WO2009114459A2 - Régime thérapeutique antinéoplasique combiné comprenant la co-disruption de la voie parp et du complexe mre11/rad50/nbs1, et compositions utiles à cette fin - Google Patents

Régime thérapeutique antinéoplasique combiné comprenant la co-disruption de la voie parp et du complexe mre11/rad50/nbs1, et compositions utiles à cette fin Download PDF

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WO2009114459A2
WO2009114459A2 PCT/US2009/036497 US2009036497W WO2009114459A2 WO 2009114459 A2 WO2009114459 A2 WO 2009114459A2 US 2009036497 W US2009036497 W US 2009036497W WO 2009114459 A2 WO2009114459 A2 WO 2009114459A2
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rad50
disruptor
nbsl
complex
cancer
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WO2009114459A3 (fr
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Bert W. O'malley
Waleed M. Abuzeid
Daqing Li
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The Trustees Of The University Of Pennsylvania
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • DNA single-strand breaks occur as part of normal cellular physiology. These SSBs are normally repaired by the poly-ADP ribose polymerase (PARP) pathway. Impairment of PARP-I enzyme function allows SSB damage to persist. To avoid replication fork stalling, the cell converts SSBs to double-strand breaks (DSBs) which are repaired by the Mrel l/Rad50/Nbsl complex through homologous recombination (HR). Nijmegen breakage syndrome 1 (Nbsl) is the most upstream DNA damage sensor currently identified and directly regulates downstream effectors of HR including Rad50 and MREl 1.
  • Chemotherapy agents including cisplatin and other platinum-based drugs are commonly used in cancer treatment.
  • radiation therapy is also a mainstay of cancer therapy.
  • DNA DSBs are key morphologic changes associated with cell death in chemotherapy and/or radiation treated tumors.
  • Chemotherapeutic agents and radiation exert a dose-dependent effect and, at higher concentrations, overwhelm DNA repair mechanisms, killing a larger fraction of tumor cells.
  • cancer cells can develop resistance to the damaging effects of chemotherapy and/or radiation through enhanced DNA damage repair. From a clinical viewpoint, increasing resistance necessitates increased cisplatin (or any chemotherapy agent) as well as radiation dosing and a subsequent risk for increased serious side-effects and treatment limiting toxicities. Alternatively, increased resistance can lead to reduced efficacy of the radiation therapy or chemotherapeutic agent for ongoing or subsequent treatment.
  • DSB DNA double-stranded break
  • HR homologous recombination
  • NHEJ non-homologous end-joining
  • MRN ATP-modulated DNA crosslinker comprised of a coiled-coil sequence interposed between the two parts of an ABC-ATPase domain containing the interaction sites for Mrel 1.
  • the Rad50 ATPase domain consists of Walker A and Walker B motifs at either end of the molecule, as well as a highly conserved loop in the C-terminal catalytic domain termed the signature motif. These sites have been shown to harbor both ATPase and adenylate kinase activity that are essential for all known functions of the MRN complex.
  • the centre of the Rad50 coiled-coil sequence harbors a conserved Cys-X-X-Cys (CXXC) binding motif within a region termed the zinc hook.
  • the zinc hook is critical for dimerization between Rad50 molecules and the resulting tethering link between sister chromatids in HR, and DNA ends in NHEJ.
  • Rad50 also has a role in telomere maintenance. MRN generates the 3 '-overhangs at the ends of chromosomes that act as replication primers for telomerase, a reverse transcriptase expressed by most cancers that counteracts the loss of telomeric DNA. These actions depend upon the molecular bridging of DNA strands via the Rad50 hook domain as well as intact adenylate kinase function via the Rad50 signature motif.
  • anti-neoplastic therapies which are effective and which minimize toxic effects associated with DNA damaging agents often utilized in anti-neoplastic therapies.
  • an anti-neoplastic therapy involves delivering to a subject at least one disruptor of a poly-ADP ribose polymerase 1 (PARP) pathway and at least one disruptor of the Mrel l/Rad50/Nbsl (MRN) complex in a combination therapy, thereby simultaneously disrupting direct or indirect single-stranded and double-stranded DNA repair.
  • PARP poly-ADP ribose polymerase 1
  • MRN Mrel l/Rad50/Nbsl
  • a method of reducing the toxic effects of an anti-neoplastic regimen involves administering to a subject at least one disruptor of a PARP pathway and at least one disruptor of the MRN complex.
  • a vector which carries a nucleic acid sequence encoding a mutant Rad50 construct comprising a functional Rad50 zinc finger domain (also termed the zinc hook region) and the CXXC motif and lacking the Walker A and B motifs and the signature motif.
  • the mutant Rad50 sequence is under the control of expression control elements which drive expression thereof.
  • composition comprising the viral vector and a pharmaceutically acceptable carrier is provided.
  • a method of disrupting the MRN complex by administering an inhibitory molecule of the MRN complex is provided.
  • a method of treating a neoplasm by delivering an effective amount of an MRN inhibitor is provided.
  • FIG. 1 illustrates the construction of a mutant Rad50 construct, in which a 326 base pair mutant Rad50 hook fragment from the wild-type Rad50 gene was cloned into a recombinant adenovirus vector to produce Ad-mutRad50.
  • the zinc hook region (residues 631 - 739 of SEQ ID NO: 1), including the CXXC binding motif (amino acid residues 680-684 of SEQ ID NO:1), is reflected by the arrow labeled Rad50 hook (spanning nucleotides 1890- 2216 of the wild-type Rad50 gene).
  • Mutant Rad50 is able to dimerize with intact Rad50 due to the intact zinc hook region but is not functional as the ATPase region is not included in the mutant construct.
  • Fig. 2 is a graph showing the results of a study of the in vivo effects of dual disruption of the MRN complex and PARP on tumor volume in a model of a human head and neck cancer.
  • the solid diamond line is the saline control
  • the solid square line is an adenoviral vector expressing a Nbsl protein
  • the solid triangle line is an adenovirus construct expressing a Rad50 protein
  • the X is a PARP inhibitor
  • the line with the star is a combination of an adenoviral vector carrying a green fluorescent protein marker gene and a PARP inhibitor
  • the line with the solid circle is the combination of the Ad-Nbsl and PARP inhibitor
  • the vertical line is a combination of the Ad-Rad50 and PARP inhibitor.
