A synthetic Rev-independent bovine immunodeficiency virus-based packaging construct

Area of Science:

• * Gene therapy
• * Virology
• * Molecular biology

Background:

• * Replication-competent lentivirus (RCL) poses a significant safety risk in lentivirus-based gene therapy.
• * Previous lentiviral vector generations, including bovine immunodeficiency virus (BIV)-based systems, retained sequence homologies that could lead to RCL generation.
• * Minimizing sequence homology between packaging and transfer constructs is crucial for enhancing lentiviral vector safety.

Purpose of the Study:

• * To engineer a safer lentiviral vector system by minimizing sequence homologies.
• * To develop a recoded gag/pol packaging construct optimized for human cells, independent of Rev/RRE.
• * To assess the safety and efficacy of the novel lentiviral vector system.

Main Methods:

• * Recoded gag/pol genes using codon optimization for human cell expression.
• * Eliminated the Rev/RRE system from the packaging construct.
• * Constructed a four-plasmid lentiviral vector system utilizing the recoded packaging construct.

Main Results:

• * The recoded gag/pol expression was Rev/RRE independent, allowing RRE removal and reducing homology by 312 bp.
• * Overall sequence homology between packaging and transfer vector constructs was significantly minimized.
• * Lentiviral vectors generated exhibited high titers (>1 x 10^6 TU/mL), comparable to previous systems.
• * Vectors demonstrated in vitro functionality and efficient in vivo transduction of rat pigment epithelial cells.

Conclusions:

• * The synthetic packaging construct significantly enhances the safety of lentiviral vectors for clinical applications.
• * Recoding gag/pol and eliminating RRE represent key advancements in generating safer lentiviral vectors.
• * This approach offers improved safety profiles for future gene therapy trials.