Research and Product Development Tools
Three-Dimensional System to Measure Lymphocyte Migration
There is a need for model systems that can be used to identify clinically relevant behaviors of cytotoxic T lymphocytes (CTL), including tumor cell lysis and active migration, as well as systems that can be used to identify chemokines and tumor antigens that influence active migration of CTL towards tumor cells. Traditionally, CTL are raised and studied in two-dimensional mixed lymphocyte tumor cell culture (MLTC) that includes either (1) long-term cultured tumor cells to stimulate peripheral blood mononuclear cells (PBMC) for CTL induction or (2) disaggregated tumor tissue with tumor infiltrating lymphocytes (TIL) where both types of cultures are two dimensional and are grown directly on plastic surfaces. Studies in melanoma and colon cancer patients have shown that characterization of CTL responses using such two-dimensional cultures do not reflect the CTL’s in vivo behavior and specifically, CTL-exerted anti-tumoral activity.
Wistar scientists have developed a three-dimensional reconstruct model containing solid layers of collagen, tumor cells and CTL that closely mimic the condition of patients in vivo, preserving in vivo semi-phenotypic and functional characteristics of the cells. This model closely mimics CTL behavior in cancer patients and allows the detection of CTL migration and identification of tumor antigens and cytokines/chemokines involved in CTL migration.
Method for Solid-Phase Amplification of DNA Using Multiarrays
Amplification of DNA is a common first step in nucleic acid diagnostics, particularly when testing for the presence or absence of an organism or a given nucleic acid sequence in a sample. For simplicity and sensitivity, DNA amplification assays have been developed wherein either the nucleic acid template or the primer is attached to a solid phase. These assays are particularly suitable for screening multiple samples in an automated system.
Wistar researchers have developed an improved assay for detecting the presence of a given test DNA in a sample, wherein the 5¹ and 3¹ primers for the test DNA are irreversibly bound to a solid phase and the DNA in the sample is reversibly bound to the solid phase. When these components are incubated under conditions that promote nucleic acid amplification, no signal is detectable unless the sample contains the test DNA. This assay may be useful for high throughput testing for pathogens (e.g. microorganisms or DNA viruses) or for detecting mutations in test samples, e.g. in multiarray systems.
Device and Method for Fractionation of Proteomes and Complex Biological Mixtures
Two-dimensional electrophoresis, which is widely used for quantitatively comparing changes in protein profiles (proteomes) of cells, is not capable of resolving all of the thousands of proteins in eukaryotic cell samples. While two-dimensional electrophoresis may resolve up to 2,000-3,000 individual proteins, most eukaryotic proteomes have over 10,000 protein components.
To address this limitation, Wistar researchers have developed a new device and method to separate proteomes and other complex mixtures into multiple discrete fractions. The subsequent fractions are then suitable for analysis by other analytical methods, such as two-dimensional electrophoresis or liquid chromatography/mass spectroscopy. Using this new device and method to pre-fractionate samples before analysis permits increase in overall protein loads (with a resulting increased sensitivity to proteins present in low concentrations), improved resolution of proteins or other components, and greater dynamic range compared to currently available pre-fractionation methods.
The new Wistar device is also useful for separation of components of other complex mixtures (e.g. nucleic acids, biological fluids) prior to analysis. The Wistar device may be used as a component of an automated system.
Stabilized Production of Ribozymes, iRNA, mRNA and Other Gene Products
Wistar researchers have developed a novel method to stabilize introns that offers the potential for stable production of ribozymes, iRNA, mRNA and related gene products. Using the Wistar technology, stabilized intron compositions are prepared with a sequence from the LAT intron of the HSV-1 virus. Polynucleotide encoding sequences are then ligated into a stabilized intron, resulting in a 105 -fold increase in stability. Unlike typical introns that rapidly degrade within seconds after excision, these stabilized introns have a half life of 24 hours (Thomas et al. 2002. J. Virol., 76, 532-540). The increased stability has been demonstrated in several models, indicating that the method is neither cell type- nor species-specific and can be applied to both eukaryotic and yeast expression systems.
These methods and compositions are useful for:
- Stabilizing a gene transcript to permit enhanced expression, and increased production, of a recombinant gene product.
- Production of vectors and gene products for research reagents, markers of gene production, and diagnostic and therapeutic compositions.
- Markers for determining whether a gene of interest is being transcribed, particularly for identification of genes transcribed only in a selected stage of the cell cycle.
RNA Editing Enzyme and Methods of Use Thereof
The ADAR (adenosine deaminase that acts on RNA) enzyme, previously known as DRADA, is involved in the RNA editing of glutamate-gated ion channels and the serotonin-2C receptor. This novel enzyme is implicated in central nervous system disorders characterized by abnormal neuronal transmission and intracellular signaling in mammalian brain such as stroke, schizophrenia, depression, and substance abuse.
A Method of Delivering Genes to the Central Nervous System
One of the limitations of gene therapy has been the lack of methods to effectively deliver genes to the central nervous system. Herpes simplex virus (HSV) is a neurotropic virus that naturally establishes latent infections in the peripheral nervous system and the central nervous system of humans. Wistar researchers have developed a novel method for using neurotropic HSV to deliver genes of therapeutic value. Additionally, the Wistar technology promotes long-term expression of the therapeutic genes, which is necessary for treatment of a genetic defect.
The Wistar technology has several advantages over other methods of delivering genes to the central nervous system (CNS). First, no helper virus is needed, and second, long-term expression of the gene is achieved using a naturally-occurring viral promoter. This technology has been used successfully in vivo in a mouse model. In this study, the gene encoding §-glucorinodase was delivered to the CNS and long-term expression (over 4 months) of the gene product was obtained.