Bicistronic Lentiviral Architecture Increases Surface Density of Membrane-Associated HIV Entry Inhibitors and Confers Tropism-Independent Protection to Modified Cells

Background: Previous studies have demonstrated the efficacy of membrane-anchored C-peptides against HIV and have shown that the surface expression level of these peptides is critical for effective protective activity. Methods: Bicistronic genetic constructs were designed for GPI-anchored expression of protective C-peptides on the cell membrane. The constructs were delivered using lentiviral vectors and tested in primary CD4+ lymphocytes and hematopoietic stem cells. Results: The bicistronic configuration significantly enhanced surface expression levels compared to monocistronic constructs and allowed simultaneous expression of two distinct protective peptides, thereby potentiating their activity. Lentiviral vectors harboring these constructs conferred robust protection to primary CD4+ lymphocytes against HIV infection irrespective of viral tropism and provided a selective advantage to transduced cells upon challenge with replication-competent HIV strains. The bicistronic construct was also suitable for lentiviral transduction of hematopoietic stem cells. Conclusions: These findings establish bicistronic GPI-anchored C-peptide expression as a promising platform for generating HIV-resistant immune cells and highlight its potential for ex vivo gene therapy.