EP0716614A1 - Traitement de tumeurs humaines par transformation genetique de cellules tumorales humaines - Google Patents

Traitement de tumeurs humaines par transformation genetique de cellules tumorales humaines

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Publication number
EP0716614A1
EP0716614A1 EP94926678A EP94926678A EP0716614A1 EP 0716614 A1 EP0716614 A1 EP 0716614A1 EP 94926678 A EP94926678 A EP 94926678A EP 94926678 A EP94926678 A EP 94926678A EP 0716614 A1 EP0716614 A1 EP 0716614A1
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EP
European Patent Office
Prior art keywords
tumor
cells
agent
producer
brain
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP94926678A
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German (de)
English (en)
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EP0716614A4 (fr
Inventor
Edward J. Oldfield
Zvi Ram
R. Michael Blaese
Kenneth W. Culver
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US Department of Health and Human Services
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US Department of Health and Human Services
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Publication of EP0716614A1 publication Critical patent/EP0716614A1/fr
Publication of EP0716614A4 publication Critical patent/EP0716614A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to the treatment of human tumors by transforming or transducing human tumor .cel s with DNA (RNA) encoding an agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of such DNA (RNA) encoding the agent.
  • RNA DNA
  • Gene transfer has been recognized, for some time as a promising avenue to therapies for cancers, among other diseases.
  • the earliest applications of gene transfer for cancer treatment have been indirect approaches focusing on enhancing anti-tumor immune responses. Thus, for instance, attempts have been made to increase the cytotoxicity of immune cells, or to enhance their proliferation.
  • TNF tumor necrosis factor
  • TIL tumor infiltrating lymphocytes
  • tumor cells have been modified in vitro with cytokine genes and reintroduced into patients in an attempt to immunize the patient to their own cancer.
  • the IL-4 gene was introduced to tumors by Tepper, et al., Cell 57: 503 (1989); the IL-2 gene by Fearon, et al., Cell 60 :397 (1990), and by Gansbacher, et al., J. Exp. Med. 172: 1217 (1990); the interferon-gamma gene by Gansbacher, et al., Cancer Res. 50: 7820 (1990); and TNF gene by Asher, et al., J. Immunol. 146: 3227 (1991).
  • Each of the animal studies demonstrated rejection of genetically altered tumors upon reimplantation, and the mice in these studies were immune to subseguent rechallenge with the same tumor.
  • Retroviral vectors currently provide the most efficient means for ex vivo gene transfer in the clinical setting, but their usefulness has been seen as limited because retroviruses stably integrate only in target cells that are actively synthesizing DNA, and integration is a prerequisite to retroviral gene expression.
  • Cancers consist of actively replicating cells, however, and are often surrounded by quiescent normal cells. Thus, the above limitation may be exploited as an advantage in treating cancers, because a retroviral vector that carries a therapeutic agent would be integrated and expressed preferentially or exclusively in the cells of the cancerous mass.
  • HSV-1 tk gene herpes simplex virus 1
  • Ezzeddine, et al. were able to use the method to define conditions in vitro for killing essentially all infected cells but not uninfected cells.
  • C6 cells were introduced subcutaneously into nude mice to form tumors and the tumor-bearing mice were treated with ganciclovir.
  • Ganciclovir inhibited the growth of tumors formed by HSV-1 tk expressing C6 cells, but did not affect tumors formed by HSV-1 tk-negative C6 cells.
  • Ezzeddine, et al. thus showed that in vitro retroviral gene transfer can be used to sensitize cells to a cytotoxic agent, which can then be used to kill the cells when they are propagated as tumors in nude mice.
  • the authors did not demonstrate any practical way to introduce an HSV-1 tk gene into tumor cells in situ, however.
  • Ezzeddine, et al. also did not show how to eradicate all neoplastic cells, a prerequisite for tumor remission, when less than all cells in the tumor would take up a tk gene, express the gene at a level sufficient to assure toxicity and, as a consequence, be killed by exposure to ganciclovir. Short, et al., J. Neurosci. Res.
  • FIG. 27: 427-33 (1990) have described the delivery of genes to tumor cells by means of grafting a retroviral vector-packaging cell line into a tumor.
  • the packaging cell line produced a replication-defective retroviral vector in which the MoMLV LTR promoter-operator was used to drive expression of ⁇ - galactosidase, which served as a marker of retroviral vector propagation.
  • ⁇ -galactosidase expression in situ was seen only in packaging cells and in proliferating tumor cells, not in normal tissue.
  • Malignant brain tumors are responsible for significant morbidity and mortality in both pediatric and adult populations. These common tumors present an enormous therapeutic challenge due to their poor outcome despite radical surgery, high dose radiotherapy and chemotherapy. Survival of patients from the time of diagnosis is measured in months and recurrence after treatment is associated with a life expectancy of weeks.
  • Culver et al.. Science. Vol. 256, pgs. 1550-1552 (June 12, 1992) and Ram, et al.. Cancer Research, Vol. 53, pgs. 83-88 (January 1, 1993), disclose the administration by intratumoral injection of fibroblasts producing a retroviral vector which includes the Herpes Simplex thymidine kinase gene to rats with cerebral glioma. After the rats were given the producer cells, they were given ganciclovir.
  • PCT Application No. W093/04167 discloses a method for transferring therapeutic genes to brain tumor cells in order to kill the cells.
