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WO1997010349A1 - Matieres et procedes de traitement de maladies neurodegeneratives et de recherche systematique d'inhibiteurs et d'inducteurs potentiels de l'apoptose - Google Patents

Matieres et procedes de traitement de maladies neurodegeneratives et de recherche systematique d'inhibiteurs et d'inducteurs potentiels de l'apoptose Download PDF

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WO1997010349A1
WO1997010349A1 PCT/US1996/014147 US9614147W WO9710349A1 WO 1997010349 A1 WO1997010349 A1 WO 1997010349A1 US 9614147 W US9614147 W US 9614147W WO 9710349 A1 WO9710349 A1 WO 9710349A1
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gene
cells
cell line
apoptosis
vector
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PCT/US1996/014147
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English (en)
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Bernard Roizman
Joany Chou
Bin He
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Arch Development Corporation
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Priority to AU69645/96A priority Critical patent/AU6964596A/en
Publication of WO1997010349A1 publication Critical patent/WO1997010349A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention is directed to methods for blocking or delaying programmed cell death for delivery of gene therapy to specific cells.
  • the present invention is also directed to assays for candidate substances which can either inhibit, induce, or potentiate programmed cell death.
  • apoptosis involves distinctive mo ⁇ hological changes including nuclear condensation and degradation of DNA to oligonucleosomal fragments.
  • apoptosis is triggered by or is preceded by changes in protein synthesis.
  • the degradation of DNA described above may be a slow process occurring days after cessation of cellular activities accompanying apoptosis.
  • Apoptosis appears to provide a very clean process for cellular destruction, in that the cells are disposed of by specific recognition and phagocytosis prior to bursting. In this manner cells can be removed from a tissue without causing damage to the surrounding cells.
  • programmed cell death is crucial in a number of physiological processes, including morphological development, clonal selection in the immune system, and normal cell maturation and death in other tissue and organ systems. It has also been demonstrated that cells can undergo apoptosis in response to environmental information.
  • Examples include the appearance of a stimulus, such as glucocorticoid hormones for immature thymocytes, or the disappearance of a stimulus, such as interleukin-2 withdrawal from mature lymphocytes, or the removal of colony stimulating factors from hemopoietic precursors (for a review of literature see Williams, Cell, 85: 1097-1098, [1991]).
  • a stimulus such as glucocorticoid hormones for immature thymocytes
  • a stimulus such as interleukin-2 withdrawal from mature lymphocytes
  • colony stimulating factors from hemopoietic precursors
  • the APO- 1 monoclonal antibody can induce apoptosis in several transformed human B and T cell lines.
  • the antibody binds to a surface protein and could act either by mimicking a positive death-inducing signal or by blocking the activity of a factor required for survival.
  • anti-FAS antibodies have similar effects, and the recent cloning and sequencing of the gene for the FAS antigen has shown that it is a 63 kilodalton transmembrane receptor. Itoh, et al. , Cell 66: 233-243 (1991). However, it is important to note that neither APO-1 nor FAS appear to function exclusively as triggers for cell death. Both are cell surface receptors that may activate quite different responses under other circumstances. Moreover, these antigens are not confined to tumor cells and their effect on normal cells is certainly an important consideration, as is the possible appearance of variants that no longer display the antigens.
  • bcl-2 gene has also been shown to inhibit cell death by apoptosis.
  • the bcl-2 gene was isolated from the breakpoint of the translocation between chromosomes 14 and 18 found in a high proportion of the most common human lymphomas, that being follicular B cell gene and immunoglobulin heavy chain locus, resulting in an aberrantly increased bcl-2 expression in B cells.
  • Henderson, et al. (Cell, 65: 1107-1115, [1991]) demonstrated that expression of latent membrane protein 1 in cells infected by Epstein-Barr virus protected the infected B cells from programmed cell death by inducing expression of the bcl-2 gene. Sentman, et al.
  • Alzheimer's disease Gshwind et al , J. Neurochem 65(l):292-300 [1995], LaFerla et al. , Nat. Genet. 9(l):21-30 [1995]
  • Huntington's disease Portera- Cailliau et al. , J. Neurosci 15(5): 3775-3787 [1995]
  • spinal muscular dystrophy Roy et al. , Cell 80: 165-178 [1995]
  • amyotrophic lateral sclerosis Alexianu et al. , J. Neurochem 63:2365-2368 [1994]).
  • Herpes simplex viruses have particular characteristics that make them useful in the study of apoptosis (see, WO 93/19591, published 14 October 1993, the disclosure of which is inco ⁇ orated herein by reference).
  • the he ⁇ es simplex virus one genes are arranged in four clusters, i.e. in a unique long (U L ) sequence, in a sequence designated as ab and its inverted repeat, ba which flank U L , in a unique short (U s ), and in the sequence ac and ⁇ a which flank U s (Sheldrick et al. , Cold Spring Harbor Symp. Quant. Biol. 39:667-678 [1975]; Wadsworth et al. , J. Virol.
  • the a sequence varies in length (reviewed in Roizman et al. , In Roizman B, ed. The Human Herpes Viruses, New York: Raven press, pages 11-68 [1993]).
  • HSV-1 strain F HSV-1(F)
  • this sequence is approximately 500 bp long and consists largely of reiterations of short sequences (Mocarski et al. , Cell 31:89-97 [1992]).
  • the a sequence serves many functions including the cleavage of mature, unit length DNA from newly synthesized DNA concatamers for packaging into preformed capsids.
  • the a sequence flanking the U L sequence also contains a promoter for a gene designated 7,34.5 (Chou et al. , J. Virol. 57:629-637 [1986]). Because it is located within inverted repeats, the ]34.5 is present in two copies per genome. Because it is a class gene, the expression of the gene is only partially induced by inhibitors of DNA synthesis.
  • mutants lacking the 7,34.5 gene are avirulent in experimental murine and guinea pig models (Chou et al , Science 250: 1262- 1266 [1990]; Whitley et al , J. Clinical Invest. 91:2837-2843 [1993]).
  • the deletion mutant replicates as well as the wild type virus in Vero cells.
  • the function of 7] 34.5 gene product appears to be to preclude the premature shutoff of total protein synthesis in selected human cells.
  • Experiments described elsewhere (Chou et al . J. Virol 66:8304-8311 [1994]) indicate that the amounts of 7,34.5 protein required for this purpose can be ver)' small and may be supplied in trans by cells carrying the viral 7,34.5 gene.
  • the 7,34.5 protein (ICP34.5) predicted to contain 263 amino acids consists of 3 domains: a 159 amino acid-amino terminal domain; 10 repeats of three amino acids (AlaThrPro), and a 74 amino acid-carboxyl terminal domain (Chou et al , J. Virol 64: 1014-1020 [1990]).
  • the number of the triplet repeats appears to vary among virus strains (Chou et al , J. Virol 64: 1014-1020 [1990]).
  • a stretch of 64 amino acids at the carboxyl terminus of the 7,34.5 is homologous to a corresponding stretch of amino acids of the carboxyl terminus of a murine protein known as MyD116 (Lord et al, Nucleic Acids Res. 18:2823 [1990]; McGeoch et al. , Nature (London) 353:609 [1991]) and a Chinese hamster protein known as GADD34 (Fornace et al , Mol. Cell. Biol 9:4196-4203 [1989]).
