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WO1992007945A1 - Alteration specifique du type cellulaire des niveaux de produits geniques dans des cellules - Google Patents

Alteration specifique du type cellulaire des niveaux de produits geniques dans des cellules Download PDF

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WO1992007945A1
WO1992007945A1 PCT/US1991/007993 US9107993W WO9207945A1 WO 1992007945 A1 WO1992007945 A1 WO 1992007945A1 US 9107993 W US9107993 W US 9107993W WO 9207945 A1 WO9207945 A1 WO 9207945A1
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virus
construct
cells
vector
cell
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PCT/US1991/007993
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Alfred I. Geller
Matthew J. During
Christine Wilcox
Howard J. Federoff
Arthur B. Pardee
Moses V. Chao
Karen O'malley
Rachael Neve
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Dana Farber Cancer Institute
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Publication of WO1992007945A1 publication Critical patent/WO1992007945A1/fr

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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13041—Tyrosine N-monooxygenase (1.14.13.41)
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    • C12Y114/16—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced pteridine as one donor, and incorporation of one atom of oxygen (1.14.16)
    • C12Y114/16002—Tyrosine 3-monooxygenase (1.14.16.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
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    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • 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

  • HSV-1 herpes simplex virus type 1 vector, pHSVlac, which contains the E. coli LacZ gene under the control of the HSV-1 Immediate Early 4/5 (IE 4/5) promoter, was recently developed
  • the pHSVlac vector has been propagated and packaged into HSV-1 virus particles, using a temperature sensitive mutant of HSV-1, the HSV-1 strain 17 ts K virus (Davison, M. J. et al . , J. Gen. Virol., 65: 859-863, (1984)), following a protocol described by Geller (Geller, A.I. Nucleic Acids Res. 16: 5690 (1988)).
  • HSV-1 Due to the broad host range of HSV-1, the defective HSV-1 vector can be introduced into a wide variety of cells. HSV-1 can also infect postmitotic cells, including neurons in adult animals, and can be maintained indefinitely in a latent state
  • the IE 4/5 promoter present in pHSVlac functions in most cell types, and upon infection of various neural cell lines (Geller et al., Abstr. Soc. Neurosci. 14: no. 254.11 (1988)) and a variety of human cell types (Boothman, D. A. et al., FEBS Lett. 258: 159-162 (1989)), the pHSVlac virus has been shown to direct expression of the
  • LacZ gene product ⁇ -galactosidase
  • the present invention relates to a method of altering the level of a gene product in a neuronal or non-neuronal cell, comprising inserting a
  • nucleotide sequence encoding the desired product into a defective Herpes virus vector such as a defective HSV-1 virus vector, such that the promoter of the vector is able to express a functional gene product upon introduction of the virus vector into the cell.
  • the resulting defective virus vector construct, encoding the gene product of interest, is packaged into virus particles by introducing the construct into a cell line together with a defective herpes virus vector, such as a defective HSV-1 virus vector, such that the promoter of the vector is able to express a functional gene product upon introduction of the virus vector into the cell.
  • the resulting defective virus vector construct, encoding the gene product of interest is packaged into virus particles by introducing the construct into a cell line together with a
  • neurotropic Herpes mutant helper virus such as an
  • Target cells i.e., cells in which expression of the gene product is desired
  • the packaged virus i.e., the packaged virus construct
  • a functional gene product is expressed.
  • a functional gene product which can be an RNA, a protein or a peptide, is one which has an activity of the desired product.
  • the invention relates to a method of packaging an HSV-1 defective virus vector into virus particles using a deletion mutant virus as helper virus.
  • the HSV-1 defective virus vector and an HSV-1 deletion mutant helper virus are introduced into a complementing cell line.
  • the cell line contains Herpes virus sequences and is able to complement the defect of the deletion mutant virus so that the HSV-1 defective virus vector is packaged into virus particles.
  • the invention further relates to a method of altering the level of a gene product in cells to alter a disease state.
  • a defective HSV-1 virus vector, pHSVth capable of expressing human tyrosine hydroxylase is described, and has been shown to increase the level of functional tyrosine hydroxylase in a cell. Production of tyrosine hydroxylase in this manner is useful for altering the level of this enzyme in cells affected by Parkinson's disease.
  • the level of a gene product can be altered in a cell by introducing into the cell a vector in which a sequence encoding the gene product is under the control of a cell type-specific promoter. Expression of the gene product is directed by the promoter in a cell-type-specific manner.
  • the human neuronal cell-specific neurofi lament L promoter can be used in a defective Herpes virus vector for this purpose.
  • the gene product can be targeted to a particular location in the cell (e.g. a
  • targeting sequence such as the amino terminal region of human GAP-43, a growth-associated protein, which is localized to nerve tissue and linked to the synaptosomal membrane.
  • the intracellular targeting sequence is incorporated into the nucleotide sequence encoding the gene product to be expressed from the defective Herpes virus vector in a location consistent with its targeting function.
  • the present invention further relates to a method of altering neurotransmitter metabolism in a cell by altering the level of a gene product, which alters neurotransmitter metabolism, in the manner described.
  • neurotransmitter a gene product that alters neurotransmitter metabolism
  • metabolism e.g., neurotransmitter release
  • non-neuronal e.g., glia, fibroblasts
  • neuronal e.g., striatal neurons, sympathetic neurons
  • the invention also pertains to the particular defective HSV-1 virus vectors useful in the present method.
  • Figure 1 illustrates the structure of the pHSVlac vector, which contains the HSV-1 IE 4/5 promoter (arrow), the intervening sequence following that promoter (triangle), the LacZ gene, SV40 early region polyadenylation site, the HSV-1 ori s (small circle) from the HSV-1 c region, the HSV-1 a
  • FIG. 2 illustrates the structure of HSV-1 DNA in M64A cells and D30EBA deletion helper virus.
  • the top line is a schematic map of the HSV-1 genome.
  • the IE-3 gene is present in two copies in the duplicated c region.
  • the second line diagrams the HSV-1 DNA present in the genome of M64A cells.
  • the fragment is flanked by Xho I (X) and Sma I (S) sites, and contains the IE-3 gene encoding 1298 amino acids.
  • the third line indicates the extent of the deletion (codons 83-1236) in the IE-3 gene in D30EBA virus.
  • the fourth line represents the 659 bp fragment used in Southern analysis of the viral DNA.
  • Figure 3 illustrates (1) calcium phosphate DNA transfection of defective HSV-1 vector DNA into M64A cells (complementing cell line), which contain the IE-3 gene; (2) superinfection of transfected cells with D30EBA helper (H) virus; (3) the IE-3 gene in M64A cells complements the D30EBA virus, resulting in productive infection and release of virus
  • H packaged D30EBA
  • V HSV-1 vector virus
  • the virus stock is used to infect cells in culture, such as neurons or glia, or to infect cells by stereotactic injection into the brain of a rat, for example.
  • Figure 4 illustrates the structure of defective HSV-1 vector pNFLlac. The location of the human neurofilament L (hNFL) promoter is indicated.
  • FIG. 5 illustrates the structure of defective HSV-1 vector pHSVGAPlac.
  • G10 indicates the
  • nucleotide sequence of the GAP-43 intracellular targeting sequence is nucleotide sequence of the GAP-43 intracellular targeting sequence.
  • Figure 6 illustrates the structure of defective HSV-1 vector pHSVth, which carries the human
  • TH tyrosine hydroxylase
  • Figure 7 illustrates the structure of defective HSV-1 vector pHSVcyr, and a schematic map of the yeast adenylate cyclase protein from the amino (N) to the carboxyl terminus (C), indicating the
  • the nucleotide sequence encoding the catalytic portion is present in pHSVcyr.
  • Figure 8 illustrates the structure of the 5'-end of the gpt-trpS-lacZ fusion of pHSVlac.
  • Figure 9 is a diagram illustrating the
  • the vector contains 2 genetic elements, the HSV ori s and pac a sites (HSV pac), that are necessary for packaging into viral particles. It also contains a
  • polyadenylation signal (SV-40 poly A).
  • FIG. 10 is a histogram illustrating the effect of injection of pHSVngf virus particles on tyrosine hydroxylase (TH) activity (pmole
  • SCG superior cervical ganglion
  • control (right SCG control from animals receiving pHSVngf virus); pNFlac injection +
  • Figure 11 is a histogram illustrating the effect of pHSVngf virus infection on the choline acetyl transferase (ChAT) activity of striatal cholinergic neurons. From left to right the bars correspond to: (a) mock infected control; (b) uninfected cells with 100 ng/ml exogenous NGF added; (c) pHSVlac virus infected cells; (d) pHSVngf virus infected striatal cells (5 ⁇ l virus stock); and (e) pHSVngf virus infected striatal cells (10 ⁇ l virus stock).
  • the present invention relates to a method of altering the level of a gene product in a cell, comprising inserting a nucleotide sequence encoding the desired product into a defective Herpes virus vector, such that the vector is able to express a functional gene product upon introduction of the vector into the cell.
  • a defective Herpes simplex type 1 (HSV-1) vector, pHSVlac which contains the E. coli LacZ gene under the control of the HSV-1 Immediate Early 4/5 (IE 4/5) promoter, was recently developed.
  • the construction and structure of the defective HSV-1 vector, pHSVlac is described in detail in U. S. Serial No. 304,619, filed February 1, 1989, and in papers by Geller and Breakefield (Geller, A. I.
  • the HSV-1 vector pHSVlac ( Figure 1) contains the E. coli Lac Z gene under the control of the
  • HSV-1 Immediate Early 4/5 (IE4/5) promoter (The intervening sequence following the IE4/5 promoter is also present.)
  • the SV40 early region polyadenylation site is incorporated 3' to the LacZ gene.
  • the backbone of the vector contains the Col E1 origin of replication and the gene conferring ampicillin resistance for propagation and selection in E. coli.
  • the pHSVlac vector was propagated and packaged into HSV-1 virus particles using a temperature sensitive (ts) mutant of HSV-1, the HSV-1 strain 17 ts K virus (Davison, J. J. et al., J. Gen. Virol. , 65:859-863 (1984)), in a protocol described by Geller (Geller, A. I. , Nucleic Aci d s Res. 16: 5690 (1988)).
  • the ts K mutant virus has a single base change in a gene essential for the HSV-1 lytic cycle, the IE-3 gene. The mutation results in an amino acid substitution in the encoded protein, which renders the virus incapable of undergoing the lytic cycle at 37-39°C.
  • the mutant virus is able to propagate at the permissive
  • the virus can be used as a helper virus to propagate and package defective HSV-1 vectors at the permissive temperature.
  • the virus can be used as a helper virus to propagate and package defective HSV-1 vectors at the permissive temperature.
  • the ts helper virus is unable to sustain a productive lytic infection, and persists without productive infection.
  • the vector is delivered to the cell and persists without a productive cytopathic infection.
  • the HSV-1 strain 17 ts K mutants revert to wild type, and the temperature sensitive phenotype is not absolute at 37 °C, making these viruses unsuitable as helpers for packaging defective HSV-1 vector constructs for gene therapy in human hosts, for example.
  • a deletion mutant virus packaging i.e., propagation and packaging
  • Herpes deletion mutant helper viruses suitable in the present invention will contain a deletion of all or part of a gene essential for productive lytic infection by the virus.
  • a deletion may be the absence of two or more nucleotides that results in an unconditional mutation, such that productive lytic infection of the deletion virus in a cell can result only when all or part of the deleted
  • HSV-1 deletion mutant helper virus with a deletion of all or part of the IE-3 gene product, the major regulatory gene of HSV-1, is preferred. After packaging, introduction of the deletion mutant helper virus into a target or other cell, alone or together with the desired defective Herpes virus vector, will not damage the cell because the deletion mutant viruses essentially do not revert.
