Bystander-mediated regression of osteosarcoma via retroviral transfer of the herpes simplex virus thymidine kinase and human interleukin-2 genes.

Current treatment of osteosarcoma produces disappointing outcomes, and innovative therapies must be investigated. We have used retroviral vectors to transfer the herpes simplex virus thymidine kinase (HSVtk) and interleukin-2 genes to human osteosarcoma cells. Each gene was stably transduced and expressed; the HSVtk gene effectively conferred ganciclovir (GCV) susceptibility to transduced cells. A strong bystander effect was observed in vitro, whereby nontransduced tumor cells in proximity to transduced cells acquired susceptibility to GCV killing. Human osteosarcoma cells were used to develop a series of experiments in athymic nude mice to treat experimental osteosarcoma. Subcutaneously implanted mixtures of tumor cells and HSVtk vector producer cells developed into tumors that completely regressed upon administration of GCV. Subcutaneously implanted mixtures of transduced and wild-type cells showed a potent bystander effect upon administration of GCV, with complete tumor ablation when as little as 10% of the cells were HSVtk+. A significant (P < .05) antitumoral response was seen against primary tumors composed of unmodified cells when a secondary tumor of transduced cells was implanted at a distance of 1 cm, suggesting a diffusible bystander factor. The presence of interleukin-2-transduced cells improved the efficacy of treatment. A significant (P < .03) antitumoral response was seen in the treatment of established osteosarcomas by the injection of HSVtk vector producer cells.

Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides.

A sequence-specific genomic delivery system for the correction of chromosomal mutations was designed by incorporating two different binding domains into a single-stranded oligonucleotide. A repair domain (RD) contained the native sequence of the target region. A third strand-forming domain (TFD) was designed to form a triplex by Hoogsteen interactions. The design was based upon the premise that the RD will rapidly form a heteroduplex that is anchored synergistically by the TFD. Deoxyoligonucleotides were designed to form triplexes in the human adenosine deaminase (ADA) and p53 genes adjacent to known point mutations. Transfection of ADA-deficient human lymphocytes corrected the mutant sequence in 1-2% of cells. Neither the RD or TFD individually corrected the mutation. Transfection of p53 mutant human glioblastoma cells corrected the mutation and induced apoptosis in 7.5% of cells.

Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells.

Intratumoral implantation of murine cells modified to produce retroviral vectors containing the herpes simplex virus-thymidine kinase (HSV-TK) gene induces regression of experimental brain tumors in rodents after ganciclovir (GCV) administration. We evaluated this approach in 15 patients with progressive growth of recurrent malignant brain tumors. Antitumor activity was detected in five of the smaller tumors (1.4 +/- 0.5 ml). In situ hybridization for HSV-TK demonstrated survival of vector-producing cells (VPCs) at 7 days but indicated limited gene transfer to tumors, suggesting that indirect, "bystander," mechanisms provide local antitumor activity in human tumors. However, the response of only very small tumors in which a high density of vector-producing cells had been placed suggests that techniques to improve delivery and distribution of the therapeutic gene will need to be developed if clinical utility is to be achieved with this approach.

Gene therapy for malignant neoplasms of the CNS.

Five different gene transfer protocols have progressed into human clinical trials for the treatment of brain tumors. Two utilize the in vivo transfer of the Herpes Simplex-thymidine kinase (HS-tk) gene by either retroviral or adenoviral gene transfer. HS-tk confers a sensitivity to the anti-herpes drug ganciclovir (GCV). Insertion of HS-tk into tumors and subsequent treatment with GCV has successfully eliminated tumors in experimental animal models despite less than a 100% gene transfer efficiency. This phenomenon, the 'bystander effect', allows the destruction of neighboring tumor cells not transduced with HS-tk. Two other approaches use ex vivo gene transfer of either the IL-2 or antisense insulin-like growth factor type 1 (IGF-1) genes into autologous tumor cells. In animal models, tumor cells genetically altered with antisense IGF-1 or IL-2 genes induce a potent cell-mediated antitumor response. The fifth approach uses the genetic modification of hematopoietic stem cells instead of tumor cells. In this approach, the multiple drug resistance (MDR-1) gene is transferred into stem cells to protect them from the toxic effects of certain chemotherapy drugs. This may allow the administration of higher doses without increasing bone marrow toxicity. Together, these clinical trials will provide critical information needed to develop improved gene transfer technologies for humans and to attain clinical benefit for cancer patients.

Measuring success in clinical gene therapy research.

Medical science is a compelling career choice, filled with the thrill of discovery, joy of learning and a meaningful purpose to lessen human suffering. These benefits and rewards of laboratory and clinical research accumulate in an asynchronous, irregular, and incremental mechanism, euphemistically known as the scientific method. Ultimate success in clinical research is the elusive 'cure'. But in progress towards that goal, success is also measured first as the 'absence of doing harm', and then by various stages of efficacy. Perhaps only in the case of smallpox has medicine achieved total 'victory'; it is now exactly 200 years since Jenner's first clinical trial.