  • the MyD116 protein is predicted to contain 657 amino acids and consists of a long amino terminal domain, 4.5 repeats of a sequence of 38 amino acids, and a relatively short carboxyl terminal domain.
  • GADD34 structurally closely related to MyD116, is also one of a subset of proteins induced following DNA damage or cell growth arrest (Fornace et al. , Mol. Cell. Biol. 9:4196-4203 [1989]; Zhan et al. , Mol. Cell Biol. 14:2361-2371 [1994]).
  • GADD34 or MyD116 alone or in association with another protein known as GADD45 is to suppress cell division during DNA repair and differentiation of lesions to preclude stress responses which could result in cell death (Selvakumaran et al , Blood 84:1036-1042 [1994]; Zhan et al , Mol. Cell. Biol 14:2361-2371 [1994]).
  • the precise function of MyD116 and of GADD34 genes is not known.
  • This invention relates to methods for the prevention or treatment of programmed cell death, or apoptosis, in neuronal cells for therapy in connection with neurodegenerative diseases, as well as methods of treatment of cancer and other tumorigenic diseases and herpesvirus infection.
  • the invention also relates to assay methodologies allowing for the identification of substances capable of modulating the effects of the 7,34.5 gene or modified 7,34.5 gene or their protein expression products (ICP34.5) or modified, i.e., substances capable of potentiating, inducing, or inhibiting their effects. Additionally, the invention also relates to assay methodologies designed to identify candidate substances able to mimic either 7,34.5 expression or the activity of ICP34.5.
  • the invention also relates to methods of delivering genes to cells for gene therapy.
  • a method of preventing or treating programmed cell death in neuronal cells in which a non-pathogenic vector (viral vectors, plasmids, or other vectors) is prepared which contains the 7,34.5 or a modified 7,34.5 gene having the same or similar biological properties. This vector is then introduced into neuronal cells which are presently undergoing or are susceptible to undergo programmed cell death.
  • a non-pathogenic vector viral vectors, plasmids, or other vectors
  • This vector is then introduced into neuronal cells which are presently undergoing or are susceptible to undergo programmed cell death.
  • HSV herpes simplex virus
  • the invention utilizes the non-pathogenic HSV virus injected into the vicinity of the synaptic terminals of affected neurons, or in the area of a peripheral wound or lesion or other appropriate peripheral locus.
  • the HSV-1 virus containing the 7,34.5 gene, under a different target-specific promoter, would then be transported into the neuronal cell body via retrograde axonal transport.
  • the invention envisions specific genomic modifications being introduced into the HSV-1 virus in order to render the virus non-cytotoxic.
  • modifications could include deletions from the genome, rearrangements of specific genomic sequences, or other specific mutations.
  • One example of such a modification comprises modification or deletion of the ⁇ 4 gene which encodes the ICP4 protein. Deletion or modification of the ⁇ 4 gene renders the HSV-1 virus unable to express genes required for viral DNA and structural protein synthesis.
  • the 7,34.5 gene placed under a suitable promoter capable of directing its expression in the absence of viral DNA and protein synthesis would be expressed, thus inducing an anti- apoptotic effect in the neuron without the potential for stress induced neurovirulence.
  • Other genes which may be modified include the ⁇ 0 gene.
  • the invention also envisions the use of other vectors including, for example, retrovirus, picorna virus, vaccinia virus, HSV-2, coronavirus, eunyavirus, togavirus or rhabdovirus vectors. Again, use of such viruses as vectors will necessitate construction of appropriate mutations so that the vectors will be safe and non-pathogenic.
  • Another method by which the invention envisions introducing the 7,34.5 gene into neuronal cells undergoing or likely to undergo programmed cell death is through the use of multi-potent neural cell lines.
  • Such lines have been shown to change phenotype in vitro and have also been demonstrated to become integrated into the central nervous system of mice and to differentiate into neurons or glia in a manner appropriate to their site of engraftment. Snyder, et al , Cell, 68: 33-51 , (1992).
  • Transplant or engraftment of multi-potent neural cell lines expressing a 7,34.5 gene into an area of the central nervous system in which cells are undergoing or are likely to undergo programmed cells death is expected to lead to reversal and inhibition of programmed cell death.
  • the invention also envisions the use of the 7,34.5 gene in the extension of the life of neuronal cells in cell culture.
  • Introduction of a non- cytotoxic vector into cultured neuronal cells will have an anti-apoptotic effect and will thereby extend the life of cell cultures. This in turn will extend the time periods over which experimentation on those cells may be conducted.
  • the invention also provides a method of preventing or treating programmed cell death in neuronal cells which involves the use of the product of expression of the 7,34.5 gene.
  • the protein expressed by 7,34.5 is called ICP34.5. Ackermann, et al. (/.
  • ICP34.5 has an apparent molecular weight of 43,500 upon SDS- polyacrylamide gel electrophoresis, appears to accumulate largely in the cytoplasm of HIV infected cells, and in contrast to many HSV-1 proteins, ICP34.5 has been demonstrated to be soluble in physiologic solutions.
  • modified ICP34.5 are intended to refer to those proteins, and their encoding nucleic acid sequences, in which certain structural changes have been made but which nonetheless are, or encode proteins evidencing some or all of the biological effects of ICP34.5.
  • Patent 4,554, 101 the disclosure of which is incorporated herein by reference.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; giutamine and asparagine; and valine, leucine and isoleucine.
  • Modified 7,34.5 genes also include chimeric genes or divergent genes which encode proteins that exhibit the same biological properties as ICP34.5.
  • chimeric 7,34.5 genes may be utilized in any of the aspect of the invention calling for expression of a ICP34.5 like activity so long as the encoded proteins maintains the salient biological features as ICP34.5.
  • An exemplary chimeric 7,34.5 gene comprises the DNA encoding the amino terminus of the ICP34.5 protein including one or more reiterations of the AlaThrPro triplet fused to the carboxyl terminus of a gene selected from the group consisting of DNA encoding the carboxyl terminus of MyD 116 or G ADD genes .
  • modified 7,34.5 genes in which the amino terminus is modified.
  • chimeric genes While maintaining an active carboxyl terminus, chimeric genes having an amino terminus derived from a different gene fused to the active carboxyl terminus of ICP34.5. Such chimeric 7,34.5 genes retain the ability to prevent the premature shut off of protein synthesis in cells undergoing apoptotic stress.
  • An exemplary chimeric gene comprises DNA encoding amino acids 1-205 of the 7,34.5 gene fused in 5 ' orientation to a DNA encoding amino acids 524- 657 of the MyD116 gene.
  • the invention also provides a method wherein ICP34.5 or a modified ICP34.5 is combined with a pharmaceutically acceptable carrier in order to form a pharmaceutical composition.
  • a pharmaceutical composition would then be administered to neurons susceptibly undergoing programmed cell death.
  • Such a composition would then be administered to neurons susceptible to undergo or undergoing programmed cell death.
  • Such a composition could be administered to an animal using intravenous or intraspinal injection; in certain circumstances, oral, intracerebral or intraventricular administration may be appropriate.
