University of Pittsburgh Cancer Institute (UPCI)
Overview
The Vector Core was established in 1991 as part of the gene therapy initiative at the University of Pittsburgh. VCF generates viral vectors and provides reagents to projects working on treatment of gene therapy for
- cancer
- gene transfer to facilitate islet transplantation
- gene transfer to myoblasts
- in utero gene transfer
- gene transfer to hepatocytes and endothelial cells
Adenoviral and Retroviral Vectors
The VCF constructs and/or propagate recombinant adenoviral vectors for gene therapy applications. The adenoviral vector that is being used at the Facility is a serotype 5 first generation vector deleted for E1 and E3. The gene of interest is introduced into the adenovirus vector using a shuttle vector that carries a CMV expression cassette with a LoxP site. Recombination between the vector backbone and the shuttle plasmid is driven by the CRE recombinase, resulting in a high frequency of the appropriate recombinants.
MFG, designed by Drs. Paul Robbins and Braydon Guild, provides high level expression of a variety of inserted genes, like cytokines, receptors, enzymes, and growth factors, following non-selective infection of cells. Further modification to enable introduction of multiple genes into the MFG vector was achieved by cloning an internal ribosome entry site (IRES) sequence from encephalomyocarditis virus (EMCV) that allows for translation of a downstream gene in a polycistronic message. The MFG-IRES vectors were shown to efficiently express two genes from the same message using two different secreted marker proteins, IL1Ra (IRAP) and growth hormone. The MFG-IRES vectors have now been used to express three genes such as both subunits of IL-12 and a neomycin resistance gene (neoR) marker gene using two IRES elements.
Lentiviral Vectors
Lentiviral vectors are available that express shRNA and cDNA. These reagents may be used for gene-discovery, drug development, and target validation studies.
