Suicide Gene Therapy For Cancer Research Articles

46 Targeted suicide gene therapy for small cell lung cancer

46 Targeted suicide gene therapy for small cell lung cancer

581. A Chimeric Fusion of the Human Achaete Scute Homolog 1 (hASH1) and the Enhancer of Zeste Homolog 2 (EZH2) Promoters Is a Promising Regulator of Suicide Gene Therapy in Small Cell Lung Cancer (SCLC)

581. A Chimeric Fusion of the Human Achaete Scute Homolog 1 (hASH1) and the Enhancer of Zeste Homolog 2 (EZH2) Promoters Is a Promising Regulator of Suicide Gene Therapy in Small Cell Lung Cancer (SCLC)

11A chimeric fusion of the human Achaete Scute Homolog 1 (hASH1) and the Enhancer of Zeste Homolog 2 (EZH2) promoters is a promising regulator of suicide gene therapy in small cell lung cancer (SCLC)

11A chimeric fusion of the human Achaete Scute Homolog 1 (hASH1) and the Enhancer of Zeste Homolog 2 (EZH2) promoters is a promising regulator of suicide gene therapy in small cell lung cancer (SCLC)

Characterization of HIV-1 TAT peptide as an enhancer of HSV-TK/GCV cancer gene therapy

Cancer suicide gene therapy based on herpes simplex virus type I thymidine kinase (HSV-TK) and ganciclovir (GCV) suffers from the lack of efficacy in clinical use, which is mostly due to low gene-transfer efficiency and absence of bystander effect in tumors. We have previously demonstrated the enhancement of GCV cytotoxicity by fusing the HSV-TK with the cell penetrating peptide from HIV-1 transactivator protein transduction domain (TAT PTD). Despite the earlier promising results, we found that the triple fusion protein HIV-1 transactivator protein transduction domain-thymidine kinase suicide gene-green fluorescent protein marker gene (TAT-TK-GFP) increased GCV cytotoxicity only in 3/12 of different human tumor cell lines. Extended GCV exposure enhanced the cytotoxic effect of HSV-TK/GCV gene therapy, but the difference between TK-GFP and TAT-TK-GFP was not statistically significant. The modest improvement on cell killing mediated by TAT PTD in Chinese hamster ovary cells appeared to be associated with cell-surface heparan sulfate proteoglycan (HSPG) composition. However, TAT-mediated increased cell death did not correlate with the density of cell-surface HSPG expression in different tumor cell lines. In conclusion, although some degree of enhancement by TAT was shown in certain tumor cells in vitro, it is unlikely that TAT peptide linked to a suicide protein could be a useful booster of in vivo gene therapy trials.

Two‐step transcriptional amplification–lipid‐based nanoparticles using PSMA or midkine promoter for suicide gene therapy in prostate cancer

A two-step transcriptional amplification system (TSTA) was used to enhance the efficacy of suicide gene therapy for treatment of prostate cancer. We designed a TSTA system and constructed two types of plasmid: one containing GAL4-VP16 fusion protein under the control of a tumor-specific promoter, the other containing luciferase or herpes simplex virus thymidine kinase (HSV-tk) under the control of a synthetic promoter. The TSTA systems using nanoparticles based on lipids were evaluated by measuring the amount of induced luciferase activity as a function of prostate-specific membrane antigen (PSMA) and midkine (Mk) promoters, specific for LNCaP and PC-3 prostate cancer cells, respectively. In LNCaP cells that were PSMA-positive, the TSTA system featuring the PSMA enhancer and promoter exhibited activity that was 640-fold greater than a system consisting of one-step transcription with the PSMA promoter. In contrast, this difference in activity did not occur in PSMA-negative PC-3 cells. In Mk-positive PC-3 cells, the TSTA system with the Mk promoter exhibited a five-fold increase in activity over one-step transcription, but such activity was not induced in Mk-negative LNCaP cells. When using HSV-tk for suicide gene therapy, TSTA systems featuring the PSMA or Mk promoter inhibited in vitro cell growth in the presence of ganciclovir. Furthermore, the TSTA system featuring the Mk promoter suppressed in vivo growth of PC-3 tumor xenografts to a greater extent than one-step transcription. These findings show that TSTA systems can enhance PSMA and Mk promoter activities and selectively inhibit PC-3 cell growth in tumors. This suggests that TSTA systems featuring tumor-specific promoters are suitable for cancer treatment by gene therapy.

