EP1513956A2 - Test zum nachweis von replikationskompetenten viren - Google Patents

Test zum nachweis von replikationskompetenten viren

Info

Publication number
EP1513956A2
EP1513956A2 EP03757465A EP03757465A EP1513956A2 EP 1513956 A2 EP1513956 A2 EP 1513956A2 EP 03757465 A EP03757465 A EP 03757465A EP 03757465 A EP03757465 A EP 03757465A EP 1513956 A2 EP1513956 A2 EP 1513956A2
Authority
EP
European Patent Office
Prior art keywords
promoter
viras
cells
replication
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03757465A
Other languages
English (en)
French (fr)
Inventor
Mark Bowe
Russette M. Lyons
Tracey Walker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis AG
Original Assignee
Novartis AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis AG filed Critical Novartis AG
Publication of EP1513956A2 publication Critical patent/EP1513956A2/de
Withdrawn legal-status Critical Current

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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention generally relates to methods and assays useful for detecting replication competent viruses in preparations of selectively replicating viruses.
  • Viruses that replicate selectively in tumor cells are being developed as anticancer agents ("oncolytic viruses"). Such oncolytic viruses amplify the input virus dose due to viral replication in the tumor, leading to spread of the virus throughout the tumor mass. In situ replication of viruses leads to cell lysis. This in situ replication may allow relatively low, non-toxic doses to be highly effective in the selective elimination of tumor cells.
  • viruses designed to preferentially replicate in tumors cells include those derived from adenoviruses (US patent 5,998,205; WO 02/067861), herpesvirus (PCT Publication number WO 96/39841), reovirus (Yin, H.S.,. J Virol Methods, 1997. 67:93- 101; Strong, J.E. and P.W. Lee,. J Virol, 1996. 70:612-616; Strong, J.E., et al, Virology, 1993.
  • Paramyxovirus e.g., Newcastle Disease, measles and mumps virus; PCT Publication number WO 99/18799
  • Togavirus e.g., Sindbis PCT Publication number WO 99/18799
  • Parvoviruses Poxvirus (e.g., Vaccinia), Picomavirus (e.g., Poliovirus), Orthonyxoviruses (e.g., influenza), and rhabdovirus (PCT Publication number WO 01/19380).
  • tissue-selective promoters to control the expression of one or more early viral genes required for replication (WO 96/17053; WO 99/25860; WO 02/067861; and WO 02/068627).
  • tissue-selective promoters to control the expression of one or more early viral genes required for replication (WO 96/17053; WO 99/25860; WO 02/067861; and WO 02/068627).
  • the adenoviruses will selectively replicate and lyse tumor cells if a gene that is essential for replication is under the control of a promoter or other transcriptional regulatory element that is tissue-selective.
  • adenoviral virus preparation is the possibility of contamination with a replication competent adenovirus (RCA) or even a non adenoviral replication competent virus.
  • RCA replication competent adenovirus
  • detection of RCA can usually be accomplished using either PCR methods or biological cell-based assays (Dion, et al., "Supernatant rescue assay vs. polymerase chain reaction for detection of wild type adenovirus-contaminating recombinant adenovirus stocks," J.
  • PCR methods utilize primers based on predicted recombination events that could lead to the production of RCA; for example, recombination events with the genome of the production cell line, wherein the complementing gene provided by the cell line is recombined into the adenovirus, thus creating an RCA.
  • PCR-based assays are limited to only detecting predicted recombination events.
  • the cell-based assays when used with a replication defective virus, comprise infecting non-complementing cell lines with the virus preparations and serially passaging the virus on the non-complementing cell line (Dion, et al., supra).
  • the desired replication defective virus should not replicate on the non-complementing cell line, whereas an RCA would amplify to a detectable level through serial passaging. Detection of the RCA can usually be determined through the appearance of cytopathic effect of the cells during the amplification steps.
  • This biological cell-based assay should theoretically detect the presence of any recombinant or contaminant RCAs, whereas the PCR-based assays only detect predicted RCAs.
  • the present invention is meant to include any selectively replicating virus and not just "oncolytic viruses.” This would include any virus that preferentially replicates in a specific tissue or cell type.
  • the present invention addresses the need for a biological assay to detect replication competent virus (RCV) in replication selective virus (a.k.a. selectively replicating; e.g., oncolytic virus) preparations.
  • RCV replication competent virus
  • selectively replicating viruses present a unique challenge with regard to testing for the presence of RCV.
  • selectively replicating adenoviruses are replication competent.
  • the degree of replication of selectively replicating viruses will depend on both the design of the virus and the cells infected. It is unlikely that cell lines can be identified that will support efficient replication of a fully replication competent virus while being absolutely non- permissive for replication of a selectively replicating virus such that testing similar to that currently used for replication defective viruses can be applied to these novel products.
  • a biological testing strategy based on the relative difference in replication between a fully replication competent virus and the selectively replicating virus is presented herein. Accordingly, the present invention provides a method for detecting a non- selective replication competent virus (RCV) in a preparation of selectively-replicating virus, comprising:
  • analyzing the virus preparation amplified in (a), thereby detecting the presence of a replication competent vims comprises one or more of the following: detecting increased cytopathic effect; detecting increased virus production; detecting increased potency to kill normal cells; detecting an altered restriction digest pattern; detecting an altered viral genome sequence; detecting anticipated recombinants with PCR amplification; and detecting acute hepatotoxicity in an animal administered the vims preparation.
  • Figure 1 shows a schematic of PCR detection promoter recombinations.
  • Figure 2 shows the potency of Ar6pAE2fE3F as compared to wild-type Ad5 on HS-68 cells.
  • the difference in potency (LD50 values) is approximately 30 fold.
  • Figure 3 shows the potency of Ar6pAE2fE3F (P0), Ar6pAE2fE3F (P4) and Ad5 wild-type on MRC-5 cells. Potency of vims is measured by cell killing with an MTS readout. Potency increased more than 2 logs after amplification on MRC-5 cells. Potency is greater than wild-type Ad5 on MRC-5 cells.
  • Figure 4 shows the potency on MRC-5 cells of Ad5 wild-type after passaging on MRC-5 cells. No significant change in Ad5 potency after amplification was observed.
  • Figure 5 shows the structure/sequence some of the RCAs generated in the assay from Ar6pAE2fE3F.
  • Right end of rearranged vector contains the packaging signal, suggesting recombination mechanisms of either intermolecular recombination or polymerase jumping.
  • These RCAs had a deletion of some or all of E2F promoter, deletion of the ⁇ (A), duplication of part or all of E4 promoter and/or duplication of the packaging signal.
  • Figure 6 shows a comparison of cell killing (potency) by MTS.
  • Figure 7 shows a comparison of replication measured by hexon FACS assay in human cells.
  • Figure 8 shows the potency on MRC-5 cells for Ar5pAE2fF and Ar6pAE2fF.
  • Figure 9 shows potency on MRC-5 and human aortic endothelial cells for Ar6pAE2fE3F and wild-type Ad5 vims.
  • FIG 10 shows schematic representations of selectively replicating, oncolytic adenoviral vectors Ar6pAE2fE3F, Ar6pAE2fF and Ar5pAE2fF.
  • the Ar6pAE2fE3F- vector is based on the human adenovims serotype 5 (Ad5) backbone, and is approximately 34kb in size (Jakubczak et al. 2002).
  • the native adenoviral E1A promoter has been replaced with the human E2F-1 promoter (E2f P).
  • the packaging signal ( ⁇ ) has been moved to the right end of the genome, and an SV40 polyadenylation sequence (pA) has been inserted after the left inverted terminal repeat (ITR).
  • the 14.7K gene of the E3 region has also been deleted.
  • the Ar6pAE2fF vector is similar to Ar6pAE2fE3F, except that the entire adenoviral E3 region (E3) has been removed.
  • the Ar5pAE2fF vector is similar to Ar6pAE2fF, except that the packaging signal remains at the left end of the genome. Schematics are not drawn to scale. El A, adenoviral El A coding sequence; E4, adenoviral E4 coding sequence.
  • Figure 1 1 shows a potency analysis by cytotoxicity assay.
  • Figure 12 shows a potency analysis by cytotoxicity assay.
  • Figure 13 shows the stracture of Ar6pAE2fE3F recombinants. Twenty-six clonal isolates are prepared from the Ar6pAE2fE3F that has been passaged 4 times on MRC-5. Each clone is sequenced. Seven individual recombinants were found (A-G). The labeled schematic indicates the potential components of the recombined left end, while the bars below indicate the sequence of each recombinant analyzed. The columns to the right indicate whether each recombinant has been isolated using PER.C6 or MRC-5 cells or both.
  • the forward (— ) and reverse ( ⁇ — ) arrows indicate positions of the forward and reverse primers, respectively, for a quantitative PCR assay that recognizes recombinants A and E (REC133 Q-PCR), while the asterisked bar (*-) indicates the position of the probe.
  • the schematics are not drawn to scale. ITR, inverted terminal repeat; pA, poly A site; E2f P, human E2F-1 promoter; El A, adenoviral El A coding sequence; E4, adenoviral E4 coding sequence; E4 P, adenoviral E4 promoter; ⁇ packaging signal; Rec P, recombinant promoter.
  • Figure 14 shows a potency analysis by cytotoxicity assay.
  • the vector indicated Ar6pAE2fF or Ar5pAE2fF
  • the vector indicated is initially cloned and produced on the cell line indicated in parentheses (defined as P0), prior to being passaged sequentially four times on MRC-5 cells (P4).
  • the vector samples are diluted to the concentrations shown (ppc; particles per cell) and applied to MRC-5 cells in 96-well plates.
  • the cells are incubated for 10 days, and then analyzed for viability using an MTS assay. Measurements of viability at each dilution are expressed as the mean ⁇ SE of four replicates.
  • EC50's are calculated by regression analysis of a four parameter logistic equation, using the pooled data for each vector.
  • PCR may be used to test for the presence of recombinants containing the deleted gene ⁇ Zhang, Koch, et al. 1995 ⁇ ⁇ Dion, Fang, et al. 1996 ⁇ ⁇ Melcher, Murphy, et al. 1999 ⁇ .
  • a more general approach, and an advisable one due to its ability to detect the unexpected recombinant, is to use a biological assay to detect the replication competent recombinant ⁇ Dion, Fang, et al. 1996 ⁇ ⁇ Hehir, Armentano, et al. 1996 ⁇ ⁇ Zhu, Grace, et al. 1999 ⁇ ⁇ Melcher, Murphy, et al.
  • a biological assay for replication competent vims in a replication defective vims preparation typically involves inoculating a sufficient quantity of the vims into one or more cell lines which are non-permissive to the replication defective vims but permissive to the parental wild-type vims or replication competent recombinant. After passaging to amplify any replication competent vims that might be present, the cultures are examined for evidence of viral infection. Cytopathic effect (CPE) is a common read-out for viral vectors in this type of assay ⁇ Lehmberg, McCaman, et al. 2002 ⁇ . These assays are endpoint assays with established limits of detection that have been determined by spike-in experiments using known quantities of replication competent positive control virus.
  • CPE Cytopathic effect
  • Readenovirus Ar6pAE2fE3F which is described in detail in PCT/US02/05280 (WO 02/068627) and PCT/US02/05300 (WO 02/067861), is a genetically modified human adenovirus designed to preferentially replicate in tumor cells, resulting in tumor cell death due to oncolysis.
