Production and Purification of Recombinant Adeno-Associated Vectors

Abstract

The use of recombinant adeno-associated virus (rAAV) vectors in gene therapy for preclinical studies in animal models and human clinical trials is increasing, as these vectors have been shown to be safe and to mediate persistent transgene expression in vivo. Constant improvement in rAAV manufacturing processes (upstream production and downstream purification) has paralleled this evolution to meet the needs for larger vector batches, higher vector titer, and improved vector quality and safety. This chapter provides an overview of existing production and purification systems used for adeno-associated virus (AAV) vectors, and the advantages and disadvantages of each system are outlined. Regulatory guidelines that apply to the use of these systems for clinical trials are also presented. The methods described are examples of protocols that have been utilized for establishing rAAV packaging cell lines, production of rAAV vectors using recombinant HSV infection, and for chromatographic purification of various AAV vector serotypes. A protocol for the production of clinical-grade rAAV type 2 vectors using transient transfection and centrifugation-based purification is also described.

Accelerating the clinical development of protein-based vaccines for malaria by efficient purification using a four amino acid C-terminal ‘C-tag’

“…This resin can be produced to be suitable for use in clinical and commercial biomanufacture. Notably, multiple different single chain antibody-based affinity resins are now available for cGMP manufacture, and the first product purified in this manner, an adeno-associated virus gene therapy product called alipogene tiparvovec (Glybera®) for lipoprotein lipase deficiency, has been licenced in Europe (Wang et al, 2011, Wang et al, 2015). Here, C-terminal fusion of this short C-tag to PfRH5 achieved >85% recovery and >70% purity in a single step purification directly from clarified, concentrated S2 cell supernatant under mild conditions.…”

Long‐term anti‐endotoxin/E. coli efficacy in mice transfected with AAV2/1‐muBPI25‐muFcγ1

“…AAV-muBPI-Fc viruses were prepared by AGTC Gene Technology Company Ltd. (Vector Gene Technology Company Ltd. Beijing, China), complying with the guidelines of SFDA and GMP facilities, according to the protocols described previously (15,(20)(21)(22). The AAV-muBPI-Fc viruses were purified and diluted to the concentration of 1 9 10 12 vector genomes (v.g.…”

An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes

“…Additionally, the limited capacity and complex manufacturing process present further obstacles to their application. 10 Similar limitations apply for the infection of hepatocytes with adenoviral vectors: gene delivery is only transient and their in vivo application induces a profound immune response in the liver. 11 However, even with transient expression, these vectors can be successfully employed to genetically alter hepatocytes in vivo either by the use of recombinases such as Cre or Flp in genetically engineered mice or by the adaptation of novel genetic tools such as CRISPR/Cas9 systems to induce stable mutations.…”

“…Although AAV vectors target hepatocytes with high efficiency, integration into the hepatocyte genome is considered to be a rare event and AAV‐mediated gene expression is usually rapidly lost in dividing hepatocytes. Additionally, the limited capacity and complex manufacturing process present further obstacles to their application . Similar limitations apply for the infection of hepatocytes with adenoviral vectors: gene delivery is only transient and their in vivo application induces a profound immune response in the liver .…”