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

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Boulikas: An overview on gene therapy secreting cells to treat Parkinson's disease, nerve growth factor for Alzheimer's disease and other diseases. Ingenious techniques under development with great future prospects for human gene therapy, include the Cre- LoxP recombinase system to rid of undesirable viral DNA sequences used for gene transfer, use of tissue-specific promoters to express a gene in a particular cell type or use of ligands, such as peptides selected from random peptide libraries, recognizing surface molecules to direct the gene vehicle to a particular cell type, designing p53 “gene bombs” that explode into tumor cells, exploit the HIV-1 virus to engineer vectors for gene transfer, the combining of viruses with polymers or cationic lipids to improve gene transfer, the attachment of nuclear localization signal peptides to oligonucleotides to direct them to nuclei, and the invention of molecular switch systems allowing genes to be turned on or off at will. Although many human tumors are non- or weakly immunogenic, the immune system can be reinforced and instructed to eliminate cancer cells after transduction of patient’s cells ex vivo with the cytokine genes GM-CSF, IL- 12, IL-2, IL-4, IL-7, IFN-γ, and TNF-α, followed by cell vaccination of the patient (e.g. intradermally) to potentiate T-lymphocyte-mediated antitumor effects (cancer immunotherapy). DNA vaccination with genes encoding tumor antigens and immunotherapy with synthetic tumor peptide vaccines are further developments in this exciting field. The genes used for cancer gene therapy in human clinical trials include a number of tumor suppressor genes (p53, RB, BRCA1, E1A), antisense oncogenes (antisense c-fos, c-myc, K-ras), and suicide genes (HSV-tk, in combination with ganciclovir, cytosine deaminase in combination with 5-fluorocytosine). Important in gene therapy are also the genes of bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, CFTR, LDL-R, TGF-β, and leptin. Reports on human clinical trials using adenoviral and retroviral injections of the p53 gene have been very encouraging; future directions might go toward the use of genes involved in the control of tumor progression and metastasis. The molecular mechanisms of carcinogenesis have been largely elucidated and improvements in gene delivery methods are likely to lead to the final victory of the human race in the fight against cancer and other deadly diseases. Abbreviations: α1-AT, α1-antitrypsin 5FC, 5-fluorocytosine 5FU, 5-fluorouracil aa, amino acid AAV, adeno-associated virus Ad, adenovirus ADA, adenosine deaminase aFGF, acidic fibroblast growth factor AIDS, acquired immunodeficiency syndrome APCs, antigen-presenting cells bFGF, basic fibroblast growth factor bp, base pairs CAT, chloramphenicol acetyltransferase CD, cytosine deaminase CDKs, cyclin-dependent kinases CEA, carcinoembryonic antigen CF, cystic fibrosis CFTR, cystic fibrosis transmembrane regulator cfu, colony forming units CMV IE, cytomegalovirus immediate-early CMV, cytomegalovirus CNS, central nervous system CNTF, ciliary neurotrophic factor CTLs, cytotoxic T lymphocytes DBD, DNA-binding domain DSBs, double-strand DNA breaks EBV, Epstein-Barr virus EGF, epidermal growth factor EGFR, epidermal growth factor receptor FH, familial hypercholesterolemia GCV, ganciclovir GFP, green fluorescent protein GM-CSF, granulocyte-macrophage colony stimulating factor HIV-1, human immunodeficiency virus type 1 HPV, human papillomavirus HSC, hematopoietic stem cells HSV, herpes simplex virus i.m., intramuscular i.p., intraperitoneal i.v., intravenous ICE, interleukin-1β converting enzyme IFN-γ, interferon-γ IGF-I, insulin-like growth factor I IGF-IR, insulin-like growth factor I receptor IL, interleukin IL-1β, interleukin-1β ITR, inverted terminal repeat LAK, lymphokine-activated killer cells LDL-R, low density lipoprotein receptor LTR, long terminal repeat mAb, monoclonal antibody MAR, matrix-attached region MeP-dR, 6-methylpurine-2’-deoxyriboside MHC, major histocompatibility complex MLV, murine leukemia virus MMTV, mouse mammary tumor virus Mo-MLV, Moloney murine leukemia virus MOI, multiplicity of infection MT, metallothionein Neo R , neomycin phosphotransferase NK, natural killer cells nt, nucleotides ODNs, oligodeoxynucleotides ORFs, open reading frames ORIs, origins of replication 2 PAI-1, plasminogen activator inhibitor-1 PARP, poly(ADP-ribose) polymerase PBL, peripheral blood lymphocytes PCNA, proliferating cell nuclear antigen PDGF, platelet-derived growth factor PGF, placenta growth factor PKC, protein kinase C PMGT, particle-mediated gene transfer PNP, purine nucleoside phosphorylase PSA, prostate specific antigen PVR, proliferative vitreoretinopathy RA, rheumatoid arthritis RA-SF, rheumatoid arthritis synovial fibroblasts rAAV, recombinant adeno-associated virus RAC, recombinant advisory committee RB, retinoblastoma RLU, relative luciferase units RSV, Rous sarcoma virus s.c., subcutaneous SCID, severe combined immunodeficient SMC, smooth muscle cell TAD, transactivation domain TGF-β, transforming growth factor-β TIL, tumor-infiltrating lymphocyte TK, thymidine kinase TNF-α, tumor necrosis factor α tPA, tissue plasminogen activator uPA, urokinase plasminogen activator VEGF, vascular endothelial growth factor VSMC, vascular smooth muscle cell VSMC, vascular smooth muscle cells VSV, vesicular stomatitis virus wt, wild-type
