Integrase competent or deficient lentiviral vector expression of microRNAs and short hairpin RNAs for messenger RNA inhibition

Lentiviral vectors are an integrating class of gene transfer vectors that allow for permanent correction of a genetic disorder. However, transgene integration can cause insertional mutagenesis and may not be required for all applications. A non-integrating lentiviral vector may be beneficial for transient gene expression. One potential application of a non-integrating vector is for RNAi expression targeting exogenously expressed viral genes. Additional RNAi expression applications can be derived from integrating lentiviral vectors.;Initially, we generated an integration deficient FIV vector by introducing a disruptive D66V mutation to integrase. We confirmed functional disruption in vitro with DNA and protein expression analysis. Although transgene expression is transient in dividing cells, gene expression can persist in slowly dividing cell types. We hypothesized that INdef FIV vectors would maintain high levels of gene expression in slowly dividing airway epithelia. While in vitro and in vivo experiments were initiated, low vector titers and repressed gene expression prevented further assessment of this system.;We proposed to use an integration deficient FIV vector to deliver miRNAs to silence exogenously expressed viral genes. We investigated inhibition of respiratory syncytial virus (RSV). Preliminary plasmid transfection studies indicated decreased RSV infection. To determine maximum RSV inhibition, we generated immortalized cell lines expressing miRNA constructs. However, only modest, non-specific silencing was observed, representative of an ineffective miRNA. We began testing shRNA construct efficacy, but studies were halted following parallel experiments demonstrating all cells in a population must express the shRNA for protection, which is unachievable through vector transductions.;We also explored applications of integrase competent FIV expression of RNAi. By including an RNAi cassette with a selectable transgene, we hypothesized that a polarized airway epithelia cell culture could be produced with all cells reduced in target gene expression. We focused on two proof-of-principle targets: lamin A/C and ACE2. Using immortalized Calu3 cells, we demonstrated target inhibition of both targets in polarized cultures. Additionally, we extended this technique for use in human primary cultures, and demonstrated that a polarized epithelia layer can be generated following vector transduction and selection.