EP1005375A1 - Stress oxydatif genere par traitement photodynamique pour l'expression temporaire et selective de genes heterologues - Google Patents

Stress oxydatif genere par traitement photodynamique pour l'expression temporaire et selective de genes heterologues

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Publication number
EP1005375A1
EP1005375A1 EP98910249A EP98910249A EP1005375A1 EP 1005375 A1 EP1005375 A1 EP 1005375A1 EP 98910249 A EP98910249 A EP 98910249A EP 98910249 A EP98910249 A EP 98910249A EP 1005375 A1 EP1005375 A1 EP 1005375A1
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Prior art keywords
gene
promoter
vector
expression
heat
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German (de)
English (en)
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Charles J. Gomer
Sam Keng Sum Wong
Angela Ferrario Nehme
Marian Coensgen Luna
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Research Development Foundation
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Research Development Foundation
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates generally to the clinical treatment of solid tumors. More specifically, the present invention relates to inducing expression of therapeutic genes in a controlled and localized manner.
  • Photodynamic therapy is a clinical treatment of solid malignancies (Fisher, A.M.R., et al., Laser Surgery Medicine 17:2- 31 (1995); Marcus, S.L. and Dugan, M.H., Laser Surgery Medicine, 12: 318-24 ( 1992); and Henderson, B .W. and Dougherty, T.J., Photochem. Photobio l., 55 :931 -48 ( 1992)) .
  • Properties of photosensitizer localization in tumor tissue and photochemical generation of reactive oxygen species are combined with precise delivery of laser generated light to produce a procedure offering effective local tumoricidal activity (Henderson, B .W.
  • Photofrin is the photosensitizer used in the majority of clinical trials.
  • Photofrin-mediated PDT has recently received FDA approval for treatment of esophageal carcinoma and this compound has also received regulatory approval in Canada, the Netherlands and Japan.
  • PDT shows considerable promise for treating tumors of the bronchus, bladder, skin, head/ neck and cervix as well as for non- malignant disorders such as psoriasis and age-related macular degeneration.
  • Second generation photosensitizers undergoing clinical trials include tin etiopurpurin (SnET2), mono-1-aspartyl chlorin e6 (NPe6) , benzoporphyrin derivative (B PD) , meso-tetra- (hydroxyphenyl) chlorin (mTHPC) and 5-amino levulinic acid (ALA).
  • SnET2 tin etiopurpurin
  • NPe6 mono-1-aspartyl chlorin e6
  • B PD benzoporphyrin derivative
  • mTHPC meso-tetra- (hydroxyphenyl) chlorin
  • ALA 5-amino levulinic acid
  • ALA is a metabolic precursor of endogenous protoporphyrin IX.
  • PDT induces expression of early response genes (c-fos, c-jun, c-myc, egr-1) as well as stress protein genes belonging to the heat shock proteins (HSP), glucose regulated proteins (GRP) and heme oxygenase families (Luna, M.C., et al, Cancer Res., 54: 1374-80 (1994); Gomer, C.J., et al., Photochem. Photobiol. , 53 :275-279 (1991 ); Gomer, C.J., et al., Cancer Res., 51 :6574-79 ( 1991 ); and Gomer, C.J., et al., Cancer Res. (In press) (1997)).
  • Apoptosis is also induced by PDT and appears to involve a signal transduction pathway originating at the cell membrane (Agarwal, M.L., et al, Cancer Res., 51 :5993-96, ( 1991); He, X.Y., et al., Photochem. Photobiol. 59:468-73 (1994) and Zaidi, S.I.A., et al., Photochem. Photobiol. 58:771-76 ( 1993)). Characteristic DNA fragmentation, chromatin condensation, and activation of a constitutive endonuclease is observed in photosensitized cells. Apoptosis has also been identified in vivo as an early event in PDT-treated tumors.
  • Heat shock is one of the best- characterized of these stress responses.
  • heat shock exposure to high but nonlethal temperatures (heat shock) and to certain chemicals causes the appearance of new puffs on the salivary gland Drosophila busckii. These puffs, clearly visible in the light microscope, represent high transcriptional activity on heat shock-inducible genes, and RNA synthesis that generates the messenger RNAs for the heat shock proteins. More recently, a wide range of organisms, from bacteria to higher vertebrates, has been shown to display similar dramatic changes in gene expression with heat shock.
