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

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(responsible for the formation of the extravascular fibrin gel) through postcapillary venules in tumor cells is responsible for generating the supporting matrix for fibroblast migration, angiogenesis, and fibroplasia (reviewed by Senger et al, 1993). Migration of macrophages and fibroblasts in the fibrin gel in tumors is determined by fibrinogen concentration and Factor XIII crosslinking of α and γ chains. Tumor cells as well as inflammatory cells in healing wounds produce a higher concentration of degradative proteases for extracellular matrix but also protease inhibitors which explains the resistance toward degradation of the fibrin gel in solid tumors (reviewed by Senger et al, 1993). The mechanism of overexpression of VEGF in solid tumors might involve its induction by hypoxia (Shweiki et al, 1992); the rapid proliferation of the cells in the center of the tumor induces an increase in the interstitial pressure and may lead to closure of capillaries by compression; inefficient vascular supply, including the compensatory development of collateral blood vessels in ischaemic tissues, leads to neovascularization via production of VEGF. A clustering of capillaries alongside VEGFproducing cells in a subset of glioblastoma cells immediately proximal to necrotic foci have been observed in intracranial brain neoplasms obtained from surgical specimens; this was thought to be the result of a local angiogenic response elicited by VEGF (Shweiki et al, 1992). VEGF expression by hypoxia was also induced in skeletal muscle myoblasts, in the fibroblast mouse L cell line, and in cells from rat heart muscle (Shweiki et al, 1992). Targeting of VEGF gene leading to its transcriptional inactivation (e.g. via triplex-forming oligonucleotides or antisense vectors) is expected to limit growth in solid tumors via inhibition in neo-vascularization; the prolonged sustenance of hypoxia in the center of the tumor is also expected to induce p53. An important concept to understand is that tumor angiogenesis results from a balance between angiogenic and anti-angiogenic factors. Expression of VEGF 165 in rat C6 glioma cells and subcutaneous injection of the transduced cells in athymic mice has shown that tumors from cells expressing VEGF grew slower than tumors developed from nontransduced C6 cells, were highly vascularized, and contained varying degrees of necrosis and eosinophilic infiltrate (Saleh, 1996). VEGF plays an important role not only in carcinogenesis but also restenosis (see below). VEGF promotes endothelial cell proliferation to accelerate reendothelialization of the artery reducing intimal thickening; up-regulation of VEGF is the desired effect for treatment of restenosis (Asahara et al, 1996; Isner et al, 1996a). The transfer of the VEGF gene demonstrates a special mission of gene therapy: how to treat one human disease by upregulating the expression of a specific gene while treating a different disease by downregulating the Boulikas: An overview on gene therapy 102 expression of the same gene. Targeting is important. Also, exploring the molecular mechanisms affected by the transfer and overexpression of the cDNA of a gene, in all aspects and at their entire spectrum, is essential for a successful gene therapy application. F. Transfer of the VEGF gene in ischemia VEGF gene transfer can improve blood supply to the ischaemic limb and is a promising approach for the treatment of acute limb ischemia. In a gene therapy approach for tissue ischemia, the VEGF 165 cDNA under the transcriptional control of the HSV immediate-early 4/5 promoter was used to transduce BLK-CL4 fibroblasts resulting in the secretion of high levels of biologically active VEGF; when the transduced cells were resuspended in basement membrane extract (matrigel) and were injected subcutaneously into syngeneic C57BL/6 mice they showed a strong angiogenic response (Mesri et al, 1995). Regional angiogenesis was induced in nonischemic retroperitoneal adipose tissue by adenoviral VEGF gene transfer supporting a 123% increase in vessel number compared to control (Magovern et al, 1997). Treatment of a 71 year-old patient with an ischaemic leg with 2 mg phVEGF 165 plasmid applied to the hydrogel polymer coating of an angioplasty balloon and reaching the distal popliteal artery resulted in an increase in collateral vessels at the knee, mid-tibial, and ankle levels, which persisted for 12-weeks (Isner et al, 1996a). Ischemia, induced in the hindlimb of rats by excision of the femoral artery, was experimentally treated by transfer of the VEGF gene; therapeutic angiogenesis produced morphologically similar, but significantly more extensive, networks of collateral microvessels (Takeshita et al, 1997). Direct i.m. injection of naked VEGF 165 plasmid DNA into the ischemic thigh muscles in rabbits resulted in more angiographically recognizable collateral vessels at 30 days posttransfection (Tsurumi et al, 1996, 1997). G. Cancer treatment with angiogenesis inhibitors On November 20, 1997 the first exciting data on clinical trials using the TNP-470, a drug extracted from fungi which inhibits angiogenesis, were reported in a speech before the National Institutes of Health by Judah Folkman (Harvard University). A woman in Texas with cervical cancer and metastasis to lungs had been tumorfree for months after treated with TNP-470; and a young girl with a slow-growing bone tumor in her jaw was cancer-free after treatment with IFN-α. Both TNP-470 and IFN-α are relatively weak inhibitors of blood vessel formation compared with angiostatin (O'Reilly et al, 1994, 1996), endostatin (O'Reilly et al, 1997), and vasculostatin