  • neuronal cells in culture could also benefit from administration of ICP34.5 directly into the medium in which the neuronal cells are grown.
  • ICP34.5 can be prepared using a nucleic acid segment which is capable of encoding ICP34.5 (i.e.
  • the 7,34.5 gene or a modified 7,34.5 gene could be expressed using, for example, a technique involving transferring the 7,34.5 segment into a host cell, culturing the host cell under conditions suitable for expression of the segment, allowing expression to occur, and thereafter isolating and purifying the protein using well established protein purification techniques.
  • the nucleic acid segment would be transferred into host cells by transfection or by transformation of a recombinant vector into the host cell.
  • a particularly important embodiment of the invenuon relates to assays for candidate substances which can either mimic the effects of the 7,34.5 gene, or mimic the effects of ICP34.5, as well as assays for candidate substances able to potentiate the function of 7,34.5 or potentiate the protective function of ICP34.5. Additionally, methods for assaying for candidate substances able to inhibit either 7,34.5 expression or the activity of ICP34.5 are also embodiments of the invention.
  • an assay testing for candidate substances that would block the expression of the anti-apoptosis gene or inhibit the activity of an anti-apoptotic protein such as ICP34.5 would proceed as follows.
  • a test plasmid construct bearing the a sequence promoter and portions of the coding sequence of 7,34.5 (or modifications thereof according to the discussion set out above) is fused to the lacZ reporter gene, or any other readily assayable reporter gene.
  • This construct is then introduced into an appropriate cell line, for example a neuroblastoma or PC12 cell line, by G418 selection, for example.
  • a clonal continuous cell line for screening pu ⁇ oses is then established.
  • a control plasmid construct bearing an HSV late promoter (a promoter which would normally not be expressed in cell lines and not induced to express by a stress factor which would normally induce apoptosis) is fused to the same or different indicator gene.
  • This construct is also introduced into a continuous clonal cell line and serves as a control for the test cell line.
  • the candidate anti-apoptosis drugs would then be applied.
  • Environmental stresses that typically trigger a sequence promoter activation and cause programmed cell death, such as UV injury, viral infection or deprivation of nerve growth factor, would then be applied to the cells. In control cells, the stress should have no effect on the cells and produce no detectable reaction in the assay.
  • the invention provides an assay for candidate substances that would mimic or potentiate the activity of ICP34.5, or which would mimic the expression of 7,34.5, and such an assay would proceed as described above.
  • a test cell line e.g. , a neuroblastoma cell line
  • a tagged cellular gene e.g. with a fluorescent tag
  • a corresponding null cell line consisting of an appropriate indicator gene, for example the a-lacZ indicator gene, and the same host indicator gene as in the test cell line is also produced.
  • a third cell line (e.g., a Vero cell line) consisting of the same indicator gene and the identical host indicator gene is also produced.
  • a third cell line e.g., a Vero cell line
  • environmental stresses that trigger programmed cell death in the absence of 7,34.5 are applied to the cells.
  • Candidate substances are also applied in order to determine whether they are able to mimic or potentiate the anti-apoptotic effects of 7,34.5 expression or the anti-apoptotic activity of ICP34.5 or biological functional equivalents thereof.
  • the invention also provides a method of delivering genes for gene therapy.
  • the method involves combining the gene used for gene therapy (e.g., 7,34.5 or modified 7,34.5) with a mutated virus such as those described above, or with the HSV-1 virus rendered non-pathogenic, thereby producing a gene therapy vector.
  • the gene and the virus are then combined with a pharmacologically acceptable carrier in order to form a pharmaceutical composition.
  • the gene therapy vector in the pharmaceutical composition is then administered in such a way that the mutated virus containing the gene for therapy, or the HSV-1 wild type virus or non-pathogenic mutant virus containing the gene, can be incorporated into cells at an appropriate area.
  • the composition when using the HSV-1 viruses, the composition could be administered in an area where synaptic terminals are located so that the virus can be taken up into the terminals and transported in a retrograde manner up the axon into the axonal cell bodies via retrograde axonal transport.
  • axonal transport Clearly, such a method would only be appropriate when cells in the peripheral or central nervous system were the target of the gene therapy.
  • Figure IA shows a schematic representation of the genome structure and sequence arrangements of the HSV-1(F) ⁇ 305.
  • Figure IB shows a schematic representation of the genome structure and sequence arrangements of R3616, R8300, and R8301.
  • Figure 2 depicts autoradiographic images of electrophoretically separated restriction endonuclease digests of the recombinant virus DNAs hybridized to labeled BamHI Q fragment.
  • Figure 3 depicts immunoblots of electrically separated lysates of infected Vero cells probed with either a GST-MyD116 antiserum or BR4 antiserum.
  • Figure 4 is a photograph of autoradiographic images of electrophoretically separated, [ 35 S]methionine-labeled proteins from lysates of cells infected with HSV-1(F) ⁇ 305 containing a wild type 7,34.5 gene at its natural location or viruses containing mutated 7,34.5 genes.
  • Figure 5 depicts immunoblots of electrophoretically separated lysates of infected SK-N-SH cells or human foreskin fibroblasts (HFF) reacted with BR4 antiserum.
  • Figure 6 depicts immunoblots of electrophoretically separated nuclear and cytoplasmic fractions of HEp-2 cells infected with 5 PFU of HSV- 1(F) ⁇ 305, R8300 or R8301 per cell.
  • CNS degenerative diseases including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), and others, the etiology of which may be traceable to a form of apoptosis, and the treatment of which is currently very poor, could be improved significantly through the use of either the 7,34.5 gene (or modified 7,34.5 genes) in gene therapy or the protein expressed by 7,34.5 as a therapeutic agent.
  • This is especially critical where the death of neuronal cells is involved, due to the fact that, as noted, such cells do not reproduce post-mitotically. Since a finite number of neurons are available it is crucial to have available methods and agents for their protection and maintenance.
  • 7,34.5 and modified 7,34.5 genes are also very useful gene for assays of substances which mimic the effect of 7,34.5 and block stress of biologically induced programmed cell death.
  • an HSV virus or other viruses can serve as a vehicle for delivery of gene therapy to neurons undergoing or about to undergo apoptosis.
  • the HSV-1 virus is present in neurons of the sensory ganglia of 90% of the world's human population. The virus ascends into neuronal cell bodies via retrograde axonal transport, reaching the axon from the site of infection by the process of neurotropism. Once in the neuronal cell body the virus remains dormant until some form of stress induces viral replication (e.g. UV exposure, infection by a second virus, surgery or axotomy).
  • HSV- 1 as a vector necessitates construction of deletion mutants to serve as safe, non-pathogenic vectors.
  • Such a virus would act as an excellent vector for neuronal gene therapy and its use would be an especially important development since few methods of gene therapy provide a means for delivery of a gene across the central nervous system's blood-brain barrier.
  • viruses such as HSV-2, picornavirus, coronavirus, eunyavirus, togavirus, rhabdovirus, retrovirus or vaccinia virus
  • HSV-2 picornavirus
  • coronavirus eunyavirus
  • togavirus eunyavirus
  • rhabdovirus retrovirus or vaccinia virus
  • vectors for 7,34.5 gene or modified 7,34.5 genes to the CNS which avoids the blood brain barrier.
