Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell

Abstract

Many applications of hematopoietic gene therapy require selection for clones with active transgene expression. However, it was unclear whether the clonal progeny of a retrovirally transduced hematopoietic stem cell would be capable of maintaining transgene expression through serial repopulation and multilineage differentiation. Such investigations require simultaneous analyses of clonality, multilineage activity and transgene copy numbers. Using a mouse model, the present study demonstrates that a single hematopoietic stem cell expressing a marker gene from one or two insertions of a simple retroviral vector actively maintains multilineage transgene expression in the vast majority (80-99%) of bone marrow and peripheral blood cells. Gene expression persisted through serial transplantations for at least 97 weeks post gene transfer and was observed in the lymphoid (B, T and NK cells), myeloid (CD11b + , Gr-1 + ), erythroid (Ter119 + , mature red blood cells) and megakaryocytic (as indicated by platelets) progeny. Therefore, a single immunoselection for hematopoietic stem cells expressing the transgene in vivo was sufficient to establish a completely chimeric hematopoiesis. These observations imply that the retroviral vectors used in this study contain cis-elements that mediate expression through massive clonal expansion and multilineage differentiation, provided the insertion occurred in genetic loci permissive for expression in hematopoietic stem cells.

Side effects of retroviral gene transfer into hematopoietic stem cells

“…On the other hand, single clones of transduced HSCs may provide a perfectly normal hematopoiesis with persistence of transgene expression in all hematopoietic lineages, at least in mice. 120,123 This supports the idea that clones with favorable insertion sites and "neutral" transgenes are not necessarily at increased risk for transformation, even when undergoing massive expansion. The minimal number of HSCs that stably support primate hematopoiesis remains to be defined.…”

“…168 These 4 examples should be sufficient to underline the importance of a systematic risk assessment of the transgenes under consideration. Special attention should be paid to molecules that are involved in cellular signaling networks, such as those required for correction of some inherited disorders 3,168,169 or those generated as surface tags 120,123,144 or artificially inducible proteins that promote cellular proliferation or differentiation decisions. [153][154][155] We would propose that such transgenes should be tested under conditions of high, intermediate, and low constitutive expression, 129 preferentially achieved with vector design and not with variation of transgene dosage.…”

“…Different variants of MLV enhancerpromoters and some cellular promoters have shown a great potential for multilineage and persistent transgene expression in hematopoietic cells in vivo, typically accounting for less than 1% of the total cellular protein content. 71,113,[119][120][121][122][123] Cellular control elements have been modified to provide lineage-specific expression with promising potency, [65][66][67]124 and inducible expression has been achieved with designer promoters. 68 The insertion site modulates all aspects of transgene expression, including duration, level, and differentiation dependence.…”

A novel ‘sort-suicide’ fusion gene vector for T cell manipulation

“…Retroviral expression of tCD34 in murine hematopoietic stem cells did not produce overt alterations of multilineage hematopoiesis in vivo in an observation period of 97 weeks after gene transfer and in three serial transplanations. 31 Importantly, T cells expressing tCD34 showed both normal levels and tissue distribution. Further studies have been initiated to explore the immunological capacity and safety of T cells modified with the fusion construct vector.…”

Clinical Stem Cell Imaging and In vivo Tracking

“…The disadvantages are the necessity to genetically manipulate stem cells ex vivo and intravenous administration of another dosage of labeling agent for each imaging session (Zhang and Wu 2007). Before reporter gene imaging can fully and safely be applied clinically, several problems must be solved, the main example of which are transfection stability, minimizing immunologic response and minimizing the potential interference of stem cell function and differentiation by vector transfection or transduction (Chung, Andersson et al 2002;Li, Fehse et al 2002;Jakobsson, Rosenqvist et al 2004;Zhang and Wu 2007;Olmer, Haase et al 2009;Tran, Lair et al 2010). Receptor-mediated targeting, which is briefly described previously, requires stable expression of a receptor not found elsewhere throughout the body and intravenous injection of a labeling ligand of the receptor.…”