Optimizing delivery and expression of designer nucleases for genome engineering

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Epigenetics

Background:

• Genome engineering relies on designer nucleases to create targeted DNA double-strand breaks.
• Integration-defective lentiviral vectors (IDLVs) offer transient expression of nucleases but suffer from epigenetic silencing.
• This silencing compromises nuclease expression and limits the efficiency of genome engineering applications.

Purpose of the Study:

• To investigate methods for overcoming epigenetic silencing of designer nucleases delivered via IDLVs.
• To evaluate the efficacy of histone deacetylase (HDAC) inhibitors in restoring IDLV-driven nuclease expression.
• To assess the impact of HDAC inhibition on targeted gene disruption using nonhomologous end-joining (NHEJ).

Main Methods:

• Utilized integration-defective lentiviral vectors (IDLVs) for delivering designer nucleases.
• Employed histone deacetylase (HDAC) inhibitors to interfere with chromatin remodeling.
• Assessed nuclease expression levels and gene disruption efficiency via nonhomologous end-joining (NHEJ).

Main Results:

• HDAC inhibition successfully restored the expression of designer nucleases from IDLVs.
• Restored nuclease expression enabled efficient targeted gene disruption through NHEJ.
• Gene disruption efficiency achieved with HDAC-inhibited IDLVs was comparable to integrating lentiviral vectors.

Conclusions:

• Interfering with chromatin remodeling via HDAC inhibition is a viable strategy to enhance IDLV-mediated genome engineering.
• This approach overcomes the bottleneck of epigenetic silencing, enabling robust transient expression of designer nucleases.
• The findings support the ex vivo application of IDLVs for gene repair and gene targeting strategies.