Gene therapy for cancer: what have we done and where are we going?

Gene-based therapies for cancer in clinical trials include strategies that involve augmentation of immunotherapeutic and chemotherapeutic approaches. These strategies include ex vivo and in vivo cytokine gene transfer, drug sensitization with genes for prodrug delivery, and the use of drug-resistance genes for bone marrow protection from high-dose chemotherapy. Inactivation of oncogene expression and gene replacement for tumor suppressor genes are among the strategies for targeting the underlying genetic lesions in the cancer cell. A review of clinical trial results to date, primarily in patients with very advanced cancers refractory to conventional treatments, indicates that these treatments can mediate tumor regression with acceptably low toxicity. Vector development remains a critical area for future research. Important areas for future research include modifying viral vectors to reduce toxicity and immunogenicity, increasing the transduction efficiency of nonviral vectors, enhancing vector targeting and specificity, regulating gene expression, and identifying synergies between gene-based agents and other cancer therapeutics.

An agent that increases tumor suppressor transgene product coupled with systemic transgene delivery inhibits growth of metastatic lung cancer in vivo.

Low levels of gene expression following systemic delivery have impaired the effectiveness of tumor suppressor gene replacement in treating metastases. We asked whether combined treatment with 2-methoxyestradiol (2-Me), which increases levels of wild-type p53 protein in cancer cells, and the systemic administration of an adenoviral vector expressing wild-type p53 (Ad-p53) would inhibit the growth of human metastatic lung cancer cells in vivo. The simultaneous administration of p53 and 2-Me resulted in a greater than additive reduction with the lung colony count reduced to 33% of its control value. These results suggest that the synergistic effect of 2-Me and Ad-p53 in combination treatment may have application in the systemic treatment of cancer.

Autocrine interleukin-1beta production in leukemia: evidence for the involvement of mutated RAS.

Interleukin (IL)-1beta is constitutively expressed in many leukemias and operates as an autocrine growth factor. To study the cellular basis for this aberrant production, we analyzed two cell lines, B1 (acute lymphoblastic leukemia) and W1 (juvenile chronic myelogenous leukemia), which express high levels of IL-1beta and have mutations in the K-RAS and N-RAS genes, respectively. Electromobility shift assays demonstrated transcription factor binding at multiple IL-1beta promoter elements [nuclear factor (NF)-IL6/CREB, NFB1, NFkappaB, and NF-IL6], consistent with the activation of an upstream signaling pathway. To determine whether activated Ras was involved, two structurally distinct classes of farnesyltransferase (FTase) inhibitors (the monoterpenes and a peptidomimetic) and an adenoviral vector expressing antisense targeted to K-RAS were used to specifically interfere with Ras function and/or expression. Treatment with the FTase inhibitors resulted in a concentration-dependent decrease in both NF-IL6/CREB binding to the IL-1beta promoter and IL-1beta protein levels, without a significant change in total cellular protein levels. Furthermore, exposure of the B1 cells to antisense against K-RAS resulted in an approximately 50% reduction in both p21Ras and IL-1beta protein levels. Growth suppression was observed after FTase inhibitor or antisense exposure, an effect that was partially reversible by the addition of recombinant IL-1beta to the cultures. Our observations suggest that mutated RAS genes may mediate autocrine IL-1beta production in some leukemias by stimulating signal transduction pathways that activate the IL-1beta promoter.

Down-regulation of bcl-2 is associated with p16INK4-mediated apoptosis in non-small cell lung cancer cells.

We introduced a functional p16 cDNA into non-small cell lung cancer (NSCLC) cell lines expressing different combinations of normal and mutated p16, p53, and Rb genes via a recombinant adenovirus to determine the effect of exogenous p16 expression on cell growth. Analysis of p16-deficient cells infected with Adv/p16 identified growth arrest of the cells in the G0 - G1 phase early on. Apoptosis was identified to occur by the 5th day after infection which corresponded with increased p16 expression, reduced Rb expression, and increased Rb hypophosphorylation, but only occurred in cells expressing functional p53. Further analysis indicated that the expression of the anti-apoptotic protein bcl-2 was greatly reduced in the NSCLC cell lines H460 and A549 (both -p16, +p53, +Rb), again only by the 5th day after Adv/p16 infection, but no affect on Bax expression was observed. H1299 cells (-p16, -p53, +Rb) infected with Adv/p16 only exhibited apoptosis by an additional infection with Adv/p53 which also corresponded with a down-regulation of bcl-2. In addition, the infection of A549 cells with Adv/p16 followed by a subsequent infection with Adv/Rb lead to a significant decrease in apoptosis which correlated with an increase in bcl-2 expression. These studies suggest that p16 is capable of mediating apoptosis in NSCLC cell lines expressing wild-type p53, through a direct down-regulation of Rb and an indirect down-regulation of the anti-apoptotic protein bcl-2.

