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- Title
442. LV Expressing MR Reporter Genes Allows In Vivo Monitoring of Stem Cell Gene Therapy.
- Authors
Amendola, Mario; S. Politi, Letterio; Cadioli, Marcello; Galli, Rossella; Binda, Elena; Falini, Andrea; Levi, Sonia; Scotti, Giuseppe; Biffi, Alessandra; Naldini, Luigi
- Abstract
Somatic stem cells (SSC) have raised interest because of their therapeutic potential in both cell-based and gene therapy applications. Towards this goal, tracking the fate of either delivered cells or of genetically modified endogenous cells is of utmost importance. Diverse imaging approaches are available for cell tracking and among these MRI shows a greater resolution and allows direct anatomic correlation and long-term studies of dynamic cell migration on living animals. Superparamagnetic iron oxide (SPIO) has been used to label SSC in vitro and to make them detectable in vivo upon transplantation. However, major limitations of this approach are the progressive dilution of the contrast media among cell progeny and the need for ex vivo SPIO loading. We thus explored an alternative strategy based on the combination of lentiviral vectors (LV), which efficiently transduce SSC both ex vivo and in vivo and allow long-term expression in their progeny, and MR reporter genes, able to increase iron uptake and accumulation into different cell types.We tested human tyrosinase and human ferritin chains in the context of bi-directional LVs carrying each MR reporter gene together with GFP.These LVs were first used on cell lines to analyze protein expression, iron accumulation and to compare the different MR reporter genes. Transduced cells became detectable in T2 and T2? weighted images, concomitantly with the appearance of melanin or iron within cellular bodies, and fluorescence for GFP expression.Similar experiments were performed on primary human SSC, like hematopoietic and neural stem cells. Viability, proliferation and differentiation capacity were preserved after gene transfer of the MR genes.The new LVs were then injected both into the mouse brain striatum to mark resident neurons, and into the sub ventricular zone (SVZ) to label neural progenitor cells (NPC) and their progeny.Injected mice underwent serial MR examinations by a 3 Telsa human scanner and were eventually analyzed for protein expression in the brain by immunofluorescence. MR reporter expression was detectable in vivo in the striatum from 1 week up to 3 months (latest time of analysis) from the time of vector injection. Pathology confirmed co expression of GFP and the reporter gene. Upon SVZ delivery, we are currently following the migration of genetically modified NPC progeny along the rostral migratory stream to the olfactory bulb. Overall these data suggest that expression of MR marker genes enables efficient cellular marking for MR localization studies and that LV transduction represents a promising strategy for in vivo long term monitoring of stem cell fate and their progeny. Furthermore, the use of MR reporter gene in a bi-directional vector together with gene of interest will allow studies coupling imaging to the administration of a bioactive molecule.Molecular Therapy (2006) 13, S170–S170; doi: 10.1016/j.ymthe.2006.08.509
- Subjects
REPORTER genes; GENE therapy; STEM cells; MAGNETIC resonance imaging; CELL migration
- Publication
Molecular Therapy, 2006, Vol 13, pS170
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.509