  • Other vectors comprising expressible 7,34.5 genes such as plasmids may also be used for the introduction of the 7,34.5 gene to cells susceptible to apoptosis.
  • the vectors may be administered using, for example, calcium phosphate precipitation, electroporation, cationic liposomes or other methods capable of delivery of an expressible gene to a host cell. (See, e.g. Feigner et al , Proc. Nat.
  • the present invention is useful for screening for agents which induce or inhibit apoptosis.
  • the Vero and human neuroblastoma SK-N-SH cell lines used were obtained from the American Type Culture Collection (ATCC).
  • the human 143TK " cells are also available from the ATCC.
  • the human foreskin fibroblast (HFF) cell strain used was obtained from Aviron, Burlingame, CA.
  • the media for propagation of cells consisted of Dulbecco's modified Eagle's medium supplemented with 10% (SK-N-SH cells) or 5 % (Vero cells), fetal bovine serum, or 5% fetal bovine serum and 100 ⁇ g of BUdR/ml (143TK- cells).
  • HSV- 1(F) was the prototype HSV-1 strain used in these studies
  • HSV-1(F) ⁇ 305 lacks the 501-bp Bglll-SacI fragment from the domain of the thymidine kinase (tk) gene contained in BamHI Q fragment (Post et al. , Cell 25:227-232 [1981]).
  • R3617, derived from HSV-1(F) ⁇ 305 lacks the 501 bp fragment from the domain of the tk gene and a 1 Kb stretch from the coding sequences of both copies of the 7,34.5 gene. The deleted sequences from tk gene in R3617 were restored in the recombinant R3616 (Chou et al , Science 250: 1262-1266 [1990] incorporated herein by reference).
  • Recombinants R8300 and R8301 were constructed by cotransfection of the intact viral DNA of R3616 with either plasmid pRB4871 or pRB4872 on rabbit skin cells.
  • the plasmids pRB4871 and pRB4872 were deposited under the Budapest Treaty with the American Type Culture Collection, 12301 Parkiawn Drive, Rockville, Maryland 20852 on September 1 , 1995 and were given ATCC accession numbers 69895 and 69896, respectively.
  • the virus R3616 (ATCC Accession No. VR2280) was also described in U.S. Patent No. 5,328,688 issued July 12, 1995, the disclosure of which is incorporated herein by reference, tk.
  • recombinants were selected on 143TK- cells in medium consisting of mixture 199 supplemented with 100 ⁇ g of bromodeoxyuridine per ml and 2% fetal calf serum as described previously (Post et al , Cell 25:227-232 [1981]).
  • Viral DNAs were extracted from cells and prepared as previously described (Tongon et al. , Microbiologic 6: 191-198 [1983]). Viral stocks and titrations of infectivity were done in Vero cells. The 2.3 Kbp EcoRI fragment containing the full length
  • MyDl 16 cDNA (Lord et al. , Nucleic Acids Res. 18:2823 [1990]) was cloned into the EcoRI site of pBluescript SK (Source).
  • Plasmid pRB4867 contained the BamHI Q fragment of HSV- 1(F) cloned into the BamHI site of pUC9.
  • Plasmid pRB3207 contained approximately 100 bp of the 7,34.5 5 ' non- coding sequence, the coding sequence of the 7,34.5 gene, and the domain of the ⁇ O gene contained in BamHI S fragment.
  • pRB4871 To construct pRB4871, pRB3207 was cleaved with Drain and BspEI and the 0.25 Kbp DNA fragment containing the coding sequences of the carboxyl terminal 58 codons of the 7,34.5 gene was replaced with the double stranded DNA oligomer linker (GTGCTGCAGGAATTCT [SEQ ID NO: 1] and its complement, CCGGAGAATTCCTGCAGCACCAG [SEQ ID NO: 2]) to yield plasmid pRB4868.
  • GTGCTGCAGGAATTCT [SEQ ID NO: 1] and its complement, CCGGAGAATTCCTGCAGCACCAG [SEQ ID NO: 2] A 474 bp Pstl-EcoRI fragment from murine MyDl 16 cDNA was inserted into the PstI and EcoRI sites of pRB4868, the resulting plasmid was designated pRB4869.
  • Plasmid pRB4870 was constructed by cloning a 887 bp Hindlll- Xbal fragment containing the U L 26.5 promoter from pRB4090 (Liu et al , J. Virol. 65:5149-5156 [1991]) into the Hindlll and Xbal sites of pRB4869. Lastly, a 2.7 Kbp SphI fragment containing the U L 26.5 promoter, the coding sequences of 7,34.5 gene for the amino terminal portion of 205 amino acid residues and the coding sequences of MyDl 16 for the carboxyl terminal portion of 133 amino acid residues was cloned into the SphI site of the tk gene in plasmid pRB4867. The resulting plasmid was designated pRB4871. In this plasmid, the expression of the 7,34.5-MyD116 chimeric gene was driven by the U L 26.5 promoter.
  • pRB4871 was cleaved with Drain and StuI to remove a 0.8 Kbp DNA fragment containing the coding sequences of MyDl 16 gene.
  • the oligomer (GTGTGACTAGTCTAGAGG [SEQ ID NO: 3] and its complement, CCTCTAGACTAGTCACACCAG [SEQ ID NO: 4]) containing stop codons was ligated into the Dralll and StuI sites.
  • the 7,34.5 gene driven by the U L 26.5 promoter lacks the coding sequences for the carboxyl-terminal 58 amino acids.
  • the two covalently linked components of HSV-1 DNA, L and S each consist of unique sequences U L and U s , respectively, flanked by inverted repeats (Sheldrick et al . Cold Spring Harbor Symp. Quant. Biol. 39:667-678 [1975]; Wadsworth et al , J. Virol. 15: 1487-1497 [1975]).
  • the reiterated sequences flanking U L designated as ab and b 'a ' are each 9 Kbp in size, whereas the repeats flanking U s designated a 'c ' and ca are 6.3 Kbp in size (Wadsworth et al , J. Virol 15: 1487-1497 [1975]).
  • TK ' identifies the position of thymidine kinase (tj ) gene and of the Bglll-SacI fragment deleted from HSV-1(F) ⁇ 305 (Post et al. , Cell 25:227-232 [1981]).
  • the location of 7,34.5 gene is shown in the expanded portions of the inverted repeat sequences b_ and b .
  • the solid bar and the arrow indicate the coding region and the direction of transcription of ⁇ ,34.5. Since the b sequence is repeated in an inverted orientation, there are two copies of 7,34.5 gene per genome.
  • Figure B shows a schematic representation of the sequence arrangements of the HSV-1 R3616 mutants and of the recombinant viruses derived from it.
  • Figure IB line 2 shows: An enlarged portion of R3616 genome showing the BamHI Q fragment of HSV-1 containing tk gene.
  • the hatched bar and arrow represent the coding region of tk gene and the direction of transcription, respectively.
  • Line 3 and 4 of Figure IB show the sequence arrangements of the recombinant viruses R8300 and R8301 which were selected from among the progeny of transfection of intact R3616 DNA with either plasmid pRB4871 or pRB4872 as described above.
