(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`5 January 2006 (05.01.2006)
`
`
`
`PCT
`
`(51) International Patent Classification:
`A61K 48/00 (2006.01)
`CIZN 15/869 (2006.01)
`
`(81)
`
`(21) International Application Number:
`PCT/US2005/0225 61
`
`(22) International Filing Date:
`
`24 June 2005 (24.06.2005)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(84)
`
`(30) Priority Data:
`60/582,714
`
`24 June 2004 (24.06.2004)
`
`US
`
`(71) Applicant (for all designated States except US): NEW
`YORK UNIVERSITY [US/US]; 70 Washington Square
`South, New York, NY 10012 (US).
`
`1 Washington Square Vil—
`(72) Inventors: MOHR, Ian;
`lage, Apt. 6K, New York, NY 10012 (US). MULVEY,
`Matthew; 323 East 14th Street, Apt. 3C, New York, NY
`10003 (US).
`
`(10) International Publication Number
`
`WO 2006/002394 A2
`
`Designated States (unless otherwise indicated, for ever
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KM, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA,
`MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI, NO, NZ,
`OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL,
`SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC,
`VN, YU, ZA, ZM, ZW.
`
`Designated States (unless otherwise indicated, for ever
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, 1E, 18, [1, LT, LU, MC, NL, PL, PT, RO,
`SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report
`
`(74) Agents: GOLDMAN, lVfichael, L. et 211.; Nixon Peabody
`LLP, Clinton Square, PO. Box 31051, Rochester, NY
`146031051 (US).
`
`For two—letter codes and other abbreviations, refer to the ”Guid—
`ance Notes on Codes and Abbreviations ” appearing at the begin—
`ning of each regular issue of the PCT Gazette.
`
`(54) Title: AVIRULENT ONCOLYTIC HERPES SIMPLEX VIRUS STRAINS ENGINEERED TO COUNTER THE INNATE
`HOST RESPONSE
`
`(57) Abstract: The present invention relates to an avirulent, oncolytic herpes simplex virus modified from a wild—type herpes
`simplex virus so that both 7134.5 genes of the virus have been deleted and each replaced with an interferon—resistance gene that is
`expressed as an immediate—early gene. The present invention also relates to a pharmaceutical composition that includes the modified
`herpes simplex virus ofthe present invention and a pharmaceutically acceptable vehicle for in situ administration to tumor cells. Also
`provided in the present invention are methods for killing tumor cells in a subject and for immunizing a subject against an infectious
`disease, cancer, or an autoimmune disease that involve administering to a subject the modified avirulent, oncolytic herpes simplex
`virus of the present invention.
`
`
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`WO 2006/002394
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`PCT/U82005/022561
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`-1-
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`AVIRULENT ONCOLYTIC HERPES SIMPLEX VIRUS STRAINS
`ENGINEERED TO COUNTER THE INNATE HOST RESPONSE
`
`[0001]
`
`This application claims benefit of U.S. Provisional Patent Application
`
`Serial No. 60/5 82,7 14, filed June 24, 2004, which is hereby incorporated by reference
`
`in its entirety.
`
`[0002]
`
`The subject matter of this application was made with support from the
`
`United States Government under the National Institutes of Health Grant No.
`
`ROlGM056927. The U.S. Government may retain certain rights.
`
`FIELD OF THE INVENTION
`
`[0003]
`
`The present invention relates to an oncolytic avirulent modified herpes
`
`simplex virus having a high level of replication in neoplastic cells, and uses thereof
`
`for immunization against and treatment of disease conditions.
