A variety of gene transfer vectors will ultimately be required to develop successful gene therapies for human disease. Each vector type requires specialized technology to optimize gene transfer and to address production issues. Although initial efforts utilized viral vectors for human gene therapy, synthetic, non-viral gene delivery systems have shown promise in clinical use. Desirable features of these vectors include improved safety for direct gene transfer in vivo and simplified production. This proposal is intended to support the development of a national vector production facility for DNA-based non-viral vectors at the UM. Investigators at the UM have demonstrated commitment and expertise in developing these vectors, having previously prepared and/or used four Food and Drug Administration (FDA)-approved non-viral vectors in human gene therapy trials. The objective of this facility will be to provide vectors for human clinical studies and to ensure that clinical grade vectors are available. The first goal is to define the identity, purity, and potency of non-viral vectors. Certification and quality control testing will be conducted on vectors to ensure that these agents are safe and standardized for use in patient testing. Preparation of master cell banks (MCBs), characterization of both a MCB and a manufacturer's cell bank, testing of the vectors and testing of final lots will be performed. In addition, reagents for DNA liposomes and particle-mediated gene delivery will be certified for human use. The facility will be comprised of three components: 1) Vector Production Unit; 2) Plasmid/Reagent Repository; and 3) Cell-Transduction Service. These plans are based upon previous experience in generating such vectors for human gene therapy trials. This facility will produce naked DNA, DNA/liposome complexes, and particle-mediated gene transfer vectors. The vector production unit will utilize existing facilities at the UM, including a HAL in the General Clinical Research Center, a separate dedicated vector laboratory, a molecular diagnostic core, and a DNA sequencing facility. These resources, together with appropriately trained and experienced personnel, will produce these vectors for gene therapy trials nationally. To certify these vectors, the investigators will implement techniques previously used and approved by the FDA for plasmid DNA in human trials. Analyses of DNA sequence, gene expression, quality assurance (QA) and quality control (QC) will be performed through existing core facilities to complete certificates of analysis. The Plasmid/Reagent Repository will maintain a bank of DNAs and delivery agents for investigators. These reagents will include liposomes and FDA-approved lots of gold particles and associated reagents for particle-mediated gene transfer. The cell transduction service will provide a service for institutions who do not have the capability to perform cell transductions by performing genetic modifications on cells transported to the UM and subsequently returned to the test site. Such arrangements would expand upon efforts which have already been instituted informally with other sites. The Vector Production Unit will also incorporate newer technologies which will continue to evolve, including the development of vectors with alternative enhancers and other regulatory elements to modulate the expression of recombinant proteins. Two vectors will be available immediately for potential use in human trials. The investigators hope to facilitate the use of non- viral gene delivery and direct gene transfer for human gene therapy trials and allow them to proceed with greater safety and efficacy.
