**2. Soluble TRAIL**

Unlike other apoptosis-inducing TNF family members, soluble TRAIL appears to be inactive against normal healthy tissue (Ashkenazi et al., 1999; Lawrence et al., 2001). A variety of preclinical data clearly show that soluble TRAIL is a cancer cell-specific molecule exerting a remarkable antitumor activity both in vitro (Ashkenazi et al., 1999; Gazitt, 1999; Jin et al., 2004; Mitsiades et al., 2001; Pollack et al., 2001; Rieger et al., 1998) as well as in vivo in athymic nude mice or in non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice (Ashkenazi et al., 1999; Daniel et al., 2007; Kelley et al., 2001).

The physiological functions of TRAIL are not yet fully understood, but mouse gene knockout studies indicate that this agent has an important role in antitumor surveillance by immune cells, mediates thymocyte apoptosis, and is important in the induction of autoimmune diseases (Cretney et al., 2002; Lamhamedi-Cherradi et al., 2003; Smyth et al., 2003).

TRAIL signals by interacting with its receptors. So far, five receptors have been identified, including the two agonistic receptors TRAIL-R1 (Pan et al., 1997b) and TRAIL-R2 (Walczak et al., 1997), and the three antagonistic receptors (Sheridan et al., 1997) TRAIL-R3 (Pan et al.,

Targeting TRAIL Receptors with Genetically-Engineered CD34+ Hematopoietic Stem Cells 663

its short half-life and the consequent short exposure of tumor cells to the molecule (Ashkenazi et al., 2008). Because of soluble TRAIL's short half-life (Ashkenazi et al., 1999; Kelley et al., 2001; Walczak et al., 1999), it seems unlikely that the recommended soluble TRAIL dose of 8 mg/kg body weight will allow adequate exposure of tumor cells at high drug concentrations (Ashkenazi et al., 2008). Strategies to enhance the therapeutic activity of soluble TRAIL include combining it with conventional chemotherapy (Ballestrero et al., 2004) or with new agents such as histone deacetylase inhibitors that upregulate TRAIL-R1

Gene therapy approaches have also been proposed to enhance TRAIL-mediated tumor cell targeting. Recently, a TRAIL-expressing adenoviral vector (Ad-TRAIL) has been shown to cause direct tumor cell killing, as well as a potent bystander effect through presentation of TRAIL by transduced normal cells (Lee et al., 2002). Thus, using Ad-TRAIL might be an alternative to systemic delivery of soluble TRAIL possibly resulting in better tumor cell targeting and increased tumoricidal activity (Armeanu et al., 2003; Griffith et al., 2000; Griffith & Broghammer, 2001; Kagawa et al., 2001; Lee et al., 2002). However, systemic Ad-TRAIL-based gene therapy requires efficient infection of target tumor cells as well as avoidance of immune clearance, and is limited by several safety and toxicity issues related to intravenous adenovector administration (Harrington et al., 2002). Intratumoral injection of TRAIL-encoding adenovectors has been successfully explored in a number of experimental models; however, this approach results in local antitumor activity and has

Alternatively, cell-based vehiculation of the full-length, membrane-bound (m)TRAIL (Griffith et al., 2009) has been proposed to achieve an optimal systemic delivery. Indeed, genetically modified stem/progenitor cells represent an innovative approach for delivery of anticancer molecules (Harrington et al., 2002; Introna et al., 2004). Due to their homing properties, systemically injected stem/progenitor cells could infiltrate both primary and metastatic tumor sites, thus allowing tumor-specific targeting (Burger & Kipps, 2006; Jin et al., 2006; Kaplan et al., 2007; Kucia et al., 2005; Loebinger et al., 2009; Najbauer et al., 2007; Rafii et al., 2002), and potentially overcoming limitations inherent to the pharmacokinetic profile of soluble drugs (Aboody et al., 2008; Griffith et al., 2009; Sasportas et al., 2009). Neural or mesenchymal stem cell-mediated mTRAIL delivery has been investigated in solid tumors (Grisendi et al., 2010; Kim et al., 2008; Loebinger et al., 2009; Menon et al., 2009;

In order to optimize the use of TRAIL-encoding adenovectors for the treatment of systemic tumors, we have recently investigated a cell-based approach using mobilized CD34+ hematopoietic cells transduced with a replication-deficient Ad-TRAIL (CD34-TRAIL+) encoding a full-length mTRAIL under the control of the CMV promoter (Carlo-Stella et al., 2006; Griffith et al., 2000). Several lines of evidence support the use of gene-modified CD34+ cells as optimal vehicles of antitumor molecules. In fact, CD34+ cells are already widely used in the clinical setting. Additionally, they can migrate from the bloodstream into tumor tissues due to the expression of adhesion receptors that specifically interact with counterreceptors on endothelial cells in the tumor microenvironment (Burger & Kipps, 2006; Kaplan et al., 2005; Verfaillie, 1998). Moreover, up-regulation of inflammatory chemo-attractants in the tumor microenvironment provides with a permissive milieu that potentially allows for homing of systemically delivered CD34-TRAIL+ cells and efficient tumor targeting (Jin et al., 2006). Using a multiplicity of infection (MOI) of 500, the transduction protocol optimized for the transduction of CD34+ cells consistently results in a transduction efficiency higher

and/or TRAIL-R2 (Inoue et al., 2004).

Mohr et al., 2008; Uzzaman et al., 2009).

little, if any, value in the treatment of disseminated tumors.

1997a), TRAIL-R4 (Degli-Esposti et al., 1997), and osteoprotegerin (OPG) (Emery et al., 1998). Both TRAIL-R1 and TRAIL-R2 are type I transmembrane proteins containing a cytoplasmic death domain (DD) motif that engage apoptotic machinery upon ligand binding (Almasan & Ashkenazi, 2003), whereas the other three receptors either act as decoys or transduce antiapoptotic signals (Wang & El-Deiry, 2003). TRAIL-R3 and TRAIL-R4 have close homology to the extracellular domains of agonistic receptors. TRAIL-R4 has a truncated, nonfunctional cytoplasmic DD, while TRAIL-R3 exists on the plasma membrane as a glycophospholipid-anchored protein lacking the cytosolic tail. The physiological relevance of OPG as a soluble receptor for TRAIL is unclear, but a recent study suggests that cancer-derived OPG may be an important survival factor in hormone-resistant prostate cancer cells (Holen et al., 2002).