`
`BACKGROUND OF THE INVENTION
`
`[0004]
`One hundred years ago, the first anecdotal observations correlating
`viral infection with tumor regression were reported, which lead to several
`
`investigations, over the course of the last century, evaluating the potential of various
`
`natural hmnan and animal viruses to treat cancer. (Sinkovics et al., “New
`
`Developments in the Virus Therapy of Cancer: A Historical Review,” Intervirology
`
`36:193-214 (1993)). These observations suggest that malignant lesions could regress
`
`in response to viral infection. To be effective in treatments of malignancies, such
`
`isolates should only grow productively and exhibit virulence in neoplastic cells, and
`
`should not be capable of propagating a productive infection through surrounding
`
`normal, terminally differentiated tissue. As virulence is governed by specific viral
`
`genes, one approach would be to genetically alter the virulence of viruses to obtain
`
`viruses which selectively destroy neoplastic cells. However, it was the achievements
`
`of the last two decades, most notably technical innovation in the area of molecular
`
`biology coupled with a heightened understanding of viral replication and pathogenesis
`
`at the genetic level, which ushered in the possibility of creating Viruses in the
`
`laboratory which were selectively pathogenic for neoplastic cells. The advent of
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`genetic engineering techniques has made it possible to selectively ablate viral
`
`virulence genes in the hope of creating a safe, avirulent virus that can selectively
`
`replicate in and destroy tumor cells. Such a virus would be able to propagate an
`
`infection throughout a tumor mass and directly kill the cancer cells, but be unable to
`
`inflict substantial damage to normal terminal differentiated cells. Although safe
`
`viruses have been created by such methodology, the attenuation process often has an
`
`overall deleterious effect on viral replication. This replication defect prevents the
`
`virus fiom completely destroying the tumor mass, and the surviving cancer cells can
`
`simply repopulate.
`
`10
`
`[0005]
`
`HSV-l is a double-stranded DNA virus which is replicated and
`
`transcribed in the nucleus of the cell. HSV—l consists of at least three groups of
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`15
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`20
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`25
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`genes, or, B, and y, whose expression is coordinately regulated and sequentially
`
`ordered in a cascade fashion. (Honess et al., “Regulation of Herpesvirus
`
`Macromolecular Synthesis, 1. Cascade Regulation of the Synthesis of Three Groups of
`
`Viral Proteins,” J Vir 14:8—19 (1974)). Five immediate early (IE or or) genes are the
`
`first genes expressed during productive infection. While one of these genes encodes
`
`the immunomodulatory protein ICP47, the remaining four 0!, genes encode infected
`
`cell proteins (ICPs) 0, 4, 22, and 27, the major regulatory proteins of the virus. These
`
`immediate early proteins then activate the expression of viral genes of the early (E or
`
`B) and late (L or 1/) classes. Once the viral lifecycle advances beyond the IE stage, the
`
`ICP4 protein also autoregulates IE gene expression by reducing transcription from IE
`
`promoters (Preston, “Control of Herpes Simplex Virus Type 1 mRNA Synthesis in
`
`Cells Infected with Wild-Type Virus or the Temperature—Sensitive Mutant tsK,” J Vir
`
`29: 275-284 (1979); Roberts et al., “Direct Correlation Between aNegative
`
`Autoregulatory Response Element at the Cap Site of the Herpes Simplex Virus Typel
`
`IE175(0(.4) Promoter and a Specific Binding Site for the IE175 (a4) Protein,” J Vir 62:
`
`4307-4320 (1988); Lium et al., “Repression of the alphaO gene by ICP4 During a
`
`Productive Herpes Simplex Virus Infection,” J Vir 70: 348 8—3496 (1996)). Proteins
`
`of the E class are responsible for viral DNA replication. The late (L) genes are
`
`30
`
`induced after DNA replication and encode the structural components and enzymes
`
`required for assembly of virus particles.
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`-3-
`
`[0006]
`
`Following infection of oral epithelial cells in its human host, HSV-l
`
`invades axons and travels to the nuclei of sensory neurons that innervate this epithelia.
`
`Here, the virus establishes a latent infection, characterized by a restricted pattern of
`
`viral gene expression. (Roizman et al., “Herpes Simplex Viruses and Their
`
`Replication,” in: Knipe et al., eds. Fields Virology Vol. 2, 4th Ed., Philadelphia,
`
`Pennsylvania: Lippincott, Williams & Wilkins, pp. 23 99—2460 (2001)). Latency
`
`results in the permanent colonization of the host by the virus, and the severely limited
`
`expression of viral genes fiinctions to shield the virus fiom host defenses.
`
`[0007]
`
`In response to a variety of stimuli, these latent infections “reactivate,”
`
`10
`
`resulting in episodes of productive viral growth characterized by expression of over
`
`80 viral open reading frames (ORFs) distributed among two unique, single copy
`
`segments or within multiple repetitive loci of the large HSV-l DNA genome.