746 ナノバブルと超音波を用いた自殺遺伝子導入法の開発(S08-4 細胞の構造とメカノバイオロジー(4),S08 細胞の構造とメカノバイオロジー)

746 ナノバブルと超音波を用いた自殺遺伝子導入法の開発(S08-4 細胞の構造とメカノバイオロジー(4),S08 細胞の構造とメカノバイオロジー)

Monocyte chemoattractant protein-1 gene delivery enhances antitumor effects of herpes simplex virus thymidine kinase/ganciclovir system in a model of colon cancer

Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system is a well-characterized tool for cancer gene therapy; however, it does not yet exhibit sufficient efficacy to cure patients of malignancies. We have reported that adenovirally delivered monocyte chemoattractant protein (MCP)-1 augmented the antitumor effects of the HSV-tk/GCV system in an athymic nude mouse model. The current study, which uses an immunocompetent mouse model of colon cancer, was designed to evaluate the antitumor effects of MCP-1 gene delivery in conjunction with this suicide gene therapy system. Subcutaneous tumor foci were directly transduced with both recombinant adenoviruses (rAds) expressing an HSV-tk gene and either of the MCP-1, CD80 and LacZ genes, followed by GCV administration. The growth of tumors was markedly suppressed by codelivery of HSV-tk and MCP-1 genes, which was exclusively associated with the recruitment of monocytes/macrophages, T helper 1 (Th1) cytokine gene expression and cytotoxic activity of the splenocytes. Furthermore, the antitumor effects were more efficient than that obtained by the combination of HSV-tk and CD80 genes. These results suggest an immunomodulatory effect of MCP-1 in the context of suicide gene therapy of colon cancer via orchestration of innate and acquired immune responses.

Highly specific expression of luciferase gene in lungs of naïve nude mice directed by prostate-specific antigen promoter

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 × 10 9 PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.

Folate-linked nanoparticle-mediated suicide gene therapy in human prostate cancer and nasopharyngeal cancer with herpes simplex virus thymidine kinase

For targeted gene delivery to human prostate cancer LNCaP and PC-3 cells and nasopharyngeal cancer KB cells, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated suicide gene therapy in the cells and xenografts with herpes simplex virus thymidine kinase (HSV-tk) and connexin 43 (Cx43). An NP-F-plasmid DNA complex (NP-F nanoplex) showed high DNA transfection efficiency in KB, LNCaP and PC-3 cells. Cell growth inhibition in the presence of ganciclovir (GCV) was enhanced with HSV-tk and Cx43 genes in LNCaP cells. In suicide gene therapy, the tumor growths of KB and LNCaP xenografts were significantly inhibited when an NP-F nanoplex of the HSV-tk gene, and HSV-tk and Cx43 genes, respectively, was injected intratumorally and GCV was administered intraperitoneally. These findings suggested that the NP-F is a potential target vector in prostate and nasopharyngeal cancer for suicide gene therapy.

315. Highly Specific Expression of the Luciferase Gene in Lungs of Naïve Nude Mice Directed by Prostate Specific Antigen Promoter

The PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy for prostate cancer models. The characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate the therapy safety. Here we constructed a adenovirus vector (AdPSA-Luc), containing firefly luciferase (Luc) gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device (CCD) video camera was used to noninvasively image the expression of luciferase in nude mice on days 3, 7, 11 after injection of 2109 PFU of AdPSA-Luc virus via tail vein. ). On days 3, the weak liver and chest signals could only be detected. On days 7, light signals in the chests and lower abdomens were apparent. The chest signals were emitted by 1.8103 , 1.210 4 and 6.010 4 RLU/min respectively from 3 to 11 days. On days 7, the outlines of lungs were imaged distinctly. As a control, AdCMV-Luc containing CMV promoter and luciferase gene was injected into normal nude mice via tail vein. The strong liver signals (5.310 6 , 3.610 7 and 4.110 7 RLU/min respectively) could be seen from 3 to 11 days after injection. On days 11, mice were sacrificed and isolated organs were imaged. The signals in the chest injected with AdPSA-Luc were found to originate from the lung. The signal in the whole lung from AdPSA-Luc injected mice displayed about 190 fold higher than from AdCMV-Luc injected mice. However, the signals in the isolated livers sacrificed mice injected with AdCMV-Luc appeared about 1.010 5 folds higher than in the lungs while the signals couldn't be detected in the isolated livers from the mice injected with AdPSA-Luc. The result showed highly specific expression of the luciferase gene in lungs of mice from days 7 while AdCMV-Luc just led to high level of luciferase expression in livers. These results may also predict PSA expression in the lungs of normal animals. The finding indicates the possible disadvantage of the suicide gene therapy of prostate cancer based on the selectivity of PSA promoter. By contrary, it has encouraging implications for the further development of vectors via PSA promoter to enable gene therapy for pulmonary disease.

