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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
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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
• • 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
  • A61P35/00—Antineoplastic agents
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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
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  • 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
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  • 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
• • C—CHEMISTRY; METALLURGY
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

• the present invention relates generally to the field of gene therapy. More particularly, it concerns methods and compositions for increasing transgene expression.
• Viral vectors are one method employed as a gene delivery system.
• a great variety of viral expression systems have been developed and assessed for their ability to tr-ansfer genes into somatic cells.
• retroviral and adenovirus based vector systems have been investigated extensively over a decade.
• adeno-associated virus AAV
• Lipid vectors including cationic lipids and liposomes also are used to deliver plasmid DNA containing therapeutic genes.
• the therapeutic treatment of diseases and disorders by gene therapy involves the transfer .and stable or transient insertion of new genetic information into cells.
• the correction of a genetic defect by re-introduction of the normal allele of a gene encoding the desired function has demonstrated that this concept is clinically feasible (Rosenberg et al, New Eng. J. Med., 323:570 (1990)).
• Rosenberg et al New Eng. J. Med., 323:570 (1990)
• preclinical and clinical studies covering a large range of genetic disorders currently are underway to solve basic issues dealing with gene transfer efficiency, regulation of gene expression, and potential risks of the use of viral vectors.
• the majority of clinical gene transfer trials that employ viral vectors perform ex vivo gene transfer into target cells which are then administered in vivo.
• Viral vectors also may be given in vivo but repeated administration may induce neutralizing antibody.
• Gene regulatory elements provide a potential answer to that question.
• Gene regulatory elements such as promoters and enhancers possess cell type specific activities and can be activated by certain induction factors via responsive elements.
• the use of such regulatory elements as promoters to drive gene expression facilitates controlled and restricted expression of heterologous genes in vector constructs.
• heat shock promoters can be used to drive expression of a heterologous gene following heat shock.
• US Patent Nos. 5,614,381, 5,646,010 and WO 89/00603 refer to driving transgene expression using heat shock at temperatures greater than 42°C. These temperatures are not practicable in human therapy as they can not be maintained for a sustained period of time without harm to the individual.
• Hyperthermia enhances the cell killing effect of radiation in vitro (H.arisiadis et al, Cancer, 41:2131-2142 (1978)), significantly enhances tumor response in animal tumors in vivo .and improves the outcome in randomized clinical trials.
• the major problem with the use of hyperthermia treatment is that the hyperthermia system can not adequately heat large and deep tumors.
• vectors that may be used at temperatures of 42°C and below, systemically or locally, to treat a patient such that the expression of the therapeutic gene(s) is activated preferentially in regions of the body that have been subjected to conditions which induce such expression.
• the present invention provides methods for effecting the inducible expression of polynucleotides in cells.
• the use of heat shock promoters in methods for effecting the inducible expression of polynucleotides in mammalian cells is taught.
• the present invention overcomes deficiencies in the prior art by providing heat shock-controlled vectors that may be used at temperatures of 42°C and below. These methods may be used to treat a patient via the inducible expression of a therapeutic gene.
• the present invention provides a method for effecting transgene expression in a mammalian cell that comprises first providing an expression construct that comprises both (i) an inducible promoter operably linked to a gene encoding a transactivating factor and (ii) a second promoter operably linked to a selected polynucleotide.
• the second promoter is activated by the transactivating factor expressed by the same construct.
• the method then includes the step of introducing the expression construct into the cell. Finally, the cell is subjected to conditions which activate the inducible promoter and result in the expression of the selected polynucleotide.
• the inducible promoter is a heat shock promoter and the conditions which activate the heat shock promoter are hyperthermic conditions.
• the hyperthermic conditions may comprise a temperature between about basal temperature and about 42°C.
• the basal temperature of the cell is defined as the temperature at which the cell is normally found in its natural state, for example, a cell in skin of a mammal may be at temperatures as low as 33°C whereas a cell in the liver of an organism may be as high as 39°C.
• the application of hyperthermia involves raising the temperature of the cell from basal temperature, most typically 37°C to about 42°C or less.
• the hyperthermic conditions may range from about 38°C to about 41°C, or from about 39°C to about 40°C.
• the heat shock promoter is optionally derived from a promoter selected from the group of the heat shock protein (HSP) promoters HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25 and HSP28.
• HSP heat shock protein
• the ubiquitin promoter may also be used as the heat-shock inducible promoter in the expression construct.
• a minimal heat shock promoter derived from HSP70 and comprising the first approximately 400 bp of the HSP70B promoter may optionally be used in the invention.
• the inducible promoter comprises a hypoxia-responsive element (HRE).
• HRE hypoxia-responsive element
• This hypoxia-responsive element may optionally contain at least one binding site for hypoxia-inducible factor- 1 (HIF-1).
• the second promoter may be selected from the group consisting of an human immunodeficiency virus-l (HIV-1) promoter and a human immunodeficiency virus-2 (HIV-2) promoter.
• the transactivating factor may be a transactivator of transcription
• the selected polynucleotide may code for a protein or a polypeptide.
• the selected polynucleotide may encode any one of the following proteins: ornithine decarboxylase antizyme protein, p53, pl6, neu, interleukin- 1 (IL1), interleukin-2 (IL2), interleukin -4 (IL4), interleukin-7 (IL7), interleukin- 12 (IL12), interleukin- 15 (IL15), FLT-3 ligand, granulocyte-macrophage stimulating factor
• GM-CSF granulocyte-colony stimulating factor
• G-CSF granulocyte-colony stimulating factor
• IFN ⁇ gamma-interferon
• IFN ⁇ alpha-interferon
• TNF tumor necrosis factor
• HSV-TK herpes simplex virus thymidine kinase
• I-CAM1 human leukocyte antigen-B7 (HLA-B7)
• TNF-3 tumor necrosis factor
• the selected polynucleotide is positioned in a sense orientation with respect to the second promoter.
• expression of the selected polynucleotide may involve transcription but not translation and produces a ribozyme.
• the selected polynucleotide is also positioned in a sense orientation with respect to the second promoter.
• the expression of the selected polynucleotide involves transcription but not translation and results in -an RNA molecule which serves as an antisense nucleic acid.
• the selected polynucleotide may be the target gene, or a fragment thereof, which is positioned in the expression construct in .an .antisense orientation with respect to said second promoter.
• the expression construct may further comprise a gene encoding a selectable marker, such as hygromycin resistance, neomycin resistance, puromycin resistance, zeocin, gpt, DHFR, green fluorescent protein or histadinol.
• a selectable marker such as hygromycin resistance, neomycin resistance, puromycin resistance, zeocin, gpt, DHFR, green fluorescent protein or histadinol.
• the expression construct may further comprise (i) a second selected polynucleotide which is operably linked to said second promoter, and (ii) an internal ribosome entry site positioned between said first and second selected polynucleotides.
• the cell may be a tumor cell, a cell located within a tumor, or a cell located within a mammal.
• the introduction of the expression construct into the cell may occur in vitro or in vivo.
• the introduction of the expression construct into the cell is mediated by a delivery vehicle selected from the group consisting of liposomes, retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, herpes simplex viruses, and vaccinia viruses.
• a method of providing a subject with a therapeutically effective amount of a product of a selected gene involves providing a first expression construct which comprises an inducible promoter operably linked to a gene encoding a transactivating factor and providing a second expression construct which comprises a second promoter operably linked to a selected polynucleotide, where the second promoter is activated by the transactivating factor encoded by the first expression construct.
• the first and second expression construct are introduced into the desired cell of said subject and that cell is subjected to conditions which activate the inducible promoter, so that expression of the selected polynucleotide is induced.
• the first .and second expression constructs . are present on the same vector.
• the inducible promoter is preferably a heat shock promoter and the activating conditions comprise a temperature below 42°C and above about basal temperature.
• the introduction of one or both of the expression constructs may be performed either in vivo or ex vivo.
• the expression product of the selected polynucleotide may optionally be deleterious to a pathogen in the subject, such as a virus, bacterium, fungus, or parasite.
• the expression product of the selected polynucleotide may inhibit the growth of the cell of the subject.
• the expression product of the selected polynucleotide replaces a deficient protein in the subject.
• the expression product of the selected polynucleotide may promote nerve regeneration.
• a method of treating cancer in a mammal comprising the steps of (a) providing an expression construct that comprises (i) an inducible promoter, preferably a heat shock promoter, which is operably linked to a gene encoding a transactivating factor; and (ii) a second promoter operably linked to a selected polynucleotide, wherein the second promoter is activated by the transactivating factor; (b) introducing said expression construct into a tumor cell; and (c) subjecting the tumor cell to conditions which activate the inducible promoter so that the selected polynucleotide is expressed in high enough quantities to inhibit the growth of the tumor cell.
• the inducible promoter is a heat shock promoter
• the activating conditions comprise a temperature below about 42°C and above about basal temperature.
• This method further may comprise treating said tumor cell with an established form of therapy for cancer which is selected from the group consisting of external beam radiation therapy, brachytherapy, chemotherapy, and surgery.
• the cancer may optionally be selected from the group consisting of cancers of the brain, lung, liver, spleen, kidney, lymph node, small intestine, p-ancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, vulva, cervix, skin, head and neck, esophagus, bone marrow and blood.
• the selected polynucleotide is ornithine decarboxylase antizyme protein.
• the method comprises (a) providing an expression construct that comprises (i) an inducible promoter, preferably a heat shock promoter, which is operably linked to a gene encoding a transactivating factor; and (ii) a second promoter operably linked to a selected polynucleotide, wherein the second promoter is activated by the transactivating factor; (b) introducing said expression construct into a cell in the mammal; and (c) subjecting the cell to conditions which activate the inducible promoter so that the selected polynucleotide is expressed highly enough to provoke an immune response in the mammal.
• the inducible promoter is a heat shock promoter
• the activating conditions comprise a temperature below about 42°C and above about basal temperature.
• the immune response which is provoked is directed against the cell in the mammal which contains the expression construct.
• the method may also optionally involve treating the cell with an established form of therapy for cancer selected from the group consisting of chemotherapy, external beam radiation therapy, brachytherapy, and surgery.
• an expression construct comprising (a) a gene encoding a transactivating factor; (b) an inducible promoter operably linked to the gene; (c) a selected polynucleotide; and (d) a second promoter which is operably linked to the selected polynucleotide.
• the second promoter of the construct is activated by the transactivating factor.
• the inducible promoter is a heat shock promoter and the expression of the selected polynucleotide can be induced by hyperthermic conditions comprising a temperature below about 42°C and above about 37°C.
• the inducible promoter of the expression construct may comprise a hypoxia-responsive element.
• the expression construct may also comprise a second selected polynucleotide which is also operably linked to the second promoter and separated by the first selected polynucleotide by a IRES.
• a cell comprising the expression construct is also provided.
• the provided expression construct can also optionally be used in a method of altering the genetic material of a mammal.
• FIG. 1 depicts the basic vector used for quantitating heat shock promoter activity.
• the plasmid contains a rninimal promoter derived from the HSP70B promoter (StressGen).
• StressGen Enhanced Green Fluorescence Protein
• MCS multiple cloning site
• the plasmid also contains the neomycin and ampicillin resistance genes for selectability in mammalian cells as well as the standard elements for growth in a bacterial system.
• the S8 plasmid comprises the plasmid shown with EGFP inserted in the multiple cloning site.
• FIG. 2 shows fluorescence activated cell sorting (FACS) histograms for DU- 145 cells stably transfected with the S8 plasmid. Fluorescence increases from left to right. The top histogram is from transfected DU-145 cells which have not been subjected to heat shock. The bottom histogram is from transfected DU-145 cells which have been subjected to a 42°C heat shock for 1 hour.
• FACS fluorescence activated cell sorting
• FIG. 3 shows FACS histograms for three different populations of S8- transfected MCF7 cells.
• the original population came from a polyclonal selected cell line. That cell line's activated (i.e., cells expressing EGFP) population was separated from the non-activated population. After the sort, the positive population was grown and then re-sorted to obtain a more purely positive cell line. In this case, the polyclonal MCF7-S8-P cells were sorted twice yielding the highly positive population MCF7-S8-PS2.
• FIG. 4 shows expression of EGFP in different cell lines assayed by FACS.
• Cell lines were transfected with the plasmid S8. The cells were then cloned or a polygonal line was grown. In some cases the cell lines were sorted for EGFP expression by FACS. The total mean fluorescence was quantified and graphed.
• FIG. 5 shows expression of EGFP in stably transfected DU-145 cells which have been twice sorted (DU-S8-PS2) following heat shock.
• the DU-S8-PS2 cells were heated at either 40°C or 42°C and allowed to recover for various times. The cells were then analyzed by FACS.
• FIG. 6 shows expression levels of EGFP in stably transfected DU-145 cells 16 hours after exposure to heat stresses.
• One population of cells (DU-S8-PS2) was stably transfected with the S8 plasmid.
• DU-V9-PS2 Another population (DU-V9-PS2) was stably transfected with the V9 plasmid, a plasmid identical to S8 except that the EGFP of the V9 plasmid is operably linked to a CMV promoter, rather than HSP 70B (see FIG. 7).
• the cells were heated at various temperatures and allowed to recover for 16 hours.
• Non-transfected DU-145 cells were included as a control.
• FIG. 7 shows a schematic diagram of the plasmid V9 which contains a CMV promoter that is operably linked to the gene encoding the Enhanced Green Fluorescence Protein (EGFP)
• EGFP Enhanced Green Fluorescence Protein
• FIG. 8 shows the basic vector design for a vector containing a second promoter which allows for amplification of the heat shock response.
• the plasmid contains a multiple cloning site (MCS) operably linked to HSP70B promoter, but also contains a therapeutic gene operably linked to a second promoter.
• MCS multiple cloning site
• the plasmid also contain the neomycin resistance gene, the ampicillin resistance gene, and standard elements for growth in bacteria.
• the second promoter is the HIV-1 long terminal repeat (LTR) and the therapeutic gene is IL2.
• LTR long terminal repeat
• IL2 therapeutic gene
• tat is inserted in the MCS
• the second promoter is the HIV-1 LTR
• the therapeutic gene is IL2.
• Another plasmid, p007 is the same as pf 12, except that the HIV-2 LTR is used as the second promoter.
• FIG. 9 shows amplifier constructs containing the therapeutic gene IL-2 driven by either the HIV-1 or the HIV-2 promoter.
• the amplifier part is controlled by either the CMV or the HSP 70 promoter driving TAT expression.
• the plasmids also contain the neomycin resistance gene and elements for growth in bacteria. These constructs were used in the amplifier studies of Examples 2 and 3.
• FIG. 9A shows a plasmid designated X14 containing a CMV-TAT-HIV-1-IL2 expression cassette
• FIG. 9B shows a plasmid designated Y15 containing a CMV-TAT-HIV-2- IL2 expression cassette
• FIG. 9A shows a plasmid designated X14 containing a CMV-TAT-HIV-1-IL2 expression cassette
• FIG. 9B shows a plasmid designated Y15 containing a CMV-TAT-HIV-2- IL2 expression cassette
• FIG. 9A shows a plasmid designated X14 containing a
• FIG. 9C shows a plasmid designated pf!2 containing an HSP-TAT-HIV-1-IL2 expression cassette
• FIG. 9D shows a plasmid designated p007 containing an HSP-TAT-HIV-2-IL2 expression cassette.
• FIG 10. shows the DNA sequence of the BamHl-Hindlll fragment of pl73OR from StressGen Biotechnology Corp. This fragment contains the approximately 0.4kb minimal HSP70B promoter fragment used in constructs of the specific examples, Example 1 and 3, below.
• Gene therapy faces two major technical problems: how to both regulate and enhance the expression of therapeutic genes in vivo.
• the present invention addresses both of these questions by combining hyperthermia treatment with inducible expression constructs.
• the inventors have demonstrated increases in the efficiency of specific, inducible gene expression.
• the ability to express therapeutic gene(s) at very high levels and the ability to control the levels of expression are important objectives in the development of gene therapy.
• the inventors have created new sets of expression vectors to address these objectives.
