A self-deletion lentiviral vector to reduce the risk of replication-competent virus formation

Major improvements have been made progressively on human immunodeficiency virus (HIV)-1 based lentiviral vectors to minimize the probability of replication-competent lentivirus formation. This includes the deletion of U3 promoter and the use of packaging cells, which has increased their potential for use in gene therapy and other in vivo applications. However, the risk of forming replication-competent lentiviruses remains.

We investigated the use of Cre-loxP mediation with the insertion of the transgene-expressing cassette in ΔU3 to remove additional parts of the HIV-1 backbone upon cre expression, after integration. This, leads to deletion of the packaging signal, primer binding site and Rev response element, including cre itself.

This approach left a split truncated form of long terminal repeat flanked by a loxP and a transgene-expressing cassette in the genome, which made replication-competent lentivirus formation almost impossible. This self-deletion vector could stably express transgenes both in cell lines and transgenic mice with only modest losses of viral titer. The maximum size of the inserts was approximately 3 kb, which was sufficient for most transgenic applications. Moreover, the addition of some enhancer blocking agents downstream of the transgene could reduce the probability of transcriptional read-through in transfected 293T cells.

Our approach could improve the biosafety of lentiviral vectors, thus improving their potential application for use in clinical trials and other in vivo applications.