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.

Keywords: Adeno-associated virus; Bioprocessing; Gene therapy; Vector production.

Fig. 1

Adeno-associated virus (AAV) vector biology. Wild-type AAV genome (a) contains Rep and Cap genes. Rep encodes four regulatory proteins that play important roles in replication and encapsidation of viral DNA, and their expression is controlled by p5 and p19 promoters. Cap encodes three capsid proteins and assembly-activating protein (AAP), regulated by p40 promoter. In an AAV vector (b), the wild-type AAV Rep and Cap genes have been replaced with the transgene of interest. Three components have to be delivered into the host cell line either by transfection or viral infection: vector AAV DNA containing the transgene of interest, Rep and Cap genes (also known as packaging construct), and helper genes from adenovirus. Rep78 and 68 promote AAV DNA rescue and subsequent replication. Cap proteins are synthesized in the cytoplasm and are shuttled to the nucleus for assembly. AAP supports assembly and maturation of the AAV capsid (Samulski and Muzyczka 2014). Rep52 and 40 interact with single-stranded DNA and pre-formed capsids to promote viral DNA encapsidation by a mechanism not yet fully understood (Ling et al. 2015). P, promoter; pA, polyadenylation sequence

Fig. 2

rAAV production-related impurities and molecular strategies aimed for their reduction. Adapted from Wright (2014)

Fig. 3

rAAV-producing systems: Production in adenovirus complementation systems (a) are traditionally performed as plasmid transfection processes, where AAV Rep/Cap genes, the ITR-flanked gene of interest (GOI), as well as AdV-helper genes are provided to a E1a/E1b-containing HEK293 cell line. HSV complementation systems (b) use two recombinant herpes viral strains to provide AAV Rep/Cap genes, GOI, and HSV-helper elements to a mammalian cell line such as BHK. Sf9—baculovirus expression systems (c) require two recombinant BV viral strains to provide the AAV-producing capability to insect cells. AAV protein expression is controlled by Sf9 natural promoters. Yeast-based systems (d) are transformed with a set of extrachromosomal plasmids that contain six AAV expression cassettes and GOI. AAV protein expression is controlled by yeast natural promoters