  • Fig. 3 provides a bar chart illustrating tumor volume change in the same study as illustrated in Fig. 2.
  • the invention provides compositions and methods which have anti-neoplastic effects.
  • a method involving the simultaneous disruption of PARP-I function and disruption of the Mrel l/Rad50/Nbsl (MRN) complex is provided.
  • the use of at least one disruptor of a poly-ADP ribose polymerase 1 (PARP) pathway and at least one disruptor of the Mrel l/Rad50/Nbsl (MRN) complex in disrupting single-stranded and double-stranded DNA repair is provided.
  • PARP poly-ADP ribose polymerase 1
  • At least one disruptor of a poly-ADP ribose polymerase 1 (PARP) pathway and at least one disruptor of the Mrel l/Rad50/Nbsl (MRN) complex in the preparation of a medicament for disrupting single-stranded and double-stranded DNA repair in a subject.
  • PARP poly-ADP ribose polymerase 1
  • MRN Mrel l/Rad50/Nbsl
  • either the disruptor of a poly-ADP ribose polymerase 1 (PARP) pathway or the disruptor of the Mrel l/Rad50/Nbsl (MRN) complex may be used in the preparation of a medicament for disrupting single-stranded and double-stranded DNA repair in a subject, wherein disrupting single-stranded and double-stranded DNA repair comprises administration of the PARP pathway disruptor and the MRN complex disruptor.
  • PARP poly-ADP ribose polymerase 1
  • MRN Mrel l/Rad50/Nbsl
  • SSBs DNA single strand breaks
  • DSBs lethal double-strand breaks
  • MRN complex inhibitor is selected from the group consisting of: a mutant Rad50 comprising a zinc finger domain and a CXXR motif and lacking a Walker A, Walker B and signature motif; a full-length Nbsl protein; a mutant Nbs protein which retains the Mrel 1 interaction domain; and mixtures thereof.
  • a method of treating a squamous cell carcinoma comprising delivering an effective amount of a MRN complex inhibitor and a PARP pathway inhibitor, wherein the MRN complex inhibitor is selected from the group consisting of: a mutant Rad50 comprising a zinc finger domain and a CXXR motif and lacking a Walker A, Walker B and signature motif; a full-length Nbsl protein; a mutant Nbs protein which retains the Mrel 1 interaction domain; and mixtures thereof.
  • At least one disruptor of a poly- ADP ribose polymerase 1 (PARP) pathway and at least one disruptor of the Mrel l/Rad50/Nbsl (MRN) complex in treating a squamous cell carcinoma, or in the preparation of a medicament therefor, is also provided.
  • either the disruptor of a poly-ADP ribose polymerase 1 (PARP) pathway or the disruptor of the Mrel l/Rad50/Nbsl (MRN) complex may be used in the preparation of a medicament for the treatment of a squamous cell carcinoma in a subject, wherein the treatment comprises administration of the PARP pathway disruptor and the MRN complex disruptor.
  • neoplasms include, without limitation, solid tumors and non-solid tumors and all cancers.
  • Such neoplasms may include, e.g., renal cancer, soft tissue cancer, breast cancer, neuroendocrine tumor of the lung, cervical cancer, uterine cancer, head and neck cancer, glioma, non-small lung cell cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma, small cell lung cancer, ovarian cancer, colon cancer, esophageal cancer, gastric cancer, leukemia, colorectal cancer, and unknown primary cancer.
  • the cancer is a squamous cell carcinoma.
  • this "dual disruption" system of SSBs and subsequently DBSs is performed in the absence of other chemotherapeutic agents or radiation.
  • the method of the invention is used in association with chemotherapy and/or radiation treatment regimens to substantially reduce or eliminate the toxicity associated therewith.
  • any agent or combination of agents which disrupts one or more of the proteins or pathways of the MRE] l/Rad50/Nbsl (MRN) complex and inhibits or prevents its ability to repair double-stranded breaks may be used.
  • an agent which disrupts the complex at any one of the 5 proteins which compose the complex Nijmegen breakage syndrome 1 (NBSl, formerly termed p95), p200, p400, MREl 1, and Rad50 may be delivered.
  • NBSl Nijmegen breakage syndrome 1
  • p200, p400, MREl 1, and Rad50 may be delivered.
  • a vector is used to deliver an RNA sequence which extinguishes expression of the targeted nucleic acid sequence.
  • RNA small interfering RNA
  • Other desirable RNA molecules may include tRNA, dsRNA, ribosomal RNA, catalytic RNAs, and antisense RNAs of a coding sequence for a protein of the MRN complex (e.g., the Rad50 or Nbsl or Mrel 1).
  • the agent functions indirectly by targeting the DNA repair pathways and mechanism associated with the MRN complex (e.g., by targeting ATM or E2F).
  • a dominant negative approach is utilized.
  • a mutant version of a protein of the normal MRN complex is delivered which interferes with the normal DSB function of the complex, e.g., by competing with the normal protein (which requires dimerization for its function) and thereby disrupting complex function.
  • two or more agents, each of which disrupts the complex at different site may be delivered.
  • a sole MRNl l/Rad50/Nbs complex disruptor or a combination of such agents are delivered in combination with a chemotherapeutic agent or chemoradiation.
  • the MRN complex is disrupted at the Rad50 gene. It is believed that this is a downstream disrupter of the MRN complex which disrupts Rad50-mediated DNA repair.
  • SEQ ID NO: 1 reproduces the Rad50 protein and identifies features therein. This sequence is incorporated by reference herein.
  • the disruptor is a Rad50 comprising a functional Rad50 zinc finger domain and the CXXC motif, with a non-functional Walker A motif (amino acids 36 - 43 of SEQ ID NO: 1 ), non-functional Walker B motif (amino acids 1227-1232 of SEQ ID NO:1), and non-functional signature motif (amino acids 1201-1210 of SEQ ID NO:1).
  • the mutant Rad50 retains Rad50 dimerization ability.
  • the mutant Rad50 is rendered nonfunctional in one or more of these motifs by virtue of an internal deletion or truncation which eliminates the motifs.
  • a mutant Rad50 protein as described herein may be delivered to a subject via a vector which expresses the protein in the target cells. While any means may be used to deliver such mutant Rad50, an adenoviral vector is illustrated below.
