Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

Abstract

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1×10¹² to 1×10¹³ vector genomes/ml.

Introduction

Gene transfer experiments in animal models have shown that dramatic differences exist in the transduction efficiency and cell specificity of recombinant adeno-associated viral (rAAV) vectors of different serotypes [1], [2], [3], [4], [5], [6]. In general, there are two different approaches for packaging rAAV vectors: “true type” and “pseudotyped” vectors. The former refers to vectors having inverted terminal repeats (ITRs), Rep proteins, and capsid proteins derived from the same wild-type virus, e.g., AAV2 [7], [8], [9]. The latter refers to vectors derived from ITRs and Rep proteins of one serotype virus and capsid proteins of another, e.g., 2 and 1 (AAV2/1) [3], [4], [6], [10].The pseudotyping of AAV2-ITR-containing vectors was pursued because more experience exists with the safety profile of these ITRs in animal models and humans. The chromosomal integration efficiency and specificity have been investigated for AAV2 ITRs [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], but few data have been generated thus far with the ITRs of other AAV serotypes.

In recent years, there have been significant improvements in production and purification of rAAV vectors. The major improvements in production have included enhanced output of the number of particles per cell and the emergence of a number of scalable systems [21], [22], [23], [24], [25], [26], [27], [28]. Several groups have independently found that the use of plasmids to express adenovirus (Ad) helper genes in transient transfection results in greater efficiency of rAAV production than infection with Ad virus, perhaps because of enhanced viability of producer cells or the lack of competition with the helper virus for DNA replication machinery [29], [30], [31], [32]. Another interesting finding is that downregulation of Rep78/68 relative to Rep52/40 and the capsid proteins results in a greater accumulation of single-stranded DNA genomes and packaged vector DNA [33]. The incorporation of these improvements into transient transfection production protocols has enhanced yields from about 1–10 IU per cell to more than 100 IU per cell. Stable producer cell lines and packaging cell lines used in combination with recombinant hybrid AAV–adenoviruses have achieved 100–300 IU per cell. Hybrid AAV–herpes vectors have achieved outputs that approach the 5000–10,000 IU per cell seen with wtAAV. Overall, these newer methods produce greater vector yields, and reduce or eliminate detectable replication-competent AAV (rcAAV) contamination.

Early reports comparing the transduction efficiencies and specificities of rAAV vector serotypes relied on CsCl gradients for purification, but this approach can generate vector stocks with large particle:infectious (P:I) ratios. Purification using affinity chromatography, based on identified cellular receptors [34], [35], is becoming more common and the more physiological conditions result in vector stocks with P:I ratios of <200. An efficient and reproducible protocol based on partial purification of an initial freeze/thaw lysate by iodixanol gradient fractionation, followed by chromatography on heparin–Sepharose for the purification and concentration of rAAV2 vectors has been reported [36]. Rabinowitz et al. [4] described the binding of AAV2 and AAV3 to heparin–Sepharose. Auricchio et al. published the binding of AAV5 vectors to mucin crosslinked to Sepharose [37]; however, this matrix is not commercially available at this time. To date, there have been no reports on the chromatographic purification of AAV1 or AAV4 vectors. Here we describe the purification of rAAV1, 2, and 5 vectors by iodixanol gradient centrifugation and anion-exchange (Q-Sepharose) chromatography. The resulting vector stocks are 99% pure with titers of 1×10¹² to 1×10¹³ vector genomes/ml.