Leszek Lisowski

Progress toward the genetic treatment of the β-thalassemias

Abstract: The β‐thalassemias are congenital anemias that are caused by mutations that reduce or abolish expression of the β‐globin gene. They can be cured by allogeneic hematopoietic stem cell (HSC) transplantation, but this therapeutic option is not available to most patients. The transfer of a regulated β‐globin gene in autologous HSCs is a highly attractive alternative treatment. This strategy, which is simple in principle, raises major challenges in terms of controlling expression of the globin transgene, which ideally should be erythroid specific, differentiation‐ and stage‐restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, May et al. demonstrated in 2000 that an optimized combination of proximal and distal transcriptional control elements permits lineage‐specific and elevated β‐globin expression, resulting in therapeutic hemoglobin production and correction of anemia in β‐thalassemic mice. Several groups have by now replicated and extended these findings to various mouse models of severe hemoglobinopathies, thus fueling enthusiasm for a potential treatment of β‐thalassemia based on globin gene transfer. Current investigation focuses on safety issues and the need for improved vector production methodologies. The safe implementation of stem cell‐based gene therapy requires the prevention of the formation of replication‐competent viral genomes and minimization of the risk of insertional oncogenesis. Importantly, globin vectors, in which transcriptional activity is highly restricted, have a lesser risk of activating oncogenes in hematopoietic progenitors than non‐tissue‐specific vectors, by virtue of their late‐stage erythroid specificity. As such, they provide a general paradigm for improving vector safety in stem cell‐based gene therapy.

Supplying clotting factors from hematopoietic stem cell-derived erythroid and megakaryocytic lineage cells.

Systemically distributed proteins such as clotting factors have been traditionally expressed from muscle or liver to achieve therapeutic, long-term expression. As long-lived cell capable of generating an abundant progeny, hematopoietic stem cells (HSCs) also merit consideration for this purpose. To be clinically relevant, this approach would require that hematopoietic cells be capable of expressing high levels of functional, secreted proteins, that the risk of insertional oncogenesis be minimized, and that sufficient stem cell engraftment be achieved following minimally invasive conditioning. Recent reports demonstrate the feasibility of expressing either factor IX (FIX) or factor VIII (FVIII) in erythroblasts and platelets using lineage-restricted vectors, resulting in effective treatments in mouse models of hemophilia. The erythroid system is especially powerful in providing high protein output, yielding FIX levels approaching 1 micro g/ml per vector copy in the plasma of long-term hematopoietic chimeras, a secretion level that vastly outperforms any other current mammalian constitutive or long-terminal repeat (LTR)-driven vector system. These early but promising studies raise the prospect of further developing these strategies for clinical investigation.

Globin gene transfer: a paradigm for transgene regulation and vector safety

The β-thalassemias and sickle cell disease are severe congenital anemias that are caused by the defective synthesis of the β chain of hemoglobin. Allogeneic hematopoietic stem cell (HSC) transplantation is curative, but this therapeutic option is not available to the majority of patients. The transfer of a regulated β-globin gene in autologous HCSs thus represents a highly attractive alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling transgene expression, which ideally should be erythroid-specific, differentiation stage-restricted, elevated, position-independent, and sustained over time. Using lentiviral vectors, we recently demonstrated that an optimized combination of proximal and distal transcriptional control elements permits lineage-specific and elevated expression of the β-globin gene, resulting in therapeutic hemoglobin production and correction of anemia in β-thalassemic mice. Several groups have now confirmed and extended these findings in various mouse models of severe hemoglobinopathies, thus generating enthusiasm for a genetic treatment based on globin gene transfer. Furthermore, globin vectors provide a paradigm for improving vector safety by restricting transgene expression to the differentiated progeny within a single lineage, thereby reducing the risk of activating oncogenes in hematopoietic progenitors. Here we review the principles underlying the genesis of regulated vectors for stem cell therapy.