Molecular design for recombinant adeno-associated virus (rAAV) vector production

Abstract

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 103 to 105 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization.Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system.

Two-Step Small Scale Purification of Recombinant Adeno-Associated Viruses

“…AAVs are preferred vectors for many in vivo gene transfer applications to non-dividing cells. In order to facilitate gene delivery for basic and clinical research, manufacturing practices for AAV production have been optimized to provide research and clinical grade preparations [10]. A myriad of AAV samples are commercially packaged and available for purchase.…”

“…Currently, there are several platforms used for production and purification of AAVs [10,11]. Biotech manufacturing and virus core facilities often rely on helper viruses such as adenovirus, herpesvirus, or baculoviruses for en mass delivery of complementary genes for large-scale and cost-efficient AAV production.…”

A rAAV2‐producing yeast screening model to identify host proteins enhancing rAAV DNA replication and vector yield

“…There are currently three production platforms available for industrial recombinant adeno‐associated viral vector (rAAV) vector production. Adenovirus, Herpesvirus, and Baculovirus complementation systems constitute robust platforms for vector production at preclinical and clinical scale . Several process optimization studies achieved a considerable improvement in vector particle formation by focusing on many bioengineering aspects such as cell growth, plasmid transfection, media optimization, and vector purification .…”

“…1 The AAV genome is~4. 7 kb with three open reading frames (ORFs), Rep, Cap, and assembly activating protein (AAP); flanked by inverted terminal repeats (ITRs). The Rep gene encodes four nonstructural proteins important for DNA replication and encapsidation (Rep40, 52, 68, and 78).…”

“…Adenovirus, Herpesvirus, and Baculovirus complementation systems constitute robust platforms for vector production at preclinical and clinical scale. 6,7 Several process optimization studies achieved a considerable improvement in vector particle formation by focusing on many bioengineering aspects such as cell growth, plasmid transfection, media optimization, and vector purification. [8][9][10][11][12] Vector volumetric yields range between 10 10 and 10 11 vector genomes (vg) per mL of culture; however, the generation of sufficient rAAV quantities to supply drug commercialization remains a significant challenge.…”