Human adult mesenchymal stem cells (hMSC) are under active investigation as

Human adult mesenchymal stem cells (hMSC) are under active investigation as cellular carriers for gene therapy. EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSC was selected based on antibiotic resistance and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSC. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly human MSC expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells and at least 7-fold increased retention in human U87-EGFRvIII expressing tumors In addition hMSC possess the plasticity and ability to differentiate into multiple cell types under appropriate cell culture conditions (Pittenger 1999; Pittenger and Martin 2004). The genetic modification of MSC has been performed using various approaches and each of these methods has advantages and drawbacks. hMSC have been shown to be prone to viral infection and as such adenoviruses encoding a protein of interest have been used for genetic engineering of MSC to express therapeutic proteins. However such an approach does not allow efficient and stable incorporation of a gene of interest in the host genome and over time expression is lost (Harui 1999). Retro- and lentiviruses on another hand are able to stably integrate a gene of interest into the genome of hMSC but the cells expressing these viral proteins could be immunogenic in animals and humans (Cherry 2000). In contrast nonviral methods such as lipofection have been shown to be inefficient at transducing hMSC (Gheisari 2008). Additionally electroporation requires the use high concentration of DNA and is not favorable for cell viability (Helledie 2008). Recently developed nucleofection technology overcomes these limitations and allows the introduction of a gene directly in the PNU-120596 nucleus of difficult to transfect primary cells and produce reasonable viability and efficiency of transfection (Aluigi 2006). To date multiple attempts to utilize MSC as cellular vehicles to deliver therapeutic molecules to tumors have been described. Our group and others have demonstrated the ability of hMSC to deliver viral loads (Sonabend 2008) interferon-β (Nakamizo 2005) IL-12 (Eliopoulos 2008) IL-2 (Nakamura 2004) cytosine deaminase (Kucerova 2008) and NK4 an antagonist of hepatocyte growth factor (Kanehira 2007) to tumors. It has been demonstrated that hMSC may persist for prolonged time within the tumor environment contribute to the stroma of tumors (Studeny 2002) and engraft in neovasculature of the tumor (Beckermann 2008; Bexell 2009). However the targeting of drugs/genes to a specific cell population remains a challenging task. Antibody mediated targeting of drugs and genes to specific cells population has been a long-term PNU-120596 interest and there has been some success in Rabbit polyclonal to ZNF101. the targeting of tumors with PNU-120596 antibodies or engineered derivatives (Kioi 2008; Modjtahedi 2003). However the fast clearance of small antibody fragments or poor penetration of antibodies through the tumor requires multiple injections and limits their therapeutic potential. The use of therapeutic scFv by engineering tumor cells or by modification of hMSC might overcome this problem (Compte 2008). Previously several attempts were made to express scFv on the surface of tumor cells. The anti-CD3 scFv was expressed on the cell surface of colon cancer cells and able to induce cytotoxic lymphocytes (Liao 2000). The anti-4-1BB scFv was successfully expressed on hepatoma cells and shown to mediate immune activity and anti-tumor effect (Liu 2008). Radiometal chelates binding single-chain antibodies were also expressed on the surface of U87 human PNU-120596 glioma cells as a reporter system for PET imaging (Wei 2008). In addition the scFv display on the surface of mammalian cells was proposed as a method for selection of high affinity antibodies (Ho and Pastan 2009). In this study we sought to investigate the feasibility of. PNU-120596