January 15, 2017 (Vol. 37, No. 2)

Range of Methods Used in Generating and Purifying AAV Vectors

Vectors derived from adeno-associated virus (AAV) provide promising gene delivery vehicles that can be used effectively in large-scale productions for preclinical target identification/validation studies, or used in large animal models and clinical trials of human gene therapy.

Why is AAV one of the most promising viral gene transfer vectors? Notably, recombinant adeno-associated virus (rAAV) vectors come in different serotypes (AAV 1–9), each with different tissue tropisms. rAAV provides a high rate of gene transfer efficiency, long-term gene expression, and natural replication deficiency. It is nonpathogenic and does not have the capability of altering biological properties upon integration of the host cell.

However, achieving preclinical efficacy testing, especially in large animal models and toxicology studies, requires vector quantities that simply cannot be produced in a laboratory setting or in most research-grade vector core facilities. Current methods for transfection require use of adherent HEK 293 cell cultures, expanded by preparing multiple culture plates. Ideally, a single large-scale suspension culture would be a replacement for multiple culture plates.

In this tutorial, we examine some of the currently available schemes used in generating rAAV from suspension cultures, and describe what it takes to achieve scalable rAAV production. 

Scalable Production

Two basic systems for growing cells in culture exist: monolayers on an artificial substrate (i.e., adherent culture) and free-floating in culture medium (i.e., suspension culture). rAAV vector production uses a triple transfection method performed in adherent HEK 293 cells, which is the most common and reliable method (Lock et al., 2010), albeit resource intensive. Due to its scalability and cost, rAAV cell suspensions are more desirable.

To simplify scalability and dramatically decrease operational costs and capital investments, use of bioreactors provides process simplification, from pre-culture to final product. Two examples are the iCELLis from Pall Life Sciences, designed for adherent cell culture applications, and the WAVE Bioreactor from GE Healthcare Life Sciences, ready for batch culture, fed-batch culture, perfusion culture, and cultivation of adherent cells.

Both are designed for convenient handling and control of cell cultures up to 25 L. Both enable rapid and scalable rAAV production. Recently, Grieger et al. (2016) showed suspension HEK293 cell lines generated greater than 1×105 vector genome-containing particles (vg)/cell or greater than 1×1014 vg/L of cell culture when harvested 48 hours post-transfection, a protocol developed and used to successfully manufacture GMP Phase I clinical AAV vectors.

Large-scale productions require consistent and reproducible. AAV produced for clinical uses must be thoroughly analyzed to identify the main purity, potency, safety, and stability factors described below.


Empty capsids typically take 50–95% of the total AAV particles generated in cell culture, depending on specific serotypes and protocols used. Empty capsids may solicit deleterious immune response against AAV (Zaiss and Muruve, 2005). It is desirable they be minimized during production and removed during purification.

AAV empty capsids are composed of an AAV capsid shell identical to that of the desired product, but lacking a nucleic acid molecule packaged within. Gradient ultracentrifugation using iodixanol is effective in separating empty capsids. Assessment and measurement can be done by either electron microscopy or A260/A280 spectrometry. It may be difficult to distinguish AAV capsids containing small fragments of DNA not readily distinguished from completely empty capsids using density centrifugation or electron microscopy.

Helper virus-dependent replication-competent AAV (rcAAV), also referred to as “wild-type” or “pseudo-wild-type” AAV, is an AAV capsid particle containing AAV rep and cap flanked by ITR. This type of AAV (rcAAV) is able to replicate in the presence of a helper virus.

Though wild-type AAV is unable to replicate autonomously and requires co-infection with helper viruses, such as adenovirus, the expression of AAV rep or cap from rcAAV present in an AAV vector increases the risk of immunotoxicity in vector-transduced tissues. Replication competent rcAAV is a rare (-8) and yet deleterious event.

To assess rcAAV generation, target DNA sequence spans left AAV2 ITR D-Sequence and AAV2 rep sequence. An intact left AAV ITR-rep gene junction is a requisite feature for AAV replication to occur in vivo in the presence of a helper virus. The assay employs sequence-specific PCR primers and a dual-labeled hybridizable probe for detection and quantification of amplified DNA junction sequence.

rcAAV DNA sequence titer is calculated by direct comparison to the fluorescent signal generated from known plasmid dilution bearing the same DNA sequence. A positive signal indicates an intact left IRT-rep gene junction has been detected and amplified, representing the maximum possible rcAAV contamination level present in the rAAV vector sample being analyzed. It does not indicate whether the DNA sequence is infectious or capable of helper-virus assisted replication.


High potency of AAV vectors is achieved by carefully selecting and isolating full capsids. Physical and functional titers can be measured to assess the actual potency of AAV production. Physical titer measures the encapsidated AAV vector genome, a key mediator and indicator of therapeutic effect. Measurement of vector genomes by quantitative real-time PCR is the closest physical indicator of rAAV vectors. Functional titer is established by measuring transgene protein expression in a dose-dependent manner, following transduction into appropriate cell lines.


Safety concerns comprise infectious agents used to generate AAV vectors. Mechanisms to inactivate infectious viruses include heat inactivation of adenoviruses and detergent inactivation of enveloped viruses. A complete list of product release tests can include adventitious virus tests of porcine, canine, and bovine viruses (Table).

Payload Increase

Developing viral vector comes with key features scientists strive for, including large payload capacity. With rAAV, the limited packaging capacity precludes the design of vectors for the treatment of diseases associated with larger genes; AAV has a packaging capacity of up to 4.5 kb for packaging foreign DNA.

Utilizing the split vector system, which exploits head-to-tail concatamerization formation, has been developed to circumvent AAV small packaging capacity. Two main approaches include  trans-splicing and  homologous recombination methods; both depend upon recombination between two vector genomes, with each genome encoding approximately half the transgene, within the same cell to achieve gene expression. ViGene has developed a trans-splicing system that can expand the payload to 8 kb.

We setup a screen for a more efficient trans-splicing system. Here, the GFP reporter was split and cloned into two AAV vectors. Based on GFP expression, after co-transfection in HEK-293 cells, we scored expression efficiency of the combination of different trans-splicing elements and included the selection of a splicing donor and acceptor sequence, and the annealing sequence in the intron. Our best vector generated about 70% expression compared to single vector (Figure 1).

Figure 1. Elements in optimization. Before optimization, the trans-splicing efficiency is about 1–5% when compared to GFP expression from single vector. After optimization, the efficiency reaches about 25–50%.

We generated and purified both vectors in AAV9 trans-splicing and efficiency and expression levels were tested in vitro and in vivo. Following 72-hours transduction in HEK-293 cells by AAV9 virus (Figure 2), GFP expression was detectable in approximately 70% of cells, about 50% when compared to single vector GFP expression. Similar efficiency and expression levels were observed in RGC neurons, following two-week intraocular injection.

A deeper understanding of the molecular basis for inherited and acquired diseases continues to drive the broader adoption of AAV as the vector of choice for treating many diseases. Numerous Phase I and II trials utilizing AAV have been performed for various inherited and acquired diseases.

A continuation to greatly increase AAV vector yield, improve AAV potency and purity, and increase payload size will further make AAV a bigger player in gene therapy.

Figure 2. Trans-splicing AAV vector expression of GFP in vitro and in vivo.

Eva Szarek, Ph.D., is scientific marketing and sales manager and Jeffrey Hung, Ph.D. ([email protected]), is chief commercial officer and GM of cGMP business at ViGene Biosciences.

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