**3. Selection of genetically modified HSCs in vivo: Negative selection**

As current efficiency of transduction of human LTR HSCs with viral vectors appears to be quite low and there are no clinically proven protocols for expansion of these cells ex vivo, the most promising solution at hand to this problem is an in vivo selection of modified cells after their transduction and re-transplantation back to a patient. Conceptually, one might distinguish negative and positive in vivo selection strategies. The first one can be defined as a strategy that is aimed at elimination of stem and progenitor cells that do not bear integrated functional constructs. Positive selection implies a strategy that does not target the construct-negative stem cells but rather provides selective survival and growth advantage to the cells that bear the inserted construct. The negative selection gains presently much of attention and seems to be better poised for a clinical advancement in the near future. Arguably, the most promising and advanced variant of negative selection is based on the use of O6-MGMT as a selection marker and various alkylating compounds as selection agents (Davis et al., 2000, Ragg et al., 2000). Using this approach and multiple rounds of selection in vivo, overall peripheral blood chimerism has been driven in mice and larger animal models to levels higher than 75%. However, the clinical applicability of this technique is as yet unclear, as recent experiments performed by two research teams with non-human primates using MGMT-mediated selection produced rather conflicting results. One team demonstrated successful implementation of this strategy in monkeys, although with selection efficiencies and chimerism rates highly variable between individual animals (Beard et al., 2010), whereas another team reported a rather negligible increase in chimerism rates upon selection in vivo (Larochelle et al., 2009).

Various implementations of negative selection strategy are listed in the Table 1.
