1. Single-Shot, Multicycle Suicide Gene Therapy by Replication-Competent Retrovirus Vectors Achieves Long-Term Survival Benefit in Experimental Glioma 
Chien-Kuo Tai, Wei Jun Wang, Thomas C. Chen, Noriyuki Kasahara 
Molecular Therapy 
Volume 12, Issue 5, Pages (November 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Replication kinetics of RCR vectors in glioma cells. (A) Structure of ACE-GFP, showing location of the IRES-GFP insert between env and 3′ UTR. CMV, cytomegalovirus promoter. (B) Glioma cells were inoculated with ACE-GFP at a multiplicity of infection of At various time points after infection, the cells were analyzed by flow cytometry to determine the percentage of cells expressing GFP. The x axis shows days after virus inoculation. (C) ACE-GFP-transduced U-87 cells were mixed with their uninfected parental cells as indicated. At 4 and 7 days postmixing, the cells were analyzed for GFP expression. The x axis shows days after cell mixing.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Cytotoxicity of ACE-CD/5-FC on tumor cells. (A) Structure of ACE-CD, showing location of IRES-CD insert between env and 3′ UTR. (B) ACE-CD-transduced U-87 cells were treated with 5-FC on day 0 at various concentrations ranging from 0 to 5 mM. Viability of cells was determined in a triplicate repeat with the MTS assay. The x axis shows days after 5-FC incubation. The y axis shows the viability percentage of the cells normalized to that of negative control, 0 mM. (C) The ACE-CD-transduced and untransduced U-87 cells were mixed at various initial ratios (0, 0.1, 1, 10, and 100% of ACE-CD-transduced cells) and seeded onto 96-well plates. The mixed cell populations were exposed to 2 mM 5-FC or to control medium without 5-FC, and viability of cells was determined with MTS assay. The values shown of viability percentage of the cells were normalized to that of negative control (0%/5-FC−). 5-FC−, without 5-FC incubation. 5-FC+, with 5-FC incubation.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Survival and histological analyses after single-cycle RCR suicide gene therapy. (A) Survival analysis of athymic mice bearing intracerebral U-87 glioma. ACE-CD or PBS was stereotactically injected into intracerebral U-87 xenografts 7 days after tumor inoculation. Eight days after injection, mice received daily intraperitoneal injections of 5-FC or PBS for 15 consecutive days. Survival curves were constructed for four treatment groups as indicated. ACE-CD/5-FC vs ACE-CD/PBS, ACE-CD/5-FC vs PBS/5-FC, and ACE-CD/5-FC vs PBS/PBS, all show significance at P < (B) Brain sections and immunohistochemical analysis. Top shows low-magnification views of whole brain sections at primary tumor inoculation site. Bottom shows immunohistochemistry using antiviral envelope antibody at primary tumor site (PBS/5-FC, ACE-CD/PBS, ACE-CD/5-FC) or ectopic tumor site (ACE-CD/5-FC). Red/brown staining indicates positive immunohistochemical signal. Original magnifications: bottom left three, ×200; bottom right, ×100. (C) Immunohistochemical staining of periventricular region in ACE-CD/PBS-treated animals. Sections in which the tumor mass is encroaching upon (left) or penetrating (right) the ventricle were stained using antiviral envelope antibody, as above. Original magnification: ×400.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Survival and histological analyses after multiple cycles of 5-FC administration. (A) Survival analysis after multicycle 5-FC treatments. Survival curves were constructed for three treatment groups as indicated. ACE-CD/5-FC vs ACE-CD/PBS and ACE-CD/5-FC vs PBS/5-FC both show significance at P < (B) Histological analysis after multicycle 5-FC treatments. Control groups PBS/5-FC and ACE-CD/PBS showed extensive growth of several large tumors, indicated by numbers, and in contrast, ACE-CD/5-FC group showed a small tumor confined within the primary inoculation site. Anti-MLV immunohistochemistry confirmed viral persistence in glioma foci (ACE-CD/PBS, ACE-CD/5-FC).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
PCR analyses of virus stability and spread. (A) Genetic stability of ACE-CD during prolonged replication in tumors. Genomic DNA of intracranial gliomas was analyzed by PCR using primers in the MLV sequence flanking the IRES-CD insert. The expected size of the full-length PCR product is approximately 1300 bp. Three tumors from each group were assayed, and only the virus-injected tumors (ACE-CD/PBS, ACE-CD/5-FC) show a detectable IRES-CD signal. M, 100-bp DNA marker. +, pACE-CD plasmid DNA. (B) Biodistribution of ACE-CD during prolonged replication in vivo. The sensitivity of the PCR assay was determined by amplification of the CD gene from serially diluted pACE-CD plasmid mixed with untransduced mouse tissue genomic DNA (top); above each lane is indicated the number of copies of the CD gene per approximately 100,000 cell genomes. Six hundred nanograms of genomic DNA isolated from intracranial tumor as well as various extratumoral tissues from the same ACE-CD-injected animal was analyzed by PCR using a primer flanking the CD transgene. The expected size of the full-length PCR product is 458 bp. A 525-bp fragment of the β-casein gene was also amplified from the same genomic DNA sample as an internal control for the PCR procedure. M, 100-bp DNA marker. Lanes 1, lung; 2, liver; 3, esophagus and stomach; 4, intestine; 5, spleen; 6, kidney; 7, skin; 8, bone marrow; 9, contralateral normal brain; 10, intracranial tumor; 11, negative control tumor (no virus injection).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions