Optimization of various envelopes for efficient gene delivery with lentiviral vectors

Authors

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

Abstract

Hematopoietic stem cell (HSC)-targeted gene therapy allows for a one-time cure of sickle cell disease (SCD), approved by the FDA in Dec 2023. HIV-1-based lentiviral vectors are used to deliver a therapeutic gene into patients’ HSCs, due to their ability of long-term gene expression. However, further development of lentiviral gene delivery remains crucial to expand gene therapy to many patients. The envelope protein of lentiviral vectors can be replaced with that of different virus to change target cells and transduction efficiency dependent on the viral origin. Vesicular stomatitis virus G (VSVG) is commonly used due to the efficient transduction of most cell types; however, the development of alternative envelopes is desired for more specific delivery. Envelope proteins are generally activated by cleavage of the cytoplasmic tail with each viral enzyme. Therefore, modification at the cytoplasmic tail of other viral envelopes may be required to allow for activation by HIV-1 enzymes. Here, we aim to optimize various viral envelopes for HIV-1-based lentiviral vectors, allowing for efficient and specific transduction in CD34 + HSCs to expand SCD gene therapy. We demonstrate that inserting a stop codon at the cleavage site in Measles, RD114, and GaLV envelopes can increase lentiviral titers about 10-fold. Also, replacing the HIV-1 gag-pol cleavage peptide or using an artificial cleavage peptide at the cytoplasmic tail in the RD114 envelope can increase lentiviral titers. This study would be beneficial to improving transduction efficiency and specificity in various types of cells, including CD34+ HSCs to apply to SCD gene therapy.

Scientific Focus Area: Molecular Biology and Biochemistry

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