**8. Novel technologies**

450 Advances in Hematopoietic Stem Cell Research

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

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

The genotoxicity issue is currently the most immediate and direct safety concern related to the gene therapy using HSCs. Several otherwise successful gene therapy trials of severe combined immunodeficiency using retroviral vectors have resulted in occurrence of leukemia in a significant percentage of patients. Substantial efforts were thus devoted to elucidation of integration patterns and clonal population structure in the hematopoietic compartment after viral transduction, both in experimental models and in clinical trials. The obtained results, although not unanimous, demonstrate nevertheless a frequent occurrence of oligoclonal hematopoiesis after gene therapy, with viral integration sites tending to concentrate in the vicinity of a limited number of genes preferentially involved in growth and proliferation control such as above mentioned Evi-1, PRDM16 or HMGA2. Although upregulation of these genes rarely led to overt neoplastic transformation, it is nevertheless clear that the patients with oligoclonal hematopoiesis are at substantial risk of acquiring

Various strategies are being currently developed to minimize the risk of neoplastic transformations of HSCs after viral transduction. The most promising approaches include using lentiviral instead of retroviral vectors, and insulators to shield cellular oncogenes from activation by strong viral promoters (Puthenveetil et al., 2004). Insulators, however, tends to significantly reduce viral titers (Nielsen et al., 2009), relatively inefficient (Uchida et al., 2011) and do not provide guarantee against insertional activation of potential oncogenes such as HMGA2 (Cavazzana-Calvo et al., 2010). Another approach is to use promoters specific for differentiated cells that are expected to produce negligible activation of oncogenes in stem cells. However, such promoters tend to provide comparably lower expression levels, and although this might be improved by addition of strong enhancers (Gruh et al., 2008), it is far from certain that such combinations would not activate nearby

Transposon vectors offer an exciting alternative to retro- and lentiviral vectors. The transposon-based gene delivery combines advantages of integrating viral vectors with those of plasmid vectors. Permanent genomic integration of transposon vectors provides longterm expression, whereas there are significantly fewer constraints on vector design and use

**7. Safety: Vector genotoxicity, transposon vectors and other issues** 

showed promising results (Masuda et al., 2009).

cells while minimizing off-target events.

leukemias at some future time point.

cellular promoters.

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

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technique may result in production of bona fide hematopietic stem cells capable of longterm reconstitution, remains to be seen. It should be noted that such a goal has not yet been achieved for ES or iPS cells. If efficient reprogramming into HSCs were possible, the perspectives would look staggering. First of all, since starting primary cell populations such as mesenchymal stem/progenitor cells can be propagated for many generations and are amenable for selection of efficient vector integration events, it will be possible to obtain cell populations in which the majority of reprogrammed HCS-like cells bear functioning transgenes, thus increasing efficiency of gene therapy many-fold. Besides, if this technology were able to generate ex vivo significantly more reprogrammed cells with HSC properties than is possible to obtain from a patient, this would establish basis for a radically increase in a level of chimerism after transplantation, thus further improving the efficiency of gene therapy. Of course, the safety issues, in particular potential epigenetic and genome instability of reprogrammed cells that might result in neoplastic transformations, must be addressed especially carefully in this case.
