Superior lentiviral vectors designed for BSL-0 environment abolish vector mobilization

Area of Science:

• Molecular Biology
• Virology
• Gene Therapy

Background:

• Lentiviral vectors, including human immunodeficiency virus type 1 (HIV-1) based vectors, are promising tools for gene therapy.
• However, lentiviral vector mobilization from transduced cells poses biosafety risks, particularly in patients and their communities.
• Current self-inactivating (SIN) vector designs reduce but do not eliminate vector mobilization.

Purpose of the Study:

• To investigate a novel strategy for abolishing lentiviral vector mobilization.
• To elucidate the underlying molecular mechanisms, including the role of host cell responses.
• To assess the efficacy and biosafety of the modified vectors in preclinical models.

Main Methods:

• Construction and characterization of SIN lentiviral vectors with internal expression cassettes in opposite orientation to the long terminal repeats (LTRs).
• Assessment of vector mobilization using quantitative PCR and viral DNA analysis.
• Investigation of host cell responses, specifically the PKR pathway, using shRNA-mediated knockdown.
• Evaluation of vector titers and transgene expression in vitro and in vivo (mouse liver models).

Main Results:

• Configuring the internal expression cassette in opposite orientation to the LTRs effectively abolished SIN lentiviral vector mobilization.
• This enhanced SIN mechanism involves the induction of the host PKR response to double-stranded RNAs generated during transcription.
• PKR activation negatively impacted vector titers, but knockdown of PKR restored titers to levels comparable to conventional SIN vectors.
• High-titer vectors with opposite orientation cassettes demonstrated efficient gene delivery and sustained transgene expression in mouse livers.

Conclusions:

• Opposite orientation expression cassettes represent a novel, PKR-dependent SIN mechanism that abolishes lentiviral vector mobilization.
• This strategy significantly enhances the biosafety profile of both integrated and episomal SIN lentiviral vectors.
• The findings have critical implications for the development of safer and more effective lentiviral vector-based gene therapies.