Investigators aimed to improve lentiviral vectors to boost therapeutic β-like globin levels without increasing the mutagenic vector load in hematopoietic stem/progenitor cells.
Investigators from Europe created a lentiviral vector able to concomitantly silence the βS-globin and express β-Alanine synthase (βAS), achieving clinically relevant levels of therapeutic hemoglobin (Hb) and efficient correction of the sickling phenotype.
Of the potential treatments for the vaso-occlusions and organ damage causef by sickle cell disease (SCD), the Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LV) expressing an anti-sickling β-globin transgene (βAS LV) remains promising.
However, it is only partially effective in SCD patients, who still present elevated HbS levels.
As such, investigators led by Anne Chalumeau, PhD, Université de Paris, Imagine Institute, Laboratory of chromatin and gene regulation during development, intended to improve LVs to boost therapeutic β-like globin levels without increasing the mutagenic vector load in HSPCs.
Chalumeau and colleagues developed 2 novel LVs expressing βAS together with an artificial microRNA (amiR) targeting either the fetal Hb (HbF) repressor BCL11A (βAS/amiRBCL11A) or the βS-globin (βAS/amiRHBB).
Regarding βAS/amiRBCL11A LV, the team inserted the amiR in multiple positions of the βAS intron 2 under the control of HBB promoter/enhancers to limit BCL11A downregulation to the erythroid lineage and reduce potential amiR-related cellular toxicity and off-target effects.
The process showed that amiR insertion site did not affect LV titer nor βAS expression in a human erythroid cell line (HUDEP2).
Downregulation of BCL11A in HUDEP2 led to γ-globin gene de-repression and a high proportion of HbF+ cells, with the total amount of therapeutic β-like globins being substantially higher in βAS/amiRBCL11A LV- than in βAS LV-transduced cells, with no impairment in cell viability or erythroid differentiation.
The team also designed 17 amiRs targeting HBB and generated the corresponding βAS/amiRHBB, before testing them in HUDEP2 and selected 2 amiRs efficiently downregulating β-globin at mRNA and protein levels.
Chalumeau and colleagues tested both βAS/amiRBCL11A and βAS/amiRHBB in HSPCs from patients with sickle cell disease.
They recorded increased HbF levels in HSPC-derived erythroid cells transduced with βAS/amiRBCL11A LV, though the HbS levels remained high.
In an effort to reduce βS-globin levels, βS-globin mRNA was targeted using the βAS/amiRHBB LV.
Efficient HSPC transduction by βASm/amiRHBB LV led to a substantial decrease of βS-globin transcripts in HSPC-derived erythroid cells compared to the βAS LV-transduced cells (RTqPCR) at a VCN/cell of 2.
Additionally, the amiR down-regulated βS-globin without affecting βAS expression, while in βASm/amiRHBB- vs βAS LV-transduced cells, βS-globin downregulation led to a significant decrease of HbS, representing 58% and 71% of the total hemoglobin.
This was followed by a significant increase of the therapeutic Hb in βASm/amiRHBB LV- vs βAS LV-transduced erythroid cells.
The investigators also observed reductions in the proportion of HbS-positive cells in βASm/amiRHBB- vs βAS LV-transduced samples (from 96% to 70%).
Overall, investigators noted that the increased incorporation of βAS in Hb tetramers and the decrease in βS-globin resulted in a better correction of the sickling phenotype in mature RBCs derived from HSPCs transduced with βASm/amiRHBB LV- compared to βAS LV (55% and 84% of sickling cells.
“Therefore, the combination of gene addition and gene silencing strategies can improve the efficacy of current therapeutic approaches, representing a novel treatment for SCD,” the team wrote.
The study, “Novel Lentiviral Vectors for Gene Therapy of Sickle Cell Disease Combining Gene Addition and Gene Silencing Strategies,” was published online for ASH2021.