`
`Activation of the productive or lytic gene expression program results in the
`
`production of Viral particles and the eventual death of the infected cell. Distinct
`
`15
`
`mRNA populations accumulate at discrete times in the productive replication cycle,
`
`resulting in the differential expression of viral genes in what has been termed a
`
`cascade pattern. (Roizman et al., “Herpes Simplex Viruses and Their Replication,”
`
`in: Knipe et al., eds. Fields Virology Vol. 2, 4th Ed, Philadelphia, Pennsylvania:
`
`Lippincott, Williams & Wilkins, pp. 2399-2460 (2001)). The process is initiated by
`
`20
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`VP16, a transcription factor carried Within the viral particle that recruits cellular
`
`transcription factors along with the RNA polymerase 11 holoenzyme to the promoters
`
`of the five viral IE genes. While one of these IE gene products dampens the host
`
`immune response by inhibiting the presentation of peptide antigens in conjunction
`
`with MHC class I molecules (U312), the remaining four IE proteins are important for
`
`25
`
`the subsequent expression of the next class of Viral genes, the early or B genes. Viral
`
`early polypeptides primarily encode functions required for nucleotide metabolism and
`
`viral DNA synthesis, the initiation of which signals entry into the final late or 7 phase
`
`of the viral life cycle. Two classes of late genes have been identified based upon their
`
`transcription in the presence of viral DNA synthesis inhibitors. While transcription of
`
`30
`
`a subset of 'y genes, the 72 class, requires viral DNA synthesis, expression of 71 genes
`
`is not completely dependent upon viral DNA replication and is only modestly reduced
`
`in the presence of inhibitors. Included among the late gene products are polypeptides
`
`critical for assembling infectious virus, virion components that function following
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`-4-
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`entry but before IE gene expression, and proteins that regulate the host response to
`
`infection. Reactivation of a latent infection in a sensory neuron results in
`
`antereograde transport of viral progeny back to the portal of entry followed by the
`
`ensuing infection of epithelial cells, mobilization of the cellular immune response,
`
`and the formation of a fever blister or cold sore. Rarely, HSV—l can enter and
`
`replicate within the CNS, causing encephalitis.
`
`[0008]
`
`Martuza and colleagues were the first to demonstrate the therapeutic
`
`promise of an engineered oncolytic HSV—l strain, the thymidine kinase (tk) negative
`
`HSV—l mutant, dlsptk. (Martuza et al., “Experimental Therapy of Human Glioma by
`
`Means of a Genetically Engineered Virus Mutant,” Science 252:854—856 (1991)). tk
`
`mutants replicate effectively in actively dividing cells such as those found in tumors,
`
`but are relatively impaired for replication in non—dividing cells, such as neurons, and,
`
`therefore, display reduced neurovirulence compared with wild-type strains upon
`
`introduction into the CNS of adult mice. (Field et al., “The Pathogenicity of
`
`Thymidine Kinase-Deficient Mutants of Herpes Simplex Virus in Mice,” J Hyg
`
`(Land) 81 :267-277 (1978); Jamieson et al., “Induction of Both Thymidine and
`
`Deoxycytidine Kinase Activity by Herpes Viruses,” J Gen Virol 242465-480 (1974);
`
`Field et al., “Pathogenicity in Mice of Strains of Herpes Simplex Virus Which are
`
`Resistant to Acyclovir In Vitro and In Vivo,” Antimicrob Agents Chemother 17:209-
`
`21 6 (1980); Tenser et a1, “Trigeminal Ganglion Infection by Thymidine Kinase-
`
`Negative Mutants of Herpes Simplex Virus,” Science 205:915-917 (1979); Coen et
`
`al., “Thymidine Kinase—Negative Herpes Simplex Virus Mutants Establish Latency in
`
`Mouse Trigeminal Ganglia But Do Not Reactivate,” Proc Natl Acad Sci USA
`
`86:4736—4740 (1989)). The tumor selected for oncolytic therapy was malignant
`
`glioma. The outcome for patients with this devastating brain tumor is grim,
`
`remaining essentially unchanged over the past 50 years despite advances in surgery,
`
`radiation, and chemotherapy. Direct injection of dlsptk into established tumors
`
`inhibited the growth of human glioma implants in athymic mice and prolonged
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`survival of mice with intracranial gliomas as well. However, fatal encephalitis was
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`still observed in 70-100 % of the treated mice despite the fact that the tk mutant was
`
`significantly less neurovirulent than wild-type HSV—l . (Martuza et al., “Experimental
`
`Therapy of Human Glioma by Means of a Genetically Engineered Virus Mutant,”
`
`Science 252:854—856 (1991)). Thus, while it proved possible to use HSV—l as an
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`oncolytic virus to destroy cancer cells, the understanding of virulence was not
`
`sufficiently advanced to render the virus safe.
