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

Mariana V Ferreira1,2, Sofia Fernandes1,2, Ana Isabel Almeida1,2

  • 1iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.

International Journal of Molecular Sciences
|July 14, 2023
PubMed
Summary
This summary is machine-generated.

New split-inteins, Cfa and Gp41-1, significantly improve dual adeno-associated viral (AAV) vector gene delivery efficiency. Higher quality vector preparations further enhance these protein trans-splicing rates, enabling larger gene delivery.

Keywords:
AAV co-transductiondual AAV vectordual AAV-intein mediated systemsfull capsid enrichmentsplit-inteins

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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.