Molecular analysis of T lymphocyte-directed gene therapy for adenosine deaminase deficiency: long-term expression in vivo of genes introduced with a retroviral vector.

Peripheral blood lymphocytes from a patient with adenosine deaminase (ADA) deficiency were transduced in vitro with a replication-defective retroviral vector containing a human ADA-cDNA. Eighteen months after the last of a series of infusions of autologous retroviral vector-treated cells, vector sequences were detectable in DNA isolated from peripheral blood mononuclear cells (PBMCs), with an average copy number approaching one per cell. Increased ADA enzyme activity reaching approximately one-quarter normal levels was found in this population of cells. Other evidence of long-term retroviral vector expression in vivo included neomycin phosphotransferase (NPT) activity and demonstration of persistent vector mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). No evidence of spontaneous reversion of either mutant endogenous ADA allele was found.

Gene therapy: socioeconomic and ethical issues. A roundtable discussion.

Gene therapy research has the potential to revolutionize the way in which many human diseases are treated. Despite its enormous potential, roundtable panelists concluded that the field needs time to mature scientifically without pressure to develop a marketable therapeutic product. In addition, health care decision makers, physicians, and the lay public need to be educated on the future medical, economic, and ethical ramifications of gene therapy.

T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Gene therapy for solid tumors.

Advances in molecular biology have proven that there is a genetic basis to the process of carcinogenesis that allows for the consideration of entirely new approaches to the treatment of cancer. The development of an ability to selectively destroy cancer cells through the manipulation of DNA may provide the opportunity to dramatically improve the quality of care and treatment of cancer patients by decreasing systemic toxicities and enhancing efficacy. These new therapies may occur through the restoration of genetic health, such as the insertion of normal tumor suppressor genes or via down-regulation of oncogene or growth factor receptor expression. Other possibilities include the targeting of genetic alterations in tumor cells that will enhance tumor immunogenicity or induce a specific sensitivity to a prodrug. In this chapter, we have reviewed the current status of gene therapy for solid tumors in the United States and evolving new approaches for this emerging clinical discipline.

Survival and toxicity of xenogeneic murine retroviral vector producer cells in liver.

Murine retroviral vector producer cells (VPC) can selectively transfer genes stably into proliferating cells. We injected LacZ gene producing VPC directly into normal rat liver. There was no measurable gene transfer into the surrounding hepatic parenchyma with X-GAL staining. Rejection of the xenogeneic murine VPC occurred 7-14 days after injection. Toxicity of this delivery method was evaluated with the herpes simplex-thymidine kinase (HS-tk) gene, which confers sensitivity to the antiherpes drug, ganciclovir (GCV). HS-tk VPC were injected and allowed to grow in normal liver for 7 days before GCV treatment. There was no clinical or histologic evidence of toxicity with GCV treatment. These findings suggest that the direct injection of murine VPC into xenogeneic human tumors may survive sufficiently long without immunosuppression to transfer genes to tumor cells in situ without attendant toxicity.

The effect of thymidine kinase transduction and ganciclovir therapy on tumor vasculature and growth of 9L gliomas in rats.

Eradication of malignant brain tumors by in situ intratumoral, retrovirally mediated transfer of the herpes simplex virus thymidine kinase (HSVtk) gene, which sensitizes the tumor cells to ganciclovir, has recently been demonstrated in animal models. The observation that tumors studied in vitro and in animals can be completely eliminated despite only partial transduction of the tumor suggests a bystander mechanism that affects nontransduced tumor cells. Such a bystander effect is not completely understood and may represent a combination of several factors that lead to tumor eradication. Endothelial cells of the tumor blood vessels were shown to occasionally integrate the retroviral vector and thus become sensitized to ganciclovir. In the presence of vector-producer cells, which continuously release infectious viral particles, diffuse multifocal hemorrhages occurred during ganciclovir administration. When the tumor was composed of cells that had been transduced with the thymidine kinase gene before inoculation, no infectious viral particles were present within the tumor, no transduction of endothelial cells occurred, and no hemorrhages were observed during ganciclovir therapy. These observations suggest that tumor regression may be due, in part, to destruction of in vivo HSVtk-transduced endothelial cells after exposure to ganciclovir, resulting in tumor ischemia as one possible bystander mechanism. The authors investigated this hypothesis using the subcutaneous 9L gliosarcoma tumor model in Fischer rats. The tumors were evaluated with Doppler color-flow and ultrasound imaging during the various phases of the study. Twenty rats received intratumoral injections of HSVtk retroviral vector-producer cells (6 x 10(7) cells/ml) 21 days after bilateral flank tumor inoculation. Ten rats were subsequently treated with intraperitoneal ganciclovir (15 mg/kg/ml twice a day) for 14 days starting on Day 7 after producer cell injection; 10 control rats received intraperitoneal saline injections (1 ml twice a day) instead of ganciclovir. Ultrasound and flow images were obtained before cell injection, before and during ganciclovir or saline administration, and after cessation of treatment. The number, location, and ultrasonographic appearance of tumor vessels and the tumor volumes were recorded. The number of blood vessels in the tumors increased over time in both groups before treatment. Intratumoral cell injection without ganciclovir administration did not influence tumor growth or intratumoral vasculature. However, tumor vasculature decreased after initiation of ganciclovir therapy in the HSVtk-transduced tumors (p < 0.05). Early patchy or diffuse necrotic changes associated with ultrasonographic evidence of scattered intratumoral hemorrhage occurred in tumors treated with ganciclovir.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene therapy for cancer.

