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Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

Published on: April 2, 2012

A truncated reverse transcriptase enhances prime editing by split AAV vectors.

Zongliang Gao¹, Sujan Ravendran¹, Nanna S Mikkelsen¹

¹Department of Biomedicine, Aarhus University, Aarhus C, Denmark.

Molecular Therapy : the Journal of the American Society of Gene Therapy

|July 9, 2022

Summary

This summary is machine-generated.

Researchers developed a smaller, more efficient prime editor (PE) for CRISPR gene editing. This optimized PE, delivered via viral vectors, shows improved efficiency and successful in vivo gene correction in mice.

Keywords:

AAV vectors CRISPR-Cas9 PASTE gene editing gene therapy in vivo delivery prime editing reverse transcriptase

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Area of Science:

Molecular Biology
Gene Editing Technologies
Biotechnology

Background:

Prime editing (PE) is an advanced CRISPR-based genome editing tool.
Current PE systems face limitations due to large component sizes, hindering efficient viral vector delivery.

Purpose of the Study:

To enhance prime editor (PE) efficiency and reduce its size for improved viral vector delivery.
To optimize the split intein PE system for better in vivo applications.

Main Methods:

Engineered the reverse transcriptase (RT) moiety of the PE.
Performed RT variant screening, codon optimization, and PE truncation.
Optimized the split intein PE system with specific Cas9 split sites.
Utilized adeno-associated virus (AAV) and lentiviral vectors for delivery.

Main Results:

Developed a codon-optimized, size-minimized PE (621 bp shorter) with 1.4-fold increased expression.
Achieved superior AAV titers and prime editing efficiency using a truncated split PE system with dual AAVs.
Demonstrated up to 6% precise editing of the PCSK9 gene in mouse liver using minimized PE delivered by AAV8 vectors.

Conclusions:

The optimized and truncated PE system significantly improves delivery efficiency and editing outcomes.
This minimized PE is valuable for in vivo gene editing applications, particularly in the liver.
The advancements pave the way for more effective therapeutic gene editing strategies.