Gene therapy approaches to promote fetal hemoglobin production for the treatment of β-hemoglobinopathies

Abstract

Different mutations in the β-globin gene cause one of the most frequent single-gene disorders worldwide known as β-hemoglobinopathies. Initial therapeutic approaches consisted of the introduction of a wild-type copy of the β-globin gene by vectors (LVs). However, the induction of fetal hemoglobin (HbF, α2γ2) by LVs and other molecular approaches, reproducing the benign condition referred to as hereditary persistence of fetal hemoglobin (HPFH), has become a promising alternative treatment for these blood disorders. There is a big disparity of opinions to determine the most efficient and reliable gene therapy for inherited diseases such as β-hemoglobinopathies. While generally LVs seem to be the strategy of choice for disorders where high expression of the transgene is needed to ensure therapeutic effects, CRISPR/Cas9 looks more favorable for diseases where controlled gene expression is essential. However, both techniques present disadvantages, including insertional mutagenesis for LVs, and possible off-target cutting for CRISPR/Cas9 gene editing. In this study, a head-to-head comparative analysis of different gene therapy approaches was performed utilizing LVs and CRISPR/Cas9 to promote HbF production and determined their limitations, efficacy, and safety profile for the treatment of β-hemoglobinopathies. LVs encoding for 1) γ-globin gene, 2) IGF2BP1, and 3) miRNA-embedded shRNA for BCL11A, were tested. To enhance viral transduction efficiency in HSCs, two novel baboon envelope proteins (BaEV) were compared to the highly used vesicular-stomatitis-virus-G protein (VSV-G). On the other hand, the CRISPR/Cas9 system was employed to knock-down KLF1 and BCL11A genes involved in HbF repression, and to disrupt the binding site of different transcription factors in the γ-globin gene (HBG1/2) promoter. Two tested VSV-G lentiviral strategies (IGF2BP1 and miRNA-embedded shRNA for BCL11A) yielded the highest HbF levels among all approaches (~50%) with clinically relevant VCNs (<2) and no impaired differentiation. VSV-G γ-globin lentivirus treatment showed 20% HbF induction and relevant VCN, whereas all BaEV-LVs induced weaker HbF resurgence. Alternatively, BCL11A and HBG1/2 CRISPR/Cas9 gene disruption approaches also generated therapeutic HbF levels (~40%), showed low off-targets effect, weak transcript impairment, and no adverse effect during hematopoiesis. KLF1 gene editing showed stronger gene dysregulation and lower HbF resurgence. Based on our findings, we endorse BCL11A and HBG1/2 gene disruption approaches as the most promising strategies to be applied in the clinic due to their safety profile and high efficacy. On the other hand, VSV-G envelope protein and the γ-globin construct might be the safest LV strategy for β-hemoglobinopathies to be implemented in clinical studies. Nonetheless, γ-globin gene addition depends on efficient transgene expression, and therefore, alternative strategies to reverse the fetal-to-adult hemoglobin switch, such as BCL11A knockdown or IGF2BP1 overexpression, need to be examined and enhanced as more efficient LV treatment options.