  • the response to heat shock entails both strong induction of genes for heat shock proteins (HSPs) and repression of most genes that were being expressed previous to the induction. There are marked changes both to patterns of gene transcription and to the way in which mRNAs are selected for translation by the protein- synthesizing machinery of the cell.
  • HSPs heat shock proteins
  • the transcriptional changes with heat shock are due to the presence of a heat shock element in heat shock gene promoters.
  • This element is a DNA sequence needed for specific induction of transcription in response to heat shock.
  • the concensus sequence for the heat shock element is CTGCCACCC at nucleotide positions -44 to -36 relative to the transcription initiation site.
  • the heat-inducible promoters of eukaryotes it is contiguous repeats of the 5-bp sequence NGAAN, arranged in alternative orientation positioned upstream of the TATA box element.
  • the heat shock response is usually induced by a temperature upshift.
  • the optimal temperature for induction is species dependent, but it is usually a degree or two above the maximum that permits growth. Also, the response is generally transient.
  • Glucose Regulated Proteins with molecular weights of 78,000 and 94,000, share sequence homology with heat shock proteins.
  • the GRP family of proteins is coordinately induced by glucose starvation, anoxia, alterations in intracellular calcium, exposure to inhibitors or glycosylation as well as by PDT-mediated oxidative stress (Gomer, C.J., et al., Cancer Res. 51 :6574-79 (1991 ); and Li, L-J., et al., J. Cell Physiol. 153:575-82 (1992)).
  • the 78,000 GRP is identical in sequence to the immunoglobulin heavy chain binding protein and both GRP78 and GRP94 are localized in the endoplasmic reticulum (ER).
  • GRP78 binds transiently to nascent, secretory and transmembrane proteins and binds permanently to abnormally folded or processed proteins in the ER. GRP78 is thought to have a protective function during and after cellular stress when protein processing in the ER is perturbed.
  • Lee et al. have shown that transcriptional activation of grp78 by tumor hypoxia can be exploited for targeted gene therapy.
  • a truncated grp78 promoter with most of the basal elements removed was shown effectively to drive high level expression of a reporter gene in hypoxic mouse tumors (Gazit, G., et al., Cancer Res. 55 : 1660-63 ( 1995)) .
  • the present invention demonstrates that PDT-mediated oxidative stress is a strong transcriptional transducer of stress proteins, specifically those belonging to the heat shock proteins.
  • the present invention is drawn to methods of targeted gene therapy using recombinant constructs with HSP- or GRP-inducible promoters to drive high-level, local expression of cytotoxins or immunomodulators to enhance PDT tumoricidal action.
  • One object of the present invention is to provide a novel method for enhancing locally the tumoricidal (or anti-angiogenic) properties of PDT (or heat) by using these therapeutic modalities to act as a molecular switch for the spatial and temporal expression of genes to enhance or act synergistically with the direct effects of PDT.
  • the present invention is superior to current strategies which attempt to deliver localized cytotoxic gene therapy using constitutively-acting promoters, i.e. viral delivery systems using tissue-specific receptors or tissue-specific enhancers that limit gene transcription to a select group of cells.
  • the temporal regulation of gene expression is not possible using constitutively-acting promoters due to the high basal expression of cytotoxic gene productions in normal cells and tissue.
  • the present invention (combining inducible gene therapy and PDT or heat) enhances local tumor control without increasing normal tissue toxicity (increasing the therapeutic gain).
  • Transcriptional activation of a PDT or heat inducible promoter is controlled within a given tissue volume and for a specific time period. This procedure, therefore, exploits both the direct cytotoxicity induced by PDT or heat and the targeting potential of PDT or heat to induce spatial and temporal regulation of cytotoxic gene transcription.
  • PDT- or heat-targeted gene therapy uses a construct containing a PDT- or heat-inducible promoter upstream of a cDNA encoding a heterologous gene which is activated transcriptionally within the PDT or heat treatment field to enhance local cytotoxicity.
  • Weichselbaum et al. has reported on a similar procedure using ionizing radiation (Weichselbaum, R.R., et al., Cancer Res. 54:4266-69 ( 1994)).
  • One object of the present invention is to provide a method of temporal and localized treatment of a target tissue in an individual comprising the steps of: administering an expression vector to said individual, wherein said vector expresses a therapeutic, heterologous gene under control of a promoter inducible by photodynamic therapy or heat, and exposing said target tissue to said photodynamic therapy or heat.