• the inventors use an amplifier strategy to drive the expression of the gene(s) of interest.
• the amplifiers consist of the human HSP70B promoter driving expression of proteins that are transcriptional activators of other promoters, which, in turn, drive reporter genes.
• These additional promoters and their operably linked reporter genes are preferably included in the same vector with the HSP70B promoter element and the gene encoding the transactivating protein.
• HSP70B alone (see specific example, Example 3, below). Constructs containing both the HSP70B promoter, upstream of the human immunodeficiency virus (HIV) tat gene, and the HIV1 or HIV2 long terminal repeats, upstream of the interleukin-2 (IL-2) gene, exhibited promoter activity at 37°C which was further amplified by heat shock. Co-transfection experiments indicated that the activities of the HSP, HSP/HIVl and HSP/HIV2 promoter expression constructs were 0.4, 6.9 and 83.3, respectively, times that of the CMV promoter expression construct in m-ammalian cells. These data indicate that, while less active than the CMV promoter by itself, this minimal heat shock promoter can be used in conjunction with a second promoter to markedly amplify gene expression while still maintaining some temperature dependence.
• HSP human immunodeficiency virus
• IL-2 interleukin-2
• the inventions described herein differ from these earlier approaches, for example, by use of 1) different heat shock promoters, (Schweinfest et al, use Drosophila promoters) 2) different modes of delivery (the present inventors have incorporated both promoters into a single construct — whereas others have used co-transfection) 3) different temperatures for induction (the earlier work used temperatures greater than 42°C, whereas the present invention advantageously operates at temperatures of 42°C and lower); and 4) use in gene therapy context rather industrial production.
• the present inventors are able to use either HIV-1 or HIV-2 promoters and the present invention shows a clear distinction in the expression levels resulting from these two promoters.
• a heat shock inducible element in a preferred aspect of the present invention, methods of effecting tr-ansgene expression in a mammalian cell by using a heat shock inducible element are provided.
• the heat shock sequence is used to drive the expression of a transactivating gene.
• the transactivating gene acts upon a second promoter which becomes activated to drive the expression of the therapeutic gene of interest.
• a promoter derived from the HSP70 promoter is employed.
• a particularly useful aspect of this promoter is that it has a low basal level of expression at ambient temperatures and is inducible.
• the present invention further provides methods of providing a subject with a therapeutically effective amount of a gene product and for inhibiting the growth of a cell or provoking an immune response.
• compositions and methods employed in order to meet the objectives of the present invention are discussed in further detail herein below.
• the heat shock or stress response is a universal response occurring in organisms ranging from plants to primates. It is a response that can be elicited as a result of not only heat shock, but also as a result of a variety of other stresses including ischemia, anoxia, glucose deprivation, ionophores glucose and amino acid analogues, ethanol, tr-ansition series metals, drugs, hormones and bacterial and viral infections. Furthermore, there is evidence that overexpression of heat shock protein genes may be associated with enhanced proliferation and stress of tumor cells (Finch et al, Cell Growth and Differentiation 3(5):269-278, 1992). This response is characterized by the synthesis of a family of well conserved proteins of varying molecular sizes that are differently induced and localized. These proteins are among the most phylogenetically conserved and .are characterized according to their weights.
• the activation of the stress genes is mediated by the conversion of a preexisting heat shock transcription factor (HSF) from an inactive to an active form.
• HSF heat shock transcription factor
• the heat shock element is a conserved upstream regulatory sequence of HSP70 to which HSF binds.
• HSF acts through a highly conserved response element found in multiple copies upstream of the heat shock gene.
• the heat shock response element is composed of three contiguous inverted repeats of a 5-base pair sequence whose consensus was defined as nGAAn and more recently defined as AGAAn.
• the regulation of HSF primarily comprises a change in activity rather than an alteration in synthesis or stability.
• Hyperthermia is intended to refer to a temperature condition that is greater than the ambient temperature of the subject to which the treatment is being administered.
• a hyperthermic temperature will typically range from between about 37°C to about 42°C.
• the temperature will range from about 38°C to about 42°C, in other embodiments, the temperature range will be from about 39°C to about 41°C, in other embodiments, the temperature will be about 40°C.
• the devices currently available for the application of hyperthermia in adjuvant therapies it is possible to maintain the temperature of hyperthermia treatment to within about 0.5°C for temperatures up to 42°C.
• the therapeutic treatments of the present invention may be carried out at 37.0°C, 37.2°C, 37.4°C, 37.6°C, 37.8°C, 38.2°C, 38.4°C, 38.6°C, 38.8°C, 39.2°C, 39.4°C, 39.6°C, 39.8°C, 40.2°C, 40.4°C, 40.6°C, 40.8°C, 41.2°C, 41.4°C, 41.6°C, 41.8°C, or 42.0°C.
• efficacy of hyperthermia required that temperatures within a tumor(s) remain above about 43 °C for 30 to 60 min, while safety considerations limit temperatures in normal tissues to below 42°C. Achieving uniform temperatures above 42°C in tumors is very difficult and often not possible.
• Tissues in mammals can be heated using a number of technologies including ultrasound, electromagnetic techniques, including either propagated wave (e.g., microwaves), resistive (e.g., radiofrequency) or inductive (radiofrequency or magnetic) procedures (Hahn, G.M., Hyperthermia and Cancer, 2nd Ed., New York, Plenum, 1982; Lehman, L.B., Postgard Med., 88(3):240-243, 1990; both herein incorporated by reference).
• tissue temperatures can be elevated using circulated hot air or water.
• U.S. Pat. No. 4,230, 129 to Le Veen refers to a method of heating body tissue and monitoring temperature changes in the tumor in real time with the aid of a scintillation detector.
• the method provides for the coupling of radiofrequency (RF) energy to the patient's body to avoid any significant heat absorption in the fatty tissues. This is obtained by focusing the RF energy on the tumor with an orbital movement of the applicator so that energy is not constantly being applied to the same confined area within the patient's body.
• RF radiofrequency
• 3,991,770 to Le Veen also herein incorporated by reference, teaches a method of treating a tumor in a human by placing the part of the human body containing the tumor in a radiofrequency electromagnetic field to heat the tumor tissue and cause necrosis of the tumor without damaging the adjacent normal tissue.
• hyperthermia is applied in combination with the gene therapy vectors disclosed herein to achieve inducible gene expression at a particular tumor site.
• the hyperthermia/gene therapy treatment regimens may be used in combination with other conventional therapies, such as the chemotherapies and radiotherapies discussed below, to effectively treat cancer.
• Other methods for inducing hyperthermia also are known in the art. Methods and devices for the regional and/or systemic application of hyperthermia are well know to those of skill in the art and are disclosed in for example, U.S. Patent Nos. 5,284, 144; 4,230,129; 4, 186,729; 4,346,716; 4,848,362; 4,815,479; 4,632, 128, all incorporated herein by reference.
• the present invention involves the manipulation of genetic material to produce expression constructs that encode therapeutic genes.
• Such methods involve the use of an expression construct containing, for example, a heterologous DNA encoding a gene of interest and a means for its expression, replicating the vector in an appropriate helper cell, obtaining viral particles produced therefrom, and infecting cells with the recombinant virus particles.
• the gene will be a therapeutic gene, for example to treat cancer cells, to express immunomodulatory genes to fight viral infections, or to replace a gene's function as a result of a genetic defect.
• the gene will be a heterologous DNA, meant to include DNA derived from a source other than the viral genome which provides the backbone of the vector.
• the virus may act as a live viral vaccine and express an antigen of interest for the production of antibodies thereagainst.
• the gene may be derived from a prokaryotic or eukaryotic source such as a bacterium, a virus, a yeast, a parasite, a plant, or an animal.
• the heterologous DNA also may be derived from more than one source, i.e., a multigene construct or a fusion protein.
• the heterologous DNA also may include a regulatory sequence which may be derived from one source and the gene from a different source.
• the selected polynucleotide of the present invention may optionally be a therapeutic gene. Any of a wide variety of therapeutic genes are suitable for use in the vectors and methods described herein. Therapeutic genes which are suitable for application of the present invention to a particular disorder, medical condition, or disease will be discernible to one skilled in the art.
