Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses

Area of Science:

• Molecular Biology
• Gene Therapy
• Biotechnology

Background:

• Adeno-associated viral (AAV) vectors are key gene delivery tools, but limited packaging capacity hinders delivery of large therapeutic genes.
• Dual-AAV systems using protein trans-splicing, like Npu DnaE split-intein, aim to overcome packaging limits but face efficiency challenges.
• Insufficient reconstitution efficiency of existing split-inteins necessitates higher vector doses, impacting therapeutic applications.

Purpose of the Study:

• To evaluate the performance of Cfa and Gp41-1 split-inteins against Npu DnaE for dual-AAV vector protein trans-splicing.
• To assess the impact of vector preparation quality on split-intein performance in vitro.
• To demonstrate the potential for reduced vector doses with improved split-intein strategies for large gene delivery.

Main Methods:

• Transient transfections and in vitro co-transductions were used to compare Npu DnaE, Cfa, and Gp41-1 split-inteins.
• Dual-AAV vector systems were employed to assess protein reconstitution and trans-splicing efficiency.
• The influence of vector preparation quality (percentage of full particles) on split-intein performance was systematically evaluated.

Main Results:

• Cfa and Gp41-1 split-inteins demonstrated over two-fold higher reconstitution rates compared to Npu DnaE, achieving 100% protein reconstitution.
• Higher-quality vector preparations (60-75% full particles) enhanced split-intein performance threefold over low-quality preparations (20-30%).
• Low-quality vector preparations were found to inhibit co-transduction, limiting split-gene reconstitution efficiency.

Conclusions:

• Cfa and Gp41-1 represent superior split-inteins for dual-AAV vector applications, significantly improving protein trans-splicing efficiency.
• Optimizing vector preparation quality is crucial for maximizing split-intein performance and overcoming co-transduction inhibition.
• Combining enhanced split-inteins with high-quality vector preparations enables reduced vector doses and facilitates the delivery of larger therapeutic genes via dual-AAV systems.