100. Development of an Integrase Deficient FIV Vector for Transient Gene Expression

Lentiviral vectors mediate long term transgene expression through integration into host genomic DNA. However, integration comes with potential risks of insertional mutagenesis. In cases where transient gene expression is desired, an integrase (IN) deficient retroviral vector might be a useful alternative. Furthermore, IN deficient vectors may provide some advantages over alternative methods of transient gene expression due to the ability to pseudotype these vectors with heterologous envelopes that target specific cell types. Class I IN mutants have point mutations in the catalytic triad amino acids (DX39-58 DX35 E) of IN that disable this protein but leave other viral proteins functional. Transient gene expression from IN deficient vectors is mediated from episomal forms of vector DNA. 1-LTR and 2-LTR circles form through homologous recombination and non-homologous end joining, respectively, of the linear form of the reverse transcribed viral DNA. Although 1-LTR and 2-LTR circles are unable to efficiently integrate in the presence of functional IN protein, they can mediate transgene expression. It was previously reported that IN deficient HIV and FIV vectors could mediate transient gene transfer. In growth arrested cells, transgene expression was maintained. However, in rapidly dividing cells transgene expression was lost, and duration of expression was related to the rate of cell division. Because some gene transfer targets are mitotically quiescent and slowly dividing cells, IN deficient mutants may promote long term, though ultimately transient, gene expression. We hypothesized that IN deficient FIV vectors can be used to maintain transient gene expression in slowly dividing cell types, such as airway epithelia. We developed IN deficient FIV vectors with single (D66V, D118A, E154G), double (D66V/D118A, D66V/E154G, D118A/E154G), and triple (D66V/D118A/E154G) mutations that disrupt the catalytic triad of the IN protein. Preliminary data indicate that the FIV D66V IN mutant has titers similar to wild type IN vectors. Furthermore, we documented the production of 1-LTR and 2-LTR circles in human cell line cultures transduced with FIV D66V IN deficient vector. Ongoing studies are comparing the persistence of transgene expression in IN deficient versus wild type IN FIV in dividing and non-dividing cells. Furthermore, to ensure that transgene expression seen in IN deficient vectors represents episomal and not integrated vector DNA, Southern blotting will be used. IN deficient lentiviral vectors may have applications for transient gene expression, long term expression in slowly dividing cells, and can be used as controls in studies with integration competent vectors.