(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`26 July 2001 (26.07.2001)
`
` (10) International Publication Number
`
`WO 01/53506 A2
`
`(51) International Patent Classification7:
`7/01, A61K 48/00, C12Q 1/02
`
`C12N 15/869,
`
`(74) Agent: WOODS, Geoffrey, Corlett; J.A. Kemp &Co., 14
`South Square, Gray’s Inn, London WC1R 51X (GB).
`
`(21) International Application Number:
`
`PCT/GB01/00229
`
`(22) International Filing Date: 22 January 2001 (22.01.2001)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR,
`HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
`NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
`TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
`
`(30) Priority Data:
`00014753
`00028548
`01002880
`01004308
`
`21 January 2000 (21.01.2000)
`8 February 2000 (08.02.2000)
`5 January 2001 (05.01.2001)
`6 January 2001 (06.01.2001)
`
`GB
`GB
`GB
`GB
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`(71) Applicant (for all designated States except US): BIOVEX
`LIMITED [GB/GB]; The Windeyer Institute, 46 Cleve—
`land Street, London WlP 6DB (GB).
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): COFFIN, Robert,
`Stuart [GB/GB]; The Windeyer Institute, 46 Cleveland
`Street, London WlP 6DB (GB).
`
`For two—letter codes and other abbreviations, refer to the "Guid—
`ance Notes on Codes andAbbreviations " appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
`
`WO01/53506A2
`
`(54) Title: VIRUS STRAINS
`
`(57) Abstract: The present invention relates to non—laboratory Virus strains, for example of herpes viruses such as HSV, with im—
`proved oncolytic and/or gene delivery capabilities as compared to laboratory virus strains.
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`VIRUS STRAINS
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`Field of the Invention
`
`The present invention relates to non-laboratory virus strains, for example of
`
`herpes viruses such as HSV, vm'th improved oncolytic and/or gene delivery
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`capabilities as compared to laboratory virus strains.
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`Background of the Invention
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`Viruses have been suggested or demonstrated to have utility in a variety of
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`applications in biotechnology and medicine on many occasions. Each is due to the
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`unique ability of viruses to enter cells at high efficiency. This is followed in such
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`applications by either virus gene expression and replicationand/or expression of an
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`inserted heterologous gene. Thus viruses can either deliver and express genes in cells
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`(either viral or other genes) which may be useful in for example gene therapy or the
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`development of vaccines, or they may be usefiJl in selectively killing cells by lytic
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`replication or the action of a delivered gene in for example cancer.
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`Herpes simplex virus (HSV) has been suggested to be of use both as a gene
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`delivery vector in the nervous system and elsewhere and for the oncolytic treatment
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`of cancer. In both applications the virus must however be disabled such that it is no
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`longer pathogenic but such that it can still enter cells and perform the desired
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`function. Thus for non-toxic gene delivery to target cells using HSV it has become
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`apparent that in most cases immediate early gene expression must be prevented/
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`minimised from the virus. For the oncolytic treatment of cancer, which may also
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`include the delivery of gene(s) enhancing the therapeutic effect, a number of
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`mutations to HSV have been identified which still allow the virus to replicate in
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`culture or in actively dividing cells in vivo (e.g. in tumours), but which prevent
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`significant replication in normal tissue. Such mutations include disruption of the
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`genes encoding ICP34.5, ICP6 and thymidine kinaSe. Of these, viruses with
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`mutations to ICP34.5, or ICP34.5 together with mutations of e.g. ICP6 have so far
`shown the most favourable safety profile. Viruses deleted for only ICP34_.5 have
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`been shown to replicate in many tumour cell types in vitro and to selectively replicate
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`in artificially induced brain tumours in mice while sparing surrounding tissue. Early
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`stage clinical trials have also shown their safety in man.