  • the disruption of the Rad50 is particularly well suited for use in combination with disruption of the PARP function, it is believed that the disruption of the Rad50 (e.g., via delivery of the mutant Rad50 described herein) has anti-neoplastic effects even when delivered as a sole agent.
  • the invention provides an anti-neoplastic therapy involving disruption of the Rad50 function. This may be achieved by delivery of a mutant Rad50 as described herein.
  • disruption of the Rad50 function is combined with a therapy involving disruption of the Nbsl protein.
  • disruption of the Rad50 function is combined with disruption of the Nbsl protein function with a PARP inhibitor.
  • these therapeutic methods may be further combined with a chemotherapeutic regimen and/or with radiation.
  • the constructs and methods of the invention advantageously allow reduction or elimination of the toxicity association with such chemotherapy and/or radiation.
  • a method of reducing the toxic effects of an anti-neoplastic, or chemotherapetic, regimen comprising administering to a subject: at least one disruptor of a po Iy-ADP ribose polymerase 1 (PARP) pathway and at least one disruptor of the Mrel l/rad50/nbsl (MRN) complex, thereby simultaneously disrupting single-stranded and double-stranded DNA repair.
  • PARP po Iy-ADP ribose polymerase 1
  • MRN Mrel l/rad50/nbsl
  • PARP poly-ADP ribose polymerase 1
  • MRN Mrel l/rad50/nbsl
  • the MRN complex is disrupted upstream by rendering the Nbsl gene non-functional, either directly or indirectly.
  • a "non-functional" Nijmegen breakage syndrome 1 (Nbsl, formerly termed p95) gene means a gene wherein the encoded protein having part or all of the primary structural conformation of the wild type Nbsl protein (reproduced in SEQ ID NO:2, which is incorporated by reference herein), i.e., possessing the biological property of participating in properly active hMREl l/hRAD50/NBSl nuclease complex has been destroyed or eliminated.
  • “non-functional” may include a deletion or truncation which eliminates sequences.
  • a mutant Nbsl protein which retains the human Mrel 1 interaction domain (located in amino acids 682 to 746 of the protein, with reference to SEQ ID NO:2) is provided, but which has a non-functional N-terminus.
  • the MRN complex is disrupted by delivery of a mutant Nbsl protein which consists of about the C-terminal 300 amino acids of Nbsl [J G Rhee, et al, Int'lJ Radiation Oncol. Biol. Physics, 67 (l):273-278 (2007)], but lacks the ability to bind to the remainder of the complex.
  • a full-length Nbsl protein may be delivered, as this full-length protein has been found to disrupt MRN complex function.
  • a nibrin as described herein may be delivered to a subject via a vector which expresses the protein in the target cells.
  • a delivery vector any genetic element may be used as a delivery vector
  • the Nbsl construct is delivered to a subject via an adenoviral vector.
  • Such a construct can be prepared using methods such as those described. See, e.g., US Patent 6,458,534.
  • an MRN complex disruptor is delivered by any biologically useful moiety that can be transferred into targeted cells.
  • suitable moieties include lipid-based delivery vehicles or any genetic element composed of nucleic acids, including DNA and RNA molecules, an enzyme, a protein, peptide, or non-proteinaceous molecule, which may include small molecules or other chemical moieties.
  • a vector may include, e.g.. plasmids, episomes, cosmids, viral vectors, phage, "naked DNA", any of which desirably contains a transgene under the control of regulatory sequences that direct expression thereof in a target cell.
  • the macromolecular complex comprises a viral vector.
  • Suitable viral vectors include, without limitation, adenoviruses, picomavirus, adeno-associated viruses, retroviruses, baculoviruses, and Antiviruses, among others. Methods of producing such viral vectors have been described [US Patent No. 6,083,716; US Patent No. 7,247,472; US Patent 7,291,498].
  • the nucleic acid sequences of Rad50, Nbsl, or RNA sequences thereof are employed to prepare a transgene or RNA (e.g., siRNA) useful in the preparation of vectors.
  • the sequences are isolated from a natural source (e.g., using primers designed based upon published sequences) or prepared synthetically based upon published sequences. [See, e.g., US Patent 5,965,427 and International Patent Publication No. WO 1997/027284 and for a description of the human Rad50 gene; see, US Patent 7,122,343, for a description of human Nbsl (formerly p95) gene; and GenBank],
  • the sequences are from a human MRN sequence.
  • a vector used in the invention carries a nucleic acid sequence encoding an single MRN complex disruptor sequence.
  • the vector may carry multiple transgenes, e.g. , mutant Rad50 and mutant Nbs 1.
  • a single transgene includes the DNA encoding more than one MRN complex disruptor, with the DNA for each product (or the RNA) separated by an internal ribozyme entry site (IRES). This is desirable when the size of the DNA encoding each of the subunits is small, e.g. , the total size of the DNA encoding the subunits and the IRES is less than five kilobases.
  • the DNA may be separated by sequences encoding a 2A peptide, which self-cleaves in a post-translational event. See, e.g., M.L. Donnelly, et al, J. Gen. Virol, 78(Pt 1): 13-21 (Jan 1997); Furler, S., et al, Gene Ther., 8(1 1):864-873 (June 2001); Klump H., et al, Gene Ther., 8(10): 811-817 (May 2001).
  • This 2A peptide is significantly smaller than an IRES, making it well suited for use when space is a limiting factor.
  • a vector also includes control elements necessary which are operably linked to the transgene in a manner that permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention.
  • operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic MRNA; sequences that enhance translation efficiency ⁇ i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • RNA processing signals such as splicing and polyadenylation (polyA) signals
  • sequences that stabilize cytoplasmic MRNA sequences that enhance translation efficiency ⁇ i.e., Kozak consensus sequence
  • sequences that enhance protein stability e.g., telomereon sequences that enhance protein.
  • a great number of expression control sequences including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
  • constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl ⁇ promoter [Invitrogen].
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • the native promoter for the transgene will be used.
  • the native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression.
  • the native promoter may be used when expression of the transgene must be regulated temporally or developmental Iy, or in a tissue- specific manner, or in response to specific transcriptional stimuli.
  • other native expression control elements such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.