  • a complementing cell line is constructed, which complements the defect of the deletion mutant helper virus, allowing
  • a defective Herpes virus vector construct encoding a gene product of interest, can be introduced into a complementing cell line by transfection (Graham, F. L. and Van der Eb, A. J., Virology 52: 456-467 (1973)) or other suitable methods.
  • the Herpes deletion mutant helper virus can be introduced into the cells by
  • Complementing cell lines may be derived from a cell line capable of supporting infection of the helper virus.
  • a BHK TK fibroblast line is suitable for use with HSV-1, as described (Davidson, I. and Stow, N. D., Virology 141: 77-88 (1985); Paterson, T. and Everett, R. D., J. Gen. Virol. 71 : 1775-1783 (1990)).
  • D30EBA HSV-1 deletion virus and complementing cell line M64A described by
  • Paterson and Everett are useful in the present method (Figure 2, Figure 3).
  • D30EBA helper virus has a deletion in the same gene that is mutated in the HSV-1 ts K helper virus.
  • the deletion in D30EBA encompasses codons 83-1236 of the 1298 codon IE-3 gene.
  • Defective HSV-1 vectors pHSVlac and pHSVth which carries the gene for human tyrosine hydroxylase
  • pNFLlac in which the human neurofilament L promoter is operably linked to Lac Z
  • pHSVGAPlac in which an intracellular targeting sequence is fused to Lac Z
  • the D30EBA helper virus reverts at a much lower frequency than the ts K helper virus.
  • pHSVlac virus i.e., pHSVlac packaged into HSV-1 particles
  • titers were 25-fold greater using the deletion mutant as helper than when the ts K helper virus was used.
  • the reversion frequency of the deletion virus is probably due to homologous recombination between the deletion virus and the HSV-1 DNA flanking the IE-3 gene in the M64A complementing cell line. This recombination could be reduced or eliminated by construction of a helper line with less extensive or no homology to the deletion mutant in the region flanking the deletion or by increasing the extent of the deletion in the helper virus, or both.
  • deletion mutants with one or more additional mutations, particularly deletions, in genes required for productive HSV-1 infection could be used to further reduce reversion.
  • the complementing cell line would be capable of complementing each virus defect.
  • Other herpes mutant viruses can be used as helper virus, such as neurotropic Herpes mutant helper viruses.
  • a neurotropic Herpes virus is one that is capable of infecting neural cells; although such a neurotropic Herpes virus (e.g. HSV-1, Herpes simplex virus type-2 (HSV-2), and pseudorabies virus) may also be able to infect non-neural cells.
  • the Herpes mutant helper virus is a mutant virus, incapable of productive lytic infection in the target cells.
  • the Herpes mutant helper virus does not revert (i.e., becomes altered in a manner that confers the ability to direct a
  • the defective Herpes virus vector can be derived from any Herpes virus, or combination of Herpes viruses, providing the vector can be encapsidated into a Herpes virus particle by a Herpes helper virus.
  • the nucleotide sequence of a desired gene product(s) is introduced into a defective HSV-1 vector backbone.
  • the gene product(s) can be RNA transcribed from the nucleotide sequence (e.g., an anti-sense RNA), protein(s) and/or peptide(s) encoded by the
  • nucleotide sequence or portions thereof.
  • the nucleotide sequence can be, for example, a foreign sequence, synthetic DNA, genomic DNA or cDNA
  • the pHSVlac virus has been shown to direct expression of the Lac Z gene product
  • the level of a gene product can be altered in a cell type-specific manner, by
  • the desired gene product when under the control of a cell type-specific promoter, can be expressed preferentially or exclusively in one or more specific cell types.
  • the range of cell types will vary depending on the nature of the promoter or promoter fragment used. However, in each case, the range of
  • expression of the gene product will be restricted (i.e., promoter .is active in fewer cell types) and/or will display an altered specificity (i.e., the level of activity is altered, either increased or reduced, in certain cell types) as compared to the IE 4/5 promoter.
  • expression of the gene product in cells may be directed to the appropriate cell types.
  • HSV-1 vector backbone to make pNFLlac ( Figure 4).
  • This defective HSV-1 vector construct was packaged by a deletion mutant helper virus, and directed cell type-specific expression of a functional gene product encoded by the nucleotide sequence under its control in the vector, upon introduction into cells pNFLlac construct by infection. Expression is preferentially activated in neurons by the
  • neurofilament L promoter in the construction.
  • VIP vasoactive intestinal peptide
  • a cell type-specific promoter that is active in dividing cells but not quiescent cells such as the promoter from an appropriate cell cycle regulated gene, can be used to drive cell type-specific expression of a cytotoxin (e.g., ricin) from a defective HSV-1 vector in neural tumor cells to kill the cells.
  • a cytotoxin e.g., ricin
  • the gene product can be targeted to a particular location in the cell (e.g., the cell body, nucleus, or neuronal processes) if desired, by use of an intracellular targeting sequence, such as the human GAP-43 targeting sequence.
  • an intracellular targeting sequence such as the human GAP-43 targeting sequence.
  • the nucleotide sequence encoding the intracellular targeting sequence is incorporated into the nucleotide
  • GAP-43 is a neuronal growth-associated protein and is linked to the synaptosomal membrane. It is also a major protein of the growth cone membrane complex. GAP-43 is thought to be attached to the growth cone membrane via fatty acylation of the protein's only two cysteine residues.
  • GAP-43 targeting sequence which is a nucleotide sequence of a portion of the human GAP-43 coding sequence, or a functional equivalent thereof, capable cf targeting a gene product to neuronal processes, could be incorporated into the nucleotide sequence of the desired gene product to direct the gene product to neuronal processes.
  • nucleotide sequences encoding variants of such a targeting sequence that retain targeting function are included in the present method, and such
  • variants are considered to be GAP-43 targeting sequences.
  • the N-terminal 10 codons of human GAP-43 can be incorporated into the nucleotide sequence of the desired gene product, as in pHSVGAPlac ( Figure 5) to target a gene product to neuronal processes.
  • a nucleotide sequence specifiying the N-terminal 10 codons of human GAP-43 is fused in frame to nucleotide sequence specifying the N- terminus of the gene product. It is possible that incorporation of the nucleotide sequence specifying a GAP-43 targeting sequence in another location of the nucleotide sequence of the desired gene product will also result in targeting to neuronal processes.
  • additional sequences from GAP-43 either contiguous or non-contiguous with the
  • N-terminal 10 codons in GAP-43 may enhance the efficiency of targeting or confer specificity of targeting to axonal processes. These sequences may be incorporated into the nucleotide s e quenc e o f the de s ire d gene pro duc t in one o r more locations consistent with the targeting function.
  • a targeting sequence derived from another molecule that is localized to a particular portion of a cell and that is capable of targeting the gene product to the desired intracellular location when incorporated into the nucleotide sequence of the gene product in one of the manners described can be used.
  • the invention further relates to a method of altering the level of a gene product in target cells to alter a disease state or an undesired or abnormal condition.
  • Expression of a desired gene product in a target cell can act, directly or indirectly, to prevent, reduce, or reverse a disease process.
  • the gene product can act on the target cell to correct a defect in that cell associated with a disease process or can alter a disease state in another cell.
  • altering the level of the gene product in the target cell can result in secretion of a substance or substances (e.g., neurotransmitters, growth factors) that act, directly or indirectly, to alter a disease state that affects the other cell or can induce cell-cell interactions that alter the disease state.
  • a substance or substances e.g., neurotransmitters, growth factors
  • defective HSV-1 vectors can be used to introduce a gene product into a wide variety of cell types, including postmitotic cells such as neural cells (e.g., neurons, glia) to affect neurological disorders such as Parkinson's disease or Alzheimer's disease.
  • postmitotic cells such as neural cells (e.g., neurons, glia) to affect neurological disorders such as Parkinson's disease or Alzheimer's disease.
  • Parkinson's disease is a neurodegenerative disorder resulting from the destruction of dopaminergic neurons in the substantia nigra pars compacta, which project into the corpus striatum (Yahr, M. D. and Bergmann, J (Eds.), Parkinson's Disease, Raven Press, New York, (1987)).
  • tyrosine hydroxylase is the rate-limiting enzyme in dopamine biosynthesis, introduction of the tyrosine
  • hydroxylase gene into neurons in, or projecting to, the striatum increases striatal dopamine levels.
  • tyrosine hydroxylase can be introduced into neural (e.g., neurons, glia) or neuronal cells to alter a disease state, such as Parkinson's disease.
  • a nucleotide sequence encoding TH can be introduced into a defective Herpes virus vector such that it is under the control of a promoter (e.g., a neuronal cell-specific promoter) in the vector, and
  • TH functional TH
  • the resulting defective Herpes virus vector is introduced into the desired cells by infection, following packaging, in the method of the present invention, and the level of the TH gene product in the cells is altered.
  • a nucleotide sequence encoding tyrosine hydroxylase, a functional equivalent, or portion thereof, could be used to produce TH.
  • Nucleotide sequences encoding variants or portions of TH that retain TH function are included in the present method, and such
  • TH tyrosine hydroxylase
  • a cDNA fragment encoding human tyrosine hydroxylase (O'Malley, K. L. et al . , Biochemistry 26: 6910-6914 (1987) is
  • pHSVth was able to direct expression of active TH in cells in culture, and in neuronal cells (neurons), such as striatal neurons, pHSVth was able to induce an increase in monoamine neurotransmitter release
  • the level of a gene product in cells which do not normally (naturally) express the product can be altered.
  • a neurotrophic factor such as nerve growth factor can be introduced into cells (e.g., neural cells) to alter (e.g., reduce or prevent) a disease state (e.g., diabetes, Alzheimer's disease) or to prevent, reduce or reverse the effects of injury to the nervous system (e.g., traumatic axon injury, neurotoxicity).
  • a defective Herpes virus construct which encodes a neurotrophic factor (e.g., nerve growth factor, brain-derived neurotrophic factor (BDNF),
  • BDNF brain-derived neurotrophic factor
  • neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary neurotrophic factor (CNTF), basic or acidic fibroblast growth factor (FGF)
  • NT-3 neurotrophin-3
  • NT-4 neurotrophin-4
  • CNTF ciliary neurotrophic factor
  • FGF basic or acidic fibroblast growth factor
  • expressing a neurotrophic factor also expresses an appropriate receptor, it too can be affected by the factor by an autocrine mechanism.
  • Each member of the family of neurotrophic factors including factors such as nerve growth factor, brain derived neurotrophic factor, and neurotrophin 3, promotes the survival of particular types of developing neurons (NGF, Thoenen, H. e t al . , Rev. Physiol. Biochem. Pharmacol. 109: 146-178 (1987); BDNF, (Lindsay, R. M. et al . , Dev. B iol. 112 : 319-328 (1985); Hofer, M. M. and Y. Barde, Nature
  • Defective HSV-1 vectors that express other neurotrophic factors, such as BDNF, neurotrophin-3, neurotrophin-4, basic or acidic fibroblast growth factor or CNTF (Stockli, K. et al . , Nature 342 : 920-922 (1989)) are useful in the present invention for maintaining neuronal phenotype and promoting neuronal survival to prevent, reduce or reverse disease or the effects of injury.
  • the ability to use such viruses to alter the levels of specific neurotrophic factors in specific regions of the brain e.g., the basal forebrain
  • a nerve growth factor (NGF) minigene was constructed and inserted into a defective HSV-1 vector to make pHSVngf. This construct was packaged and used to infect cells which do not normally or naturally express NGF
  • the cells produced biologically active NGF, which was able to prolong the survival of sympathetic neurons.
  • the superior cervical ganglion (SCG) of adult rats contains neuronal and non-neuronal cells
  • sympathetic neurons and surrounding glia which do not express NGF.