  • Ar6pAE2fE3F utilizes the E2F-1 promoter to control the expression of El a.
  • the E2F-1 promoter is permanently de-repressed in Rb-defective tumor cells, it is transiently de-repressed in normal, proliferating cells. Therefore, a low level of replication of replication- selective viruses controlled by the E2F-1 promoter may be expected in normal cells in vitro.
  • the E2F-1 promoter takes advantage of multiple defects in the Rb pathway, a common feature of many tumor cells, that results in hyper-phosphorylated Rb, release of Rb from E2F/DP-1 complexes and concomitant activation of promoters containing E2F binding domains, such as the E2F-1 promoter itself.
  • the E2F-1 promoter is activated in Rb-defective tumor cells due to mutation or loss of RB pathway checkpoint proteins, RB is also transiently active during the cell cycle of normal cells. Therefore, a low level of replication of Ar6pAE2fE3F may be expected in normal cells in vitro.
  • the RB pathway is tightly regulated. Without being bound by theory, the inventors believe that the mechanism of action is as follows. In GO and GI, the ternary complex of E2F-DP-l-pRB represses transcription from promoters containing E2F binding sites, including the E2f-1 promoter in Ar6pAE2fE3F. pRB is temporally regulated by phosphorylation during the cell cycle. Phosphorylation reversibly inactivates pRB, resulting in transcriptional activation by E2F-DP-1 dimers and entry into S phase of the cell cycle. Dephosphorylation of pRB after mitosis causes re-entry into GI phase.
  • any one or several of the cell cycle checkpoint proteins may be modified and lead to cell cycle deregulation and unrestricted cell cycling.
  • Loss of the pRB-E2F-DP-l interaction, or abundance of "free E2F,” results in derepression/activation of promoters having E2F sites.
  • Derepression of the E2f-1 promoter in Ar6pAE2fE3F leads to transcription of El a, viral replication, and oncolysis.
  • the RCVs that can be generated can be unpredictable.
  • the characteristics of each RCV and how they were generated will vary based on the specific design of the particular selctively-replicating vims. It is the entire viral genome that must be considered, as each element, whether engineered into the vims or an endogenous viral sequence, may influence the nature of any recombinant that is formed.
  • These data in the provided examples suggest that a combination of factors contribute to the generation of RCV in a background of replication-selective vims. The relative importance of each of these factors may vary with each virus construct.
  • the degree of replication of replication-selective vimses will depend on both the design of the vims and the cells that are infected. It is unlikely that cell lines can be identified that will support efficient replication of a non-selective replication competent vims while being absolutely non-permissive for replication of a selectively replicating viral vector. Therefore, biological assays that rely solely on the presence of, for example, CPE would be unlikely to be able to differentiate easily and objectively between a replication-selective vector and a replication competent vims.
  • the present invention provides a method for detecting a non-selective replication competent virus in a viral preparation.
  • the method includes passaging the viral preparation at least once on cells.
  • the cells can be any cells that are selected prior to performing the assay and they are selected for their ability to differentially amplify the selectively-replicating vims versus an RCV.
  • RCV may be defined as a recombinant or contaminant virus which lacks the selectivity of the replication-selective virus.
  • the viral preparation is comprised of a vims from the group consisting of adenovirus, herpesvirus, reoviras, paramyxovirus, Sindbis, parvovirases, poxvirus, picomavirus, orthonyxovirases, and rhabdovirus.
  • the viral preparation is an adenoviral preparation.
  • detected RCV would most likely be a replication competent adenovirus (RCA), although the assay will detect a non- adenovirus RCV.
  • RCA replication competent adenovirus
  • the vims is a tissue-specific replication-conditional virus.
  • the replication-conditional virus comprises a heterologous tissue-specific transcriptional regulatory sequence operatively linked to the coding region of a gene that is essential for replication of the virus, wherein the transcriptional regulatory sequence functions in the cell so that replication of the virus occurs in the cell.
  • the selectively-replicating virus comprises a heterologous tissue-specific transcriptional regulatory sequence operatively linked to the coding region of a gene that is essential for replication of the virus.
  • the transcriptional regulatory sequence is selected from the group consisting of an E2F-responsive promoter, a human telomerase reverse transcriptase (hTERT) promoter, an osteocalcin promoter, a carcinoembryonic antigen (CEA) promoter, a DF3 promoter, an ⁇ -fetoprotein promoter, an ErbB2 promoter, a surfactant promoter, a tyrosinase promoter, a MUC1/DF3 promoter, a TK promoter, a p21 promoter, a cyclin promoter, an HKLK2 promoter, a uPA promoter, a HER-2neu promoter, a prostate specific antigen (PSA) promoter, a probasin promoter, a glandular kallikrein transcriptional regulatory element (U.S.
  • hTERT human telomerase reverse transcriptase
  • CEA carcinoembryonic antigen
  • DF3 promoter an ⁇ -feto
  • Patent Application Publication No. US 2002/0136707 Al a uroplakin II-derived transcriptional regulatory element
  • U.S. Patent Application Publication No. US 2002/0120117 Al a melanoma cell specific transcriptional regulatory element (TRE), e.g. a tyrosinase, tyrosinase-related protein 2 (TRP2), MIA, microphthalmia associated transcription factor (MITF); melanocyte-specific gene 1; melanocyte-specific tyrosinase-related protein-1, or MART-1 derived TRE (all disclosed in U.S. Patent Application Publication No. US 2003/0039633 Al).
  • TRP2 tyrosinase
  • MIA microphthalmia associated transcription factor
  • melanocyte-specific gene 1 melanocyte-specific tyrosinase-related protein-1
  • MART-1 derived TRE all disclosed in U.S. Patent Application Publication No. US 2003/0039633 Al.
  • the transcriptional regulatory sequence is an E2F-1 promoter.
  • the virus further comprises a second heterologous tissue-specific transcriptional regulatory sequence operatively linked to the coding region of a second gene that is essential for replication of the virus, wherein the second transcriptional regulatory sequence functions in the cell so that replication of the virus occurs in the cell.
  • the first and second heterologous tissue-specific transcriptional regulatory sequences are the same or different.
  • the selectively-replicating vims comprises first and second genes co-transcribed as a single mRNA, wherein the first and the second genes are under transcriptional control of a heterologous, target cell- specific TRE, wherein the second gene has a mutation in or deletion of its endogenous promoter and is under translational control of an internal ribosome entry site (IRES) (WO 01/73093).
  • IRES internal ribosome entry site
  • the virus further comprises a heterologous coding sequence.
  • the product of the coding sequence provides anti-tumor activity in the cells of a target tissue.
  • the heterologous coding sequence encodes a GM-CSF.
  • the virus is replication-competent in a neoplastic cell and overexpresses an adenovirus death protein.
  • the tissue-specific replication- conditional virus is an adenovirus.
  • the tissue-specific replication- conditional adenovirus is tumor-specific.
  • tissue-specific replication-conditional virus or the tumor-specific replication-conditional virus is an adenovirus.
  • the tumor-specific replication- conditional adenovims comprises a mutation or deletion in the Elb gene, wherein the encoded Elb protein lacks the capacity to bind p53.
  • the tumor-specfic replication- conditional adenovims comprises a mutation or deletion in the Elb gene.
  • the encoded Elb protein lacks the capacity to bind p53.
  • This modification of the Elb region may be combined with vimses where all or a part of the E3 region is present.
  • the tumor-specific replication- conditional adenovirus comprises a mutation or deletion in the Ela gene.
  • the encoded Ela protein lacks the capacity to bind RB. This modification of the Ela region may be combined with vimses where all or a part of the E3 region is present.
  • the selectively-replicating vims comprises (a) an E3 sequence; and (b) an El A gene under transcriptional control of a probasin transcriptional regulatory element and an EIB gene under transcriptional control of a prostate specific antigen transcriptional regulatory element.
  • the adenovims vector comprises the entire E3 region
  • the probasin transcriptional regulatory element is the rat probasin promoter
  • the prostate specific antigen transcriptional regulatory element is the human prostate- specific enhancer/promoter.
  • the replication-conditional adenovirus comprises a mutation or deletion in the E3 region.
  • all or a part of the E3 region may be preserved or re-inserted. See, e.g., U.S. Pat. No. 6,495,130. Presence of all or a part of the E3 region may decrease the immunogenicity of the virus. It also may increase cytopathic effect in tumor cells and decrease toxicity to normal cells. Preferably, the vims expresses more than half of the E3 proteins.
  • the transcriptional regulatory sequence is a promoter or an enhancer.
  • the adenovirus coding region that is operatively linked to the transcriptional regulatory sequence is an El, E2, or E4 coding region.
  • the El coding region is an Ela or Elb coding region.
  • the E2 coding region is an E2a or E2b coding region.
  • the adenovirus comprises an E2F-1 promoter operatively linked to the Ela coding region. In another embodiment of the invention, the adenovirus comprises an E2F-1 promoter operatively linked to the Ela coding region and an hTERT promoter operatively linked to the E4 coding region.
  • the adenovirus comprises an adenovims early gene essential for propagation under the transcriptional control of a prostate cell specific response element for transcription of prostate specific antigen comprising an enhancer and promoter specific for a prostate cell.
  • the adenovims comprises at least one of the genes El A, EIB, or E4 under the transcriptional control of a prostate cell specific response element
  • the adenovims comprises a transgene under the transcriptional control of a prostate cell specific response element and lacking at least one of El A, EIB, or E4 as a functional gene.
  • the adenovirus is replication competent only in mammalian cells expressing prostate specific antigen.
  • the adenovirus comprises an adenovirus gene essential for propagation under transcriptional control of a prostate specific response element, said prostate cell specific response element comprising an enhancer specific for prostate specific antigen and a promoter.
  • the adenovirus comprises El A and EIB, wherein E1A and EIB arc both under transcriptional control of separate a fetoprotein transcription regulatory elements .
  • the adenovims comprises a first adenovims gene under transcriptional control of a first heterologous transcriptional regulatory element (TRE) and at least a second gene under transcriptional control of a second heterologous TRE, wherein the first heterologous TREs is cell-specific, the first heterologous TRE is different from the second heterologous TRE, and the heterologous TREs are functional in the same cell.
  • TRE heterologous transcriptional regulatory element
  • the adenovirus comprises an adenovims gene under transcriptional control of an alpha-fetoprotein transcription regulatory element .
  • the adenovims comprises an adenovims gene under transcriptional control of a transcriptional regulatory element comprising a cell status- specific TRE.
  • the adenovims comprises (a) an adenovims gene under transcriptional control of a target cell-specific transcriptional regulatory element; and (b) an E3 sequence.
  • the adenovims comprises g (a) an E3 sequence; and (b) a first adenoviras gene under transcriptional control of a first target cell-specific transcriptional response element and a second gene under transcriptional control of a second target cell-specific TRE.