`
`[0009]
`
`The breakthrough in creating attenuated HSV—l strains resulted from
`
`characterizing Viruses containing engineered mutations in the y,34.5 genes.
`
`Embedded within a repetitive genome component, the y,34.5 gene is expressed with 'yl
`
`late kinetics and is not required for growth in cultured monkey kidney cells (Vero
`
`cells). Strikingly, its impact on viral neurovirulence is greater than any single HSV—l
`
`gene identified to date. (Chou et al., “Mapping of Herpes Simplex Virus-1
`
`Neurovirulence to Gamma (1) 34.5, a Gene Nonessential for Growth in Culture,”
`
`Science 250:1262-1266 (1990); Maclean et al., “Herpes Simplex Virus Type 1
`
`Deletion Variants 1714 and 1716 Pinpoint Neurovirulence—Related Sequences in
`
`Glasgow Strain 17+ Between Immediate Early Gene 1 and the 'a' Sequence,” J Gen
`
`Virol 72:631-639 (1991); Bolovan et al., “ICP34.5 Mutants of Herpes Simplex Virus
`
`Type 1 Strain 17syn+ Are Attenuated for Neurovirulence in Mice and for Replication
`
`in Confluent Primary Mouse Embryo Cell Cultures,” J Viral 68:48—55 (1994)). While
`
`the LDso of many wild-type (WT) HSV—l strains is less than 300 pfiJ following
`
`intracranial delivery, it is not possible to accurately measure the LDSO for 734.5
`
`mutant viruses. Indeed, upwards of 106-107 pfu of 734.5 mutant viruses have been
`
`safely injected intracranially into mouse, non—human primate, and human brains
`
`(Chou et al., “Mapping of Herpes Simplex Virus—1 Neurovirulence to Gamma (1)
`
`34.5, a Gene Nonessential for Growth in Culture,” Science 250:1262—1266 (1990);
`
`Maclean et al., “Herpes Simplex Virus Type 1 Deletion Variants 1714 and 1716
`
`Pinpoint Neurovirulence-Related Sequences in Glasgow Strain 17+ Between
`
`Immediate Early Gene 1 and the 'a' Sequence,” J Gen Virol 72:631—639 (1991);
`
`Bolovan et al., “ICP34.5 Mutants of Herpes Simplex Virus Type 1 Strain 17syn+ Are
`
`Attenuated for Neurovirulence in Mice and for Replication in Confluent Primary
`
`Mouse Embryo Cell Cultures,” J Viral 68:48-55 (1994); Mineta et al., “Attenuated
`
`Multi—Mutated Herpes Simplex Virus—1 for the Treatment of Malignant Gliomas,” Nat
`
`Med 1:93 8—943 (1995); Hunter et al., “Attenuated, Replication-Competent Herpes
`
`Simplex Virus Type 1 Mutant G207: Safety Evaluation of Intracerebral Injection in
`
`Nonhuman Primates,” J Viral 73:6319-6326 (1999); Markert et al., “Conditionally
`
`Replicating Herpes Simplex Virus Mutant, G207 for the Treatment of Malignant
`
`Glioma: Results of a Phase I Trial,” Gene Ther 7:867-874 (2000); Rampling et al.,
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`-6—
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`“Toxicity Evaluation of Replication-Competent Herpes Simplex Virus (ICP 34.5 Null
`
`Mutant 1716) in Patients With Recurrent Malignant Glioma,” Gene Ther 7:85 9-866
`
`(2000); Sundaresan et a1., “Attenuated, Replication—Competent Herpes Simplex Virus
`
`Type 1 Mutant G207: Safety Evaluation in Mice,” J Viral 74:3 832-3 841 (2000)). In
`
`studies designed to examine the efficacy with which 1134.5 mutants were able to
`
`destroy human or murine gliomas implanted into mice, not only had the attenuation
`
`problem been solved, but the treated mice survived longer than their untreated
`
`counterparts and no longer developed viral encephalitis. Long-term surviving animals
`
`(usually in the vicinity of 60—80 days) were produced with efficiencies ranging from
`
`10—50% of the treated animals depending on the tumor model and treatment regimen.
