**6. Pre-conditioning and transplantation regimens**

A common practice in the field of HSC gene therapy is a transduction of HSCs using viral vectors in the ex vivo setting. The advantages of this strategy include elimination of non-target transduction events, higher transduction efficiency and better control over the overall process. However, the opposite side of the coin in this case is the necessity for transduced cells to compete with the bone marrow-resident ones, which is likely to lower significantly the degree of chimerism after gene therapy. For efficient repopulation of hematopoietic system with genemodified HSCs, extensive myeloablative treatments eliminating resident HSCs are usually performed. However, since these treatments are of generalized character and connected with substantial risks of morbidity and mortality, especially for elderly patients, they should preferably be avoided whenever possible. A combination of nonmyeloablative preconditioning of the recipient animals with in vivo selection strategy can be used to achieve substantial degrees of chimerism (Davis et al., 2000, Zielske et al., 2003). Additional ways to develop more appropriate pretreatment conditions involve the use of molecules that disrupt key signaling pathways within HSCs or niche components thus inducing HSC loss, as was shown for the case of inactivation of c-kit or mpl signaling by neutralizing antibodies (Czechowicz et al., 2007; Yoshihara et al., 2007), and for combined poly(I:C)/5-fluorouracil (5- FU) treatment (Sato et al., 2009). The other approach for nonmyeloablative HSC transplantation is based on disruption of HSC-niche interactions thus aiding in the stem cell mobilization (Chen et al., 2006). This alternative might grow into clinically relevant technique if the efficiency of current protocols for mobilization of HSCs is further improved. The more HSCs are mobilized into circulation and used for viral transduction, the higher is ratio of transduced vs. resident stem cells and better chances to achieve significant engraftment and chimerism of gene-modified cells without resorting to drastic myeloablative regimens. Although current combinations of mobilizing agents (Ramirez et al, 2009) demonstrate much higher mobilization rates than the initially used G-CSF, there is still a long way to go before this strategy may equal or surpass myeloablative pre-conditioning in its efficiency.

Quite promising developments occur currently in the field of 3-D culture (Yuan et al., 2011; Tan et al., 2010; Miyoshi at el., 2011). Despite a relative paucity of data related to the 3-D culture of HSCs, available publications demonstrate significant advantages of this technique and indicate that in combination with correctly chosen or gene-modified stromal cell layers, 3-D culturing may eventually lead to creation of artificial niche that will be able to support

A question of paramount importance for the field is whether specific combinations of soluble factors will be able to attain a bone fide ex vivo expansion of HSCs, or this goal can only be achieved if specific cell surface proteins produced by the niche cells are also employed in the process, or perhaps the only way to the eventual success is the use of supporting stromal cell layers for ex vivo culture? As a number of molecules that contribute to the maintenance of HSCs in vitro and in vivo continues to rise, and there is a steady improvement in techniques for culturing HSCs, chances are that within a matter of a few years, key combination(s) of specific factors and modes of their application that can produce robust self-renewal and expansion of human HSC ex vivo will be identified. Table 3 provides a list, albeit incomplete, of factors and chemicals that, in addition to "classical"

A common practice in the field of HSC gene therapy is a transduction of HSCs using viral vectors in the ex vivo setting. The advantages of this strategy include elimination of non-target transduction events, higher transduction efficiency and better control over the overall process. However, the opposite side of the coin in this case is the necessity for transduced cells to compete with the bone marrow-resident ones, which is likely to lower significantly the degree of chimerism after gene therapy. For efficient repopulation of hematopoietic system with genemodified HSCs, extensive myeloablative treatments eliminating resident HSCs are usually performed. However, since these treatments are of generalized character and connected with substantial risks of morbidity and mortality, especially for elderly patients, they should preferably be avoided whenever possible. A combination of nonmyeloablative preconditioning of the recipient animals with in vivo selection strategy can be used to achieve substantial degrees of chimerism (Davis et al., 2000, Zielske et al., 2003). Additional ways to develop more appropriate pretreatment conditions involve the use of molecules that disrupt key signaling pathways within HSCs or niche components thus inducing HSC loss, as was shown for the case of inactivation of c-kit or mpl signaling by neutralizing antibodies (Czechowicz et al., 2007; Yoshihara et al., 2007), and for combined poly(I:C)/5-fluorouracil (5- FU) treatment (Sato et al., 2009). The other approach for nonmyeloablative HSC transplantation is based on disruption of HSC-niche interactions thus aiding in the stem cell mobilization (Chen et al., 2006). This alternative might grow into clinically relevant technique if the efficiency of current protocols for mobilization of HSCs is further improved. The more HSCs are mobilized into circulation and used for viral transduction, the higher is ratio of transduced vs. resident stem cells and better chances to achieve significant engraftment and chimerism of gene-modified cells without resorting to drastic myeloablative regimens. Although current combinations of mobilizing agents (Ramirez et al, 2009) demonstrate much higher mobilization rates than the initially used G-CSF, there is still a long way to go before

cytokines, are being used for maintenance and expansion of HSCs ex vivo.

this strategy may equal or surpass myeloablative pre-conditioning in its efficiency.