  • the adenovirus comprises a therapeutic gene and a disease specific gene regulatory region operationally linked to at least one replication gene wherein the cancer cells activate the tumor specific gene regulatory region causing the adenoviral vector to replicate.
  • the adenovims possesses enhanced infectivity towards a specific cell type due to a modification or replacement of the fiber of a wildtype adenovims, said modification or replacement resulting in enhanced infectivity relative to said wildtype adenovirus, and wherein said infectivity-enhanced conditionally-replicative adenoviras has at least one conditionally regulated early gene, said early gene conditionally regulated such that replication of said infectivity- enhanced conditionally-replicative adenovims is limited to said specific cell type.
  • the adenovims is derived from a human adenoviras 5 genome or a human adenovims 35 genome.
  • virus and “viral particle” are used to include any and all vimses that infect a human or anima cells.
  • vims preparation and “viral preparation” are used interchangeably and refer to any batch of a vims. This includes batches prepared for research, preclinical or clinical use.
  • a viral preparation may be purified, concentrated, non-purified or partially purified.
  • adenoviras and "adenoviral particle” are used to include any and all vimses that may be categorized as an adenoviras, including any adenovims that infects a human or an animal, including all groups, subgroups, and serotypes.
  • Adenoviras serotypes 1 through 47 are currently available from ATCC and the invention contemplates the production of any other serotype of adenoviras available from any source.
  • the adenoviruses that can be produced according to the invention may be of human or non-human origin.
  • an adenoviras can be of subgroup A (e.g., serotypes 12, 18, 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35), subgroup C (e.g., serotypes 1, 2, 5, 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-47), subgroup E (serotype 4), subgroup F (serotype 40, 41), or any other adenoviral serotype.
  • Preferred serotypes are adenoviras serotypes 2(Ad2), 5 (Ad5) and 35 (Ad35).
  • adenovirus and “adenovirus particle” refer to the virus itself or derivatives thereof and cover all serotypes and subtypes and both naturally occurring and recombinant forms, except where indicated otherwise.
  • adenoviruses are ones that infect human cells.
  • Such adenoviruses may be wildtype or may be modified in various ways known in the art or as disclosed herein. Such modifications include modifications to the adenoviras genome that is packaged in the particle in order to make an infectious viras. Such modifications include deletions known in the art, such as deletions in one or more of the Ela, Elb, E2a, E2b, E3, or E4 coding regions.
  • Such modifications also include deletions of all of the coding regions of the adenoviral genome.
  • adeno vimses are known as "gutless" adenovimses.
  • the terms also include replication-conditional adenoviruses; that is, viruses that preferentially replicate in certain types of cells or tissues but to a lesser degree or not at all in other types.
  • the adenoviral particles replicate in abnormally proliferating tissue, such as solid tumors and other neoplasms. These include the vimses disclosed in EP0514603 U.S. Patent Nos.
  • viruses are sometimes referred to as "cytolytic” or “cytopathic" viruses (or vectors), and, if they have such an effect on neoplastic cells, are referred to as "oncolytic" viruses (or vectors).
  • adenovims vector and "adenoviral vector” are used interchangeably and are well understood in the art to mean a polynucleotide comprising all or a portion of an adenoviras genome.
  • An adenoviral vector of this invention may be in any of several forms, including, but not limited to, naked DNA, DNA encapsulated in an adenovims capsid, DNA packaged in another viral or viral- like form (such as herpes simplex, and AAV), DNA encapsulated in liposomes, DNA complexed with polylysine, complexed with synthetic polycationic molecules, conjugated with transferrin, complexed with compounds such as PEG to immunologically "mask” the molecule and/or increase half-life, or conjugated to a non-viral protein.
  • vector As used herein, the terms “vector,” “polynucleotide vector,” “polynucleotide vector construct,” “nucleic acid vector constract,” and “vector construct” are used interchangeably herein to mean any nucleic acid constract for gene transfer, as understood by those skilled in the art.
  • viral vector is used according to its art-recognized meaning. It refers to a nucleic acid vector construct that includes at least one element of viral origin and may be packaged into a viral vector particle. The viral vector particles may be utilized for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo.
  • Viral vectors include, but are not limited to, retro viral vectors, vaccinia vectors, lentiviral vectors, herpes viras vectors (e.g., HSV), baculoviral vectors, cytomegalovirus (CMV) vectors, papillomavirus vectors, simian viras (SV40) vectors, Sindbis vims vectors, semliki forest vims vectors, phage vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors.
  • the viral vector is preferably an adenoviral vector.
  • viral particles are used interchangeably and are to be understood broadly as meaning infectious viral particles that are formed when, e.g., a viral vector of the invention is transduced into an appropriate cell or cell line for the generation of infectious particles.
  • Viral particles according to the invention may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo.
  • these terms preferably refer to adenovimses, including recombinant adenovimses formed when an adenoviral vector of the invention is encapsulated in an adenoviras capsid.
  • the term "Replication Competent Viras” or “RCV” is understood broadly as meaning any viras in a viral preparation that does not retain the same selective replication pattern as the original (parental) selectively-replicating virus. This includes a viras that does not retain the same selective replication pattern as the originally constructed selectively-replicating viras.
  • the originally constructed virus has mutated, so that viruses in the preparation are different from the parental virus and the different viras has a replication selectivity profile different from the parental vims.
  • An RCV may be of the same type as the parental viras (e.g. both the parental virus and the RCV are based on adenovimses) or may be different types (e.g. the parental viras is based on a herpes viras and the RCV is an adenovirus).
  • Replication Competent Adenoviras or "RCA” is an RCV that is based on any adenoviras.
  • replica-conditional conditionally replicating
  • replication-selective and “replication restricted viruses” are use interchangeably and understood to mean any viras that has been designed to selectively replicate in a type or types of cells. It is not meant to encompass standard replication defective viruses that are not used as replicating viruses, except for the purpose of amplifying more virus.
  • Replication defective vimses usually have a certain function mutated or deleted, which is meant to eliminate or substantially reduce replication of the virus in the cells that are to be infected. Although, deletion of a viral function that may reduce viral replication in a cell, but is intended to allow replication in another cell type would be by definition and is still considered herein as selectively replicating.
  • the adenoviras Addll520 has the Elb region deleted, but is being used as a selectively-replicating vims for killing tumor (cancer) cells (WO 94/189992; Ganly, et al., "A Phase I Study of Onyx-015, an EIB Attenuated Adenoviras, Administered Intratumorally to Patients with Recurrent Head and Neck Cancer," Clinical Cancer Research, 6:798-806 (March 2000)).
  • Tumor-specific, tumor-selective, “tumor-selective replication-conditional” or “Tumor-specific replication-conditional” vimses are understood to be selectively- replicating viruses that preferentially replicate in at least one tumor cell type as compared to another cell type.
  • Tumor-specific virus uses tumor-selective regulatory elements to control the expression of early viral genes essential for replication.
  • Such oncolytic viruses will selectively replicate and lyse tumor cells if the gene that is essential for replication is under the control of a promoter or other transcriptional regulatory element that is tumor- selective.
  • tumor-specific replication-conditional adenoviruses have one or more mutations or deletions in the El a and/or Elb genes such that the El a protein lacks the capacity to bind RB and such that the Elb protein lacks the capacity to bind p53. (See, e.g., U.S. Pat. No. 5,677,178.)
  • a gene essential for replication refers to a nucleic acid sequence whose transcription is required for a viral vector to replicate in a target cell.
  • a gene essential for replication may be one or more of the Ela, Elb, E2a, E2b, or E4 genes, such as the Ela gene or the Ela and E4 genes.
  • Regulatory elements are sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements include promoters, enhancers, and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
  • promoter refers to an untranslated DNA sequence usually located upstream of the coding region that contains the binding site for RNA polymerase II and initiates transcription of the DNA.
  • the promoter region may also include other elements that act as regulators of gene expression.
  • Promoters and other transcriptional regulatory elements that are tumor- selective include, but are not limited to, an E2F responsive promoter such as the E2F- 1 promoter, a human telomerase reverse transcriptase (hTERT) promoter, an osteocalcin promoter, a carcinoembryonic antigen (CEA) promoter, a DF3 promoter, an ⁇ -fetoprotein promoter, an ErbB2 promoter, a surfactant promoter, a tyrosinase promoter, a MUC1/DF3 promoter, a TK promoter, a p21 promoter, a cyclin promoter, an HKLK2 promoter, a uPA promoter, a HER-2neu promoter, a prostate specific antigen (PSA) promoter, a probasin promoter, a glandular kallikrein transcriptional regulatory element (U.S.
  • E2F responsive promoter such as the E2F- 1 promoter, a human telome
  • Patent Application Publication No. US 2002/0136707 Al a uroplakin Il-derived transcriptional regulatory element (U.S. Patent Application Publication No. US 2002/0120117 Al), and a melanoma cell specific transcriptional regulatory element (TRE), e.g. a tyrosinase, tyrosinase-related protein 2 (TRP2), MIA, microphthalmia associated transcription factor (MITF); melanocyte-specific gene 1; melanocyte-specific tyrosinase-related protein- 1, or MART-1 derived TRE (all disclosed in U.S. Patent Application Publication No. US 2003/0039633 Al).
  • TRE melanoma cell specific transcriptional regulatory element
  • cancer As used herein, the terms “cancer,” “cancer cells,” “neoplastic cells,” “neoplasia,” “tumor,” and “tumor cells” (used interchangeably) refer to cells that exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. Neoplastic cells can be malignant or benign.
  • coding sequence and "coding region” refer to a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.
  • RNA is then translated in a cell to produce a protein.
  • expression refers to the transcription and/or translation of an endogenous gene or a transgene in a cell. In the case of an antisense constract, expression may refer to the transcription of the antisense DNA only.
  • gene refers to a defined region that is located within a genome and that, in addition to the aforementioned coding sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of expression, i.e., transcription and translation of the coding portion.
  • a gene may also comprise other 5' and 3 ' untranslated sequences and termination sequences. Depending on the source of the gene, further elements that may be present are, for example, introns.
  • heterologous and “exogenous” as used herein with reference to nucleic acid molecules such as promoters and gene coding sequences refer to sequences that originate from a source foreign to a particular viras or host cell or, if from the same source, are modified from their original form.
  • a heterologous gene in a vims or cell includes a gene that is endogenous to the particular viras or cell but has been modified through, for example, codon optimization.
  • codon optimization also includes non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence.
  • the terms refer to a nucleic acid segment that is foreign or heterologous to the viras or cell, or homologous to the virus or cell but in a position within the host viral or cellular genome in which it is not ordinarily found.
  • nucleic acid molecule refers to a nucleic acid sequence naturally associated with a host viras or cell.
  • native refers to a gene that is present in the genome of wildtype viras or cell.
  • Naturally occurring or wildtype is used to describe an object that can be found in nature as distinct from being artificially produced by man.
  • a protein or nucleotide sequence present in an organism which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof ("polynucleotides”) in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid molecule/polynucleotide also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • conservatively modified variants thereof e.g. degenerate codon substitutions
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al, J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al, Mol. Cell. Probes 8: 91-98 (1994)).
  • the nucleic acid molecule/polynucleotide is preferably a segment of DNA. Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G).
  • a nucleic acid sequence is "operatively linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or regulatory DNA sequence is said to be “operatively linked” to a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the promoter or regulatory DNA sequence affects the expression level of the coding or structural DNA sequence.
  • Operatively linked DNA sequences are typically, but not necessarily, contiguous.
  • the te ⁇ n "ORF" means Open Reading Frame.
  • the term “Ar5" refers to the virus Ar5 ⁇ AE2fF which is shown in Figure 10.
  • Ar5pAE2fF is the same as Ar6pAE2fF except that the packaging sequence is located on the "left" end of the viral genome.
  • Ar6pAE2fE3F utilizes the E2F-1 promoter to control the expression of Ela. Selective activation of the E2F-1 promoter in tumor cells is based on the repression of E2F-1 promoter activation in normal cells and both the derepression and activation of the E2F-1 promoter in tumor cells.
  • Arl7pAE2fFTrtex which is described in detail in PCT/US02/05300 (WO 02/067861), is a tumor-selective oncolytic adenoviras designed to be delivered systemically for the treatment of a broad range of cancer indications.
  • the replication of Arl7pAE2fFTrtex is engineered to be dependent on the presence of the two most common alterations in human cancer, namely defects in the Rb-pathway ( ⁇ 85% of all cancers) and overexpression of telomerase ( ⁇ 85% of all cancers).
  • Arl7pAE2fFTrtex utilizes the E2F-1 promoter to control expression of the adenoviral Ela gene.
  • Arl7pAE2fFTrtex is controlled by the hTERT (human telomerase reverse transcriptase) promoter.
  • hTERT human telomerase reverse transcriptase
  • an adenovirus produced according to the invention further comprises at least one heterologous coding sequence, such as a therapeutic gene coding sequence.
  • the therapeutic gene coding sequence for example in the form of cDNA, can be inserted in any position that does not adversely affect the infectivity or replication of the virus. Preferably, it is inserted in the E3 region in place of at least one of the polynucleotide sequences coding for the E3 proteins.
  • the therapeutic gene coding sequence may be inserted in place of the 19kD or 14.7 kD E3 gene.
  • a therapeutic gene coding sequence can be one that exerts its effect at the level of RNA or protein.
  • Therapeutic gene coding sequences that may be introduced into the adenoviras include a factor capable of initiating apoptosis, antisense or ribozymes, which among other capabilities may be directed to mRNAs encoding proteins essential for proliferation, such as structural proteins, transcription factors, polymerases, etc., sequences encoding cytotoxic proteins, sequences that encode an engineered cytoplasmic variant of a nuclease (e.g. RNase A) or protease (e.g. trypsin, papain, proteinase K, carboxypeptidase, etc.), or encode the Fas gene, and the like.
  • RNase A e.g. RNase A
  • protease e.g. trypsin, papain, proteinase K, carboxypeptidase, etc.
  • Immunostimulatory genes include, but are not limited to, genes that encode cytokines (GM-CSF, IL1, IL2, IL4, IL5, IFN ⁇ , IFN ⁇ , TNF ⁇ , IL12, IL18, and flt3), proteins that stimulate interactions with immune cells (B7, CD28, MHC class I, MHC class II, TAPs), tumor-associated antigens (immunogenic sequences from MART-1, gpl00(pmel-17), tyrosinase, tyrosinase-related protein 1, tyrosinase-related protein 2, melanocyte-stimulating hormone receptor, MAGE1, MAGE2, MAGE3, MAGE12, BAGE, GAGE, NY-ESO- 1, ⁇ -catenin, MUM-1, CDK-4, caspase 8, KIA 0205, HLA-A2R1701, ⁇ -
  • Anti-angiogenic genes include, but are not limited to, genes that encode METH-1, METH -2, TrpRS fragments, proliferin-related protein, prolactin fragment, PEDF, vasostatin, various fragments of extracellular matrix proteins and growth factor/cytokine inhibitors.
  • Various fragments of extracellular matrix proteins include, but are not limited to, angiostatin, endostatin, kininostatin, fibrinogen-E fragment, thrombospondin, tumstatin, canstatin, and restin.
  • Growth factor/cytokine inhibitors include, but are not limited to, VEGF/VEGFR antagonist, sFlt-1, sFlk, sNRPl, angiopoietin/tie antagonist, sTie-2, chemokines (IP- 10, PF-4, Gro-beta, IFN-gamma (Mig), IFN ⁇ , FGF/FGFR antagonist (sFGFR), Ephrin Eph antagonist (sEphB4 and sephrinB2), PDGF, TGF ⁇ and IGF-1.
  • VEGF/VEGFR antagonist include, but are not limited to, VEGF/VEGFR antagonist, sFlt-1, sFlk, sNRPl, angiopoietin/tie antagonist, sTie-2, chemokines (IP- 10, PF-4, Gro-beta, IFN-gamma (Mig), IFN ⁇ , FGF/FGFR antagonist (sFGFR
  • a "suicide gene” encodes a protein that itself can lead to cell death, as with expression of diphtheria toxin A, or the expression of the protein can render cells selectively sensitive to certain drags, e.g., expression of the Herpes simplex thymidine kinase gene (HSV-TK) renders cells sensitive to antiviral compounds, such as acyclovir, gancyclovir and FIAU (l-(2-deoxy-2-fluoro- ⁇ -D-arabinofuranosil)-5- iodouracil).
  • HSV-TK Herpes simplex thymidine kinase gene
  • suicide genes include, but are not limited to, genes that encode carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella zoster viras thymidine kinase (VZV-TK), and xanthine-guanine phosphoribosyl transferase (XGPRT).
  • CPG2 carboxypeptidase G2
  • CA carboxylesterase
  • CD cytosine deaminase
  • cyt-450 cytochrome P450
  • dCK deoxycytidine kinase
  • NR nitroreductase
  • PNP purine nucleoside phosphorylase
  • the therapeutic gene can exert its effect at the level of RNA, for instance, by encoding an antisense message or ribozyme, a protein that affects splicing or 3' processing (e.g., polyadenylation), or a protein that affects the level of expression of another gene within the cell, e.g. by mediating an altered rate of mRNA accumulation, an alteration of mRNA transport, and/or a change in post- transcriptional regulation.
  • the addition of a therapeutic gene to the virus results in a viras with an additional antitumor mechanism of action.
  • a single entity i.e., the viras carrying a therapeutic transgene is capable of inducing multiple antitumor mechanisms.
  • the therapeutic gene coding sequence encodes thymidine kinase, Nos, FasL, sFasR (soluble Fas receptor), or granulocyte macrophage colony stimulating factor (GM-CSF; U.S. Patent No. 5,908,763).
  • the therapeutic gene coding sequence is under the control of a suitable promoter.
  • suitable promoters include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter and/or the E3 promoter; or heterologous promoters, such as the cytomegaloviras (CMV) promoter; the Rous Sarcoma Viras (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; and a tissue-selective promoter such as those disclosed in PCT/EP98/07380 (WO 99/25860).
  • CMV cytomegaloviras
  • RSV Rous Sarcoma Viras
  • inducible promoters such as the MMT promoter, the metallothionein promoter
  • heat shock promoters such as the albumin promoter
  • ApoAI promoter the ApoAI
  • the invention further contemplates combinations of two or more transgenes with synergistic, complementary and/or nonoverlapping toxicities and methods of action.
  • the resulting oncolytic adenoviras would retain the viral oncolytic functions and would, for example, additionally have the ability to induce immune and antiangiogenic responses, etc.
  • adenoviruses produced according to the invention further comprise a targeting ligand included in a capsid protein of the particle.
  • the capsid protein is a fiber protein and the ligand is in the HI loop of the fiber protein.
  • the adenoviral vector particle may also include other mutations to the fiber protein. Examples of these mutations include, but are not limited to those described in US application no. 10/351,890, WO 98/07877, WO 01/92299, and US Patent Nos. 5,962,311, 6,153,435, and 6,455,314.
  • adenoviral vector particles produced according to the invention may also contain mutations to other viral capsid proteins. Examples of these mutations include, but are not limited to those described in US Patent Nos. 5,731,190, 6,127,525, and 5,922,315. Other adenoviruses that can be produced according to the invention are described in U.S. Patent Nos. 6,057,155, 5,543,328 and 5,756,086.
  • Viruses are made by transferring vectors into packaging cells by techniques known to those skilled in the art.
  • Packaging cells may complement functions deleted from the wild-type virus genome. The production of such particles requires that the vector be replicated and that those proteins necessary for assembling an infectious viras be produced.
  • the packaging cells are cultured under conditions that permit the production of the desired viral particle. The particles are recovered by standard techniques.
  • a biological assay is described herein for unintended recombinant adenoviruses which are no longer replication-selective, but rather replicate non-selectively much like parental wild-type viruses. This biological assay or a variation thereof is broadly applicable to selectively replicating viral products.
  • a biological assay has a distinct advantage in testing for replication competent virus (as is well illustrated in the examples below by the discovery of the REC133 RCV and related RCVs in the Ar6pAE2fE3F vector particle preparation).
  • the biological assays of the present invention are capable of detecting recombinants due not only to predicted but also due to unpredicted recombination events.
  • the assay detected RCVs that are not predicted (e.g. REC133) and would not have been detected by PCR assays that would have been developed in the anticipation of predicted recombinants (e.g. between the vector and packaging sequences).
  • the biological assay for RCV can be considered to have two phases.
  • the first phase is the relative amplification of the RCV compared to the intended selectively-replication viras by passaging the viral sample on the cell line (e.g. MRC-5 in the case of Ar6pAE2fE3F as described in the examples).
  • the second phase includes all appropriate analyses of the amplified material in order to detect and confirm the presence of an RCV Increased CPE, increased viras production, increased potency to kill normal cells, altered restriction digest patterns or other means of characterization of the viras may be used in any combination as detection methods to indicate the presence or absence of an RCV that is relatively amplified in the cells selected for the assay.
  • the MRC-5 cells do allow some replication of the Ar6pAE2fE3F viras and some CPE is observed at the time of optimum harvest.