`
`(Markert et a1., “Reduction and Elimination of Encephalitis in an Experimental
`
`Glioma Therapy Model with Attenuated Herpes Simplex Mutants That Retain
`
`Susceptibility To Acyclovir,” Neurosurgeiy 32:597-603 (1993); Chambers et a1.,
`
`“Comparison of Genetically Engineered Herpes Simplex Viruses for the Treatment of
`
`Brain Tumors in a SCID Mouse Model of Human Malignant Glioma,” Proc Natl
`
`Acad Sci USA 92:1411-1415 (1995); Kesari et a1., “Therapy of Experimental Human
`
`Brain Tumors Using a Neuroattenuated Herpes Simplex Virus Mutant,” Lab Invest
`
`73 2636-648 (1995); Andreansky et a1., “The Application of Genetically Engineered
`
`Herpes Simplex Viruses to the Treatment of Experimental Brain Tumors,” Proc Natl
`
`Acad Sci USA 93 :1 13 13—1 1318 (1996); Andreansky et a1., “Evaluation of Genetically
`
`Engineered Herpes Simplex Viruses as Oncolytic Agents for Human Malignant Brain
`
`Tumors,” Cancer Res 57:1502—1509 (1997); Randazzo et al., “Treatment of
`
`Experimental Intracranial Murine Melanoma With a Neuroattenuated Herpes Simplex
`Virus 1 Mutant,” Virology 211194-101 (1995) of Human Malignant Glioma,” Proc
`
`A
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`Natl Acad' Sci USA 92(5):1411-1415(1995)). 734.5 mutants were also tk+ and,
`
`therefore, retained their sensitivity to acyclovir, which could be used, if necessary, to
`
`control viral encephalitis. To further restrict viral replication to actively dividing
`
`cells, additional mutations in the UL39 ribonucleotide reductase gene or the ULZ uracil
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`DNA glycosidase gene were introduced into the 734.5 mutant background (Mineta et
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`a1., “Attenuated Multi-Mutated Herpes Simplex Virus—1 for the Treatment of
`
`Malignant Gliomas,” Nat Med 1:93 8-943 (1995); Kramm et a1., “Therapeutic
`
`Efficiency and Safety of a Second-Generation Replication—Conditional HSVl Vector
`
`for Brain Tumor Gene Therapy,” Hum Gene Ther 82057-2068 (1997); Pyles et a1.,
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`“A Novel Multiply—Mutated HSV—l Strain for the Treatment of Human Brain
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`Tumors,” Hum Gene Ther 82533-544 (1997)). Although each of these non—
`
`neurovirulent, multi—mutated Viruses could still reduce subcutaneous tumor growth
`
`and extend the survival of mice with intracranial tumors, more than 80% of the treated
`
`subjects still succumbed, emphasizing a different problem limiting the efficacy and
`
`outcome of treatment. While encephalitis was no longer observed in animals treated
`
`with any of these y,34.5 mutant derivatives, the y,34.5 deletion, either alone or in
`
`conjunction with additional mutations, impaired the replicative ability of these Viruses
`
`in many human tumor cells, allowing the growth of residual glioma cells that
`
`ultimately killed the animals. Thus, the successful attenuation of HSV-1 left in its
`
`wake another problem for investigators to grapple with: engineered mutants that were
`
`sufficiently safe had lost a substantial amount of their replicative efficacy, impairing
`
`their oncolytic ability.
`
`.
`
`[0010]
`
`The reduced oncolytic ability of 31134.5 mutant derivatives results from
`
`their inability to counter an innate host defense designed to inhibit protein synthesis in
`
`Virus — infected cells. After the initial reports established that the y,34.5 gene was a
`
`major determinant of HSV-1 neurovirulence non— essential for growth in cultured
`
`monkey kidney cells, flirther investigation revealed that 31134.5 mutants actually
`
`behaved like classical Viral host range mutants, exhibiting restricted growth in some
`
`lines of cultured cells but not others. Thus, while a standard line of monkey kidney
`
`cells were permissive or supported the replication 7,345 mutants, many human tumor
`
`cells were non-permissive, or did not support the growth of y134.5 mutant derivatives.