293. Optimizing the Efficacy of Spliceosome-Mediated RNA Trans-Splicing (SMaRT™) for Suicide Gene Therapy of Cervical Cancer

SMaRT|[trade]| is a platform technology that has the ability to reprogram genetic information at the mRNA level. SMaRT involves the trans-splicing of a pre-trans-splicing molecule (PTM) to a target pre-mRNA. PTMs are RNAs that are designed to contain a binding domain (BD) complementary to a target pre-mRNA, splice elements necessary for efficient and specific trans-splicing and the coding domain. Target specificity is obtained by tethering the PTM to the target pre-mRNA through the BD in the PTM. SMaRT can be used to replace or repair 5' exon(s), internal exon(s), and 3' terminal exon(s) and to make chimeric mRNAs. We are investigating the use of SMaRT to specifically target the expression of a therapeutic molecule to cervical cancer, which is associated with oncogenic human papillomaviruses (HPVs). We have designed HPV-specific PTMs that target the pre-mRNAs encoding E6 and E7 oncoproteins and have shown efficient trans-splicing to HPV pre-mRNAs. We are using this system to study the factors that affect trans-splicing efficiency and specificity. Previously, we have shown that the BD confers both target specificity as well as splice site specificity. Trans-splicing efficiency is very high (80%) in cotransfection experiments and the amount of trans-spliced product is directly proportional to the amount of target over at least a 300-fold range. The efficiency of trans-splicing to endogenous targets is generally 10-fold lower. We investigated SMaRT by cotransfecting GFP and PTM expression vectors into cells stably expressing a mini-gene HPV pre-mRNA target and sorting cells for high and low GFP expression using flow cytometry. Those cells that expressed the most GFP also expressed the highest levels of PTM and had higher levels of trans-splicing (|[sim]|17%). In contrast, trans-splicing efficiency in low GFP-expressing cells was only 4%. We conclude from these experiments that trans-splicing to endogenous targets can be improved significantly with efficient delivery and high expression of PTMs. Cervical cancer cell lines such as CaSki and SiHa express endogenous HPV-16 pre-mRNAs, but have been refractory to efficient transfection. We have now generated recombinant adenoviral vectors expressing HPV PTMs and are using them to efficiently deliver PTMs to cervical cancer cells. Experiments are in progress to assess trans-splicing efficiency and potential therapeutic effects as a function of multiplicity of infection.

Virus Specific Immune Responses after Human Neoadjuvant Adenovirus-mediated Suicide Gene Therapy for Prostate Cancer

Purpose: Neoadjuvant gene therapy potentially improves the outcome of primary treatment of prostate cancer by radical prostatectomy in patients with high risk of recurrence. We conducted a Phase I escalating dose study with a replication-defective adenovirus expressing the herpes simplex virus-thymidine kinase gene (Adv-HSV-tk vector). The primary end point was toxicity, while the evaluation of the patients’ cellular and humoral immune responses served as a secondary endpoint. Material and methods: The Adv-HSV-tk vector was injected into the prostate in two doses (2 × 10 10 to 2 × 10 11 viral particles), followed by ganciclovir twice daily for 14 days and retropubic radical prostatectomy on day 21. Adenovirus-specific neutralizing, IgG and IgA antibodies were evaluated. Peripheral blood mononuclear cells (PBMC) were stimulated by Adv-HSV-tk and analysed for IFN-γ production and 3H-thymidine incorporation. Prostate specimens were immunostained for B (CD20 +) and for T (CD3 +) lymphocytes. Results: Toxicity was minor in all 8 patients treated. In the prostate, no virus related cytopathic effect could be observed. Dose-dependent infiltration of T and B lymphocytes in the whole prostate and in tumor areas was observed. Boosting of adenovirus-specific antibody responses was observed in 7 patients, and an increased adenovirus-specific PBMC proliferation and IFN-γ production was seen after Adv-HSV-tk stimulation. Conclusion: Neo-adjuvant adenovirus-mediated cytotoxic gene therapy prior to prostatectomy for prostate cancer is feasible and safe in an outpatient setting for intraprostatic vector doses up to 2 × 10 11 viral particles. Activation of the immune system was observed. Application of higher vector doses may be considered.