• the selected polynucleotide is the gene encoding for omithine decarboxylase antizyme protein.
• ODC omithine decarboxylase
• the omithine decarboxylase (ODC) antizyme protein is an important component of feedback regulation of intracellular poly.amine pool sizes (Hayashi et al, Trends in Biochemical Sciences 21(l):27-30, 1996, herein incorporated by reference). The levels of this protein are directly related to levels of intracellular polyamines, which stimulate translation of antizyme message.
• Antizyme protein targets ormthine decarboxylase, the first and often rate-limiting enzyme in poly.amine synthesis, for degradation. This protein also suppresses polyamine uptake.
• the radioprotector WR-33278 (N,N"-(dithiodi-2, l-ethanediyl)bis-l,3- propanediamine) is a disulfide-containing polyamine analog, which is taken up by cells using the polyamine transporter (Mitchell et al, Carcinogenesis, 16:3063- 3068, 1995, herein incorporated by reference).
• This tr-ansporter is inhibited by antizyme.
• Evidence from animal models indicates that this radioprotector is taken up by at least some normal tissues to a greater extent than some tumors (Ito et al., International Journal of Radiation Oncology, Biology, Physics 28:899-903, 1994).
• Agents like WR-33278 have been used in clinical radiotherapy in attempts to protect dose-limiting normal tissues from toxicity, without reducing the tumor control effectiveness of radiotherapy (Spencer and Goa, Drugs, 50(6): 1001-31, 1995, herein incorporated by reference). Rationale for the difference in uptake of WR-33278 may be that proliferating tumor cells often contain higher levels of polyamines than do non-proliferating cells in normal tissues. Thus, tumors would express higher levels of antizyme than would normal tissues.
• the inventors have placed an antizyme cDNA lacking the sequences necessary for polyamine-dependent regulation under the control of the human heat shock 70B promoter.
• the inventors have stably transfected human prostate cancer derived DU-145 cells with this construct and have selected clones which display heat-inducible suppression of polyamine uptake (indicating heat-inducible antizyme activity).
• the therapeutic application of this gene therapy (HSP70B promoter regulation of antizyme expression) will be put to use in future clinical trials in men with localized prostate cancer. Patients are treated with this gene therapy, administered intratumorally, combined with systemic WR-33278 and localized radiotherapy. Expression of antizyme intratumorally is then activated by localized hyperthermia.
• WR-33278 Dose-limiting normal tissues adjacent to these prostate tumors will not express antizyme in response to hyperthermia and will take up the radioprotector WR-33278, while the tumor tissue will not take up the radioprotector because they will express antizyme in response to hyperthermia.
• This strategy will allow higher doses of radiotherapy to be given to the prostate, with the intent to improve local control of prostate cancers.
• other metabolic products of the cytoprotective drug ethyol also known as amifostine, WR-2721, or S-2-(3- aminopropylamino)ethylphosphororthioic acid
• WR- 1065 (2-(3-aminopropylamino)ethanethiol) may be instead used as the radioprotector.
• the vectors of the present invention may be used to transfer tumor suppressors, antisense oncogenes and prodrug activators, such as the HSV-TK gene (Rosenfeld et al, Annals of Surgery, 225:609-618, 1997; Esandi et al, Gene Therapy, 4:280-287, 1997), for the treatment of cancer.
• HSV-TK gene Rosenfeld et al, Annals of Surgery, 225:609-618, 1997
• Esandi et al Gene Therapy, 4:280-287, 1997)
• genes which could optionally be used in the expression constructs of the present invention include p53, pl6, p21, p27, C-CAM, HLA-B7 (Gleich, et al, Arch Otolaryngol Head Neck Surg, 124: 1097-104, 1998; Heo et al, Hum. Gene Ther. 9:2031-8, 1998; Nabel et al, Proc. Nat. Acad.
• p53 currently is recognized as a tumor suppressor gene (Montenarh, Crit. Rev. Oncogen, 3:233-256, 1992). High levels of mutant p53 have been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses, including SV40.
• the p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and already is documented to be the most frequently-mutated gene in common human cancers. It is mutated in over 50% of human NSCLC and in a wide spectrum of other tumors.
• P16 mK4 belongs to a newly described class of CDK-inhibitory proteins that also includes pl6 B , p21 WAF1 ' CIP1 ' SDI1 , and p27 KIP1 .
• the pl6 mK4 gene maps to 9p21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the pl6 mK4 gene are frequent in human tumor cell lines. This evidence suggests that the pl6 mK4 gene is a tumor suppressor gene. This interpretation has been challenged, however, by the observation that the frequency of the pl ⁇ 11 ⁇ 4 gene alterations is much lower in primary uncultured tumors th-an in cultured cell lines.
• C-CAM is expressed in virtually all epithelial cells.
• C-CAM with an apparent molecular weight of 105 kD, originally was isolated from the plasma membrane of the rat hepatocyte by its reaction with specific antibodies that neutralize cell aggregation.
• Ig immunoglobulin
• CEA carcinoembryonic antigen
• CAMs Cell adhesion molecules, or CAMs are known to be involved in a complex network of molecular interactions that regulate organ development and cell differentiation (Edelman, Annu. Rev. Biochem., 54: 135-169, 1985). Recent data indicate that aberrant expression of CAMs may be involved in the tumorigenesis of several neoplasms; for example, decreased expression of E-cadherin, which predominantly is expressed in epithelial cells, is associated with the progression of several kinds of neoplasms.
• the selected polynucleotide may be any one of the following genes: retinoblastoma (Rb); adenomatous polyposis coli gene (APC); deleted in colorectal carcinomas (DCC); neurofibromatosis 1 (NF-1); neurofibromatosis 2 (NF-2); Wilm's tumor suppressor gene (WT-1); multiple endocrine neoplasia type 1 (MEN-1); multiple endocrine neoplasia type 2 (MEN- 2); BRCA1; von Hippel-Lindau syndrome (VHL); mutated in colorectal cancer (MCC); pl6; p21; p57; p27; and BRCA2.
• Rb retinoblastoma
• APC adenomatous polyposis coli gene
• DCC deleted in colorectal carcinomas
• NF-1 neurofibromatosis 1
• NF-2 neurofibromatosis 2
• WT-1 Wilm's tumor suppressor gene
• the methods and vectors of the present invention may be used to promote regeneration processes, such as nerve regeneration, by stimulating the production of growth factors or cytokines.
• the selected polynucleotide may be a neurotrophic factor.
• the selected polynucleotide may encode ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), or glial cell line-derived neurotrophic factor (GDNF) (Mitsumoto et al, Science, 265: 1107-1110, 1994 and Gash et al, Ann. Neurol, 44(3 Suppl 1):S121-125, 1998, both herein incorporated by reference).
• CNTF ciliary neurotrophic factor
• BDNF brain-derived neurotrophic factor
• GDNF glial cell line-derived neurotrophic factor
• the selected polynucleotide of the expression construct may optionally encode tyrosine hydroxylase, GTP cyclohydrolase 1, or aromatic L-amino acid decarboxylase (Kang, Mov. Disord., 13 Suppl 1:59-72, 1998, herein incorporated by reference).
• the therapeutic expression construct may express; a growth factor such as insulin-like growth factor-I (IGF-I) (Webster, Mult. Scler., 3: 113-120, 1997, incorporated herein by reference).
• IGF-I insulin-like growth factor-I
• diseases for which the present vectors are useful include but are not limited to hyperproliferative diseases and disorders, such as rheumatoid arthritis or restenosis by transfer of therapeutic genes, e.g., gene encoding angiogenesis inhibitors or cell cycle inhibitors.
• Oncogenes such as ras, myc, neu, raf erb, src, fins, jun, trk, ret, gsp, hst, bcl and ⁇ bl also are suitable targets. However, for therapeutic benefit, these oncogenes would be expressed as an antisense nucleic acid, so as to inhibit the expression of the oncogene.
• the term "antisense nucleic acid” is intended to refer to the oligonucleotides complementary to the base sequences of oncogene-encoding DNA and RNA. Antisense nucleic acid, when expressed in a target cell, specifically bind to their target nucleic acid and interfere with transcription, RNA processing, transport and/or translation. Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double- helix formation.