`
`Upon infection of a non-permissive human tumor cell with a y,34.5 mutant strain, all
`
`of the events in the Viral lifecycle proceeded normally up to and including viral DNA
`
`replication and the accumulation of 1/2 late mRNA transcripts. These viral late
`
`mRNAs encoding key structural proteins required to complete the Viral lifecycle and
`
`assemble the next generation of viral progeny, however, were never translated due to
`
`a block at the level of protein synthesis, effectively interrupting the viral lifecycle
`
`prior to the assembly and release of viral particles (Chou et al., “The Gamma (1) 34.5
`
`Gene of Herpes Simplex Virus 1 Precludes Neuroblastoma Cells From Triggering
`
`Total Shutoff of Protein Synthesis Characteristic of Programmed Cell Death in
`
`Neuronal Cells,” Proc Natl Acad Sci USA 89:3266—3270 (1992)). Subsequent
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`biochemical analysis demonstrated that the y,34.5 gene product was required to
`
`prevent accrual of phosphorylated eIF2, a critical translation initiation factor required
`
`to bring the initiator tRNA to the ribosome. Phosphorylation of eIF2 on its alpha
`
`subunit inactivates this translation factor and inhibits protein synthesis (Chou et al.,
`
`“Association of a M(r) 90,000 Phosphoprotein With Protein Kinase PKR in Cells
`
`Exhibiting Enhanced Phosphorylation of Translation Initiation Factor eIF—2 Alpha
`
`and Premature Shutoff of Protein Synthesis After Infection With Gamma (1) 34.5-
`
`Mutants of Herpes Simplex Virus 1,” Proc Natl Acad Sci USA 92:10516—10520
`
`(1995)). Thus, the inability of 334.5 mutants to sustain protein synthesis in tumor
`
`cells limits their potential efficacy as replicating oncolytic viruses.
`
`[0011]
`
`As obligate intracellular parasites, viruses are completely dependent
`
`upon the translational machinery resident in their host cells. It is not surprising,
`
`therefore, that a major innate host defense component centers on impeding viral
`
`mRNA translation. Indeed, the double—stranded RNA—dependent protein kinase PKR,
`
`an eIF20t kinase, is induced by the antiviral cytokines interferon oc/B. It has been
`
`proposed that abundant dsRNA, a replicative intermediate formed in the replication of
`
`RNA Viruses and a by-product of overlapping transcription units on opposite DNA
`
`strands of DNA viruses, is a signature of viral infection. PKR binds dsRNA and, in
`
`the presence of this activating ligand, forms a dimer, whereupon each subunit
`
`phosphorylates the other. It is this activated, phosphorylated form of PKR that then
`
`goes on to phosphorylate other substrates, including eIFZOL, the regulatory subunit of
`
`eIF2 (Kaufman RJ, “Double-Stranded RNA-Activated Protein Kinase PKR,” In:
`
`Sonenberg eds. Translational Control. Cold Spring Harbor, NY: Cold Spring Harbor
`
`Laboratory Press, 503—528 (2000)). Should cells initially infected succeed in
`
`inhibiting translation, the viral invader would effectively be stopped in its tracks,
`
`denied access to the cellular translational apparatus it needs to complete its life—cycle.
`
`This arm of the innate host response, then, is designed to sacrifice the initially
`
`infected cells for the benefit of the larger population. Any effective oncolytic virus
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`must be able to thwart host defenses if it is to propagate an infection throughout a
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`tumor causing regression and, ultimately, the destruction of the tumor. Failure to
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`mount an effective response against both innate and acquired host defenses is likely to
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`prevent viral replication and subsequent spread throughout the tumor tissue. The
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`WO 2006/002394
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`PCT/U82005/022561
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`-9-
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`present invention describes how to engineer an attenuated 7,345 mutant Virus that is
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`capable of countering both the acquired immune response and the innate host
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`reSponses mediated by interferon.
`
`[0012]
`
`At least two herpes simplex virus gene products have been implicated
`
`in the virus’ s ability to resist the pleiotropic effects of interferon (IFN). One of these
`
`is the product of the ICPO gene, a multifunctional polypeptide produced very early in
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`the viral life cycle that transactivates viral gene expression (Everett, R.D., “ICPO, A
`
`Regulator of Herpes Simplex Virus During Lytic and Latent Infection,” Bioessays
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`22:761—770 (2000)).