  • the solid bars indicate the amino terminal portion of 7 j 34.5 gene encoding amino acids 1 to 205, whereas the hatched bar indicates the carboxyl terminus of MyDl 16 gene encoding amino acids 524 to 657.
  • the thin line on the left side (5 ') of the 7,34.5 gene represents the promoter sequences from U L 26.5 of HSV- 1(F).
  • the arrow indicates the site of transcription initiation and the direction of transcription.
  • the thin line on the right side of MyDl 16 or 7,34.5 gene indicates the DNA sequences downstream of 7,34.5 coding region of BamHI S fragment. Restriction sites shown in Figure IB are indicated as follows: B, BamHI; Bg, Bglll; S, SphI; D, Drain.
  • rabbit skin cells were cotransfected with intact R3616 viral DNA and plasmid pRB4871 containing the tk gene into which was inserted the coding sequence of the chimeric gene 7,34.5 -MyDl 16 driven by the U L 26.5 promoter.
  • the progeny viruses of the transfection was plated on 143TK- cells overlaid with medium containing BUdR as described above.
  • the tk ⁇ progeny viruses were plaque purified and their DNAs were analyzed for the presence of the chimeric gene as follows: Viral DNAs were digested with restriction endonucleases, electrophoretically separated on 0.8% agarose gels, transferred to a nitrocellulose sheet, hybridized to 32 P-labeled BamHI Q DNA fragment, and exposed to Kodak X-ray film. (See, Ackermann et al , J. Virol. 58:843-850 [1986]; Chou et al , Science 250: 1262-1266 [1990]; Chou et al , Proc. Natl. Acad. Sci.
  • Lanes 1 and 3 show, BamHI-Dralll digests of DNAs of recombinant viruses R8301 and R8300, respectively; lanes 2, 4 and 5 show, BamHI-Bglll digests of DNAs from recombinant viruses R8301, R8300 and R3616 respectively; lane 6 shows a, BamHI digest of R3616 DNA.
  • the lower case letters identify the DNA sequences expected to be present in each band as predicted in Figure 1.
  • Recombinant virus R8301 contained the codons 1 through 205 of the 7,34.5 gene linked to the U L 26.5 promoter.
  • the BamHI-Dralll digest of R8301 yielded (lane 1), a 2.7 Kbp band (yt) from a portion of the BamHI Q fragment (y) and a portion of BamHI S fragment (t), and a 1.37 Kbp band (mnp) containing a portion of the BamHI Q fragment (m plus n) and a portion of the U L 26.5 promoter sequence (p).
  • the BamHI-Bglll digest of R8301 yielded (lane 2) a 3.4 Kb band (yqt) consisting of a portion of BamHI Q fragment (y) and a portion of the BamHI S fragment (q plus t), a 0.7 Kbp band (m) and a 0.4 Kbp (n) band, both derived from BamHI Q.
  • Recombinant virus R8300 contained the chimeric 7,34.5- MyD116 gene consisting of codons 1 through 205 of 7,34.5 gene fused in frame to the codons 524 to 657 of the MyDl 16 gene and driven by the U L 26.5 promoter. This gene was inserted into the SphI site of the tk gene.
  • the BamHI-Dralll digest of R8300 yielded (lane 3), a 3.4 Kbp band (yr) consisting of a portion of the BamHI Q fragment (y), a portion of the BamHI S fragment and of the MyDl 16 coding sequences (r), a 1.37 Kbp (mnp) described above.
  • the BamHI-Bglll digest of R8300 yielded (lane 4) a 4.2 Kbp band (yqr) consisting a portion of the BamHI Q fragment (y) and a portion of the BamHI S fragment (q plus r), a 0.8 Kbp band (m) and a 0.44 Kbp band (n), both derived from the BamHI Q fragment.
  • the R3616 DNA digest served as a control for the purity of the recombinant viruses constructed from it and as a marker.
  • the BamHI-Bglll digest of R3616 (lane 5) yielded a 2.8 Kbp band (ny) and a 0.8 Kbp band (m), whereas the BamHI digest (lane 6) yielded an intact BamHI Q fragment (mny).
  • the BamHI-Bglll digest ( Figure 2, lane 4) revealed the presence of the predicted fragment gr . (4.2 Kbp) consisting of a portion of BamHI Q, MyDl 16, and 7,34.5, and the fragments m (0.8 Kbp) and n (0.44 Kbp) derived from BamHI Q.
  • the parent virus with an intact BamHI Q fragment yielded a single 3.6 Kbp band on digestion with BamHI ( Figure 2 lane 6) and the predicted two 2.8 Kbp and 0.8 Kbp bands on digestion with BamHI and Bglll ( Figure 2 lane 5)
  • recombinant virus R8301 (FigJB, line 4) rabbit skin cells were co-transfected with intact R3616 viral DNA and the plasmid pRB4872. This plasmid contains the amino terminal 205 codons of 7,34.5 [7,34.5( ⁇ C) gene] linked to the U L 26.5 promoter.
  • the recombinant virus (R8301) obtained and analyzed as described above was found to be as predicted in Figure IB, lines 2 and 4).
  • Rabbit polyclonal antibody BR4 to the 10 repeats of AlaThrPro triplet in HSV-1(F) 7,34.5 protein was described by Ackermann et al , J. Virol 58:843-850 (1986).
  • Rabbit polyclonal antibody to MyDl 16 was prepared as follows. An AccI-EcoRI fragment encoding the carboxyl- terminal 174 amino acids of the MyDl 16 cDNA was inserted into the vector pGEX3X (Pharmacia, Uppsala, Sweden and Piscataway, New Jersey) cleaved with BamHI and EcoRI and the BamHI site was made blunt-ended with Klenow fragment before ligation. The resulting plasmid pRB4873 was predicted to encode E.
  • GST glutathione synthetase fused in frame with the carboxyl terminal portion of MyD 116.
  • Thejunction of GST and MyD 116 was sequenced to verify that the fusion was in frame.
  • E. coli BL21 cells transformed with pRB4873 were induced to produce the fusion protein by induction with isopropyl-3-D-thio- galactopyranoside (IPTC), followed by affinity purification of the fusion protein from bacterial lysates on agarose beads conjugated with glutathione.
  • IPTC isopropyl-3-D-thio- galactopyranoside
  • the fusion protein was used for immunization of rabbits for production of polyclonal antibody.
  • Vero cells were infected with 5.0 plaque forming units of R8301 , R8300, as HSV-l(F) ⁇ 305 and the proteins produced were analyzed on immunoblots as follows. At 15 hrs. after infection, viral infected cells were rinsed, harvested, solubilized, denatured by boiling and the lysates were subjected to electrophoresis in 12% sodium dodecyl sulfate-polyacrylamide gels, electrically transferred to nitrocellulose, blocked with 5% non-fat milk, reacted with either the GST-MyD116 (Anti MyDl 16 Ab) antiserum or BR4 antibody (Anti-7,34.5 Ab) both described above, rinsed, and reacted with anti-rabbit immunoglobulin coupled to alkaline phosphatase as recommended by the manufacturer (Bio-Rad, Hercules, California) and the results are shown in Figure 3A and B.
