Downstream Processing of Oncoretroviral and Lentiviral Gene Therapy Vectors
Development*of*the*NovelFN3*Displayed*Lentiviral Vectors...
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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
Transcript of Development*of*the*NovelFN3*Displayed*Lentiviral Vectors...
Development of the Novel FN3 Displayed Lentiviral Vectors for CD4-‐Targeted In Vivo Gene DeliveryHsu-‐Yu (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 ResultsI. CD4-targeted LVs specifically target CD4+ T cells in unstimulated PBMC
II. CD4-targeted LVs specific target CD4+ T cells and transduce resting and memory T cells in CD34 humanized mice
Figure 1. Comparing different vectors titer andspecificity. (a) Titer of 100-fold concentrated CD4-targetedMeV or NiV with FN3 or DARPin were tested on Molt-4 cells.(b) unstimulated PBMC were transduced with VSV-G orselected CD4-targeted LVs at a MOI of 2. Both CD4 targetedvectors had a markedly enhanced ability to transduce restingT cells compared to the control VSV-G pseudotyped vectors,and this was specific for CD4 expressing cells.
Figure 2. Gene delivery efficiency and specificity of MeV-DARPin and NiV-FN3in CD34 humanized mouse model. (a) NSG mice were engrafted with CD34+cells as pups. Resting and memory T cells were identified with activation andsubtype markers and analyzed by FACS. (b) Vectors were i.v. injected into 16 wksold CD34 humanized mice at the indicated dose. Tissues were harvest one weekafter injection. GFP expression in human CD4+T cells from different tissues wereanalyzed by FACS. (c) Representative FACS plots from the spleen shows CD4target specificity (d) and (e) GFP expression in different T cell subtypes showedboth vectors are able to transduce resting and memory T cells in vivo.
(a) (b) (c)
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Part I & II. Long term transgene delivery by CD4-targeted lentiviral vectors (LV) in vitro and in vivoParamyxovirus 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 vitroTransient expression forsafe gene therapy
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(a) (b)
III. MeV-DARPin based CD4-targeted VLPs and vesicles deliver GFPtransiently and specifically to CD4+ T cells in unstimulated PBMC • Paramyxovirus entry mechanism allows a re-
targeting strategy
• Both MeV-DARPin and NiV-FN3 LVs can target resting and memory CD4+ T cells in vitro and in vivo
• FN3 shows promise as a targeting ligand
• Genome-less targeted VLPs and Vesicles have potential for transient delivery of proteins, including gene editing tools.
SummaryNovel for targeted LV engineering
Figure 3. Transient transgenedelivery to resting PBMC. CD4-targeted VLPs and Vesicles weregenerated to evaluate their ability todeliver GFP as protein instead ofexpressed from a vector genome.Unstimulated PBMC were transducedwith 20ul of CD4-targeted LV, VLP orvesicle. GFP expression was measuredat day 1 and day 6 to examine bothshort and long term expression.
Panel of particle testedwith GFP as reporter
FN3 DARPin FN3100
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Tite
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MeV NiV
Targeting Ligand