Transcriptional tumor‐selective promoter targeting of E. coli purine nucleoside phosphorylase for pancreatic cancer suicide gene therapy

Pancreatic cancer remains a rapidly fatal disease. Suicide gene therapy has been shown to be an effective tool for pancreatic tumor cell destruction, but a cell-specific gene delivery is required to limit host toxicity. The objective of this study was both to design recombinant vectors in which the suicide gene E. coli purine nucleoside phosphorylase (ePNP) is under the control of either CEA or MUC1 promoter sequences and to investigate on experimental pancreatic carcinomas the selective killing effects of the conditional ePNP/prodrug (MePdR) system. Transcriptional activities of CEA and MUC1 promoter sequences were analyzed using luciferase reporter gene constructions. Thereafter, recombinant vectors expressing ePNP under control of the most promising pCEA and pMUC1 sequences were designed and used to establish stable tumor cell transfectants from two human pancreatic cell lines, respectively tumor-marker positive (BxPc3) or negative (Panc-1), then applied for in vitro and in vivo experiments. Transient experiments indicated that CEA and MUC1 promoter sequences confer specificity while preserving high transcriptional activities. The MePdR treatment induced a high in vitro cytotoxicity on the sole CEA- and MUC1-producing cell lines (i.e. BxPc3-CEA and -MUC1/ePNP). In the same way, prodrug treatment induced a significant tumor regression on the sole tumor-marker-positive BxPc3 xenografts, whilst the Panc1-CEA and -MUC1/ePNP tumors were not affected. These data confirm and extend the antitumor efficacy of the ePNP/MePdR killing system and demonstrate the feasibility of the transcriptional targeting strategy under tumor marker promoter control and thereby a preferential killing of CEA- and MUC1-producing pancreatic tumor cells. Thus, efficient in vivo gene delivery and transcriptional targeting constitute the major future clinical challenge for a selective pancreatic cancer suicide gene strategy.

A polymer library approach to suicide gene therapy for cancer

Optimal gene therapy for cancer must (i) deliver DNA to tumor cells with high efficiency, (ii) induce minimal toxicity, and (iii) avoid gene expression in healthy tissues. To this end, we generated a library of >500 degradable, poly(beta-amino esters) for potential use as nonviral DNA vectors. Using high-throughput methods, we screened this library in vitro for transfection efficiency and cytotoxicity. We tested the best performing polymer, C32, in mice for toxicity and DNA delivery after intratumor and i.m. injection. C32 delivered DNA intratumorally approximately 4-fold better than one of the best commercially available reagents, jetPEI (polyethyleneimine), and 26-fold better than naked DNA. Conversely, the highest transfection levels after i.m. administration were achieved with naked DNA, followed by polyethyleneimine; transfection was rarely observed with C32. Additionally, polyethyleneimine induced significant local toxicity after i.m. injection, whereas C32 demonstrated no toxicity. Finally, we used C32 to deliver a DNA construct encoding the A chain of diphtheria toxin (DT-A) to xenografts derived from LNCaP human prostate cancer cells. This construct regulates toxin expression both at the transcriptional level by the use of a chimeric-modified enhancer/promoter sequence of the human prostate-specific antigen gene and by DNA recombination mediated by Flp recombinase. C32 delivery of the A chain of diphtheria toxin DNA to LNCaP xenografts suppressed tumor growth and even caused 40% of tumors to regress in size. Because C32 transfects tumors locally at high levels, transfects healthy muscle poorly, and displays no toxicity, it may provide a vehicle for the local treatment of cancer.

Relationship between the expression of connexin43 and bystander effect of suicide gene therapy in ovarian cancer.

The relationship of connexin43 (Cx43) and bystander effect in ovarian tumor cells in herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in vitro was explored and the effect of all-trans retinoic acid (RA) on the expression of Cx43 and bystander effect investigated. The Cx43 expression was detected by flowcytometry, Western blot, and immunofluorescence in two ovarian tumor cell lines OVCAR3, CaOV3 before and after RA treatment. Bystander effect was determined by the cells growth inhibitory rate with methyl thiazolyl tetrazolium. Following exposure to ganciclovir, there was much greater bystander killing in OVCAR3 than that in CaOV3 (P<0.05). The expression of Cx43 was detected in OVCAR3 by flowcytometry and Western blot, but it could not be detected in CaOV3. The expression of Cx43 in both cell lines could be induced by RA. Immunofluoresence staining showed that Cx43 protein of OVCAR3 was located on membrane surface, whereas CaOV3 in cytoplasm. RA could not change the location of Cx43 protein in both cell lines. There is relationship between Cx43 expression and HSV-TK/GCV bystander effect. HSV-TK/GCV bystander effect can be enhanced by RA in ovarian cancer.

The use of gene therapy in cancer research and treatment.

Gene therapy involves identifying a gene of interest and then manipulating the expression of this gene through a variety of techniques. Here we specifically address gene therapy's role in cancer research. This paper will encompass thoroughly investigated techniques such as cancer vaccines and suicide gene therapy and the latest advancements in and applications of these techniques. It will also cover newer techniques such as Antisense Oligonucleotides and small interfering RNAs and how these technologies are being developed and used. The use of gene therapy continues to expand in cancer research and has an integral role in the advancement of cancer treatment.

Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference.

Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.

GENIS: gene expression of sodium iodide symporter for noninvasive imaging of gene therapy vectors and quantification of gene expression in vivo

With the goal of optimizing adenovirus-mediated suicide gene therapy for prostate cancer, we have developed a method based on the human sodium iodide symporter (hNIS) that allows for noninvasive monitoring of adenoviral vectors and quantification of gene expression magnitude and volume within the prostate. A replication-competent adenovirus (Ad5-yCD/mutTKSR39rep-hNIS) coexpressing a therapeutic yeast cytosine deaminase (yCD)/mutant herpes simplex virus thymidine kinase (mutTKSR39) fusion gene and the hNIS gene was developed. Ad5-yCD/mutTKSR39rep-hNIS and a replication-defective hNIS adenovirus (rAd-CMV-FLhNIS) were injected into contralateral lobes of the dog prostate and hNIS activity was monitored in live animals following administration of Na99mTcO4 using gamma camera scintigraphy. Despite the close proximity of the urinary bladder, 99mTcO4− uptake was readily detected in the prostate using viral dose levels (1010 to 1012 viral particles) that have been safely administered to humans. Due to its rapid clearance and short physical half-life (6 h), it was possible to obtain daily measurements of 99mTcO4− uptake in vivo, allowing for dynamic monitoring of reporter gene expression within the prostate as well as biodistribution throughout the body. High-resolution autoradiography of prostate sections coupled with 3D reconstruction of gene expression demonstrated that the magnitude and volume of gene expression could be quantified with submillimeter resolution. Implementation of the GENIS (gene expression of Na/I symporter) technology in the clinic will facilitate optimization of future human gene therapy trials.

739. Mammary Gland-Specific Promoters for Targeted Breast Cancer Suicide Gene Therapy with Retroviral Vectors

739. Mammary Gland-Specific Promoters for Targeted Breast Cancer Suicide Gene Therapy with Retroviral Vectors

Adenovirus-mediated gene transfer of enhanced Herpes simplex virus thymidine kinase mutants improves prodrug-mediated tumor cell killing.

The Herpes simplex virus 1 (HSV) thymidine kinase (tk) suicide gene together with ganciclovir (GCV) have been successfully used for the in vivo treatment of various solid tumors and for the ablation of unwanted transfused stem cells in recent clinical trials. With the aim of improving this therapeutic system, we compared the potential efficacy of adenoviral (Ad) vectors expressing enhanced tk mutants in vitro and in vivo. The previously created HSV-tk mutants dm30 and sr39, created by random sequence mutagenesis, were inserted into a standard Ad.RSV E1(-)E3(-) backbone using homologous recombination. GCV killing of Ad.HSV-tk, Ad.dm30-tk and Ad.sr39-tk was assessed in various tumor cell lines with a cell proliferation assay. Cells expressing the two TK mutants were two-to-five-fold more sensitive to GCV when compared with Ad.HSV-tk transduced cells in all cell lines tested (five human mesotheliomas, one human lung cancer, a human cervical carcinoma, a mouse fibrosarcoma, and a rat glioma line) at equal TK expression levels. Flank tumor models, including cell-mixing studies, assessed the in vivo efficacy of the engineered viruses in BALB/C and SCID mice. In all animal studies, Ad.dm30-tk and Ad.sr39-tk showed more tumor growth inhibition than Ad.HSV-tk when GCV was administered. The use of adenovirus-mediated gene transfer of both tk mutants dm30-tk and sr39-tk for cancer suicide gene therapy should provide a more effective and safer alternative to wild-type HSV-tk.