• ds double-stranded
• Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene.
• Antisense RNA constructs, or DNA encoding such antisense RNAS may be employed to inhibit gene transcription or tr-anslation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
• Nucleic acid sequences comprising "complementary nucleotides” are those which are capable of base-pairing according to the standard Watson-Crick complementary rules.
• the larger purines will base pair with the smaller pyrimidines to form only combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T), in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
• the terms "complementary" or "antisense sequences” mean nucleic acid sequences that are substantially complementary over their entire length and have very few base mismatches. For example, nucleic acid sequences of fifteen bases in length may be termed complementary when they have a complementary nucleotide at thirteen or fourteen positions with only single or double mismatches. Naturally, nucleic acid sequences which are "completely complementary” will be nucleic acid sequences which are entirely complementary throughout their entire length and have no base mismatches.
• any sequence 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence.
• shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs will be used.
• antisense constructs which include other elements, for example, those which include C-5 propyne pyrimidines.
• Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al, Science, 260: 1510-1513, 1993, herein incorporated by reference). c) Ribozyme constructs
• targeted ribozymes may be used.
• the term "ribozyme” refers to .an RNA-based enzyme capable of targeting and cleaving particular base sequences in DNA or, more typically, RNA.
• ribozymes are introduced into the cell as an expression construct encoding the desired ribozymal RNA.
• the targets of the ribozymes are much the same as described for antisense nucleic acids.
• ribozymes are known to catalyze the hydrolysis of phosphodiester bonds under physiological conditions.
• the ribozymes of the present invention catalyze the sequence specific cleavage of a second nucleic acid molecule, preferably an mRNA transcript, and optionally an mRNA transcript of an oncogene.
• ribozymes bind to a target RNA through the target binding portion of the ribozyme which flanks the enzymatic portion of the ribozyme.
• the enzymatic portion of the ribozyme cleaves the target RNA. Strategic cleavage of a target RNA destroys its ability to directly or indirectly encode protein. After enzymatic cleavage of the target has occurred, the ribozyme is released from the target and searches for another target where the process is repeated.
• the ribozyme is a hammerhead ribozyme, a small RNA molecule derived from plant viroids (Symons, Ann. Rev. Biochem. 61 : 641-671, 1992; Clouet-D'Orval and Uhlenbeck, RNA, 2:483-491, 1996; Haseloff and Gerlach, Nature 334:585-591, 1988; Jeffries and Symons, Nucleic Acids Res. 17: 1371-1377, 1989; Uhlenbeck, Nature 328:596-600, 1987; all herein incorporated by reference).
• the ribozyme may be a group I intron, a hairpin ribozyme, VS RNA, a hepatitis Delta virus ribozyme or an Rnase P-RNA ribozyme (in association with an RNA guide sequence).
• hairpin motifs are described by Hampel et al., Nucleic Acids Res. 18:299, 1990 and Hampel and Tritz, Biochemistry 28:4929, 1989; an example of the hepatitis delta virus motif is described by Perrotta and Been, Biochemistry 31 : 16, 1992; an example of the RNAseP motif (associated with an external guide sequence) is described by Yuan et al., Patent No.
• ribozymes that may be utilized herein comprise a specific substrate binding site which is complementary to a target mRNA. Such ribozymes also comprise an enzymatic portion which imparts RNA cleaving activity to the molecule. The enzymatic portion resides within or surrounds the substrate binding site.
• the therapeutic vectors of the present invention contain nucleic acid constructs whose expression may be identified in vitro or in vivo by including a marker in the expression construct.
• markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
• a drug selection marker aids in cloning and in the selection of transformants.
• genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
• enzymes such as herpes simplex virus thymidine kinase (tk) may be employed. Immunologic makers also can be employed.
• selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product.
• selectable markers include reporters such as EGFP, ⁇ -gal and chloramphenicol acetyltransferase (CAT).
• CAT chloramphenicol acetyltransferase
• IRES elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites. IRES elements from two members of the picanovirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, Nature, 334:320-325, 1988), as well as an IRES from a mammalian message (Macejak and Sarnow, Nature, 353 :90-94, 1991). IRES elements can be linked to heterologous open reading frames.
• each open reading frame can be transcribed together, each separated by an IRES, creating polycistronic messages.
• IRES element By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can thus be efficiently expressed using a single promoter/enhancer to transcribe a single message.
• Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins encoded by independent genes, intracellul-ar or membrane-bound proteins and selectable markers.
• the polynucleotide encoding the therapeutic gene will be under the transcriptional control of a promoter and a polyadenylation signal.
• a "promoter” refers to a DNA sequence recognized by the synthetic machinery of the host cell, or introduced synthetic machinery, that is required to initiate the specific transcription of a gene.
• a polyadenylation signal refers to a DNA sequence recognized by the synthetic machinery of the host cell, or introduced synthetic machinery, that is required to direct the addition of a series of nucleotides on the end of the MRNA transcript for proper processing and trafficking of the transcript out of the nucleus into the cytoplasm for translation.
• under transcriptional control means that the promoter is in the correct location in relation to the polynucleotide to control RNA polymerase initiation and expression of the polynucleotide.
• promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
• promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
• At least one module in each promoter functions to position the start site for
• RNA synthesis The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the st-art site itself helps to fix the place of initiation.
• promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
• the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements -are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
• a human cell it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
• a promoter might include either a human or viral promoter.
• Table 1 A list of promoters is provided in Table 1.
• NCAM Neural Cell Adhesion Molecule
• SAA Human Serum Amyloid A
• the particular promoter that is employed to control the expression of the therapeutic gene is not believed to be critical, so long as it is capable of being activated by the gene product linked to the inducible promoter.
• the transactivating protein is tat
• the promoter which is operably linked to the therapeutic gene is the HIV-1 or HIV-2 LTRs.
• a promoter element containing an AP-1 site would respond to the inducible expression of the c-jun or c-fos proteins.
• Other suitable transactivating factor/promoter combination would be known by one skilled in the art.
• the promoter which controls expression of the gene encoding the transactivating factor must be an inducible promoter.
• An inducible promoter is a promoter which is inactive or exhibits relatively low activity except in the presence of an inducer substance.
• Some examples of promoters that may be included as a part of the present invention include, but are not limited to, MT II, MMTV, collagenase, stromelysin, SV40, murine MX gene, ⁇ -2-macroglobulin, MHC class I gene h-2kb, proliferin, tumor necrosis factor, or thyroid stimulating hormone ⁇ gene.
• the associated inducers of these promoter elements are shown in Table 2.
• the Egr-1 promoter and the multidrug resistance gene (MDR1) promoter are also options for inducible promoters.
• the inducible promoter is heat shock inducible and is derived from one of the following promoters: HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25, ubiquitin, and HSP28.
• the inducible promoter comprises a hypoxia- responsive element, such as those responsive to HIF-1. It is understood that any inducible promoter may be used in the practice of the present invention and that all such promoters would fall within the spirit and scope of the claimed invention.
• the tat protein is used as the transactivating factor.
• the genome of HIV-1 and HIV-2 share a great deal of similarities with the Simian immunodeficiency viruses (SIVS) and they have been extensively studied. It was discovered that in addition to the gag, env, pol genes that are common to all retroviruses, there are a number of regulatory genes that are important in HIV transcription.
• the viral tat protein is one such regulatory factor and it is characterized by its ability to greatly increase the activity of the HIV-1 and HIV-2 promoter (Sodroski et al, J.
• Tat is thought to bind with the transactivation response element (TAR) in the HIV LTR and increase the steady state levels of the HIV specific RNA.
• TAR transactivation response element
• Tat and adenovirus transactivator EIA can act synergistically in increasing the levels of steady state RNA (Laspia et al, Genes Dev., 4(12B):2397-2408, 1990, herein incorporated by reference).
• a way to increase further the activity of the HIV-LTR/TAT constructs is to incorporate EIA into the same construct.
• polyadenylation signal to effect proper polyadenylation of the gene transcript.
• the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed.