  • tissue-specific promoter is utilized. Promoters that are tissue-specific are known for a variety of cell types, including a variety of cancer cell types. For example, the squamous cell carcinoma antigen 1 and antigen 2 promoters, the human telomerase reverse transcriptase (hTERT) promoter, NF- kappa B-CEA enhancer-promoter, L-plastin promoter, amongst others, may be useful in targeting cancer cells.
  • hTERT human telomerase reverse transcriptase
  • vectors may include an origin of replication. Selection of these and other promoters and vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, and references cited therein].
  • an adenoviral vector may be utilized to deliver a transgene of the invention.
  • Methods for preparing adenoviral vectors have been described, as have a variety of sources of adenoviral sequences.
  • a variety of adenovirus sequences are available from the American Type Culture Collection, Manassas, Virginia, or available by request from a variety of commercial and institutional sources.
  • the sequences of many such strains are available from a variety of databases including, e.g., PubMed and GenBank.
  • Homologous adenovirus vectors prepared from other simian or from human adenoviruses are described in the published literature [see, for example, US Patent No. 5,240,846].
  • the DNA sequences of a number of adenovirus types are available from
  • GenBank including type Ad5 [GenBank Accession No. M73260].
  • the adenovirus sequences may be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and further including any of the presently identified human types.
  • adenoviruses known to infect non-human animals e.g., simians
  • adenoviruses have been described for delivery of heterologous molecules to host cells. See, US Patent 6,083,716, which describes the genome of two chimpanzee adenoviruses. Simian adenoviruses, C5, C6 and C7, have been described in US Patent No. 7,247,472 as being useful as vaccine vectors. Yet other adenoviruses are described in WO 2005/1071093 as being useful for making adenovirus vaccine carriers. See, also, US 7,291,498. The selection of the adenoviral sequences present in vector is not a limitation of the present embodiment.
  • an adenoviral vector used is an adenoviral particle which is replication - defective.
  • the adenoviral particles are rendered replication-defective by deletions in the EIa and/or EIb genes.
  • the adenoviruses are rendered replication-defective by another means, optionally while retaining the EIa and/or EIb genes.
  • the adenoviral vectors can also contain other mutations to the adenoviral genome, e.g., temperature-sensitive mutations or deletions in other genes.
  • an adenoviral vector contains the 5' end of an adenoviral genome and/or the
  • the 3' end of an adenoviral genome contains the 5' cis-elements necessary for packaging and replication; i.e., the 5' inverted terminal repeat (ITR) sequence (which function as origins of replication) and the native 5' packaging enhancer domains (that contain sequences necessary for packaging linear Ad genomes and enhancer elements for the El promoter).
  • the 3 ' end of the adenoviral genome includes the 3' cis-elements (including the ITR) necessary for packaging and encapsidation.
  • a recombinant adenovirus contains both 5' and V adenoviral cis-elements and the minigene is located between the 5' and 3' adenoviral sequences.
  • a range of adenovirus nucleic acid sequences can be employed in the vectors.
  • all or a portion of the adenovirus delayed early gene E3 may be eliminated from the simian adenovirus sequence which forms a part of the recombinant virus.
  • the function of simian E3 is believed to be irrelevant to the function and production of the recombinant virus particle.
  • Simian adenovirus vectors may also be constructed having a deletion of at least the ORF6 region of the E4 gene, and more desirably because of the redundancy in the function of this region, the entire E4 region.
  • Still another vector of this invention contains a deletion in the delayed early gene E2a. Deletions may also be made in any of the late genes Ll through L5 of the simian adenovirus genome. Similarly, deletions in the intermediate genes TX and IVa 2 may be useful for some purposes. Other deletions may be made in the other structural or non-structural adenovirus genes. The above discussed deletions may be used individually, i.e., an adenovirus sequence for use as described herein may contain deletions in only a single region.
  • deletions of entire genes or portions thereof effective to destroy their biological activity may be used in any combination.
  • the adenovirus sequence may have deletions of the El genes and the E4 gene, or of the El, E2a and E3 genes, or of the El and E3 genes, or of El, E2a and E4 genes, with or without deletion of E3, and so on.
  • deletions may be used in combination with other mutations, such as temperature-sensitive mutations, to achieve a desired result.
  • An adenoviral vector lacking any essential adenoviral sequences may be cultured in the presence of the missing adenoviral gene products which are required for viral infectivity and propagation of an adenoviral particle.
  • helper functions may be provided by culturing the adenoviral vector in the presence of one or more helper constructs (e.g., a plasmid or virus) or a packaging host cell. See, for example, the techniques described for preparation of a "minimal" human Ad vector in International Patent Application WO96/13597, published May 9, 1996, and incorporated herein by reference.
  • Vectors are generated using the techniques and sequences provided herein, in conjunction with techniques known to those of skill in the art. Such techniques include conventional cloning techniques of cDNA such as those described in texts, use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.
  • the vectors described herein may be formulated in a suitable physiologically compatible carrier for delivery by any suitable route.
  • the vectors are suspended in a physiologically compatible saline solution and delivered via direct injection into a tumor, parenterally, or intraperitoneally. Further examples of suitable delivery routes are provided below. Where desired, the vector are delivered in an amount of about 1 x 10 9 to about 1 x IO 12 genomes/mL. However, higher or lower amounts may be utilized, taking into account such factors as the vector selected and the age, weight, and condition of the patient. PARP
  • the MRN complex is disrupted at the same time that the PARP function is inhibited.
  • PARP inhibitors have been described in the literature and several are presently in clinical trial. Examples of suitable PARP inhibitors include, without limitation, AG014699 [a nicotinomide, Pfizer, NY], KU59436 [AstraZeneca/KuDOS, London, UK, EPl 397350], ABT-888 [Abbott Laboratories, IL], BSI-201 [BiPar, Brisbane, CA], lNO-1001 [Inotek/Genentech, Beverly, MA], and GPI21016 [MGI Pharma, Bloomington, MN].
  • the PARP-I inhibitor is selected from a nicotinamide: NUl 025; 3-aminobenzamide; 4-amino-l,8- naphthalimide; 1,5-isoquinolinediol; 6(5H)-phenanthriddinone; 1,3,4,5,- tetrahydrobenzo(c)(l,6)- and (c)(l,7)-naphthyridin- 6-ones; adenosine substituted 2,3-dihydro- IH-isoindol-t-ones; AG14361 ; AGOl 4699; 2-(4-chlorophenyl)-5-quinoxalinecarboxamide; 5- chloro-2-[3-(4-phenyl-3,6-dihydro- I(2H)-pyr
  • PARP inhibitor is not a limitation of the present invention. Still other PARP inhibitors may be selected by one of skill in the art or identified by one of skill in the art. Assays for determining whether a compound has PARP or PARP-inhibitory activity have been described. Further, a kit for performing such an assay is commercially available [TREVIGEN®].
