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

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Boulikas: An overview on gene therapy Figure 14. Structure of retroviral vectors LoxP-HyTK-large T and Cre-puro allowing expression of T antigen and Cre/LoxPcatalyzed deletion of T antigen cDNA. The 4 small arrows indicate the primers used for PCR analysis; the large arrow above the NLS- CRE box indicates the Cre recombinase mediating LoxP specific deletion. LTR are the long terminal repeats of Moloney Murine Leukemia virus; LOX is the 34 bp sequence identical to the recognition site of Cre recombinase; HyTK is the hygromycin/thymidine kinase fusion gene; NLS-CRE is the Cre recombinase gene targeted to the nucleus by a nuclear localization signal (NLS); SV40 is the SV40 promoter; Puro is a puromycin selection marker. From Li LP, Schlag PM, Blankenstein T (1997) Transient expression of SV 40 large T antigen by Cre/LoxP-mediated site-specific deletion in primary human tumor cells. Hum Gene Ther 8, 1695-1700. Reproduced with the kind permission of the authors and Mary Ann Liebert, Inc. 38
Gene Therapy and Molecular Biology Vol 1, page 39 Figure 15. Large T antigen-immortalized breast cancer cells change morphology and lose expression of T antigen after infection with Cre-Puro retrovirus: after prolonged culture the cells were infected with Cre-Puro retrovirus (B, D) or mock-infected (A, C), selected for Puromycin resistance (A, B, C, D) and resistance to ganciclovir (B, D) and analyzed by light microscopy (A, B) and staining with a large T-antibody (C, D). From Li LP, Schlag PM, Blankenstein T (1997) Transient expression of SV 40 large T antigen by Cre/LoxP-mediated site-specific deletion in primary human tumor cells. Hum Gene Ther 8, 1695-1700. Reproduced with the kind permission of the authors and Mary Ann Liebert, Inc. Reversible immortalization of primary cells was achieved by Westerman and Leboulch (1996) using retrovirus-mediated transfer of an oncogene that could be subsequently excised by site-specific Cre/LoxP recombination; the FLP/FRT recombination was not efficient in primary cells. Pure populations of cells in which the oncogene was permanently excised were obtained which reverted to their preimmortalized state. Using the Cre/LoxP recombination strategy primary cells could be cultured and expanded; the method was proposed to be applicable for facilitating gene transfer to cells unresponsive to exogenous growth factors. A retroviral vector, containing both a neomycin resistance expression unit flanked by loxP sites and GM- CSF cDNA, was used to transduce the human hematopoietic K-562 cell line. Superinfection of K562 cell clones with a retrovirus containing a Cre recombinase expression unit and molecular analyses of 30 doubly transduced subclones showed a strict correlation between Cre expression and LoxP-flanked selectable cassette excision; excision of the selectable cassette resulted in a significant increase of GM-CSF transcription driven by the retroviral promoter (Fernex et al, 1997). Novel retroviral vectors for gene transfer were developed by Bergemann et al (1995) by inserting two LoxP sites into a retroviral vector also containing the HSV-tk gene; Cre expression in cells infected with this vector was followed by BrdU selection for cells in which site-specific recombination took place. Furthermore, replacement of the enhancer/promoter elements in both LTRs by Lox sequences led to the development of retroviral suicide vectors for gene therapy. Vanin et al (1997) have used the Cre/LoxP recombinase system to generate high-titer retroviral producer cell lines; incorporation of LoxP sites at the flanks of a Neo R -HSV-tk cassette in the proviral DNA allowed excision of these selectable markers through expression of Cre recombinase and the production of a high-titer producer cell line containing a single LoxP site flanked by the viral LTRs. Retransfection of this cell line with a plasmid containing a gene of interest flanked by LoxP sites and the Cre expression vector allowed insertional LoxP/LoxP recombination of the gene into the favorable preexisting site in the genome and the generation of a new line with a 39 titer equivalent to that of the parental producer cell line. The Cre/LoxP recombinase strategy has been used to generate retroviral vectors with the ability to excise themselves after inserting a gene into the genome (Russ et al, 1996). Bushman and Miller (1997) fused retroviral integrase enzymes to sequence-specific DNA-binding domains and investigated target site selection by the resulting proteins. A fusion protein composed of HIV integrase linked to the DNA-binding domain of λ repressor was able to direct selective integration of retroviral cDNA in vitro into target DNA containing λ repressor binding sites. A fusion of HIV integrase to the DNA binding domain of the zinc finger protein Zif268 also directed increased integration near Zif268 recognition sites. Introduction of foreign DNA into cell nuclei with recombinase cDNA and appropriate sequences to promote recombination may promote nor only insertion of a therapeutic gene into a specific chromosomal site but also chromosomal rearrangements that could convert therapeutically transduced cells into malignant. There is a great deal of knowledge to be derived from these very promising strategies of gene therapy before they can be successfully applied to humans. XIII. Fate of the transgene in the nucleus A. How to sustain transgene expression? A major drawback in gene therapy applications is loss in gene activity within a few days from gene transfer although all previous steps were successful. In other words, the transferred gene is transiently expressed for 1-4 days and its expression thereafter declines dramatically. This is due (i) to the degradation of the gene in the nucleus; (ii) the dilution of the plasmid during replication of the cells from its inability to replicate; (iii) its inactivation by position effects from chromatin surroundings after its integration into the chromosomal DNA; (iv) the elimination of the therapeutic cells expressing the transgene by the immune system of the organism either because of the antigenicity of the expressed protein or because of the antigenicity of viral
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 and 20: defective HSV virus (DeLuca and Sch
Page 21 and 22: complex into the cytosol from the e
Page 23 and 24: Gene Therapy and Molecular Biology
Page 25 and 26: delivered by Sendai virus-liposome
Page 27 and 28: plasmids with the cell surface, tra
Page 29 and 30: infected by adenovirus alone; infec
Page 33 and 34: B. Transcription factor binding sit
Page 35 and 36: A closely related family of ubiquit
Page 37: cells. I-CreI is a member of this c
Page 41 and 42: C. Considerations of chromatin stru
Page 45 and 46: A. The molecular basis of cancer im
Page 47 and 48: fragments, or heat shock proteins c
Page 49 and 50: ecombinant vaccinia virus expressin
Page 51 and 52: HIV-1 envelope DNA construct develo
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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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Page 67 and 68: Figure 23. A pathway leading to the
Page 69 and 70: Excessive necrotic death in cells o
Page 71 and 72: implantation, and similar processes
Page 73 and 74: normal cells in the surroundings. T
Page 79 and 80: A bicistronic retroviral vector (Ha
Page 81 and 82: C. Antisense ras gene transfer for
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Page 85 and 86: protein (e.g. Smith et al, 1995; Fo
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IL-1 - receptor antagonist protein
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p53 lung cancer retroviru s MHC cla
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Gene Therapy and Molecular Biology
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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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