• polyadenylation signals as that from SV40, bovine growth hormone, and the herpes simplex virus thymidine kinase gene have been found to function well in a number of target cells.
• the therapeutic constructs of the present invention e.g., various genetic (i.e., DNA) constructs must be delivered into a cell.
• the introduction of the expression construct into a cell is mediated by non- viral means.
• Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell. Biol, 7:2745-2752, 1987; Rippe et al, Mol. Cell Biol, 10:689-695, 1990) DEAE-dextran (Gopal, Mol Cell Biol, 5: 1188-1190, 1985), electroporation (Tur-Kaspa et al, Mol Cell Biol., 6:716-718, 1986; Potter et al, Proc. Nat. Acad. Sci.
• the nucleic acid encoding the therapeutic gene may be positioned and expressed at different sites.
• the nucleic acid encoding the therapeutic gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
• the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
• the expression construct may be entrapped in a liposome.
• Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
• the lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers.
• the addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules.
• DNA-lipid complexes are potential non-viral vectors for use in gene therapy.
• Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
• Nicolau et al (Methods Enzymol, 149: 157-176, 1987, herein incorporated by reference) accomplished successful liposome-mediated gene transfer in rats after intravenous injection. Also included are various commercial approaches involving "lipofection" technology.
• the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA.
• HVJ hemagglutinating virus
• the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1).
• HMG-1 nuclear nonhistone chromosomal proteins
• the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
• receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a therapeutic gene into cells. These take advantage of the selective uptake of macromolecules by receptor- mediated endocytosis in almost all eukaryotic cells. Because of the cell type- specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, Adv. Drug Delivery Rev., 12: 159-167, 1993).
• Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
• ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, J. Biol Chem., 262:4429-4432, 1987) and transferrin (Wagner et al, Proc. Natl. Acad. Sci. 87(9):3410-3414, 1990).
• the delivery vehicle may comprise a ligand and a liposome.
• a ligand and a liposome For example, Nicolau et al Methods Enzymol, 149: 157-176, 1987, employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
• a nucleic acid encoding a therapeutic gene also may be specifically delivered into a cell type such as prostate, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes.
• the human prostate-specific antigen (Watt et al, Proc. Natl Acad.
• the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is applicable particularly for transfer in vitro, however, it may be applied for in vivo use as well. Dubensky et al. (Proc. Nat. Acad. Sci. USA, 81:7529-7533, 1984), successfully injected polyomavirus DNA in the form of CaPO 4 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
• Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, Nature, 327:70-73, 1987, herein incorporated by reference).
• Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, Proc. Natl. Acad. Sci. USA, 87:9568-9572, 1990).
• the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
• Another method of achieving gene transfer is via viral transduction using infectious viral particles as a delivery vehicle, for example, by transformation with an adenovirus vector of the present invention as described herein below.
• retroviral or bovine papilloma virus may be employed, both of which permit permanent transformation of a host cell with a gene(s) of interest.
• viral infection of cells is used in order to deliver therapeutically signific-ant genes to a cell.
• the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus.
• adenovirus is exemplified, the present methods may be advantageously employed with other viral vectors, as discussed below. Such methods will be familiar to those of ordinary skill in the art.
• Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity.
• the roughly 36 kB viral genome is bounded by 100- 200 base pair (bp) inverted terminal repeats (ITR), in which are contained cis- acting elements necessary for viral DNA replication and packaging.
• ITR inverted terminal repeats
• the early (E) and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA replication.
• the El region encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
• the expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan, 1990).
• the products of the late genes (LI, L2, L3, L4 and L5), including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP).
• MLP located at 16.8 map units
• TL tripartite leader
• adenovirus In order for adenovirus to be optimized for gene therapy, it is necessary to maximize the carrying capacity so that large segments of DNA can be included. It also is very desirable to reduce the toxicity and immunologic reaction associated with certain adenoviral products.
• the two goals are, to an extent, coterminous in that elimination of adenoviral genes serves both ends. By practice of the present invention, it is possible to achieve both these goals while retaining the ability to manipulate the therapeutic constructs with relative ease.
• Plasmids containing ITR's can replicate in the presence of a non-defective adenovirus. Therefore, inclusion of these elements in an adenoviral vector should permit replication.
• the packaging signal for viral encapsulation is localized between 194-385 bp (0.5-1.1 map units) at the left end of the viral genome.
• This signal mimics the protein recognition site in bacteriophage ⁇ DNA where a specific sequence close to the left end, but outside the cohesive end sequence, mediates the binding to proteins that are required for insertion of the DNA into the head structure.
• El substitution vectors of Ad have demonstrated that a 450 bp (0-1.25 map units) fragment at the left end of the viral genome could direct packaging in 293 cells.
• adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line. There also have been reports of complementation of replication deficient adenoviral vectors by "helping" vectors, e.g., wild-type virus or conditionally defective mutants.
• Replication-deficient adenoviral vectors can be complemented, in trans, by helper virus. This observation alone does not permit isolation of the replication- deficient vectors, however, since the presence of helper virus, needed to provide replicative functions, would contaminate any preparation. Thus, an additional element was needed that would add specificity to the replication and/or packaging of the replication-deficient vector. That element, as provided for in the present invention, derives from the packaging function of adenovirus.
• helper viruses that are packaged with varying efficiencies.
• the mutations are point mutations or deletions.
• helper viruses with low efficiency packaging are grown in helper cells, the virus is packaged, albeit at reduced rates compared to wild-type virus, thereby permitting propagation of the helper.
• helper viruses are grown in cells along with virus that contains wild-type packaging signals, however, the wild-type packaging signals are recognized preferentially over the mutated versions.
• the virus containing the wild-type signals are packaged selectively when compared to the helpers. If the preference is great enough, stocks approaching homogeneity should be achieved.
• the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription.
• the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
• the integration results in the retention of the viral gene sequences in the recipient cell and its descend-ants.
• the retroviral genome contains three genes - gag, pol and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
• a sequence found upstream from the gag gene, termed ⁇ functions as a signal for packaging of the genome into virions.
• Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter .and enhancer sequences and also are required for integration in the host cell genome.
• a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
• a packaging cell line containing the gag, pol and env genes but without the LTR and ⁇ components is constructed.
• the ⁇ sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media.
• Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression of many types of retroviruses require the division of host cells.
• AAV utilizes a linear, single-stranded DNA of about 4700 base pairs.
• Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non- structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription.
• the three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, pl9 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced.
• the splice site derived from map units 42-46, is the same for each transcript.
• the four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.
• AAV is not associated with any pathologic state in humans.
• AAV requires "helping" functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus.
• the best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAV replication.
• Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block.
• the terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV genome (Sarnulski et al J. Virol, 61(10):3096-3101, 1987, herein incorporated by reference), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV.
• the ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, /. e. , stable and site-specific integration.
• the ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration.
• AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, .and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo .and in vivo.
• AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, Ann. N Y. Acad. Sci., 770:79-90, 1995; Flotte et al, Proc. Natl. Acad. Sci. USA, 90: 10613-10617, 1993).
• viral vectors may be employed as expression constructs in the present invention.
• Vectors derived from viruses such as vaccinia viruses (Ridgeway, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez RL, Denhardt DT. ed., Stoneham: Butterworth, pp. 467-492, 1988; Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum Press, pp. 117- 148, 1986; Coupar et al, Gene, 68: 1-10, 1988) canary pox viruses, lentivirases and herpes viruses may be employed.
• the methods .and vectors of the present invention may be used to target a wide variety of cells, organs, and tissues within a mammal.
• the expression constructs described herein . are used to treat cancer.
• the cell which is targeted may be either a tumor cell, a cell within a tumor, or a cell near a tumor.
• the tumor may optionally be in the brain, lung, liver, spleen, kidney, bladder, lymph node, small intestine, pancreas, colon, stomach, breast, endometrium, prostate, testicle, vulva, cervix, ovary, skin, head and neck, esophagus, bone marrow, or blood.
• One of ordinary skill in the art will be able to readily discern an appropriate therapeutic gene to be expressed in a given tumor type.
• a medical condition other than cancer is being treated.
• the present invention provides for highly effective protein replacement therapy.
• a specific type of cell, tissue, or organ may be targeted for expression of a protein which is underexpressed in the subject, especially if the activity of the protein is limited to that specific cell type, tissue, or organ.
• the activity of the protein is limited to that specific cell type, tissue, or organ.
• the expression construct may be introduced into the cell of interest through an in vitro, ex vivo, or in vivo method. Much gene therapy is currently performed ex vivo, since the transfection or transduction of an isolated cell is often more efficient.
• the choice of method of introduction will be dependent upon the cell type, tissue, or org.an being t.argeted, as well as the particular delivery vehicle chosen. One of ordinary skill in the art can readily navigate such a choice.
• the expression constructs of the present invention require induction to be active, in many cases the expression construct may be delivered to a larger part of the subject's body than just the cell, tissue, or organ in which expression is desired. Exposure of the subject to the activating conditions which induce expression of the tr.ansferred expression constructs can then be limited to achieve specificity of expression. In many cases, this is preferred. For inst-ance, exposure of a subject to radiation therapy is preferably limited to only those areas necessary. Application of hyperthermia will generally also be limited in its range. In other embodiments, the activating conditions may be conditions inherent to the targeted cell itself. For instance, the hypoxic environment of a tumor will trigger expression when the expression construct has an inducible promoter containing a hypoxia- responsive element. In such cases, the resulting expression, will be by its very nature, very localized, even if delivery of the expression construct was not
• the expression constructs of the present invention may advantageously be combined with one or more traditional clinical therapies.
• One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy.
• One way is by combining such traditional therapies with gene therapy.
• the herpes simplex-thymidine kinase (HS-tk) gene when delivered to brain tumors by a retroviral vector system, successfully induces susceptibility to the antiviral agent ganciclovir.
• HS-tk herpes simplex-thymidine kinase
• the effective use of gene therapy in combination with traditional cancer therapies has been hindered by the need for clinically significant expression of the genes once they have been transferred to the target cell.
• compositions of the present invention To kill cells, inhibit cell growth, inhibit metastasis, decrease tumor size and otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present invention, one would generally introduce an expression construct of the present invention into the "target" cell and induce expression by the application of hyperthermia or other conditions which activate the inducible promoter.
• This gene therapy may be combined with compositions comprising other agents effective in the treatment of cancer. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell.
• This process may involve introducing the expression construct and the agent(s) or factor(s) into the cell at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by exposing the cell to two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the agent.
• the gene therapy/hyperthermia treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
• the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell.
• both agents are delivered to a cell in a combined amount effective to kill the cell.
• Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell.
• Such agents and factors include radiation and waves that induce DNA damage such as, ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like.
• the radiation therapy which is combined with the gene therapy constitutes external beam radiation.
• the external beam radiation treatment typically delivers high-energy radiation, such as high- energy x-ray beams.
• brachytherapy may be used in combination with the gene therapy.
• Methods of delivering brachytherapy include intracavitary or interstitial placement of radiation sources, instillation of colloidal solutions, and parenteral or oral administration. Sealed sources are encapsulated in a metal, wire, tube, needle, or the like. Unsealed radioactive sources are prepared in a suspension or solution.
• Encapsulated radioactive elements are placed in body cavities or inserted directly into tissues with suitable applicators.
• the applicator is usually placed into the body cavity or tissue surgically or using fluoroscopy.
• the applicators usually plastic or metal tubes, may be sutured into or near the tumor to hold them in place.
• the radioactive isotope is later placed into the applicator ("afterloading").
• Radiative implants are used in the treatment of cancers of the tongue, lip, breast, vagina, cervix, endometrium, rectum, bladder, and brain. Encapsulated sources may also be left within a patient as permanent implants. "Seeding" with small beads of radioactive material is an approach used for the treatment of localized prostate cancers, and localized, but inoperable, lung cancers.
• Chemotherapeutic agents function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein.
• Chemotherapeutic agents contemplated to be of use include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.
• the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
• the tumor cells in treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct and induce the expression of the gene by application of hyperthermia.
• This may be achieved by applying hyperthermia locally at the tumor site or systemically to the individual.
• This treatment may be in combination with irradiation of the tumor with radiation such as X-rays, UV-light, gamma-rays or even microwaves.
• the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin.
• the agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a therapeutic expression construct, as described above.
• Agents that directly cross-link nucleic acids, specifically DNA are envisaged to facilitate DNA damage leading to a synergistic, antineoplastic combination with the expression constructs of the present invention.
• Agents such as cisplatin, and other DNA alkylating agents may be used.
• Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
• Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation.
• Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, veraparnil, podophyllotoxin, and the like.
• these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m at 21 day intervals for adriamycin, to 100 mg/m 2 for etoposide intravenously or double the intravenous dose orally.
• nucleic acid precursors and subunits also lead to DNA damage.
• nucleic acid precursors have been developed.
• agents that have undergone extensive testing and are readily available are particularly useful.
• agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells.
• 5-FU is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 450-1000 mg/m /day being commonly used.
• ⁇ -rays X-rays
• X-rays X-rays
• UV-irradiation UV-irradiation
• Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens.
• Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
• the regional delivery of the therapeutic expression constructs of the present invention to patients with cancers is a preferred method for delivering a therapeutically effective gene to counteract the clinical disease being treated.
• the chemo- or radiotherapy may be directed to a particular, affected region of the subject's body.
• systemic delivery of expression construct and/or the agent may be appropriate in certain circumstances, for example, where extensive metastasis has occurred.
• combination of multiple gene therapies will be advantageous.
• targeting of p53 and pl6 mutations at the same time may produce an improved anti-cancer treatment.
• Any other tumor-related gene conceivably can be targeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BRCA2, pl6, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, rafi erb, sre, fins, jun, trk, ret, gsp, hst, bcl and abl
• compositions of the present invention may be administered, in vitro, ex vivo or in vivo.
• this will entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals.
• One also will generally desire to employ appropriate salts and buffers to render the complex stable and allow for complex uptake by target cells.
• the compositions of the present invention comprise an effective amount of the expression construct or a viral vector or liposome carrying the expression construct, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
• Such compositions also can be referred to as inocula.
• phrases "pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be inco ⁇ orated into the compositions.
• Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the therapeutic compositions of the present invention may include classic pharmaceutical preparations for use in therapeutic regimens, including their administration to humans.
• Administration of therapeutic compositions according to the present invention will be via .any common route so long as the target tissue or cell is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical.
• administration will be by orthotopic, intradermal subcutaneous, intramuscular, intraperitoneal, or intravenous injection.
• Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
• direct intratumoral injection injection of a resected tumor bed, regional (e.g., lymphatic) or systemic administration is contemplated. It also may be desired to perform continuous perfusion over hours or days via a catheter to a disease site, e.g., a tumor or tumor site.
• compositions of the present invention are administered advantageously in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
• a typical composition for such pu ⁇ ose comprises a pharmaceutically acceptable carrier.
• the composition may contain about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
• Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like may be used. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
• Intravenous vehicles include fluid .and nutrient replenishers.
• Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to well known par-ameters.
• Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
• the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
• the route is topical, the form may be a cream, ointment, salve or spray.
• An effective .amount of the therapeutic agent is determined based on the intended goal, for example (i) inhibition of tumor cell proliferation, (ii) elimination or killing of tumor cells, or (iii) gene transfer for short- or long-term expression of a therapeutic gene.
• unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, t.e., the appropriate route and treatment regimen.
• the quantity to be administered both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the result desired. Multiple gene therapeutic regimens are contemplated by the present inventors
• a vector encoding a therapeutic gene is used to treat cancer patients.
• Typical amounts of a viral vector used in gene therapy of cancer is 10 6 -10 15 PFU/dose (e.g., 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 and 10 15 ) wherein the dose is divided into several injections at different sites within a solid tumor.
• the treatment regimen also involves several cycles of administration of the gene transfer vector over a period of 3-10 wk. Administration of the vector for longer periods of time from months to years may be necessary for continual therapeutic benefit.
• a viral vector encoding a therapeutic gene may be used to vaccinate humans or other mammals.