  • a retrovirus containing a selectable marker and at least one gene required for its replication is introduced into producer cells such that integration of the proviral DNA corresponding to the retrovirus into the genome of the producer cell results in the generation of a modified retrovirus wherein at least one of the genes required for replication of the retrovirus is replaced by the therapeutic gene or genes.
  • Producer cells then are selected in which the modified retrovirus is incorporated as part of the genome of the producer cells.
  • the producer cells then are grafted in proximity to the dividing tumor cells in order to infect the tumor cell with the modified retrovirus, thereby transferring the therapeutic gene or genes to the tumor cells.
  • the cells then are killed by administering a substance that is metabolized by the therapeutic gene transferred to the tumor cells into a metabolite that kills the cells.
  • the therapeutic gene may be the Herpes Simplex thymidine kinase gene, and the substance which is metabolized by Herpes Simplex thymidine kinase to kill the tumor cells may be ganciclovir or acyclovir.
  • the cited PCT application shows only (i) that a replication-defective retrovirus which carried an HSV tk gene and a G418 resistance gene could be transduced stably, via G418 selection, into a glioma cell line in vitro; (ii) that the viral tk gene in the transformed cells rendered them about 20-fold more sensitive to ganciclovir than control glioma cells; and (iii) that some glioma tumor cells which formed tumors when implanted in rat brains also expressed a ⁇ -galactosidase marker when the tumors were injected with a producer cell line which produced a retroviral vector with the marker gene.
  • the vector in the described experiments did not carry a tk gene, and there was no systemic administration of a chemotherapeutic agent.
  • the PCT application in question does not show that tumor cells can be rendered sensitive in vivo to any such agent.
  • a method of treating a tumor in a human host preferably a brain tumor.
  • the method comprises transducing tumor cells in vivo with a nucleic acid (DNA or RNA) sequence encoding an agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of the nucleic acid sequence encoding the agent.
  • a nucleic acid DNA or RNA
  • the nucleic acid sequence which encodes the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of the nucleic acid sequence is contained in an appropriate expression vehicle which transduces the tumor cells.
  • expression vectors include, but are not limited to, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors), and viral vectors.
  • the expression vector is a viral vector.
  • Viral vectors which may be employed include, but are not limited to, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and Herpes virus vectors.
  • the viral vector is a retroviral vector.
  • a packaging cell line is transduced with a viral vector containing the nucleic acid sequence encoding the agent which provides for the inhibition, prevention, or destruction of the tumor cells upon expression of the nucleic acid sequence encoding the agent to form a producer cell line including the viral vector.
  • the producer cells then are administered to the tumor, whereby the producer cells generate viral particles capable of transducing the tumor cells.
  • the viral vector is a retroviral vector.
  • retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.
  • the retroviral vector is an infectious but non-replication competent retrovirus.
  • replication competent retroviruses may also be used.
  • Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells.
  • Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
  • Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
  • a packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
  • the retroviral vector may be one of a series of vectors described in Bender, et al., J. Virol. 61:1639-1649 (1987), based on the N2 vector (Armentano, et al., J. Virol. , 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed. In the first of these vectors, LNL-XHC, there was altered, by site-directed mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point.
  • MoMuLV Moloney murine leukemia virus
  • pPr809 a 9 another glycosylated protein
  • MoMuSV Moloney murine sarcoma virus
  • the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV.
  • the 5' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells.
  • Miller has eliminated extra env sequences immediately preceding the 3' LTR in the LN vector (Miller, et al., Biotechniques. 7:980-990, 1989).
  • Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector.
  • Miller, et al. have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging- defective helper genome (i.e. LN with pPAM3).
  • the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and Miller, et al. (1989).
  • Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
  • the term "mutated” as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.
  • the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs.
  • Preferred cloning sites are selected from the group consisting of NotI, SnaBI, Sail, and Xhol.
  • the retroviral vector includes each of these cloning sites. Such vectors are further described in U.S. Patent Application Serial No. 919,062, filed July 23, 1992, and incorporated herein by reference.
  • a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, Sail, and Xhol located on the retroviral vector.
  • the shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
  • the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
  • the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems.
  • the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria.
  • the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
  • the vector includes one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters).
  • CMV cytomegalovirus
  • Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the vector then is employed to transduce a packaging cell line to form a producer cell line.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2 , ⁇ -AM, PA12, T19- 14X, VT-19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines.
  • the vector containing the nucleic acid sequence encoding the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of the nucleic acid sequence encoding the agent may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • the producer cells then are administered directly to or adjacent to the tumor in an amount effective to inhibit, prevent, or destroy the growth of the tumor.
  • the producer cells are administered in an amount of at least 2.5 x 10 8 cells per cc of tumor.
  • the amount of cells administered does not exceed 4 x 10 8 cells per cc of tumor; however, greater amounts may be used.
  • the exact amount of producer cells to be administered is dependent upon various factors, including but not limited to, the type of the tumor and the size of the tumor.
  • the producer cells are administered directly to or adjacent to the tumor by injection.
  • the cells may be administered by use of a CT or MRI guided stereotaxic system which permits representation of a tumor mass on a two dimensional implantation grid, such as one containing 89 holes.
  • the system can provide the exact coordinates, positions and injection tracts to optimize the distribution of producer cells into a tumor mass.
  • the producer cells are administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient.
