In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations. However, the conventional purification procedure with resin-based packed-bed chromatography suffers from a number of limitations, including variations in pressure, slow pore diffusion, and large bed volumes. Here we have employed disposable high-performance anion- and cation-exchange membrane adsorbers to effectively purify recombinant viruses. As a result of isoelectric focusing analysis, the isoelectric point of empty particles was found to be significantly higher than that of packaged virions. Therefore, AAV vector purification with the membrane adsorbers was successful and allowed higher levels of gene transfer in vivo without remarkable signs of toxicity or inflammation. Electron microscopy of the AAV vector stocks obtained revealed highly purified virions with as few as 0.8% empty particles. Furthermore, the membrane adsorbers enabled recovery of AAV vectors in the transduced culture supernatant. Also, the ion-exchange enrichment of retroviral vectors bearing the amphotropic envelope was successful. This rapid and scalable viral purification protocol using disposable membrane adsorbers is particularly promising for in vivo experimentation and clinical investigations.

Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin ␣9␤1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for ␣9␤1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin ␣9 subunit in leukocytes, indicating that ␣9␤1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN. Osteopontin (OPN),3 an integrin-binding cytokine, plays critical roles in physiological and pathological processes, including inflammation, immunomodulation, tissue remodeling, fibrosis, mineralization (1), stem cell retention (2), and tumor metastasis (3). These functions are exerted through interactions with nine integrins and CD44 (4). OPN undergoes many types of post-translational modification, including phosphorylation (5), glycosylation, transglutamination (6), and proteolytic cleavage. Among these post-translational modifications, transglutamination (7) and thrombin-cleavage (8, 9) enhance interactions with integrins. Thrombin-cleaved N-terminal fragment of OPN (nOPN) has been found to play roles in rheumatoid arthritis (10, 11), autoimmune hepatitis (12), and stem cell retention in the bone marrow niche (2). Although OPN was found as a substrate for transglutaminase 2 (TG2; EC 2.3.2.13) earlier than thrombin, little is known about the functional role of the product of transglutamination, polymeric OPN. TG2 is a cross-linking enzyme that catalyzes isopeptide bonding between Gln and Lys residues (13) with specificity for amino acid sequences containing Gln (...

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Osteopontin (OPN) is a cytokine and ligand for multiple members of the integrin family. OPN undergoes the in vivo polymerization catalyzed by cross-linking enzyme transglutaminase 2, which consequently increases the bioactivity through enhanced interaction with integrins. The integrin ␣9␤1, highly expressed on neutrophils, binds to the sequence SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence appears to be cryptic in intact OPN because ␣9␤1 does not recognize intact OPN. Because transglutaminase 2-catalyzed polymers change their physical and chemical properties, we hypothesized that the SVVYGLR site might also be exposed on polymeric OPN. As expected, ␣9␤1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with ␣9-transfected cells and by detection of direct binding of recombinant soluble ␣9␤1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombincleaved OPN, a ligand for ␣9␤1, has been reported to attract neutrophils, we next examined migration of neutrophils to polymeric OPN using time-lapse microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which was clearly inhibited by anti-␣9␤1 antibody. Unexpectedly, mutagenesis studies showed that ␣9␤1 bound to polymeric OPN independently of the SVVYGLR sequence, and further, SVVYGLR sequence of polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize polymeric OPN. These results demonstrate that polymerization of OPN generates a novel ␣9␤1-binding site and that the interaction of this site with the ␣9␤1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.Acidic phosphorylated secreted glycoprotein osteopontin (OPN), 4 known as a cytokine, has multiple functions, including roles in tissue remodeling, fibrosis, mineralization, immunomodulation, inflammation, and tumor metastasis (1-3). OPN is also an integrin ligand. At least nine integrins can function as OPN receptors. ␣5␤1, ␣8␤1, ␣v␤1, ␣v␤3, ␣v␤5 (1), and ␣v␤6 (4) recognize the linear tripeptide RGD, and ␣9␤1, ␣4␤1, and ␣4␤7 recognize the sequence, SVVYGLR (5), adjacent to RGD but only after OPN has been cleaved by the protease, thrombin (Fig. 1).The overlap of receptors for OPN does not necessarily mean that these integrins play redundant roles in cellular responses to OPN because the patterns of integrin expression and utilization vary widely among cell types. In addition, interactions of different integrins with a single ligand can exert distinct effects on cell behavior in a single cell type. For example, we have previously reported that signals by ligation of ␣v␤3, ␣v␤6, or ␣9␤1 to a single ligand, tenascin-C, differently affected cell adhesion, spreading, and proliferation of the colon cancer cell line, SW480 (6). Furthermore, intact OPN or thrombin-or matrix metalloproteinase-cleaved OPN interact with distinct subsets of integrins and exhibit distinct effects on cell behavior (4,7,8). Collectively, some of the functional diversity...

Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality. Multipotent mesenchymal stromal cells (MSCs) are of interest because of their ability to differentiate to form myogenic cells in situ. In the present study, methods were developed to expand cultures of MSCs and to promote the myogenic differentiation of these cells, which were then used in a new approach for the treatment of DMD. MSC cultures enriched in CD271(+) cells grew better than CD271-depleted cultures. The transduction of CD271(+) MSCs with MyoD caused myogenic differentiation in vitro and the formation of myotubes expressing late myogenic markers. CD271(+) MSCs in the myogenic cell lineage transplanted into dog leukocyte antigen (DLA)-identical dogs formed clusters of muscle-like tissue. Intra-arterial injection of the CD271(+) MSCs resulted in engraftment at the site of the cardiotoxin (CTX)-injured muscle. Dogs affected by X-linked muscular dystrophy in Japan (CXMD(J)) treated with an intramuscular injection of CD271(+) MSCs similarly developed muscle-like tissue within 8-12 weeks in the absence of immunosuppression. In the newly formed tissues, developmental myosin heavy chain (dMyHC) and dystrophin were upregulated. These findings demonstrate that a cell transplantation strategy using CD271(+) MSCs may offer a promising treatment approach for patients with DMD.