  • the sympathetic neurons in the SCG of adult rats depend on target-derived nerve growth factor for maintenance of tyrosine hydroxylase levels and the noradrenergic neurotransmitter system.
  • Axotomy of a SCG results in NGF deprivation, causing a decline in TH activity in the sympathetic neurons; however, continuous local application of NGF can prevent this decline in TH activity.
  • HSV-1 vectors can be used to prevent deleterious effects (e.g., decline in TH levels) of nerve injury (axon injury) in vivo.
  • the ability to increase the neurotrophic factor supply to neurons, such as neurons deprived of neurotrophic factors, by the use of defective HSV-1 vectors capable of expressing genes encoding neurotrophic factors provides a method of preventing, reducing or reversing the effects (i.e., treating) of peripheral neural injury. Disruption of neurotrophic factor supply in the central nervous system (CNS) may also produce disease.
  • CNS central nervous system
  • acetyltransferase decreases. Disruption of this NGF supply may be involved in pathophys iology of Alzheimer's Disease, which is characterized by a progressive loss of cognitive function which is correlated with degeneration of cholinergic neurons (affected cells) in the basal forebrain (reviewed in Hefti, F. et al., Neurobiol. Aging 10: 515-533
  • NGF neuropeptide
  • deprivation may be a direct or indirect cause of toxic or metabolic neuropathy, as well as neuropathy secondary to injury.
  • supplies of the neurons affected in these conditions by the method of the present invention can prevent, reduce or reverse damage to the neurons associated with these conditions.
  • nerve growth factor can be introduced into neural cells to alter a disease state such as Alzheimer's disease.
  • a nucleotide sequence encoding nerve growth factor can be introduced into a defective Herpes virus vector under the control of a promoter in the vector, and functional NGF can be expressed from the resulting construct.
  • the packaged virus construct is used to infect target cells, and the level of NGF in those cells is altered.
  • NGF can affect the target cell (e.g., an affected cell, or a cell which can secrete NGF to alter a disease state affecting another cell) to reduce the symptoms of the disease.
  • Nucleotide sequences encoding variants or functional portions of NGF are useful in the present method and are included in the designation "nerve growth factor".
  • nerve growth factor "minigene” described in the examples is one such nucleotide sequence.
  • the method of the present invention further relates to a method of altering neurotransmitter metabolism, directly or indirectly, by altering the level of a gene product in a cell.
  • neurotransmitter metabolism can be altered in neuronal (e.g., striatal neurons) or non-neuronal cells (e.g., glia, fibroblasts).
  • Alterations in metabolism of classical (e.g., GABA , monoamines such as dopamine, norepinephrine) or peptide (e.g., somatostatin, enkephalins, vasoactive intestinal peptide (VIP)) neurotransmitters can occur presynaptically, postsynaptically, and/or at
  • alterations can occur in neurotransmitter biosynthesis, release, uptake, action and/or breakdown, for example.
  • neurotransmitter metabolism is introduced into a defective HSV-1 vector such that the gene product is functionally expressed from a promoter in the defective HSV-1 vector.
  • the defective HSV-1 vector construct, specifying production of the gene product is packaged with helper virus and introduced into a target cell by infection.
  • Certain gene products can affect more than one aspect of neurotransmitter metabolism.
  • gene products capable of altering neurotransmitter biosynthesis can alter neurotransmitter release as well.
  • a defective HSV-1 vector construct capable of
  • expressing tyrosine hydroxylase (TH), such as pHSVth is introduced into a cell.
  • TH tyrosine hydroxylase
  • non-neuronal cell such as a fibroblast
  • the level of tyrosine hydroxylase which is a rate-limiting enzyme for neurotransmitter biosynthesis, is altered by introduction of the construct, and monoamine neurotransmitter release is altered (increased).
  • Herpes virus vector construct capable of expressing tyrosine
  • TH hydroxylase
  • defective Herpes virus constructs encoding neurotrophic factors can also be used to modulate neurotransmitter metabolism.
  • Neurotrophic factors play a pivotal role in the development and maintenance of neurons, both in the peripheral and central nervous system (reviewed in Barde, Y. Neuron 2: 1525-1534 (1989); Snider, W. D. and E.M. Johnson, Ann. Neurol. 26: 489-506 (1989); Thoenen, H. and Barde, Y. -A. Physiol.
  • NGF neurotrophic factor
  • Depletion of NGF in developing animals results in the death of sympathetic neurons and many sensory neurons, indicating that NGF promotes neuronal survival.
  • NGF acts to maintain neuronal phenotype, such as the noradrenergic characteristics of sympathetic neurons (e.g., synthesis of tyrosine hydroxylase, a catecholamine biosynthetic enzyme), and the cholinergic phenotype of some CNS neurons (e.g., synthesis of acetyl choline transferase, a cholinergic biosynthetic enzyme) by stimulating the synthesis of
  • NGF neurotransmitter metabolic enzymes.
  • NGF is involved in modulating the levels of gene products involved in neurotransmitter metabolism.
  • noradrenergic neurotransmitter system depends on stimulation of synthesis of the catecholamine biosynthetic enzyme TH.
  • expression of NGF in cells which do not normally express NGF can prevent the decline in TH levels usually observed on axotomy, indicating that delivery of a gene product to cells by HSV virus constructs is an effective approach to treatment of peripheral nerve damage.
  • the TH levels were increased in cells infected by an HSV-1 vector encoding NGF relative to the control.
  • Increases in the levels of enzymes involved in the synthesis of neuro- transmitters can also increase the release of neurotransmitters (e.g., monoamine, cholinergic).
  • cholinergic neurons are infected with a defective Herpes virus construct such as pHSVngf containing a sequence which encodes nerve growth factor (e.g., a nerve growth factor minigene).
  • a defective Herpes virus construct such as pHSVngf containing a sequence which encodes nerve growth factor (e.g., a nerve growth factor minigene).
  • nerve growth factor e.g., a nerve growth factor minigene
  • ChAT neurotransmitter
  • neurotransmitter e.g., acetyl choline
  • retroviral vector has been used to express NGF in transplanted fibroblasts; however, retroviral vectors require at least one round of host cell DNA replication for integration and the resulting stable expression. Consequently, retrovirus vectors are not effective for gene transfer into postmitotic neurons and glia. In contrast, defective HSV-1 virus vectors are useful for therapy in post-mitotic cells and provide a powerful approach to gene therapy of neurological disorders.
  • a gene product that participates in a signal transduction or second messenger pathway i.e., a signal transduction factor
  • a signal transduction or second messenger pathway i.e., a signal transduction factor
  • a signal transduction factor such as adenylate cyclase, cAMP-dependent protein kinase, protein kinase C, the calcium- calmodulin dependent protein kinase II, or parval- bumin (a calcium binding protein)
  • signal transduction or second messenger pathway gene products have been implicated in regulating the frequency of action potentials (Madison, D. V. and Nicoll, R. A., J. Physiol. 372: 245-259, 1986)) and neurotransmitter release (Nichols, R.A. et al . ,
  • the entire gene product or an active or regulatory fragment thereof can be expressed.
  • discrete catalytic and regulatory domains have been recognized in a number of these gene products, such as yeast adenylate cyclase.
  • Other signal transduction enzymes with defined catalytic domains are known, such as cyclic nucleotide
  • phosphodiesterases both serine/threonine and tyrosine protein kinases and protein phosphatases
  • phospholipases e.g., phospholipase C
  • transcription regulation factors e.g., the cAMP response element binding protein CREB , fos, jun
  • neurotransmitter release machinery e.g., synapsins.
  • the catalytic domains of adenylate cyclase such as yeast adenylate cyclase (Kataoka, T. et al . , Cell 43 : 493-505 (1985)), protein kinase C
  • a nucleotide sequence encoding an active catalytic fragment of yeast adenylate cyclase is introduced into a defective HSV-1 virus vector, to make pHSVcyr ( Figure 7) such that a functional gene product is expressed when pHSVcyr is introduced into a target cell.
  • a nucleotide sequence encoding an active catalytic fragment of a protein kinase is introduced into a defective HSV-1 virus.
  • a nucleotide sequence which encodes the calcium binding protein parvalbumin is inserted into a defective HSV-1 virus vector, to make pHSVparv, such that a functional gene product is expressed when pHSVparv is
  • calcium binding protein such as parvalbumin participates in a signal transduction or second messenger pathway and is a "signal
  • Parvalbumin which is aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl-N-transduction factor
  • GABAergic neurons e.g., GABAergic neurons
  • GABAergic neurons e.g., GABAergic neurons
  • receptors e.g., neurotransmitter receptors, growth factor receptors, neurotrophic factor receptors.
  • a number of cell surface receptors for growth factors e.g., a number of cell surface receptors for growth factors and
  • neurotrophic factors are protein tyrosine kinases (i.e., tyrosine kinase receptors), which activate signals triggering trophic effects upon binding of the appropriate ligand. Therefore, receptors such as the EGF receptor, fibroblast growth factor receptors 1 and 2, or neurotrophin receptors TrkA, TrkB and TrkC, represent another kind of "signal transduction factor”.
  • Another "signal transduction factor” is the low affinity nerve growth factor receptor, which belongs to a class of receptors which is distinct from the tyrosine kinases.
  • catalytic fragment of a receptor such as the EGF receptor or an FGF receptor is inserted into a defective HSV-1 vector and expressed in neural cells. Expression of an unregulated portion of the receptor with tyrosine kinase activity can activate the signal transduction pathway associated with the selected receptor.
  • a nucleotide sequence encoding a version of the low affinity NGF receptor which lacks the extracellular ligand binding domain is inserted into a defective HSV-1 vector to make a construct such as pDB3.
  • the receptor fragment encoded by pDB3 is designed to trigger the effects of its corresponding ligand (e.g., NGF) in the absence of the ligand.
  • NGF its corresponding ligand
  • This construct can be used to bypass or supplement the requirement of a cell for NGF. For example, cells which do not receive sufficient quantities of target-derived NGF due to injury or disease (e.g., Alzheimer's disease) can be infected with a packaged pDB3 virus construct.
  • Expression and activity of the encoded fragment can mimic the effects of NGF (e.g., maintenance of the phenotype of cholinergic neurons or the noradrenergic features of sympathetic neurons) even in the absence of NGF.
  • infection with pDB3 can permit maintenance or restoration of levels of choline acetyl transferase or tyrosine hydroxylase, thereby altering neurotransmitter release.
  • introduction of pDB3 into a cell could alter the cell so as to express cholinergic or adrenergic features (e.g., induce expression of neurotrasmitter metabolic enzymes such as TH and ChAT) which it did not previously express.
  • the GAP-43 targeting sequence is incorporated into the nucleotide
  • nucleotide sequence is operably linked to a neuronal cell-specific promoter in the vector.
  • the gene product such as an active fragment of adenylate cyclase, is functionally expressed and targeted to neuronal processes.
  • the effect of the product on neurotransmitter metabolism can be altered by targeting the gene product to a specific region of the cell, where its activity can be enhanced, for example.
  • pHSVcyr and other HSV-1 vector constructs capable of increasing cAMP levels in a cell can be used to alter neurotransmitter metabolism at the transcriptional level.
  • CRE cAMP response element
  • the presence of the cAMP response element (CRE) in the regulatory region of the genes for tyrosine hydroxylase and peptide neurotransmitters somatos tatin and VIP causes these genes to be transcr iptionally activated by cAMP in neural cell lines (Montimony, M. R. et al., Trends in Neurosci. 13: 184-188 (1990)).
  • components of the neurotransmitter machinery such as synapsin I or synaptophysin, can be selected as gene products for use in altering neurotransmitter metabolism, particularly of
  • Defective HSV-1 vector constructs such as pHSVcyr, pHSVpkc ⁇ , pHSVCaCK, pHSVparv, pHSVngf, and pHSVth, which are capable of altering
  • neurotransmitter metabolism can be introduced into neural or neuronal cells to alter a disease state.
