Dual AAV vectors for efficient delivery of large transgenes

Area of Science:

• Gene Therapy
• Molecular Biology
• Biotechnology

Background:

• Adeno-associated viral (AAV) vectors are standard for in vivo gene delivery but have limited cargo capacity (<5 kb).
• Delivering large therapeutic genes and CRISPR tools necessitates overcoming AAV size constraints.

Purpose of the Study:

• To provide a comprehensive protocol for designing, producing, and evaluating dual AAV vectors.
• To facilitate the efficient in vivo delivery of large DNA fragments using dual AAV technology.

Main Methods:

• Guidelines for selecting dual AAV strategies and designing/cloning therapeutic genes.
• In vitro evaluation of expression efficiency and dual AAV production.
• Assessment in human cellular models (iPSC-derived retinal organoids) and in vivo studies in mice.
• Analysis of transgene expression (RNA and protein) in various tissues.

Main Results:

• The protocol offers a structured approach to dual AAV vector development.
• Demonstrates successful application in human organoid models and murine systems.
• Provides a framework for optimizing in vivo delivery of large DNA fragments.

Conclusions:

• Dual AAV vectors are a powerful tool for overcoming cargo limitations in gene therapy.
• This protocol supports the advancement of gene delivery for large genetic payloads.
• The methodology is adaptable across model organisms and human organoid cultures.