  • the MRN complex and PARP are disrupted at the same time.
  • this does not necessarily require simultaneous delivery of the PARP disruptor(s) and MRN complex disruptor(s). Rather, delivery of these agents can take into consideration a variety of factors which will be readily understood by one of skill in the art, e.g., any delay time in protein expression following delivery of a viral vector, the circulating half-life of such a protein, a PARP inhibitor, or other factors.
  • the effective amount of the PARP-I inhibitor is between about 10 and 500 mg/day. Alternatively, the effective amount of the PARP-I inhibitor can be between about 100 and 250 mg/day.
  • compositions described herein can be administered in multiple doses over prolonged periods of time.
  • the PARP inhibitor compounds are administered for periods up to about one week, and even for extended periods longer than one month or one year. In some instances, administration of the compounds can be discontinued and then resumed at a later time.
  • a daily dose of the compounds can be administered in several doses, or it can be given as a single dose.
  • the amount of PARP-I inhibitor administered is between about 10 to about 500 mg/day. However, in each case, the dose depends on the activity of the administered compound. Appropriate doses for any particular host can be readily determined by empirical techniques well known to those of ordinary skill in the art.
  • the compounds can be administered separately or as a single composition (combined). If administered separately, the compounds may be given in a temporally proximate manner. More particularly, the compounds can be given within one to twenty-four hours of each other.
  • the administration can be by either local or by systemic injection or infusion. Other methods of administration can also be suitable.
  • compositions described herein maybe oral, intravenous, respiratory (e.g., nasal or intrabronchial), infusion, parenteral (besides Lv., such as intralesional, intraperitoneal and subcutaneous injections), intraperitoneal, transdermal (including all administration across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues), and vaginal (including intrauterine administration).
  • Other routes of administration are also feasible, such as via liposome- mediated delivery; topical, nasal, sublingual, uretheral, intrathecal, ocular or otic delivery, implants, rectally, intranasally.
  • a product or pack according to the invention may contain a PARP-inhibitor, for delivery by a different route than that of the MRN complex disruptor, e.g., one or more of the components maybe delivered orally, while one or more of the others are administered intravenously.
  • other active components may be delivered by the same or different routes as the PARP-inhibitor or the MRN complex disruptor.
  • Other variations would be apparent to one skilled in the art and are contemplated within the scope of the invention.
  • the combination regimen can be given simultaneously or can be given in a staggered regimen, with the PARP-inhibitor being given at a different time during the course of therapy than the MRN complex disruptor.
  • This time differential may range from several minutes, hours, days, weeks, or longer between administration of the at least two agents. Therefore, the term combination (or combined) does not necessarily mean administered at the same time or as a unitary dose, but that each of the components are administered during a desired treatment period.
  • the agents may also be administered by different routes.
  • Example 1 Head and neck cancer natively expresses wild-type Mrel 1, Rad50, and Nbsl proteins
  • the disruption of the Rad50 is a viable approach to anti-tumor therapy, as illustrated by the mutant Rad50 construct.
  • the absence of ATPase domains in the mutant Rad50 construct disrupts MRN-mediated telomere maintenance such that G- overhangs are not generated and telomerase function is impaired.
  • Combination treatment with cisplatin and Ad-mutRad50 caused significantly greater telomere shortening than either treatment alone.
  • Ad-mutRad50 compromises telomerase activity, enhancing the telomere-damaging effects of cisplatin, and inducing cytotoxicity in tumors.
  • cisplatin alone did not demonstrate anti-tumor efficacy, as defined by the National Institutes for Cancer (NCI), when used on xenografts mirroring the resistance to cisplatin observed clinically.
  • NCI National Institutes for Cancer
  • cisplatin-induced tumor volume reduction was dramatically enhanced by combination therapy with Ad-mutRad50 which acted to overcome chemoresistance, resulting in a significant antitumor effect according to NCI criteria. This effect could be partially attributable to the observed increase in apoptosis.
  • Tumors treated with a combination of Ad-mutRad50 and 3 mg/kg of cisplatin were, on average, over 30% smaller than tumors treated with cisplatin 5 mg/kg alone.
  • HNSCC human head and neck squamous cell cancer
  • the MRN complex is highly conserved but mutations in Nbsl, Mrel 1 and Rad50 genes which produce chromosomal instability syndromes and an inherent susceptibility to DNA damaging agents have been identified [Tauchi, H., et al, Oncogene 21 : 8967-8980; Fukuda, T., et al, Cancer Res 61 :23-26; Heikkinen, K., et al, 2006, Carcinogenesis 27:1593- 1599].
  • Cells were cloned from the original parental cell lines for use in all experiments. Cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 0 C in 5% CO 2 .
  • Example 2 Ad-mutRad50 produces a dominant negative down-regulation of MRN A. Construction of Ad-mutant Rad50
  • a novel recombinant serotype 5 adenoviral vector, Ad-mutRad50, containing a gene sequence encoding a fragment of the Rad50 zinc hook region with an intact CXXC motif but completely missing the ATPase region was designed.
  • AdEasyTM system was used to construct the adenoviral vector [He, T.C, 1998, Proc Natl Acad Sci USA 95:2509-2514].
  • a plasmid containing a fragment of the Rad50 hook situated at the hinge domain of the wild-type Rad50 gene was cloned into a pAdTrack-CMV shuttle vector.
  • the plasmid was linearized by Pme ⁇ digestion, co- transformed into E. coll BJ5183 cells with the pAdEasy-1TM adenoviral backbone plasmid and then transfected into HEK293 cells to generate the recombinant adenovirus, Ad-mutRad50.
  • Ad-mutRad50 was amplified, purified and titers were determined using standard plaque assay.
  • the control adenovirus, DL312 with EIa region deletion was obtained from Dr. Tom Shenk, Princeton University, Princeton, NJ.