Insulin-like growth factor binding protein-6 activates programmed cell death in non-small cell lung cancer cells.

Insulin-like growth factor binding proteins (IGFBPs) are secreted into the extra-cellular matrix and inhibit cell growth through IGF-dependent and -independent mechanisms. In this study, we investigated the role of IGFBP-6, a relatively unexplored member of the IGFBP family, in the proliferation of non-small cell lung cancer (NSCLC) cells. Infection of NSCLC cell lines in vitro with an adenovirus expressing human IGFBP-6 under the control of a CMV promoter (Ad5CMV-BP6) reduced NSCLC cell number through activation of programmed cell death, as shown by cell staining with Hoechst 33342 or DNA end-labeling with bromodeoxyuridine triphosphate. The growth regulatory effect of IGFBP-6 was investigated in vivo by intratumoral injection of Ad5CMV-BP6 in NSCLC xenografts established in nu/nu mice. A single injection of Ad5CMV-BP6 reduced the size of NSCLC xenografts by 45%. These findings indicate that IGFBP-6 is a potent inducer of programmed cell death in cancer cells and support investigations into IGFBP-6 as a potential target in cancer therapeutics.

Up-regulation of the proapoptotic mediators Bax and Bak after adenovirus-mediated p53 gene transfer in lung cancer cells.

Overexpression of wild-type p53 in cancer cells by adenovirus-mediated p53 gene transfer can result in the induction of apoptosis. To identify the potential mediators of this p53-induced apoptosis, we examined apoptotic protein levels in human lung cancer cells after Adp53 gene transfer. We observed up-regulation of Bax and Bak protein levels 18-36 h after transduction with Adp53 in H1299, H358, and H322 lung cancer cells. Contrary to expected observations, no changes in Bcl-2 and Bcl-X(L) protein levels were observed. Morphological cell changes and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining showed evidence of apoptosis in all cell lines 48 h after transduction with Adp53. These results indicate that the induction of apoptosis by adenovirus-mediated p53 transfer may be mediated by the induction of proapoptotic mechanisms rather than suppression of antiapoptotic mechanisms.

Adenoviral-mediated wild-type p53 gene expression sensitizes colorectal cancer cells to ionizing radiation.

Wild-type p53 gene transfer into the SW620 colorectal carcinoma cell line was performed using the replication-defective adenovirus Ad5/CMV/p53 to evaluate the effect of wild-type p53 expression on radiation sensitivity. The results indicated that infection with Ad5/CMV/p53 sensitized the cells. The survival at 2 Gy was reduced from 55 to 23%. Flow cytometric analysis of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay-labeled cells and in situ TUNEL staining of xenograft tumors demonstrated an increase in labeled cells with combination treatment, indicating increased apoptosis in cells treated with Ad5/CMV/p53 before irradiation. A significant enhancement of tumor growth suppression by this combination strategy was observed in a s. c. tumor animal model compared to p53 gene therapy alone. The delay in regrowth to control tumor size of 1000 mm3 was 2 days for 5 Gy, 15 days for Ad5/CMV/p53, and 37 days for Ad5/CMV/p53 + 5 Gy, indicating synergistic interactions. These data indicate that the delivery of wild-type p53 to cells with p53 mutations increases their radiation sensitivity, and this may be accomplished by adenoviral-mediated gene therapy.

Epidermal growth factor mediated DNA delivery into lung cancer cells via the epidermal growth factor receptor.

