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

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eplication and packaging in the presence of defective helper virus carrying a deletion in the IE3 gene in the E5 cell line expressing the IE3 gene; (iii) the latent oriP of EBV and (iv) the EBNA-1 cDNA allow episomal replication of the infectious vector in the E5 cell line so that viral stocks of high titer can be made. Infection of tumor-derived fibroblast and epithelial cell lines in culture and local injection of human liver tumors in nude mice was used to demonstrate 95-99% efficiency of infection and transfer of the reporter β-galactosidase gene. Genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) were used to efficiently deliver genes into cultured hepatocytes of different origin; delivery into human hepatocytes with baculovirus vectors approached 100% efficiency in cell culture and expression levels were high when mammalian promoters were chosen. A number of drawbacks preclude their direct application in vivo; nevertheless gene transfer was feasible in ex vivo perfused human liver tissue (Sandig et al, 1996; Hofmann et al, 1998 this volume). VIII. Liposomal gene delivery Abbreviations: DC-CHOL: 3β [N-(N',N'dimethylaminoethane)carbamoyl]cholesterol DDAB: dimethyldioctadecyl ammonium bromide DMRIE: N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2hydroxyethyl) ammonium bromide DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane DOGS: Dioctadecylamidoglycylspermine (Transfectam, Promega) DOPE: dioleyl phosphatidylethanolamine DOSPA: 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,Ndimethyl -1-propanaminium trifluoroacetate DOTAP: N-(1-(2,3-dioleoyloxy)propyl)-N,N,Ntrimethylammonium chloride DOTMA: N-[1-(2,3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride DPTAP: 1,2- dipalmitoyl-3-trimethylammonium propane DSTAP: 1,2-disteroyl-3-trimethylammonium propane Lipofectin: DOTMA:DOPE 1:1 (GIBCO BRL) A. Immune responses and toxicity of cationic lipid-DNA complexes Cationic lipids have been widely used for gene transfer; a number of clinical trials (34 out of 220 total RAC-approved protocols as of December 1997) use cationic lipids (see Table 4 in Martin and Boulikas, 1998, this volume, pages 203-206). Although many cell culture studies have been documented, systemic delivery of genes with cationic lipids in vivo has been very limited. All clinical protocols use subcutaneous, intradermal, intratumoral, and intracranial injection as well as intranasal, intrapleural, or aerosol administration but not Boulikas: An overview on gene therapy 20 i.v. delivery because of the toxicity of the cationic lipids and DOPE (see Table 4 in Martin and Boulikas, 1998, this volume, pages 203-206). Liposomes formulated from DOPE and cationic lipids based on diacyltrimethylammonium propane (dioleoyl-, dimyristoyl-, dipalmitoyl-, disteroyl-trimethylammonium propane or DOTAP, DMTAP, DPTAP, DSTAP, respectively) or DDAB were highly toxic when incubated in vitro with phagocytic cells (macrophages and U937 cells), but not towards non-phagocytic T lymphocytes; the rank order of toxicity was DOPE/DDAB > DOPE/DOTAP > DOPE/DMTAP > DOPE/DPTAP > DOPE/DSTAP; the toxicity was determined from the effect of the cationic liposomes on the synthesis of nitric oxide (NO) and TNFα produced by activated macrophages (Filion and Phillips, 1997). Another factor to be considered before i.v. injections are undertaken is that negatively charged serum proteins can interact and cause inactivation of cationic liposomes (Yang and Huang, 1997). Condensing agents used for plasmid delivery including polylysine, transferrinpolylysine, a fifth-generation poly(amidoamine) (PAMAM) dendrimer, poly(ethyleneimine), and several cationic lipids (DOTAP, DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE) were found to activate the complement system to varying extents. Strong complement activation was seen with long-chain polylysines, the dendrimer, poly(ethyleneimine), and DOGS; complement activation was considerably reduced by modifying the surface of preformed DNA complexes with polyethyleneglycol (Plank et al, 1996). B. Mechanism of liposome entry to cells Cationic lipids increase the transfection efficiency by destabilizing the biological membranes including plasma, endosomal, and lysosomal membranes; indeed, incubation of isolated lysosomes with low concentrations of DOTAP caused a striking increase in free activity of βgalactosidase, and even a release of the enzyme into the medium demonstrating that lysosomal membrane is deeply destabilized by the lipid; the mechanism of destabilization was thought to involve an interaction between cationic liposomes and anionic lipids of the lysosomal membrane, allowing a fusion between the lipid bilayers; the process was less pronounced at pH 5 than at pH 7.4 and anionic amphipathic lipids were able to prevent partially this membrane destabilization (Wattiaux et al, 1997). In contrast to DOTAP and DMRIE which were 100% charged at pH 7.4, DC-CHOL was only about 50% charged as monitored by a pH-sensitive fluorophore; this difference decreases the charge on the external surfaces of the liposomes and was proposed to promote an easier dissociation of bilayers containing DC-CHOL from the plasmid DNA and an increase in release of the DNA-lipid
complex into the cytosol from the endosomes (Zuidam and Barenholz, 1997). C. Tissue targets using cationic liposomes in vivo Although cationic lipids have been used widely for the delivery of genes very few studies have used systemic i.v. injection of cationic liposome-plasmid complexes because of the toxicity of the lipid component and certainly in animal models, not humans. Administration by i.v. injection of two types of cationic lipids of similar structure, DOTMA and DOTAP, has shown that the transfection efficiency was determined mainly by the structure of the cationic lipid and the ratio of cationic lipid to DNA; the luciferase and GFP gene expression in different organs was transient, with a peak level between 4 and 24 hr, dropping to less than 1% of the peak level by day 4 (Song et al, 1997). Figure 5 shows the effect of cationic lipid:DNA ratio on transfection efficiency after i.v. tail injection. Luciferase activity was detected in all organs examined with the highest level in lung. In the absence of neutral lipid both DOTMA and DOTAP promoted a linear increase in