  • increasing neurotransmitter synthesis or expression of neurotransmitter biosynthetic gene products e.g., TH , ChAT
  • the desired defective HSV-1 virus particles e.g., pHSVth virus
  • the HSV-1 vector or construct is introduced into cells to alter the level of the desired gene product and acts to prevent, reduce, or reverse a disease process.
  • virus can be delivered directly into the organ of interest by injection or into the brain by stereotactic
  • virus can be delivered into the brain or organ of interest by intravenous or subcutaneous administration, following incorporation of virus particles into liposomes (Ostrom, M. J., Liposomes: From Biophysics to Therapeuties, Marcel Dekker, New York, (1987)) or polymers (Brown, L. et al . , Diabetes 35: 692-697 (1986)).
  • stereotactic injection of pHSVth virus into the striatum can be used to introduce the pHSVth vector and encoded tyrosine hydroxylase gene into striatal neurons and neurons projecting into the striatum by infection of those cells.
  • a construct which directs the expression of NGF can be introduced into the septal nuclei which contain deteriorating cholinergic neurons or into the pyramidal neurons of the hippocampus, for example, to maintain the cholinergic phenotype of CNS neurons (e.g., basal forebrain cholinergic neurons).
  • the location of the cells infected with the packaged virus contruct is determined by several factors, such as the site of injection, the location of neurons which project to the site of injection, the number of virus particles administered, and the extent of diffusion of the particles.
  • Another possible mode of administration involves the implanation of genetically modified post-mitotic cells which express the desired gene product from a defective Herpes virus vector.
  • a defective HSV-1 vector can be used to deliver genes into neurons in culture and the cells can subsequently be
  • dopamine agonists such as bromocryptine (Yahr, M. D. and Bergmann, J. (Eds.),
  • HSV-1 vectors encoding human tyrosine hydroxylase in the method of the present invention provides a method of gene therapy for Parkinson's disease, providing an attractive alternative to oral administration of L-DOPA, which loses its effectiveness over time, and to tissue transplantation, which has technical and practical difficulties.
  • the present method is applicable to animal models of disease, such as Parkinson's disease.
  • injection of the neurotoxin 6-OH-dopamine into the substantia nigra of rats results in
  • Parkinsonian syndrome which is characterized by dopamine depletion in the nigrostriatal system, can be induced by the neurotoxin MPTP (Langston, J. W. et al., Science 219: 979-980 (1983)).
  • Different defective HSV-1 vectors could be assayed using behavioral tests for recovery of dopaminergic function in these animal models.
  • Herpes virus vectors can be introduced into the brain by stereotactic injection (Pellegrino, L. J., and Cushman, A. J., Methods in Psychobiology, pp.
  • HSV-1 vector construct encoding a signal transduction factor was tested in an assay for apomorphine- induced rotational behavior in rats (Hefti, et al . , Pharmacol. Biochem. Behav. 12: 185-188 (1980)).
  • Example 1 Animals stereotactically injected with the packaged defective HSV-1 vector constructs and deletion helper virus were healthy. This supports the safety and effectiveness of defective HSV-1 vectors in delivering a gene into neural cells of a mammal by the method of the present invention.
  • the vectors can direct expression of a gene inserted into the vector to alter the level of the encoded gene product in target cells, and can alter neurotransmitter metabolism (e.g., neurotransmitter release) in the mammal.
  • the invention is further and more specifically described in the following examples which are not intended to be limiting in any way.
  • Example 1 Example 1
  • pHSVth Directs Expression of Active TH in
  • HTH-2 human tyrosine hydroxylase (TH) cDNA
  • the cDNA was inserted into a variety of vectors, including pHSVlac (Geller, A. I. and Breakefield, X. O., Science 241: 1667-1669
  • pHSVth DNA was packaged into HSV-1 virus particles by the method of Geller, using HSV-1 strain 17 ts K as helper virus. (Geller, A. I.,
  • Oligonucleotide primers OhTH-116 and OhTH-193 were derived from the human TH cDNA sequence (HTH-1) and were synthesized by the Protein Chemistry
  • Oligonucleotide OhTH-116 (5'-dGGGCTTCCGCAGGGCCGTGTCTGAGCTGGA) is identical to the coding sequence of nucleotides 36-65, and OhTH-193
  • Control and infected cells were lysed in a PCR-compatible buffer containing nonionic detergents and proteinase K. After incubation at 60 oC for one hour, the proteinase K was inactivated by heating and an aliquot of the mixture was added to an amplification reaction.
  • 32 P-end labeled primers from exons 1 and 2 (OhTH-116, OhTH-193) of the human
  • TH gene were used to amplify TH cDNA in the mixture.
  • the predicted size of the PCR fragment obtained with primers OhTH-116 and OhTH-193 is 161 base pairs.
  • pHSVtk infected CV1 cells (multiplicity of infection of 0.1) were assayed for TH
  • glycerol (1:1) containing 0.4% n-propyl gallate 0.4% n-propyl gallate.
  • TH-positive cells are easily detected in the background of surrounding negative cells.
  • uninfected cells were negative for staining.
  • a nonenzymatic coupled decarboxylation assay was used to measure TH activity in infected
  • TH activity exhibited a level of TH activity (pmoles DOPA/ ⁇ g protein/hr) approximately 40% that of rat striatal cells assayed in parallel.
  • pHSVth was faithfully packaged into virus particles using the HSV-1 strain 17 ts K as helper virus.
  • the packaged pHSVth was able to infect fibroblasts, and to direct production of active TH in these cells as monitored by immunofluorescence and enzymatic assays.
  • Example 1 Each 35-mm dish contained about 1 X 10 5 cells at the time of infection, and about 10% of the cells were neurons.
  • Mouse antibody specific for TH (Boeringer Mannheim Biochemicals #1017-381) and an IgG fraction of rabbit anti-neurofilament - 200 (Sigma Chem. Corp., St. Louis, Mo., #N-4142) were used as primary antibodies. Secondary antibodies were fluorescein isothiocyanate - conjugated goat F(ab') 2 antibody against mouse F(ab') 2 (1:200 dilution) and rhodamine isothiocyanate-conjugated goat F(ab') 2 antibody to rabbit F(ab') 2 (1:25) dilution.
  • CV1 fibroblasts were maintained and infected as described in Example 1. Primary cultures of 1 to 4-day-old rat striatal neurons were prepared and infected as described in Example 2. Cultures were infected with pHSVth, pHSVpUC, HSV-1 ts K virus, or were mock infected. HSV-1 strain 17 ts K was used as helper virus in packaging. pHSVpUC is a derivative of pHSVlac (c. f., Example 1), in which the Eco
  • RI -Hind III fragment encoding lacZ of pHSVlac was replaced by the polylinker of pUCl9 to make pHSVpUC.
  • the cell culture medium was removed and the cells were washed in release buffer and then incubated in 200 ⁇ l of the release buffer for 15 minutes. This release buffer was then aspirated off the cells, cooled on ice water (in prechilled tubes), and centrifuged for 5 minutes (1400 rpm) to remove any cellular debris. 20 ⁇ l of 2M HClO 4 and 20 ⁇ l of 1% Na 2 S 2 O 5 were added, and the samples stored at -70°C until analysis by HPLC.
  • the release buffer was of the following composition: 135mM NaCl, 3mM KCl, ImM MgCl 2 , 1.2mM CaCl 2 , 2mM NaPO 4 , 200 ⁇ M ascorbate and 10 ⁇ M glucose.
  • the following drugs were added to the release buffer where indicated: tyrosine (1mM), the tyrosine hydroxylase co-factor, tetrahydrobiopterin (BH 4 , ImM), tetrodotoxin (1 ⁇ M), veratridine (5mM), calcium-free, where 0.1 mM EGTA replaced the calcium and high potassium buffer to depolarize cells where the KCl was increased to 56mM and NaCl reduced to 80mM to maintain osmolarity.
  • Sample analysis was performed using HPLC with a series array of 16 coulometric electrode sensors (CEAS, Model 55-0650, ESA, Inc. Bedford, MA) (Matson, W. R. et al., Clin. Chem.
  • the coulometric electrodes fully oxidize at 100% for a given potential, the following sensors are essentially independent. This allows a compound to be defined not only by its elution time, but also by its specific oxidation pattern. For example, at 60 millivolt (mM) incremental settings, dopamine has a dominant response on electrode 2 (60mV versus a palladium reference electrode), the response on detector 1 (OmV) or detector 3 (120mV) is only
  • Two mobile phases were used, "A” was 0.1M NaH 2 PO 4 with 10mg/L of dodecyl sulfonic acid, and 100nM nitrilotriacetic acid, adjusted to pH 3.45 with phosphoric acid; the B mobile phase was 0.1M NaH 2 PO 4 (pH 3.35) with 50 mg/L of dodecylsulfonic acid and 100nM nitriloacetic acid, 50% methanol v/v.
  • Several different electrode settings were used.
  • the primary potential settings were 60mV increments from 0 to 900mV, in addition, a 50mV incremental system was used with electrodes 1 to 4 set at increments from 50 to 250mV. Finally, for selected samples a gate-cell array was used where the electrodes were set at oxidizing
  • Dopamine production was also measured. 0.08 Pg/ ⁇ l of dopamine was detected in release buffer from mock infected CV1 cells, and 0.06 pg/ ⁇ l of dopamine was detected in release buffer of HSV-1 ts K virus infected cells. In contrast, 0.56 pg/ ⁇ l of dopamine was detected in release buffer from pHSVth virus infected CV1 cells.
  • K + -dependent component of L-Dopa release as glia would not be expected to show K + -dependent release.
  • pHSVcyr caused increases in the cAMP concentration in the cell body, in protein phosphorylation, and in monoamine neurotransmitter release. Construction of pHSVcyr, pHSVpUC, and Packaging Into HSV-1 Particles
  • pHSVcyr was constructed from pHSVlac using standard recombinant DNA techniques (Maniatis et al., Mo lecular Cloning, Cold Spring Harbor
  • Hind III linkers were ligated to the fragment, and it was cloned into the unique Hind III site of pHSVpUC.
  • HSV-1 vectors e.g. pHSVpUC and pHSVcyr
  • HSV-1 particles were packaged into HSV-1 particles as described (Geller, A. I., Nucleic Acids Res. 16 : 5690, 1988), using HSV-1 strain 17 ts K as helper virus.
  • the titer of the virus stock was 2 X 10 6 plaque forming units (pfu)/ml ts K and 9 X 10 5 infectious particles/ml pHSVcyr.
  • CV1 monkey fibroblasts were grown in Dulbecco's modified minimum essential medium (DMEM) with 10% fetal bovine serum.
  • PC12 rat pheochromocytoma cells (Greene, L. A. and Tischler, A. S., Proc. Natl. Acad. Sci. USA 7 3 : 2424-2428, 1976) were grown in RPMl 1640 containing 10% horse serum and 5% fetal bovine s erum.
  • PC12 cells (1X10 4 cells/ml) were plated in 24 well plates, when the cell density reached 2x10 5 cells/0.5 ml, the cultures were infected with pHSVcyr (7.5 ⁇ l) or pHSVpUC (7.5 ⁇ l); and one day later assays were performed.
  • pHSVcyr 7.5 ⁇ l
  • pHSVpUC virus 7.5 ⁇ l
  • six days later parallel wells were infected and one day later assays were performed (seven days and one day after infection, respectively).
  • Each well in a 24 well plate contained approximately 2x10 5 cells in 0.5 ml at the time of infection and
  • 2x10 7 CV1 cells were infected with 2x10 6 infectious particles of pHSVcyr or pHSVpUC virus, and incubated for 24 hours at 37°C.
