Lentiviral System for Gene Delivery into Resting T-cells

Abstract

Current gene delivery systems suffer from poor genetic delivery into resting T-cells and therefore, compensate for this by using multiple viral challenges, by activating and expanding the cells ex vivo. However, cells activated and expanded in this manner have lower engraftment, proliferative potential and potency. In contrast, the use of resting T-cells overcomes many of these short-comings, but comes with restrictions: Firstly, at the plasma membrane, due to the lack of receptor for the commonly used lentiviral envelope pseudotype, VSVg. Secondly, at reverse transcription (RTN), by intracellular restriction factor, SAMHD1.

At the membrane restriction, we focused on the abundant expression of CXCR4 on resting T-cells, by testing a panel of lentiviral systems expressing various CXCR4-dependent, CD4-independent HIV envelopes. Results revealed that the above panel fused poorly or not at all within resting T-cells. The lead envelope was initially derived from HIV-2 VCP isolate, which was dependent on HIV-2 Gag to maintain high fusogenicity. Whilst HIV-2 based lentiviral systems are advantageous; they are not as developed as HIV-1 systems. Thus, we re-investigated a HIV-1 system based on a derivate of iR3A isolate that enters resting T-cells primarily through CXCR4, but to do so, it required modifications to its matrix.

Fortunately, SIV and HIV-2 have evolved with the accessory gene, vpx, that directly targets SAMHD1 for degradation and can be readily incorporated into lentiviral particles. Yet it is presently unknown if all vpx genes are equal in their ability to antagonise SAMHD1 and/or enhance RTN. Through bioprospecting a genetically diverse panel of vpx, we tested SAMHD1 antagonism across several cell types. Majority of the Vpx proteins could overcome SAMHD1 restriction in dendritic cells and macrophages, but only one lead Vpx could efficiently achieve this in resting T-cells, and this was due to an additional Lysine within its nuclear localisation signal. Results further revealed that, in resting T-cells, lentiviral genetic cargo is prone to heavy deletions during the process of RTN. Inclusion of Vpx within lentiviral particles increased preservation of intact gene cargo and supports a role for SAMHD1 in viral restriction, through promoting deletions in incoming reversed transcribed DNA prior to genomic integration. Taken together, these observations serve as a viable platform to build towards the generation of resting T-cell product for use in vivo.