Initiation of clinical trials of gene therapies for cancer has been made possible by two major technological advances: the ability to clone genes that constitute the genetic basis of carcinogenesis or that have therapeutic potential, and the development of an increasing number of gene transfer methods. As a result, 30 experimental trials of gene therapy for the treatment of human cancer have been approved in the United States of America. Here, we discuss the current status of gene therapy for cancer together with future directions for its development.

Clinical applications of gene therapy for cancer.

The number of gene therapy protocols for the treatment of cancer is growing rapidly. The most common type of approved clinical trial for cancer gene therapy involves the ex vivo gene transfer of cytokine genes (e.g., tumor necrosis factor, interleukin-2, granulocyte-macrophage colony-stimulating factor) into tumor cells. The idea behind this approach is to use gene transfer to induce a patient's tumor to become more immunogenic. The genetically altered tumor cells are reinjected into the patient in an effort to induce a systemic antitumor immune response against residual tumor cells. In other trials, investigators are using in situ gene transfer to selectively destroy cancer cells, sparing normal tissues. Continuing advances in molecular biology are likely to allow the development of new cancer treatments and methods of cancer prevention that will redefine cancer therapy.

Gene therapy for the treatment of malignant brain tumors with in vivo tumor transduction with the herpes simplex thymidine kinase gene/ganciclovir system.

Murine retroviral vectors can infect a wide variety of proliferating mammalian cell types (e.g. lymphocytes). Non-proliferating tissues (e.g. neurons) are not transduced by murine retroviral vectors. These findings suggest that this type of vector may be useful for the selective introduction of genes into growing tumors in the brain, since the tumor is essentially the only tissue that will integrate and express the vector genes.

In vivo transfer of the human interleukin-2 gene: negative tumoricidal results in experimental brain tumors.

The authors have recently shown the feasibility of eradicating brain tumors using in vivo retroviral-mediated transduction of tumors with the herpes simplex thymidine kinase (HStk) gene and ganciclovir therapy. However, thymidine kinase-transduced subcutaneous tumors in immunocompromised (athymic) mice were less responsive to this therapy than in immunocompetent animals, suggesting a role of the immune system in the process of tumor eradication. Broad suppression of humoral and cell-mediated immunity is found in patients with malignant gliomas. Interleukin-2 (IL-2) production and IL-2 receptor expression are decreased in gliomas patients. These findings and the proposed association between lymphocytic infiltration of brain tumors and survival suggest that immune response modifiers may be useful in treating glioma patients. To evaluate the role of local cytokine expression by tumor cells, alone or combined with HStk gene transfer and ganciclovir therapy, the authors investigated the efficacy of tumor (9L gliosarcoma) eradication in Fischer rats by in vitro and in vivo tumor transduction with the IL-2 gene alone or with a combined vector carrying both the HStk and IL-2 genes. Tumors injected with HStk vector-producer cells alone, with or without ganciclovir, and rats inoculated in the brain and subcutaneously with 9L cells that had previously been transduced in vitro served as controls. Murine vector-producer cells (3 x 10(6)/50 microliters) were injected into the brain tumors 7 days after tumor inoculation. Ganciclovir (15 mg/kg) was administered intraperitoneally twice daily for 10 days to animals that received HStk with or without IL-2 vector-producer cells, starting 5 days after producer-cell injection. The experiment was repeated with continuous daily treatment of all rats with oral dexamethasone (0.5 mg/kg). Rats were sacrificed 21 days after tumor inoculation, and the brains were removed for histological and immunohistochemical analysis for IL-2. Within each experimental group, tumors were found in a similar proportion in the dexamethasone-treated and untreated rats. Large brain tumors developed in all 10 rats that had been inoculated with 9L cells which had been pretransduced in vitro with the IL-2 gene, whereas only three of eight rats receiving subcutaneous inoculation of similar cells developed palpable tumors. No enhancement of tumor eradication was observed by adding the IL-2 gene in the HStk vector construct compared to the use of the vector with HStk alone. Lymphocytic infiltration was absent in all dexamethasone-treated rats but was observed in all treatment groups not receiving steroids.(ABSTRACT TRUNCATED AT 400 WORDS)