Suicide Gene Therapy Approach Research Articles

Abstract P32: Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease

Abstract Cell type-specific gene delivery which restricts gene transfer to therapy-relevant cells, hence reducing unwanted side effects caused by the ectopic expression of transgenes, remains one of the biggest challenges. To mitigate potential off-target effects, we developed a programmable cell type-specific gene therapy using an RNA trans-splicing-based approach for gene replacement therapy or suicide gene therapy that can selectively express therapeutic proteins or suicide signals based on the cell’s endogenous pre-mRNA expression profile. Using the liver as a medical target organ, we developed a hepatocellular carcinoma (HCC)-specific trans-splicing based Herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy approach. We rationally designed suicide RNAs that can target multiple HCC-related pre-mRNA biomarkers thereby increasing the overall concentration of targetable pre-mRNA. For delivery, non-viral redosable dumbbell-shaped DNA vectors were designed, manufactured, and tested in tissue culture cells, in 3D patient-derived xenograft spheroids and in mouse model. For cell-type specific delivery, the conjugation of dumbbell vectors with GalNAc3 residues enabled targeted delivery into hepatoblastoma-derived human tissue culture cells. To deliver nucleic acids into the mitochondria of human cells, we studied the long non-coding β2.7 RNA of the human cytomegalovirus which was previously reported to colocalise with mitochondrial complex I. Using thermodynamic profiling, we identified four thermodynamically conserved structural subdomains within the β2.7 RNA that are responsible for its mitochondrial targeting activity. A vector comprising eight mitochondrial targeting domains exhibited a three times higher mitochondrial targeting activity as compared with the parental β2.7 RNA. The amalgamation of mitochondrial targeting RNA and trans-splicing allows cell type-specific transcription of mRNA which can be delivered into mitochondria for mitochondrial gene therapy. Trans-splicing based gene therapy represent a technology that is highly translational offering promising perspectives towards suicide gene therapy of any cancer types or incurable infections with integrating viruses and may enable cell type-specific mitochondrial gene therapy. Citation Format: Pei She Loh (Rachel), Sushmita Poddar, Kaustav Chatterjee, Chiu Jing Wen, Derrick Wee Zan Liang, Fritz Lai, Chen Qingfeng, Volker Patzel. Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P32.

Peptide-Based Nanoparticles for αvβ3 Integrin-Targeted DNA Delivery to Cancer and Uterine Leiomyoma Cells

Uterine leiomyoma is the most common benign tumor of the reproductive system. Current therapeutic options do not simultaneously meet the requirements of long-term efficiency and fertility preservation. Suicide gene delivery can be proposed as a novel approach to uterine leiomyoma therapy. Non-viral vehicles are an attractive approach to DNA delivery for gene therapy of both malignant and benign tumors. Peptide-based vectors are among the most promising candidates for the development of artificial viruses, being able to efficiently cross barriers of DNA transport to cells. Here we described nanoparticles composed of cysteine-crosslinked polymer and histidine-arginine-rich peptide modified with iRGD moiety and characterized them as vehicles for plasmid DNA delivery to pancreatic cancer PANC-1 cells and the uterine leiomyoma cell model. Several variants of nanoparticles were formulated with different targeting ligand content. The physicochemical properties that were studied included DNA binding and protection, interaction with polyanions and reducing agents, size, structure and zeta-potential of the peptide-based nanoparticles. Cytotoxicity, cell uptake and gene transfection efficiency were assessed in PANC-1 cells with GFP and LacZ-encoding plasmids. The specificity of gene transfection via αvβ3 integrin binding was proved in competitive transfection. The therapeutic potential was evaluated in a uterine leiomyoma cell model using the suicide gene therapy approach. The optimal formulation was found to be at the polyplex with the highest iRGD moiety content being able to transfect cells more efficiently than control PEI. Suicide gene therapy using the best formulation resulted in a significant decrease of uterine leiomyoma cells after ganciclovir treatment. It can be concluded that the application of iRGD-modified peptide-based nanoparticles has a high potential for cellular delivery of DNA therapeutics in favor of uterine leiomyoma gene therapy.

Aptamer-Driven Toxin Gene Delivery in U87 Model Glioblastoma Cells.

A novel suicide gene therapy approach was tested in U87 MG glioblastoma multiforme cells. A 26nt G-rich double-stranded DNA aptamer (AS1411) was integrated into a vector at the 5′ of a mammalian codon-optimized saporin gene, under CMV promoter. With this plasmid termed “APTSAP”, the gene encoding ribosome-inactivating protein saporin is driven intracellularly by the glioma-specific aptamer that binds to cell surface-exposed nucleolin and efficiently kills target cells, more effectively as a polyethyleneimine (PEI)-polyplex. Cells that do not expose nucleolin at the cell surface such as 3T3 cells, used as a control, remain unaffected. Suicide gene-induced cell killing was not observed when the inactive saporin mutant SAPKQ DNA was used in the (PEI)-polyplex, indicating that saporin catalytic activity mediates the cytotoxic effect. Rather than apoptosis, cell death has features resembling autophagic or methuosis-like mechanisms. These main findings support the proof-of-concept of using PEI-polyplexed APTSAP for local delivery in rat glioblastoma models.

Suicide gene therapy-mediated purine nucleoside phosphorylase/fludarabine system for in vitro breast cancer model with emphasis on evaluation of vascular endothelial growth factor promoter efficacy.

In this study, a suicide gene therapy approach was optimized by a non-viral polyplex system based on pEGFP-N1 vector harboring purine nucleoside phosphorylase gene conducted by vascular endothelial growth factor promoter for an in vitro breast cancer model (4T1 cell line). The VEGF promoter and purine nucleoside phosphorylase gene were cloned into the vector from the source of 4T1 and E. coli genomic DNA, respectively. A gene construct was developed by replacing VEGF promoter instead of CMV promoter in pEGFP-N1vector. PNP gene was integrated in to the multiple cloning site of the obtained vector. On the other hand, a construct from pEGFP-N1 harboring PNP gene under the control of the original CMV promoter was developed. The transfection method using cationic polymer was optimized based on N/P ratio, cell cytotoxicity, polyplex size, zeta potential and the green fluorescent protein (GFP) expression by fluorescent microscopy and flowcytometry. Also, the effect of hypoxia condition induced by 0.5mM H2O2 on the promoter efficiency was investigated. The results showed that the performed gene delivery system is capable of the gene transfection to more than 30% of the cancer cells with both VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1 plasmids. The hypoxia condition did not show a significant effect on the VEGF promoter. But, it revealed that bystander effect can improve the efficacy of this system and reduce drug IC50 to 2 and fourfold for plasmids VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1, respectively. These results showed that the bystander effect could almost compensate the low efficiency of non-viral gene delivery systems. We suggest that the tumor-specific gene expression system mediated by the VEGF promoter can be especially useful in the present model of breast cancer gene therapy.

ERK-dependent suicide gene therapy for selective targeting of RTK/RAS-driven cancers

Suicide gene therapies provide a unique ability to target cancer cells selectively, often based on modification of viral tropism or transcriptional regulation of therapeutic gene expression. We designed a novel suicide gene therapy approach wherein the gene product (herpes simplex virus thymidine kinase or yeast cytosine deaminase) is phosphorylated and stabilized in expression by the extracellular signal-regulated kinase (ERK), which is overactive in numerous cancers with elevated expression or mutation of receptor tyrosine kinases or the GTPase RAS. In contrast to transcriptional strategies for selectivity, regulation of protein stability by ERK allows for high copy expression via constitutive viral promoters, while maintaining tumor selectivity in contexts of elevated ERK activity. Thus, our approach turns a signaling pathway often coopted by cancer cells for survival into a lethal disadvantage in the presence of a chimeric protein and prodrug, as highlighted by a series of invitro and invivo examples explored here.

Abstract 4069: Selective ablation of solid tumors using a p53-targeted FAST-LNP gene therapy

