Distinguishing between DNA-Loaded Full and Empty Capsids of Adeno-Associated Virus with Atomic Force Microscopy Imaging

Abstract

Recently, miraculous therapy approaches involving adeno-associated virus (AAV) for incurable diseases such as spinal muscular atrophy and inherited retinal dysfunction have been introduced. Nonreplicative, nonpathogenic, low rates of chromosome insertional properties and the existence of neutralizing antibodies are main safety reasons why the FDA approved its use in gene delivery. To date, AAV production always results in a mixture of nontherapeutic (empty) and therapeutic (DNA-loaded) full capsids (10–98%). Such existence of empty viral particles inevitably increases viral doses to human. Thus, the rapid monitoring of empty capsids and reducing the empty-to-full ratio are critical in AAV science. However, transmission electron microscopy (TEM) is the primary tool for distinguishing between empty and full capsids, which creates a research bottleneck because of instrument accessibility and technical difficulty. Herein, we demonstrate that atomic force microscopy (AFM) can be an alternative tool to TEM. The simple, noncontact-mode imaging of AAV particles allows the distinct height difference between full capsids (∼22 nm) and empty capsids (∼16 nm). The sphere-to-ellipsoidal morphological distortion observed for empty AAV particles clearly distinguishes them from full AAV particles. Our study indicates that AFM imaging can be an extremely useful, quality-control tool in AAV particle monitoring, which is beneficial for the future development of AAV-based gene therapy.

Enhancing the production of adeno‐associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design‐of‐experiment optimization of triple plasmid ratio

“…To prepare a control, HEK293 cells were transfected with three different plasmids at widely used ratios: weight ratio of 1:1:1 and weight ratio of 2:1:1 (molar ratio of approximately 1:1:1). [ 1,2,17,21–24 ]…”

Use of Atomic Force Microscopy in UVB-Induced Chromosome Damage Provides Important Bioinformation for Cell Damage Assessment

“…AFM shows great potential in high-resolution imaging and quantitative mechanical characterization of biological samples under near-physiological conditions. – Moreover, this technology provides unique insights into the characterization of chromosome structure and provides a more comprehensive understanding of the relationship between its structure and functions. – For example, the G-band structure of chromosomes has been clearly imaged by AFM . Fan et al studied the ultrastructure of G 1 -phase chromosomes by AFM and obtained the 30 nm chromatin fiber structure.…”