  • the positions of the bands containing the chimeric protein 7,34.5 (DC) MyDl 16, the truncated protein 7,34.5 (AC), and wild type 7,34.5 are shown.
  • the viruses used in this study were all tk ⁇ in as much as the chimeric 7,34.5( ⁇ C)-MyD116, and the truncated 7,34.5( ⁇ C) genes were inserted into the viral tk gene disrupting it.
  • the BR4 antibody reacted with a relatively abundant band containing the 7,34.5( ⁇ C)-MyD116 protein with an apparent M r of 52,000 in the lane containing the lysates of R8300, the 7,34.5 protein in the lane containing the lysate of cells infected with HSV-1(F) ⁇ 305, and with a fast migrating band with an apparent IVL of 36,000 expected to contain the amino terminal domain of the 7,34.5 [7,34.5 ⁇ C)] gene in lysates of cells infected with R8301 ( Figure 3B).
  • the serum did not react with the lysate of cells mock infected or infected with the R3617 virus.
  • R8300 and R8301 contain and express the 7,34.5 gene derivatives described above. It is noteworthy that the lanes shown in this and other figures in these studies contain identical amounts of protein. There appears to be an overload of the chimeric 7,34.5( ⁇ C)-MyDl 16 protein, lesser amounts of the truncated protein and relatively small amounts of 7,34.5 protein.
  • the 7,34.5 protein was made from mRNA transcribed from its natural promoter. The U L 26.5 promoter utilized in this and other studies appeared to be a much stronger promoter than that of the natural 7,34.5 gene.
  • HFF FIBROBLASTS
  • the antibody reacted with bands corresponding to full length and truncated 7,34.5( ⁇ C) protein in human foreskin fibroblasts and with a single band of predicted full length in lysates of SK-N-SH cells.
  • the chimeric 7,34.5( ⁇ C)-MyD116 protein formed multiple bands including one of expected size in both cell lines infected with R8300 recombinant virus.
  • the cytoplasmic and nuclear fractions were solubilized in disruption buffer (50mM Tris-HCl pH7.2, 2%SDS, 2.75% sucrose, and 5% 3-mercatoethanol and subjected to electrophoresis in denaturing gels as previously described (Ackermann et al , J. Virol 58:843- 850 [1986]; Chou et al , Science 250: 1262-1266 [1990]; Chou et al , Proc. Natl Acad. Sci. USA 89:3266-3270 [1992]; Chou et al , Proc. Natl. Acad. Sci. USA 91:5247-5251 [1994]; Chou et al , J.
  • the HSV "borrowed" a portion of the MyD116/GADD34 genes to prevent a similar cell response, but why not the whole gene. It may be that the amino terminal of the MyD116/GADD34 proteins perform a different function than the amino terminal domain of 7,34.5 protein. Another plausible possibility is that the 7,34.5 amino terminal domain brings together in one complex the proteins interactive with the carboxyl terminus and other viral proteins needed to preclude the shutoff of protein synthesis. Experimental results suggest that the premature shutoff of protein synthesis is not overcome by inserting only the carboxyl terminus of MyDl 16 into the viral genome. These results, however, were not readily inte ⁇ retable since the amounts of the MyDl 16 carboxyl terminus peptide were very small and the protein may have been also very unstable.
  • mutated HSV-1 virus is proposed as a vector for introduction of the gene into neuronal cells undergoing or about to undergo programmed cell death.
  • this embodiment of the invention could be practiced using alternative viral vectors, including retroviral vectors and vaccinia viruses and other viruses whose genome has been manipulated in alternative ways so as to render the virus non-pathogenic.
  • the methods for creating such a viral mutation are set forth in detail in U.S. Patent No. 4,769,331 , the disclosure of which is incorporated herein by reference.
  • this embodiment of the present invention could be practiced using any gene whose expression is beneficial in gene therapy, and use of the non-HSV viruses would allow gene therapy in non-neural systems.
  • He ⁇ es simplex virus has a natural tropism for human CNS tissue. Under wild type conditions, the virus is capable of replicating and multiplying in the nervous system and is neurovirulent. The virus can also establish latent infection in the neurons and can be occasionally reactivated.
  • an HSV vector To establish a vector system for delivery of genes into neurons, the proposed construct of an HSV vector must satisfy the following criteria; (1) such a vector should have a natural tropism for CNS and brain tissue; (2) such a vector should be non-pathogenic; that is, totally avirulent and not reactivatable to cause an infection; (3) such a vector should consist of constitutive expression of 7,34.5 (or a modified 7,34.5 in accordance with the description set out above) to prevent cell death in cells undergoing neurodegeneration; and (4) such a vector so proposed above is suitable for additional foreign gene insertion for gene therapy.
  • a modified viral HSV vector with a mutational lesion in the a4 gene is constructed.
  • the viral vector virus will no longer be able to replicate, multiply and reactivate from latent infection in the CNS.
  • the virus can, in the absence of ⁇ 4 gene, establish a latent infection in the neuron.
  • This virus is obtained by co-transfection of viral DNA with plasmid containing a ⁇ 4 gene from which a significant portion had been deleted, into a cell line expressing ICP4, the protein product of the ⁇ 4 gene.
  • ⁇ 4 expressing cell lines and the virus have been reported previously. DeLuca et al , J. Virol 56:558-570 (1985).
  • the plasmid used for co-transfection with the virus may also comprise a ⁇ 4 gene having insertions or mutations that render it incapable of expressing an active ICP4. Additionally, such an HSV vector with 7,34.5 gene under a constitutive expression promoter is also envisioned.
  • This constitutive expression promoter can be the HSV LAT promoter, the LTR promoter of retroviruses, an adenosine deaminase promoter or any other foreign promoter specific for neuronal gene expression.
  • Such a viral vector properly introduced is suitable for prevention of cell death in neuronal cells undergoing apoptosis.
  • HSV vector with foreign genes inserted at a neutral location on the viral genome is suitable for delivery of foreign genes into target neurons and for CNS gene therapy.
  • the procedures to generate the above recombinant viruses are those published by Post and Roizman (Cell 25:227 [1991]) and U.S. Patent No. 4,769,331 , the disclosure of which is incorporated herein by reference. See also, U.S. Patent Nos. 5,328,688 and 5,288,641, the disclosures of which are incorporated herein by reference.
  • the virus can be combined with a pharmaceutically acceptable carrier such as buffered saline and injected at the site of peripheral nerve endings whose axons originate from neural cell bodies undergoing or about to undergo apoptosis.
  • a pharmaceutically acceptable carrier such as buffered saline
  • the amount of virus will vary depending upon several factors, including the vector's ability to target the cells requiring treatment, the extent to which the gene is expressed int he target tissue, and the activity of the expressed protein, among others.
  • An inoculum containing approximately IO 4 - 10 5 viruses in phosphate buffered saline or skim milk has produced successful results in mice.
  • Virus so injected is taken up into the peripheral nerve endings and is then transported via retrograde axonal transport to the neuronal cell bodies.
  • peripheral injection is not useful or appropriate, localized intraspinal or intraventricular injection, or direct microinjection of the virus could be utilized.