Abstract While chemotherapy is a key treatment strategy for many solid tumors, it is rarely curative as patients will eventually become resistant. In this study, we sought to develop an effective suicide gene therapy approach for solid tumors that specifically exploits their unique transcriptional activation state. The tumor suppressor p53 is frequently mutated or dysregulated in cancer, and as a result the upstream signaling pathways activating p53 transcription are strongly upregulated. RNA-seq analysis has demonstrated that p53 transcription is significantly upregulated in almost all forms of cancer. Additionally, HCT116 cells lacking functional p53 display a 6-fold increase in p53 promoter activity when compared to its wild type p53 parent cell line. To exploit this, we have developed a Fusogenix FAST-LNP formulation to deliver a p53-driven inducible suicide gene, iCasp9, to solid tumors and destroy them upon activation with small molecule dimerizer, Rapamycin. To establish a proof-of-concept, plasmid encoding iCasp9 and luciferase under control of the p53 promoter was constructed and evaluated in a panel of cancer cell lines. While LNPs administered without Rapamycin or Rapamycin administered alone had no impact on cell viability, we observed greater than 90% apoptotic cell death when both were employed in a wide range of cancer cell lines with p53 deletions or mutations, as measured using cell viability assays, imaging assays, as well as Annexin V and TUNEL flow cytometry. Induction of iCasp9 protein expression and caspase-mediated apoptosis was confirmed using Western blot. No cell death was observed in cells with intact p53 such as human umbilical vein endothelial cells or the fibroblast cell line IMR-90. Next, we assessed the efficacy of FAST-LNPs containing p53-iCasp9 in xenograft PC-3 and H1299 models of human prostate cancer and lung cancer respectively. In some experiments, tumors were implanted subcutaneously in the flanks of 30 mice and allowed to grow to 500 mm3 before treatment by intravenous doses of 100 µg LNP twice per week during continuous low dosing of Rapamycin. We observed a rapid and dramatic reduction in tumor volume averaging 87% over the following 48 hours, with durable response. Tumors in control mice continued to grow exponentially. Overall survival of mice was extended 250% in the PC-3 cohort and 300% in the H1299 cohort. Optimization of number and concentration of LNP doses should allow for long term control of both localized and systemic disease. In conclusion, we describe a novel LNP gene therapy approach for the treatment of cancer with high selectivity for tumors with dysregulated p53 transcriptional activation. This approach has the potential to provide a highly efficacious alternative to current therapies for localized and advanced solid tumors. Citation Format: Douglas Wilson Brown, Arun Raturi, Prakash Bhandari, Deborah Sosnowski, Liliya Grin, Ping Wee, Hector Vega, Jennifer Gyoba, Maryam Hejazi, Jailal Ablack, Matthew Scholz, John D. Lewis. Selective ablation of solid tumors using a p53-targeted FAST-LNP gene therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4069.

Suicide gene therapy for the treatment of high-grade glioma: past lessons, present trends, and future prospects.

Suicide gene therapy has represented an experimental cancer treatment modality for nearly 40 years. Among the various cancers experimentally treated by suicide gene therapy, high-grade gliomas have been the most prominent both in preclinical and clinical settings. Failure of a number of promising suicide gene therapy strategies in the clinic pointed toward a bleak future of this approach for the treatment of high-grade gliomas. Nevertheless, the development of new vectors and suicide genes, better prodrugs, more efficient delivery systems, and new combinatorial strategies represent active research areas that may eventually lead to better efficacy of suicide gene therapy. These trends are evident by the current increasing focus on suicide gene therapy for high-grade glioma treatment both in the laboratory and in the clinic. In this review, we give an overview of different suicide gene therapy approaches for glioma treatment and discuss clinical trials, delivery issues, and immune responses.

Suicide Gene Therapy for Oral Squamous Cell Carcinoma.

Suicide gene therapy has been tested for the treatment of a variety of cancers, including oral cancer. Among the various suicide gene therapy approaches that have been reported, the Herpes Simplex Virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system is one of the most extensively studied systems, holding great promise in cancer therapy. In this chapter, we describe methods to use the HSV-tk/GCV system to achieve antitumor activity, both in cultured oral cancer cells and in orthotopic and subcutaneous murine models of oral squamous cell carcinoma, using ligand-associated lipoplexes for enhancing therapeutic delivery.

RNA Structure Design Improves Activity and Specificity of trans-Splicing-Triggered Cell Death in a Suicide Gene Therapy Approach

Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3′ exon replacement (3’ER), 5′ exon replacement (5’ER) correlated with the thermodynamic stability of the tsRNA 3′ end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy.

