Abstract

Abstract 3625

Poster Board III-561

The hematopoietic microenvironment plays an important role during stem cell homeostasis and in disease. With an improved understanding, new ways to modulate cellular and molecular targets in the microenvironment have become increasingly important. Cell-derived microvesicles (exosomes) have recently attracted interest in immune and cancer biology for their ability to transmit protein and nucleic acid content between cells. We described the ability of VSV-G pseudotyped lentivector to access intracellular multivesicular bodies (MVB) in primary (hematopoietic and non-hematopoietic) cells, with subsequent transfer to secondary (2°) cells (Skinner et. al., PLoS One, 2009). Specifically, we demonstrated that during vector trafficking in non immune target cells, a minority of replication incompetent particles are sequestered in a non-canonical, tetraspanin-associated cytoplasmic compartment. Particles are refractory to envelope (VSV-G) neutralization, susceptible to pharmacological inhibition of PI-3 kinase and capable of transmission to 2° cells. We therefore hypothesized that lentivector particles captured in exosomes and delivered to the bone marrow via homed hematopoietic carriers could be used for the deliberate genetic modification of the hematopoietic microenvironment. To ascertain trafficking of lentivector in exosomes, we imaged particles within vesicles using electron microscopy of vector-exposed human K562 myeloid leukemia cells. In addition, we exposed K562 cells to GFP-vpr fusion protein tagged vector particles and the exosome tracer N-rhodamine- phosphatidylethanolamine (N-Rh-PE), purified exosomes and observed colocalization of GFP-vpr (particles) with N-Rh-PE (exosomes) by deconvolution fluorescent microscopy. We next wished to test the exosomal delivery for the genetic marking of murine stromal cells in situ. We isolated whole bone marrow from CD45.1 mice, exposed these cells to GFP transfer vector for 1 hour (MOI 5), removed residual surface-bound particles and injected cells into tail veins of five non-irradiated, congenic CD45.2 animals. At the time of sacrifice, whole bone marrow, spleen, peripheral blood, and multiple other organs were analyzed by quantitative real time PCR, revealing preferential vector marking in the hematopoietic organs. To determine if lentivector particles were delivered to non-hematopoietic elements in the bone marrow, adherent stromal cells were isolated 15 weeks after transplantation, plated at varying densities per well in 6-well plates, and cultured in vitro. We generated 13-15 CFU-F colonies per 1×105 cells and used fluorescent microscopy to image GFP expression. We observed GFP-expressing stromal cells from an initial cohort of 5 animals, ranging in frequency from 24-87% of pooled CFU-F cells examined. Using semi-quantitative PCR, we amplified GFP sequence from individual colonies, confirming that these stromal cells were genetically modified in all animals. To confirm that the isolated stromal cells were of host origin, we developed a single nucleotide polymorphism (SNP) PCR strategy to differentiate cells of donor versus host origin. Additional cohorts are undergoing analysis. In conclusion, we propose a model in which lentivector is trafficked into exosomes and delivered to hematopoietic microenvironment via homed carrier cells. Future experiments will optimize the efficiency of delivery. Successful cellular delivery of rationally designed vector particles to the hematopoietic microenvironment may offer one potential strategy to facilitate manipulation of the stem cell niche in vivo.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.