  • the CPE observed can be variable, and 'scoring' it is subjective. This does not exclude that assays developed for other selectively-replicating virases may be able to use CPE as observable indication of RCV. Parameters may be optimized whereas selectively-replicating virases only develop CPE (or an observable difference in CPE) when an RCV is present at what is considered unacceptable amounts.
  • the RCV detection assays of the invention are not meant to detect every viras present in a preparation that has even a slightly altered pattern of selectivity or is present in acceptable amounts, but to detect RCVs that may pose problems for the desired purpose of the viral preparation.
  • Acceptable amounts is meant to be an amount that does not significantly effect the intended use of the viras.
  • a contaminating viras present in the preparation that has only a slight change in selectivity that would cause the killing of a limited amount of noncancerous cells would be acceptable.
  • a low level contamination of a potent RCV maybe acceptable.
  • the level contamination would probably be considered acceptable, since theoretically less than 0.1% of the experiments would ever be compromised by the contaminating RCV.
  • the sensitivity to detect RCV will likely depend on both the nature of the replication-selective viras and that of the RCV. A greater difference in their relative selectivity should allow a greater sensitivity of detection by these methods.
  • Another read out for the assays of the invention is total viral particle production.
  • total viral particle production may be a more quantitative and robust indicator.
  • the cytotoxicity potency assay is another very useful indicator of amplified RCV, and in some cases may be more reliable than even viral particle production.
  • Restriction digests provide the most concrete visual indication of altered stracture, but are dependent upon: (1) selecting a restriction endonuclease that yields a detectably different restriction pattern for the recombinant, and (2) amplification of the recombinant to a frequency high enough that it is detected by this relatively insensitive method. Banding patterns may be misleading if an aberrant fragment is not adequately resolved from expected fragments.
  • the REC133 recombinants in Ar6pAE2fE3F amplified to more than 10% of the total genomes. However, they were resolved with only one of the five endonucleases chosen.
  • Another read out in vivo toxicity For example, both the original viras preparation (P0) and a preparation from assay amplification (e.g., P4) can be injected in an animal in vivo and various data collected and compared. For example, inject the virus intravenously into mice and measure parameters of toxicity. For example, serum may be collected at various time points after injection and liver enzyme measured, which may be indicators of hepatotoxicity.
  • Increased hepatotoxicity in the amplified preparation (e.g., P4) as compared to the original (P0) preparation may indicate the presence of an RCV.
  • the use of multiple detection methods for an RCA Bioamplification Assay of the present invention is contemplated and in fact will probably increase the sensitivity of the assay.
  • Analysis of the amplified material can be extended to include some characterization of the recombinants detected.
  • One concern with a biological amplification assay such as this is that a previously absent recombinant might be generated during the amplification on MRC-5 cells.
  • the method of detection may be based on PCR.
  • a developed PCR assay for the identified recombinant sequence REC133 is able to confirm that the recombinant RCA was indeed present in the initial (P0) Ar6pAE2fE3F material at a frequency exceeding 1 copy in 1 x 10 7 vp.
  • Ar5pAE2fF that is cloned and produced on HeLa-S3 cells shows no change in any of the read-out methods, indicating that no RCV is present in the starting material.
  • the disclosure presented herein describes a biological assay for detection of RCV in selectively-replicating viras products, and that these products may contain RCVs with genomes that cannot be predicted.
  • a biological assay such as this that permits differential amplification of a RCV and the selectively-replicating virus is of general utility for testing selectively-replicating viral products, regardless of the arrangement of the viras genome.
  • a Bioamplification Assay for Replication- Selective Vectors has been successfully developed to detect RCV in a preparation of selectively-replicating viras.
  • the examples describe the assay for detecting RCV in an adenoviral preparation, but the teachings described herein are not limited to an assay to detect RCAs or to performing the assay on only adenoviral preparations.
  • EXAMPLE 1 Cell culture and virus production.
  • the tumor cell line derivative HeLa-S3 and the normal diploid human fibroblast cell lines MRC-5, Hs68 and WI-38 are obtained from American Type Culture Collection (Manassas, VA). MRC-5 and WI-38 cells are cultured in EMEM containing 10% fetal bovine serum (10% EMEM). HeLa-S3 and Hs68 cells are cultured in DMEM supplemented with 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • SAEC Small airway epithelial cells
  • NHBE bronchial epithelial cells
  • HRE renal epithelial cells
  • HMEC mammary epithelial cells
  • HAEC aortic endothelial cells
  • HMVEC microvascular endothelial cells
  • NHLF lung fibroblasts
  • PrEC prostate epithelial cells
  • PER.C6 is also an adenoviral vector complementing cell line (Cracell, Leiden, The Netherlands; ⁇ Fallaux, Bout, et al. 1998 ⁇ ) and is cultured in DMEM supplemented with 10% FBS and 10 mM MgC12. All cells are cultured at 37°C and 5% CO2, except Hs68, HeLa-S3 and PER.C6 cells, which are cultured at 37°C and 10% CO2.
  • Adenoviral vector particles are produced in the indicated cell line and purified on CsCl gradients using methods described previously ⁇ Jakubczak, Rollence, et al. ⁇ ⁇ Jakubczak, Ryan, et al. 2002 ⁇ .
  • EXAMPLE 2 Limiting dilution cloning of virus.
  • tissue culture plates are seeded with 5 x 10 3 PER.C6, MRC-5 or HeLa-S3 cells/well as indicated, in 100 ⁇ l/well.
  • three dilutions of the viral vector particles are prepared and added to 10 plates/dilution, in a volume of 100 ⁇ l/well.
  • the three dilutions of viral vector particles are chosen such that the middle dilution is calculated to yield ⁇ 3 positive wells per 10 plates (e.g. an infecting concentration of 0.03 pfu/ml), with the other two dilutions differing by 10-fold higher and lower.
  • the 31st plate for each vector is infected with higher concentrations of the viral vector particles to serve as positive controls for observable CPE.
  • the virus-infected cells are incubated for 10-14 days and then scored for CPE. Whenever cloning on HeLa-S3 and sometimes when cloning on PER.C6, scoring of positive wells is achieved with a secondary infection of S8 cells. In these cases, S8 cells are seeded on 96-well tissue culture plates at 5 x 10 4 cells/well in Richter's medium with 10% FBS, 2 mM glutamine, arid 0.5 ⁇ M dexamethasone added (100 ⁇ l/well).
  • Secondary infection is performed after 1 freeze and thaw of the primary infection plates, by transferring 50 ⁇ l of the contents of each well of the primary infection plates to the corresponding well of the S8 plates.
  • the primary infection plates are refrozen and stored at -80°C. After three days, the secondary infection plates are observed for CPE.
  • Primary infection plates corresponding to secondary infection plates containing CPE-positive wells are thawed and the clonal isolates in the primary infection plates are then amplified, preferably in the same cell line in which they had been cloned, first in 6-well plates and then in T- 175 flasks.
  • Crude viral lysates (CVL's) are prepared from the T-175 flasks and the clonal isolates are analyzed by restriction digest and agarose gel electrophoresis. Based on the restriction digest analysis of genome structure, selected clones are chosen for production of purified vector particle lots and further analysis.
  • This example is one of many methods to clone a viras by limiting dilution (e.g. plaque purification) that are known to one skilled in the art.
  • the secondary hexon titer assay is an indirect measure of adenoviral titer.
  • Samples are applied to a cell line, AE1-2A, that is permissive to viral vector replication. After 3 days, the cells are analyzed by an ELISA for total adenoviral hexon produced. The hexon signal is proportional to the input viras titer.
  • the titer of unknown samples is determined by generating a standard curve using dilutions of a reference standard vector.
  • AE1-2A cells are seeded into 96-well tissue culture plates on the day of infection at 5 x 10 4 cells/well (in 100 ⁇ l), in Richter's medium supplemented with 10% FBS, 2 mM glutamine, and 1 ⁇ M dexamethasone. Only the inner 60 wells of each plate are seeded with cells, while the outer wells of each plate are filled with 200 ⁇ l DPBS. Serial dilutions of standards and unknowns are prepared (1 :2 for known concentrations of Ad5 and Ar6pAE2fE3F to serve as the standard curves and 1 :4 for each unknown production sample) in the same medium.
  • AE1-2A cells Secondary infection of the AE1-2A cells takes place by adding 100 ⁇ l of each dilution of production sample or standard curve to triplicate wells of AE 1-2 A cells and incubating the plates for three days at 37°C with 5% CO2, with no further media changes. After three days, the secondary infection plates are harvested and three freeze-thaws are performed.
  • An ELISA is performed on these samples by first coating the inner 60 wells of the appropriate number of Immulon 96-well flat-bottom plates. Plates are coated overnight at 4°C with 100 ⁇ l anti-hexon purified antibody (2.5 ⁇ g/ml; Chemicon #MAB8052, diluted in 20 mM Tris + 150 mM NaCI). The next day, the plates are washed three times (20 mM Tris, 150 mM NaCI, 0.05% Tween 20), blocked with Pierce SuperBlock (Pierce Cat #37545), and washed again.
  • CVLs from the secondary infection plates are diluted 1:500 with dilution buffer (20 mM Tris, 150 mM NaCI, 1% bovine albumin fraction V), and 100 ⁇ l of each diluted lysate is added to the pre- coated wells. The plates are incubated at room temperature (R.T.) for 1 hour, followed by washing as above. Anti-hexon biotinylated antibody (2.5 ⁇ g/ml, diluted in dilution buffer) is then added to each well (100 ⁇ l/well) and the plates are incubated at room temperature (R.T.) for 1 hour.
  • dilution buffer 20 mM Tris, 150 mM NaCI, 1% bovine albumin fraction V
  • a research viral seed lot of Ar6pAE2fE3F is generated on AE1-2A cells, a derivative of A549 cells (Gorziglia MI, Kadan MJ, Yei S, Lim J, Lee GM, Luthra R, Trapnell BC, "Elimination of both El and E2 from adenoviras vectors further improves prospects for in vivo human gene therapy.” J Virol 1996 Jun;70(6):4173-8). Following limiting dilution cloning on PER.C6 cells, a single clone of Ar6pAE2fE3F is expanded on PER.C6 cells to generate an Accessducy Viras Bank (AcVB).
  • the AcVB Is purified by centrifugation on a cesium chloride gradient. Molecular and biologic characterization indicates that the AcVB is acceptable for generation of further viral banks. It should be noted that limiting dilution cloning followed by production of an AcVB may require a significant number of viral passages.
  • EXAMPLE 5 Procedure for screening cell lines for differential amplification of "wild-type" versus selectively replicating adenoviruses
  • normal cell lines are evaluated for increased production of an RCA compared to the selectively-replicating adenoviras, i.e. "relative amplification”.
  • Cell lines that may be evaluated include, but are not limited to, the fibroblast cell lines MRC-5, Hs68 and WI-38, as well as primary human Small Airway Epithelial Cells, SAEC (Clonetics).
  • a wild-type Ad5 is used to model potential RCA
  • the oncolytic viras Ar6pAE2fE3F ( Figure 10) is used as the test article. Production tests are performed in 75 cm tissue culture flasks, with cells cultured to 50-70% confluence. Multiple multiplicities of infection (0.4-50 particles per cell; ppc) and times of harvest (3-12 days post-infection) are assessed.
  • a cell pellet is formed by centrifugation, and a crude viral lysate (CVL) is prepared by: (1) resuspending the cell pellet in a small volume of Dulbecco's phosphate buffered saline (DPBS), (2) three cycles of freeze-thawing, and (3) clarification by low speed centrifugation.