  • Various embodiments of this particular object of the invention include generating heat by thermal laser, microwaves, ultrasound or radiofrequency waves.
  • a method of temporal and localized treatment of a target tissue in an individual comprising the steps of: administering a photosensitizer for photodynamic therapy to said individual; administering an expression vector to said individual, wherein said vector expresses a therapeutic, heterologous gene under control of a promoter inducible by photodynamic therapy; allowing said photosensitizer and said expression vector to be taken up by said target tissue; and exposing said target tissue to light, wherein said light combined with said photosensitizer will generate reactive oxygen species to induce said promoter inducible by photodynamic therapy, causing said therapeutic, heterologous gene to be expressed.
  • a particular embodiment of this object of the present invention includes a photosensitizer is selected from the group of photofrin, tin etiopurpurin, mono-1- aspartyl chlorin e6, benzoporphyrin derivative, meso-tetra- (hydroxyphenyl)chlorin and 5-amino levulinic acid.
  • both objects of the method of the present invention include administering the vector systemically or locally.
  • Preferred embodiments of the vector of the present invention include cases wherein said vector is a retroviral vector, an adeno-associated viral vector, or a liposomal DNA vector.
  • Particular preferred embodiments of both objects of the present invention are where said promoter is a heat shock protein (hsp) promoter or a glucose regulated protein promoter.
  • preferred embodiments include wherein said heterologous gene is an immunomodulatory gene, particularly a cytokine.
  • embodiments include where said heterologous gene is a tumor suppressor gene, an anti-sense DNA, or an anti-angiogenic gene.
  • a preferred embodiment includes where said target tissue is a tumor or an area of abnormal tissue growth.
  • Figures 1-5 demonstrate the selective and temporal expression of the beta-galactosidase ( ⁇ -gal) reporter gene in RIF HB-3 cells exposed to either heat or PDT:
  • Figure 1 Beta-galactosidase activity (measured by using ONPG (o-nitrophenyl-pyranogalactose) as a substrate) in transfected RIF HB-3 cells treated with heat (45° C) for 1.5, 10, 20, 30 and 40 minutes. Samples were collected six hours after hyperthermia. Each data point represents the average of at least three separate experiments ⁇ S.E.
  • Beta-galactosidase activity (measured by using ONPG as a substrate) in RIF HB-3 cells treated with NPe6- mediated PDT at doses ranging from 600 Joules/cm ⁇ to 5400 Joules/cm ⁇ . Samples were collected six hours after light exposure. Each data point represents the average of at least three separate experiments + S.E.
  • FIG. 3 Kinetics of Beta-galactosidase expression (measured by using ONPG as a substrate) in RIF HB-3 cells treated at 45°C for 20 minutes. Each data point represents the average of at least three separate experiments +_ S.E. Enzyme activity was detected three hours after heat treatment (19 milliunits/mg protein). The peak values of ⁇ -gal activity (100 and 125 milliunits/mg protein) were reached at six and twelve hours following heat exposure.
  • Figure 4 Kinetics of Beta-galactosidase expression (measured by using ONPG as a substrate) in RIF HB-3 cells treated with NPe6- PDT at a light dose of 3000 Joules/cm ⁇ . Each data point represents the average of at least three separate experiments ⁇ S.E. A minimal level of enzyme activity was detected three hours after treatment. Peak activity was reached between six and twelve hours after NPe6 mediated PDT.
  • Beta-galactosidase activity (measured by using ONPG as a substrate) from RIF HB-3 tumors growing in C3H/HeJ mice and treated with either heat or NPe6-PDT. Heat treatment consisted in 20 minutes exposure at 45 °C. Three doses of light were tested for PDT. Each column represents the average of five distinct measurements +. S.E. The largest response was observed following hyperthermia (8.25 milliunits/mg protein). NPe6-PDT induced ⁇ -gal enzyme activity ranged between 1.08 and 3.48 milliunits/mg protein. No measurable levels were recorded for control tumors nor for tumors treated only with light or NPe6.
  • Figures 6 through 12 demonstrate the selective and temporal expression of the chloramphenicol acetyl transferase (CAT) reporter gene in RIF HC-2 cells and RIF RHC-7 cells exposed to either heat or PDT Figure 6.