Simultaneous detection of 5-fluorocytosine and 5-fluorouracil in human cells carrying CD/5-FC suicide gene system by using capillary zone electrophoresis

A well-known suicide gene therapy approach, cytosine deaminase (CD) in combination with prodrug 5-flurocytosine (5-FC), has become an effective strategy of tumor treatment. However, there are short of simple and convenient detection methods to evaluate the efficiency of 5-FC conversion to 5-fluorouracil (5-FU) in human cells carrying various CD/5-FC systems. In this study, we developed an effective capillary zone electrophoresis (CZE) method to simultaneously measure 5-FC and 5-FU in cells carrying CD/5-FC suicide gene system. Under the condition of 60 mM borate buffer (pH 9.5) and 25 kV separation voltage with 0.5 psi × 15 s injection in 210 nm, the separation of 5-FC and 5-FU could be completely achieved within 15 min. The linearity of the calibration curve of standard 5-FC and 5-FU was in the range from 1 to 1000 μM (r2 > 0.999) and their recoveries were 98.4% and 96.0%, respectively. Due to the simple sample preparation and easy detection, this method is suitable for the study of the conversion efficiency of CD/5-FC suicide gene system. It aims to intuitively evaluate CD/5-FC systems and helps to guide the improvement of more effective CD/5-FC suicide gene systems.

Can we be SMaRT-er in our approach to cancer therapy?

Can we be SMaRT-er in our approach to cancer therapy?

422. In a Suicide Gene Therapy Approach to Cancer, γ-Irradiation Can Completely Abolish the Bystander Effect of the PA-STK Ovarian Tumour Cell Line Due to Its Radiation Sensitivity, Resulting in Necrotic Cell Death

The effect of irradiation on HSV-TK-expressing tumour cells and their bystander killing has been examined. The data presented shows that the irradiation substantially reduced the ability of PA-STK ovarian cells to induce the bystander killing of unmodified tumour cells. PA-STK cells were found to be very sensitive to irradiation and appeared to die too rapidly (even after a low dose of 500-rad) to enable metabolic conversion of GCV to its phosphorylated products, and/or fail to form gap-junctions with their neighbouring cells. Characterisation of tumour cell death showed that PA-STK cells undergo picnosis (a form of necrosis) after irradiation. In contrast, the TK-modified human and mouse mesothelioma cells were found to retain their in vitro bystander killing effect after irradiation. These results suggest that PA-STK cells are not suitable for clinical trials if the cells need to be lethally irradiated before in vivo use. However, the human mesothelioma cell line CRL-5830-TK appears to retain its bystander killing potential after lethal irradiation. In clinical trials, it may therefore be a suitable vehicle for HSV-TK suicide gene therapy for mesothelioma. The low IC50 of GCV in these cells (about 1μμ) makes them particularly attractive.

A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing

The limitations of the ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV1 TK: EC 2.7.1.21) system as a suicide gene therapy approach have been extensively studied over the years. In our study, we focused on improving the cytotoxic profile of the GCV/equine herpes virus-4 thymidine kinase (EHV4 TK: EC 2.7.1.21) system. Our approach involved the structure-guided mutagenesis of EHV4 TK in order to switch its ability to preferentially phosphorylate the natural substrate deoxythymidine (dT) to that of GCV. We performed steady-state kinetic analysis, genetic complementation in a thymidine kinase-deficient Escherichia coli strain, isothermal titration calorimetry, and analysis of GCV-induced cell killing through generation of HEK 293 stable cell-lines expressing EHV4 TK mutants and wild-type EHV4 TK. We found that the EHV4 TK S144H-GFP mutant preferentially phosphorylates GCV and confers increased GCV-induced cytotoxicity compared to wild-type EHV4 TK.

Spliceosome-Mediated RNA Trans-Splicing Facilitates Targeted Delivery of Suicide Genes to Cancer Cells

Patients suffering from recessive dystrophic epidermolysis bullosa (RDEB), a hereditary blistering disease of epithelia, show susceptibility to develop highly aggressive squamous cell carcinoma (SCC). Tumors metastasize early and are associated with mortality in the 30th-40th years of life in this patient group. So far, no adequate therapy is available for RDEB SCC. An approach is suicide gene therapy, in which a cell death-inducing agent is introduced to cancer cells. However, lack of specificity has constrained clinical application of this modality. Therefore, we used spliceosome-mediated RNA trans-splicing technology, capable of replacing a tumor-specific transcript with one encoding a cell death-inducing peptide/toxin, to provide tumor-restricted expression. We designed 3' pre-trans-splicing molecules (PTM) and evaluated their efficiency to trans-splice an RDEB SCC-associated target gene, the matrix metalloproteinase-9 (MMP9), in a fluorescence-based test system. A highly efficient PTM was further adapted to insert the toxin streptolysin O (SLO) of Streptococcus pyogenes into the MMP9 gene. Transfection of RDEB SCC cells with the SLO-PTM resulted in cell death and induction of toxin function restricted to RDEB SCC cells. Thus, RNA trans-splicing is a suicide gene therapy approach with increased specificity to treat highly malignant SCC tumors.

