458. Development of Non-Integrating and Site- Specifically Integrating Lentiviral Vectors
Authors: Luis Apolonia, Simon Waddington, Mary Collins, Adrian Thrasher, Nicola Philpott
Journal: Molecular Therapy (2006)

The lentiviral genome non-specifically integrates into the human genome and this can lead to insertional mutagenesis and potentially cell transformation. We are using two strategies to eliminate the risk of insertional mutagenesis thereby improving the safety and efficacy of lentiviral gene therapy vectors. The first is to develop non-integrating lentiviral vectors and the second is to target integration to a non-pathogenic site in the human genome.We have generated several integration deficient lentiviral vectors and all express eGFP from extrachromosomal lentivector DNA at 2 days post-infection. In contrast to integrating vectors that show stable eGFP expression in dividing cells, non-integrating vector DNA is lost with cell division; this correlates with a decrease in eGFP expression over time. Therefore, integration deficient lentivectors will be particularly useful in non-dividing cells, such as muscle. We have therefore tested such vectors in myoblast cultures. Although eGFP expression in dividing myoblasts decreases over time, expression persists in differentiated, non-dividing myoblasts until at least 18 days post-infection. The non-integrating vectors were then used to deliver an eGFP transgene to post-mitotic tibialis anterior muscle. EGFP is expressed in muscle transduced by vectors containing the wt and D64V integrase at 1 week post–intramuscular injection. Furthermore, assessment of muscle at 3 months post-injection shows that stable transgene expression was achieved from transduction of an integrase-deficient lentiviral vector. DNA analyses are currently being used to confirm that viral DNA is being maintained episomally.To obtain stable and safe gene expression in dividing cells the transgene could be integrated into a site that does not induce cell transformation. Adeno-associated virus (AAV) site-specifically integrates into the human genome and importantly this event is not associated with any pathogenicity. AAV integration is mediated by Rep in trans and the p5 integration efficiency element (p5IEE) in cis. Therefore, we are using these AAV elements to target lentiviral vector integration. Results show that cells infected with p5IEE-containing lentivectors maintain higher eGFP expression in the presence of Rep than in its absence. Experiments are being carried out to determine whether Rep-induced transgene persistence is caused by site-specific integration events. The lentiviral genome non-specifically integrates into the human genome and this can lead to insertional mutagenesis and potentially cell transformation. We are using two strategies to eliminate the risk of insertional mutagenesis thereby improving the safety and efficacy of lentiviral gene therapy vectors. The first is to develop non-integrating lentiviral vectors and the second is to target integration to a non-pathogenic site in the human genome. We have generated several integration deficient lentiviral vectors and all express eGFP from extrachromosomal lentivector DNA at 2 days post-infection. In contrast to integrating vectors that show stable eGFP expression in dividing cells, non-integrating vector DNA is lost with cell division; this correlates with a decrease in eGFP expression over time. Therefore, integration deficient lentivectors will be particularly useful in non-dividing cells, such as muscle. We have therefore tested such vectors in myoblast cultures. Although eGFP expression in dividing myoblasts decreases over time, expression persists in differentiated, non-dividing myoblasts until at least 18 days post-infection. The non-integrating vectors were then used to deliver an eGFP transgene to post-mitotic tibialis anterior muscle. EGFP is expressed in muscle transduced by vectors containing the wt and D64V integrase at 1 week post–intramuscular injection. Furthermore, assessment of muscle at 3 months post-injection shows that stable transgene expression was achieved from transduction of an integrase-deficient lentiviral vector. DNA analyses are currently being used to confirm that viral DNA is being maintained episomally. To obtain stable and safe gene expression in dividing cells the transgene could be integrated into a site that does not induce cell transformation. Adeno-associated virus (AAV) site-specifically integrates into the human genome and importantly this event is not associated with any pathogenicity. AAV integration is mediated by Rep in trans and the p5 integration efficiency element (p5IEE) in cis. Therefore, we are using these AAV elements to target lentiviral vector integration. Results show that cells infected with p5IEE-containing lentivectors maintain higher eGFP expression in the presence of Rep than in its absence. Experiments are being carried out to determine whether Rep-induced transgene persistence is caused by site-specific integration events.