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`However, while promise has been shown for various viruses including HSV
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`for gene delivery/therapy or for the oncolytic treatment of cancer, the majority of this
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`work has used virus strains which have been maintained in tissue culture cells for
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`many years. In applications where the virus merely needs enter cells to deliver genes
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`this may not prove problematical as maintenance in cell culture also requires the
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`virus to enter cells, albeit often cells of a different type or species in comparison to
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`the likely target cells for a vector. However, in applications where other properties
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`are required, the use of laboratory virus strains may not allow the filll potential of a
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`virus in a particular application to be utilised.
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`HSV has the unique ability amongst viruses currently under development as
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`vectors in that it has naturally evolved to infect and remain latent in neurons. HSV
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`has also evolved to be highly efficiently transported along nerves from the site of
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`infection, usually at the periphery, to the neuronal cell body, usually in the spinal
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`ganglia. Such capabilities are not required in cell culture and as such capabilities
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`require specific evolved properties of HSV, further adaption to growth in culture may
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`have resulted in optimally efficient axonal transport capabilities to have been lost.
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`HSV vectors for gene delivery to the central or peripheral nervous system are likely
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`to show maximum effectiveness if axonal transport properties have been retained at
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`maximum efficiency. Here, inoculation at a peripheral site would then allow
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`maximally efficient gene delivery to peripheral neuron cell bodies, and inoculation in
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`the brain would allow maximally efficient gene delivery to multiple connected sites.
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`Current vectors based on laboratory strains of HSV may not allow this to occur at the
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`maximum efficiency possible. Indeed, because of HSV’S high capacity to be
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`transported along nerves, there is potentially a particularly large discrepancy between
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`the properties which it is desired to conserve and those likely to be retained in
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`culture.
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`HSV and other Viruses such as adeno- or rheovirus also have potential utility
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`in the oncolytic treatment of cancer. However, again viruses under development for
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`such purposes have previously been extensively maintained in culture. As the
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`oncolytic treatment of cancer requires active replication in often relatively slowly
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`growing human tumour cells, it would be anticipated that adaptation of laboratory
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`virus strains to growth in particular cultured cells may have reduced the efficiency
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`with which such lytic replication in human tumour cells, or infection of human
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`tumour cells, could optimally occur.
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`Summary of the Invention
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`The present invention provides the opportunity to deve10p viruses with
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`improved in vivo capabilities of gene transfer and/or lytic destruction of tumour cells.
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`Here, virus strains are constructed appropriate for these purposes based on recent
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`clinical isolates of the appropriate virus rather than the serially passaged laboratory
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`strains which have previously been used. The present invention therefore has the
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`potential to provide viruses with improved capabilities of infecting human cells in
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`vivo, improved replicative/lytic capability in such cells, and (in the case of HSV)
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`improved abilities of trafficking along nerves from the inoculation site to the
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`neuronal cell body. The invention is exemplified using HSV but is equally
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`applicable for other viruses currently under development as vectors and/or for the
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`oncolytic destruction of cancer cells.
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`We have shown that two clinical isolates of HSVl (strains J81 and BLl) have
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`enhanced replication in some human tumour cell lines as compared to HSVl strain
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`17+ (a standard laboratory strain).
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`We have deleted ICP34.5 from the clinical isolate J 81 strain and again
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`compared replicative potential in human tumour cell types in comparison to HSVl
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`strain 17+ (a standard laboratory strain) in which ICP34.5 was also deleted. This
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`strain (JSl/lCP34.5-) is a modified strain derived from a clinical isolate, and is thus a
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`modified non-laboratory strain of the invention.
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`JS1 with ICP34.5 deleted showed enhanced growth in some human tumour
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`cells tested as compared to HSVl ICP34.5 deleted strain 17+, i.e. a laboratory strain
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`virus with the same modification. However, as compared to the laboratory strain
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`derived from strain 17+, cell killing capabilities were enhanced with the
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`JSl/ICP34.5- virus in all tumour cell lines tested.
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`Thus, the use of non-laboratory virus strains can be seen to enhance the anti-
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`tumour capabilities of such viruses and was evident in all the tumour cell lines tested
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`so far. This will have applicability for cancer treatment in human patients.
