Adeno-associated viral vector transduction of human mesenchymal stem cells

Abstract

Mesenchymal stem cells (MSCs) have received considerable attention in the emerging field of regenerative medicine. One aspect of MSC research focuses on genetically modifying the cells with the aim of enhancing their regenerative potential. Adeno-associated virus (AAV) holds promise as a vector for human gene therapy, primarily due to its lack of pathogenicity and low risk of insertional mutagenesis. However, the existing data pertaining to AAV transduction of MSCs is limited.The objective of this work was to examine the efficiency and kinetics of in vitro transduction using AAV serotype 2 in human MSCs and to assess whether AAV transduction affects MSC multipotentiality. The results indicated that human MSCs could indeed be transiently transduced in vitro by the AAV2 vector with efficiencies of up to 65%. The percentage of GFP-positive cells peaked at 4 days posttransduction and declined rapidly towards 0% after day 8. The level of transgene expression in the GFP-positive population increased 4-fold over a 10,000 fold viral dose increase. This dose-response contrasted with the 200-fold increase observed in similarly transduced 293-cells, indicating a relatively restricted transgene expression in MSCs following AAV mediated gene delivery. Importantly, transduced MSCs retained multipotential activity comparable to untransduced controls.

High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

“…In contrast, a vector dose-dependent enhancement in transduction efficiency of 1-65% has been reported in human MSCs, but those authors concluded that host cellular barriers may contribute to the relatively restricted transgene expression observed in MSCs (Stender et al, 2007). Importantly, the AAV2 vector-transduced MSCs in this study retained their multipotential activity compared with the untransduced controls (Stender et al, 2007). Similarly, in fibroblasts, we and others have reported *30-fold lower levels of AAV2 DNA replication when compared with 293 or HeLa cells (Hansen et al, 2000;Bhrigu and Trempe, 2009).…”

“…In murine fibroblasts, the ratelimiting step for AAV-mediated transgene expression appears to be the intracellular trafficking and capsid uncoating steps, as we have demonstrated earlier (Hansen et al, 2000). And, in MSCs, the efficiency of AAV2 vector-mediated transduction has so far been not optimal ( Ju et al, 2004;Kumar et al, 2004;McMahon et al, 2006;Stender et al, 2007). Thus, it is critical to develop novel AAV vectors that can target and transduce both of these cell types at highefficiency, for their widespread applicability in combinatorial gene and cell transfer applications.…”

“…However, despite continuous improvement in vector technology, efficient and specific delivery of a therapeutic gene to the target cells still remains a major block in clinical gene transfer studies. Alternatively, stem cells, including the hematopoietic stem cells (HSCs), MSCs, and more recently iPS cells, are considered attractive targets for ex vivo modification with gene therapy vectors for the potential cellular and gene therapy of inherited and acquired disorders (Takahashi and Yamanaka, 2006;Zhong et al, 2006;Stender et al, 2007). These cells possess the properties of self-renewal, are capable of differentiating into multiple lineages, have the ability to home to sites of injury, and, most importantly, are hypoimmunogenic when ex vivo transduced, making allogeneic transplantation of these cells a possibility.…”

“…A comparative study of viral and nonviral vectors showed that AAV serotypes 1, 2, 4, 5, and 6 were least effective in rat and rabbit MSCs compared with other means of gene transfer (McMahon et al, 2006). In contrast, a vector dose-dependent enhancement in transduction efficiency of 1-65% has been reported in human MSCs, but those authors concluded that host cellular barriers may contribute to the relatively restricted transgene expression observed in MSCs (Stender et al, 2007). Importantly, the AAV2 vector-transduced MSCs in this study retained their multipotential activity compared with the untransduced controls (Stender et al, 2007).…”

Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells

“…[1][2][3][4][5] The multipotential of MSCs, their relatively easy isolation, culture and ex vivo expansive potential have attracted considerable attention in efforts to develop cell and gene therapies and have made these cells an attractive therapeutic tool in a wide range of clinical applications. 6 During the past decade many different viral vectors including retrovirus, 7 adenovirus, [8][9][10][11][12][13] lentivirus [13][14][15][16] and adeno-associated virus 13,[17][18][19][20] have been utilized to deliver genes to or modify genes in MSCs. Although the viral gene delivery to MSCs has made an impressive advancement toward clinical applications, 21 several drawbacks exist.…”