We have developed a targeted DNA delivery system for the treatment of lung cancer by gene therapy. This system uses epidermal growth factor (EGF), which has receptors that are overexpressed on lung cancer cells, and couples receptor-mediated endocytosis with an endosomal lysis agent for targeted gene delivery. Recombinant human EGF was chemically modified to allow for its attachment to DNA through the use of the poly-cation poly-L-lysine (PLL). The EGF/PLL conjugate was able to bind DNA and when incubated with several different lung cancer cell lines, high levels of gene expression resulted when uptake was performed in the presence of replication-defective adenovirus, which was used only as an endosomal lysis agent. When the adenovirus was coupled directly to the EGF/DNA complex, the levels of gene expression were up to fourfold higher. Depending on the lung cancer cell line and the type of complex used, approximately 14% to 99% of the cells showed DNA uptake, whereas a colon adenocarcinoma cell line that does not express the EGF receptor at the cell surface showed little or no DNA uptake. As a result, the EGF/DNA complex can deliver therapeutic genes to lung cancer cells by targeting to specific receptors.

Gene delivery by an epidermal growth factor/DNA polyplex to small cell lung cancer cell lines expressing low levels of epidermal growth factor receptor.

In the present study, we wanted to determine whether efficient gene delivery using an epidermal growth factor (EGF)/DNA polyplex could be accomplished in small cell lung cancer (SCLC) cell lines expressing low EGF receptor (EGFR) levels. EGFR expression levels and transduction efficiencies with polyplexes were examined in five SCLC cell lines and two controls. EGFR expression was examined by binding assays and demonstrated low EGFR levels ranging from 3.6 to 87.4 fmol/mg protein. The SCLC cell lines exhibited high sensitivity to adenovirus infection, which was an important determinant for transduction efficiency when adenovirus was used as an endosomolytic agent. The transduction efficiencies with EGF/DNA polyplexes ranged from 41% +/- 3.5% to 73% +/- 4.6% in the EGFR-positive SCLC cell lines. In the controls lacking EGFRs, only 5% +/- 1.0% and 8% +/- 1.8% of the cells were transduced. Furthermore, the transduction efficiency could be reduced from 50% +/- 4.9% to 18% +/- 1.1% when excess EGF was added to compete with the EGF/DNA polyplexes. In the present study, receptor-targeted gene delivery to SCLC cell lines has been demonstrated for the first time. Our results indicate that even low receptor expression levels in the target cells are sufficient for efficient and specific in vitro gene delivery with EGF/DNA polyplexes.

Gene delivery into malignant cells in vivo by a conjugated adenovirus/DNA complex.

Current viral delivery systems suffer from disadvantages that may limit the rate at which therapeutic gene expressing constructs can be tested both in vitro and in vivo. In this study, our focus was to develop a simple gene delivery system for the rapid and reproducible testing of therapeutic genes in cancer cells both in vitro and in vivo. We report here that a delivery system based on using a conjugated adenovirus in complex from with a DNA plasmid can be used for not only delivering genes in vitro but also for efficient and reproducible delivery in vivo. Replication defective adenoviral particles were chemically modified by covalent attachment of poly-L-lysine (PLL) to the viral capsid, allowing for direct interaction with DNA. The adenovirus/PLL conjugate (Adv/PLL) was used to deliver the plasmid pCMV/beta-gal to several different cancer cell lines (i.e., lung, cervical) in vitro and resulted in transduction efficiencies as high as 52% as determined by histochemical staining. On direct intralesional injection of the Adv/PLL/DNA complex into subcutaneous tumors, transduction efficiencies greater than 35% could also be achieved. As a result, this system provides a simple method for delivering and testing therapeutic genes in cells both in vitro and in vivo, prior to the further development of gene therapy vectors for both malignant and benign disease.

High levels of gene transduction in human lung tumors following intralesional injection of recombinant adenovirus.

