Directed evolution of hyperactive integrases for site specific insertion of transgenes

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics Engineering

Background:

• Current gene delivery methods for biomedical interventions face challenges with low insertion efficiency and reliance on double-strand DNA breaks.
• Serine integrases offer a promising alternative for inserting large DNA payloads at specific genomic attachment (att) sites, avoiding double-strand breaks when combined with genome targeting technologies like prime editing.

Purpose of the Study:

• To systematically improve the activity of PhiC31 and Bxb1 serine integrases for efficient, targeted delivery of large transgenes.
• To develop a novel directed evolution strategy for enhancing enzyme function in gene delivery applications.

Main Methods:

• Phage-assisted continuous evolution (PACE) was employed to perform extensive mutagenesis, generating a library of serine integrase variants.
• The developed method, named IntePACE, was used to rapidly evolve PhiC31 and Bxb1 integrases.
• Synergistic mutations were identified and combined to create hyperactive integrase variants.

Main Results:

• Novel hyperactive serine integrase mutants were generated with significantly improved DNA integration efficiency.
• Integration of a multi-gene cargo, including a 15.7 kb therapeutic DNA encoding von Willebrand Factor, was achieved at rates as high as 80% of target chromosomes.
• The IntePACE method demonstrated rapid and systematic improvement of integrase activity.

Conclusions:

• The engineered hyperactive integrases and the IntePACE directed evolution strategy represent a significant advancement in gene delivery technology.
• This approach has the potential to accelerate the development of gene delivery therapeutics by enabling efficient insertion of large therapeutic payloads.
• The directed evolution strategy is adaptable for improving other novel integrases found in nature.