4.0 software for Windows (commercially available from Media Cybernetics, Inc., 8484 Georgia Avenue, Suite 200, Silver Spring, MD 20910-5611 USA) to calculate the pixel of pre- treatment bladder area, pre-treatment GFP area, post-treatment bladder area and post- treatment GFP area. Subsequently, the percentages of pre-treatment GFP area over pre- treatment bladder area and post-treatment GFP area over post-treatment bladder area were calculated. We performed a nonparametric test (Kruskal-Wallis test) to assess differences in the percent change in tumor size among the 7 treatments as described in Kruskal and Wallis, Journal of the American Statistical Association, 47:583-621, Conover WJ, Practical
- MTT assays were performed in substantial accordance with the teaching of Zhang et al, Cancer Res., 63:760-765 (2003). Briefly, the bladder tumor cells were infected with a given adenovirus at a 50 and 100 MOI for 2.5 h. At different time points the medium was removed, and 200 ⁇ l of medium were added containing 1 mg/ml MTT. After 3 hours, the reaction was stopped with 200 ⁇ l of N,N-dimethylformamide lysis buffer, and the resultant solution read at A 595 with a microplate reader. Additional 60 mm dishes were treated similarly or with Intron A and harvested for flow cytometry and caspase activity.
- Example 7 Confocal microscopy analysis for active caspase and IFND localization and their correlation with nuclear morphological changes.
- Human bladder cell lines were cultured on coverslips in DMEM supplemented with 10% fetal calf serum then infected with Ad- ⁇ -gal, IFN- ⁇ 2 ⁇ l or Ad-IFN- ⁇ 2b at a 50 and 100 MOI.
- Alexa-488 labeled goat anti rabbit and Alexa-546 labeled goat anti-mouse antibodies were then added together with the DNA dye, 633-Topro-3 (commercially available from Molecular Probes) for another hour.
- Mounted slides were imaged using a confocal microscope (Zeiss model LSM-510) and pixels were analyzed in each channel using Argon and He Ne lasers equipped with appropriate optical filters. The images are representative for at least ten microscopic fields analyzed for each condition. Each experiment was performed in duplicate and repeated at least three times.
Abstract
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WO2007070392A2 (en) * | 2005-12-12 | 2007-06-21 | Canji, Inc. | Adenoviral expression vectors having an expression cassette in the e1 region and an inactivated e2b polymerase |
WO2007070392A3 (en) * | 2005-12-12 | 2008-04-10 | Canji Inc | Adenoviral expression vectors having an expression cassette in the e1 region and an inactivated e2b polymerase |
JP2009519038A (en) * | 2005-12-12 | 2009-05-14 | カンジ,インコーポレイテッド | Adenovirus expression vector |
US8398968B2 (en) | 2005-12-12 | 2013-03-19 | Canji Inc. | Adenoviral expression vectors |
US11311487B2 (en) * | 2016-04-14 | 2022-04-26 | Trizell Ltd. | Viral vector stabilization |
US11446249B2 (en) | 2016-04-14 | 2022-09-20 | Trizell Ltd. | Viral vector stabilization |
US11547668B2 (en) | 2016-04-14 | 2023-01-10 | Trizell Ltd. | Viral vector stabilization |
WO2021186246A1 (en) | 2020-03-19 | 2021-09-23 | Trizell Ltd. | Temperature-responsive virus storage system |
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