1268 Side Population Cells (SP) in the Human Epidermis A Novel Candidate for Keratinocyte Stem Cells Molecular Therapy Vol 5, No 5, May 2002, Part 2 of 2 Parts Copyright © The American Society of Gene[.]
TISSUE TARGETED GENE EXPRESSION AND IMAGING Active chromatin domains are compartmentalized in the nucleus Scaffold / matrix attachment regions (S/MARs) are the DNA modules, that connect the chromatin loops to the nuclear matrix Besides their importance for structural reasons they regulate higher order gene expression interacting with scaffold / matrix attachment factors (SAFs) We have developed episomal, adenoviral and retroviral vectors, which incorporate S/MARs from the human interferon beta gene locus, the human HPRT gene locus and the chicken lysozyme gene locus and evaluated their expression profiles with respect to expression levels and kinetics in different cell culture systems using flow cytometric analysis and biochemical assays Episomal S/MAR vectors combine an CMV enhancer/promoter driven transgene with a S/MAR-module Moderate prolonged expression profiles can be demonstrated in CHO cell culture systems after lipoplex-mediated gene transfer of the enhanced GFP and the human iNOS gene, which is applied in cardiovascular gene therapy The Adenoviral S/MAR vector Ad-SAR1 incorporates the human interferon beta scaffold attachment region between the ITR and the promoter/transgene region This vector has a prolonged expression profile as demonstrated by infection of a COS7 cell culture system The adenoviral vector Ad-SAR1-VE1 contains the human interferon beta SAR and the human vascular endothelial VE-cadherin-1 promoter In contrast to a vector lacking the SAR this vector permits improved endothelial specific expression in human umbilical vein endothelial cells (HUVEC), demonstrating insulation-capacity of the human interferon beta SAR in an adenoviral environment Retroviral S/MAR vectors have been developed in order to reduce position-effect-variegation and silencing of retroviral transgene expression A FMEV-based retroviral vector incorporating the lysozyme locus MAR in the retroviral LTR shows reduced variability of transgene expression in transduced, independently established NIH3T3 cell clones Incorporation of scaffold / matrix - attachment - regions in expression vectors for therapeutic applications can modulate and insulate therapeutic gene expression and improve the systems concerning expression persistence and specificity 1266 Development of Prostate Specific Promoter for Gene Therapy Against Androgen-Independent Prostate Cancer Souichi Furuhata,1 Kazuteru Hatanaka,1 Hisamitsu Ide,1 Teruhiko Yoshida,1 Kazunori Aoki.2 Genetics Division; 2Section for Studies on Host-immune Response, National Cancer Center Research Institute, Tokyo, Japan Prostate cancer is one of the most common malignancies in men, and the incidence is increasing especially in developed countries Androgen ablation has been the standard treatment for metastasized prostate cancer In most cases, however, prostate cancer cells eventually lose androgen dependency and become refractory to the conventional endocrine therapy Although the molecular basis for the development of androgenindependent prostate cancer is poorly understood, androgen-independent prostate cancer is characterized by a heterogeneous loss of androgen receptor (AR) expression among tumor cells Prostate specific promoters such as prostate specific antigen (PSA) and rat probasin (rPB) promoters have been examined in the development of gene therapy targeted to prostate cancer However, those promoters require binding of the androgen-AR complex to the androgen response element (ARE) and are active only in the androgen-dependent prostate cancer cell line but not in the androgen-independent cell line In order to target transgene expression in androgen-independent prostate cancer, we designed prostate-specific promoter that is activated by the retinoids-retinoid receptor complex instead of the androgen-AR complex since retinoid receptors are ubiquitously expressed in human tissues While the tailored rPB promoters lost any responsiveness to synthetic androgen in androgen-dependent prostate cancer cell, they expressed transgenes in response to retinoid (all trans retinoic acid: ATRA) in both androgen-dependent (LNCaP) and independent prostate cancer cells (PC3 and TSUPr-1), but not in other cancer cell lines (HCT-15, MCF-7 and MIAPaCa-2) or in human normal cells (human umbilical vascular endothelial cells, hepatocytes and smooth muscle cells) in vitro Next, to determine whether transgene expression under the tailored rPB promoter was restricted to prostate cells in vivo, Molecular Therapy Vol 5, No 5, May 2002, Part of Parts Copyright © The American Society of Gene Therapy PC3 subcutaneous tumors were injected with the adenovirus encoding alkaline phosphatase (AP) gene under the regulation of the tailored rPB promoter, which led to the expression of the AP transgene in 60-70% of the cells after administration of ATRA, but in its absence the AP gene expression was significantly suppressed Furthermore, the combination of retinoid treatment and adenovirus-mediated gene transfer of the tailored rPB-driven HSV-tk gene resulted in a significant growth suppression of the androgen-independent prostate cancer cells in the presence of the prodrug ganciclovir The growth inhibitory effect of retinoids per se has been documented in a wide variety of