  • Figure 7. CAT activity in transfected RIF RHC-7 cells treated with heat (45°C) for 1.5, 10, 20, and 40 minutes.
  • FIG. 9 Kinetics of CAT activity in transfected RIF HC-2 cells treated at 45° C for 20 minutes. Samples were collected 3-48 hours after heat exposure. Conversion of chloramphenicol to acetylated chloramphenicol was calculated by counting radioactivity from resulting TLC plates.
  • FIG. 10 Kinetics of CAT activity in transfected RIF RHC-7 cells treated at 45°C for 20 minutes. Samples were collected 3-48 hours after heat exposure. Conversion of chloramphenicol to acetylated chloramphenicol was calculated by counting radioactivity from resulting TLC plates.
  • FIG 11. CAT activity from RIF HC-2 cells and RIF HC-2 tumors growing in C3H/HeJ mice and treated with either water bath heat for cells or laser induced heat for tumors. Heat treatment consisted of 20 minutes exposure at 45 °C. Cell culture samples were collected 24 hours after heating and tumor samples were collected 3, 6 or 24 hours after heating No measurable CAT activity levels were recorded for control tumors.
  • Figure 12. CAT activity from RIF HC-2 and RIF RHC-7 tumors growing in C3H/HeJ mice and treated with either laser induced heat, NPe6-PDT or PH-PDT. Heat treatment consisted in 20 minutes exposure at 45°C. No measurable levels were recorded for control tumors or for tumors treated only with light, NPe6 or PH.
  • the term "photodynamic therapy” or “PDT” refers to the treatment of solid tumors with visible light (usually generated by non-thermal lasers) following the systemic administration of a tumor localizing photosensitizer (see Fisher, A.M.R., et al., Laser Surgery Medicine 17:2-31 (1995); Marcus, S.L. and Dugan, M.H., Laser Surgery Medicine, 12: 318-24 (1992); and Henderson, B .W. and Dougherty, T.J., Photochem. Photobiol. , 55 :931 -48 ( 1992)).
  • PDT is used clinically to treat various types of solid tumors (esophagus, bronchus, bladder, brain, eye, head/neck, skin, cervical as well as non-malignant diseases such as age related macular degeneration and psoriasis.
  • Various photosensitizers including Photofrin (PH), tin etiopurpurin (SnET2), mono-1- aspartyl chlorin e6 (NPe6), benzoporphyrin derivative (BPD), meso-tetra-(hydroxyphenyl) chlorin (mTHPC) and 5-amino levulinic acid (ALA) are used in PDT.
  • PH Photofrin
  • SnET2 tin etiopurpurin
  • NPe6 mono-1- aspartyl chlorin e6
  • BPD benzoporphyrin derivative
  • mTHPC meso-tetra-(hydroxyphenyl) chlorin
  • ALA 5-amino levulinic acid
  • heat shock gene refers to a gene which is transcribed at a high level in response to elevated temperature .
  • CAT refers to an assay used to assess in vivo effectiveness of eukaryotic promoter sequences.
  • the CAT gene codes for CAT, which inactivates the antibiotic chloramphenicol by acetylating the drug at one or both of its two hydroxyl groups. Because eukaryotic cells do not synthesize CAT, the gene has been exploited as a reporter gene for analysis of promoters, particularly in mammalian cells.
  • CAT is assayed by a method based on thin-layer chromotography in which [ 14 C] chloramphenicol can be separated from the acetylated and inactive derivatives which are only synthesized in the presence of the CAT enzyme.
  • ⁇ -gal or " ⁇ -galactosidase assay” refers to an assay used to assess in vivo effectiveness of eukaryotic promoter sequences.
  • ⁇ -galactosidase is an enzyme which hydrolyzes ⁇ -galactosides such as lactose into component sugars by hydrolysis of terminal nonreducing ⁇ -galactose residues.
  • the E. coli LacZ gene is used as a reporter gene in studies of promoter action as a translational in-frame fusion between a gene of interest and the LacZ gene puts lacZ expression under the control of the promoter under investigation. The activity of the promoter can then be assayed by measuring the ⁇ -galactosidase activity using o-nitrophenyl-pyranogalactose as a substrate.
  • glucose regulated protein As used herein, the term "glucose regulated protein" or
  • GRP refers to the family of proteins that is induced coordinately by glucose starvation, anoxia, alterations in intracellular calcium, exposure to inhibitors or glycosylation as well as by PDT mediated oxidative stress conditions.
