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[.]
Trang 1Molecular Therapy Vol 5, No 5, May 2002, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
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TISSUE TARGETED GENE EXPRESSIONAND 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
1Genetics 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
androgen-independent prostate cancer is poorly understood, androgen-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 androgendependent (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,
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
1National 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
1Dermatology 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 dye-low phenotype can be attributed to cellular pump activity Although a
Trang 2Molecular Therapy Vol 5, No 5, May 2002, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy S414
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1269 Comparison of T
1269 Comparison of Transgene Expression from ransgene Expression from
CMV
CMV, SV40 V , SV40 V , SV40 Viral Promoters and PgK, UBB, and Skeletal iral Promoters and PgK, UBB, and Skeletal
α
αα
αα-Actin Cellular Promoters in Muscle and Liver Tissues -Actin Cellular Promoters in Muscle and Liver Tissues
Using In Vivo In Vivo Electroporation Electroporation
Haiping Hao,1 David W Potter.1
1 RHeoGene, Spring House, PA, United States
1270 A Cyclooxygenase-2 Promoter Based Conditionally Replicating Adenovirus with Enhanced Infectivity for T
Infectivity for Treatment of Ovarian reatment of Ovarian reatment of Ovarian Adenocarcinoma Adenocarcinoma
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
1 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center;
2 Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, AL, United States