I. Introduction Monumental progress in several fields including DNA replication, transcription factors and gene expression, repair, recombination, signal transduction, oncogenes and tumor suppressor genes, genome mapping and sequencing, and on the molecular basis of human disease are providing the foundation of a new era of biomedical research aimed at introducing therapeutically important genes into somatic cells of patients. The main targets of gene therapy are to repair or replace mutated genes, regulate gene expression and signal transduction, manipulate the immune system, or target malignant and other cells for destruction (reviewed by Anderson, 1992; Nowak, 1995; Boulikas, 1996a,b; Culver, 1996; Ross et al, 1996). Two main approaches have been pursued for gene transfer to somatic cells (i) direct gene delivery using murine retroviruses, adenoviruses, adeno-associated virus, HSV, EBV, liposomes, polymers, or direct plasmid injection (gene therapy in vivo); and (ii) ex vivo gene therapy involving removal of syngeneic cells from a specific organ or tumor of an individual, genetic correction of the defect in cell culture (ADA deficiency, LDL-R for FH) or transfer of a different gene (IL-2 to tumor infiltrating lymphocytes to potentiate the cytotoxicity to tumors, cytokine genes to tumor cells from a patient for cancer immunotherapy, multidrug resistance gene transfer to render bone marrow cells resistant to certain antineoplastic drugs), followed by reimplantation of the cells. The reimplanted cells produce the therapeutic protein. Several key factors or steps appear to be involved for the effective gene transfer to somatic cells in a patient or animal model: (i) the type of vehicle used for gene delivery (liposomes, adenoviruses, retroviruses, AAV, HSV, EBV, polymer, naked plasmid) which will determine not only the half-life in circulation, the biodistribution in tissues, and efficacy of delivery but also the route through the cell membrane and fate of the transgene in the nucleus; (ii) interaction of the genevehicle system with components in the serum or body fluids (plasma proteins, macrophages, immune response cells); (iii) targeting to the cell type, organ, or tumor, and binding to the cell surface; (iv) port and mode of entrance to the cell (poration through the cell membrane, receptormediated endocytosis), (v) release from cytoplasmic compartments (endosomes, lysosomes), (vi) transport across the nuclear envelope (nuclear import); (vii) type and potency of regulatory elements for driving the expression of the transferred gene in a particular cell type including DNA sequences that determine integration versus maintenance of a plasmid or recombinant virus/retrovirus as an extrachromosomal element; (viii) expression (transcription) of the transgene producing heterogeneous nuclear RNA (HnRNA) which is then (ix) spliced and processed in the nucleus to mature mRNA and Gene Therapy and Molecular Biology Vol 1, page 3 3 is (x) exported to the cytoplasm to be (xi) translated into protein. Additional steps may include posttranslational modification of the protein and addition of a signal peptide (at the gene level) for secretion. All steps can be experimentally manipulated and improvements in each one can enormously enhance the level of expression and therapeutic index of a gene therapy approach. It has been proposed that the plasmid vector is unable to translocate to the nucleus unless complexed in the cytoplasm with nuclear proteins possessing nuclear localization signals (NLSs). NLSs are short karyophilic peptides on proteins destined to function in the nucleus used for binding to specific transporter molecules in the cytoplasm, mediating their passage through the pore complexes to the nucleus (see Boulikas, 1998, this volume). NLS are present on histones, transcription factors, nuclear enzymes, and a number of other nuclear proteins; nascent chains of DNA-binding polypeptides could bind to the supercoiled plasmid in the cytoplasm mediating its translocation to the nucleus. During delivery of foreign DNA in vivo vehicles may be attacked by macrophages, lymphocytes, or other components of the immune system and the vast majority will be cleared from blood, intracellular, or other body fluids before it is given the chance to reach the membrane of the cell target; the half-life of naked plasmids injected intravenously into animals is about 5 min (Lew et al, 1995). Cationic lipids, other than being very toxic, mediate efficient gene delivery passing through biological membranes; those lipid-DNA complexes surviving the immediate neutralization by serum proteins in the blood can reach the lung, heart and other tissues after vein or artery injection with one heart beat and transform endothelial vascular cells (reviewed by Boulikas, 1996d). A variety of viral vectors have been developed to exploit the characteristic properties of each group to maintain persistence and viral gene expression in infected cells. Retroviral vectors and AAV integrate into target chromosomes and the transgene they carry can be inactivated from position effects from chromatin surroundings. Vectors with persistence/integration functions may not result in high levels of gene delivery in vivo. Adenoviruses and retroviruses which are of the most frequently used vehicles for gene transfer can accommodate up to 7kb of total foreign DNA into their genome because of packaging limitations. This precludes their use for the transfer of large genomic regions. Transfer of intact yeast artificial chromosome (YAC) into transgenic mice will enable the analysis of large genes or multigenic loci such as human β-globin locus (reviewed by Peterson et al, 1997). A small portion of plasmid molecules crossing the cell membrane will escape degradation from nucleases in the lysosomes and become released to the cytoplasm; even a
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inding sites A GYYY oriented in opp
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Gene Therapy and Molecular Biology
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Prives, 1994). Whereas the wild-typ
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mutant p53 (mutations on p53 are wi
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master regulator of the cell cycle
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Work from several groups has shown
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chromatin condensation, drop in pH,
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Figure 23. A pathway leading to the
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Excessive necrotic death in cells o
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implantation, and similar processes
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normal cells in the surroundings. T
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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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