![]() In contrast, none of the ligands affected AAV2 and AAV8 transduction ( Figure 2B), even at lower MOIs as shown for AAV2 ( Figure 2C). The neutralizing potential of the affinity ligands in 100-fold excess per binding site on the capsid, analyzed by a transduction assay, showed AAV1 neutralized by AVB and CSAL8 while CSAL9 had little to no effect ( Figure 2B). Although the affinity ligands are covalently linked to resins, small quantities of ligand leakage can occur during the purification process (information provided by Thermo Fisher Scientific). While the intention was to determine each resin’s ability to purify a given AAV serotype, the yields obtained in the elution fractions were consistent with what is advertised (data not shown). The binding capacities of AVB, CSAL8, and CSAL9 are specified by the manufacturer as >10 12, >10 13, and >10 14 capsids per 1 mL of resin, respectively. CSAL9 specifically purified only AAV9 vectors with high efficiency ( Figure 2A). CSAL8 efficiently purified AAV1 (and AAV6, data not shown) and AAV8 vectors but was unable to purify AAV2, AAV5, and AAV9 ( Figure 2A). Quantitative PCR (qPCR) quantification of vector titer, in the flowthrough, wash, and elution fractions, showed that AVB effectively purifies AAV1, AAV2, and AAV5, had low efficiency for AAV8, and was unable to purify AAV9 ( Figure 2A). The purification efficiencies for AAV1, AAV2, AAV5, AAV8, and AAV9, starting from clarified cell lysates, were compared for AVB, CSAL8, and CSAL9 ( Figure 2A). To our knowledge, there is no published comparative analysis of the newer affinity resins CSAL8 and CSAL9. This information is critical and could be generally applicable in the development of novel AAV vectors amenable to affinity column purification. Neutralization was observed for AAV1 and AAV5 with AVB, for AAV1 with CSAL8, and for AAV9 with CSAL9, associated with regions that overlap with epitopes for neutralizing monoclonal antibodies against these capsids. The data show that only a few residues within the epitopes served to block affinity ligand binding. The AAV contact residues required for ligand binding, and thus AAV purification, and the ability of the ligands to neutralize infection were analyzed. The AAV-ligand complex structures showed that AVB and CSAL9 bound to the 5-fold capsid region, although in different orientations, and CSAL8 bound to the side of the 3-fold protrusion. In this study, we utilized cryo-electron microscopy and 3D image reconstruction to map the binding sites of these affinity ligands on the capsids of several AAV serotypes, including AAV1, AAV2, AAV5, AAV8, and AAV9, representing the range of sequence and structure diversity among AAVs. These include AVB Sepharose (AVB) and POROS CaptureSelect affinity ligand for AAV8 (CSAL8) and AAV9 (CSAL9). ![]() This method utilizes camelid single-domain antibodies recognizing AAV capsids. Gene Editing: Technology & ApplicationsĪffinity-based purification of adeno-associated virus (AAV) vectors has replaced density-based methods for vectors used in clinical settings. ![]()
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