Development of the Novel FN3 Displayed Lentiviral Vectors for CD4Targeted In Vivo Gene Delivery HsuYu (Camille) Chen, G. Nick Llewellyn, Geoffrey L. Rogers, Krishna Patel, Paula M. Cannon Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. Background Results I. CD4targeted LVs specifically target CD4+ T cells in unstimulated PBMC II. CD4targeted LVs specific target CD4+ T cells and transduce resting and memory T cells in CD34 humanized mice Figure 1. Comparing different vectors titer and specificity. (a) Titer of 100fold concentrated CD4targeted MeV or NiV with FN3 or DARPin were tested on Molt4 cells. (b) unstimulated PBMC were transduced with VSVG or selected CD4targeted LVs at a MOI of 2. Both CD4 targeted vectors had a markedly enhanced ability to transduce resting T cells compared to the control VSVG pseudotyped vectors, and this was specific for CD4 expressing cells. Figure 2. Gene delivery efficiency and specificity of MeVDARPin and NiVFN3 in CD34 humanized mouse model. (a) NSG mice were engrafted with CD34+ cells as pups. Resting and memory T cells were identified with activation and subtype markers and analyzed by FACS. (b) Vectors were i.v. injected into 16 wks old CD34 humanized mice at the indicated dose. Tissues were harvest one week after injection. GFP expression in human CD4+T cells from different tissues were analyzed by FACS. (c) Representative FACS plots from the spleen shows CD4 target specificity (d) and (e) GFP expression in different T cell subtypes showed both vectors are able to transduce resting and memory T cells in vivo. (a) (b) (c) (e) Part I & II. Long term transgene delivery by CD4 targeted lentiviral vectors (LV) in vitro and in vivo Paramyxovirus engineering to create targeted LV Zhou et al. 2015 Compare MeV and NiV for targeted LV engineering Ligands for targeted LV engineering Part III. Transient transgene delivery by CD4 targeted VLP and vesicle in vitro Transient expression for safe gene therapy (d) (a) (b) III. MeVDARPin based CD4targeted VLPs and vesicles deliver GFP transiently and specifically to CD4+ T cells in unstimulated PBMC • Paramyxovirus entry mechanism allows a re targeting strategy • Both MeVDARPin and NiVFN3 LVs can target resting and memory CD4+ T cells in vitro and in vivo • FN3 shows promise as a targeting ligand • Genomeless targeted VLPs and Vesicles have potential for transient delivery of proteins, including gene editing tools. Summary Novel for targeted LV engineering Figure 3. Transient transgene delivery to resting PBMC. CD4 targeted VLPs and Vesicles were generated to evaluate their ability to deliver GFP as protein instead of expressed from a vector genome. Unstimulated PBMC were transduced with 20ul of CD4targeted LV, VLP or vesicle. GFP expression was measured at day 1 and day 6 to examine both short and long term expression. Panel of particle tested with GFP as reporter FN3 DARPin FN3 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 Titer (TU/ml) MeV NiV Targeting Ligand