The long-term objective of Dr. Sinn is to pursue an academic career at a major researchuniversity studying the biology and development of lentiviral vectors. This will be achieved with the aid ofDrs. McCray and Voytas and the support of the Dept. of Pediatrics at the University of Iowa. The immediateinterest of Dr. Sinn is the general safety and utility of lentiviral vectors for use in a broad range of genetherapy applications, such as cystic fibrosis. Gene therapy for cystic fibrosis, a disease that has pulmonaryand digestive manifestations, is directly applicable to the mission of the NIDDK. To persistently expressatherapeutic transgene, a retroviral vector must integrate into a host cell chromosome. This critical processmakes lentiviral vectors an attractive tool to achieve life-long gene delivery; however, the nonspecific natureof retroviral integration presents inherent hazards and variations in gene expression. If integration could berestricted to preferred genomic loci, the safety and utility of lentiviral vectors would be vastly improved. Inthis proposal a novel strategy is investigated to modify the integrase (IN) gene of a feline immunodeficiencyvirus (FIV)-based lentiviral vector to achieve restricted integration. A 'tethering' model is proposed in whichan engineered hybrid protein that contains a protein binding domain and a DNA binding domain will directthe lentiviral vector integration complex to preferred sites on chromosomal DNA.
Three aims are proposed:1) determine the capacity to which FIV IN can be modified and still retain its function, as determined bytitering assays as well as in vitro catalytic and integrase assays; 2) confirm high affinity protein-proteininteractions of modified FIV IN to the tethering protein by yeast two-hybrid assays and demonstrate thatthese complexes can mediate restricted integration in a cell-system. These studies will focus the ultimategoal to, 3) map integration sites of the modified vector into genomic DNA and demonstrate a restrictedpattern of integration in those cells in which the tethering protein is co-expressed.Relevance: Lentiviral vectors have the potential to persistently correct genetic diseases. However, suchvectors integrate nonspecifically into the host genome and therefore present a risk of disrupting normal genefunction at the site of insertion. Successful site-restricted lentiviral vector integration into human genomicDNA would have exciting and broad applications in the gene therapy field.
|Leonard, Vincent H J; Sinn, Patrick L; Hodge, Gregory et al. (2008) Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed. J Clin Invest 118:2448-58|
|Sinn, Patrick L; Arias, Ariadna C; Brogden, Kim A et al. (2008) Lentivirus vector can be readministered to nasal epithelia without blocking immune responses. J Virol 82:10684-92|
|Sinn, Patrick L; Goreham-Voss, Jessica D; Arias, Ariadna C et al. (2007) Enhanced gene expression conferred by stepwise modification of a nonprimate lentiviral vector. Hum Gene Ther 18:1244-52|