  • the carrier may be a liquid carrier such as, for example, a saline solution or a buffer solution or other isomolar aqueous solution.
  • the producer cells Upon administration of the producer cells to the tumor, the producer cells generate viral particles.
  • the viral particles then transduce the surrounding tumor cells. Because tumor cells, and in particular cancerous tumor cells, in general are actively replicating cells, the retroviral particle would be integrated into and expressed preferentially or exclusively in the tumor cells as opposed to normal cells.
  • Tumors which may be treated in accordance with the present invention include malignant and non-malignant tumors.
  • Malignant (including primary and metastatic) tumors which may be treated include, but are not limited to, those occurring in the adrenal glands; bladder; bone; breast; cervix; endocrine glands (including thyroid glands, the pituitary gland, and the pancreas); colon; rectum; heart; hematopoietic tissue; kidney; liver; lung; muscle; nervous system; brain; eye; oral cavity; pharynx; larynx; ovaries; penis; prostate; skin (including melanoma); testicles; thy us; and uterus.
  • the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of such agent is a negative selective marker; i.e., a material which in combination with a chemotherapeutic or interaction agent inhibits, prevents or destroys the growth of the tumor cells.
  • a negative selective marker i.e., a material which in combination with a chemotherapeutic or interaction agent inhibits, prevents or destroys the growth of the tumor cells.
  • Negative selective markers which may be employed include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase; and cytosine deaminase.
  • thymidine kinase such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase
  • cytosine deaminase include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase.
  • the negative selective marker is a viral thymidine kinase selected from the group consisting of Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase.
  • the interaction or chemotherapeutic agent preferably is a nucleoside analogue, for example, one selected from the group consisting of ganciclovir, acyclovir, and 1-2-deoxy- 2-fluoro-3-D-arabinofuranosil-5-iodouracil (FIAU) .
  • Such interaction agents are utilized efficiently by the viral thymidine kinases as substrates, and such interaction agents thus are incorporated lethally into the DNA of the tumor cells expressing the viral thymidine kinases, thereby resulting in the death of the tumor cells.
  • the negative selective marker is cytosine deaminase.
  • cytosine deaminase is the negative selective marker
  • a preferred interaction agent is 5-fluorocytosine. Cytosine deaminase. converts 5- fluorocytosine to 5-fluorouracil, which is highly cytotoxic. Thus, the tumor cells which express the cytosine deaminase gene convert the 5-fluorocytosine to 5- fluorouracil and are killed.
  • the interaction agent is administered in an amount effective to inhibit, prevent, or destroy the growth of the transduced tumor cells.
  • the interaction agent may be administered in an amount from 5 mg to 10 mg/kg of body weight, depending on overall toxicity to a patient.
  • the interaction agent preferably is administered systemically, such as, for example, by intravenous administration, by parenteral administration, by intraperitoneal administration, or by intramuscular administration.
  • tumor cells which were not originally transduced with the nucleic acid sequence encoding the negative selective marker may be killed upon administration of the interaction agent.
  • the transformed tumor cells may be producing a diffusible form of the negative selective marker that either acts extracellularly upon the interaction agent, or is taken up by adjacent, non-transformed tumor cells, which then become susceptible to the action of the interaction agent. It also is possible that one or both of the negative selective marker and the interaction agent are communicated between tumor cells.
  • a packaging cell line is transduced with a retroviral vector, such as those hereinabove described, which includes the Herpes Simplex Virus thymidine kinase gene.
  • the transduced packaging cells are administered j_n vivo to the tumor in an acceptable pharmaceutical carrier and in an amount effective to inhibit, prevent, or destroy the growth of the tumor.
  • the producer cells Upon administration of the producer cells to the tumor, the producer cells generate viral particles including a gene encoding the negative selective marker. Such viral particles transduce the adjacent tumor cells.
  • the human host then is given an agent such as ganciclovir.
  • acyclovir or l-2-deoxy-2-fluoro-3- D-arabinofuranosil-5- iodouracil (FIAU), which interacts with the Herpes Simplex Virus thymidine kinase to kill the transduced tumor cells.
  • FIAU Herpes Simplex Virus thymidine kinase
  • a coordinated system of diagnostic imaging, computer-image analysis and stereotaxic surgical manipulations is employed to administer a producer cell to a tumor with the least damage to the patient and with maximum therapeutic effect.
  • a set of multiple, parallel microinjection trajectories can be determined, and producer cells are deposited at sites along the trajectories. In this manner, the producer cells are more evenly distributed, preferably to effect an equi-volumetric, homogeneous distribution of the producer cells in the tumor.
  • data for a three-dimensional image of the tumor in situ first are obtained, preferably via a diagnostic imaging technique such as computerized tomography (CT) or magnetic resonance (MR) imaging.
  • CT computerized tomography
  • MR magnetic resonance
  • An image employed in this context should contain markers, called “fiducials,” which appear as spots on the image and which relate in a precise manner to the anatomy of the individual patient.
  • the fiducials provide a means for precise translation of co-ordinates in the images into co-ordinates on a stereotaxic instrument for neuroinjection into the individual patient.
  • the use of fiducials for this purpose is a well-known expediency in neurosurgical technique.
  • the data from the CT or MR image preferably are translated for use by a software program that facilitates interactive analysis to determine sites and trajectories for microinjecting a producer cell, such as retroviral producer cells, into the tumor.