  • tumor suppressor gene refers to a class of genes believed to be involved in different aspects of normal control of cellular growth and division. The common characteristic of these genes is that it is their inactivation, usually by genetic means, which contributes to tumor development.
  • immunomodulatory gene refers to a gene the expression of which modulates the course of an immune reaction to a specific stimulus or a variety of stimuli.
  • cytokine refers to a small protein produced by cells of the immune system that can affect and direct the course of an immune response to specific stimuli.
  • anti-angiogenic gene refers to genes coding for proteins which reduce or terminate the formation of blood vessels.
  • antisense DNA refers to DNA which codes for an antisense RNA.
  • antisense RNA has the potential to form an RNA-RNA duplex with the natural 'sense' mRNA transcript of a gene, thereby preventing its translation.
  • Antisense RNA provides a means of inactivating the expression of specific genes and can be applied to both simple and complex eukaryotes .
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a vector is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient mammal.
  • agent is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a change in the physiology of a recipient mammal. For example, in the treatment of retroviral infection, a compound which decreases the extent of infection or of physiologic damage due to infection, would be considered therapeutically effective.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • An "origin of replication” refers to those DNA sequences that participate in DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences , such as promoters , enhancers , polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease S I), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine- Dalgarno sequences in addition to the - 10 and -35 consensus sequences.
  • Various promoters may be used to drive vectors.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a “signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes .
  • the present invention is directed to a novel method for enhancing locally the tumoricidal (or anti-angiogenic) properties of PDT (or heat) by using these therapeutic modalities to act as a molecular switch for the spatial and temporal expression of genes to enhance or act synergistically with the direct effects of PDT.
  • the present invention is superior to current strategies which attempt to deliver localized cytotoxic gene therapy using constitutively-acting promoters, i.e. viral delivery systems using tissue-specific receptors or tissue-specific enhancers that limit gene transcription to a select group of cells.
  • the temporal regulation of gene expression is not possible using constitutively- acting promoters due to the high basal expression of cytotoxic gene productions in normal cells and tissue.
  • the present invention enhances local tumor control without increasing normal tissue toxicity (increasing the therapeutic gain) .
  • Transcriptional activation of a PDT or heat inducible promoter is controlled within a given tissue volume and for a specific time period. This procedure, therefore, exploits both the direct cytotoxicity induced by PDT or heat and the targeting potential of PDT or heat to induce spatial and temporal regulation of cytotoxic gene transcription.
  • PDT- or heat-targeted gene therapy uses a construct containing a PDT- or heat-inducible promoter upstream of a cDNA encoding a heterologous gene which is activated transcriptionally within the PDT or heat treatment field to enhance local cytotoxicity.
  • the present invention provides a method of temporal and localized treatment of a target tissue in an individual comprising the steps of: administering an expression vector to said individual, wherein said vector expresses a therapeutic, heterologous gene under control of a promoter inducible by photodynamic therapy or heat, and exposing said target tissue to said photodynamic therapy or heat.
  • the present invention describes a method for the selection and temporal expression of heterologous genes.
  • the invention consists of two methods to obtain selective and temporal expression of heterologous genes both in cells grown in culture and for cells grown in- vivo as solid tumors.
  • the human heat shock protein (hsp) promoter is used to express genes of interest under conditions of laser induced heating or Photodynamic Therapy- (PDT-) induced oxidative stress.
  • Selective and temporal expression of heterologous genes (such as cytokines, toxins, tumor suppressor genes, antisense molecules and anti-angiogenic factors) are of significant therapeutic benefit in the treatment of tumors, vascular proliferation and tissue hypertrophy.
  • Gene therapy targeted by laser induced heating, other heating sources such as microwave, ultrasound or radiofrequency induced currents) enhances treatment effectiveness by inducing expression of therapeutic genes in a controlled and localized manner.
  • REF Radiation-induced fibrosarcoma cells
  • Plasmids from StressGen Biotech Corp p2500- CAT (providing inducible expression of chloramphenicol acetyl transferase (CAT) under the control of the hsp70 promoter) and pl730R (providing inducible expression of beta galactosidase ( ⁇ - gal) under the control of the hsp70 promoter) as well as plasmid pMC lNeo from Stratagene (providing constitutive expression of the neomycin resistance gene under the control of a thymidine kinase promoter) were grown in supercompetent E. coli, isolated following alkaline lysis and purified.
