Brian E. Huber

Virus‐Directed Enzyme/Prodrug Therapy (VDEPT) Selectively Engineering Drug Sensitivity into Tumors

A gene therapy approach has been described that generates a tumor-selective qualitative difference in the metabolic capability in tumor cells. This is the result of the selective expression of a nonmammalian enzyme in tumor cells. Selective expression is achieved by utilization of a chimeric gene composed of the TRS from a tumor-associated marker gene linked to the coding domain of a gene encoding a nonmammalian enzyme. We have described the application of this approach for the treatment of metastatic CRC. This approach involves creation of a chimeric gene composed of the CEA TRS linked to the coding domain of the CD gene. Selective expression of CD in the tumor cells will allow the selective conversion of the prodrug 5-FCyt to 5-FCyt in the tumor while sparing normal cells. Most importantly, delivery and expression of CD into a small fraction of tumor cells may be sufficient to achieve a significant antitumor effect.

VDEPT: An enzyme/prodrug gene therapy approach for the treatment of metastatic colorectal cancer

Abstract Colorectal carcinoma (CRC) remains a significant medical challenge with an expected 350000 new cases per year. Although the primary cancer can be successfully controlled by surgical resection, metastatic disease to the liver is the most common demise of the CRC patient. New innovative approaches must be developed for the treatment of CRC hepatic metastasis if the overall 2- and 5-year survival rates and quality of life assessments are to improved. We now describe an innovative gene therapy approach for the treatment of metastatic CRC, an approach called VDEPT. In this approach, an artificial chimeric gene is created which consists of two components: (1) the transcriptional regulatory sequence (TRS) of the human carcinoembryonic antigen gene (CEA); and (2) the protein coding domain of the nonmammlian cytosine deaminase gene (CD). This artificial gene will express CD only in cells which naturally express CEA. Expression of CD in CEA-positive cells is, by itself, nontoxic. However, CD can convert the nontoxic prodrug, 5-fluorocytosine (5-FCyt), to the toxic anabolite, 5-fluorouracil (5-FUra). Hence, the toxic compound, 5-FUra, will be selectively produced in cells which express CD. Since expression of CD is restricted to CEA-positive cells, 5-FUra will be selectively produced in CEA-positive cells. Hence, tumor-specific expression of CD permits the tumor-specific production of 5-FUra at high concentrations for extended periods of time directly at the tumor site. The artificial, chimeric gene can be delivered to CEA-positive tumors via a replication-defective retroviral vector. Chimeric genes composed of the human CEA promoter and the coding sequence of CD were created and engineered into a retroviral gene delivery vector. These chimeric genes selectively expressed CD in CEA-positive cells which resulted in the selective conversion of 5-FCyt to 5-FUra in the CEA-positive tumor cells. Human tumor xenografts demonstrated that expression of CD in solid tumors can generate complete cures if only 4% of the solid tumor cell mass expressed this enzyme. In vivo gene transfer has indicated that retroviral vectors can delivery and express CD chimeric genes in liver tumors at this 4% level.

Activity of an NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase in normal tissue, neoplastic cells, and oncogene-transformed cells.

As an extension of the previously reported observation concerning the existence of NAD-dependent 5,10-methylenetrahydrofolate dehydrogenase in transformed cells a variety of tissues and cell lines have been assayed for this activity. This activity was found in all assayed transformed cells. Results with rat liver derived epithelial (RLE) cells transformed with a series of oncogenes (v-raf, v-raf/v-myc (J2), v-myc (J5), and v-Ha-ras (pRNR16)) indicated that expression of activity correlates with the extent of transformation and was independent of the oncogene used for transformation. Compared to previously reported values for normal tissue, surprisingly high levels of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase were found in the rat adrenal cortex. This activity was not seen in mouse or bovine adrenal. Enzymatic activity was also detected in mouse bone marrow and was strain dependent. The levels of activity in mouse bone marrow were lower than previously reported. The NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase activity in rat adrenal and RLE cells may represent tools for studying the regulation of expression of this activity.

Gene Therapy Strategies for Treating Neoplastic Disease

Over the last few decades, overwhelming evidence has accumulated that indicates that neoplastic transformation, by nature, is a genetic disease. Normal genes can be quantitatively or qualitatively altered or new genetic information can be contributed by viruses. These genetic changes can result in the many, many different phenotypic traits that we collectively call neoplastic cellular transformation. These varied phenotypic traits associated with cellular transformation may involve: altered signaling pathways from the cell surface to the cell nudeus; altered cell-cell communication and interactions; altered cellular responses to external signals or internal fail-safe mechanisms; and other characteristics, all of which can interfere with the fundamental processes of growth, differentiation, and apoptosis. Taken collectively, these varied and numerous phenotypic characteristics of a neoplastic cell ultimately result from a genetic component(s). For these reasons, neoplastic transformation is a genetic disease by nature. The genetic lesions associated with neoplastic transformation can be grouped into three very broad categories. They are:

Metabolism of a novel antitumor agent, crisnatol, by a human hepatoma cell line, Hep G2, and hepatic microsomes: Characterization of metabolites

Metabolism of the anticancer agent crisnatol was investigated using a human hepatoma cell line, Hep G2, and human liver microsomes. Crisnatol was metabolized extensively by both systems. The TLC/autoradiographic analysis showed that the crisnatol metabolite profile was similar for both systems and the major metabolites were shown to have structural characteristics similar to those formed by the rat. The Hep G2 cells formed three isomeric dihydrodiols; one of these has been identified by GC/MS and 1H-NMR as the crisnatol 1,2-dihydrodiol. Human liver microsomes also formed two isomeric dihydrodiols with 1,2-dihydrodiol as the major isomer and, in addition, produced 1-hydroxycrisnatol. Crisnatol concentrations of 1.3 micrograms/mL completely inhibited the replication of Hep G2 cells as measured by thymidine incorporation and cell growth kinetics and, at this concentration, cell viability decreased by only 35% as determined by vital staining of cells using neutral red dye.