An Improved Bicistronic CD20/tCD34 Vector for Efficient Purification and In Vivo Depletion of Gene-Modified T Cells for Adoptive Immunotherapy

T-cell-based adoptive immunotherapy is widely used to treat graft rejection and relapse after stem cell transplantation (SCT). However, this approach is hampered by a high risk of life-threatening graft-versus-host-disease (GvHD). Clinical trials have demonstrated the value of suicide genes to modify T cells for the effective control of GvHD. Herewith, we show that the combination of a codon-optimized B-cell antigen (CD20op) with a selection marker based on a cytoplasmic truncated version of the human stem cell antigen CD34 (tCD34) allows the generation of highly enriched gene-modified T cells. We demonstrate coordinate co-expression of both transgenes and high expression of CD20op resulting in an increased susceptibility to Rituximab (RTX)-induced cell death. In addition, T cells partially retained their alloreactive potential and their CD4/CD8 ratio after transduction and expansion. Long-lasting transgene expression was sustained in vivo after adoptive transfer into Rag-1(-/-) mice. Moreover, gene-modified T cells were quickly and efficiently depleted from peripheral blood (PB) and secondary lymphoid organs of transplanted animals after RTX treatment. These results warrant further steps toward a clinical application of CD20op as a suicide gene for adoptive immunotherapy.

Alteration of the carbohydrate for deoxyguanosine analogs markedly changes DNA replication fidelity, cell cycle progression and cytotoxicity

Nucleoside analogs are efficacious cancer chemotherapeutics due to their incorporation into tumor cell DNA. However, they exhibit vastly different antitumor efficacies, suggesting that incorporation produces divergent effects on DNA replication. Here we have evaluated the consequences of incorporation on DNA replication and its fidelity for three structurally related deoxyguanosine analogs: ganciclovir (GCV), currently in clinical trials in a suicide gene therapy approach for cancer, D-carbocyclic 2′-deoxyguanosine (CdG) and penciclovir (PCV). GCV and CdG elicited similar cytotoxicity at low concentrations, whereas PCV was 10–100-fold less cytotoxic in human tumor cells. DNA replication fidelity was evaluated using a supF plasmid-based mutation assay. Only GCV induced a dose-dependent increase in mutation frequency, predominantly GC→TA transversions, which contributed to cytotoxicity and implicated the ether oxygen in mutagenicity. Activation of mismatch repair with hydroxyurea decreased mutations but failed to repair the GC→TA transversions. GCV slowed S-phase progression and CdG also induced a G2/M block, but both drugs allowed completion of one cell cycle after drug treatment followed by cell death in the second cell cycle. In contrast, PCV induced a lengthy early S-phase block due to profound suppression of DNA synthesis, with cell death in the first cell cycle after drug treatment. These data suggest that GCV and CdG elicit superior cytotoxicity due to their effects in template DNA, whereas strong inhibition of nascent strand synthesis by PCV may protect against cytotoxicity. Nucleoside analogs based on the carbohydrate structures of GCV and CdG is a promising area for antitumor drug development.