  • CAT chloramphenicol acetyl transferase
  • pl730R providing inducible expression of beta galactosidase ( ⁇ - gal) under the control of the hsp70 promoter
  • plasmid pMC lNeo from Stratagene (providing constitutive expression
  • E ach S tre s s G en expression plasmid was co-transfected into RIF-1 cells along with pMC lNeo (5 to 1 ratio) using the calcium phosphate DNA precipitation technique. The cells were grown in 600 ⁇ g/ml G418 and resulting colonies were picked using cloning rings. G418 resistant clones were expanded and examined for ⁇ -gal or CAT activity using heat as a positive inducer. Positive clones were then tested for activity following PDT. Individual RIF-1 clones which expressed either ⁇ -gal or CAT activity following PDT were obtained.
  • the 3 cell lines used to prove that heat or PDT could be used as a switch for turning on heterologous reporter genes are: 1 .
  • RIF HC-2 cells which express CAT under the control of a 2.5 kb hsp promoter;
  • RIF HB-3 cells which express beta galactosidase under the control of a 2.5 kb hsp promoter; and 3. RIF RHC-7 cells which were obtained by transducing a modified GINA retroviral vector (containing a 300 bp hsp promoter ligated to the chloramphenicol acetyl transferase gene plus the phosphotransferase (neo resistance) gene constitutively expressed from the 5' LTR). Stable transfectants were established by growth in G-418 and then screened for heat induced expression of chloramphenicol acetyl transferase activity.
  • All three of these cell lines have stably integrated expression vectors and all cell lines are injected into the flank of C3H/HeJ mice to produce solid fibrosarcoma tumors.
  • Photofrin PH
  • mono-1-aspartyl chlorin e6 Npe6
  • tin etio purpurin Sn ET2
  • Cells (2xl0 6 ) were seeded in 100 mm Petri dishes 24 hours prior to a 16 hour incubation with one of the photosensitizers.
  • Photosensitizer incubation concentrations were 25 ⁇ g/ml for PH and N ⁇ e6 and 0.75 ⁇ g/ml for SnET2.
  • Cells were incubated with the photosensitizer in the dark for 16 hours in media containing 5% serum. The cells were then rinsed for 30 minutes in media containing 15% serum and then exposed to graded doses of red light.
  • Hyperthermia treatments of tumors measuring 6-7 mm in diameter consisted of a 20 minute exposure to 44.5-45 °C . These temperatures were achieved by irradiating the tumors with a diode laser emitting 810 nm laser light at power density of 270 mW/cm ⁇ .
  • Photodynamic therapy (PDT) treatments of tumors measuring 6-7 mm in diameter consisted of an intravenous injection of either PH or Npe6 at 5 mg/kg or SnET2 at 1.5 mg/kg.
  • Non-thermal laser light exposure of the tumors was initiated either 4-5 hours (for Npe6) or 24 hours (for PH and Sn ET2) after drug administration.
  • Red light at 630 nm was used for PH- mediated PDT and 664 nm light was used for Sn ET2- and Npe6- mediated PDT.
  • Light dose rates were 75 mW/cm ⁇ .
  • the reaction was stopped by adding 500 ⁇ l of 1 M Sodium Carbonate, and the absorbance at 420 nm was read with a spectrophotometer. Beta-galactosidase activity was expressed in milliunits/mg of protein (determined using the BIO- RAD protein assay).
  • Cell and Tissue CAT assays For cells, 10 ⁇ g of cellular protein (obtained by freeze/thawing of cells) was combined with 35 ⁇ l of 1M TrisCl pH 7.8, 10 ⁇ l of 6.0 mg/ml acetyl CoA, 2.5 ⁇ l of C-14 chloramphenicol (ICN Pharmaceuticals, Inc., Costa Mesa, CA, catalogue no. 12060) and water to a final volume of 75 ⁇ l. The reaction mixture was incubated for 30 minutes in a 37° C water bath. Acetylated chloramphenicol was extracted from the reaction mixture by the addition of 1 ml ethyl acetate and vortexed for 1 minute and centrifuged at maximum speed for 5 minutes.
  • 1M TrisCl pH 7.8 10 ⁇ l of 6.0 mg/ml acetyl CoA
  • C-14 chloramphenicol ICN Pharmaceuticals, Inc., Costa Mesa, CA, catalogue no. 12060
  • CAT assays were performed according to "Molecular Cloning", Sambrook, Fritsch, Maniatis, Cold Spring Harbor Laboratory Press (1989), with slight modifications. 65.5 ⁇ g of protein sample was incubated at 65° C for 10 minutes to inactivate deacetylases. To each sample, 50 ⁇ l of 1 M TrisCl pH 7.8, 10 ⁇ l of 6.0 mg/ml acetyl CoA, 4.3 ⁇ l of C-14 chloramphenicol (ICN Pharmaceuticals, Inc., Costa Mesa, CA) and water were added to a final volume of 130 ⁇ l.
  • Starting plasmids are obtained from StressGen (p2500CAT) containing the hsp promoter and ATCC (pUC-R10173) containing the complete coding sequence of TNF- ⁇ .
  • the CAT gene from p2500CAT is removed by digestion with Hindlll and BamHl . Resulting ends are modified using commercial adapters from New England Biolabs.
  • the Hindlll end is converted to EcoRl using previously annealed adapters 1105 (EcoRl - XmnI) and 1107 (Hindlll - XmnI).
  • the BamHl end is converted to EcoRl using adapters 1 105 (EcoRl - XmnI) and 1 106 (BamHl - XmnI).
  • the vector is ligated to the purified EcoRl fragment of pUC-R10173.
  • the circularized plasmid is used to transform competent E.coli. Minipreps of selected colonies are cut with Aval and Bglll to determine insert orientation. A colony with the correct insert is expanded and the resulting plasmid (pHspTNF) is transfected along with pMClNeo in RIF cancer cells. Tumor cell lines containing the stably integrated hsp promoter/TNF- ⁇ expression vector, pHspTNF, is isolated.

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Abstract

Procédé pour la sélection et l'expression temporaire de gènes hétérologues. L'invention porte sur des procédés d'obtention d'une expression sélective et temporaire de gènes hétérologues dans des tissus cibles. Des promoteur inductibles par traitement photodynamique ou par la chaleur sont utilisés pour exprimer des gènes d'intérêt dans des conditions de stress oxydatif induit par chauffage ou par traitement photodynamique (TPD). Cette expression sélective et temporaire de gènes hétérologues (tels que des cytokines, des toxines, des gènes suppresseurs tumoraux, des molécules antisens et des facteurs anti-angiogéniques) présente un avantage thérapeutique significatif dans le traitements des tumeurs, la prolifération vasculaire et l'hypertrophie tissulaire. Une thérapie génique dirigée par un chauffage induit par laser ou par d'autres sources de chaleur (telles que des courants induits par hyperfréquences, ultrasons, ou radiofréquences) ou par TPD, accroît l'efficacité du traitement en induisant l'expression de gènes thérapeutiques de manière contrôlée et localisée.
EP98910249A 1997-03-10 1998-03-09 Stress oxydatif genere par traitement photodynamique pour l'expression temporaire et selective de genes heterologues Withdrawn EP1005375A1 (fr)

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US6811788B2 (en) * 2000-01-19 2004-11-02 Baofa Yu Combinations and methods for treating neoplasms
US7125542B2 (en) * 2000-02-10 2006-10-24 Massachusetts Eye And Ear Infirmary Methods and compositions for treating conditions of the eye
US20050048109A1 (en) * 2003-08-26 2005-03-03 Ceramoptec Industries, Inc. Non-polar photosensitizer formulations for photodynamic therapy
CA2606858A1 (fr) * 2005-05-03 2006-11-09 Veterinarmedizinische Universitat Wien Capsules permeables
CN101138634A (zh) 2006-09-07 2008-03-12 于保法 用于治疗肿瘤的组合物
US11110303B2 (en) 2014-11-26 2021-09-07 Baofa Yu Hapten-enhanced chemoimmunotherapy by ultra-minimum incision personalized intratumoral chemoimmunotherapy
US20220313823A1 (en) * 2019-05-15 2022-10-06 Dong Sung Pharm. Co., Ltd. Photodynamic therapy method mediated by chlorin e6 photosensitizer composite for treating and preventing obesity

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NZ337693A (en) 2001-07-27
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