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`Further enhanced activity may also be anticipated if these viruses are then
`used to deliver genes with anti-tumour activity. Such genes include those encoding
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`pro-drug activators, tumour suppressor or pro-apoptotic factors, or immune
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`stimulatory proteins.
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`For this purpose, we have produced an ICP34.5 deleted clinical isolate of
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`HSV] which expresses human GMCSF. This virus is designed to enhance anti-
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`tumour immune responses following intra-tumoral injection.
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`The invention also provides viruses of the invention which carry a
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`heterologous gene/genes. The term heterologous gene is intended to embrace any
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`gene not found in the viral genome. The heterologous gene may be any allelic
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`variant of a wild—type gene, or it may be a mutant gene. Heterologous genes are
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`preferably operably linked to a control sequence permitting expression of said
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`heterologous gene in a cell in vivo. Viruses of the invention may thus be used to
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`deliver a heterologous gene/genes to a cell in viva where it will be expressed. For
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`oncolytic virus therapy, such genes typically encode proteins capable of enhancing
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`the tumour destroying properties of the virus. These genes may encode proteins
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`which are themselves cytotoxic, are pro-drug activating, or which may be capable of
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`stimulating lenhancing an anti-tumour immune response. For gene delivery to the
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`peripheral nervous system using HSV, the heterologous gene/genes may encode a
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`polypeptide capable of modifying responses to painful stimuli or reducing chronic
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`pain, for example a protein capable sequestering e.g. nerve growth factor, other pain
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`modulating neurotrophic factor or neurotrophic factor-like molecules, or substance P
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`i or other neuropeptides. The heterologous gene/genes may also encode a polypeptide
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`capable of stimulating the re-growth of damage nerves or preventing the further
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`degeneration of nerves in degenerative conditions. In the central nervous system,
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`heterologous genes may include those potentially beneficial in neurodegenerative
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`disease such as Parkinson’s disease or Alzheimer’s disease and might typically
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`include genes encoding neurotrophic factors and/or enzymes capable of enhancing
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`the activity of remaining cells in such diseases. In all cases, single or multiple
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`heterologous genes may be carried by a single virus.
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`Accordingly the invention provides:
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`Use of a modified, oncolytic, non-laboratory virus strain in the manufacture of a
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`medicament for the oncolytic treatment of cancer;
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`Use of a modified, replication incompetent, non-laboratory virus strain comprising a
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`heterologous gene in the manufacture of a medicament for the delivery of said gene
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`to a subject;
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`A method of determining whether a gene has an effect on a phenotype associated
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`with a peripheral nervous system disorder or on a cell of the peripheral nervous
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`system which is relevant to a peripheral nervous system disorder, which method
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`comprises:
`
`(i)
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`inoculating a replication incompetent herpes virus of the invention
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`comprising a heterologous gene into a peripheral nerve; and
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`(ii)
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`monitoring a phenotype of said disorder or an effect of expression of
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`said gene on said cell to determine thereby whether said gene has an
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`effect relevant to said disorder;
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`A method of determining whether a gene has an effect on a phenotype associated
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`with a central nervous system disorder or on a cell of the central nervous system
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`which is relevant to a central nervous system disorder, which method comprises:
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`(i)
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`inoculating into a cell of the central nervous system with a replication
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`incompetent herpes virus of the invention; and
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`(ii)
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`monitoring a phenotype of said disorder or an effect of expression of
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`said gene on said cell to determine thereby whether said gene has an
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`effect on said cell or said phenotype;
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`A method of determining whether a gene encodes an antigen associated with a
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`pathogenic infection or cancer, which method comprises infecting a dendritic cell or
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`a macrophage with a replication incompetent virus of the invention comprising a
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`heterologous gene encoding an antigen and monitoring antigen presentation of the
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`polypeptide product of said gene, or an effect of expression of said gene, or a
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`phenotype of said pathogenic infection or cancer to determine thereby whether said
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`gene encodes an antigen associated with said infection or cancer and which itself has
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`therapeutic potential or is a target for therapeutic intervention;
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`A method of determining the suitability of a non-laboratory virus strain for
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`modification into a modified strain as defined herein , comprising:
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`(i)
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`(ii)
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`optionally, isolating a non-laboratory virus strain from a host;
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`providing said non-laboratory virus strain;
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`(iii)
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`assessing the properties of the virus in respect of one or more
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`desirable characteristics; and, optionally,
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`(iv)
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`selecting for modification virus strains with desirable properties;
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`A method of determining the suitability of a non-laboratory virus strain for
