Cesium Chloride Gradient Sedimentation
Adeno-associated virus (AAV) vectors have rapidly advanced to the forefront of gene therapy in recent years. In the past decade, several naturally occurring AAV serotypes with over 100 AAV variants from different animal species have been isolated and greatly promote the development of AAV vector-based gene therapy. The use of these vectors in clinical gene transfer has increased the demand for their characterization, production, and purification. As a leading company in the gene therapy area, Creative Biolabs is devoted to helping global researchers purify AAV vectors using our advanced cesium chloride gradient sedimentation technology.
Cesium Chloride Gradient Sedimentation
With the rapid development of gene therapy, high purity of vector preparations is a key goal of AAV manufacturing both for research and clinical applications. Currently, two gradient centrifugation methods, namely cesium chloride (CsCl) density gradient ultracentrifugation and iodixanol gradient centrifugation, have been applied for AAV vectors purification. CsCl density gradient ultracentrifugation is the standard method of AAV vectors purification. By spinning the virus preparations in an ultra-centrifuge at high G-forces in solutions containing various CsCl concentrations, we can establish a density gradient which allows separation of empty, partially packaged, and fully packaged viral vector particles from cellular debris, proteins, and nucleic acids in the crude viral lysate.
Figure 1. Efficient removal of empty virions from rAAV8 preparations by CsCl gradient centrifugation. (Gao, 2014)
Process of CsCl Gradient Sedimentation
AAV vectors purified by CsCl gradient centrifugations have been evaluated in many preclinical studies both in small and large animals. This method is suitable for small-scale purification and is less expensive than column chromatography or commercial purification kits. In general, the purification of AAV by CsCl density gradient is easy to perform and yields highly pure viral preparations suitable for research applications. The typical CsCl protocol contains multiple purification steps, such as cell lysis, precipitation of DNA and proteins, ultracentrifugation, dialysis, and concentration. For animal experimentation, rAAV purification should include multiple CsCl gradient centrifugation steps.
Services
With years of experience in gene therapy, Creative Biolabs has successfully established a top CsCl gradient centrifugation platform for AAV vectors production and purification. Using our CsCl gradient centrifugation assay, our experienced scientists are happy to help you separate highly pure AAV vectors from their empty particles in the most cost-effective way.
Creative Biolabs is devoted to accelerating the development of gene therapy programs. In addition to CsCl gradient centrifugation, we also provide AAV vectors purification service by iodixanol gradient centrifugation. Our expertized staff will help you choose the most appropriate method for your AAV vector's purification. If you are interested in the service we offer, please feel free to contact us.
Reference
- Gao, K.; et al. (2014). Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects. Molecular Therapy-Methods & Clinical Development. 1: p.9.
