Expandig Lentiviral Vectors cargo capacity with protein trans-splicing

Abstract

In the last decades Lentiviral vectors (LVs) have been widely used as vehicles to stably transfer and express genes in mammalian cells in the context of monogenic diseases in both dividing and non-dividing or slowlyproliferating cells, as human CD34+ stem cells (CD34+ HSCs). However LVs cargo capacity, limited to around 8 kilobases (kb), prevents their application for the treatment of conditions requiring the delivery of larger DNA sequences and the stable expression of the transgene in actively proliferating cells. Recent studies have shown that protein trans-splicing is a powerful means to ably reconstitute large full-length proteins upon coinfection of a host cell with different vectors, each expressing a split-intein flanked portion of the full lenght- protein. The aim of our study is to translate the same technology to the platform of LVs. As proof-of-principle developed lentiviral expression plasmids (pLenti), each encoding either the N- or the C-terminal half of the EGFP reporter protein fused to the N- and C-terminal halves of the DnaE split-inteins from Nostoc punctiforme, under the control of EF1A promoter. We co-transfected HEK293 cells and we demonstrated the reconstitution of a functional full-lenght protein of the proper size. We are currently generating LVs from these pLenti that we will use to infect both lineage-negative cells from mouse bone marrow and CD34+ HSCs in order to assess if the platform of LVs is as efficient as AAVs. This system will be helpful to develop a gene therapy approach for specific inborn errors of immunity, including LRBA deficiency and Ataxia Telangiectasia, caused by mutations in genes larger than 8 kb.