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`modification into a modified oncolytic strain of the invention, comprising:
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`(i)
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`(ii)
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`optionally, isolating a non-laboratory virus strain from a host;
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`assessing the growth of the virus in one or more types of tumour cell;
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`and optionally
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`(iii)
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`selecting for modification virus strains with a high growth rate or cell
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`killing capability;
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`A method of determining the suitability of a non-laboratory virus strain for
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`modification into a modified oncolytic strain, comprising:
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`(i)
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`providing a non-laboratory virus strain, optionally one selected by a
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`method as just defined;
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`(ii)
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`modifying said strain such that it becomes oncolytic; and
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`(iii)
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`assessing the ability of said modified, oncolytic non-laboratory strain
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`to kill tumour cells; and optionally
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`(iv)
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`selecting strains that exhibit high tumour cell-killing capacity for
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`further modifications; and optionally
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`(v)
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`carrying out further modifications;
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`A method of determining whether a gene enhances the anti-tumour effects of a virus
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`comprising:
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`(i)
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`(ii)
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`providing a modified, oncolytic non-laboratory strain of the invention;
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`inserting said gene into said virus as a heterologous gene; and
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`(iii)
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`assessing the ability of said modified, oncolytic non-laboratory strain
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`to kill tumour cells compared to the ability of the precursor strain
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`provided in step (i);
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`A method of producing a modified, oncolytic, non-laboratory virus strain
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`comprising:
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`(i)
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`(ii)
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`isolating from a host a non—laboratory strain of a virus;
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`optionally determining its suitability for modification as defined
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`above; and
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`(iii) modifying it to render it oncolytic, and optionally
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`(iv)
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`carrying out further modifications;
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`A method of producing a modified non-laboratory virus strain comprising:
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`(i)
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`(ii)
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`providing a non-laboratory strain of a virus;
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`modifying it to render it replication incompetent and,
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`(iii)
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`inserting a heterologous gene;
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`A modified, oncolytic, non-laboratory virus strain as defined herein;
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`A modified non-laboratory virus strain comprising a heterologous gene, as defined
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`herein
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`Use of a gene identified as having an effect on a phenotype associated with a
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`peripheral nervous system disorder or on a cell of the peripheral nervous system
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`which is relevant to a peripheral nervous system disorder by a method as defined
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`above, or of a gene product encoded by said gene in the manufacture of a
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`medicament for the treatment of a peripheral nervous system disorder;
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`Use of a gene identified as having an effect on a phenotype associated with a central
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`nervous system disorder or on a cell of the central nervous system which is relevant
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`to a central nervous system disorder by a method as defined above, or of a gene
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`product encoded by said gene in the manufacture of a medicament for the treatment
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`of a central nervous system disorder;
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`Use of a gene identified as encoding an antigen associated with a pathogenic
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`infection or cancer by a method as defined above, or of an antigen encoded by said
`
`gene in the manufacture of a medicament for the treatment or prevention of said
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`infection or cancer;
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`A non-laboratory virus strain identified by, or produced in the course of, a method of
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`the invention;
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`Use of a gene identified as enhancing the anti-tumour effects of a Virus by a method
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`as defined above, or of a gene product encoded by said gene, in the manufacture of a
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`medicament for the treatment or prevention of cancer;
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`A modified non—laboratory virus strain obtained or obtainable by a method of the
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`invention;
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`HSVl strain JSl as deposited at the European Collection of Cell Cultures (ECACC)
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`under provisional accession number 01010209, or an HSV] strain derived therefrom,
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`a pharmaceutical composition comprising such a virus; such a virus for use in the
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`treatment of the human or animal body;
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`A method of treating a tumour in an individual in need thereof by administering to
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`said individual an effective amount of an oncolytic virus of the invention;
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`A method of delivering a gene to an individual in need thereof by administering to
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`said individual an effective amount of a non-oncolytic virus of the invention; and
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`A method of treating or preventing a central peripheral nervous system disorder by
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`administering to a peripheral nerve of an individual in need thereof an effective
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`amount of a neurotrophic virus of the invention.
