01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

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E1A/liposomes in normal mice and at cumulative doses 5 to 40 times the DNA-lipid starting dose proposed for the phase I clinical trial (Xing et al, 1997). A Phase I multicenter study of intratumoral E1A gene therapy using cationic liposome gene transfer is also in course for patients with unresectable or metastatic solid tumors that overexpress HER-2 /neu (protocol 209, see page 205). Delivery of an anti-erbB-2 single chain (sFv) antibody gene for previously treated ovarian and extraovarian cancer patients is in clinical trials using adenoviral gene delivery (protocol #133). A clinical trial for tumor vaccination with HER-2 /Neu using a B7 expressing tumor cell line prior to treatment with HSV-tk genemodified cells is in phase I for ovarian cancer (protocol #96, page 165). XXIII. Suicidal genes for cancer therapy (prodrug gene therapy) A. Molecular mechanism of cell killing with HSV-tk gene and ganciclovir (GCV) Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy (Table 3). Direct suppression of tumor growth by cytotoxic gene therapy is a successful gene transfer approach. This approach has promise for a variety of other applications where excess cell proliferation is detrimental and has also been used to restrict intimal hyperplasia of the arterial wall and smooth Boulikas: An overview on gene therapy Table 3. Prodrugs and enzymes used for their activation 72 muscle cell growth to limit restenosis after artery angioplasty (see below). Cancer cells can be induced to be conditionally sensitive to the antiviral drug ganciclovir after their transduction with the thymidine kinase (tk) gene from the herpes simplex virus (HSV); ganciclovir (GCV) is the 9- {[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl}guanine (Field et al, 1983); it is converted by HSV-tk into its monophosphate form which is then converted into its triphosphate form by cellular enzymes and is then incorporated into the DNA of replicating mammalian cells leading to inhibition in DNA replication and cell death (Moolten, 1986; Borrelli et al, 1988; Moolten and Wells, 1990). It is only viral TK, not the mammalian enzyme, that can use efficiently ganciclovir as a substrate and this drug has been synthesized to selectively inhibit herpes virus replication (Field et al, 1983); indeed, the mammalian TK has a very low affinity for this guanosine analog. The toxicity of ganciclovir is manifested only when cells undergo DNA replication and it is not harmful to normal nondividing cells. This treatment strategy has been used for hepatocellular carcinoma (Huber et al, 1991; Su et al, 1996), fibrosarcoma, glioma (Culver et al, 1992, see below), adenocarcinoma (Osaki et al, 1994), prostate cancer (Eastham et al, 1996) and many other cancers. B. Treatment gliomas in rats with HSV-tk plus ganciclovir Brain tumors have the privilege of escaping immunologic rejection; therefore brain tumors are inaccessible to cancer immunotherapy. Culver and cowor- Prodrug-activating enzyme Prodrug Toxic substance it is converted to Thymidine kinase from HSV 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine or ganciclovir (GCV) GCV monophosphate Cytosine deaminase (CD) from E. coli 5-fluorocytosine (5FC) 5-fluorouracil (5FU) Purine nucleoside phosphorylase (PNP) from E. coli Purine nucleoside phosphorylase (PNP) from E. coli 6-methylpurine-2’-deoxyriboside (MeP-dR) 6-methylpurine (a very toxic adenine analog) Arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP) commercially known as fludarabine A very toxic adenine analog Human deoxycytidine kinase (dCK) Cytosine arabinoside (ara-C) A toxic drug inducing lethal strand breaks in DNA Nitroreductase from E. coli 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) kers (1992) took advantage of the fact that retroviral vectors require DNA synthesis for stable integration into the host genome to target gliomas in rats. Murine fibroblasts were transduced with a retroviral vector expressing the HSV-tk gene (see above); the tumor cell A potent dysfunctional alkylating agent which crosslinks DNA mass was then infiltrated by intratumoral injection of the HSV-tk−producing fibroblasts. This treatment gave a continuous local infusion of retroviral vector from the injected fibroblasts, integrating into the dividing cells of the growing brain tumor but not into the nondividing
normal cells in the surroundings. Treatment of rats at day 5 after transplantation with ganciclovir (GCV) resulted in the complete regression of the tumor cell mass; this was thought to be induced by killing of cells that respond to signals promoting angiogenesis in the immediate vicinity of the tumor; vascular endothelial cells in the normal brain tissue, exhibiting cycling at a low rate, apparently were not affected. Other proliferating tissues, such as intestinal epithelium, thymus, and bone marrow, which might also uptake the retroviral HSV-tk vector and then be destroyed during GCV treatment were not affected by this approach over a 30 day period of treatment with GCV (Culver et al, 1992). A replication-defective, highly purified retroviral vector at titers of 10 8 colony forming units/mL was used to treat 9L gliosarcoma cells in rat brain. Animals with established 9L tumors treated with intralesional injection of the HSV-tk retrovirus followed by GCV treatment showed at day 26 that 29% (4/14) had no tumor and 50% (7/14) of the animals had < 1% tumor volume; substantial numbers of CD4 + Gene Therapy and Molecular Biology Vol 1, page 73 and CD8 + lymphocytes infiltrated the tumors of animals treated with HSV-tk and GCV; the former tumor bed in cured animals contained cell debris, immune cells, and fibroblasts without signs of damage to the adjacent brain tissue (Kruse et al, 1997). C. The bystander effect of HSV-tk/GCV During HSV-tk/GCV treatment of brain tumors products from the dying cells in the brain tumor killed nearby non-HSVtk-transduced cancer cells without affecting normal cells, an effect described as "bystander" antitumor effect (Culver et al, 1992). The bystander effect of the HSV-tk plus GCV system appears to be powerful and significant, circumventing the low efficiency of transduction in vivo with recombinant retroviruses. Because of this effect, the low-level percentage of cells that can be transduced with a retrovirus can cause the elimination of a much larger percentage of proliferating cells in their surroundings (Kimura et al, 1996). In vitro, the “bystander” effect works by transfer of cytotoxic small molecules between cells via gap junctions. In order to understand the “bystander effect” mechanism during which adjacent nontransduced tumor cells are killed, Yamamoto et al (1997) used Renca cells from a renal carcinoma cell line transduced with a retroviral vector bearing the HSV-tk gene to inoculate BALB/c mice. After complete regression of inoculated tumors with GCV treatment, the animals were challenged with nontransduced tumor cells. In these animals, tumorspecific cytotoxic CD8 + T cells were efficiently induced which promoted the rejection or significant growth inhibition of challenged tumor cells. 