A HIGH-THROUGHPUT, QUANTITATIVE, & RAPID CHARACTERIZATION OF AAV PROTEINS

AAV: The Gene Therapy of the Future?

Adeno-associated virus (AAV) particles are one of the primary gene delivery vehicles used in clinical research because they facilitate long-term transgene expression in animal models with little associated toxicity and have low immunogenicity in humans. These particles could potentially be used to deliver protein receptors or nucleic acids to treat diseases caused by mutant genes. AAV particles belong to the parvovirus family and can only reproduce in vitro with the help of co-infection with other viruses. AAV consists of a protein shell encapsulating a 4.7 kb size genome containing the following genes and genetic material being administered:

  • Rep (replication)
  • Cap (capsid)
  • Assembly genes

The Cap gene produces three viral proteins (VP) known as VP1, VP2, and VP3 which form the protective outer shell of the virus.

Although AAV particles have great potential in gene therapy, the development of these methods is hampered due to a long development process which requires many iterations. Any time saving method in this process will greatly help researchers unlock the potential of gene therapy with AAV particles.

AAV
AAV8 analysis by SDS-PAGE

SDS-PAGE with Silver Stain: The Qualitative Study

To study AAV particles, researchers often use SDS-PAGE with silver stain, a time and labor-intensive process which generally has low reproducibility and only yields qualitative data. To complicate matters further, SDS-PAGE can have many sources of error such as:

  • Gel overheating
  • Gel overloading
  • Inadequate standardization of staining
  • Variation in the densitometry

The Labchip® GX Touch protein characterization system offers an automated alternative to gel electrophoresis increasing both throughput and data quality. This innovative instrument supports many assays for characterization of proteins and nucleic acids. Even novice users can run samples quickly and effectively with the instruments’ intuitive touchscreen interface.

LabChip® GXII Touch System and ProteinEXact Assay: The Quantitative Alternative

The LabChip® GXII Touch HT protein characterization system utilizes microfluidic capillary electrophoresis in the presence of SDS (CE-SDS) to aspirate approximately 150 nL of sample into a quartz chip. In the chip, sample proteins are separated electrophoretically and individual peaks are detected through laser induced fluorescence. The system further determines the size and concentration of each peak using a ladder and internal markers.

The ProteinEXact assay for the LabChip® GXII Touch system enables high precision reproducibility and improved sensitivity with a sample analysis of 65 seconds. This high-throughput assay can be run with up to 96 samples at a time and delivers comparable results to traditional capillary electrophoresis.

For more information on how the LabChip® GXII Touch HT protein characterization system can study AAV particles see:

ProteinEXact

AAV8 Capsid Protein Characterization

The LabChip® Reviewer software creates an electropherogram (egram) with quantitative analysis of size, concentration and percent contribution of each individual protein. The denatured molecular weights were determined to be:

Electropherogram of fully denatured AAV8

X axis is expressed as size (kDa). Y axis is fluorescence (RFU) and corresponds to concentration. LM is lower marker while Xsys are system peaks. Major peaks are additionally labeled with concentration (ng/μL). Contaminating proteins represented 28% of the total protein concentration and were evident as small peaks in a rough baseline.

For research use only. Not for use in diagnostic procedures.