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`Brief Description of the Drawings
`
`Fig 1. Viruses
`
`From top to bottom, diagrams show: laboratory HSVl strain 17+, clinical
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`strain BLl, clinical strain .181, 17+/ICP34.5-, JSl/ICP34.5-, JS]/ICP34.5-/ICP47-/
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`hGMCSF.
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`Fig 2. Clinical isolates show enhanced growth in tumour cells
`
`(1)
`
`Growth of 17+, BLl and J81. Left hand diagram: U87 cells. Right-hand
`
`diagram: LNCaP cells.
`
`(2)
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`Growth of ICP34.5- 17+ and .1S1 on tumour cells. Left-hand diagram:
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`LNCaP cells. Right-hand diagram: MDA—MB—231 cells.
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`JSl/34.5- does not grow on cells non-permissive for HSV ICP34.5 mutants.
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`Left-hand diagram: 3T6 cells — 17+, J81. Right-hand diagram: 3T6 cells —
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`17+, JSl ICP34.5—.
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`Fig 3. An ICP34.5 deleted HSV clinical isolate shows enhanced lysis in all
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`tumour cells tested
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`Tumour cell lines were either mock infected, infected with HSVl strain l7+/
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`34.5—, or infected with HSVl strain JSl/34.5— at the indicated M01 and stained
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`with crystal violet at time points after infection to allow Visualisation of cells.
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`Each block of photographs relates to a cell type. From top to bottom, these are
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`HT29 colorectal adenocarcinoma, LNCaP.FGC prostate adenocarcinoma, MDA-
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`MB-231 breast adenocarcinoma, SK-MEL-28 malignant melanoma and U-87
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`MG glioblastoma astrocytoma. Left-hand blocks relate to results for HSVl
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`strain 17+/34.5-. Right—hand blocks relate to results for HSVl strain JSl/34.5-.
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`Central blocks represent mock infected cells. Within each block, the top row
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`represents a 24 hour time-point, the second a 48 hour time-point and the third at
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`72 hour time-point within each block, the left-hand column represents MOI=0.2,
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`the central column MOI=0.1 and the right—hand column MOI=5.
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`Detailed Description of the Invention
`
`A.
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`Viruses
`
`Virus Strains of the Invention
`
`The invention is applicable to viruses in general. Preferably, a virus strain of
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`the invention will be a strain of a herpes virus, adenovirus, picornavirus, retrovirus or
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`alphavirus. More preferably, a virus strain of the invention will be a strain of a
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`herpes virus. Still more preferably, a virus strain of the invention will be a strain of a
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`herpes simplex virus (HSV), typically a strain of HSVl or HSV2.
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`When the virus of the invention is a herpes simplex virus, the virus may be
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`derived from, for example HSVl or HSV2 strains, or derivatives thereof, preferably
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`HSVl. Derivatives include inter—type recombinants containing DNA from HSVl
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`and HSV2 strains. Such inter-type recombinants are described in the art, for example
`
`in Thompson et al (1998) and Meignier et a] (1988). Derivatives preferably have at
`
`least 70% sequence homology to either the HSVl or HSV2 genomes, more
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`preferably at least 80%, even more preferably at least 90 or 95%. More preferably, a
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`derivative has at least 70% sequence identity to either the HSVl or HSV2 genome,
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`more preferably at least 80% identity, even more preferably at least 90%, 95% or
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`98% identity.
