Tissue-specific Gene Therapy Research Articles

Identification of tissue- and cancer-selective promoters for the introduction of genes into human ovarian cancer cells.

One potential limitation of gene therapy for epithelial tumors is the lack of tissue or tumor specificity of treatment. Tumor-selective expression of gene therapies may avoid deleterious side effects and improve the efficacy of the treatment. The aim of this study was to evaluate the tissue and tumor specificity of four different potential gene therapy promoters, to determine their usefulness in tissue-specific gene therapy of epithelial ovarian carcinomas. Three potential epithelial cell-selective (hESE1, SLP1, OSP1) and one potential tumor-selective (hTERT) promoter were placed upstream of a luciferase construct to determine relative activity in a wide variety of normal and malignant cell lines. Transient transfection and luciferase assays were carried out in 12 epithelial ovarian (3 SV40 T antigen-transfected normal and 9 malignant) and 8 control cell lines. Luciferase assays revealed that the hTERT promoter presented the highest tumor selectivity. hESE1 and SLP1 promoters showed strong epithelial cell selectivity (hESE1, 16/17; SLP1, 15/17), with the OSP1 (11/17) promoter exhibiting lower epithelial selectivity. Of the potential promoters for gene therapy, hTERT promoter exhibited the strongest transcriptional activity in most of the tumor cell lines. None of the promoters exhibited strict ovarian epithelium selectivity. The hTERT promoter may be an optimal promoter for a univector gene therapy approach based on its high tumor selectivity. Utilization of multiple epithelial cell-specific promoters may result in a more tissue-selective gene therapy approach. Using a combination of promoters may prevent potential problems due to expression in nonepithelial stem cells that may constitutively express hTERT.

Tissue-specific gene therapy directed to tumor angiogenesis.

Gene therapy directed specifically to the vascular wall, particularly to angiogenic endothelial cells is a prerequisite in vascular disease treatment. Angiogenesis is a major feature in many pathological conditions including wound healing, solid tumors, developing metastases, ischemic heart diseases and diabetic retinopathy. In the present study we developed a tissue-specific gene therapy to the angiogenic blood vessels of tumor metastasis using an adeno-based vector containing the murine preproendothelin-1 (PPE-1) promoter. Genes activated by the PPE-1 promoter were highly expressed in bovine aortic endothelial cells in vitro. Systemic injection of the adenoviral vectors AdPPE-1-luciferase and AdCMV-luciferase to normal C57BL/6 mice, resulted in higher activity of PPE-1 promoter compared with CMV promoter in the aorta and vascularized tissues such as heart, kidney, lung and pancreas. Systemic administration of the adenoviral vector, in mice bearing Lewis lung carcinoma, resulted in high and specific activity of PPE-1 in the new vasculature of primary tumors and lung metastasis. Cellular distribution of the delivered gene revealed highest expression of GFP in angiogenic endothelial cells of the metastasis. We expect that this approach of 'vascular-directed' gene therapy will be applicable to both vascular diseases and cancer.

Tissue-specific promoters in gene therapy for the treatment of prostate cancer.

Delivery of therapeutic toxic genes to and their expression in tumor cells through the use of tissue-specific promoters could decrease their toxic effect on neighboring normal cells when virus-mediated gene delivery results in their infection. We have demonstrated the utility of two prostate cancer-specific promoters, long PSA and osteocalcin, for tissue-specific toxic gene therapy for prostate cancer. The two promoters were highly active in both androgen-dependent and androgen-independent prostate cancer cells. We also introduce the Phase I trial of osteocalcin promoter-based toxic gene therapy for bone metastases of prostate cancer, which is in progress at the University of Virginia.

DEVELOPMENT OF PROSTATE-SPECIFIC ANTIGEN PROMOTER-BASED GENE THERAPY FOR ANDROGEN-INDEPENDENT HUMAN PROSTATE CANCER

Purpose The goal of this study is to develop a tissue-specific toxic gene therapy utilizing the prostate specific antigen (PSA) promoter for both androgen-dependent (AD) and androgen-independent (AI) PSA-secreting prostate cancer cells. Ideally this gene therapy would be effective without the necessity of exposing the target cells to circulating androgens. Materials and Methods An AI subline of LNCaP, an AD PSA-secreting human prostate cancer cell line, C4-2, was used in this study. Castrated mice bearing C4-2 tumors secrete PSA. A transient expression experiment was used to analyze the activity of two PSA promoters, a 5837 bp long PSA promoter and a 642 bp short PSA promoter, in C4-2 cells. A recombinant adenovirus (Ad-PSA-TK) carrying thymidine kinase under control of the long PSA promoter was generated. The tissue-specific activity of Ad-PSA-TK was tested in vitro and in vivo. Results The long PSA promoter had superior activity over short PSA promoter, and higher activity in C4-2 cells than in LNCaP cells. High activity of Ad-PSA-TK was observed in C4-2 cells in an androgen deprived condition. In vitro, Ad-PSA-TK was further demonstrated to induce marked C4-2 cell-kill by acyclovir in medium containing 5% FBS. No cell-kill was observed in control WH cells (a human bladder cancer cell line). In vivo, Ad-PSA-P-TK with acyclovir significantly inhibited subcutaneous C4-2 tumor growth and PSA production in castrated animals. Conclusion The 5837 bp long PSA promoter was active in the androgen free environment and could be used to target both androgen-dependent and independent PSA-producing prostate cancer cells in vitro, and prostate tumors in castrated hosts.