  • 7,34.5 or a modified 7,34.5 gene, i.e. 7,34.5/MyD116 gene alone or in combination with other genes of interest into the cells and engraft.
  • a modified 7,34.5 gene i.e. 7,34.5/MyD116
  • Such a procedure allows the delivery of the genes to its natural site.
  • Proper expression of the 7,34.5 gene in these neurons should result in prevention of cell death in neurodegeneration and preserving cells carrying foreign genes suitable for gene therapy.
  • Cerebellar cell lines are generated as described by Ryder et al. , J. Neurobiol 21:356-375 (1990). Lines are grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Gibco), 5 % horse serum (Gibco), and 2mM giutamine on poly-L-lysine (PLL) (Sigma) (10 ⁇ gml)-coated tissue culture dishes (Corning). The lines are maintained in a standard humidified, 37°C, 5 % CO 2 -air incubator and are either fed weekly with one-half conditioned medium from confluent cultures and one-half fresh medium or split (1 : 10 or 1 :20) weekly or semiweekly into fresh medium. Transduction of cerebellar progenitor lines with 7,34.5 gene
  • a representative dish from each subclone was stained directly in the culture dish using X-gal histochemistry (See, Snyder et al , 1992). The percentage of blue cells expressing was determined under the microscope. Subclones with the highest percentage of blue cells (ideally > 90% ; at least > 50%) were maintained, characterized, and used for transplantation. Tests for Virus Transmission The presence of helper virus is assayed by measurement of reverse transcriptase activity in supernatants of cells lines as described by Goff et al. (1981) and by testing the ability of supernatants to infect NIH 3T3 cells and generate G418-resistant colonies of X-gal + colonies (detailed in Cepko, 1989a, 1989b). All cerebellar cell lines used for transplantation are helper virus- free as judged by these methods.
  • Newborn CD-I or CF-1 mice were cryoanesthetized, and the cerebellum is localized by transillumination of the head.
  • Cells are administered either via a Hamilton 10 ⁇ l syringe with a beveled 33-gauge needle or a drawn glass micropipette with a 0.75 mm inner diameter and 1.0 mm outer diameter generated from borosilicate capillary tubing (FHC,
  • the injection of BAG virus was performed as described for the cell suspension.
  • the BAG virus stock (8 x IO 7 G418-resistant cfu/ml) contained, in addition to trypan blue, and polybrene at 8 ⁇ g/ml.
  • neuronal cells undergoing or susceptible to programmed cell death can also be treated with the protein expressed by the 7,34.5 gene, i.e. ICP34.5.
  • ICP34.5 may be modified according to the description set out above.
  • ICP34.5 is isolated from cells expressing the protein and purified using conventional chromatography purification and immunoaffinity purification methods described by Ackerman et al. , J. Virol. 58:843-850 (1986), the disclosure of which is incorporated herein by reference.
  • the antibodies described in Example 3 may be used for isolation and purification.
  • Antibodies directed to other chimeric or modified ICP34.5 proteins may also be prepared as described in Example 3.
  • the purified protein is next combined with a pharmaceutically appropriate carrier, such as buffered saline or purified distilled water.
  • the proteins may also be modified by, for example, pegylation or by the covalent attachment of other molecules which may aid in solubility, bioavailability, stability, and/or efficacy.
  • the pharmaceutical composition can be injected in one of several ways, as appropriate: (i) intraspinal injection; (ii) intraventricular injection; (iii) direct injection into the area containing the neurons undergoing or about to undergo programmed cell death or any other appropriate method of administration understood by those skill in the art.
  • Such treatment would be particularly appropriate in the surgical repair of severed peripheral nerves, and the use of proteins ad therapeutic gents is well within the current level of skill in the medical arts in light of the present specification.
  • the 7,34.5 gene of he ⁇ es simplex virus enable the virus to replicate, multiply and spread into the central nervous system and the brain so that the virus is neurovirulent to the host.
  • Recombinant virus lacking the gene lost this ability to penetrate the CNS of the host and become totally avirulent.
  • the mutant virus lacking the gene exhibited a total translation shutoff phenotype characteristic of programmed cell death.
  • This mechanism of programmed cell death afforded by the host cell greatly reduced the ability of the virus to multiply and spread.
  • the function of 7,34.5 in the virus therefore is to inactivate the programmed death of the cell (anti-apoptosis) thereby restoring translation and enabling the virus to replicate to full potential in the host.
  • This anti-apoptotic effect of 7,34.5 was further reexamined and its ability to protect neural cells from other environmental stresses which lead to apoptosis was discovered. These environmental stresses include UV, nerve grown factor deprivation and neuronal cell differentiation.
  • This Example describes the use of the 7,34.5 gene (or modified genes) and its protective function to screen for pharmaceutical agents and drugs that mimic the in vivo function of 7,34.5 to prevent neurodegeneration.
  • Such a screening procedure constitutes construction of cell lines expressing 7,34.5 and a null cell line without the gene and measurement of the viability of the cells after stress treatment by induction of a reporter gene. This can be a host gene promoter tagged by a fluorescence indicator or any other easily assayable marker to signal viability.
  • a test neuroblastoma cell line is established constituitively expressing 7,34.5 and containing a fluorescence tag (e.g. , the a sequence promoter fused to lacZ) cellular gene, or any tag that provides the easily assayable marker to signal viability.
  • a neuroblastoma null cell line consisting of a-lacZ indicator gene and a second indicible promoter indicator gene is also established, along with a Vero cell line consisting of a-lacZ indicator gene and the same host indicator gene.
  • Other reporter indicator genes include but are not limited to luciferase, (Bronstein et al , Anal. Biochem.
  • the promoter for the gene 7 ,34.5 lies in the a sequence of HSV and, at time of stress, the promoter is activated. It can be assumed that the a sequence promoter contains apoptosis responsive elements and cellular factors (transcription factors in particular) that mediate the expression of anti-apoptosis gene are apoptotic in nature. These cellular factors are therefore the targets of the present assay to screen for drugs or agents that would inactivate their ability to induce the anti- apoptosis gene.
  • the assay involves the use of the a sequence promoter and its inducibility by conditions which induce apoptosis as an indicator assay which screens for therapeutic agents and drugs capable of blocking the expression of the anti-apoptosis gene and therefore allow the cell to die of programmed cell death.
  • a test plasmid construct bearing the a sequence and coding sequence up to the 28th amino acid of 7,34.5 is fused to the lacZ reporter gene or any other readily assayable reporter gene.
  • the construct was introduced into a neuroblastoma or PC 12 cell line by G418 selection and a clonal and continuous cell line for screening purposes is established.
  • a control plasmid construct bearing an HSV late promoter, a promoter which would normally not be expressed in cell lines and which further would best be induced to express by apoptosis-inducing stress is fused to the same indicator gene.
  • This construct is also introduced into a continuous clonal cell line and serves as a control for the test cell line.
  • the assay of the invention allows the screening and identification of pharmaceutically appropriate drugs and agents targeted at various cellular factors that induce the expression of anti-apoptosis gene. By inactivating essential cellular factors, these agents should be able to allow programmed cell death to occur. Such positive candidates would then be appropriately administered (via intravenous, intrathecal, or direct injection, or via oral administration) in order to induce programmed cell death in tumor cells, in neurons infected with the he ⁇ es virus, or in cells infected with a virus the virulence of which is dependent upon an anti-apoptotic effect.
  • test candidate substances are added to the medium in individual wells containing both the test and control cell lines set up in 96 well dishes.
  • the cells are then briefly exposed to UV or other stresses such as those described above. 8 hr. post stress induction, cells are washed with PBS-A twice, and fixed with 0.5 ml containing 2% (v/v) formaldehyde and 0.2% glutaraldehyde in PBS for 5 min. at room temperature.
  • the cells are rinsed again with PBS and then stained with 2 ml 5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide, 2 mM MgCl 2 and 1 mg/ml X-gal (diluted from a 40mg/ml stock solution in dimethyl sulfoxide) in PBS.
  • Cells expressing ⁇ -galactosidase were stained blue after incubation at 37°C for 2-3 days.
  • the construct described above with the lacZ reporter gene was introduced into PC 12 cell line.
  • a new cell line W5 was clonally established by G418 selection.
  • the W5 cell line was then tested for activation of the a sequence promoter under suboptimal conditions names in 3 above.
  • the results are: (a) The above cells, when exposed briefly to UV for two minutes, turn blue upon staining the fixed cells with X-gal at 6-10 hr. post UV exposure; (b) the above cells, when exposed to HSV- 1(F) virus at multiplicity of infection of 5. also turn blue upon staining at 8 hr.
  • the above cells turn light blue when nerve growth factor (rat, 7S) is introduced into the medium to allow differentiation processes; (d) the cells turn darker blue when Nerve Growth Factor is removed from the medium after differentiation is complete and the cells have become dependent on nerve growth factor for survival; (e) little or no difference in color development is seen in cells starved for serum (0% fetal bovine serum) and those fully supplied in 10% fetal bovine serum; and (f) the above experiments are repeated with control promoter fusion elements to control for the true inhibition of anti-apoptosis gene expression rather than toxicity-induced cell death.
  • nerve growth factor rat, 7S
  • the positive candidates that can induce cell death in cells will therefore render the following phenotypes: (i) Introduction of stress to the test cell line in the absence of this substance will give rise to blue colored cells; and (ii) Introduction of stress to the test cell line in the presence of same substance will give rise to white cells, (iii) Introduction of stress to control cell lines with or without this putative substance will have no effect on the color of cells.

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Abstract

Cette invention concerne des procédés de prophylaxie ou de traitement de la mort programmée des cellules, ou apoptose, dans des cellules neuronales en vue d'élaborer une thérapie adaptée aux maladies neurodégénératives; ainsi que des procédés de traitement du cancer et d'autres maladies tumorigènes et de l'infection provoquée par l'herpèsvirus. Cette invention concerne également des méthodologies d'analyse permettant d'identifier des substances capables de moduler les effets du gène η134,5 ou du gène η134.5 modifié ou de leurs produits d'expression protéiques (ICP 34,5) modifiés ou non, c'est-à-dire des substances permettant de potentialiser, d'induire ou d'inhiber leurs effets. En outre cette invention concerne des méthodologies d'analyse prévues pour identifier des substances d'intérêt potentiel capables d'imiter l'expression de η134,5 ou l'activité de ICP 34,5; ainsi que des procédés d'apport de gènes à des cellules pour effectuer une thérapie génique.
PCT/US1996/014147 1995-09-11 1996-08-29 Matieres et procedes de traitement de maladies neurodegeneratives et de recherche systematique d'inhibiteurs et d'inducteurs potentiels de l'apoptose WO1997010349A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
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WO1998041873A1 (fr) * 1997-03-14 1998-09-24 The University Court Of The University Of Glasgow Procede permettant d'identifier des regulateurs de cycle cellulaire
WO1998055863A1 (fr) * 1997-06-05 1998-12-10 Idun Pharmaceuticals, Inc. Methodes rapides d'identification des modificateurs de l'activite apoptotique cellulaire
WO1998046637A3 (fr) * 1997-04-16 1999-01-28 Arch Dev Corp Us3 et icp4 du virus de l'herpes simplex en tant qu'inhibiteurs de l'apoptose
WO1999010741A3 (fr) * 1997-08-25 1999-05-27 Univ Mcgill Procedes et reactifs permettant d'identifier les modulateurs de l'apoptose neuronale
WO1999010998A3 (fr) * 1997-08-25 1999-09-02 Univ Mcgill Utilisation de shp-1 et shp-2 pour deceler des composes impliques dans la survie neuronale
WO2000044894A1 (fr) * 1999-01-29 2000-08-03 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede pour l'isolation de sequences adn induisant l'apoptose et systeme de detection
US6248320B1 (en) 1996-07-26 2001-06-19 University College London HSV strain lacking functional ICP27 and ICP34.5 genes
US6719982B1 (en) 1998-01-29 2004-04-13 Biovex Limited Mutant herpes simplex viruses and uses thereof

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6248320B1 (en) 1996-07-26 2001-06-19 University College London HSV strain lacking functional ICP27 and ICP34.5 genes
US6641995B2 (en) 1997-03-14 2003-11-04 The University Court Of The University Of Glasgow Methods for identifying cell cycle regulators
WO1998041873A1 (fr) * 1997-03-14 1998-09-24 The University Court Of The University Of Glasgow Procede permettant d'identifier des regulateurs de cycle cellulaire
US6235467B1 (en) 1997-03-14 2001-05-22 Medical Research Council Methods for identifying cell cycle regulators
US6218103B1 (en) 1997-04-16 2001-04-17 Arch Development Corporation Herpes simplex virus US3 and ICP4 as inhibitors of apoptosis
WO1998046637A3 (fr) * 1997-04-16 1999-01-28 Arch Dev Corp Us3 et icp4 du virus de l'herpes simplex en tant qu'inhibiteurs de l'apoptose
US6251614B1 (en) 1997-06-05 2001-06-26 Idun Pharmaceuticals, Inc. Rapid methods for identifying modifiers of cellular apoptosis activity
US6270980B1 (en) 1997-06-05 2001-08-07 Idun Pharmaceuticals, Inc. Rapid methods for identifying modifiers of cellular apoptosis activity
US6518032B1 (en) 1997-06-05 2003-02-11 Idun Pharmaceuticals, Inc. Rapid methods for identifying modifiers of cellular apoptosis activity
WO1998055863A1 (fr) * 1997-06-05 1998-12-10 Idun Pharmaceuticals, Inc. Methodes rapides d'identification des modificateurs de l'activite apoptotique cellulaire
WO1999010998A3 (fr) * 1997-08-25 1999-09-02 Univ Mcgill Utilisation de shp-1 et shp-2 pour deceler des composes impliques dans la survie neuronale
WO1999010741A3 (fr) * 1997-08-25 1999-05-27 Univ Mcgill Procedes et reactifs permettant d'identifier les modulateurs de l'apoptose neuronale
US6719982B1 (en) 1998-01-29 2004-04-13 Biovex Limited Mutant herpes simplex viruses and uses thereof
WO2000044894A1 (fr) * 1999-01-29 2000-08-03 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede pour l'isolation de sequences adn induisant l'apoptose et systeme de detection

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