Human Hematopoietic (CD34+) Stem Cells Possess High Affinity Receptors for Adenovirus Type 11p

ADENOVIRUS BIOLOGY AND DISEASE APPLICATIONS purpose of this study is to demonstrate the feasibility and efficacy of adenovirus-mediated gene transfer into bone cells in vitro and in vivo. HeLa, human embryonic palatal mesenchymal (HEPM), and primary human osteoblast cells from cancellous bone were transduced with adenovirus expressing green fluorescent protein (GFP) or murine erythropoietin (mEPO). The efficiency of adenoviral transduction was determined by calculating the percentage of GFP positive cells using FACS analysis. To determine the duration of expression, media from the cells expressing mEPO were collected over thirty days and gene expression was quantitatively determined by ELISA. For in vivo studies, mouse tibia was directly injected intra-bone marrow with adenovirus expressing either mEPO or β-galactosidase (lacZ). The hematocrit of the mice was measured once a week for three weeks. Based on the data from FACS analysis, our studies show that HeLa, HEPM and human osteoblasts can be transduced with adenovirus. The transduction efficiency of HeLa cells by adenovirus is known to be high. In comparison to HeLa cells, the transduction efficiency of both HEPM and human osteoblasts is low. HEPM had less than half the number of positive GFP cells than HeLa (40.1%), and human osteoblasts had less than one seventh (13.7%). The results of the ELISA assay for mEPO show that gene expression in human osteoblasts is more persistent than in HEPM cells. The concentration of mEPO expression in HEPM peaked by day 10 (517.7mIU/ML) and returned to near baseline by day 31. In contrast, the concentration of mEPO secreted by human osteoblasts stayed near its peak level (350mIU/mL) throughout the study period. The injection of mice tibia bone with adenovirus expressing mEPO resulted in a gradual increase in hematocrit from 48.6% to 83.9% at 3 weeks in all mice, while control Ad-lacZ injected mice remained at baseline level. Our results show that adenovirus-mediated gene transfer to osteoblasts can be a useful method for gene therapy in bone diseases.

766. Human Hematopoietic (CD34+) Stem Cells Possess High Affinity Receptors for Adenovirus Type 11p Ya-Fang Mei,1 Anna Segerman,1 Kristina Lindman,1 Per Hörnsten,2 Anders Wahlin,2 Göran Wadell.1 1 Virology; 2Internal Medicine, Umeå University, Sweden. Gene transfer into human hematopoietic stem cells using Ad5 has been shown to be inefficient due to lack of the primary receptor CAR and the secondary receptors avβ3 integrin and avβ5 integrin, and due to the high seroprevalence of Ad5 antibodies in most adults, resulting in diminished gene transduction. In the present study, we screened 6 species (species A-F) of adenovirus, displaying different tropisms for interaction with CD34+ cells, at the level of virus attachment and expression. Virus particles were biotinylated and their binding capacity was determined by FACS analysis using streptavidin-FITC. Ad11p, Ad35 and Ad3 (species B) showed high binding affinity, while Ad7, Ad11a (species B) and Ad37 (species D) displayed intermediate affinity. Virions of Ad4 (species E), Ad5 (species C), Ad31(species A) and Ad41(species F) hardly bound to hematopoietic progenitor cells. By using a double-labeling system, we demonstrated that adenoviruses did bind to quiescent CD34+ cells. Ad11p virions showed the highest affinity. We further confirmed that virus fiber-specific receptors were present on the hematopoietic progenitor cell surface, since both recombinant fiber of Ad11p and specific antiserum against rfiber could block virus attachment. The ability of Ad11p, Ad35 and Ad5 to infect hematopoietic cells was studied by immunofluorescence staining. Ad11p and Ad35 showed higher infectivity than Ad5. Thus, we have confirmed that these cells have high affinity receptors for species Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

B:2 human adenoviruses Ad11p and Ad35, and these viruses may be used as candidate vectors to target therapeutic genes to hematopoietic stem cells.

767. Intact Microtubules Are Required for AdVector Induction of Chemokine Genes in Epithelial Cells Daniel A. Muruve,1 Lindsay R. White.1 Medicine, University of Calgary, Calgary, AB, Canada.

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Upon entry in epithelial cells, adenovirus vectors (Ad-vectors) activate a cascade of signaling pathways including p38 MAPK (p38) and extracellular-signal regulated kinase (ERK). These signaling events occur within minutes and are triggered directly by the viral capsid. The activation of signaling by Ad-vectors leads to the expression of chemokine inflammatory genes such as IP-10. Shortly following internalization, Ad-vectors interact with microtubules that are required to mediate viral translocation to the nucleus. To better understand the role of microtubules in Ad vector induced signaling and chemokine gene expression, epithelial cells (REC) were treated with the microtubule depolymerizing reagent nocodazole (20mM). Nocodazole substantially reduced transduction of REC cells by a first generation Ad-vector, AdΒgal, as determined by Β-galactosidase expression 24 hours post-transduction. Vector internalization into REC however was unaffected as measured by flow cytometry confirming that microtubules were required to mediate nuclear translocation of Ad-vectors. To determine the effect of microtubule depolymerization on the induction of chemokine mRNA expression, RANTES and IP-10 gene expression was determined at 6 hours following transduction with AdΒgal. Compared to untreated cells, nocodazole blocked the AdΒgal induction of RANTES and IP-10 expression. Phosphorylated ERK or p38 was increased by nocodazole in the absence of Ad-vector transduction, a response that was not changed following the addition of AdΒgal. Although indirect, this finding suggests that p38 and ERK signaling is linked to microtubules and is required but not sufficient to induce the expression of IP-10 and RANTES. In contrast, experiments with the microtubule-stabilizing agent, paclitaxel, did not affect AdΒgal transduction of REC cells. Furthermore, AdΒgal induction of p38/ ERK phosphorylation and IP-10/RANTES gene expression was unchanged following paclitaxel suggesting that microtubule treadmilling or dynamic instability was not required for Ad-vector induction of host immune responses. These preliminary data suggest that intact microtubules are required for the Ad-vector induction of host inflammatory genes in epithelial cells. Understanding the biology of Ad-vector induced intracellular signaling will contribute significantly to the development of novel recombinant vectors for gene therapy.

768. Activity of the Extracellular Domain of the Coxsackie and Adenovirus Receptor Katherine J. D. A. Excoffon,1 Geri L. Traver,1 Nicholas Gansemer,1 Joseph Zabner.1 1 Department of Internal Medicine, University of Iowa, Iowa City, IA. The Coxsackie virus and Adenovirus Receptor (CAR) plays a dual role as a viral receptor and a homotypic junctional adhesion protein. CAR is a transmembrane protein and a member of the Immunoglobulin superfamily with two extracellular Ig-like domains. According to x-ray crystallographic data, the most distal Ig-like domain (D1) mediates the homophilic interaction. This same domain is also responsible for the high affinity binding of the adenovirus (Ad) fiber protein. Currently no activity has been ascribed to the

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