To effect gene transfer into large solid malignancies for the purpose of clinical application, new treatment strategies using intralesional administration of adenovirus were studied. Replication-deficient adenovirus Ad5LacZ, containing the Escherichia coli beta-galactosidase (beta-gal) gene (LacZ), was injected directly into 1-cm x 1-cm subcutaneous xenograph tumors of human large cell lung cancers (H460 and H1299). Each tumor received a single injection or three injections of purified virus, diluted in 200 microL of phosphate-buffered saline. The tumors were harvested 3 days after the last injection, serially sectioned, and stained with X-gal. The cells expressing beta-gal were counted by using digital image analysis and the percentage of tumor cells transduced was calculated. After a single viral injection of 1 x 10(9) PFU, 5 x 10(9) PFU, or 1 x 10(10) PFU solid tumor transduction increased significantly with dose escalation. At a dose of 1 x 10(10) PFU, transduction of the H1299 and H460 tumors was 80.2% and 46.7%, respectively. Dividing the viral dose into three injections given on alternating days had no significant effect on viral transduction. These data demonstrate that a large portion of an established human lung cancer cell line tumor undergoes gene transduction after a single intralesional injection of recombinant adenovirus.

Growth inhibitory effect of anti-K-ras adenovirus on lung cancer cells.

An adenoviral vector carrying a 2-Kb fragment of the K-ras proto-oncogene inserted in antisense orientation with respect to the cytomegalovirus promoter was constructed and used to infect H460a lung cancer cells (codon 61 K-ras mutation). The gene was efficiently transferred, and a high level of expression of antisense K-ras was achieved. At a multiplicity of infection to achieve 65% transduction of cells, the expression of K-ras protein was reduced by 70% in the lung cancer cell line H460a as compared with cells infected with control vectors or noninfected cells. This reduction produced a 47% inhibition of monolayer growth and a 90% inhibition of colony formation. At a similar level of transduction in the cell line H358 (codon 12 K-ras mutation), a 59% inhibition of monolayer growth compared with control vectors occurred; however the inhibition of H322 cells (wild-type k-ras) growth was no different than control vector infected cells. These data suggest that the adenoviral K-ras H322a antisense vector may have therapeutic potential in tumors in which K-ras is mutated.

Delivery of the p53 tumor suppressor gene into lung cancer cells by an adenovirus/DNA complex.

An adenovirus/DNA complex was constructed by chemically linking poly-L-lysine to the capsid of the replication-defective adenovirus dl312, allowing for coupling with plasmid DNA by an ionic interaction. We have previously demonstrated that this adenovirus/DNA complex can efficiently transduce malignant cells with a plasmid expressing the beta-galactosidase gene both in vitro and in vivo. In this report, we show that this system can deliver a therapeutic gene that encodes for the tumor suppressor protein p53 to lung cancer cells, both in vitro and in vivo, leading to significant biological effects. Transfection of the p53-negative human lung cancer cell line H1299 with the adenovirus/DNA complex carrying a plasmid expressing the p53 gene resulted in high levels of p53 protein and induction of apoptosis. Injection of the complex carrying the p53 gene to subcutaneous tumor sites 5 days after tumor cell implantation resulted in a significant inhibition of tumorigenicity as measured by the number and size of tumors that developed 21 days after treatment. Three and six injections of the complex carrying the p53 gene into H1299 subcutaneous tumor nodules led to significant dose-related tumor growth suppression 18 days after the first injection compared with control-treated tumors. This adenovirus/DNA complex, therefore, is capable of efficiently delivering the p53 gene into malignant cells in vitro and in vivo and now provides a general gene delivery vector that is simple to construct and capable of testing therapeutic genes in malignant cells.

Cellular and humoral immune responses to adenovirus and p53 protein antigens in patients following intratumoral injection of an adenovirus vector expressing wild-type. P53 (Ad-p53).

The immune responses of 10 patients with advanced non-small cell lung cancer receiving monthly intratumoral injections of a recombinant adenovirus containing human wild-type p53 (Ad-p53) to adenovirus and transgene antigens were studied. The predominate cellular and humoral immune responses as measured by lymphocyte proliferation and neutralizing antibody (Ab) formation were to adenovirus serotype 5 vector antigens, with increased responses in posttreatment samples. Consistent alterations in posttreatment cellular and humoral immune responses to p53 epitopes were not observed, and cytotoxic Abs to human lung cancer cells were not generated. Patients in this study had evidence of an antitumoral effect of this treatment with prolonged tumor stability or regression; however, neither Abs to p53 protein nor increased lymphocyte proliferative responses to wild-type or mutant p53 peptides have been consistently detected.

Targeted, non-viral gene delivery for cancer gene therapy.