Induction by phenobarbital in McA-RH7777 rat hepatoma cells of a polycyclic hydrocarbon inducible cytochrome P450

The metabolism of 2-acetylaminofluorene (AAF) to its six oxidative metabolites has been used to study cytochrome P450 monooxygenase activity in two rat hepatoma cell lines, McA-RH7777 and Reuber H4-II-E. McA-RH7777 cells exhibited considerably higher basal activities than H4-II-E cells for all metabolic pathways studied. Phenobarbital induced AAF metabolite formation in McA-RH7777 cells to a similar extent as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but was only a weak inducer of these activities in H4-II-E cells. Northern blot analysis utilizing specific phenobarbital or 3-methylcholanthrene inducible cytochrome P450 cDNA probes indicated that there was at least a 10-fold increase in a 3-methylcholanthrene inducible cytochrome P450 transcript in phenobarbital treated McA-RH7777 cells. These data suggest that in this transformed cell line phenobarbital behaves as a polycyclic hydrocarbon-like inducer.

Regulated Expression of Artificial Chimeric Genes Contained in Retroviral Vectors: Implications for Virus-Directed Enzyme Prodrug Therapy (VDEPT) and other Gene Therapy Applications

Replication-defective retroviral vectors were created that contained chimeric genes composed of either the albumin (ALB) or the alpha-fetoprotein (AFP) transcriptional regulatory sequences linked to the coding domain of the thymidine kinase gene from Varicella zoster virus (VZV TK). These viruses were used to infect the human hepatoblastoma cell line, HepG2. Subsequent to infection, the infected cells were single-cell cloned. The level of expression of VZV TK from the chimeric genes correlated with the level of endogenous expression of ALB or AFP in most clones, indicating that the transcription of the chimeric VZV TK gene is controlled in a similar manner to the endogenous ALB or AFP genes, and that sites of viral integration are less important to overall gene expression. Most importantly, as the expression of the endogenous ALB gene was modified, so was expression of VZV TK from the ALB/VZV TK chimeric gene. This demonstrates that retroviruses can deliver a chimeric gene containing tissue-specific transcriptional regulatory sequences that can respond to endogenous cell regulatory signals resulting in regulated gene expression.

Characterization of the “hepatic” asialoglycoprotein receptor in rat late-stage spermatids and epididymal sperm

Northern blot analysis of rat testicular (Te) poly(A)+RNA reveals that a transcript homologous to the major form of the asialoglycoprotein receptor (ASGP-R), designated RHL-1, is expressed as early as one week postnatally and that steady-state levels are approx. 8-times higher in the Te of an 8-week-old rat (sexually mature) as compared to an 84-week-old rat (aged). Partial cDNAs encoding RHL-1 and the minor form of the ASGP-R, designated RHL-2/3, have been cloned from two rat Te/epididymal (Ep) cDNA libraries and rat Te poly(A)+RNA. Sequence analysis of the Te/Ep RHL-1 cDNA and the Te/Ep RHL-2/3 cDNA indicates that these cDNAs are identical to the forms expressed in rat liver. Western blot analysis demonstrates the presence of a 49-kDa Te/Ep RHL-1-related protein band and a 54-kDa Te/Ep RHL-2/3-related protein band in both rat Te membrane fractions (MF) and rat Ep sperm MF. The RHL-1-related protein has been localized to late-stage Te spermatids at the time of release from the seminiferous tubules and to Ep sperm in the region of the sperm tail, referred to as the middle piece. Taken collectively, these data indicate that the authentic RHL-1 and RHL-2/3 genes of the ASGP-R are expressed in late-stage spermatids; however, the Te/Ep RHL-1-related protein differs in size from the hepatic RHL-1 polypeptide, possibly indicating a specific function of the RHL-1-related protein in spermatogenesis.

Gene therapy for the treatment of inherited and acquired diseases of the liver

Publisher Summary This chapter discusses the clinical evaluation of gene addition type gene therapy protocols directed at the inherited or acquired diseases of the liver. Gene therapy can be divided into two categories: cellular therapy and gene therapy. The distinction between these two types of therapy is that the former involves the removal of particular target cells (e.g., hepatocytes) from a patient or an appropriate donor, ex vivo gene transfer and genetic alteration, and the reintroduction of the genetically altered cells back into the patient. The latter, gene therapy, involves the direct in vivo genetic alteration of a patients target cells. One can envision two distinctly different strategies for liver-directed gene therapy: gene replacement (repair) or excision therapy and gene addition therapy. In gene replacement scenario, one can envision a mutated hepatic gene(s) producing an aberrant hepatic protein that ultimately results in or contributes to a disease state. The pathogenicity of the hepatic gene may be qualitative or quantitative in nature resulting in either an altered hepatic protein or a hepatic protein, which is inappropriately expressed, respectively. More practical strategy is gene addition therapy. For this therapy, a complete copy of a normal gene is delivered to the hepatic target cells. Depending on the gene delivery system used, this complete copy of a normal gene can either be randomly integrated into the genome of the liver cell or it may remain extrachromosomal. An important application of the gene addition strategy is the theoretical ability to replace important genetic information that has been functionally lost through mutational events.