Inexpensive, serotype-independent protocol for native and bioengineered recombinant adeno-associated virus purification

Abstract

Recombinant adeno-associated virus (AAV) is a valuable and often used gene therapy vector. With increased demand for highly purified virus comes the need for a standardized purification procedure that is applicable across many serotypes and includes bioengineered viruses. Currently cesium chloride banding or affinity chromatography are the predominate forms of purification. These approaches expose the final purified virus to toxic contaminants or are highly capsid dependent and may require significant optimization to isolate purified AAV. These methods may also limit crude viral lysate processing volume resulting in a significant loss of viral titer. To circumvent these issues, we have developed an AAV purification protocol independent of toxic compounds, supernatant volume and capsid moiety. This purification method standardizes virus purification across native serotype and bioengineered mosaic capsids.

Keywords: PEG8000; adeno-associated virus; centrifugation; inexpensive; purification.

Figure 1.

Comparison of purified AAV2/6 vector genome captured using heparin column chromatography and our PEG/Spin protocol. Using identical transfection and clarification procedures, the PEG/Spin captured on average 30 x more packaged genome as determined by qRT-PCR.

Figure 2.

Total protein tracking and capture through centrifugations and final pellet suspension. Fractions collected as 1.0 ml aliquots. Aqueous layer A is PEG/NaCl treated supernatant immediately following 3000 × g centrifugation. Crude Suspension represents the PEG/NaCl pellet resuspended in 50 ml 250 mM NaCl prior to 10,000 × g centrifugation. Aqueous Layer B represents the suspension buffer immediately following the final 149,000 × g centrifugation. AAV pellet is suspended in 7.0 ml 250 mM NaCl.

Figure 3.

SDS-PAGE electrophoresis on 20 microliter aliquots taken at different stages of purification. Lane: (1) Marker (Bio-Rad Precision Plus), (2) Crude supernatant pre-enzymatic digest, (3) Crude supernatant post filtration, (4) PEG/NaCl treated supernatant pre-3,000 × g centrifugation, (5) Aqueous layer post 3,000 × g centrifugation, (6) Crude PEG/NaCl pellet suspended in 40 ml Suspension Buffer pre-10,000 × g spin, (7) Virus containing aqueous layer post 10,000 × g spin.

Figure 4.

SDS-PAGE electrophoresis of purified and suspended AAV2/6. Lane: (M) Marker (Bio-Rad Precision Plus), (1) Viral pellet post 149,000 × g centrifugation suspended in 10ml buffer, (2) AAV2/6 viral suspension dialyzed overnight in suspension buffer to demonstrate further purification steps are not required and may result in titer loss, (3) heparin column purified AAV2/6 control, space indicates extraneous wells removed.

Figure 5.

Adult C57BL/6 mice imaged for luciferase subsequent to AAV2/6 & AAV2/41 systemic delivery. Mice were tail vein injected with 2 × 10¹² vg and imaged eight weeks after viral delivery. Our PEG/Spin protocol can successfully isolate functional naturally occurring and engineered AAV virions.

Figure 6.

Work flow diagram outlining stepwise progression through our protocol.