73 In a similar experiment, set to assess the “bystander effect” in vivo, mixtures of HSV-tk−transduced and nontransduced oral squamous carcinoma cells were implanted subcutaneously in the left flank of nude mice, and naive HSV tk − cells were implanted subcutaneously in the right flank. Treatment with GCV eradicated the tumors in the left flank consistent with a predicted bystander effect but also resolved or arrested the growth of the naive tumors in the right flank. The histology of regressing tumors from the right flank showed an infiltration of lymphoid cells suggesting that an immune-related antitumor response accounted for the distant bystander effect (Bi et al, 1997; see also Ramesh et al, 1998 this volume). The induction of higher levels of HSV-tk expression does not augment the sensitivity to GCV: adenoviral vectors that expressed HSV-tk at different efficiencies from CMV versus RSV promoters did not display a significant difference in antitumor effects; thus, increasing the HSV-TK enzyme levels per cell above a minimal threshold level will not be effective in cell killing with GCV. To enhance the therapeutic responses of the HSVtk/GCV system one needs to improve other parameters such as to use higher doses of GCV, to enhance the "bystander effect," to engineer mutant HSV-tk genes with higher substrate affinities, or to discover vectors with increased transduction efficiencies (Elshami et al, 1997). Suicide gene therapy may be useful not only for shortterm tumor regression mediated by direct cell killing and bystander effect, but may also exert a therapeutic vaccination effect resulting in long-term tumor regression and prevention of recurrence (Yamamoto et al, 1997). D. Additional examples of tumor eradication with HSV-tk/GCV Chen et al (1996) used a recombinant adenoviral vector containing the HSV-tk gene for the treatment of metastatic colon carcinoma in the mouse liver; the HSV-tk alone exhibited substantial regression, although all treated animals suffered from subsequent relapses. Delivery of the HSV-tk + mouse IL-2 genes in adenoviral vectors to the hepatic tumors induced an effective antitumor immune response which nevertheless waned with time, and the treated animals eventually succumbed to hepatic tumor relapse; however, after combination treatment with HSVtk, mouse IL-2, and mouse GM-CSF a fraction of the animals developed long-term antitumor immunity and survived for more than 4 months without tumor recurrence (Chen et al, 1996). Microinjection of the HSV-tk gene, under control of αfetoprotein enhancer and albumin promoter, in a linear form flanked by the adeno-associated virus ITRs into pronuclei of mouse embryos led to transgenic animals
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Gene Ther Mol Biol Vol 1, 1-172. Ma
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I. Introduction Monumental progress
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The use of retroviral vectors in hu
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displaying the cleavable EGF domain
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molecules in the nucleus (Lowe and
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sensitive mutations which allow pro
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processed as described in panel A s
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On the contrary, the payload capaci
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in K562 human erythroleukemia cells
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defective HSV virus (DeLuca and Sch
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Page 25 and 26: delivered by Sendai virus-liposome
Page 27 and 28: plasmids with the cell surface, tra
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Page 33 and 34: B. Transcription factor binding sit
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Page 41 and 42: C. Considerations of chromatin stru
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Page 57 and 58: Prives, 1994). Whereas the wild-typ
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Page 63 and 64: Work from several groups has shown
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The peptide kinin, after binding to
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intake when administered to adrenal
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Introgen Therapeutics (Austin and H
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When a subfragment of only 513 bp o
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Armentano D, Zabner J, Sacks C, Soo
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Brady HJM, Miles CG, Pennington DJ,
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Chen J, Stickles RJ, Daichendt KA (
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Day ML, Wu S, Basler JW (1993) Pros
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Farmer G, Bargonetti J, Zhu H, Frie
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Giovannangeli C, Perrouault L, Escu
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the neoplastic phenotype by replace
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integrate in a site-specific fashio
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Li R, Waga S, Hannon GJ, Beach D, S
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adiation-induced apoptosis in the g
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Nakaya T, Iwai S, Fujinaga K, Sato
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Polyak K, Xia Y, Zweier JL, Kinzler
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macrophages from simian immunodefic
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intimal hyperplasia in a rat caroti
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Trono D, Feinberg MB, Baltimore D (
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Wattiaux R, Jadot M, Warnier-Pirott
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similar to mammalian interleukin-1
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Gene Therapy and Molecular Biology
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24. Gene Marking /Infectious Diseas
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Drug 50. Gene Therapy /Phase I /Can
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76. Gene Therapy /Phase I /Cancer /
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99. Gene Therapy /Phase II /Cancer
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120. Gene Therapy /Phase I /Monogen
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Gene Therapy and Molecular Biology
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cancer not specified) /Immunotherap
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