The ability to mediate targeted and specific delivery of therapeutics to cancer cells remains one of the most important hurdles in effectively treating cancer. This aspect also remains as one of the greatest limitations of gene therapy as well. Targeted vectors based on the use of DNA-binding agents attached to cell specific ligands or "molecular conjugates" were created with the goal of over-coming this hurdle. Since being conceived, many different ligands have been utilized as molecular conjugates, targeting the resulting Protein/DNA polyplex to cells efficiently in vitro while mediating limited delivery in vivo. This limited delivery is due to many reasons such as the need to identify non-viral agents that can aide in escaping endosome entrapment as well as decreasing the complexity that has evolved in the creation of these "synthetic viruses". This review will discuss the current status and the future of molecular conjugates as targeting vectors as well as the positive and negative attributes of this vector in relation to other viral and non-viral vectors that are currently used in many gene therapy strategies.

Gene therapy for lung cancer: enhancement of tumor suppression by a combination of sequential systemic cisplatin and adenovirus-mediated p53 gene transfer.

A more effective gene therapy strategy for lung cancer using sequential cisplatin administration and adenovirus-mediated p53 gene transfer was developed on the basis of our previous observation of enhanced expression of a reporter gene in malignant cells exposed to cisplatin before gene transfer. Transfer of the normal (wildtype) p53 gene into cisplatin-treated H1299 cells, in which p53 is homozygously deleted, resulted in up to a 60% further inhibition of cell proliferation in vitro than p53 transfer into untreated H1299 cells. The cisplatin plus p53 gene transfer strategy yielded significantly greater apoptosis and tumor growth suppression in an animal model of subcutaneous H1299 tumor nodules than wildtype p53 gene transfer alone. The timing of cisplatin administration and p53 gene transfer was shown to be critical: cisplatin administration simultaneous with or subsequent to p53 gene transfer was less effective than cisplatin-first sequential treatment. Moreover, the in vivo inhibition of tumor growth was maintained by repeated cycles of treatment. This gene therapy strategy has been incorporated into a phase I clinical trial for the treatment of lung cancer and provides a basis for the development of improved therapeutic protocols.

Viral and non-viral vectors for cancer gene therapy.

BACKGROUND: Our research has focused on developing improved delivery vectors for treating cancer by gene therapy using the tumor suppressor p53 gene. MATERIALS AND METHODS: Recombinant viral and non-viral vectors were used to deliver the p53 gene into non-small cell lung cancer (NSCLC) cells either in culture or as a subcutaneous tumor. Transduction of tumor cells was measured by beta-gal expression while tumor cell proliferation was used to measure the effect of p53. RESULTS: High level transduction was obtained in vitro and in vivo with a recombinant adenoviral vector, resulting in tumor cell growth inhibition in both models. A targeted, non-viral gene delivery vector based on the use of an EGF/DNA polyplex also resulted in efficient (as high as 66% transduction) and specific gene delivery in vitro when replication defective adenovirus was used as an endosome release agent. CONCLUSION: These vectors now provide improved methods to deliver therapeutic genes for cancer treatment by gene therapy.

In vivo adenovirus-mediated p53 tumor suppressor gene therapy for colorectal cancer.

BACKGROUND: The p53 tumor suppressor gene is altered in up to 70% of colorectal cancers. MATERIALS AND METHODS: We infected the colorectal cancer cell lines SW620 and KM12L4, in which p53 is mutated, with the replication-defective adenovirus Ad5/CMV/p53 to evaluate the effects of adenovirus-mediated wild-type p53 gene transfer. Gene transduction was measured by cytochemical staining of cells infected with the Ad5/CMV/beta-gal virus and expression of the wildtype p53 protein in these cells was demonstrated by immunoblotting. RESULTS: Significant suppression of in vitro cell proliferation and induction of apoptosis (as measured by TUNEL assay labeling) were observed following Ad5/CMV/p53 infection. More importantly, similar effects were observed in vivo in an established nude mouse subcutaneous tumor model; significant suppression of tumor growth (60%-70%) and induction of apoptosis were observed following intratumoral injections of Ad5/CMV/p53. CONCLUSION: This form of therapy may provide a novel approach to colorectal cancer.