  • Productivity of virus is determined by titering CVL's using a secondary hexon titer assay. For example, for detecting RCA in a preparation of Ar6pAE2fE3F, these initial production tests are performed with wild-type Ad5 and Ar6pAE2fE3F in separate flasks. The goal of these initial experiments is to select conditions for further development using mixtures of Ad5 and and the test article (e.g. Ar6pAE2fE3F).
  • Criteria used to select a cell line and culture conditions for further development include the relative production of Ad5 wild-type compared to the test article (e.g. Ar6pAE2fE3F), the absolute viras productivity, the general growth characteristics of the cells, and the overall feasibility of assay development.
  • the required viras yield is set at approximately 1 x 10 12 purified viral particles (vp) to enable molecular analyses such as quantitative PCR, restriction digest mapping and sequencing, if warranted. More or less virus may suffice depending on which diagnostic assays are needed.
  • EXAMPLE 6 Screening cell lines and initial development of an RCA bioamplification assay for Ar6pAE2fE3F preparations
  • the principle criteria used to select a cell line and culture conditions for further development is the relative production of Ad5 wild-type compared to Ar6pAE2fE3F, the absolute vims productivity, the general growth characteristics of the cells, and the overall feasibility of assay development.
  • the required viras yield is set at approximately 1 x 10 12 purified viral particles (vp) to enable molecular analyses such as quantitative PCR, restriction digest mapping and sequencing, if warranted. In general, longer times before harvest or greater MOI increase total productivity, but also decrease the difference in the amount of Ad5 produced compared to Ar6pAE2fE3F.
  • MRC-5 cells are selected for assessment of biological amplification of Ad5 spiked into Ar6pAE2fE3F to determine whether this method can be used to detect low levels of RCA in an oncolytic adenoviral virus preparation.
  • the harvest time is set to 6 days post-infection, after infecting with an MOI of 10-50 ppc.
  • MRC5 cells are selected for assessment of biological amplification of Ad5 spiked in Ar6pAE2fE3F.
  • the assay is designed to include 4 passages of virus on MRC5 cells with PCR detection of only 2 predicted classes of potential RCA. These predicted RCAs are based on the potential for recombination between viral vector and packaging sequences in the cell line ( Figure
  • the RCA Bioamplification Assay is initially tested at full scale using Ar6pAE2fE3F, either alone or spiked with wild-type Ad5 at ratios ranging from 1 plaque forming unit (pfu) Ad5 (wild-type) per 3 x 10 10 vp Ar6pAE2fE3F to 1 x 10 4 pfu Ad5 per 3 x 10 10 vp Ar6pAE2fE3F.
  • One viral production passage is defined as including infection of the MRC-5 cells with a known amount of viras, culturing for 6 days with one change of medium, and harvesting the viras produced. Each viras sample is passaged sequentially four times in MRC-5 cells.
  • the total number of adenoviral particles present is determined using an OD260/SDS method, and the number of copies of Ad5 present is determined using a quantitative PCR assay specific for the wild-type Ela promoter (absent in Ar6pAE2fE3F).
  • the ratio of Ad5 to total adenoviral particles is compared between each passage to determine whether relative amplification of Ad5 (the model RCA) compared to the test viras is achieved.
  • CPE cytopathic effect
  • the assay was designed to include 4 passages of viras on MRC5 cells with PCR detection of only 2 classes of potential RCA. These predicted RCAs are based on the potential for recombination between viras and packaging plasmid sequences ( Figure 1). However, during the development of the amplification portion of the assay, a biological change in the MRC5 cells was observed after the third passage of Ar6pAE2fE3F even though no model RCA had been spiked into the initial sample. The yield of viras increased at passage 3 and by passage 4 an obvious cytopathic effect was observed.
  • MRC5 cells are used for the amplification steps of the assay.
  • the present invention is not meant to be limited to a particular cell line.
  • selectively-replicating (e.g. oncolytic) viruses may for the purposes of the assay be passaged ("amplified") on any "normal" or non-target cell type. Normally several of these non-target cell types are screened based on the differential or selective amplification of the RCA as compared to a selectively-replicating virus.
  • the number of passages on the non-target cells may be increased or decreased to optimize for the differential amplification of the selective replicating viras as compared to an RCA. See example 5.
  • the "read-out" of the assay may include any of the many factors or combinations thereof including: observation of CPE, measurement of total viras or yield, analysis for molecular stracture (e.g. restriction digestion analysis, nucleotide sequencing), measurement of viral potency (e.g. cell killing assays). These factors may be measured/observed at the end of the amplification process or at each passage during this process. Then measurements/observations may be compared to those made from the first passage or starting viral preparation.
  • EXAMPLE 8 General procedure for characterizing an RCA in a preparation of adenovirus
  • the RCA may be molecularly characterized.
  • One way to characterize the RCA is to perform a restriction digest on viral DNA isolated from one of the passages, preferably the last passage, of the assay. If an uncharacteric restriction pattern is displayed, the unexpected fragment is cloned and sequenced.
  • a PCR assay is developed to specifically detect the rearranged viral vector.
  • this PCR assay has a sensitivity of at least 10-100 rearranged vector in 10 8 viral vectors. Using this assay, viral preparations are tested.
  • the unexpected fragment is cloned and sequenced.
  • the sequence analysis suggests that viral vector rearrangement has occurred resulting in a loss of most of the E2F-1 promoter and replacement with most of the viral E4 promoter ( Figure 5) such that the predicted tumor selectivity of Ela expression and viral replication are lost.
  • the assay detected an RCA that was not predicted by the inventors and therefore would not have been detected by the PCR designed to detect the two classes of predicted RCA ( Figure 1).
  • a PCR assay is developed to specifically detect the rearranged vector.
  • this PCR assay has a sensitivity of at least 10-100 rearranged viral vectors in 10 viral vectors.
  • the AcVB, Master Viras Bank (MVB) and Pre-Clinical Material are tested.
  • MVB Master Viras Bank
  • all of these viras banks and product lots are positive at levels between 30-200 RCA vectors per 10 8 vector particles (Table 1).
  • Table 1 Quantitation of Recombinant in Ar6pAE2fE3F by Q-PCR
  • PCR results are the average of triplicate determinations.
  • the initial test material grown in standard production cells is defined as viral passage 0 ("P0"). Subsequent viral passages on either cell line are numbered relative to the initial test material.
  • RCAs were detected in preparations of Ar6pAE2fE3F using a cell- based biological assay that involved serially passaging the virus.
  • the RCAs were fortuitously detected while developing the assay.
  • the recombinants that were detected through this assay were unexpected recombinants that were not predicted by the inventors.
  • a PCR-based assay based on predicted recombinants would not have detected these recombinants. This demonstrates a clear advantage of the cell-based biologic amplification assay over the PCR detection methods for detecting replication competent viruses.
  • a key characteristic of selectively replicating virases is decreased potency to kill normal or non-target cells.
  • the potencies of the P0 and P4 viras particles to kill normal cells are compared, using a cytotoxicity assay with MTS readout.
  • the Ar6pAE2fE3F MRC-5 P4 viral particles are more potent than the P0 viral particles by greater than 2 logs ( Figures 11 and 12).
  • the P4 viras is also more potent than wild-type Ad5.
  • a panel of normal primary human cell types are evaluated with varying results ranging from no difference in potency between the P0 and P4 viral particles to a 3+ log increase in potency of the P4 viral particles compared to P0 (Table 2). On no cell type tested is the potency of the P0 viral particles greater than that of the P4 viral particles.
  • Table 2 Relative potency of Ar6pAE2fE3F, P4 compared to P0.
  • Log(dose ratio) is calculated as: log [EC 50 of P4] - log [EC 50 of P0]
  • restriction digest analyses of the P0 and P4 viral vectors are performed.
  • a panel of restriction endonucleases are selected to provide banding patterns that together might indicate genetic changes in many of the critical regions of the vector. All five digests result in the expected pattern with the exception of SspBl digestion of the P4 viras, which yields a fragment of unexpected size.
  • the altered potency and restriction digest pattern of the P4 vectors compared to the starting vector strongly suggests that the P4 material contains a significant portion of vector particles with altered molecular structure and decreased replication selectivity.
  • the RCA Bioamplification Assay of Ar6pAE2fE3F virus at PO including analysis by potency assay and restriction digest, is then repeated in its entirety, which yields equivalent results, confirming the initial finding.
  • the unexpected restriction fragment of the P4 viras is extracted from the gel, cloned into a plasmid, and sequenced. Sequence analysis of the fragment, and subsequently of cloned recombinant virases described below, indicates that a rearrangement of the viral vector genome has occurred resulting in a duplication of the right end of the vector in place of the left end. This results in the loss of most or all of the E2F-1 promoter previously driving El A expression, and replacement with part or all of the adenoviral E4 promoter.
  • a quantitative PCR assay capable of detecting one of the recombinant sequences, termed "REC133".
  • This PCR assay has a sensitivity of 10 rearranged vector copies in 10 8 vector particles.
  • PCR analysis for REC133 is performed on Ar6pAE2fE3F viras before and after passaging on MRC-5.
  • REC133 is present in Ar6pAE2fE3F at PO at a frequency of 20 copies per 1 x 10 vp Ar6pAE2fE3F (Table 3).
  • the RCA Bioamplification Assay detects a recombinant that was present in the original Ar6pAE2fE3F test sample.
  • the frequency of REC133 increased dramatically with each passage on MRC-5 cells, indicating that "relative amplification" of this non-selective recombinant viras compared to the Ar6pAE2fE3F was achieved as hypothesized in the design of the assay. It was further postulated that in a cell line in which this selective advantage was reduced or eliminated, such as PER.C6, the relative amplification of REC133 would be reduced or absent.
  • PCR results are the average of triplicate determinations. c b.d. - below detection. Limit of detection for REC133 is 10 copies per 1 X 10 8 vp. Limit of detection for PGK promoter sequence is 50 copies per 1 X 10 10 vp.
  • Ar6pAE2fE3F was originally cloned and produced on PER.C6 cells, which provide El A to support viral replication. During the RCA Bioamplification Assay REC133 would likely co-infect cells with parental Ar6pAE2fE3F. Therefore it is possible that the recombinant observed is not truly replication competent but is dependent upon El A provided by either the cells when grown in PER.C6, or helper virus, in this case Ar6pAE2fE3F, when passaged in MRC-5. To more fully characterize the RCA, clonal isolates were prepared from the P4 viras.
  • Clones are successfully isolated on both PER.C6 cells and MRC-5 cells. Nineteen clones from PER.C6 and seven from MRC- 5 are analyzed by restriction enzyme digest and also by sequencing. The clones isolated include the parental (non-recombined) Ar6pAE2fE3F viras (3 of 26 analyzed).