  • the analysis can be carried out on a high-end graphics workstation or a more powerful computer to permit real-time interactive development of the icroinjection plan.
  • a variety of commercially available software and hardware can be modified for use in accordance with this aspect of the present invention.
  • Software that has been developed specifically for planning stereotaxically guided procedures may be adapted most readily for this purpose.
  • Exemplary of such software is the stereotaxic planning system commercialized by BrainLAB (Munich, Germany), implemented in the BrainSCAN software.
  • the BrainSCAN software provides for manipulation of three-dimensional tumor images acquired, for example by CT or MR imaging.
  • the software provides convenient utilities for viewing images, identifying and visualizing a tumor mass in an organ, calculating a volume, calculating a physical center, plotting microinjection trajectories, displaying the passage of trajectories through a tumor and, by the fiducials in an image, translating co-ordinates in an image into co-ordinates for use on a stereotaxic instrument. Accordingly, the software represents a powerful tool for a physician's identifying the most effective set of microinjection trajectories. Its use in planning a therapeutic procedure in accordance with the present invention is illustrated below.
  • the CT or MR data provide two- dimensional, sectional views of the imaged area of the patient which combined computationally, yield a three- dimensional view.
  • the tumor is identified in the two- dimensional views by the physician, who outlines the contour of the tumor(s) using a light pen or similar device. After entering the therapeutic targets into the image analysis program in this way, the physician can use the software utilities to view and assess the efficacy of different neurological approaches.
  • BrainSCAN software has been adapted to depict a grid which has been developed for guiding the microinjections. The physician in effect can place the grid anywhere on the surface of the brain.
  • the software on command, will display a microinjection trajectory for each needle guide in the grid, which accommodates many needles.
  • the program illuminates trajectories that pass through a tumor and shows their path.
  • the software summarizes which needles will pass through the tumor and the percentage of the tumor that will be covered by the intersecting trajectories.
  • the sites of cell deposition should be centered as much as possible on roughly equal volumes of the tumor. That is, the distribution of the producer cells should be equivolumetric, insofar as practical for the shape and accessibility of the tumor mass(es). Chosen to achieve this end, therefore, will be (i) the entry position, exposed by craniotomy in accordance with the present invention, where the grid will be placed on the brain; (ii) a set of parallel trajectories and, correspondingly, of needle positions in the grid; and (iii) the deposition sites along each trajectory.
  • the entry site should be convenient to the surgical procedure, which must expose the brain for the multiple injections.
  • a craniotomy is performed and the entry point is assessed for convenience in relation to the surgical procedure.
  • the trajectories also are chosen to avoid blood vessels. Another objective in planning the surgical procedure is to avoid passing a needle through any area particularly sensitive to mechanical damage.
  • the grid placement and the trajectories may be chosen to provide the least invasive procedure.
  • the program provides a listing of stereotaxic coordinates for carrying out the microinjection.
  • the co-ordinates correspond exactly to device markings that are used in the surgery. Included in the listing are the depths in each microinjection trajectory for depositing cells.
  • the procedure may be executed according to a plan developed in this manner, using well-known neuro ⁇ urgical procedures, as exemplified below.
  • This methodology contemplates trajectories and deposition sites that are more closely spaced than is characteristic of conventional neurosurgical techniques.
  • the production of multiple, parallel tracks along microinjection trajectories determined as described above requires, for properly positioning the needles, exposing a relatively large area of the brain by craniotomy.
  • the grid for guiding the needles in a particularly preferred embodiment of the present invention is placed directly onto the surface of the exposed brain.
  • FIG. 9 is a photograph which shows a typical stereotaxic instrument for neurosurgery equipped with a grid system for guiding needles along multiple parallel microinjection trajectories. Surgical devices, including needles, can be seen in their parallel paths in the grid.
  • the depicted " device provides a plurality of closely spaced guides for microinjection.
  • the device provides 89 guide holds with a center-to-center spacing of 3mm.
  • the guides are attached to a stereotaxic instrument which permits precise positioning of the injection needles to follow the trajectories determined during by image analysis of the tumor, described above.
  • the injection may be carried out using commercially available equipment.
  • the needles and syringes may be those generally employed in neurosurgery. Narrow gauge needles are preferred. Standard syringes may be employed, such as 100 microliter Hamilton syringes.
  • the needle may be inserted into the brain manually. Delivery of fluid also may be controlled manually directly by the syringe plunger.
  • a needle In delivering the producer cells into the brain, preferably a needle first is inserted along its trajectory to the maximum extent planned and cells are there deposited. Then the needle is withdrawn incrementally, with stops made to deposit cells at predetermined points along the path. In this regard it has been found practical to move the needle in increments of millimeters. The same procedure is used for each trajectory until all the planned depositions have been made.
  • the spacing of trajectories, the distribution of injection sites along the trajectories, and the volume and concentration of producer cells which will be optimal for different tumors, distinguished by type, stage, site, patient characteristics and the like, and for different vectors and interaction agents, ultimately will be determined empirically, i.e., by reference to therapeutic efficacy.
  • treatment experience will be incorporated into the foregoing procedures to provide more productive therapeutic designs against an increasingly wider variety of human tumors.
  • the method of the present invention is particularly useful when the targeted tumor is in a tissue made up of cells which are relatively quiescent mitotically, such as liver, skin, bone, brain muscle, bladder, prostate, kidney, adrenal, pancreas, heart, blood vessel and thyroid tissues, among others.
