Delivering Transgenic DNA Exceeding the Carrying Capacity of AAV Vectors

Area of Science:

• Gene Therapy
• Molecular Biology
• Virology

Background:

• Recombinant adeno-associated virus (rAAV) vectors are effective for gene delivery, showing success in treating diseases like hemophilia B.
• rAAV vectors offer high transduction efficiency and long-term transgene expression with a strong safety profile in humans.
• A significant limitation of rAAV is its small capsid size, restricting the packaging of large genomes (approx. 5 kb), hindering therapeutic applications for many genetic disorders.

Purpose of the Study:

• To address the packaging capacity limitation of rAAV vectors for large gene delivery.
• To review and discuss the design, production, and verification of leading rAAV large gene delivery strategies.
• To compare split rAAV and fragment rAAV (fAAV) genome reassembly approaches.

Main Methods:

• Split rAAV vector approach: Involves splitting large transgenes into two vectors for intracellular reconstruction via homologous recombination (HR) or nonhomologous end joining (NHEJ).
• Fragment rAAV (fAAV) genome reassembly: Utilizes heterogeneous single-strand genome fragments packaged into multiple fAAV particles, which then anneal and are reconstructed within the cell.
• Three split rAAV strategies are identified: overlapping, trans-splicing, and hybrid trans-splicing.

Main Results:

• Split rAAV vectors leverage natural genome concatemerization and HR for large gene reconstruction.
• fAAV strategy relies on annealing of packaged genome fragments followed by host-mediated DNA synthesis.
• The choice between split rAAV and fAAV may depend on factors like target tissue and transgene sequence.

Conclusions:

• Split rAAV and fAAV are key strategies to overcome the packaging limitations of rAAV vectors for large gene transfer.
• Both approaches facilitate the delivery of therapeutic genes exceeding the standard rAAV packaging capacity.
• Further research and consideration of application-specific factors are crucial for selecting the optimal large gene delivery strategy.