Specific gene delivery to liver sinusoidal and artery endothelial cells

Abstract

Key Points• CD105-mediated cell entry using targeted lentiviral vectors leads to specific gene transfer of LSEC upon systemic administration.Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors. (Blood. 2013;122(12):2030-2038

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

“…The resulting CHO-EpCAM bulk cell population was stained for EpCAM expression and a single clone named CHO-EpCAM H1 was selected for further experiments. For the generation of MDA-MB-453-luc cells, MDA-MB-453 cells were transduced with lentiviral vectors transferring the luciferase gene 44 at a multiplicity of infection of 0.5. A single-cell clone was analysed for luciferase and Her2/neu expression and was used for in vivo experiments.…”

Exclusive Transduction of Human CD4+ T Cells upon Systemic Delivery of CD4-Targeted Lentiviral Vectors

“…The in vivo distribution of CD4-LV is likely determined by CD4 receptor densities and blood circulation kinetics. We know from previous work (14,17) that transduction efficiency positively correlates with the cell-surface density of the targeted receptor. This holds true also for CD4-LV, because the transduced LN-, spleen-, and blood-derived CD4 + T cells express high CD4 densities, whereas thymus-derived CD4 + cells that are lower in CD4 densities (Fig.…”

“…The MV hemagglutinin (H) is fused to a targeting domain that can be a single-chain Ab (scFv) or a designed ankyrin repeat protein (DARPin) (9) recognizing a defined Ag expressed on the surface of the cell type of interest. Different receptor-targeted LVs have been generated being specific for cell types such as CD8 + T cells (10), B cells (11,12), dendritic cells (13), endothelial cells (14), hematopoietic stem cells (HSCs) (8,15), neurons (8), and tumor cells (16,17). Besides the high flexibility for targeting, a unique property of MV pseudotyped LVs is their capacity to efficiently transduce resting lymphocytes, whereas LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-LV) are unable to do so due to lack of receptor expression (11,(18)(19)(20)(21)(22).…”

“…HIV-1-derived transfer vector plasmids encoding GFP (pSEW) (12), membrane-associated peptide C46 (maC46) and GFP (M589) (25), luciferase (luc) and GFP (pS-luc-GFP-W) (14), or ErbB2-specific chimeric Ag receptor (pS-ErbB2-CAR-W) (26) have been described previously. For the generation of plasmids encoding FOXP3 and ΔNGFR, the reading frames of human FOXP3 (Sino Biological) and ΔNGFR (kindly provided by Dr. Ute Modlich, Paul-Ehrlich-Institut) were PCR amplified and inserted into pS-luc-GFP-W by sticky-end-ligation using BamHI/SbfI and RsrII/ NdeI restriction sites resulting in plasmid pS-FOXP3-ΔNGFR-W.…”

“…In vivo images were taken using an IVIS Spectrum device (Caliper Life Science) and analyzed using the Living Image 4 software (PerkinElmer) as described previously (14).…”