  • the inventive approach also should be useful against tumors located in the subarachnoid space, in the peritoneum, and in the pleural cavity.
  • Particularly preferred targets are brain tumors, which display several features making them especially susceptible to treatment in accordance with the present invention.
  • Neurons and most other stationary cells in the brain are quiescent and do not regularly synthesize DNA.
  • Vascular endothelial cells in the brain may be cycling at a low rate, but among those most likely to be in cycle would be cells responding to angiogenesis promoting signals often localized in the vicinity of a tumor. Such vessels would most likely be part of the blood supply of the tumor and therefore their destruction would also be desirable.
  • the principal mitotically active cells would be tumor cells or cells necessary for its support. Accordingly, retroviral vectors introduced into the brain principally will integrate into and affect only tumor cells or cells associated with tumor vascularization.
  • Brain tumors often are localized and yet are often inoperable because of their location in relationship to adjacent critical structures. Accordingly, a technique within the present invention, whereby delivery of a toxic product to the tumor is effected without surgical resection, is very useful. .Another advantage of targeting brain tumors in the present invention is that the brain is an immunologically privileged site and, thus, may permit retroviral vector-producer cells which are histoincompatible to persist for a significant period without immunologic rejection.
  • Direct injection of the producer cells also minimizes undesirable propagation of the virus in the body, especially when replication-competent retroviral vectors are used. Because most cells of the body express receptors for amphotropic retroviral vectors, any vector particle which escapes from the local environment of the tumor should immediately bind to another cell. Most cells are not in cycle, however, and therefore will not integrate the genes carried by the vector and will not express any genes which it contains. Thus, the proportion of potential target cells which are in cycle at the time of exposure will be small, and systemic toxic effects on normal tissues will be minimized.
  • An alternative and preferred embodiment of the present invention employs surgical resection and implantation of a reservoir (sometimes hereinafter referred to as an "Ommaya Reservoir") into the tumor bed of the brain and thereby provides an access port to the brain for introduction of producer cells of the type hereinabove described.
  • the cells are injected through the overlying skin and into the reservoir.
  • Example 1 Gene Therapy for the Treatment of Brain Tumors Using Intratumoral Transduction with the Thymidine Kinase Gene and Intravenous Ganciclovir
  • Brain tumors are a major cause of morbidity and mortality in the population. New brain tumors develop in approximately 35,000 adult Americans each year. They comprise the third leading cause of death from cancer in persons 15 to 34 years of age. Mahaley, et al., J. Neurosur ⁇ . ⁇ Vol. 71, pgs. 826-836 (1989). Recent evidence indicates that the prevalence of primary brain tumors is increasing, especially in the elderly. Saleman, et al., in Apuzzo, ed.. Malignant Cerebral Glioma. Park Ridge, 111., Association of Neurological Surgeons, pgs. 95-110 (1990).
  • the astroglial brain tumors including the highly malignant glioblastoma multiforme (GBM), are the most common primary brain tumors.
  • GBM highly malignant glioblastoma multiforme
  • a mirati et al., Neurosurgery. Vol. 21, pgs. 607- 614 (1989). Although controversial, it appears that neither the quality nor time of survival is significantly improved when chemotherapy is added to surgery and radiation. Walker, et al., J. Neurosurg.. Vol. 49, pgs. 333-343 (1978).
  • Cerebral metastases are a frequent complication of systemic cancer occurring in 20 to 30 percent of patients with cancer, Cairncross, et al., in Walker, ed.. Cancer Treatment and Research, Vol. 12, pgs. 341-377, Boston, Martinus Nighoff (1983) (there are 1.1 million new cases of cancer per year in the United States).
  • the metastatic disease is localized to the central nervous system.
  • a subset of patients may even be cured of their primary cancers only to succumb to the isolated metastatic disease in the brain.
  • the central nervous system has several advantages of safety and efficacy for in vivo gene transfer.
  • retroviral vectors only integrate and therefore express vector genes in proliferating cells.
  • the tumor is the most mitotically active cell, with only macrophage-derived cells, blood cells, and endochelial cells at minimal risk. Therefore, the possibility of specific transduction of the tumor is enhanced.
  • the brain is a partially immunologic privileged site, which should allow a somewhat longer survival of the xenogeneic murine cells in the brain and a greater transduction frequency of the growing tumor.
  • a special feature of human gliomas is their ability to depress local immunity.
  • This vector contains the Thymidine Kinase (hTK) gene from herpes simplex virus I regulated by the retroviral promoter and the bacterial gene, neomycin phosphotransferase (Neo R ) driven by an SV40 promoter.
  • hTK Thymidine Kinase
  • the hTK gene confers sensitivity to the DNA analogs acyclovir and ganciclovir, while the Neo R gene product confer resistance to the neomycin analogue, G418.
  • pGlTkSvNa To make pGlTkSvNa, a three step cloning strategy was used. First, the herpes simplex thymidine kinase gene (Tk) was cloned into the Gl plasmid backbone to produce pGlTk. Second, the Neo R gene (Na) was cloned into the plasmid pSvBg to make pSvNa. Finally, SvNa was excised from pSvNa and ligated into pGlTk to produce pGlTkSvNa.
  • Tk herpes simplex thymidine kinase gene
  • Na Neo R gene
  • SvNa was excised from pSvNa and ligated into pGlTk to produce pGlTkSvNa.