MLH1 deficiency enhances tumor cell sensitivity to ganciclovir

Suicide gene therapy with herpes simplex virus thymidine kinase and ganciclovir is notable for producing multi-log cytotoxicity in a unique pattern of delayed cytotoxicity in S-phase. Because hydroxyurea, a ribonucleotide reductase inhibitor that activates mismatch repair, can increase sensitivity to ganciclovir, we evaluated the role of MLH1, an essential mismatch repair protein, in ganciclovir cytotoxicity. Using HCT116TK (HSV-TK-expressing) colon carcinoma cells that express or lack MLH1, cell survival studies demonstrated greater ganciclovir sensitivity in the MLH1 deficient cells, primarily at high concentrations. This could not be explained by differences in ganciclovir metabolism, as the less sensitive MLH1-expresssing cells accumulated more ganciclovir triphosphate and incorporated more of the analog into DNA. SiRNA suppression of MLH1 in U251 glioblastoma or SW480 colon carcinoma cells also enhanced sensitivity to high concentrations of ganciclovir. Studies in a panel of yeast deletion mutants confirmed the results with MLH1, and further suggested a role for homologous recombination repair and several cell cycle checkpoint proteins in ganciclovir cytotoxicity. These data suggest that MLH1 can prevent cytotoxicity with ganciclovir. Targeting mismatch repair-deficient tumors may increase efficacy of this suicide gene therapy approach to cancer treatment.

Hypoxia response element‐driven cytosine deaminase/5‐fluorocytosine gene therapy system: a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro

We proposed to exploit hypoxia-inducible factor (HIF)-1alpha overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1alpha to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells. We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evaluated in 22Rv1 and DU145 cells in vitro. The CD protein as selectively detected in lysates of transiently transfected 22Rv1 and DU145 cells following hypoxic exposure. This is the first evidence of GAPDH HREs being used to control a suicide gene therapy strategy. Detectable CD levels were sustained upon reoxygenation and prolonged hypoxic exposures. Hypoxia-induced chemoresistance to 5-FU was overcome in both cell lines treated with this suicide gene therapy approach. Hypoxic transfectants were sensitized to prodrug concentrations that were ten-fold lower than those that are clinically relevant. Moreover, the surviving fraction of reoxygenated transfectants could be further reduced with the concomitant delivery of clinically relevant single radiation doses. This strategy thus has the potential to sensitize the hypoxic compartment of prostate tumours and improve the outcome of current therapies.

Bystander Killing of Malignant Glioma by Bone Marrow–derived Tumor-Infiltrating Progenitor Cells Expressing a Suicide Gene

Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells. However, their clinical use is limited by a low passaging capacity that impedes large-scale production. In the present study, a bone marrow-derived, highly proliferative subpopulation of mesenchymal stem cells (MSCs)-here termed bone marrow-derived tumor-infiltrating cells (BM-TICs)-was genetically modified for the treatment of malignant glioma. Upon injection into the tumor or the vicinity of the tumor, BM-TICs infiltrated solid parts as well as the border of rat 9L glioma. After intra-tumoral injection, BM-TICs expressing the thymidine kinase of herpes simplex virus (HSV-tk) and enhanced green fluorescent protein (BM-TIC-tk-GFP) were detected by non-invasive positron emission tomography (PET) using the tracer 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG). A therapeutic effect was demonstrated in vitro and in vivo by BM-TICs expressing HSV-tk through bystander-mediated glioma cell killing. Therapeutic efficacy was monitored by PET as well as by magnetic resonance imaging (MRI) and strongly correlated with histological analysis. In conclusion, BM-TICs expressing a suicide gene were highly effective in the treatment of malignant glioma in a rat model and therefore hold great potential for the therapy of malignant brain tumors in humans.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Gene therapy for Fabry disease, a deficiency in alpha-galactosidase A (alpha-gal A) activity, has the potential to provide a cure for the disorder with a single treatment. Despite modifications to existing vectors, concerns have arisen regarding the risk of genotoxicity associated with the use of retroviruses. To address safety concerns, we propose that expression of a cell surface protein, human CD25 (huCD25) in a bicistronic format, with any therapeutic gene such as alpha-gal A can provide a target that can be used to kill transduced cells selectively should transformative events occur. We show that an anti-CD25 antibody and immunotoxin can specifically target and eliminate transduced leukemia cells expressing CD25. In a murine leukemia model, antibody treatment reduced tumor burden 32-fold and increased survival compared with untreated mice. Furthermore, after a bone marrow transplant of therapeutically transduced cells into Fabry mice, antibody treatment reduced the number of retrovirally transduced huCD25-expressing cells in the peripheral blood. A systemic loss of transduced cells with functional consequences was also evident in the liver and spleen. This proof-of-principle study demonstrates that a targeted antibody can reduce tumor burden and selectively clear bicistronically transduced hematopoietic cells that express a target antigen, thus acting as a built-in safety mechanism.