which eliminated tumors in small animals. A combination therapy with angiostatin and endostatin was even more effective in tumor eradication; furthermore, these drugs have no apparent side effects and there is virtually no resistance of tumors to these drugs (see below). However, the number of human patients treated with angiogenesis inhibitors is too small and a larger number of cases need to be examined. H. Angiostatin and endostatin Angiostatin is a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, which is generated by cancer-mediated proteolysis of plasminogen to a 38 kDa plasminogen fragment; angiostatin selectively instructs endothelium to become refractory to angiogenic stimuli (O'Reilly et al, 1994, 1996). A number of enzymes including metalloelastase, pancreas elastase, plasmin reductase, and plasmin collaborate in the conversion of plasminogen to angiostatin. Systemic administration of angiostatin, but not intact plasminogen, inhibited neovascularization in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases (O'Reilly et al, 1994; Gately et al, 1997); human angiostatin inhibited almost completely the growth of three human and three murine primary carcinomas in mice without detectable toxicity or resistance (O'Reilly et al, 1996); these studies have developed the “dormancy therapy” for cancer based on that malignant tumors are regressed by prolonged blockade of angiogenesis to microscopic dormant foci in which tumor cell proliferation is balanced by apoptosis (O'Reilly et al, 1996). Human angiostatin, administered to mice with s.c. hemangioendothelioma and associated disseminated intravascular coagulopathy (Kasabach-Merritt syndrome), significantly reduced tumor volume and increased survival (Lannutti et al, 1997). PC-3 human prostate carcinoma cells release uPA and free sulfhydryl donors that converted plasminogen to angiostatin; these two components were sufficient for angiostatin generation (Gately et al, 1997); reduction of one or more disulfide bonds in the serine proteinase, plasmin, by a reductase secreted by Chinese hamster ovary cells triggered proteolysis of plasmin, generating fragments with the domain structure of angiostatin; two reductases (protein disulfide isomerase and thioredoxin) although able to produce biologically active angiostatin from plasmin and to inhibit proliferation of human dermal microvascular endothelial cells, were not the reductases secreted by cultured cells; instead the plasmin reductase factor secreted was a different one requiring reduced glutathione for activity (Stathakis et al, 1997). Two members of the human matrix metalloproteinase (MMP) family, matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9), hydrolyzed human plasminogen to generate angiostatin fragments; 58-, 42- and 38-kDa angiostatin fragments were generated; these studies Gene Therapy and Molecular Biology Vol 1, page 103 103 implicated MMP-7 and MMP-9 in regulation of new blood vessel formation by cleaving plasminogen and generating angiostatin molecules (Patterson and Sang, 1997). Recent studies exploring the mechanism responsible for the in vivo production of angiostatin that inhibits growth and metastasis in Lewis lung carcinoma have shown that angiostatin is produced by tumor-infiltrating macrophages whose metalloelastase expression is stimulated by tumor cell-derived GM-CSF (Dong et al, 1997). Endostatin is a 20 kDa C-terminal fragment of collagen XVIII produced by hemangioendotheliomas which specifically inhibits endothelial cell proliferation, angiogenesis and tumor growth (O'Reilly et al, 1997). Reports on the transfer of the angiostatin cDNA for the treatment of malignancies are about to appear (Toshihide Tanaka, personal communication). XXXII. Gene therapy of restenosis A. Pathophysiology of restenosis The pathological situation, described as recurrent narrowing of a blood vessel after a successful revascularization procedure, has been termed restenosis (from the Hellenic stenos=narrow); the most frequent revascularization procedure has been the percutaneous transluminal angioplasty (PTA) used to treat atherosclerotic obstructions in the coronary and peripheral vascular circulations; PTA is achieved using a tiny balloon mounted on a catheter which is advanced under x-ray guidance to the site of a blocked artery. The most frequent artery suffering restenosis is the superficial femoral artery (SFA)/popliteal artery of the leg and the iliac arteries. One of the factors contributing to restenosis is the intimal hyperplasia of the arterial wall; among others, the mechanism for intimal hyperplasia involves increasing the tissue levels of TGF-β following injury; injection of antibodies directed against TGF-β has blocked restenosis in a rat model (reviewed by Border and Noble, 1995). Transfer of the TGF-β gene into porcine arteries caused restenosis (Nabel et al, 1993a,b). Arterial injury has pleiotropic effects at the molecular level; for example, injury of rat arteries led to an increase in FGF receptors in vascular smooth muscle cells. Atherosclerosis and restenosis following balloon angioplasty are characterized by two steps: during the thrombotic phase in the arterial wall following injury fibrin networks are synthesized with platelet depositions; this phase is followed by smooth muscle cell proliferation. Both the synthesis of fibrin from fibrinogen, as well as the proliferation of platelet and smooth muscle cells are upregulated by the protease thrombin. Inhibition of the action of thrombin in the arterial wall is a potential target against arterial disease. The most potent and specific
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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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complex into the cytosol from the e
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Gene Therapy and Molecular Biology
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delivered by Sendai virus-liposome
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plasmids with the cell surface, tra
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infected by adenovirus alone; infec
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B. Transcription factor binding sit
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A closely related family of ubiquit
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cells. I-CreI is a member of this c
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C. Considerations of chromatin stru
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A. The molecular basis of cancer im
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fragments, or heat shock proteins c
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ecombinant vaccinia virus expressin
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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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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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cancer not specified) /Immunotherap
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