Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison

Area of Science:

• Molecular Biology
• Gene Therapy
• Virology

Background:

• Recombinant adeno-associated (rAAV) viral vectors are promising for gene therapy.
• Limited packaging capacity (<5 kb) restricts rAAV applications for diseases like cystic fibrosis and Duchenne muscular dystrophy.

Purpose of the Study:

• To compare two distinct methods for overcoming rAAV packaging limitations.
• To evaluate the efficacy of trans-splicing versus overlapping vector approaches.

Main Methods:

• The study compared trans-splicing (requiring intermolecular concatamerization and splicing) with an overlapping vector approach (using homologous recombination).
• The beta-galactosidase gene was used as a template to assess full-length transgene product generation.
• The impact of adenoviral E4 and E2a gene products on trans-splicing efficiency was investigated.

Main Results:

• Trans-splicing was significantly more effective than the overlapping vector approach, yielding threefold higher full-length transgene products in fibroblasts and 12-fold higher in muscle tissue.
• Adenoviral E4 gene products augmented trans-splicing efficiency by 1185-fold, with a less pronounced effect on the overlapping vector approach (12-fold).
• Despite improvements, trans-splicing efficiency remained moderate (~4.3% in muscle, ~7% in fibroblasts) compared to single-vector systems.

Conclusions:

• Both trans-splicing and overlapping vector approaches are viable strategies to enhance the packaging capacity of rAAV vectors.
• Trans-splicing, particularly when enhanced by adenoviral factors, demonstrates superior efficacy in generating full-length transgenes.
• Further optimization is needed to improve the overall efficiency of these methods for broader gene therapy applications.