`
`ICPO deficient mutants are hypersensitive to IFN, as viral
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`mRNAs do not accumulate in IFN treated Vero cells, Whereas cellular mRNAs
`
`encoding IFN induced gene products increase in abundance (Eidson et al.,
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`“Expression of Herpes Simplex Virus ICPO Inhibits the Induction of Interferon —
`
`Stimulated Genes by Viral Infection,” J Viral 76:2180—2191 (2002), Mossman et al.,
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`“Herpes Simplex Virus Triggers and Then Disarms a Host Antiviral Response,” J
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`Viral 75:750—758 (2001), Mossman et a1., “Herpes Simplex Virus ICPO and ICP34.5
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`Counteract Distinct Interferon — Induced Barriers To Virus Replication,” J Viral
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`76:1995—1998 (2002), Nicholl et a1., “Activation of Cellular Interferon ~ Responsive
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`Genes After Infection of Human Cells With Herpes Simplex Type 1,” J Gen Virol
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`81 :2215—2218 (2000)). This particular IFN antiviral effect requires the cellular
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`promyelocytic leukemia (PML) gene product, and it has been proposed that the
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`disassembly of PML bodies observed in HSV-l infected cells, which requires the
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`ubiquitin E3 ligase activity of ICPO (Boutell et al., “Herpes Simplex Virus Type 1
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`Immediate-Early Protein ICPO And Its Isolated RING Finger Domain Act As
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`10
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`15
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`20
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`Ubiquitin E3 Ligases in vitro,” J Virol 762841-850 (2002), Van Sant et al., “The
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`25
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`Infected Cell Protein 0 of Herpes Simplex Virus 1 Dynamically Interacts With
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`Proteasomes, Binds And Activates The cdc34 E2 Ubiquitin—Conjugating Enzyme,
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`And Possesses In Vitro E3 Ubiquitin Ligase Activity,” Proc Natl Acad Sci USA
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`98:8815-20 (2001)), enables the virus to prevent the induction of IFN responsive
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`genes (Chee et al., “Promyelocytic Leukemia Protein Mediates Interferon — Based
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`30
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`Anti — Herpes Simplex Virus 1 Effects,” J Viral 77:7101—7105 (2003)). While the
`
`ICPO polypeptide prevents the transcriptional induction of cellular IFN responsive
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`genes, the 3/1345 gene, when altered, results in an IFN hypersensitive virus, encodes a
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`product that operates by preventing host defenses fiom inactivating the critical
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`WO 2006/002394
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`PCT/U82005/022561
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`-10-
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`translation initiation factor eIF2 (Cerveny et a ., “Amino Acid Substitutions In The
`
`Effector Domain of The y134.5 Protein of Herpes Simplex Virus 1 Have Differential
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`Effects On Viral Response To Interferon—0c,” Virology 307:290—300 (2003), Cheng et
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`al., “Val193 and Phe195 of The 7134.5 Protein of Herpes Simplex Virus 1 Are Required
`
`For Viral Resistance To Interferon Ot/B,” Virology 290: 1 15—120 (2001), Mossman et
`
`al., “Herpes Simplex Virus ICPO and ICP34.5 Counteract Distinct Interferon —
`
`Induced Barriers To Virus Replication,” J Virol 76: 1995—1998 (2002)).
`
`[0013]
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`Upon binding the catalytic subunit of protein phosphatasel on (PPloc),
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`the 7134.5~PP1 0c holoenzyme prevents the accumulation of phosphorylated, inactive
`
`eIF20c in infected cells, preserving viral translation rates (He et al., “The
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`Gamma(l)34.5 Protein of Herpes Simplex Virus 1 Complexes With Protein
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`Phosphatase 1 Alpha To Dephosphorylate The Alpha Subunit of Eukaryotic Initiation
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`Factor 2 And Preclude The Shutoff of Protein Synthesis By Double-Stranded RNA-
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`Activated Protein Kinase,” Proc Natl Acad Sci USA 94:843-848 (1997)). However, in
`
`many established human cell lines infected with a y134.5 mutant virus, the onset of
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`viral DNA synthesis and the accumulation of y; late viral mRNA transcripts are
`
`accompanied by the complete cessation of cellular and viral protein synthesis (Chou
`
`et al., “The 'y34.5 Gene of Herpes Simplex Virus 1 Precludes Neuroblastoma Cells
`
`From Triggering Total Shutoff of Protein Synthesis Characteristic of Programmed
`
`Cell Death In Neuronal Cells,” Proc Natl Acad Sci USA 89:3266-3270 (1992)). Thus,
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`7134.5 mutants are not only deficient in functions intrinsic to the 31134.5 gene product,
`
