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  1. A. M. Skinner1,
  2. L. O'Neill1,
  3. Y.-W. Pan1,
  4. P. Kurre1
  1. 1Departments of Pediatrics and Cell and Developmental Biology, Oregon Health & Science University, Portland, OR.


Human immunodeficiency virus (HIV)-based lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are attractive vehicles for gene transfer to hematopoietic stem cells due to their broad host range and efficient cellular uptake. Ex vivo transduction culture of target cells and vector particles results in cellular uptake and successful genomic integration, or degradation. We have recently described the persistence of VSV-G lentivector particles in hematopopietic cells after ex vivo exposure and their ability to transduce secondary targets (Pan et al, J. Virol, in press). To distinguish cellular retention by prolonged adherence from actual cellular uptake, SupT1 (ie, “carrier”) cells were exposed for 1 hour to GFP-expressing lentivector at 37°C or 4°C (preventing uptake), followed by wash in PBS and subsequent co-culture with 293T fibroblasts. GFP expression was seen in 10% of 293T cells, confirming prolonged adherence of particles to carrier cells. This stands in contrast to vector exposure followed by pronase treatment (for proteolytic degradation of surface-bound particles), which resulted in GFP expression in only 0.1 to 1% of co-cultured 293T cells exposed to vector at 37°C, yet no discernable marking after vector exposure at 4°C and pronase treatment. Studying the kinetics of particle transfer and secondary transduction under these conditions, we observed a steady increase in secondary transduction (up to 10%) correlating with extended vector exposure times (up to 12 hours) of the SupT1 carrier cells. These data argue that viral particles remain cell bound, are progressively released from carrier cells, and maintain infectivity to secondary targets. Based on their extended half-life after ex vivo exposure to cells and differential secondary transduction after vector exposure at 4°C versus 37°C, a small number of particles also appear to be internalized and subsequently released. To explore the potential intracellular uptake and of particles postexposure to SupT1 cells, we next generated lentivector particles tagged with a vpr-GFP construct and detected persisting, cytoplasmically located particles inside cells by deconvolution microscopy. Taken together, our observations suggest that alternate fates, in addition to integration or degradation, exist for VSV-G vector particles after ex vivo exposure of hematopoietic cells. These particles persist intracellularly and remain capable of secondary transduction events upon release. Our observations have important implications for models using lentiviral vectors as research or therapeutic tools.

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