  • the resulting mutant Rad50 protein retains RadSO dimerization ability but is non-functional due to absence of the Walker A and B motifs and the signature motif. These motifs are known to be essential for MRN-mediated DNA DSB repair and telomere maintenance.
  • JHU012 cancer cells were either untreated or infected with Ad-mutRad50.
  • Recombinant adenovirus was introduced into JHUO 12 cell cultures at a multiplicity of infection (MOI) of 10 for 4 hours at 37°C.
  • the primary antibodies used were a polyclonal rabbit anti-human Rad50 (zinc hook domain) (Novus Biologicals, Littleton, CO), which recognizes both wild-type and mutant Rad50, polyclonal rabbit anti-human Mrel 1 (Novus Biologicals) and polyclonal rabbit anti-human Nbsl (Novus Biologicals). All primary antibodies were used at a concentration of 1 : 1000.
  • High-resolution images of the protein bands were captured using a Canon Digital Rebel XTI camera (Canon U.S.A., Inc., Lake Success, NY) and transferred to a desktop computer for analysis. Densitometry was performed using ImageJ software according to standard NIH gel analysis guidelines.
  • Ad-mutRad50 infected cells demonstrated a significant down-regulation of wild-type Mrel 1 (p ⁇ 0.005), Nbsl (p ⁇ 0.04) and RadSO (pO.OOl) proteins compared to control cells. Furthermore, only Ad-mutRad50 infected cells showed expression of the 13 kDa mutant Rad50 protein (p ⁇ 0.003). These results indicate that transgene expression of the non-functional mutant RadSO induces a dominant negative down-regulation of the MRN complex.
  • Example 3 - Mutant Rad50 transgene expression enhances the cytotoxicity of cisplatin in vitro
  • This cisplatin concentration represents the IC50 dose for each cell line.
  • Control and DL312-treated cells exhibited persistent logarithmic growth.
  • Cisplatin, alone or with DL312 control virus, produced a reduction in cell growth relative to controls in both cell lines (p ⁇ 0.01).
  • Ad-mutRad50 monotherapy also produced a significant reduction in cell proliferation without the need for an external DNA damaging agent (p ⁇ 0.01).
  • Combination therapy with Ad-mutRad50 and cisplatin persistently suppressed cell growth within 48 hours of treatment initiation, and this cytotoxic effect was significant compared to either treatment alone (p ⁇ 0.01).
  • Example 4 Enhanced cytotoxicity is associated with increased DNA DSB damage
  • JHUO 12 cells were assessed for DSB damage. JHU012 cancer cells that were not treated and cells treated with the replication defective DL312 empty virus served as controls. The other four groups were: cisplatin alone (0.3 ⁇ g/mL), Ad-mutRad50 alone, DL312 with cisplatin (0.3 ⁇ g/mL) and Ad-mutRad50 with cisplatin (0.3 ⁇ g/mL). An MOI of 10 was used for all adenovirus infections. Tumor cells from the JHUO 12 cell line were grown as a monolayer in 24 well tissue culture plates. Cells were transfected with either Ad-Nbsl or DL312 (MOI 10) over 4 hours at 37°C.
  • Ad-Rad50 and cisplatin produced a greater MTM (4258) than non- treatment and DL312 controls (p ⁇ 0.001), cisplatin alone or with DL312 (p ⁇ 0.001) and Ad- Rad50 monotherapy (p ⁇ 0.02).
  • the potent DNA-damaging effect associated with combination Ad-Rad50 and cisplatin correlates with the observed cytotoxicity of this treatment.
  • Example 5 Enhanced cytotoxicity is associated with telomere shortening
  • telomere PNA FISH peptide nucleic acid fluorescence in situ hybridization
  • JHUO 12 cancer cells were used for this analysis.
  • Treatment groups were identical to those used for the neutral comet assay as above and adenoviral vectors were introduced at a MOI of 10.
  • Cisplatin was used at the IC50 dose for the JHU012 cell line (0.3 ⁇ g/mL). Cells were incubated for 96 hours. Culture medium was supplemented with 10 ⁇ g/mL Colcemid (Gibco BRL 5 Grand Island, NY) for 2 hours.
  • Telomere staining intensity is linearly proportional to telomere length. Quantification of telomere staining indicated that non-treatment control JHU012 cells had the highest staining intensity of 759.0 and, thus, the longest telomeres. Cisplatin is known to preferentially bind to runs of 2 or more guanine residues. Consequently, the (TTAGGG) n (n ⁇ 2) human telomeric sequence forms an ideal target for cisplatin. Human telomeric sequences typically extend approximately 5 to 15 kb in length. Indeed, cisplatin is known to induce telomere shortening and apoptosis in human cells [Ishibashi, T., and Lippard, S.J. 1998.
  • telomere staining intensity 248.8 vs. 759.0, p ⁇ 0.01.
  • Ad-mutRad50 monotherapy MOI 10
  • MRN telomere maintenance function also produced a marked reduction in telomere staining intensity to 187.0.
  • Combination therapy with Ad-mutRad50 (MOI 10) and cisplatin reduced telomere staining intensity to 65.0 indicating that telomeres were shorter than in all other groups (p ⁇ 0.01).
  • telomere shortening impairment of Rad50 telomere maintenance function can facilitate cisplatin-mediated telomere shortening. This effect is likely to have contributed to the observed cytotoxic effect of combination treatment since telomere shortening beyond a critical threshold induces cellular senescence.
  • Example 6 - Rad50 disruption enhances the outcome of cisplatin-based chemotherapy in a mouse model of human head and neck cancer
  • a mouse model of human head and neck cancer was used to further evaluate whether the in vitro cytotoxicity caused by the disruption of normal MRN function translates into a beneficial effect on in vivo tumor growth.
  • Animal experiments were conducted in 8-10 week old, athymic, BALB/c nu/nu nude mice. Use of all animals was in accordance with the guidelines of the University of Pennsylvania School of Medicine Institutional Animal Care and Use Committee.
  • mice Sixty nude mice were randomized into 6 groups of 10 mice each. Mice were anesthetized with 8 mg tribromoethanol via intraperitoneal injection. Depth of anesthesia was determined by toe pinch. The right dorsal flank of each mouse was injected subcutaneous Iy with 1 x 10 7 JHUO 12 cells (day 0). After the establishment of palpable tumors, mouse body weight and external tumor volume was determined every 48 hours throughout the duration of the in vivo study. External tumor diameter was measured using digital calipers and tumor volume was calculated using the formula A 2 x B x 0.536, where A represents the smallest diameter and B equals the largest diameter.
