Efficient gene targeting mediated by a lentiviral vector-associated meganuclease

Area of Science:

• Molecular Biology
• Gene Therapy
• Virology

Background:

• Gene targeting utilizes nucleases to create double-strand breaks (DSBs) for donor sequence integration.
• Lentiviral vectors are employed for delivering genetic material, but controlling nuclease expression is crucial for therapeutic safety.

Purpose of the Study:

• To investigate delivering nucleases as proteins with lentiviral vectors for enhanced gene targeting.
• To assess the impact of protein-based nuclease delivery on therapeutic applications and expression control.

Main Methods:

• Incorporation of I-SceI meganuclease, fused with HIV Vpr protein, into lentiviral particles.
• Utilizing integration-deficient lentiviral vectors carrying donor sequences and the I-SceI fusion protein.
• Testing in reporter cells measuring gene targeting via puromycin resistance gene repair.

Main Results:

• Protein-based delivery of I-SceI resulted in a 2-fold higher frequency of targeted recombination compared to vector-encoded delivery.
• Observed varying integration patterns, including tandem and unrelated loci integrations, dependent on nuclease delivery method.
• Distinct recombination processes were suggested based on observed integration patterns.

Conclusions:

• Delivering nucleases as proteins associated with lentiviral vectors is an effective strategy for gene targeting.
• This approach offers better control over endonuclease delivery, mitigating risks of sustained toxic expression.
• The method shows promise for improving the safety and efficiency of gene therapy applications.