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`For example the UWGCG Package provides the BESTFIT program which
`
`can be used to calculate homology (for example used on its default settings)
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`(Devereux et al. (1984) Nucleic Acids Research 12, p387-395). The PILEUP and
`
`BLAST algorithms can be used to calculate homology or line up sequences (typically
`
`on their default settings), for example as described in Altschul (1993) J. Mol. Evol.
`
`$290600; Altschul et al. (1990) J. Mol. Biol. fiz403—10.
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`Software for performing BLAST analyses is publicly available through the
`
`National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ 1. This
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`algorithm involves first identifying high scoring sequence pair (HSPs) by identifying
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`short words of length W in the query sequence that either match or satisfy some
`
`positive-valued threshold score T when aligned with a word of the same length in a
`
`database sequence. T is referred to as the neighbourhood word score threshold
`
`(Altschul et al., 1990). These initial neighbourhood word hits act as seeds for
`
`initiating searches to find HSPs containing them. The word hits are extended in both
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`directions along each sequence for as far as the cumulative alignment score can be
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`increased. Extensions for the word hits in each direction are halted when:
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`the
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`cumulative alignment score falls off by the quantity X from its maximum achieved
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`value; the cumulative score goes to zero or below, due to the accumulation of one or
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`more negative-scoring residue alignments; or the end of either sequence is reached.
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`The BLAST algorithm parameters W, T and X determine the sensitivity and speed of
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`the alignment. The BLAST program uses as defaults a word length (W) of 11, the
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`BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.
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`Sci. USA Q: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4,
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`and a comparison of both strands.
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`The BLAST algorithm performs a statistical analysis of the similarity
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`between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci.
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`USA fl: 5873-5787. One measure of similarity provided by the BLAST algorithm is
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`the smallest sum probability (P(N)), which provides an indication of the probability
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`by which a match between two nucleotide or amino acid sequences would occur by
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`chance. For example, a sequence is considered similar to another sequence if the
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`smallest sum probability in comparison of the first sequence to the second sequence
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`is less than about 1, preferably less than about 0.1, more preferably less than about
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`0.01, and most preferably less than about 0.001.
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`A derivative may have the sequence of a HSVl or HSV2 genome modified
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`by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100
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`substitutions. The HSVl or HSV2 genome may alternatively or additionally be
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`modified by one or more insertions and/or deletions and/or by an extension at either
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`or both ends.
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`Properties of Virus Strain of the Invention
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`Virus strains of the invention are “non-laboratory” strains. They can also be
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`referred to as “clinical” strains. A person of skill in the art will readily be able to
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`distinguish between a laboratory strain and a non-laboratory, or clinical, one. Further
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`guidance on the properties likely to be exhibited by virus strains is given below.
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`The key distinction between a laboratory and non-laboratory strain is that
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`laboratory strains currently in common use have been maintained for long periods,
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`many years in some cases, in culture. The culture of viruses such as HSV involves a
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`technique known as serial passage. To grow and maintain viruses, suitable cells are
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`infected with the virus, the Virus replicates within the cell and the virus is then
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`harvested; fresh cells are then re-infected. this process constitutes one cycle of serial
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`passage. Each such cycle may take, for example, a few days in the case of HSV. As
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`discussed above, such serial passaging may lead to changes in the properties of the
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`virus strain, in that selection takes places for properties that favour growth in culture
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`(6. g. rapid replication), as opposed to properties useful for practical applications, e.g.
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`maintenance of the capacity to travel along axons in the case of HSV.
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`Virus strains of the invention are non-laboratory strains in that they are
`
`derived from strains recently isolated from infected individuals. Strains of the
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`invention are modified compared to the original clinical isolates, and may have spent
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`a time in culture, but any time spent in culture will be comparatively short. Strains of
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`the invention are prepared in such a manner as to retain substantially the desirable
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`properties of the original clinical isolates from which they are derived.
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`A virus of the invention is capable of efficiently infecting target human cells.
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`Such a virus is recently isolated from an infected individual and then screened for the
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`desired ability of enhanced replication in tumour and/or other cells in vitro and/or in
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`vivo in comparison to standard laboratory strains, or (in the case of neurotrophic
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`viruses such as HSV) for an enhanced ability to traffic along nerves as compared to
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`standard laboratory strains using an in vivo model. Such viruses with improved
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`properties as compared to laboratory Virus strains are viruses of the invention.