  • mice were re-anesthetized 13 days after tumor injection when the average externally measured tumor volume was 100 mm 3 .
  • Tumors were exposed by raising a skin flap using precise surgical dissection, taking care not to disturb the tumor or its blood supply. The exposed tumors were measured in three dimensions (maximal length x maximal width x maximal height) using calipers to accurately assess tumor volume as previously described [Rocco, J.W., et al, 1998, Clin Cancer Res 4:1697-1704].
  • the intratumoral dose of Ad-Rad50 and DL312 was 8.5 x 10 s pfu in 50 ⁇ L volume.
  • the six intervention groups were: intratumoral saline, intraperitoneal cisplatin (3 mg/kg), intratumoral DL312 and intraperitoneal cisplatin (5 mg/kg), intratumoral Ad-Rad50, intratumoral Ad- Rad50 and intraperitoneal cisplatin (3 mg/kg) and intratumoral Ad-Rad50 with intraperitoneal cisplatin (5 mg/kg). Skin incisions were closed with 4-0 nylon suture. The internal tumor volume was re-measured 1 1 days after treatment.
  • Control group tumors exhibited sustained growth following subcutaneous cancer cell injection and, between days 13 and 24, showed a 186% increase in tumor volume relative to pre-treatment size.
  • Ad-mutRad50 is able to reverse the inherent cisplatin resistance of JHUO 12 squamous cell cancer. This effect is secondary to transgene expression of the mutant Rad50 gene and not a consequence of adenovirus-mediated cytotoxicity since the combination of cisplatin with the DL312 control El -deleted adenovirus was significantly less efficacious than combination cisplatin with Ad-mutRad50.
  • TUNEL staining was used on xenograft frozen sections and the percentage of apoptotic cells was determined using IPLab software. Tumors were harvested from each mouse after size measurement. Harvested tumors were bisected and snap frozen in liquid nitrogen, embedded in optimal cutting tissue (OCT) matrix, sectioned into 7 ⁇ M thick samples and mounted on superfrosted glass slides. TUNEL apoptosis staining using an Apoptag Plus kit (Chemicon, Temecula, CA) was performed according to manufacturer instructions. After staining, five tumor specimens were randomly selected from each treatment group. Ten 4Ox high-powered fields from each specimen were randomly selected. Images were acquired using the imaging system described above. IPLab software was used to determine the percentage of each high powered field consisting of DAB-stained apoptotic cells.
  • Non-treatment controls had low levels of apoptosis per high-powered field (0.12%). Cisplatin induced significantly more apoptosis, alone and combined with DL312 (1.26% and 1.42%, respectively; p ⁇ 0.001). Ad-mutRad50 infection also enhanced apoptosis (0.84%; p ⁇ 0.005). Combining Ad-mutRad50 with cisplatin markedly increased the amount of apoptosis in mice subject to both the 3 mg/kg and 5 mg/kg dose of cisplatin (3.46% and 4.43%, respectively) relative to all other groups (pO.001). Within the combination treatment groups, there was no significant difference in apoptosis between the low and higher doses of cisplatin, consistent with the similar tumor volume reduction observed in these groups.
  • HNSCC Head and neck squamous cell carcinoma
  • Ad-Nbsl-300 was designed as described [JG Rhee, et al, InVl J radiation Oncol Biol Physics, 67 (l):273-278 (2007)], which is incorporated by reference herein.
  • Nbsl cDNA comprising the last 300 amino acids of the C-terminal was cloned into a recombinant adenovirus vector (Ad-Nbs 1 -300).
  • the mutant Nbs 1 protein contains the Mrel 1 interaction domains and, thus, retains the ability to bind to the Mrel l/Rad50 complex.
  • the mutant Nbsl protein is missing the N-terminal FHA/BRCT functional domains of the wild-type protein, eliminating the ability of Nbsl to localize to the site of DNA DSBs and to amplify the DNA damage response through the recruitment of Mrel 1 and Rad50. Consequently, infection of cells with Ad-Nbsl-300 induces a dominant negative suppression of the MRN complex.
  • the Ad-mutRad50 construct consists of a 326 base pair gene sequence encoding a fragment (amino acid residues 631 - 739) of the wild-type Rad50.
  • This mutant construct contains the Rad50 zinc hook region including the CXXC binding motif (amino acid residues 680-684), but is missing the ATPase Walker A (residues 36 - 43) and Walker B (residues 1227 - 1232) functional domains, as well as the signature motif (residues 1201 - 1210) and Mrel 1 interaction sites. Consequently, the encoded mutant Rad50 protein is capable of dimerization but does not contain the functional regions necessary for intact DNA repair. This induces a dominant negative down-regulation of the MRN DNA repair complex. Cells were incubated with the adenoviral vectors at an MOT of 10 for 4 hours at 37 0 C.
  • the PARP-I inhibitor was administered at a concentration of 8 ⁇ M and 15 ⁇ M for the two HNSCC cell lines [JHU012 and JHU022], respectively. These concentrations represent the IC50 for the PARP-I inhibitor in the cell lines as determined by standard pharmacologic assay. Cell growth was assessed over 5 days using standard MTT assay technique. Cell growth was measured using a photometric plate reader to quantify optical density (570 nm reference).
  • Combination treatment with Ad-Nbsl-300 and a PARP-I inhibitor produced a 78.7%, 56.7% and 69.4% reduction in JHU012 cell density and an 84.1%, 79.0% and 76.3% decline in JHU022 cell number relative to controls, Ad-Nbsl-300 monotherapy and PARP-I inhibitor monotherapy groups, respectively (p ⁇ 0.001).
  • Co-disruption utilizing Ad-mutRad50 and a PARP-I inhibitor reduced JHUOl 2 cell density by 88.6%, 74.7% and 84.2% and decreased JHU022 cell density by 90.3%, 80.1% and 87.1% versus control, Ad-mutRad50 and PARP-I inhibitor groups, respectively (pO.001 ).
  • the anti-neoplastic effect of co-disruption was immediate after initiation of treatment and was sustained throughout the duration of the MTT cell growth assay.
