Abstract: Gene therapy of SCO is based on the transplantation of genetically modified HSCs. Several LV approaches based on gene addition consist in transducing patient HSCs with a lentiviral vector expressing an anti-sickling ?-like globin chain such as use of ?AS3 HBB anti-sickling variants. Here, the inventors have improved the design of the LV-AS3 vector to treat SCO patients. These LVs allow the simultaneous expression of the potent anti-sickling ?AS3-globin and an artificial miR (amiR) silencing the ?S-globin. The reduction of ?S-globin levels will increase the incorporation of ?AS3-globin in Hb tetramers, which should increase the proportion of corrected RBCs in SCO patients. The inventors selected the best-performing miRs, and modified the therapeutic ?AS3-globin transgene by inserting silent mutations to avoid the recognition by the amiR and the silencing of the transgene.

Abstract: The clinical history of ?-hemoglobinopathies shows that the severity is mitigated by the synthesis of the fetal ?-globin in adulthood, typically associated with genetic variants the HBB cluster known as hereditary persistence of fetal hemoglobin (HPFH) mutations. The inventors identified that most of the known HPFH mutations in the ?-globin promoters (C>T, G>A, T>C or A>G) can be recapitulated using CBE- and ABE-mediatedbase-editing approaches. In particular, the inventors designed gRNAs that, when combined with CBEs or ABEs, generate HPFH mutations, and either disrupt binding sites for transcriptional repressors (-200 and -115 sites) or generate de novo DNA motifs recognized by transcriptional activators (e.g., -198 T>C, the -175 T>C and -113 A>G).

Abstract: The #?-hemoglobinopathies #?-thalassemia (BT) and sickle cell disease (SCD) are the most frequent genetic disorders worldwide. These diseases are caused by mutations causing reduced or abnormal synthesis of the ?-globin chain of the adult hemoglobin (Hb) tetramer. Here, the inventors intend to improve HSC-based gene therapy for ?-thalassemia and SCD by developing an innovative, highly infectious LV vector expressing a potent anti-sickling ?-globin transgene and a second biological function either increasing fetal ?-globin expression (for ?-thalassemia and SCD). More particularly, the inventors have designed a novel lentivirus (LV), which carry two different functions: ?AS3 gene addition and gene silencing. This last strategy allows the re-expression of the fetal ?-globin genes (HBG1 and HBG2) and production of the endogenous fetal hemoglobin (HbF).

Abstract: This invention relates to recombinant viral vectors, preferably retroviral (RV), lentiviral (LV) or adeno-associated viral (AAV) vectors, compositions thereof, the use of the recombinant viral vectors or the compositions thereof, kits of parts comprising said recombinant viral vectors or compositions thereof and a catalytically active Cas9 or Cpf1 protein, methods for modifying the genome of a cell, and the cells obtainable by such methods.

Abstract: The clinical severity of ?-hemoglobinopathies is alleviated by the co-inheritance of genetic mutations causing a sustained fetal ?-globin chain production at adult age, a condition termed hereditary persistence of fetal hemoglobin (HPFH). Here, the inventors have compared the extent of fetal hemoglobin (HbF) de-repression following CRISPR/Cas9-mediated targeting of different regions of the HBG1 and HBG2 promoters in an adult erythroid cell line (HUDEP-2). They achieved a potent and pancellular HbF re-activation upon disruption of binding sites for ?-globin repressors located in both HBG1 and HBG2 genes. They validated these findings in Red Blood Cells (RBCs) derived from genome edited Sickle Cell Disease (SCD) patient hematopoietic stem/progenitor cells. Overall, this study identified a binding site for an HbF repressor as a novel and potent target for the treatment of ?-hemoglobinopathies.

Abstract: This invention relates to recombinant lentiviral vectors, compositions thereof, the use of the vectors or the compositions thereof, kits of parts comprising said vectors or compositions thereof and a catalytically active Cas9 or Cpf1 protein, methods for modifying the genome of a hematopoietic stem/progenitor cell (HSPC), and the HSPC obtainable by such methods.