• an amount of virus effective to produce the desired effect in this case vaccination, would be a ninistered to a human or other mammal so that long term expression of the transgene is achieved and a host immune response develops.
• a series of injections for example, a primary injection followed by two booster injections, would be sufficient to induce an long term immune response.
• a typical dose would be from 10 6 to 10 15 PFU/injection depending on the desired result.
• Low doses of antigen generally induce a strong cell-mediated response, whereas high doses of .antigen generally induce an antibody-mediated immune response.
• Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. 9. Examples
• HSP70 promoter To study the ability of the HSP70 promoter to induce gene expression, either a minimal heat shock (HS) promoter or a minimal CMV promoter was inserted upstream of a reporter gene in a plasmid containing neomycin and ampicillin selectable markers.
• HS minimal heat shock
• CMV minimal CMV promoter
• M5 was constructed by replacing the CMV promoter in pcDNA3.0 (Invitrogen, Inc.) with a minimal HSP70B promoter (SEQ ID NO: l, Figure 10), a 0.4 kb fragment (Hindlll-BamHl) of the human heat shock protein 70B (HSP70B) promoter, obtained from StressGen, Inc.
• HSP70B human heat shock protein 70B
• the S8 plasmid derived from the M5 vector of Figure 1, contains the minimal HSP70B promoter operably linked to the gene encoding Enh-anced Green Fluorescence Protein (EGFP).
• EGFP Enh-anced Green Fluorescence Protein
• S8 was constructed by inserting the EGFP gene from pEGFP-1 (Clonetech, Inc.) into the multiple cloning site (MCS) of M5.
• One positive clone, clone 4, and a polyclonal line were selected with geneticin from the MCF7 cells' transfection.
• One polyclonal line was selected with geneticin from the DU-145 cell's transfection. (In each case, the cells were selected with geneticin for 2 weeks.) The selected lines were then analyzed and sorted by FACS.
• MCF7 breast carcinoma parental cell line.
• Dul45 prostate carcinoma parental cell line.
• MCF7-S8-P MCF7 cells transfected with the S8 plasmid, polyclonal line.
• MCF7-S8-PS1 MCF7-S8-P cells that were sorted for EGFP expression by FACS once.
• MCF7-S8-PS2 MCF7-S8-PS 1 cells that were resorted for EGFP expression by FACS.
• MCF7-S8-4 Clone 4 of the MCF7 S8 transfection.
• MCF7-S8-4S1 MCF7-S8-4 cells sorted once for EGFP expression.
• Dul45-S8-P Dul45 cells transfected with the S8 plasmid, polyclonal line.
• the CMV promoter was inserted upstream of either the tat gene or a multiple cloning site (MCS) and either the HIV1 or HIV2 long terminal repeats (LTRs) was inserted upstream of the mouse interleukin-2 (IL-2) gene.
• the plasmids X14 and Y15 are shown schematically in Figure 9A and 9B.
• the L-27 plasmid served as a reference.
• IL-2 was measured from tissue culture supematants by ELISA using the IL-2 EASIA kit (Medgenix Diagnostics, Fleurus, Belgium). The sensitivity of the kit is estimated at 0.1IU IL-2/ml.
• SW480 cells were tr-ansfected with the lipid Dosper (see the transfection protocol of Example 3, below.).
• MCF-7 cells were transiently transfected with a series of vectors, including pC8, pfl2, and p007 ( Figures 8 and 9).
• the minimal heat shock promoter was inserted upstream of either the tat gene or a multiple cloning site (MCS) and either the HIV1 or HIV2 long terminal repeats (LTRs) was inserted upstream of the mouse interleukin-2 (IL-2) gene.
• the plasmids also each carried neomycin and ampicillin selectable markers.
• the plasmid fl l was first created by inserting a 0.5 kb EcoRI fragment, containing the interleukin-2 (IL-2) coding region (see GenBank accession no.
• the plasmid C8 was constructed by inserting a 1 kb
• the vector fl2 ( Figure 9) was then constructed by inserting a 0.4 kb NotI fragment, containing the coding region for the HIV tat gene, into the NotI site of C8.
• An intermediate vector D10 was constructed by inserting the 1 kb BamH I fragment containing the minimal HSP70B promoter into plasmid MNP-7 (Tsang et al, 1996, and Tsang et al, 1997), which contains the HIV2 LTR upstream of the IL-2 coding region.
• Plasmid 007 ( Figure 9) was created by inserting the 0.4 kb Not I fragment, encoding the tat gene, into the Not I site of D 10.
• Transfection Protocol Transfection were performed according to the published procedure (Stopeck, et al, Cancer Gene Therapy, 5: 119-126, 1998.) MCF-7 cells were plated in either a 6 well or 12 well plates. The next day, cells were washed with Hanks Buffered Saline Solution and replaced with a 1 ml transfection solution.
• the transfection solution was a 4: 1 lipid to DNA mass ratio of either Dosper (l,3-Di-Oleoyloxy-2-(6-Carboxy-spermyl)-propylamid, from Boehringer Mannheim) or Dmrie C (l,2-dimyristyloxypropyl-3dimethyl-hydroxy ethyl ammonium bromide, from Gibco BRL) with either 1.25 ⁇ g or 2.5 ⁇ g of plasmid DNA in serum-free OptiMEM (from Gibco BRL). Fetal bovine serum (FBS) was immediately added to each well to a final concentration of 10% (vol/vol). Dmrie C was determined to be a better lipid then Dosper. Cells were incubated for 24 hours before heating and 24 hours after heating prior to IL-2 quantitation.
• Dosper l,3-Di-Oleoyloxy-2-(6-Carboxy-spermyl)-propylamid, from Boehringer Mannheim
• HIV1-IL2 fl2 HSP-TAT 107.6 347.4 3.2 6.9 (17) HIV1-IL2
• the HIV1 promoter in the absence of tat expression, was similar to that of the CMV promoter and was nearly independent of heat shock. However, when the minimal heat shock promoter was used to express tat, reporter gene expression was dramatically increased after 42°C heat shock. In cells transiently transfected with heat shock promoter/t ⁇ t and HIV1/IL-2, IL-2 production was similar to that for heat shock promoter/MCS and HIV1/IL-2 in cells maintained at 37°C. This activity was increased over 3 fold, and to levels nearly 7 fold greater than CMV promoter activity by itself, after 42°C HS.
• the HS promoter/t ⁇ t and HIV2/IL-2 transfected cells showed substantial reporter gene expression in cells maintained at both 37 and after 42°C heat shock.
• Relative promoter activity, measured by IL-2 production was over 80 fold higher than that for the CMV promoter, alone.
• Temperature regulation was reduced, with reporter gene expression approximately 2 times higher after 42°C heat shock compared to the same activity in cells maintained at 37°C.
• reporter gene expression was not influenced by the presence of a second promoter.
• reporter gene expression in cells transiently transfected with the minimal heat shock promoter/t ⁇ t and HIV2/IL-2 containing plasmid, increased in a temperature-dependent manner between 37 and 44°C.
• MCF7 breast cancer cells were transiently transfected with vectors as shown; heat shocked for 1 hr 24 hrs later; media were collected and IL2 measured 24 hrs after heat shock
• SCID mice are injected with human tumor cells stably transfected with reporter constructs in which the HSP70B promoter is driving the expression of TAT and in which the HIV-1 or HIV-2 promoter is driving either EGFP or IL-2 expression.
• the tumors After growing the tumors to an appropriate measurable size of for example, 1 cm in diameter, the tumors .are heated using ultrasound to temperatures up to about 42°C. Gene expression is quantitated at various times after heating by either removing the tumor, making tissue slices and measuring fluorescence from EGFP or measuring tumor tissue levels of IL2 using ELISA.
• human tumor cells are injected into SCID mice.
• the tumors grown to .an appropriate measurable size and injected with DNA-lipid complexes.
• Tumors are heated using ultrasound and gene expression measured at times after heating. The efficacy of these treatments is indicated by a decrease in the size of the tumor, a decrease in metastatic activity, a decrease in cell proliferation or a halt in the tumor growth as a result of the administration of the therapeutic compositions of the present invention.

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