An Optimized Lentiviral Vector Efficiently Corrects the Human Sickle Cell Disease Phenotype

Area of Science:

• Hematology
• Gene Therapy
• Molecular Biology

Background:

• Sickle cell disease (SCD) is a genetic blood disorder caused by a mutation in the hemoglobin beta (HBB) gene.
• Current treatments for SCD are limited, highlighting the need for innovative therapeutic approaches.
• Gene therapy using lentiviral vectors (LV) to express anti-sickling HBB variants is a promising strategy for SCD.

Purpose of the Study:

• To develop and optimize lentiviral vectors (LV) for gene therapy of sickle cell disease (SCD).
• To evaluate the efficacy of different combinations of DNase I hypersensitive sites (HSs) from the locus control region (LCR) in enhancing anti-sickling HBB transgene expression in hematopoietic stem progenitor cells (HSPCs).

Main Methods:

• Generated LV constructs with an anti-sickling HBB transgene (HBBAS3), a minimal HBB promoter, and varying combinations of LCR HS elements (HS2, HS3, HS4).
• Transduced HSPCs from SCD patients with optimized LVs (β-AS3 and β-AS3 HS4).
• Assessed vector titer, infectivity, transgene expression, and reduction in red blood cell (RBC) sickling under hypoxic conditions.

Main Results:

• The LV containing HS2 and HS3 (β-AS3) efficiently transduced SCD HSPCs, achieving high levels of anti-sickling HBB expression (up to ~60% of total HBB-like chains).
• The inclusion of HS4 (β-AS3 HS4) significantly reduced vector efficiency without improving transgene expression.
• Efficient HBBAS3 expression led to a 50% reduction in RBC sickling frequency under hypoxic conditions.

Conclusions:

• A high-titer lentiviral vector (LV) incorporating HS2 and HS3 elements effectively expresses a potent anti-sickling HBB transgene.
• This optimized gene therapy approach ameliorates the cellular phenotype of sickle cell disease (SCD) by reducing red blood cell sickling.
• These findings support the advancement of this LV-based gene therapy towards clinical trials for SCD treatment.