  • the restriction digest analyses indicate the presence of multiple recombinants isolated from both PER.C6 and MRC-5 cells, which is confirmed by sequence analysis ( Figure 13). Sequencing demonstrates that all of the sequenced RCAs shared the characteristic of a right end duplication to the left end of the vector, with an altered promoter containing all or part of the viral E4 promoter presumably driving El A expression. In some recombinants a chimeric E4/E2F-1 promoter is present, while in others a complete E4 promoter is present at that position. The clones are also analyzed using the REC133 PCR assay.
  • RCA Bioamplification Assay it is possible to detect an RCA in the presence of a replication-selective adenoviral vector particle preparation.
  • the assay is performed using two additional virases, Ar6pAE2fF and Ar5pAE2fF ( Figure 10).
  • Each viras is cloned and produced on two different cell lines, PER.C6 and HeLa-S3.
  • PER.C6 is a derived cell line that expresses the adenoviral Ela gene under the control of the human PGK promoter [ ⁇ Fallaux, Bout, et al. 1998 ⁇ ].
  • PER.C6 cells were originally developed to support production of El -deleted replication defective adenoviral virases.
  • the presence of the Ela sequence preceded by the PGK promoter could allow recombination with the vector to generate a viras with Ela under the control of the PGK promoter, which is constitutive and non-selective [ ⁇ Murakami, Pungor, et al. ⁇ ].
  • the other cell line used for production, HeLa-S3 is a clonal derivative of the tumor cell line HeLa. This transformed cell line supports replication of E2F-1 promoted oncolytic adenoviruses, and so also serves as an excellent production platform for these virases.
  • the virus particle concentrations of the purified virus samples are determined by an OD260 method, and the total particles produced at each passage are calculated.
  • the patterns in productivity are similar to the observable patterns of CPE.
  • Productivity increases at P4 with all of the Ar6pAE2fF samples regardless of producer cell platform, and to a smaller degree with the Ar5pAE2fF virus sample that has been cloned and produced in PER.C6 cells.
  • no change in productivity during sequential passaging on MRC-5 cells is observed in the Ar5pAE2fF sample that has been cloned and produced in HeLa-S3 cells. Restriction digest analyses are also performed using the same enzymes as are used previously.
  • Ar6pAE2fF Aberrant restriction patterns are observed after 4 passages on MRC-5 with Ar6pAE2fF that has been cloned and produced on either PER.C6 or HeLa-S3, and with Ar5pAE2fF that has been cloned and produced on PER.C6.
  • Each value represents total viral particles purified at viral passage 1 -4 (P 1 , P2...P4) from one experiment.
  • Murakami and colleagues [ ⁇ Murakami, Pungor, et al. 2002 ⁇ ] have described adenoviral recombinants that have inco ⁇ orated the PGK-promoted Ela of the PER.C6 cell line when using that cell line for production of virases containing overlap with the Ela sequence of the cell line.
  • the PO and P4 viral particles are analyzed by quantitative PCR for both the PGK promoter and REC133.
  • Ar6pAE2fF and Ar5pAE2fF that are originally cloned and produced on PER.C6 cells do contain PGK sequence at moderate frequencies prior to passaging on MRC-5 cells. In both viral preparations, the frequency of PGK sequence in the viral genomes increases after passaging on MRC-5 cells. Neither viras cloned and produced on HeLa-S3 cells contains detectable PGK sequences.
  • Ar6pAE2fF viras that is produced on either PER.C6 or HeLa-S3 cells contain sequences detected by the REC133 PCR.
  • Ar5pAE2fF produced on either cell line contained no sequences detected by the REC133 PCR. Further characterization was not performed on the PGK promoter-containing recombinant(s) detected in our assay.
  • molecular structures of the REC133-related recombinants could have been the result of intermolecular recombination, or intramolecular polymerase jumping.
  • the formation of the REC133-related recombinants may have happened due to at least three structural factors within the Ar6pAE2fE3F and Ar6pAE2fF virases: (1) the location of the packaging signal on the right end of the molecule; (2) the replacement of the endogenous viral El promoter with the human E2F-1 promoter; and (3) the endogenous viral E4 promoter (adjacent to the packaging signal).
  • EXAMPLE 12 A general description of an RCA bioamplification assay for selectively replicating adenovirus preparations on MRC-5 cells.
  • the biological assay comprises of four sequential passages of each viras in 20 roller bottles of MRC-5 cells per virus, with CsCl purification of virus at each passage.
  • roller bottles (RB) are seeded at 2 x 10 7 cells/RB and incubated at 37°C with 5% CO2.
  • the cells are infected at 50 vp/cell with an estimated cell concentration of 4 x 10 7 cells/RB (for a total of 4 x 10 10 vp tested per sample).
  • Each roller bottle receives 60 ml infection medium (viras diluted in 10% EMEM).
  • Roller bottles are incubated at 37°C with 5% CO2 for 4 hours and then further filled with 240 ml/RB 10% EMEM supplemented with antibiotics (100 ug/ml streptomycin, 100 U/ml penicillin, 2.5 ug/ml Fungizone; BioWhittaker, Walkersville, MD). Two days after infection, all roller bottles receive a medium change with 300 ml/RB fresh 10% EMEM supplemented with antibiotics.
  • antibiotics 100 ug/ml streptomycin, 100 U/ml penicillin, 2.5 ug/ml Fungizone; BioWhittaker, Walkersville, MD.
  • Harvesting the infected cells may be performed by various methods. For example, one method is to aspirate the medium and add 50 ml Versene (0.2 mg/ml EDTA in phosphate buffered saline) to each roller bottle. After 15 to 20 min incubations at 37°C, cells are sha ⁇ ly banged off of the roller bottles and the bottles are rinsed with 10% EMEM. This method is preferred when the cells show lower levels of CPE when infected and incubated in the manner described, and are not easily removed from the surface of the roller bottles. Another harvest method is to simply shake the roller bottles with the medium in them to remove the cells.
  • This method is preferred when the cells show more CPE and/or are already starting to detach from the roller bottles.
  • cells are pelleted by low-speed centrifugation and resuspended in DPBS twice.
  • a crude viral lysate (CVL) is formed by subjecting the cell suspension to 5 freeze-thaw cycles and removing cellular debris by low speed centrifugation.
  • CVL from each viras passage is purified through two rounds of CsCl gradients and dialyzed in THPG dialysis buffer (200 mM Tris, 50 mM HEPES, phosphoric acid to pH 8.0, 10% glycerol) to remove the CsCl.
  • EXAMPLE 13 A detailed description of an RCA bioamplification assay for selectively replicating adenovirus preparations on MRC-5 cells. 1.0 SEEDING CELLS
  • MRC-5 cells have a finite life span and should not be used at the point where the growth starts to decline. ATCC estimates that they are capable of attaining 42 to 46 population doublings.
  • the infection medium for each group of 20 roller bottles contains 1260mL of EMEM + 10% FBS. Each roller bottle is infected with 60mL infection medium and this allows an excess of 1 roller bottle.
  • the average number of cells per roller bottle has been determined to be 4xl0 7 cells on the day of infection.
  • each roller bottle will be infected with 2xl0 9 viras particles.
  • the total volume of viras that has been collected from the discontinuous gradients in a sterile 50mL tube should be divided by seven for the number of gradients that will be set up for the overnight gradient.
  • Beakers are placed at 2-8°C on a stir plate set on medium-low speed.
  • MRC-5 cells are seeded in 96-well tissue culture dishes at 10,000 cells per well in 100 ⁇ l 10% EMEM. The next day, adenoviruses are serially diluted 1 :3 in 10% EMEM. The growth medium from each plate is removed and 100 ⁇ l of each viral dilution are added to the appropriate wells on each of four plates.
  • the dilution ranges tested are 3.3 x 10 8 to 7.0 x 10 1 vp/mL (or 3.3 x 10 3 to 7.0 x 10 "4 particles per cell, ppc).
  • MTS assay Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay
  • Absorbance units are converted to percent uninfected control values (% Max Control) and replicates are averaged ( ⁇ SD) between the four plates.
  • a sigmoidal dose- response curve of average % Max Control plotted versus vector viral particle dose (ppc) in logarithmic scale is fit to the data and an EC50 value is determined for each virus tested, using GraphPad Prism software.
  • EXAMPLE 15 Determination of viral vector particle concentration by OD260/SDS procedure.
  • a quantitative real-time PCR is designed to detect the wild-type El A promoter.
  • a standard curve is generated using 1.2 ⁇ g (3 x 10 10 viral genomes) of Ar6pAE2fE3F viral genomic DNA as background mixed with known amounts of Ad5 viral genomic DNA, ranging from 10 to 1 x 10 6 viral genomes.
  • a negative control of 1.3 ⁇ g (3 x 10 10 plasmid copies) of plasmid containing the Ar6pAE2fE3F sequence is included in each assay.
  • Each 100 ⁇ l reaction of standard or unknown contains: 1.2 ⁇ g DNA (3 x 10 10 viral genomes), lx TaqMan Universal PCR Master Mix (Applied Biosystems), 0.3 ⁇ M of each primer (forward primer 5'-GACCGTTTACGTGGAGACTCG-3' (SEQ ID NO:l) and reverse primer 5'-TCGGAGCGGCTCGGA-3' (SEQ ID NO:2)) and 0.1 ⁇ M of the probe (5'-FAM-TTTTCCGCGTTCCGGGTCAAAGT-TAMRA-3* ((SEQ ID NO:3)).
  • the PCR reactions are performed under the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 60 cycles of 95°C for 15 sec and 60°C for 1.5 min.
  • Adenoviruses can be grown (amplified) on PerC.6 cells.
  • PerC.6 cells contain an expression cassette with Ad5 Ela operably linked to a PGK promoter. If adenoviruses are grown on a PerC.6 cell line there is a potential for the PGK promoter to be inco ⁇ orated in to the adenoviras.
  • the PGK promoter has been reported to inco ⁇ orate into the adenoviras gemone between the ITR and the El region in such a way that it is operably linked to the adenoviras El coding regions. Thus replacing or interfering with any promoter that may have been operably linked to the El coding region to selectively control replication ofthe viras.
  • the PCR assay is designed to detect adenoviral sequences containing the PGK promoter/El A junction as found in PER.C6 cells.
  • a standard curve is generated using a background of 5 x 10 10 PGK promoter-negative adenoviral genomes (2 ug of adenoviral genomic DNA) mixed with 50 to 1 x 10 6 copies of a plasmid containing the PGK/E1 a junction sequence.
  • Each 100 ⁇ l reaction of either standard or unknown sample contains: 2 ug adenoviral genomic DNA, lx TaqMan Universal PCR Master Mix, 0.3 ⁇ M of each primer (forward primer 5'- CCGCACGTCTCACTAGTACCC-3' (SEQ ID NO:4) and reverse primer 5 > - ACACGATCGAATTCGGAACG-3' (SEQ ID NO:5)) and 0.1 ⁇ M ofthe probe (probe 5'-6FAM-CGGAGCGGGATCGAGCCCTCT-TAMRA-3' (SEQ ID NO:6)).
  • the PCR reactions are carried out under the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 60 cycles of 95°C for 15 sec and 60°C for 1.5 min. In one of these reactions the limit of detection of the assay was 50 copies of PGK Ela per 5 x 10 10 adenoviral genomes.
  • a standard curve is generated using 10 to 1 x 10 6 copies of a plasmid containing a cloned REC133 restriction fragment in a background of 1 x 10 8 adenoviral genomes (4 ng adenovioral genomic DNA).
  • Each 100 ⁇ l PCR reaction contains: 4 ng adenoviral genomic DNA, lx TaqMan Universal PCR Master Mix, 1 ⁇ M of each primer (forward primer 5'-TTTCTGGGCGTAGGTTCGC-3' (SEQ ID NO:7) and reverse primer 5'-GGTAATAACACCTCCGTGGCA-3' (SEQ ID NO:8)) and 0.2 ⁇ M of the probe (probe 5'-6FAM-CGGAGCGGGATCGAGCCCTCT- TAMRA-3' (SEQ ID NO:9)).
  • the PCR reactions are carried out under the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 64°C for 1 min. In one of these reactions tbe limit of detection of the assay was 10 copies of REC133 per 1 x 10 8 adenoviral genomes.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
EP03757465A 2002-06-07 2003-06-09 Test zum nachweis von replikationskompetenten viren Withdrawn EP1513956A2 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US38725102P 2002-06-07 2002-06-07
US387251P 2002-06-07
US45937503P 2003-03-31 2003-03-31
US459375P 2003-03-31
PCT/US2003/018243 WO2003104476A2 (en) 2002-06-07 2003-06-09 Assay to detect replication competent viruses

Publications (1)

Publication Number Publication Date
EP1513956A2 true EP1513956A2 (de) 2005-03-16

Family

ID=29739937

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03757465A Withdrawn EP1513956A2 (de) 2002-06-07 2003-06-09 Test zum nachweis von replikationskompetenten viren

Country Status (4)

Country Link
EP (1) EP1513956A2 (de)
AU (1) AU2003261076A1 (de)
CA (1) CA2488778A1 (de)
WO (1) WO2003104476A2 (de)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6900049B2 (en) 1998-09-10 2005-05-31 Cell Genesys, Inc. Adenovirus vectors containing cell status-specific response elements and methods of use thereof
AU2003265873A1 (en) * 2003-08-28 2005-04-14 Cell Genesys, Inc. Oncolytic adenoviral vectors encoding gm-csf
US7482156B2 (en) 2003-10-15 2009-01-27 Cell Genesys, Inc. Hepatocellular carcinoma specific promoter and uses thereof
US12152281B2 (en) * 2019-05-30 2024-11-26 Trizell Ltd. Gene therapy vector contamination assay
CN115583994B (zh) * 2022-11-04 2024-09-24 四川农业大学 一种猕猴桃AcVB病毒的多克隆抗体及其制备方法和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6265212B1 (en) * 1995-06-15 2001-07-24 Introgene B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
US6248514B1 (en) * 1996-07-09 2001-06-19 Canji, Inc. Methods for measuring viral infectivity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03104476A3 *

Also Published As

Publication number Publication date
CA2488778A1 (en) 2003-12-18
WO2003104476A3 (en) 2004-03-04
AU2003261076A8 (en) 2003-12-22
AU2003261076A1 (en) 2003-12-22
WO2003104476A2 (en) 2003-12-18

Similar Documents

Publication Publication Date Title
US20240191255A1 (en) Adenoviral Vectors
US7510868B2 (en) Chimeric adenoviruses for use in cancer treatment
Jogler et al. Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species
Kruyt et al. Toward a new generation of conditionally replicating adenoviruses: pairing tumor selectivity with maximal oncolysis
Gao et al. Human adenovirus type 35: nucleotide sequence and vector development
Rivera et al. Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo
US20050095705A1 (en) Method for production of oncolytic adenoviruses
US20200157567A1 (en) Viral Vector
Ugai et al. Thermostability/infectivity defect caused by deletion of the core protein V gene in human adenovirus type 5 is rescued by thermo-selectable mutations in the core protein X precursor
Xie et al. Viral vector‐based cancer treatment and current clinical applications
Carette et al. Replication‐dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice‐acceptor site
US7078030B2 (en) Oncolytic adenovirus
EP1513956A2 (de) Test zum nachweis von replikationskompetenten viren
Lu et al. Enhanced growth of recombinant human adenovirus type 41 (HAdV-41) carrying ADP gene
Murakami et al. Chimeric adenoviral vectors incorporating a fiber of human adenovirus 3 efficiently mediate gene transfer into prostate cancer cells
US7396679B2 (en) Oncolytic adenovirus
Davydova et al. Oncolytic adenoviruses: design, generation, and experimental procedures
Gao et al. A qPCR-Based method for quantification of RCA contaminants in oncolytic adenovirus products
Zhu et al. Development of an optimized conditionally replicative adenoviral agent for ovarian cancer
Sia et al. Hybrid herpes simplex virus/Epstein–Barr virus amplicon viral vectors confer enhanced transgene expression in primary human tumors and human bone marrow‐derived mesenchymal stem cells
Wu et al. Generation of a replication-deficient recombinant human adenovirus type 35 vector using bacteria-mediated homologous recombination
Xie et al. Construction of recombinant adenovirus-5 vector to prevent replication-competent adenovirus occurrence
Möritz et al. E1A genes of adenovirus type 2 and type 5 are expressed at different levels
Yeung et al. 2. HSV VECTORS 2.1. Introduction Two general strategies are used to construct HSV vectors. In one strategy, the gene of interest is inserted into the genome of the vector, either wild-type virus or, more often, virus with deletions in genes essential or nonessential for viral replication and growth. This chapter focuses on a second
To A New Type of Adenovirus Vector That

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20041220

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20050623