Identifying Viral Vector Characteristics by Nanopore Sensing.

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Biology

Background:

• Gene therapy relies on viral vectors, necessitating stringent quality control.
• Current methods for inspecting viral vector genomes are often destructive or lack single-molecule resolution.

Purpose of the Study:

• To develop a non-destructive nanopore sensing method for inspecting the genomes within viral vectors.
• To enable single-molecule level quality control of adeno-associated virus (AAV) vectors.

Main Methods:

• Utilized solid-state nanopore technology for translocation of AAV vectors.
• Employed ionic current measurements to track vector movement and analyze ion blockage characteristics.
• Correlated electrical signals with the physical features of packaged DNA (length and volume).

Main Results:

• Successfully motion-tracked AAV vectors through a nanopore using ionic current.
• Demonstrated that longer DNA molecules within AAV vectors resulted in slower translocation speeds due to electrophoretic forces.
• Showed that ion blockage characteristics correlated with packaged DNA length, revealing volume differences up to ~3600 nm³.
• Achieved electrical discrimination of AAV vectors based on their gene-derived physical properties.

Conclusions:

• Nanopore sensing offers a promising, non-destructive tool for AAV vector quality control.
• This technique enables the screening of empty, intermediate, and full AAV vectors at the single-particle level.
• Facilitates enhanced quality assurance for gene therapy products.