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Multigene lentiviral vectors based on differential splicing and translational control
1Department of Microbiology & Immunology, University of Rochester Cancer Center, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Summary
This study demonstrates that lentiviral vectors can express multiple genes using splicing or IRES strategies without reducing viral titers or infectivity. These multigene vectors are feasible for gene therapy applications.
Area of Science:
- Molecular Biology
- Gene Therapy
- Virology
Background:
- Lentiviral vectors are typically engineered for single gene expression.
- Gene therapy applications often necessitate the expression of multiple genes simultaneously.
Purpose of the Study:
- To investigate the feasibility of creating lentiviral vectors capable of expressing two marker genes.
- To evaluate two distinct strategies for multigene expression from a single lentiviral vector.
Main Methods:
- Designed lentiviral vectors utilizing HIV-1 natural splicing signals to generate multiple mRNAs.
- Constructed polycistronic lentiviral vectors incorporating the encephalomyocarditis virus internal ribosome entry site (IRES).
- Introduced mutations in tat and rev genes to assess the impact on reporter gene expression levels and developed a truncated tat mutant.
Main Results:
- Achieved successful expression of multiple genes from single lentiviral vectors.
- Demonstrated that multigene lentiviral vectors maintain high virus titers and infect non-dividing cells.
- Showed that splicing strategies and IRES elements are effective for multigene expression.
- Found that a truncated tat mutant does not affect virus generation titers.
Conclusions:
- Lentiviral vectors can be engineered for efficient multigene expression, expanding their utility in gene therapy.
- Splicing and IRES-mediated translation are viable approaches for constructing multigene lentiviral vectors.
- The developed multigene lentiviral vectors are stable and functional for gene delivery applications.