Gene therapy for human colorectal carcinoma using human CEA promoter contro led bacterial ADP-ribosylating toxin genes human CEA: PEA & DTA gene transfer.

AIM:To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes. METHODS:Pseudomonas exotoxin A domain [II+III (PEA) was cloned from genomic DNA of Pseudomonas aeruginosa.PEA and diphtheria toxin A chain gene (DTA) were modified to express eukaryotically. After sequencing, the toxin genes under the control of human carcinoembryonic antigen (CEA)promoter were cloned into retroviral vectors to construct CEAPEA and CEADTA respectively. In vitro cotransfection of the constructs with luciferase vectors and in vivo gene transfer in nude mice were subsequently carried out. RESULTS:Both CEAPEA and CEADTA specifically inhibited the reporter gene expression in the CEA positive human colorectal carcinoma (CRC) cells in vitro. Direct injection of CEAPEA and CEADTA constructs into the established human tumors in BALB/c nude mice led to significant and selective reductions in CRC tumor size as compared with that in control groups.CONCLUSION:The toxin genes, working as therapeutic genes, are suitable for the tissue-specific gene therapy for colorectal carcinoma.

575 Isolation of vasopressin (AVP) promoter and enhancers for tissue specific gene therapy in SCLC

575 Isolation of vasopressin (AVP) promoter and enhancers for tissue specific gene therapy in SCLC

Targeted expression of the neuropeptide calcitonin gene-related peptide to beta cells prevents diabetes in NOD mice.

To investigate whether the immunosuppressive neuropeptide calcitonin gene-related peptide (CGRP) was a potential candidate for tissue-specific gene therapy, we engineered nonobese diabetic (NOD) mice to produce CGRP in beta cells by placing the modified calcitonin gene under the control of the rat insulin promoter. CGRP inhibits CD4 T cell production of the cytokines that have been implicated in the pathogeny of type I diabetes. Three transgene-positive mouse lines were obtained, two of which expressed immunoreactive CGRP in beta cells (NOD-CGRP mice). Isolated islets from one of these two transgene-positive founders produced active CGRP, whereas islets from transgene-negative littermates did not. The production of CGRP by beta cells prevented insulin-dependent diabetes mellitus in male and reduced its incidence by 63% in female mice. This prevention was due to a local immunosuppressive effect of CGRP as no difference was detected between NOD-CGRP and NOD littermate lymph node, spleen, and thymus cells by either FACS analysis or proliferative response to stimulation by Ag, alloantigen or anti-CD3. These data suggest that CGRP is a potential therapeutic molecule to prevent or treat diabetes and possibly other diseases and conditions in which immune cells are involved. These data also suggest that endogenous CGRP concentrated in sensory nerve endings may regulate locally the immune response, further strengthening the importance of the functional neuroimmune link.

Tissue-specific gene therapy

Tissue-specific gene therapy

A system for tissue-specific gene targeting: transgenic mice susceptible to subgroup A avian leukosis virus-based retroviral vectors.

Avian leukosis viruses (ALVs) have been used extensively as genetic vectors in avian systems, but their utility in mammals or mammalian cell lines is compromised by inefficient viral entry. We have overcome this limitation by generating transgenic mice that express the receptor for the subgroup A ALV under the control of the chicken alpha sk-actin promoter. The skeletal muscles of these transgenic animals are susceptible to efficient infection by subgroup A ALV. Because infection is restricted to cell lineages that express the transgene, the method has utility for studies of development and oncogenesis and will provide models for tissue-specific gene therapy.

Use of transgenic animals to study human retroviruses.

The purpose of transgenic technology is to introduce a gene into the germline of an animal in order to investigate its proper expression in the appropriate cell types and its effect on cellular functions. This technology has been used to explore a wide variety of genetic and biological issues including stage- and tissue-specific gene expression, development, immunology, oncology, and gene therapy. The intent of this article is to discuss the value of gene transfer technology for the study of mechanisms of viral pathogenesis. We will focus mainly on our experience with two experimental systems involving human T-lymphotropic virus Type 1 (HTLV-1) and human immunodeficiency virus Type 1 (HIV-1).