  • Example 8 Co-disruption of the MRN complex and PARP inhibits tumor progression and provides tumor regression in an in vivo mouse model of human head and neck cancer
  • mice Seventy nude mice were randomized into 7 groups of 10 mice each. Mice were anesthetized with 8 mg tribromoethanol via intraperitoneal injection. Depth of anesthesia was determined by toe pinch. The right dorsal flank of each mouse was injected subcutaneously with 1 x 10 7 head and neck squamous cell carcinoma cells [from a cell line, JHU012, available from Johns Hopkins University]. After the establishment of palpable tumors, mouse body weight and external tumor volume was determined every 72 hours from the time at which the tumor was first palpable. External tumor diameter was measured using digital calipers and tumor volume was calculated using the formula A 2 x B x 0.536, where A represents the smallest diameter and B equals the largest diameter.
  • mice were re-anesthetized 12 days after tumor injection when the average externally measured tumor volume was 118 mm 3 .
  • Tumors were exposed by raising a skin flap using precise surgical dissection, taking care not to disturb the tumor or its blood supply. The exposed tumors were measured in three dimensions (maximal length x maximal width x maximal height) using calipers to accurately assess tumor volume as previously described [Rocco, J.W., et al., 1998, Clin Cancer Res 4: 1697-1704].
  • Adenoviral vectors were used to introduce genes encoding non-functional, mutant forms of Nbsl or Rad50 into head and neck squamous cell carcinoma xenografts.
  • the vectors were prepared as described in preceding examples.
  • the seven intervention groups were: intratumoral saline, intratumoral Ad-Nbsl-300, intratumoral Ad-mutRad50, orally administered PARP-I inhibitor, intratumoral Ad-GFP control virus with oral PARP-I inhibitor, intratumoral Ad-Nbsl-300 with oral PARP-I inhibitor and intratumoral Ad- mutRadSO with oral PARP-I inhibitor.
  • Therapeutic adenoviral constructs (Ad-mutRad50 and Ad-Nbsl-300), as well as the control virus (Ad-GFP) which contains no therapeutic gene, were administered at a dose of 3.5 x 10 s pfu in 50 ⁇ L volume.
  • Intratumoral infiltration of the tumor was achieved by loading the virus into a Hamilton syringe and injecting the exposed tumor mass with a 27 gauge needle. Skin incisions were closed with 4-0 nylon suture.
  • the internal tumor volume was re- measured 9 days after treatment.
  • the dynamic tumor volume change derived from external tumor volume measurements was determined.
  • the change in internal tumor volumes between treatment initiation and conclusion of the experiment 9 days later was determined.
  • Prior to treatment initiation the mean tumor volume was not significantly different between intervention groups and tumor xenografts showed sustained volume growth. Between treatment initiation on day 12 and animal sacrifice on day 21 , control tumors showed a 179% increase in volume.
  • Co-disruption of MRN and PARP function using either Ad-Nbsl-300 or Ad- mutRad50 in combination with a PARP-I inhibitor produced a dramatic and significant anti- neoplastic effect. Indeed, tumors treated with our co-disruption approach showed a 40% and 29% volume reduction relative to pre-treatment levels when treated with a PARP-I inhibitor and Ad-Nbsl-300 or Ad-mutRad50, respectively. This anti-neoplastic effect was significant compared to controls (p ⁇ 0.001), to monotherapy with a PARP-I inhibitor (p ⁇ 0.01) and to monotherapy with either Ad-Nbsl-300 (p ⁇ 0.02) or Ad-mutRad50 (p ⁇ 0.05).

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Abstract

La présente invention concerne un procédé antinéoplasique comprenant une thérapie combinée qui comprend l'administration d'un inhibiteur de PARP dans un régime thérapeutique avec un disrupteur du complexe MRE11/RAD50/NBS1. Le disrupteur du complexe MRE11/RAD50/NBS1 peut être une construction NBS1 ou une construction RAD50 mutante, et l'une ou l'autre, ou les deux, peuvent être administrées via un vecteur viral qui transporte les séquences de codage pour une de ces constructions, ou les deux. La présente invention concerne également un adénovirus déficient pour la réplication contenant la construction RAD50 mutante.
PCT/US2009/036497 2008-03-10 2009-03-09 Régime thérapeutique antinéoplasique combiné comprenant la co-disruption de la voie parp et du complexe mre11/rad50/nbs1, et compositions utiles à cette fin WO2009114459A2 (fr)

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Cited By (11)

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Publication number Priority date Publication date Assignee Title
WO2010081778A1 (fr) * 2009-01-17 2010-07-22 Universität Zürich Bloqueurs de parp pour la prévention et le traitement d'un cancer gastrique induit par helicobacter pylori
US20120258181A1 (en) * 2009-12-23 2012-10-11 Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. Anticancer combination of artemisinin-based drugs and other chemotherapeutic agents
US9023861B2 (en) * 2009-12-23 2015-05-05 Sigma-Tau Industrie Farmaceutiche Riunite, S.P.A. Anticancer combination of artemisinin-based drugs and other chemotherapeutic agents
US9359367B2 (en) 2012-07-09 2016-06-07 Lupin Limited Tetrahydroquinazolinone derivatives as PARP inhibitors
CN109985240A (zh) * 2017-12-29 2019-07-09 广州威溶特医药科技有限公司 Parp抑制剂和溶瘤病毒在制备抗肿瘤药物的应用
CN113811333A (zh) * 2019-05-14 2021-12-17 诺维逊生物股份有限公司 靶向抗癌核激素受体的化合物
US11826430B2 (en) * 2019-05-14 2023-11-28 Nuvation Bio Inc. Anti-cancer nuclear hormone receptor-targeting compounds
CN113811333B (zh) * 2019-05-14 2024-03-12 诺维逊生物股份有限公司 靶向抗癌核激素受体的化合物
US11952349B2 (en) 2019-11-13 2024-04-09 Nuvation Bio Inc. Anti-cancer nuclear hormone receptor-targeting compounds
US11834458B2 (en) 2021-03-23 2023-12-05 Nuvation Bio Inc. Anti-cancer nuclear hormone receptor-targeting compounds
US12006314B2 (en) 2021-05-03 2024-06-11 Nuvation Bio Inc. Anti-cancer nuclear hormone receptor-targeting compounds

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