Multimodal production of adeno-associated virus

Adeno-associated virus (AAV) is an increasingly popular tool in the research laboratory, and use of this viral vector clinically is occurring at an accelerated pace. Nevertheless, despite its popularity, AAV is a relatively cumbersome virus to produce; however, significant efforts have been invested to develop, optimize, and simplify methodology that allows the generation of high-quality AAV with significantly increased production yields. Here we describe multiple modalities for production and purification of AAV particles produced in HEK293 cell cultures using an iodixanol density gradient. We include two methods adapted for harvesting virus from the culture media: Tangential flow filtration (TFF) and polyethylene glycol precipitation (PEGylation). Moreover, we also describe the protocol for anion exchange chromatography, which can be used after the iodixanol gradient as an additional purification step. Last, we provide various protocols for determining virus titer

Basic Concepts in Viral Vector-Mediated Gene Therapy

Today any researcher with the desire can easily purchase a viral vector. However, despite the availability of viral vectors themselves, the requisite knowledge that is absolutely essential to conducting a gene therapy experiment remains somewhat obscure and esoteric. To utilize viral vectors to their full potential, a large number of decisions must be made, in some instances prior to even obtaining the vector itself. For example, critical decisions include selection of the proper virus, selection of the proper expression cassette, whether to produce or purchase a viral vector, proper viral handling and storage, the most appropriate delivery method, selecting the proper controls, how to ensure your virus is expressing properly, and many other complex decisions that are essential to performing a successful gene therapy experiment. The need to make so many important decisions can be overwhelming and potentially prohibitive, especially to the novice gene therapist. In order to aid in this challenging process, here we provide an overview of basic gene therapy modalities and a decision tree that can be used to make oneself aware of the options available to the beginning gene therapist. This information can be used as a road map to help navigate the complex and perhaps confusing process of designing a successful gene therapy experiment