`but, by failing to translate the viral yz mRNAs, they are also deficient in all the
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`activities encoded by this entire class of genes as well. Importantly, when dealing
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`with phenotypes ascribed to a deficiency in the 31134.5 gene, it is fair to question
`
`whether the failure to translate these late yz viral mRNAs contributes to the observed
`
`phenotype. One of these late 'yz mRNAs encodes the Usll polypeptide, a dsRNA
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`binding (Khoo et al., “Characterization of RNA Determinants Recognized by The
`
`Arginine— and Proline—Rich Region of Us] 1, A Herpes Simplex Virus Type 1—
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`Encoded Double—Stranded RNA Binding Protein That Prevents PKR Activation,” J
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`Viral 76:1 1971-11981 (2002)), ribosome—associated protein (Roller et al., “The
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`Herpes Simplex Virus 1 RNA Binding Protein Usll Is A Virion Component And
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`Associates With 608 Ribosomal Subunits,” J Viral 66:3624—3632 (1992)) that
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`10
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`15
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`20
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`25
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`30
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`WO 2006/002394
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`PCT/US2005/022561
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`-11-
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`physically associates with PKR (Cassady et al., “The Herpes Simplex Virus Type 1
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`Usll Protein Interacts With Protein Kinase R In Infected Cells and Requires a 30
`
`Amino Acid Sequence Adjacent to a Kinase Substrate Domain,” J Viral 76:2029—
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`2035 (2002); Poppers et al., “Identification of a Lytic-Cycle Epstein-Barr Virus Gene
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`Product That Can Regulate PKR Activation,” J Viral 77:228-236 (2003)) and can
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`prevent PKR activation in response to dsRNA and PACT, a cellular protein that can
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`activate PKR in an RNA independent manner (Peters et a ., “Inhibition of PACT—
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`Mediated Activation of PKR By The Herpes Simplex Virus Type 1 Usll Protein,” J
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`Viral 76:1 1054—11064 (2002)). Furthermore, Usl 1 can preclude the premature
`
`cessation of protein synthesis observed in cells infected with a y134.5 mutant when it
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`is expressed at immediate-early, as opposed to late times, post-infection (Mohr et al.,
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`“A Herpesvirus Genetic Element Which Affects Translation In The Absence of The
`
`Viral GADD34 Function,” EMBO J 15 :4759—4766 (1996); Mulvey et al., “A
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`Herpesvirus Ribosome Associated, RNA-Binding Protein Confers A Growth
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`Advantage Upon Mutants Deficient in a GADD34-Re1ated Function,” J Viral
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`73:3375-3385 (1999)). Recently, it was demonstrated that the premature cessation of
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`translation observed in cells infected With a y134.5 mutant actually results from the
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`combined loss of 31134.5 fimction along with the failure to translate the Us11 mRNA,
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`10
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`15
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`establishing that HSV—1 utilizes different mechanisms to regulate eIF20t
`phosphorylation at discrete phases of the viral life cycle (Mulvey et al., “Regulation
`
`20
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`of eIF20L Phosphorylation By Different Functions That Act During Discrete Phases In
`
`The HSV—1 Lifecycle,” J Virol 77:10917—10928 (2003)).
`
`[0014]
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`Numerous replication—competent, attenuated herpes simplex virus—1
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`(HSV-1) derivatives that contain engineered mutations into the viral 734.5 virulence
`
`25
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`gene have been used as oncolytic agents. (U.S. Patent Nos. 5,328,688 and 6,071,692
`
`to Roizman; U.S. Patent No. 5, 824,318 to Mohr et al.; Mulvey et al., “Regulation of
`
`eIF20L Phosphorylation By Different Functions That Act During Discrete Phases In
`
`The HSV—1 Lifecycle,” J Viral 77:10917—10928 (2003); (Taneja et a1., “Enhanced
`Antitumor Efficacy of a Herpes Simplex Virus Mutant Isolated by Genetic Selection.
`
`30
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`in Cancer Cells,” Proc Natl Acad Sci USA 98:8804-08 (2001); Markert et a1.,
`
`“Genetically Engineered HSV in the Treatment of Glioma: A Review,” Rev Med
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`Virol 10(1): 17-30 (2000); Martuza et a1., “Conditionally Replicating Herpes Vectors
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`for Cancer Therapy,” J Clin Invest 105(7):841—846 (2000); Andreansky et al., “The
`
`Application of Genetically Engineered Herpes Simplex Viruses to the Treatment of
`
`Experimental Brain Tumors,” Proc Natl Acad Sci USA 93(21):113 13—8 (1996); Kes