  • Total cellular RNA was prepared as described (Chirgwin et al . ,
  • RNA secondary structure was synthesized from each section in a 50 ⁇ l in situ transcription reaction (50 mM Tris pH 8.2 at 42°C, 50 mM KCl, 6 mM MgCl 2 , 10 mM DTT , 1000 u/ml Promega Biotech RNAsin, 1 ug 3' primer, 5 mM DNTPs, 1000 u/ml Life Sciences AMV reverse transcriptase) at 42oC for 2 hours. The reaction was placed at 65oC for 10 minutes to inactivate the reverse transcriptase, 2 ⁇ l of each reaction was transferred to a 100 ⁇ l polymerase chain reaction (PCR) mix (Perkin Elmer Gene Amp kit).
  • PCR polymerase chain reaction
  • the cDNA was subjected to 40 cycles of PCR (94°C, 1 min; 60°C, 2 min; 72°C, 3 min). 40 ⁇ l of each reaction was elec trophoresed on a 1% agarose/1% Nusieve gel in tris acetate EDTA buffer and transferred to Hybond (Amersham) in
  • the 5' primer used in the PCR reaction was a 27 base
  • antibodies were rabbit anti-yeast adenylate cyclase (Heideman, W. et al., J. Cell Biochem. 42 : 229-242, 1990; provided by Dr. Heideman; U1 used at a 1:50 dilution) (ovalbumin) or a rabbit anti-cAMP (1:50 dilution; Chemicon) and monoclonal mouse anti-rat neurofilament (1:800 dilution; SMI-33, Cappel).
  • the rabbit anti-cAMP antibody was preabsorbed with 1.0 mM cAMP for 30 minutes at 4 oC.
  • the secondary antibodies were fluorescein
  • PC12 cells were cultured and infected (moi 0.1) as described above, except 60 mm plates containing 5 ml of medium were used. One day after infection, the media was removed (all subsequent manipulations and solutions were at 4oC), and the cells were washed once with 2 ml of PBS. The cells were lysed in 1 ml of 5% TCA 0.1 M HCl by shaking for 5
  • the cell lysates were centrifuged (1400 rpm) for 5 minutes, the supernatants were frozen in dry ice-ethanol, and stored at -70oC. The samples were extracted five time with diethyl-ether,
  • the media was removed, and the cells were incubated for 30 minutes in 0.3 ml of 32 P PO 4 medium
  • PC12 cells 2.4 ml DMEM-PO 4 (source), 0.4 ml dialyzed horse serum, .02 ml dialized fetal bovine serum, and 1 ml 32 P PO 4 (2 mCi, 8500 Ci/mmol, New
  • Lysis buffer contained 100 mM NaPO 4 pH 7.0, 50 mM KF, 20 mM EDTA, 5% TX-100, and protease Inhibitors (1 uM pepstatin A, 1 mM 1,10- phenanthroline, 0.1 mM PMSF, 1 mM iodoacetamide, 1 ⁇ g/ml aprotinin, and 1 ⁇ g/ml leupeptin). The lysis buffer was removed, centrifuged at 1400 rpm for 5 minutes, and the supernatant was analyzed by TCA precipitation and SDS polyacrylamide gel (8%
  • the cell culture medium was removed, cells were washed once in release buffer, and then incubated in 200 ⁇ l release buffer for 15 minutes.
  • the release buffer was then aspirated off the cells, cooled in ice water for 5 minutes (in prechilled tubes), and centrifuged for 5 minutes (1400 rpm) to remove any cellular debris.
  • 20 ⁇ l of 2 M HClO 4 and 20 ⁇ l 1% Na 2 S 2 O 5 were added, and the samples were stored at -70oC prior to analysis by HPLC.
  • Release buffer 135 mM NaCl, 3 mM KCl, 1 mM MgCl 2 , 1.2 mM CaCl 2 , 2 mM NaPO 4 pH7.4, 200 ⁇ m ascorbate and 10 ⁇ M Glucose.
  • the following drugs were added to the release buffer where indicated: Forskolin (1 mM), bt 2 cAMP (2 mM), tetrodotoxin (1 ⁇ M), and veratadine (5 mM).
  • Release buffer without calcium contained 0.1 mM EGTA in place of the CaCl 2 . (When this release buffer was used, the wash before incubation in release buffer was also performed with this buffer.)
  • Release buffer to depolarize cells contained 56 mM KCl, 80 mM NaCl, and the other components of release buffer.
  • dopamine has a dominant response on electrode 2 (60 mV versus a palladium reference electrode), the response on detector 1 (0 mV) or detector 3 (120 mV) is only 10-30% of its response on the dominant 60 mV electrode.
  • coeluting compound is likely to alter the peak ratio.
  • a gradient method which had been optimized by Dr. I. N. Acworth of ESA Inc. was used for the resolution of catecholamines.
  • Two mobile phases were used, "A” was 0.1 M NaH 2 PO 4 with 10 mg/L of dodecycl sulfonic acid, and 0.1 ⁇ M nitrilotriacetic acid, adjusted to pH 3.35 with H 3 PO 4 ; the "B” mobile phase was 0.1 M NaH 2 PO 4 , pH 3.35) with 50 mg/L of dodecysulfonic acid, and 0.1 ⁇ M nitrilotriacetic acid, 50% methanol (vol./vol.).
  • Several different electrode settings were used.
  • the primary potential settings were 60 mV increments from 0 to 900 mV, in addition, a 50 mV incremental system was used with electrodes 1 to 4 set at increments from 50 to 250 mV. Finally for selected samples, a gate-cell array was used where the electrodes were set at oxidizing potentials alternating with reducing (negative) potentials. The concept behind this system is that only those compounds which reversibly oxidize and reduce at the defined potentials will pass the "gate”. An 8 cm by 0.45 cm, 3 ⁇ m C18 ESA HR80 (Teflon) column was used for the majority of assays with a 15 cm by 0.45 cm, 5 ⁇ m Nikko Bioscience
  • pHSVcyr virus expresses cyr RNA and protein
  • pHSVpUC DNA was also properly packaged into HSV-1 particles (not shown).
  • pHSVcyr The ability of pHSVcyr to express cyr RNA was examined.
  • CV1 cells were infected with pHSVcyr virus, pHSVpUC virus or mock infected, and one day later total cellular RNA was isolated.
  • cyr cDNA was synthesized using reverse transcriptase and a primer homologous to the 3' end of the cyr transcript, the cyr products were amplified using the polymerase chain reaction with primers homologous to the cyr transcript, and displayed on a polyacrylamide gel.
  • pHSVcyr, but not pHSVpUC or mock infected cells contained the expected 1.5kb band.
  • pHSVlac expresses an RNA of the predicted size
  • PC12 cells (Greene and Tischler, PNAS
  • Cultures infected with pHSVcyr virus contained cells with prominent cyr immunoreactivity (cyr-IR). Cultures infected with pHSVpUC virus, or mock infected cultures, lacked cells with cyr-IR. Cultures infected with pHSVcyr virus and subjected to immunohistochemistry using preimmune rabbit serum lacked cells with rhodamine fluoresence. The results demonstrate that pHSVcyr DNA was properly packaged into HSV-1 particles and that pHSVcyr virus expresses cyr RNA and cyr protein in PC12 cells.
  • pHSVcyr Virus Causes an Increase in cAMP
  • PC12 cells were infected with pHSVcyr virus, one day later a TCA extract was prepared, and the amount of cAMP was determined using a radioimmunoassay for cAMP. Cultures infected with pHSVcyr virus
  • pHSVcyr caused about a 20X increase in cAMP
  • the antibody was visualized with the rhodamine conjugated goat anti-rabbit IgG antibody (same secondary antibody used to assay cyr-IR).
  • cAMP-immunoreactivity cAMP-IR
  • longer exposures detected low levels of background cAMP-IR present in all cells.
  • Cultures infected with pHSVpUC virus or mock infected cultures lacked cells with elevated levels of cAMP-IR and cultures infected with pHSVcyr virus and subjected to immunohistochemistry using rabbit anti-cAMP antibody (preabsorbed with cAMP) lacked cells with cAMP-IR.
  • PC12 cells were infected with pHSVcyr virus, pHSVpUC virus, or mock infected; one day later, these cells were incubated for 30 minutes with 32 p
  • pHSVcyr and bt 2 cAMP produced significant increases in protein phosphorylation compared to mock infected cultures. In contrast, the extent of protein phosphorylation produced by pHSVpUC was similar to mock infected cultures.
  • Ca ++ /calmodulin dependent protein kinase II also produced an increase in protein phosphorylation, but the pattern of bands observed on SDS -polyacrylamide gels was clearly different from that obtained with pHSVcyr and bt 2 cAMP.
  • Cells infected with pHSVcyr were incubated for one day prior to analysis, whereas the cells treated with bt 2 cAMP were
  • Pharmacological agents that stimulate adenylate cyclase activity are known to transiently increase neurotransmitter release from PC12 cells; therefore, the ability of pHSVcyr to direct an analogous increase in monoamine neurotransmitter release
  • PC12 cells were infected with pHSVcyr, pHSVpUC, or mock infected, and one day later, the cells were washed once in release buffer, and then incubated for 15 minutes in 200 ⁇ l release buffer containing physiological concentrations of ions (135 mM NaCl, 3 mM KCl, 1 mM MgCl 2 , 1.2 mM CaCl 2 , 2 mM NaPO 4 pH 7.4, and 10 ⁇ M glucose). The amount of monamines
  • phosphorylation and monoamine neurotransmitter release were increased. Since undifferentiated PC12 cells do not extend processes, the Increase in cAMP levels caused by pHSVcyr occurred throughout the cell. It is possible that the increase in cAMP could act directly affect the neurotransmitter release machinery, to alter the amount of release, consistent with the observation that the increase in neurotransmitter release required calcium, which is required for fusion of synaptic vesicles to the plasma membrane. However, the increase in
  • Typical release buffer except 56 mM KCl and 80 mM NaCl.
  • pHSVpUC does not contain a gene.
  • Cultured superior cervical ganglia contain a
  • Nf-IR neurofilament immunoreactivity
  • a mouse anti-Nf antibody and a fluorescein conjugated goat anti-mouse IgG antibody previously described in Geller, A. I. and Breakefield, X. O., Science 241: 1667-1669, 1988.
  • Cultures infected with pHSVcyr virus contained some neurons with prominent cyr-IR localized to the cell body; cyr-IR cells were absent from cultures infected with pHSVpUC and from mock infected cultures.
  • cyr-IR was localized to the cell bodies of neurons. Since cyr is a yeast protein, it is unlikely to contain signals directing its transport to neuronal
  • E. coli ⁇ -galactosidase is observed primarily in cell bodies and proximal processes following infection with pHSVlac virus (Geller, A. I. and Breakefield, X. O., Science 241 : 1667-1669, 1988). These observations indicate that pHSVcyr virus can stably express cyr in sympathetic neurons and the cyr protein remains localized to the cell body.
  • pHSVcyr The ability of pHSVcyr to increase cAMP levels in neurons was investigated. Cultures of neurons were infected with pHSVcyr virus, pHSVpUC virus, or mock infected, and one day later the cAMP-IR and the Nf-IR were visualized. Cultures infected with pHSVcyr contained neurons with dramatically elevated levels of cAMP-IR localized to the cell bodies compared to neurons in mock infected cultures;
  • parallel cultures infected with the same amount of pHSVcyr virus (moi. 0.1) produced similar numbers of cyr-IR and cAMP-IR neurons (approximately 10% of each), suggesting that the neurons expressing the cyr protein produced the increased cAMP levels.