**6. Pre-conditioning and transplantation regimens** 

substantial expansion of human HSCs ex vivo.


Table 3. Proteins and compounds affecting ex vivo maintenance and expansion of HSCs ("classical" cytokines not listed)

Gene Therapy of Hematopoietic and Immune Systems: Current State and Perspectives 451

of various function elements like insulators. Transposon systems are inherently less immunogenic than viral delivery systems, whereas their cargo capacity generally exceeds that of retro- and lentiviral vectors (Zayed *et al.*, 2004). Initial experiments with transposons were plagued by low efficiency of integration, but continuous improvements in molecular design of transposases have significantly increased the efficiency of integration process (Mátés et al., 2009). Currently, transposons based on Sleeping Beauty (SB) system represent the most advanced version of this technology (reviewed by Ivics Izsvák, 2011), although other system such as piggyBac are also being perfected (Yusa et al., 2011) and may offer some advantages, such as larger cargo capacity, over the SB system (Lacoste et al., 2009).

Although stable SB transposon-mediated gene transfer into hematopoietic cells was reported (Xue et al., 2009), efficient vector delivery to HSCs remains poorly resolved issue, which is currently being addressed by using electroporation or hybrid lentiviral-transposon vectors (Staunstrup et al., 2009). Although certain undesired effects such as SB transposase cytotoxicity were observed, it seems that they might be minimized by controllable mRNA delivery (Galla et al., 2011). Compared to lenti- and retroviral vectors that show preferential integration near active genes, SB transposon vectors demonstrate nearly random integration profiles (Moldt et al., 2011), although this property might not be shared by other transposon

Another serious safety concern is a direct consequence of a current low efficiency of transduction of LTR HSCs, which necessitates the use of myeloablative pre-conditioning and negative selection strategies to eliminate competing endogenous HSCs and increase chimerism levels. Negative selection strategies using in particular alkylating drugs place a significant stress upon hematopoietic system. However, as demonstrated by Xie et al., 2010, repetitive hematopoietic stress by busulfan administration in a nonhuman primate may rapidly lead to reduction of polyclonality and eventually to cytopenia. In addition, potential long term mutagenic effects of alkylating agents are largely unknown, thus adding more uncertainty as to correct assessment of risks and benefits of this strategy. Apparently, in order to tackle efficiently the problem of low transduction efficiency, it is not sufficient to rely on the use of negative selection only, but is also important to achieve substantial improvements in ex vivo stem cell culturing, expansion and transduction efficiency. Promising approaches also involve use of positive ex vivo and in vivo selection and in situ

In the recent few years, a group of new exciting and very powerful technologies, namely cell reprogramming using specific combinations of transcription factors and/or micro RNAs appeared (Takahashi & Yamanaka, 2006; Miyoshi et al., 2011). Much hope is invested into development of strategies aiming at derivation of patient-specific induced pluripotent (iPS) cells similar to embryonic stem (ES) cells, with their subsequent differentiation into hematopoetic cells capable of long-term hematopoiesis. In addition to this indirect reprogramming strategy, methods for direct reprogramming that bypass derivation of iPS cells are also being elaborated. There is one report stating that ectopic expression of Oct4 transcription factor in human fibroblasts is sufficient to convert them into hematopoietic cells with in vivo engraftment capacity (Szabo et al., 2010). However, whether the published

systems (Huang et al., 2010).

transduction strategies.

**8. Novel technologies** 

There are reports indicating that the engraftment of gene-modified stem cells might be significantly improved by their direct intra-bone transplantation (Mazurier et al., 2003). As irradiation commonly used for preconditioning also damages hematopoietic niche, in particular mesenchymal stem cells, HSC co-transplantation with MSCs was tested and showed promising results (Masuda et al., 2009).

Even a more radical departure from the accepted strategies for HSCs would be in situ transduction of HSCs using systemic or intra-bone delivery of viral vectors (McCauslin et al., 2003, Pan, 2009). Currently, this is a rather hypothetical approach due to serious safety concerns connected with potential off-target modifications of non-hematopoetic cells. However, this strategy alleviates the need for hazardous pre-conditioning treatments and will become a viable alternative with further development of modified viral envelops (Zhang X & Roth, 2010) that target vectors specifically to hematopoietic stem and progenitor cells while minimizing off-target events.