  • Plasmid pGlTkSvNa was derived from plasmid PG1 ( Figure 3). Plasmid pGl was constructed from pLNSX (Palmer, et al.. Blood. Vol. 73, pgs. 438-445. The construction strategy for plasmid pGl is shown in Figure 1. The 1.6kb EcoRI fragment, containing the 5' Moloney Murine Sarcoma Virus (MoMuSV) LTR, and the 3.0kb EcoRl/Clal fragment, containing the 3' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately.
  • MoMuSV Moloney Murine Sarcoma Virus
  • pGl Figure 3
  • pGl Figure 3
  • MCS 54 base pair multiple cloning site
  • the MCS was designed to generate a maximum number of unique insertion sites, based on a screen of non-cutting restriction enzymes of the pGl plasmid, the neo r gene, the J3-galactosidase gene, the hygromycin r gene, and the SV40 promoter.
  • the structure of the 5' linker was as follows: 5' - 1/2 Ndel - SphI - NotI - SnaBI - Sail - SacII - AccI - Nrul - Bglll - III 27 bp ribosomal binding signal - Kozak consensus sequence/Ncol - first 21 bp of the lacZ open reading frame - 1/2 BamHI - 3' .
  • the structure of the 3' linker was as follows: 5' - 1/2 mutated EcoRI - last 55 bp of the lacZ open reading frame - Xhol
  • the Kozak consensus sequence (5'-GCCGCCACCATGG-3' ) has been shown to signal initiation of mRNA translation (Kozak, Nucl.Acids Res. 12:857-872, 1984).
  • the Kozak consensus sequence includes the Ncol site that marks the ATG translation initiation codon.
  • pBR322 (Bolivar et al. Gene 2:95, 1977) was digested with Ndel and EcoRI and the 2.1 kb fragment that contains the ampicillin resistance gene and the bacterial origin of replication was isolated.
  • the ligated 5' linker - lacZ - 3' linker DNA described above was ligated to the pBR322 Ndel/EcoRI vector to generate pBg.
  • pBg has utility as a shuttle plasmid because the lacZ gene can be excised and another gene inserted into any of the restriction sites that are present at the 5' and 3' ends of the lacZ gene. Because these restriction sites are reiterated in the pGl plasmid, the lacZ gene or genes that replace it in the shuttle plasmid construct can easily be moved into pGl.
  • a 1.74 kB Bglll/PvuII fragment containing the Herpes Simplex Virus Type I thymidise kinase gene (GenBank accession no. V00467, incorporated herein by reference) was excised from the pXl plasmid (Huberman, et al., Exptl. Cell Res. Vol. 153, pgs 347-362 (1984) incorporated herein by reference), blunted with the large (Klenow) fragment of DNA polymerase I, and inserted into the unique SnaBI site in the pGl multiple cloning site, to form plasmid pGlTK. ( Figure 5) .
  • a producer cell line was made from vector plasmid and packaging cells.
  • the PA317/GlTkSvNa producer cell was made by the same techniques used to make previous clinically relevant retroviral vector producer cell lines.
  • the vector plasmid pGlTkSvNa DNA was transfected into a ecotropic packaging cell line, PE501. Supernatant from the PE501 transfected cells was then used to transinfect the amphotropic packaging cell line (PA317).
  • Clones of transinfected producer cells were then grown in G418 containing medium to select clones that contain the Neo R gene. The clones were then titered for retroviral vector production. Several clones were then selected for further testing and finally a clone was selected for clinical use.
  • PE501 cells 5 x 10 s PE501 cells (Miller, et al., Biotechniques, Vol. 7, pgs. 980-990 (1989), incorporated herein by reference) were plated in 100 mm dishes with 10 ml high glucose Dulbecco's Modified Essential Medium (DMEM) growth medium supplemented with 10% fetal bovine serum (HGD10) per dish. The cells were incubated at 37°C, in 5% C0 2 /air overnight. The plasmid pGlTKSvNa then was transfected into PE501 cells by CaP0 4 precipitation using 50 ⁇ g of DNA by the following procedure.
  • DMEM Dulbecco's Modified Essential Medium
  • HFD10 fetal bovine serum
  • a culture dish(es) with optimum precipitate following the overnight incubation then was (were) selected.
  • the dish(es) then was (were) washed again with PBS to remove the salt and the salt solution.
  • 10 ml of HGD10 medium then was added to the dish(es), and the dish(es) incubated at 37°C in a 5% C0 2 atmosphere for about 48 hrs.
  • PE501 supernatarits from such colonies of PE501 cells were collected in volumes of from about 5 to 10 ml, placed in cryotubes, and frozen in liquid nitrogen at -70°C.
  • PA317 cells (Miller et al. Mol. Cell. Biol. 6:2895- 2902 (1986)) then were plated at a density of 5 x 10 4 cells per 100 mm plate on Dulbecco's Modified Essential Medium (DMEM) including 4.5 g/1 glucose, glutamine supplement, and 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Essential Medium
  • FBS fetal bovine serum
  • the PE501 supernatant then was thawed, and 8 ⁇ g/ml of polybrene was added to the supernatant.
  • the medium was aspirated from the plates of PA317 cells, and 7 to 8 ml of viral supernatant was added and incubated overnight.
  • the PE501 supernatant then was removed and the cells refed approximately 18-20 hours with fresh 10% FBS.
