HIV-1 inhibitory sequences improve vector titers and transduction efficiency with the minimal size of lentiviral vectors

Authors

• AC Bradley
• A Le
• J Ball
• S Demirci
• X Liu
• M Hsieh
• JF Tisdale
• N Uchida

Abstract

Gene therapy can cure various genetic diseases, including sickle cell disease (SCD), and HIV-1-based lentiviral vectors are used to introduce a curative gene into hematopoietic stem cells (HSCs). The therapeutic capabilities are limited by the vector size (~10kb), which must include curative genes, as well as several HIV-1 components essential for gene delivery. The goal of this project is to shrink the size of these essential components to increase the available space for curative genes. We first reduced vector size by using only core sequences of known HIV-1 components, which led to decreasing vector titers and lentiviral transduction in human primary CD34+ HSCs. We hypothesized that the HIV-1 inhibitory sequence (INS), which prevents HIV-1 nuclear export, is crucial for maintaining high titers and efficient transduction in shortened vectors. To determine the impact of the HIV-1 INS, we developed shortened vectors with escalating numbers of INS. Shortened vectors with added INS achieved a high titer similar to the standard-size vector while reducing the size by ~ 0.7kb, and these shortened vectors transduced CD34+ HSCs more efficiently than shortened counterparts without added INS. To analyze the potential therapeutic benefit for SCD, we generated a curative βT87Q-globin-expressing vector with a shortened backbone and additional INS. This construct allowed for more efficient transduction and increased βT87Q-globin output in CD34+ cell-derived erythroid cells. These data demonstrate that INS sequences are crucial to improving lentiviral titers and transduction in human CD34+ cells while minimizing vector size, thereby expanding the potential for gene therapy.

Scientific Focus Area: Molecular Biology and Biochemistry

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