luciferase activity in the lung with increasing lipid:DNA from 12:1 to 36:1 nmol lipid: µg of DNA. DOTMA was 10 times more efficient than DOTAP (10 6 versus 10 7 relative luciferase units (RLU) per mg protein. Cholesterol (Chol) mixed with DOTMA (1:1 molar ratio) decreased the level of gene expression in the lung whereas cholesterol did not affect the transfection efficiency of DOTAP liposomes. Inclusion of DOPE into either DOTAP or DOTMA liposomes significantly decreased the transfection efficiency by 100-fold in the lung. When a group of four cationic lipids with identical head group but of different fatty acyl chains were tested for their transfection efficiencies (Figure 6); these included DOTAP, DMTAP, DPTAP, and DSTAP. The C 14 acyl chain-lipid DMTAP had a similar transfection efficiency as DOTAP which has 18 carbon atoms in the acyl chain and one double bond (C 18Δ 9); on the contrary, the transfection efficiencies of DPTAP (C 16) was 10-100 fold lower and that of DSTAP (C 18) was 100 to 1000 fold lower. Confocal microscopy of lung tissue after injection of 25 µg pCMV-GFP plasmid DNA complexed with Gene Therapy and Molecular Biology Vol 1, page 21 21 DOTMA liposomes to mice (Figure 7) has shown that the type of cells that express the transgene are the endothelial cells that have typical characteristics of neighboring multiple air-sac structures (Figure 7D). A number of different organs in vivo can be targeted after liposomal delivery of genes or oligonucleotides. Intravenous injection of cationic liposome-plasmid complexes by tail vein in mice targeted mainly the lung and to a smaller extend the liver, spleen, heart, kidney and other organs (Zhu et al, 1993). Intraperitoneal injection of a plasmid-liposome complex expressing antisense K-ras RNA in nude mice inoculated i.p. with AsPC-1 pancreatic cancer cells harboring K-ras point mutations and PCR analysis indicated that the injected DNA was delivered to various organs except brain (Aoki et al, 1995). A number of factors for DOTAP:cholesterol/DNA complex preparation including the DNA:liposome ratio, mild sonication, heating, and extrusion were found to be crucial for improved systemic delivery; maximal gene expression was obtained when a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size were used. Cryo-electron microscopy showed that the DNA was condensed on the interior of invaginated liposomes between two lipid bilayers in these formulations, a factor that was thought to be responsible for the high transfection efficiency in vivo and for the broad tissue distribution (Templeton et al, 1997). Steps to improve for successful liposome-mediated gene delivery to somatic cells include persistence of the plasmid in blood circulation, port of entry and transport across the cell membrane, release from endosomal compartments into the cytoplasm, nuclear import by docking through the pore complexes of the nuclear envelope, expression driven by the appropriate promoter/enhancer control elements, and persistence of the plasmid in the nucleus for long periods. A number of strategies for liposomal delivery and for enhancing the efficiency of uptake by the cells and release from endosomal compartments of plasmid or oligonucleotide DNA are reviewed in the following article (Martin and Boulikas, 1998).
Page 1 and 2: Gene Ther Mol Biol Vol 1, 1-172. Ma
Page 3 and 4: I. Introduction Monumental progress
Page 5 and 6: The use of retroviral vectors in hu
Page 7 and 8: displaying the cleavable EGF domain
Page 9 and 10: molecules in the nucleus (Lowe and
Page 11 and 12: sensitive mutations which allow pro
Page 13 and 14: processed as described in panel A s
Page 15 and 16: On the contrary, the payload capaci
Page 17 and 18: in K562 human erythroleukemia cells
Page 19: defective HSV virus (DeLuca and Sch
Page 23 and 24: Gene Therapy and Molecular Biology
Page 25 and 26: delivered by Sendai virus-liposome
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Page 33 and 34: B. Transcription factor binding sit
Page 35 and 36: A closely related family of ubiquit
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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 67 and 68: Figure 23. A pathway leading to the
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implantation, and similar processes
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normal cells in the surroundings. T
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Gene Therapy and Molecular Biology
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A bicistronic retroviral vector (Ha
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C. Antisense ras gene transfer for
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equal to 6.0 showed S1 hyperreactiv
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protein (e.g. Smith et al, 1995; Fo
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transduced primary mouse fibroblast
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IL-1 - receptor antagonist protein
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p53 lung cancer retroviru s MHC cla
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designed by immunization with porti
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Isolation of an RNA aptamer that ca
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induction of human apoE in neonate
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atrium in heart, endothelial cells
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which eliminated tumors in small an
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C. Transfer of other genes against
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significant reduction in neointima
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(Prehn et al, 1996); this implies a
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B. Approaches to gene therapy of RA
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of human immunoglobulin and antigen
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A. Etiology and mechanisms of destr
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However, CsA also inhibits the acti
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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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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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