Erythroid Specificity and Safety of Globin-Encoding Lentiviral Vectors

The beta-thalassemias and sickle cell disease are the most common monogenic inherited blood disorders, both arising from mutations affecting the beta-globin locus. Recent clinical trials utilizing lentiviral-mediated beta-globin gene transfer in autologous CD34+ cells have shown encouraging results in patients with non-beta-zero thalassemias and sickle cell anemia. A case report of insertional mutagenesis in a beta-thalassemia patient resulted in a prolonged clonal expansion that eventually regressed without progressing to leukemic transformation. Nonetheless, this occurrence and the need to insert multiple vector copies per cell when using globin vectors that provide insufficient globin expression to achieve curative responses from a single integrated copy per cell, raise some questions about the erythroid specificity and safety of globin vectors. We thus found it imperative to investigate globin vector expression in hematopoietic progenitors and in non-erythroid cells. To this end, we investigated what regulatory elements, including locus control region (LCR) hypersensitive sites (HS) and others, achieve the highest beta-globin expression per vector copy while at the same time minimizing non-erythroid transcriptional activity. We developed an in vivo assay to track the enhancer/promoter activity of different HS elements in hematopoietic progenitor and differentiated cell subsets. We designed lentiviral vectors expressing hrGFP under the control of a short β-globin promoter (137bp) controlled by a set of LCR HS elements and/or a novel biliverdin (BLV) enhancer. Analysis of hrGFP expression in bone marrow cells derived from C57BL6 mice transplanted with transduced syngeneic bone marrow cells revealed that many of the vectors encompassing elements thought to encode erythroid-specific elements were in fact expressed in long term-HSC (LT-HSC), short term-HSC (ST-HSC), multipotent progenitors (MPP), pre-GM, Granulocyte/macrophage progenitors (GMP) and megakaryocyte progenitors (MKP). These findings confirmed that ectopically integrated erythroid regulatory elements can serve as transcriptional enhancers in non-erythroid cells. This transcriptional leakiness was confirmed in therapeutic globin vectors harboring a combination of LCR elements. To avoid these effects, we flanked the globin transcription unit with a small human enhancer-blocking element called A1, kindly provided by the late George Stamatoyannopoulos (Liu, M. et al. 2015). The insulated vector showed markedly reduced non-erythroid expression in non-erythroid cells including ST-HSC, MPP, Pre-GM and GMP. Expression in mature erythroid cells was unchanged. Our findings underscore the benefits of selecting lineage-specific regulatory elements with the least lineage promiscuity for therapeutic vectors, with the eventual addition of enhancer-blocking elements. Based on these results, we are now investigating new globin expression vectors with optimal combination of HS elements. Such novel globin vectors should minimize the risk of oncogene trans-activation in hematopoietic progenitor cells and therefore improve the safety of globin gene therapy while providing lineage-specific high-level expression. Disclosures Sadelain: Mnemo: Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Atara: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties.