Single-shot, multicycle suicide gene therapy by replication-competent retrovirus vectors achieves long-term survival benefit in experimental glioma

Abstract

Achieving therapeutically efficacious levels of gene transfer in tumors has been a major obstacle for cancer gene therapy using replication-defective virus vectors. Recently, replicating viruses have emerged as attractive tools for cancer therapy, but generally achieve only transitory tumor regression. In contrast to other replicating virus systems, transduction by replication-competent retrovirus (RCR) vectors is efficient, tumor-selective, and persistent. Correlating with its efficient replicative spread, RCR vector expressing the yeast cytosine deaminase suicide gene exhibited remarkably enhanced cytotoxicity in vitro after administration of the prodrug 5-fluorocytosine. In vivo, RCR vectors replicated throughout preestablished primary gliomas without spread to adjacent normal brain, resulting in profound tumor inhibition after a single injection of virus and single cycle of prodrug administration. Furthermore, stable integration of the replicating vector resulted in persistent infection that achieved complete transduction of ectopic glioma foci that had migrated away from the primary tumor site. Thus, efficient and stable integration of suicide genes represents a unique property of the RCR vector that achieved multiple cycles of synchronous cell killing upon repeated prodrug administration, resulting in chronic suppression of tumor growth and prolonged survival.

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 0.05. 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.

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.

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 < 0.0001. (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.

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 < 0.0001. (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).

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 α-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).