  • the increase in cAMP The increase in cAMP
  • pHSVcyr produced by pHSVcyr was quantitated by two methods, radioimmunoassay and incubation of cells with
  • pHSVcyr The ability of pHSVcyr to Increase protein phosphorylation in neurons, presumably by activating the A kinase, was investigated. Cultures were infected with pHSVcyr virus, pHSVpUC virus, or mock infected; one day or one week after infection, the cells were incubated for 30 minutes with 32 P PO 4 ; and protein extracts were prepared. The amount of protein kinase activity was measured by determining the amount of 32 P PO 4 incorporated into protein by
  • pHSVcyr causes a stable increase in protein kinase activity in sympathetic neurons.
  • pHSVcyr Stably Increases Monoamine Neurotransmitter Release from Sympathetic Neurons, and the Increase
  • Norepiniphrine the neurotransmitter used by adult sympathetic neurons, was measured in initial release experiments. However, subsequent experiments measured dopamine, because it is easier to detect in this assay, and because it is a significant
  • pHSVcyr causes an increase in neurotransmitter release from cultured sympathetic neurons; the increase in
  • neurotransmitter release requires calcium and physiological activity, and is stable for at least one week.
  • Cultured sympathetic neurons prepared from newborn rats can be induced to change from the monoaminergic neurotransmitter system to the
  • tyrosine hydroxylase immunoreactivity TH-IR
  • CAT-IR choline acteyltransferase immunoreactivity
  • pHSVcyr increased the frequency of action potentials, which originate in the cell body, thereby increasing the amount of neurotransmitter released. Consistent with this mechanism is the observation that depolarizing agents, which substitute for action potentials in affecting neurotransmitter release, cause the same amount of neurotransmitter release from pHSVcyr, pHSVpUC, and mock infected cells. If, on the other hand, pHSVcyr directly affected the neurotransmitter release machinery, then differences in the amount of neurotransmitter release between pHSVcyr and mock infected cells might still be observed in the presence of depolarizing agents.
  • Dissociated neuronal cultures were prepared from superior cervical ganglia of 4 day old rats. Two weeks after plating cultures (approximately 5 x 10 5 cells) were infected with the indicated virus. One day or one week later, the medium was removed, cells were washed once in release buffer, and then incubated in 200 ⁇ l release buffer for 15 minutes. Dopamine levels were measured by HPLC. Release buffer: 135 mM NaCl, 3mM KCl, 1 mM MgCl 2 , 1.2 mM CaCl 2 , 2 mM NaPO 4 pH7.4, and
  • HSV-1 strain 17 D30EBA virus Paterson, T. and Everett, R. D., J. Gen. Virol., 71: 1775-1783 (1990)
  • the complementing M64A cells containing the IE3 gene Davidson, I. and Stow, E. C., J. Gen. Virol., 67: 2571-2585 (1986); Paterson, T. and Everett, R. D., J. Gen. Virol., 71 : 1775-1783 (1990)
  • Dr. Everett Universality of Glasgow, Glasgow, Scotland.
  • Figure 2 shows the extent of the deletion in D30EBA, which removes codons 83 to 1236 of the 1298 codons of the IE3 gene (McGeoch, D. J. et al . , Nucleic Acids Res. 14: 1727-1745 (1986)).
  • Figure 2 also shows the region of HSV-1 DNA containing the IE3 gene that is present in M64A cells.
  • M64A cells contain the HSV-1 strain 17 IE3 gene and the a sequence, from nucleotide 844 in the short repeat region (McGeoch, D. J. et al . , Nucleic Acids Res. 14: 1727-1745 (1986)) to nucleotide 123,018 in the long repeat region (Perry, L. J. and McGeoch, D. J. J. Gen. Virol. 69: 2831-2846 (1988)).
  • M64A cells were constructed by transfection of BHK tk- cells with the plasmid p65, which contains the IE3 gene and the HSV-1 tk gene, and subsequent isolation by HAT selection as described (Paterson, T. and Everett, R. D., J. Gen. Virol. , 71: 1775-1783 (1990); Davidson, I. and Stow, E. C., J. Gen. Virol. , 67: 2571-2585 (1986))).
  • M64A cells were grown in Dulbecco's modified minimum essential medium with 10% fetal bovine serum; M64A cells were maintained in HAT medium until just before use.
  • the HSV-1 vector pHSVlac ( Figure 1; Geller, A. I. and Breakefield, X. O., Science 241: 1667-1669 (1988)), was packaged into HSV-1 particles using a deletion virus and complementing cell line.
  • the D30EBA virus contains a deletion in the IE3 gene
  • the M64A complementing cell line contains the HSV-1 1E3 gene.
  • M64A cells 1.5x10 5 M64A cells were seeded on a 60 mm plate. The following day, the M64A cells were transfected (Graham, F. L. and Van der Eb, A. J., Virology 52:456-467 (1973)) with a 0.5 ml calcium phosphate co-precipitate containing 1 ⁇ g pHSVlac DNA and 9 ⁇ g salmon sperm DNA. Four hours later, the cells were treated with 15% glycerol (Parker, B. A. and Stark, G. R., J. Virol. 31:
  • D30EBA virus was titered on M64A cells and revertants to wild type were detected on CV1 monkey fibroblasts.
  • PC12 cells were grown in RPMl 1640 containing 10% horse serum and 5% fetal bovine serum (Greene, L. A. and Tischler, A. S., Proc. Natl. Acad.
  • CV1 cells were grown in Dulbecco's modified minimum essential medium with 10% fetal bovine serum.
  • the IE3 gene in M64A cells complements the deletion in the IE3 gene in D30EBA virus, resulting in a productive HSV-1 infection.
  • the progeny virus from this experiment included both D30EBA virus and pHSVlac virus, since pHSVlac contains the sequences required for packaging into HSV-1 particles (Geller, A. I. and Breakefield, X. O., Science 241 : 1667-1669 (1988)).
  • pHSVlac is maintained in an HSV-1 virus stock due to its growth advantage over the helper virus and no genetic selection is required; pHSVlac contains 1 HSV-1 ori in 8.1 kb, while HSV-1 contains 3 ori in 150 kb, or 1 ori in 50 kb (Spear, P. G. and Roizman, B. In: DNA Tumor Viruses, Tooze, J., Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 615-746
  • pHSVlac becomes a larger fraction of the virus particles.
  • the virus stock from the initial packaging was passaged three additional times on M64A cells.
  • the titer and reversion frequency of the D30EBA virus was determined (Table 5). D30EBA virus grew efficiently in the presence of pHSVlac DNA, and the reversion frequency of D30EBA was about 5x10 -5 , comparable to D30EBA virus alone. By comparison, the ts K virus has an apparent reversion frequency of about 2x10 -3 at 37o C; the true restrictive temperature of ts K is 39o C (Davison, M. J. et al . , J. Gen. Virol. 65: 859-863 (1984)), su the apparent reversion frequency includes virus produced due to imcomplete penetrance of the ts K allele at 37 oC.
  • pHSVlac virus was compared to the amount of D30EBA virus in the virus stocks, pHSVlac represented one half to two thirds of the virus stock.
  • Previous analyses have shown that the ratio of pHSVlac to ts K was 0.8 (Geller, A. I. and Breakefield, X. O., Science 241: 1667-1669 (1988); Geller, A. I. and Freese, A., Proc. Natl. Acad. Sci. USA 87: 1149-1153 (1990)).
  • the ratio of pHSVlac to helper virus in the virus progeny is similar for ts K and D30EBA.
  • pHSVlac is more efficiently packaged into HSV-1 particles using the deletion mutant D30EBA as helper virus as compared to packaging with the ts K helper virus.
  • the reversion frequency of the deletion virus is 40-fold lower than that of ts K.
  • transfection/superinfection used to initiate packaging pHSVlac DNA into HSV-1 particles.
  • Four transfections were performed, pHSVlac-1 through pHSVlac-4.
  • p-1, p-2, and p-3 are the subsequent serial passages of each virus stock.
  • the reversion frequency is the titer of D30EBA virus on CV1 cells divided by the titer of D30EBA virus on M64A cells.
  • the titer of pHSVlac virus was divided by the titer of D30EBA virus on M64 cells to give the ratio of pHSVlac virus to D30EBA virus.
  • pHSVlac DNA 5 ⁇ g of DNA or 10 ng of pHSVlac DNA (isolated from E. coli HB101 as standard), were digested with 12.5 units of Eco RI overnight and resolved on 0.7% agarose gels. Following transfer to Nytran membrane (Schleicher and Schuell, Keene, NH) , hybridization was performed as described (Southern,
  • D30EBA DNA was detected by the same procedure except DNA was digested with Eco RI and Xho I
  • the probe was the 5.9 kb Eco RI fragment from the plasmid pCHHO (Hall, C. V. et al . , J. Molec. App. Genet. 2: 101-109 (1983)). This fragment contains the pBR sequences and most of the Lac Z gene, except for 133 bp at the 3' end.
  • pHSVlac contains three Eco RI sites, one at each end of the pBR segment and a third in the LacZ gene, 133 bp from the 3' end of the fragment ( Figure 1).
  • the 4.3 kb Eco RI fragment which contains most of the
  • pHSVlac DNA was properly and efficiently packaged into HSV-1 particles using the deletion mutant packaging system.
  • D30EBA DNA was detected by the same procedure, except that DNA was digested with Eco RI and Xho I, and the probe was a 659 bp fragment from the HSV-1 IE3 gene (nucleotides 1065 to 1724;
  • D30EBA contains a 3462 bp deletion in the IE3 gene
  • the expected size of the fragments are 5.1 kb (8.5 kb in ts K) and 2.1 kb (5.5 kb in ts K).
  • pHSVlac Virus Stably Expresses ⁇ -Galactosidase in Cultured Rat Sympathetic Neurons and Glia
  • pHSVlac virus prepared using the deletion mutant packaging system, to stably express ⁇ -galactosidase in neurons and glia was determined.
  • Cultured rat sympathetic neurons were infected with pHSVlac virus, and one week later an in situ assay for ⁇ -galactosidase was performed.
  • Dissociated neuronal cultures from superior cervical ganglia were prepared from four day old rats (Hawrot, E. and Patterson, P. H., Method in Enzymol. 58: 574-584 (1979)). Five days after plating, cultures were treated with 40 ⁇ M cytosine arabinoside for 24 hours to prevent glial overgrowth. One to two weeks later, cultures were infected with pHSVlac virus; at the time of infection, a culture contained approximately 5x10 5 cells and approximately 20% of the cells were neurons.
  • the ⁇ -galactosidase positive cells could arise from pHSVlac persisting in one cell for a week or from horizontal transmission of pHSVlac from one cell to another. If horizontal transmission occurred, then virtually all the cells in a culture would contain pHSVlac DNA and express ⁇ -galactosidase, and both D30EBA and pHSVlac virus would be present in the culture medium. In contrast, approximately 90% of the cells were ⁇ -galactosidase negative.
  • the culture medium contained less than 10 infectious particles of pHSVlac/ml and less than 10 pfu/ml D30EBA, below detection levels.
  • wild type HSV-1 kills all the cells in a culture in less than 24 hours.
  • pHSVlac packaged using ts K stably persists in cultured peripheral and CNS neurons for at least two weeks (Geller, A. I. and Breakefield, X. O., Science 241: 1667-1669 (1988);
  • pHSVlac virus prepared using the deletion mutant packaging system, efficiently infects and stably expresses ⁇ -galactosidase in cultured sympathetic neurons and glia.
  • pHSVth With the Deletion Mutant System and Expression of TH in Fibroblasts pHSVth was packaged using the D30EBA deletion virus and the M64A complementing cell line as described in Example 12, substituting pHSVth DNA for pHSVlac DNA. Titers were similar to those of pHSVlac packaged in the deletion mutant packaging system, in which the titers were about 10X higher than packaging with ts K helper virus.
  • CV1 cells were infected with pHSVth and were assayed one day post-infection for TH immunoreactivity using mouse anti-human TH antibody as primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse F(ab') 2 as secondary antibody essentially as described in Example 1. However, the multiplicity of infection was 0.1. The results were similar to those observed when the ts K virus was used to package pHSVth (Example 1). Uninfected cells and cells infected with a vector control, pHSVpUC (pHSV with the pUC19 polylinker replacing the TH insert), showed no significant staining above background.
  • pHSVpUC pHSVpUC
  • pHSVth vector was packaged into virus particles by the D30EBA virus and the complementing M64A cell line. pHSVth packaged by the deletion virus system infected fibroblasts, and directed production of TH as determined by immunoreactivity. Further, the observation that the percentage of cells stained was proportional to the m.o.i. indicates that expression occurred in the majority of cells that were infected by virus.
  • pIEA15 which contains the ICPO promoter of HSV-1 strain KOS, was digested with Nco I, and treated with deoxynucleotide triphosphates and Klenow fragment of DNA polymerase I to fill in the Nco I overhang.
  • the linearized vector was then digested with Hind III, and an approximately 1 Kb fragment containing the ICPO promoter was isolated. This fragment was ligated into the Sma I- Hind III site of bluescript, ablating the Sma I site, to make plasmid pO-Bst.
  • pO-Bst was digested with Nco I, treated with mung bean nuclease to ablate the Nco I site, and religated to make pO-1-Bst.
  • Defective Herpes virus vector pNFLlac was derived from vector pOHSVlac, which carries the ICPO promoter and adjacent HSV ori S (described above).
  • the ICPO promoter was removed by digesting pOHSVlac with restriction enzymes Hind III and Not I and the resulting 8 kb fragment isolated.
  • a Hind III-Not I fragment containing 2.2 kb of the human neurofilament L promoter Julien, J. P. et al. Genes and Dev. 1:
  • CV1 monkey fibroblasts, rat PC12 cells, and primary cultures of rat superior cervical ganglia (SCG) were infected with pNFLlac virus or control virus.
  • pNFLlac virus and control viruses, pOHSVlac and pHSVlac were packaged in the deletion mutant virus packaging system described in Example 12.
  • SCG superior cervical ganglia
  • the ratio of positive neurons to positive glia in pHSVlac virus infected cultures of superior cervical ganglia was about 1:4, which closely parallels the ratio of neurons to glia in the culture and suggests that the HSV-1 IE4/5 promoter in pHSVlac is expressed equally well in either cell type.
  • SCG cells there was about a
  • the neuronal protein GAP-43 is thought to be attached to the growth cone membrane via fatty acylation of the proteins's only two cysteine
  • the 5' end of the lacZ gene in pHSVlac consists of a gpt-trpS-lacZ fusion, as shown in Figure 8.
  • the gpt portion of the fusion gene was replaced by the coding sequence of the first 10 amino acids of human GAP -43 (Kosik, K. S. et al., Neuron 1: 127-132, (1988)) as follows. pHSVlac was digested to completion with Hind III. The Hind III cut pHSVlac was then partially digested with Asp718 and the appropriate restriction fragment was purified from a gel. This fragment was ligated with G10, which consisted of two
  • oligonucleotides annealed with each other that contain the coding sequence for the first 10 amino acids of human GAP-43 with Hind III and Asp718
  • cells were fixed with 0.5% glutaraldehyde for 15 minutes, washed three times for five minutes each with phosphate buffered saline. Subsequently, they were reacted with X-gal (5-bromo-4-chloro-3- indolyl-beta-D-galactopyranoside), a chromogenic substrate for beta-galactosidase, which generates a dark blue reaction product at the site of
  • Lac Z was also monitored by immunofluorescence as described (Geller, A. I. and Breakefield, X. O., Science 241: 1667-1669, (1988); Geller, A. I. and Freese, A., PNAS 87: 1149-1153 (1990)).
  • PC12 cells were differentiated for 6 days in NGF, and were infected with pHSVGAPlac. Virus was added to the culture medium two days before cells were fixed and incubated with antibody to
  • beta- galactosidase Following treatment with a rhodamine-conjugated secondary antibody, cultures were viewed under epifluorescence.
  • beta-galactosidase enzyme activity was demonstrated by X-gal staining (Geller, A. I. and Breakefield, X.
  • Example 12 packaged using the deletion virus packaging system described in Example 12, or with HSV-1 ts k as helper virus.
  • hippocampal neurons were prepared as described in Example 1 and were maintained for 12-21 days, then infected with virus two days before fixing and immunostaining as described (Geller, A. I. and
  • beta-galactosidase immunoreactivity was visualized with a rhodamine - conj ugated second antibody.
  • MAP-2 is a marker of dendritic processes.
  • fusion of the amino-terminal 10 amino acids of GAP-43 to beta-galactosidase targets the chimeric protein to neuronal processes. Fusion of the aminoterminal 10 amino acids of GAP-43 to
  • neuronal proteins provides a means of targeting recombinant molecules to the presynaptic membrane. This technology may be useful for enhancing the effects of recombinant presynaptic molecules
  • pHSVpkc ⁇ , pHSVpary, and pHSVCaCK Increase Monoamine and Excitatory Neurotransmitter Release in
  • Vectors containing the full length or catalytic domain of PKC were constructed.
  • the coding regions of the full length clone or the catalytic domain were fused to a ten amino acid peptide which is recognized by an antibody (Flag).
  • Flag an antibody
  • a synthetic duplex encoding a 10 amino acid Flag peptide was introduced into vector pHSVpUC to make pHSVflag.
  • pHSVlac was digested with Eco RI and Hind III and the vector fragment was purified. The removal of the Eco RI-Hind III fragment results in the excision of most of the lacZ gene from the pHSV vector.
  • a fragment encoding the pUC19 polylinker (Hind III-Eco RI) was inserted into the Eco RI and Hind III sites of the vector to make pHSVpUC.
  • a synthetic duplex of the following sequence was assembled and
  • the resulting duplex encodes the 10 amino acid Flag peptide (shown above) and has 5'-overhangs compatible with Hind III and Eco RI cut DNA.
  • pHSVpUC was cut with Hind III and Eco RI, and the vector portion was isolated and ligated to the synthetic duplex shown above to make pHSVflag.
  • the flag peptide is fused in frame via the Cla I linker to the coding region of PKC, beginning at nucleotide 994.
  • rat protein kinase C ⁇ -II, coding sequence was inserted into an HSV-1 vector.
  • PKC-II Knopf, J. L. et al., Cell 46: 491-502 (1986)
  • Cla I 8-mer linkers Cla I 8-mer linkers (New England Biolabs) were phosphorylated and ligated to the Fnu Dll-cut plasmid.
  • parvalbumin cDNA into an HSV-1 vector was BPV-CaMPV (Rasmussen, C. D. and A. R. Means, Molec. Endocrinol. 3(3): 588-595 (1989)).
  • BPV-CaMPV was digested to completion with Hind III, and partially digested with EcoRI. The Hind III-Eco RI fragment spanning the rat parvalbumin coding region was isolated.
  • Vector pHSVpUC was digested with Hind III and EcoRI, the vector portion was isolated and was ligated the to the Hind III-Eco RI parvalbumin fragment to make pHSVparv.
  • a cDNA encoding the catalytic domain of the ⁇ -subunit of the calcium/calmodulin dependent protein kinase type II (CaM-K- ⁇ ) from rat brain was cloned into an HSV-1 vector.
  • the HSV-1 construct encodes the amino terminal portion of CaM-K- ⁇ , from the nucleotides encoding the initiator methionine (codon 1) to the Xmn I site, which cuts within codon 292 (out of 478 amino acids).
  • the fragment was inserted Into pUC18, fusing the 3' end of the fragment to Hinc II-cleaved vector, thereby fusing the open-reading frame of the CaM-K fragment with the sequence of the polylinker.
  • This procedure introduces an in frame Arg-Leu-Stop sequence to produce an open reading frame with the following structure: (CaM-K- ⁇ Met 1 -Lys 291 )-(Arg-Leu)-Stop (numbering as in Lin, C.R. et al., Proc. Natl. Acad. Sci. USA 84: 5962-5966 (1987)).
  • a fragment encoding this gagated version of the gene was inserted downstream of the IE 4/5 promoter, replacing the lacZ sequence.
  • the resulting construct is named pHSVCaCK.
  • pHSVpkc, pHSVpck ⁇ , pHSVparv, and pHSVpUC constructs were packaged into HSV-1
  • pHSVCaCK was packaged using a
  • Amino acids were analyzed using a BAS 200A binary gradient high pressure liquid chromatography (HPLC) system (Bioanalytical Systems Inc., West Lafayette, IN) with a CMA200 autoinjector (Carnegie Medicin, Sweden) (Shea, P. A. and W.A.
  • HPLC high pressure liquid chromatography
  • the amino acids were derivatized using the autoinjector prior to injection.
  • BAS Phase II 100 x 3.2 mm 3 micron C 18 column.
  • the mobile phases used to achieve separation were 0.1 M acetic acid [pH 5.9], with an increasing
  • acetonitrile from 12% to 30% and tetrahydrofuran from 1.2 to 15 %.
  • chromatograms were complete within 15 minutes with separation of the major transmitter amino acids including aspartate, glutamate, taurine and GABA , with resolution in the majority of samples of serine, glycine, alanine, asparagine, threonine, histidine, methionine, and valine also.
  • Combination dual electrochemical 600 mV vs. a Ag/AgCl reference electrode (detector 1) and 700 mV (detector 2) and for selected samples ultraviolet (330 nm) detectors in series were used, with peak heights recorded on a chart recorder and compared to standards.
  • Assay sensitivity with a signal to noise ration of 5:1 ranged from 25 to 75 femtomoles.
  • pHSVpkc ⁇ rat parvalbumin
  • pHSVparv rat parvalbumin
  • pHSVCaCK catalytic domain of the rat ⁇ calcium/calmodulin protein kinase II
  • pHSVpkc and pHSVpck ⁇ were properly packaged into virus particles as determined by Southern analysis.
  • PLC protein kinase C
  • the coding regions were fused via a linker to the C-terminal end of a ten amino acid peptide (Flag), which is recognized by an anti-Flag antibody (e.g., M2, M5).
  • Flag ten amino acid peptide
  • M2, M5 anti-flag antibody e.g., M5 anti-flag antibody
  • expression of both the full length and catalytic domains from pHSVpkc and pHSVpek ⁇ , respectively, in both cultured sympathetic and cortical neurons was confirmed.
  • the pkc ⁇ protein produced by pHSVpkc ⁇ was expressed for at least 1 week in cultured sympathetic and occiptal cortex neurons, and was predominantly localized to cell bodies.
  • pHSVpkc ⁇ The ability of pHSVpkc ⁇ to affect neurotransmitter release in both sympathetic and cortical neurons suggests that PKC can alter some general, and therefore conserved, aspect of neuronal function which is likely to operate in most neurons.
  • the full-length protein kinase C had no observable effect on the cells in this assay because the wild type portien kinase C is strictly
  • Sympathetic and cortical neurons infected with the catalytic domain of the calcium/calmodulin dependent protein kinase II displayed a similar pattern of neurotransmitter release (i.e., increased neurotransmitter release in the presence of
  • parvalbumin a vector expressing parvalbumin, pHSVparv, was constructed and used to introduce parvalbumin into neurons which do not normally contain it. Expression of parvalbumin in cultured sympathetic and cortical neurons was detected using an antibody directed against parvalbumin. Table 8 shows the effect of parvalbumin on neurotransmitter release in these cell types. As shown in Table 8, parvalbumin directed a long term increase in neurotransmitter release from both sympathetic and cortical neurons in both the basal state and following depolarization.