tumor cell types including prostate cancer, and the compounds are already being used clinically Thus, the combination of ATRA and prostate specific gene therapy may be a reasonable and realistic choice for prostate cancer 1267 Targeting Gene Expression to the p53 Defective Tumor Cells Jingde Zhu.1 National Laboratory for Oncogene and Related Genes, Shanghai Cancer Institute, Shanghai, China The tumor suppressor protein p53 can positively or negatively regulate the expression of its downstream genes that participate in the control of cell growth or apoptosis Over 50% of human tumors are p53 defective by either genetic or epigenetic mechanisms We have exploited the specific defect of p53 in tumor cells, namely in the control of transcription of its downstream genes, to develop a novel tumor targeting strategy which maximizes expression of the potential therapeutic gene(s) in tumors while simultaneously down-regulates the same gene(s) in normal cells There are two genetic Units in this system, the promoter function of which are repressed by (Unit I) and enhanced (Unit II) by the wild-type p53 but not by mutant p53, respectively For instance, a therapeutic gene in Unit I may be placed under the control of a promoter such as the HSP70 gene promoter, the gene of which is over-expressed in many tumor cells The product(s) of the gene(s) capable of suppressing the expression of the gene in Unit I is put under the control of a minimal promoter in conjunction with a p53 responsive element The difference in the expression level of the therapeutic gene in Unit I would be magnified between the normal cells having a wild-type p53 function and the tumor cells where p53 function is defective when both Units rather than Unit I on its own are involved In this report, we have provided the proof of principle with both the luciferase gene and HSVtk/GCV system in cell cultures Furthermore, the interference between the promoter activities from two different Units has been eliminated by using the insulator elements from the chicken beta globin gene locus Out results demonstrate that this dual control system would offer a universal tumor targeting strategy at a level of gene expression 1268 Side Population Cells (SP) in the Human Epidermis: A Novel Candidate for Keratinocyte Stem Cells Atsushi Terunuma,1 Kimberly L Jackson,1 Veena Kapoor,2 William G Telford,2 Jonathan C Vogel.1 Dermatology Branch, National Cancer Institute, NIH, Bethesda, MD, United States; 2Medicine Branch, National Cancer Institute, NIH, Bethesda, MD, United States Epidermis, as a renewing tissue, is maintained by keratinocyte stem cells (KSC) KSC are attractive targets for skin gene therapy in order to achieve long-term expression of therapeutic genes in a high percentage of keratinocytes However, unique cell surface markers for KSC that allow their identification and manipulation are not known In the bone marrow, side population cells (SP) that represent a very primitive population of hematopoietic stem cells (HSC) have recently been described Bone marrow SP are detected by FACS analysis for their ability to exclude Hoechst 33342 dye (dye-low) after staining cell suspensions Surprisingly, we found that SP are also present in epidermal suspensions: SP represented a small population in epidermal suspensions (0.3% of the total); SP consisted of keratinocytes (89%) and increased percentage of melanocytes (10%); and SP were enriched for cells in G0/G1 phase of cell cycle Additionally, the epdidermal SP phenotype disappears following verapamil treatment, similar to bone marrow SP, suggesting that the dyelow phenotype can be attributed to cellular pump activity Although a S413 !!"7 " "!"! '2 '2 , (! (! 1 @ "!@ ?'( '2 , (! 3 (! A( % A( αB β8 3 (! (! (! A( 1 (! A( 1269 Comparison of T ransgene Expression from Transgene CMV iral Promoters and PgK, UBB, and Skeletal CMV,, SV40 V Viral α-Actin Cellular Promoters in Muscle and Liver Tissues Using In Vivo Electroporation Haiping Hao,1 David W Potter.1 RHeoGene, Spring House, PA, United States ( 3 ' " 3 3 C 3 # (.69 "8 -!A/ 41 2 -422/ α" -α" / 1 &;2D%B C - (>'!/ $*' 3 1 ! (>'! S (>'! # 3 3 3 422 (.69 α" 3 " #. !A 3 " # #. 422 α" ! 3 &; (.69 3 (.69 3 # #. 422 3 #. 3 ! 8%89 3 #. α" 3 C #. 3 (.69 !A 3 (>'! 3 422 8%& 3 &; ? 3 #. 422 α" !A 3 D #. (.69 ! 422 " 3 S414 1270 A Cyclooxygenase-2 Promoter Based Conditionally Replicating Adenovirus with Enhanced Infectivity for T reatment of Ovarian Adenocarcinoma Treatment Anna Kanerva,1 John T Lam,1 Masato Yamamoto,1 Gerd J Bauerschmitz,1 Mack N Barnes,2 Ronald D Alvarez,2 David T Curiel,1 Akseli Hemminki.1 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center; Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, AL, United States -@' / @' 1 1 3 " @' 3 3 " -'@/ 4 3 '@ ) '@ @ $"6 C" @ $"6 '@ αβ" "'@ @ $" @' 3 ", -3" ,D/ >8' -@ $@'$3",@/ 3", 3 ? G 3 @' 3 >8 3",D @ $@'$3",@ 1 3",-J/ '&65 @' -' / @' 3",-"/ 6;6 " ' ! G 4 @ $@'$3",@ 1 !@ ? @ $@'$3",@ @ $" @' 3",D >8' ' " @' Molecular Therapy Vol 5, No 5, May 2002, Part of Parts Copyright © The American Society of Gene Therapy ... Expression from Transgene CMV iral Promoters and PgK, UBB, and Skeletal CMV,, SV40 V Viral α-Actin Cellular Promoters in Muscle and Liver Tissues Using In Vivo Electroporation Haiping Hao,1 David W Potter.1... (! A( 1 (! A( 1269 Comparison of T ransgene... Liver Tissues Using In Vivo Electroporation Haiping Hao,1 David W Potter.1 RHeoGene, Spring House, PA, United States ( 3