CAG-GFP Adeno-associated virus(AAV Serotype 9)

Adeno-associated viruses (AAV) are promising tools for gene therapy due to their safety and efficacy in delivering therapeutic genes or gene-editing sequences to various tissues and organs. Among AAV serotypes, AAV serotype 9 (AAV9) stands out for its ability to effectively target a variety of tissues and, therefore, has great potential for clinical application. However, existing methods for purifying AAV are cumbersome, expensive, and often produce inconsistent results. Here, researchers explored a novel purification strategy utilizing Dynabeads^TM CaptureSelect^TM magnetic beads. AAV9 magnetic beads captured AAV9 with high specificity and recovery rates between 70% and 90%, whereas AAVX magnetic beads did not bind to AAV9. By continuously interacting with AAV in solution, these beads enhanced the clearance of genomic DNA and plasmids even in the absence of endonucleases. The beads can be regenerated at least eight times, and used beads can be stored for up to six months and reused without significant loss in recovery. The potency of the AAV9-purified vectors in vivo was comparable to that of iodixanol purified vectors.

To confirm the potency of magnetic affinity bead-purified AAV9, male C57BL/6JInv mice were injected with 5 × 1010 vg/mouse of magnetic affinity bead-purified AAV9-CAG-GFP and AAV9-CAG-Luc via the tail vein. The same dose of the corresponding iodixanol-purified AAV was also used as a positive control. Nine weeks after AAV injection, livers were harvested and subjected to ex vivo green fluorescent protein (GFP) imaging (Figure 1A) and quantification of GFP signals (Figure 1B). The results showed that AAV9-CAG-GFP purified using magnetic affinity beads was functional, with the quantified GFP signal being approximately 1.65-fold lower than that of iodixanol-purified AAV9-CAG-GFP. The gene copy number in the livers of mice injected with magnetic bead-purified AAV9-CAG-GFP was lower than that of iodixanol-purified AAV9-CAG-GFP (Figure 1C). The researchers used non-invasive imaging to monitor luciferase signals in AAV-injected mice (Figure 1D). Unlike the results of GFP imaging, the luciferase signals between magnetic bead-purified AAV9-CAG-Luc and iodixanol-purified AAV9-CAG-Luc showed more similar intensities in vivo (Figure 1E). This was further confirmed by quantifying the AAV genome copy number in the liver for AAV9-CAG-Luc vectors (Figure 1F).

Figure 1. In vivo bioactivity of AAV9-purified by magnetic affinity beads compared to iodixanol-purified AAV9. (Sia K C, et al., 2024)