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`Identified viruses with such desired improved properties can then be engineered such
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`that they can selectively kill tumour cells by the mutation of appropriate gene(s), or
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`mutated such that they can deliver a gene(s) to target tissue without toxic effect in
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`non-oncolytic applications. These modified viruses are also viruses of the invention.
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`Alternatively, virus strains may be isolated from an infected individual and mutations
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`anticipated to be appropriate for oncolytic therapy and/or gene delivery made. These
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`modified viruses are then screened for the desired improved properties as compared
`
`to laboratory strains, viruses with such improved properties providing further viruses
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`of the invention.
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`Further guidance on the likely properties of the virus strains of the invention
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`is provided as follows.
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`Preferably, a virus strain of the invention has undergone three years or less in
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`culture since isolation of its unmodified clinical precursor strain from its host. More
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`preferably, a strain of the invention has undergone one year or less in culture, for
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`example nine months or less, six months or less, three months or less, or two months
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`or less, one month or less, two weeks or less, or one week or less. By these
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`definitions of time in culture, is meant time actually spent in culture. Thus, for
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`example, it is a common practice to freeze virus strains in order to preserve them.
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`Evidently, preserving by freezing or in an equivalent manner does not qualify as
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`maintaining the strain in culture. Thus, time spent frozen or otherwise preserved is
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`not included in the above definitions of time spent in culture. Time spent in culture
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`is typically time actually spent undergoing serial passage, i.e. time during which
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`selection for undesirable characteristics can occur.
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`Preferably, a virus strain of the invention has undergone 1,000 or less cycles
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`of serial passage since isolation of its unmodified clinical precursor strain from its
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`host. More preferably, it has undergone 500 or less, 100 or less, 90 or less, 80 or
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`less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less
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`such cycles.
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`Preferably, a virus of the invention has a greater ability, as measured by
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`standard statistical tests, than a reference laboratory strain with the equivalent
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`modifications to perform certain functions useful in the application at hand. For
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`example, in the case of an oncolytic virus for tumour treatment, a virus strain of the
`
`invention will preferably have a greater ability than a reference laboratory strain with
`
`equivalent modifications to infect or replicate any tumour cell, to kill tumour cells or
`
`to spread between cells in tissue. More preferably, such greater ability is a
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`statistically significantly greater ability. For example, according to the invention, a
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`may have up to 1.1 fold, 1.2 fold, 1.5 fold, 2 fold, 5 fold, 10 fold, 20 fold, 50 fold, or
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`100 fold the capacity of the reference strain in respect of the property being tested.
`
`Preferably, a virus of the invention has, i.e. retains, substantially the ability of
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`its unmodified clinical precursor strain in respect of one or more of the properties
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`characteristic of usefulness in the application at hand. For example, in the case of an
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`oncolytic virus intended for the treatment of tumours, a Virus strain of the invention
`
`preferably has substantially the ability of its unmodified clinical precursor strain to
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`infect or replicate a tumour cell, kill tumour cells or to spread between cells in tissue.
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`Preferably, according to the invention, a virus retains substantially the
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`properties of its unmodified clinical precursor strain if, in a quantitative test, it retains
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`75%, more preferably 80, 90, 95, 98, 99 or 100% of the capacity of the unmodified
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`clinical precursor strain in respect of the property being tested. More preferably, in
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`respect of the property being tested, any differences between the unmodified clinical
`
`precursor strain and the modified strain of the invention will not be statistically
`
`significant.
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`Statistical analysis of the properties described herein may be carried out by
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`standard tests, for example, t-tests, ANOVA, or Chi squared tests. Typically,
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`statistical significance will be measured to a level of p = 0.05 (5%), more preferably
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`p = 0.01, p = 0.001, p = 0.0001, p = 0.000001.
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`Modifications
`
`Viruses of the invention are typically modified as compared to their precursor
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`clin