Hematopoietic stem cell gene therapy for the treatment of β-hemoglobinopathies

Abstract

β-hemoglobinopathies, including β-thalassemia and sickle cell disease (SCD), are autosomal recessive inherited disorders caused by various mutations in the β-globin gene. The most effective curative therapy involves allogenic hematopoietic stem cell transplantation (HSCT) from an immunologically-matched donor. However, this approach presents some limitations in terms of finding a suitable donor and transplantation related risks, such as a graft-versus-host disease (GvHD). In this thesis, we strongly emphasized that autologous HSCT in combination with gene therapy tools will develop novel treatments for β-hemoglobinopathies. A promising technique for gene editing has emerged in recent years based on the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated RNA-guided endonuclease Cas9 (CRISPR/Cas9) technology. This revolutionary tool enables to perform gene disruption, gene correction, and gene addition in a specific locus of interest with high reliability and efficiency. Through this technology, in our first study, we demonstrated that targeting KLF1 and BCL11A, which control the expression of γ-globin, as well as the promoter region of HBG1 and HBG2 (HBG1/2), resulted in upregulation of γ-globin gene expression and fetal hemoglobin (HbF). Furthermore, after a deep comparison between the three gene editing strategies, we confirmed that BCL11A approach, which is currently in clinical phase, is the safest gene therapy treatment for β-hemoglobinopathies. Nevertheless, HBG1/2 strategy also holds potential for clinical translation. Alternatively, since β-globin gene correction is also a feasible gene therapy approach, in our second study, we attained successfully gene addition in HSPCs by inserting a NheI-tag at the common aberrant splicing mutation point, HBB IVS1-110 by means of Cas9 mRNA and ssODN electroporation. In the last project, we thoroughly compared three lentiviral transgenes encoding for IGF2BP1, shRNA BCL11A, and ꝩ-globin to reactivate HbF production in HSPCs. Also, we assessed whether baboon envelope proteins (BaEV and BaEV-RLess) have a beneficial advantage over the regularly used vesicular-stomatitis-virus-G envelope protein (VSV-G). Our results showed 4 that all treatments using VSV-G envelope proteins resulted in therapeutic levels of HbF. In addition, baboon envelopes, especially BaEV-RLess, achieved decent levels of HbF with less viral particles, which might ameliorate the symptoms of the disease. Finally, even though IGF2BP1 and BCL11A approaches induced higher HbF levels than ꝩ-globin strategy, their role in gene regulation might cause undesired iatrogenic effects. Therefore, we considered ꝩ-globin the best lentiviral gene therapy strategy for the treatment of β-hemoglobinopathies. During this thesis we evidenced that both gene therapy tools, lentiviral gene transfer and genome editing, provide a successful platform for gene treatment of blood disorders. In addition to the current clinical trial approaches, we strongly believe that resurgence of HbF is the most straight forward strategy together with β-globin gene correction. However, due to the low occurrence of gene correction events in HSPCs, further investigation is required.