The utility of adenoviral vectors for gene therapy is currently limited due, in part, to the widespread distribution of the cellular receptor for the adenovirus fiber that precludes the targeting of specific cell types. In order to develop a targeted adenovirus, it is therefore necessary both to ablate endogenous viral tropism and to introduce novel tropism. We hypothesized that these two goals could be achieved by employing a neutralizing anti-fiber antibody, or antibody fragment, chemically conjugated to a cell-specific ligand. To test this concept, we chose to target the folate receptor, which is overexpressed on the surface of a variety of malignant cells. Therefore, we conjugated folate to the neutralizing Fab fragment of an anti-fiber monoclonal antibody. This Fab-folate conjugate was complexed with an adenoviral vector carrying the luciferase reporter gene and was shown to redirect adenoviral infection of target cells via the folate receptor at a high efficiency. Furthermore, when complexed with an adenoviral vector carrying the gene for herpes simplex virus thymidine kinase, the Fab-folate conjugate mediated the specific killing of cells that overexpress the folate receptor. This work thus represents the first demonstration of the retargeting of a recombinant adenoviral vector via a non-adenoviral cellular receptor.

Spaceflight, aging, and disuse lead to reduced BMD. This study shows that overexpression of constitutively active RhoA restores actin cytoskeletal arrangement, enhances the osteoblastic phenotype, and suppresses the adipocytic phenotype of human mesenchymal stem cells cultured in modeled microgravity.Introduction: Reduced BMD during spaceflight is partly caused by reduced bone formation. However, mechanisms responsible for this bone loss remain unclear. We have previously shown reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells (hMSCs) cultured in modeled microgravity (MMG). The small GTPase, RhoA, regulates actin stress fiber formation and has been implicated in the lineage commitment of hMSCs. We examined the effects of MMG on actin cytoskeletal organization and RhoA activity and the ability of constitutively active RhoA to reverse these effects. Materials and Methods: hMSCs were seeded onto plastic microcarrier beads at a density of 10 6 and allowed to form aggregates in DMEM containing 10% FBS for 7 days. Aggregates were incubated in DMEM containing 2% FBS for 6 h with or without an adenoviral vector containing constitutively active RhoA at a multiplicity of infection (moi) of 500 and allowed to recover in 10% FBS for 24 h. Cells were transferred to the rotary cell culture system to model microgravity or to be maintained at normal gravity for 7 days in DMEM, 10% FBS, 10 nM dexamethasone, 10 mM ␤-glycerol phosphate, and 50 M ascorbic acid 2-phosphate. Results: F-actin stress fibers are disrupted in hMSCs within 3 h of initiation of MMG and are completely absent by 7 days, whereas monomeric G-actin is increased. Because of the association of G-actin with lipid droplets in fat cells, the observed 310% increase in intracellular lipid accumulation in hMSCs cultured in MMG was not unexpected. Consistent with these changes in cellular morphology, 7 days of MMG significantly reduces RhoA activity and subsequent phosphorylation of cofilin by 88 ± 2% and 77 ± 9%, respectively. Importantly, introduction of an adenoviral construct expressing constitutively active RhoA reverses the elimination of stress fibers, significantly increases osteoblastic gene expression of type I collagen, alkaline phosphatase, and runt-related transcription factor 2, and suppresses adipocytic gene expression of leptin and glucose transporter 4 in hMSCs cultured in MMG. Conclusion: Suppression of RhoA activity during MMG represents a novel mechanism for reduced osteoblastogenesis and enhanced adipogenesis of hMSCs.

Adenoviral (Ad) vectors are promising gene therapy vehicles due to their in vivo stability and efficiency, but their potential utility is compromised by their restricted tropism. Targeting strategies have been devised to improve the efficacy of these agents, but specific targeting following in vivo systemic administration of vector has not previously been demonstrated. The distinct aim of the current study was to determine whether an Ad-targeting strategy could maintain fidelity upon systemic vascular administration. We used a bispecific antibody to target Ad infection specifically to angiotensin-converting enzyme (ACE), which is preferentially expressed on pulmonary capillary endothelium and which may thus enable gene therapy for pulmonary vascular disease. Cell-specific gene delivery to ACE-expressing cells was first confirmed in vitro. Administration of retargeted vector complex via tail vein injection into rats resulted in at least a 20-fold increase in both Ad DNA localization and luciferase transgene expression in the lungs, compared to the untargeted vector. Furthermore, targeting led to reduced transgene expression in nontarget organs, especially the liver, where the reduction was over 80%. Immunohistochemical and immunoelectron microscopy analysis confirmed that the pulmonary transgene expression was specifically localized to endothelial cells. Enhancement of transgene expression in the lungs as a result of the ACE-targeting strategy was also confirmed using a new noninvasive imaging technique. This study shows that a retargeting approach can indeed specifically modify the gene delivery properties of an Ad vector given systemically and thus has encouraging implications for the further development of targetable, injectable Ad vectors.

Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

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