  • the medium was changed to 10% FBS and G418 (800 ⁇ g/ml).
  • the plate then was monitored, and the medium was changed to fresh 10% FBS and G418 to eliminate dying or dead cells as necessary.
  • the plate was monitored for at least 10 to 14 days for the appearance of G418 resistant colonies.
  • the cells were tyrpsinized and incubated into wells in a six well dish in 5 ml of HGD10 plus lx hypoxanthine aminopterin thymidine (HAT) .
  • clones grew to confluency, they were trypsinized and incubated in a 100 ml dish. As a clone in the 100 ml dish approached confluency, its amphotropic vector-containing supernatant was removed and centrifuged at 1,200 to 1,500 rpm for 5 minutes to pellet out cells.
  • FIG. 7 A schematic representation for producing pGlTKlSvNa shown in Figure 7, which was prepared to remove the partial open reading frame from pGlTKSvNa ( Figure 6).
  • Generation of pSPTK5 '
  • DNA from the plasmid pGlNaSvTk was digested with restriction enzymes Bglll and S al and the 1163 base pair (bp) Herpes thymidine kinase (TK) fragment was fractionated by agarose gel electrophoresis and isolated. This fragment contains 56 bp of the TK 5'-untranslated region and 1107 bp of the TK translation open reading frame.
  • the 1163 bp TK fragment was ligated to the plasmid vector pSP73 (Promega Corporation, Madison, WI) that had been digested with restriction enzymes Bglll and S al.
  • pSPTK5 The resulting ligated plasmid construct was named pSPTK5' because it contains the 5' portion of the TK open reading frame but lacks the last 21 bp of the open reading frame and the translation termination codon.
  • the linearized pGlNaSvTK was used as a template for polymerase chain reaction (PCR) using a forward primer that contains the first 17 bases of the TK open reading frame (5'-GCACCATGGCTTCGTACCCCTGC-3' ) and a reverse primer that contains complementary sequence for an Xhol site, the TK translation termination codon, and the last 19 bp of the TK open reading frame (5'-PCR) using a forward primer that contains the first 17 bases of the TK open reading frame (5'-GCACCATGGCTTCGTACCCCTGC-3' ) and a reverse primer that contains complementary sequence for an Xhol site, the TK translation termination codon, and the last 19 bp of the TK open reading frame (5'-PCR) using a forward primer that contains the first 17 bases of the TK open reading frame (5'-GCACCATGGCTTCGTACCCCTGC-3' ) and a reverse primer that contains complementary sequence for an Xhol site, the TK translation termination codon, and the last 19 bp of the
  • PCR products were fractionated on an agarose gel and the expected 1215 bp fragment that includes the full-length TK open reading frame was isolated.
  • the isolated fragment was digested with restriction enzymes PstI and Xhol, digestion products were fractionated on an agarose gel, and the 420 bp fragment was isolated. This fragment extends from the PstI site at the nucleotides encoding amino acids 249-250 of the TK open reading frame through the Xhol site immediately downstream of the TGA translation termination codon.
  • pSPTK5' was digested with PstI and the 3993 bp fragment that contains the pSP73 vector and the 5' portion of the TK open reading frame was isolated following agarose gel electophoresis. This 3993 bp fragment was ligated to the PCR-generated 420 bp Pstl/Xhol fragment that contains the 3' end of the TK open reading frame (above) .
  • Ligated plasmid DNA was transformed into E. coli DK5 competent cells (Gibco/BRL, Gaithersburg, MD) and DNA from ampicillin-resistant colonies was screened by restriction enzyme digestion. Plasmids that appeared to contain the full-length TK open reading frame were termed pSPTKl.
  • pSPTKl DNA was digested with Bglll and the 5' overhanging ends were repaired by incubation of the digested DNA with deoxy nucleotides and Klenow fragment of E. coli DNA poly erase I.
  • the DNA was then digested with Xhol to generate a 1225 bp fragment that contains 56 bp of TK 5'-untranslated region and the full-length TK open reading frame.
  • This blunt/xhol fragment was ligated to pGlXSvNa DNA that had been digested with SnaBI and Sail.
  • pGlXSvNa the 1.2 kb SvNa fragment was excised from pSvNa (Part A above) with Sail and Hindlll. This fragment was ligated to pGl that had been digested with Sail and Hindlll.
  • the ligated plasmid was termed pGlXSvNa where the "X" denotes a multiple cloning region.
  • pGlTKlSvNa Plasmids that appeared to contain the TK fragment by diagnostic restriction enzyme digestion were termed pGlTKlSvNa. Clone #2 was dideoxy sequenced from the beginning of the 5'-LTR through the end of the 3'-LTR and was found to contain the intact TK open reading frame.
  • pGlTKlSvNa was used to produce producer cell lines by combination with PA 317 by the hereinabove described method (Part B above). The titers of the viral particles produced by the cell lines were examined. One high-titer producing cell line, designated as producer cell line PA 317/GlTKlSvNa.7, was chosen for clinical development. D. Administration of Producer Cells to Human Patients
  • the gadolinium(Gd)-enhanced tumor mass was considered to represent one or more spheres (according to the configuration of the tumor). Each of these spheres then received 5 to 7 stereotaxic trajectories (creating columns of injected cells within the tumor mass) to deliver the producer cells in a homogeneous manner.
  • Each injection was performed using a 100 ⁇ l Hamilton syringe and, depending on the total volume of the tumor and the available number of cells, each mm of tumor along the tract was injected with 50 - 100 ⁇ l of cell suspension (5x10° - 10 7 producer cells in Plasma-Lyte® A Injection, an electrolytic solution, pH 7.4, produced by Baxter Health Care Corporation , Deerfield, Illinois. The length of each column varied at different areas of the tumor. Injections were initially performed via a single burr hole in the skull.
  • the technique was modified after the first two patients were injected. Cells were seen exuding along the injection tract and a concentration gradient was created due to the expanding trajectories from a single point origin of injection. Accordingly, a craniectomy (removal of skull) replaced a single burr hole, thus exposing a larger surface of the brain and enabling multiple injections at parallel trajectories.
  • ganciclovir (Cytovene, Syntex Corp.) intravenously, in a dose of 5 mg/kg body weight, daily for 14 days.
  • MRI scans were performed frequently at the early stages of the treatment and at two to 4 weeks intervals thereafter.
  • FDG radioactive glucose
  • a craniotomy is performed and a frozen section is taken to confirm viable glioblastoma cells and optimal tumor removal is attempted.
  • the patient's tumor is cryopreserved.
  • the surgical margin of the cavity is infiltrated at multiple sites with the GlTklSvNa.7 vector producer cells (day 0) to a maximum volume of 10 ml.
  • Vector producer cells suspended in Plasma Lyte A at a concentration of 1-2 times 10 8 cells/ml are inoculated slowly in 0.25 to 0.5 ml inocula at sites distributed as evenly around the tumor sites as possible.
  • An Ommaya reservoir is placed into the tumor bed to allow future access to this area.
  • a diagram of the sites of inoculation and volumes of cells delivered at each site is kept as part of the permanent research records.
  • An MR scan is performed within 48 hours following surgery to define the size of the residual tumor.
  • the Ommaya reservoir has sealed into the patient's brain and surrounding tissues.
  • skin overlying the Ommaya reservoir is cleansed with betadine.
  • a needle is inserted through the overlying skin into the reservoir with the patient awake.
  • the Ommaya reservoir is gently irrigated with normal saline to clarify the contents of the tumor cavity and to confirm the estimate of tumor cavity volume.
  • Additional vector producer cells in suspension are delivered following the attempted removal of an equivalent amount of fluid from the tumor cavity.
  • Ganciclovir is administered twice a day by IV infusion over one hour (dose of 5 mg/kg) starting on the 21st post ⁇ operative day for 14 days (days 21 to 34), followed by a 7- day rest period. This completes the initial treatment cycle.
  • MR scans are done at the end of each cycle. Only patients with responding or stable disease will continue treatment. If assessment of the MR scan leaves an uncertainty as to whether an increased size of an enhancing lesion is due to tumor progression or inflammatory changes, a biopsy is taken to make a definitive diagnosis.
  • Each repeat cycle consists of vector producer cells injected through the Ommaya reservoir on day 0 of the retreatment cycle.
  • Ganciclovir treatment commences on day 14 and continues for 14 days (days 14 to 27). No treatment is given for 7 days (days 28 to 34). Upon completion of the patient's last cycle of ganciclovir, the patient is followed at post- treatment months 1, 2, 3, 5, 7, 9, and 12, then every three months for the second year, and then at least annually for life.
  • Factors such as tumor size, location and the pre- operative neurological condition of the patient will determine the injectable volume.
  • the volume of injected cells is, preferably, not in excess of 10 ml.
  • the final cell concentration will be adjusted to 1-2 times 10 8 cells/ml.
  • All patients receive a single dose of an antibiotic, such as Vancomycin, just prior to the initial surgical procedure and prior to the insertion of the Ommaya reservoir. All patients may also receive Dexamethasone starting 7 days prior to the initial surgery. Following the surgery, the dose will be tapered based upon the clinical condition of the patient in order to wean the patient off Dexamethasone treatment. Patients .may receive mannitol during the surgical procedure at 1 g/kg, and the dose may be repeated t.i.d.
  • Anticonvulsant therapy is administered according to the usual neurosurgical guidelines. Pain medications may include acetaminophen, preferably in a dosage of 650 to 1000 mg q 4 hours. Patients may also receive G-CSF support for neutropenia (less than 500 cells/mm 3 ).

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Abstract

Procédé de traitement d'une tumeur chez un hôte humain selon lequel on effectue la transduction in vivo des cellules tumorales avec une séquence d'acide nucléique (ADN ou ARN) codant un agent qui est capable d'assurer l'inhibition, la prévention ou la destruction du développement des cellules tumorales lors de l'expression de la séquence d'acide nucléique qui code ledit agent. Dans une forme d'exécution, on effectue ce traitement en administrant à la tumeur in vivo des cellules productrices transduites avec un vecteur retroviral comprenant un gène codant un marqueur sélectif négatif. Lorsque les cellules productrices sont administrées à la tumeur, elles génèrent des particules virales infectieuses qui infectent les cellules tumorales. Lorsqu'on administre ensuite un agent d'interaction qui interagit avec le marqueur sélectif négatif pour produire un agent qui est toxique pour les cellules tumorales, les cellules tumorales transduites sont détruites. Dans une forme d'exécution préférée, on utilise ce procédé pour traiter les tumeurs cérébrales chez l'homme.
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