Cynthia A. Richards

Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways.

MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurode- generative diseases such as Alzheimers disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

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.

Nucleotide sequence of the dihydrofolate reductase gene of Saccharomyces cerevisiae

The nucleotide sequence of a DNA fragment that contained the Saccharomyces cerevisiae gene DFR coding for dihydrofolate reductase (DHFR) was determined. The DHFR was encoded by a 633-bp open reading frame, which specified an Mr24264 protein. The polypeptide was significantly related to the DHFRs of chicken liver and Escherichia coli. The yeast enzyme shared 60 amino acid (aa) residues with the avian enzyme and 51 aa residues with the bacterial enzyme. DHFR was overproduced about 40-fold in S. cerevisiae when the cloned gene was present in the vector YEp24. As isolated from the Saccharomyces library, the DFR gene was not expressed in E. coli. When the gene was present on a 1.8-kb BamHI-SalI fragment subcloned into the E. coli vector, pUC18, weak expression in E. coli was observed.

O3-02-07: Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways

MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurodegenerative diseases such as Alzheimers disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

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.