  • Ungerstedt and Arbuthnott originally described asymmetric rotation of rats following the unilateral lesioning of the substantia nigra (Ungerstedt, U. and G. W. Arbuthnott, Brain Res. 24: 485-493 (1970)).
  • DA post-synaptic dopamine
  • amphetamine produces ipsilateral turning, as it produces a much greater increase in extracellular dopamine in the intact striatum as compared with the lesioned side.
  • the striatal dopamine system is able to downregulate these receptors in response to
  • transduction factor such as the catalytic domain of protein kinase C
  • the transduction factor into the substantia nigra can cause an increase neuronal activity and stimulated dopamine release sufficient to result in
  • constructs pHSVpkc ⁇ and pHSVpUC were packaged into virus particles using the D30EBA deletion virus and M64A helper cell line. The virus was concentrated approximately 80-fold by the following procedure
  • Rats were tested for rotational behavior. Rats were administered apomorphine (1 mg/kg) intraperi toneally and placed in a hemispherical plexiglass rotometer. This dose of apomorphine elicits rotational
  • the rat NGF gene was kindly provided by Dr. G. Heinrich, Boston University; exon IV encodes all of pre-pro NGF.
  • a hybrid mouse/rat NGF mini-gene was constructed which lacks the sequences from 90 bp into the first intron to a point within intron III which is 277 bp before the start of exon IV.
  • the rat NGF mRNA contains an AU rich sequence in its 3' untranslated region (Scott et al., Nature 302: 538-540 (1983)) that may
  • the 3' untranslated (3' UT) region was replaced with the 3' UT region from the human growth hormone (HGH) gene, whose mRNA is long lived.
  • HGH human growth hormone
  • the NGF minigene used in pHSVngf is composed of three parts from 5' to 3': (1) a non-coding Exon
  • pGEM7z was digested with Xho I, the Xho I site was made blunt by a fill-in reaction with Klenow and dNTPs , and the product was digested with Bam HI.
  • mice Exon I-Intron I fragment was excised with Hae II and Bam HI and inserted into pGEM7z at the blunted Xba I site and Bam HI site.
  • the resulting pGEM7z construct was then cut with Xba I and Bam HI to release a 118 bp fragment carrying 107 bases of mouse NGF Exon I- Intron I.
  • nucleotide 277 bp before exon IV which encodes rat NGF Intron III-Exon IV region was cleaved with Pst I (at a site 1024 bp downstream of the 5'-Eco RI site), and made blunt by removing the 3' overhang to make a 1022 bp fragment.
  • This fragment was ligated to a human growth hormone Bgl II- Eco RI fragment (nucleotides 1859-2657), which had been made blunt at the Bgl II site.
  • the fusion fragment was
  • pHSVngf was packaged into HSV-1 particles using the D30EBA deletion mutant and M64A helper cell line packaging system (Example 12).
  • the titer of the pHSVngf virus stock was 5 X 10 5 infectious
  • NIH 3T3 cells were cultured in DMEM containing 10% fetal calf serum and 5% horse serum, at 37oC in an atmosphere of 5% CO 2 .
  • PC12 cells were cultured in 90% DMEM and 10% fetal calf serum.
  • Cells were plated in wells (5 x 10 4 cells/cm 2 )) and allowed to grow for 48 hours before infection. Cells were infected with 20 ⁇ l of unconcentrated virus. Eight hours later the virus containing media was removed, and 1.0 ml of fresh media was added.
  • the media was harvested twenty four hours later; alternatively, the media was harvested 48 hours later for determining the ability of the secreted NGF to support sympathetic neuron survival in a bioassay. pHSVngf Virus Can Direct the Synthesis of NGF in Cultured Fibroblasts
  • the media contained ⁇ 12 ng/ml of NGF
  • the 2-site ELISA was also used to determine the rate of NGF production in pHSVngf-in-ected 3T3 fibroblasts. Infection of 3T3 cells with 20 ⁇ l of pHSVngf virus, containing approximately 50,000 infectious particles (determined by immunostaining
  • a neonatal sympathetic neuron survival assay (Lindsay, R. M., Nature 282: 80-82 (1979)) was used to measure the amount of biologically active NGF produced after infection of cultured cells with pHSVngf virus.
  • pheochromocytoma cells were cultured and infected with pHSVngf virus or mock infected as described above. The inoculum was replaced with fresh medium, and 48 hours later the media was harvested and assayed for NGF bioactivity in the neonatal
  • SCG superior cervical ganglia
  • NGF exogenous NGF
  • the conditioned medium from pHSVngf-infected 3T3 cells or pHSVngf-infected PC12 cells dramatically increased sympathetiic neuron survival as compared with media from mock infected cultures.
  • the data indicate that infection of both PC12 and NIH 3T3 cells with increasing amounts of pHSVngf virus particles results in progressively greater amounts of secreted bioactive NGF, as assayed by sympathetic neuron survival.
  • the neuron counts represent the mean of duplicate plates. Infection with pHSVngf Increases Survival of Primary Neonatal Sympathetic Neurons in Culture
  • surviving neurons were present in pHSVngf virus infected wells, while in the mock infected or pHSVlac virus infected control wells, only about 2500 surviving neurons were present. At 6 days, about 5000 surviving neurons were present in pHSVngf virus infected wells compared with about 1000 surviving neurons in the mock infected or pHSVlac virus infected control wells. The numbers of neurons surviving at either 4 or 6 days in pHSVngf infected cultures were significantly greater (P ⁇ 0.05) than mock or pHSVlac infected cells. Positive control cultures containing 100 ng/ml NGF had mean neuron survival numbers of 3,400 and 3,550 on days 4 and 6, respectively.
  • pHSVngf infection can prolong the survival of primary sympathetic neurons.
  • pHSVngf was packaged into HSV-1 particles using the D30EBA deletion mutant and M64A helper cell line packaging system (Example 12).
  • the titer of the pHSVngf virus stock was 5 X 10 5 infectious
  • ganglion served as an internal control. Ten days later, both the ipsilateral (experimental) and contralateral (control) ganglia were removed and assayed for TH activity. Measurement of Tyrosine Hydroxlase Activity
  • Each ganglion was homogenized in 75 ⁇ l of distilled water in a glass/teflon homogenizer. Ten ⁇ l of each homogenate were assayed for TH activity by previously published methods (Kessler, J. A. and I. B. Black, Brain Res. 171: 415-424 (1979)) using tetrahydrobiopterin as cofactor. pHSVngf Virus Can Prevent Some of the Effects of Axotomy of SCG in vivo
  • NGF is synthesized and secreted by target tissues of sympathetic neurons. NGF is taken up by sympathetic neurons and
  • noradrenergic neurotransmitter system in part by stimulating TH synthesis.
  • SCG ganglion
  • the SCG of the adult rat contains the cell bodies of sympathetic neurons whose axons project to target tissues in the head and neck which produce NGF.
  • Unilateral axotomy of a SCG interrupts its NGF supply and results in an ipsilateral decline in TH activity over a 10 day period.
  • NGF can prevent the decline in TH activity observed in the axotomized ganglion, the effect of direct injection of a SCG with pHSVngf virus was studied for a similar protective effect. Virus was packaged and concentrated as
  • concentrated pNFlac (Example 17), another defective HSV-1 vector which expresses E. coli ⁇ -galactosidase instead of NGF, or with saline.
  • a total of 10 rats were injected with pHSVngf virus and 9 rats were injected with pNFlac virus.
  • neurons different cell types including neurons and
  • pheochromocytoma cells SCG cells
  • enzyme levels e.g., TH levels
  • NGF-producing hippocampal neurons When these cholinergic neurons are disconnected from their NGF-producing target cells by axotomy, they are disconnected from their NGF-producing target cells by axotomy.
  • ChAT choline acetyl- transferase
  • HSV-1 virus constructs were made encoding (1) the human low affinity nerve growth factor receptor (NGFR), (2) a truncated mutant NGFR which has a premature termination signal shortly after the transmembrane domain coding sequence, and (3) a mutant version of the NGFR which contains a large deletion in the ligand binding domain.
  • NGFR human low affinity nerve growth factor receptor
  • Hempstead et al. have described the construction of a mutant cDNA of the NGFR having a stop codon four amino acids after the transmembrane domain at amino acid 940 (pXba; Hempstead, B. L. et al., J. Biol.
  • a third construction was designed which encodes a mutant form of the low affinity NGF receptor which lacks the presumptive ligand binding domain.
  • the 154 amino acids (462 base pairs) between residues 7 and 162 of the NGFR are deleted (7 and 162 are present).
  • the 2.3 kb Eco RI-Eco RI fragment containing the p75 NGFR coding sequence was cloned into the Eco RI site of pT7/T3 (Bethesda Research Laboratories).
  • the resulting plasmid was digested with Stu I and was partially digested with Sau 3A.
  • the Sau 3A overhang was filled in using Klenow fragment and dNTPs.
  • the construct was then religated to delete the region between the StuI and Sau 3A sites.
  • pDB3 was also confirmed by DNA sequencing.

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Abstract

Procédé d'altération du niveau d'un produit génique dans une cellule neuronale ou non neuronale, dans laquelle une séquence de nucléotides codant le produit génique désiré est insérée dans un vecteur d'Herpès virus défectueux, tel qu'un vecteur de HSV-1 déflectueux, de manière à ce que l'expression du produit génique soit sous le contrôle du promoteur. Le vecteur de virus défectueux ainsi obtenu est encapsidé en particules vitales par introduction dudit vecteur obtenu dans une lignée cellulaire en même temps qu'un virus auxiliaire mutant d'Herpès, tel qu'un virus auxiliaire mutant HSV-1, et en permettant au virus de se propager. Des cellules cibles sont infectées par le virus encapsidé et le produit génique codé est exprimé. En particulier, un procédé de production de tyrosine hydroxylase, de facteur de croissance des neurones et de plusieurs facteurs de transduction de signaux dans des cellules neuronales est décrit.
PCT/US1991/007993 1990-10-30 1991-10-23 Alteration specifique du type cellulaire des niveaux de produits geniques dans des cellules WO1992007945A1 (fr)

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EP2270175A1 (fr) 2001-03-27 2011-01-05 Novartis Vaccines and Diagnostics S.r.l. Acides nucléiques et protéines de Streptococcus pneumoniae
EP2314697A1 (fr) 2001-03-27 2011-04-27 Novartis Vaccines and Diagnostics S.r.l. Acides nucléiques et protéines de Streptococcus pneumoniae
EP2278010A1 (fr) 2001-03-27 2011-01-26 Novartis Vaccines and Diagnostics S.r.l. Acides nucléiques et protéines de Streptococcus pneumoniae
EP2270176A1 (fr) 2001-03-27 2011-01-05 Novartis Vaccines and Diagnostics S.r.l. Acides nucléiques et protéines de Streptococcus pneumoniae
EP2335723A1 (fr) 2001-12-12 2011-06-22 Novartis Vaccines and Diagnostics S.r.l. Immunisation contre la Chlamydia trachomatis
EP2335724A1 (fr) 2001-12-12 2011-06-22 Novartis Vaccines and Diagnostics S.r.l. Immunisation contre la Chlamydia trachomatis
EP2279746A2 (fr) 2002-11-15 2011-02-02 Novartis Vaccines and Diagnostics S.r.l. Proteines de surface de neisseria meningitidis
EP2267005A1 (fr) 2003-04-09 2010-12-29 Novartis Vaccines and Diagnostics S.r.l. Toxine ADP-ribosylante de Listeria monocytogenes
WO2005093064A1 (fr) 2004-03-29 2005-10-06 Galpharma Co., Ltd. Protéine de modification de galectine-9 novatrice et utilisation de celle-ci
WO2008020335A2 (fr) 2006-06-09 2008-02-21 Novartis Ag Compositions immunogènes pour streptococcus agalactiae

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