We demonstrate the rapid and reliable quantification of physical AAV-2 (adeno-associated virus type 2) particles via a novel ELISA based on a monoclonal antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at least 80% of all particles were empty, compared with a maximum of 50% in wild-type AAV-2 stocks, indicating that the recombinant genomes were less efficiently encapsidated. This finding was confirmed upon titration of CsCl gradient fractions from recombinant and wild-type AAV-2 stocks. ELISA-based measurement of capsid numbers revealed a large number of physical particles with low densities corresponding to empty capsids in the recombinant, but not in the wild-type AAV-2 preparations. Moreover, additional expression of VP proteins during rAAV production was found to result in an excessive capsid formation, whilst yielding only minor increases in DNA-containing or transducing rAAV particles. We conclude that encapsidation of viral genomes rather than capsid assembly can be limiting for rAAV production, provided that a critical level of VP expression is maintained. The feasibility of quantifying AAV-2 capsid numbers via the ELISA allows determination of physical to DNA-containing or infectious particle ratios. These are important parameters which should help to optimize and standardize the production and application of recombinant AAV-2.
Gene therapy vectors derived from the human parvovirus AAV-2 (adeno-associated virus type 2) have gained attention owing to a unique combination of attractive features. Wild-type AAV-2 is nonpathogenic in humans and naturally defective, requiring coinfection with a helpervirus (eg adenovirus) for a productive infection.1 Recombinant AAV-2 can infect both dividing and nondividing cells in vitro and in vivo2,3,4,5,6 and have the potential for site-specific integration into chromosome 19.7,8,9 Long-term expression of heterologous genes transduced by rAAV has been observed in a variety of human cells and tissues, such as muscle,10,11 lung,12 central nervous system,13,14,15 retina16 and liver.17
An important prerequisite for the testing of AAV-2 vectors in preclinical and clinical studies is the accurate and reliable titration of the recombinant virus particles. Precise information on rAAV titers is not only crucial for the careful planning and execution of such studies, but also for comparing results amongst laboratories. In brief, presently available methods for rAAV titration can be divided into biological and physical assays. Biological assays rely on infection of cultured cells followed by events that depend on the biological functionality of the rAAV vector, ie either replication of the recombinant genomes in cells in the presence of AAV-2 and adenoviral helper functions, or expression of the transduced heterologous gene.18,19,20 These two types of assay yield titers of infectious or transducing particles, respectively. In contrast, physical methods are independent of biological functions of the recombinant viruses. Typically, viral DNA is extracted from the rAAV particles via enzymatic digestion of the capsids and quantified using serially diluted plasmid DNA bearing the transgene as standard.18,21 The number of DNA-containing particles is then calculated assuming that each virion carries one single-stranded DNA molecule with a defined size.
In spite of being rather simple in concept and execution, all common rAAV titration methods display distinct disadvantages. Biological methods are highly dependent on the properties of the vectors and the particular assay conditions, which are prone to differ among various laboratories. For example, quantification of functional transducing rAAV particles is influenced by the cell type used, the promotor driving the transgene or the transgene itself.22,23 Another crucial parameter is the helper virus, which can increase the efficiency of rAAV-mediated gene transduction and consequently the functional rAAV titers by three orders of magnitude.23,24,25 Similarly, physical methods to measure DNA-containing particles involve a number of steps, in particular exhaustive enzymatic treatment of the virions, which are possibly inconsistent in different laboratories and thus may provoke variable results. As a consequence, rAAV titers as well as the ratios of physical to functional particles, which are considered as important indices for the quality of rAAV preparations, show a great variation, thus making direct comparisons of results from different studies quite difficult. Moreover, whilst quantification of encapsidated viral genomes is usually considered to yield total particle titers, this method of course fails to detect empty virions which are devoid of DNA. In fact, there is a lack of methods for quantification of total numbers of physical rAAV particles, ie the sum of assembled packaged or empty virions. Yet, in view of the possibility that AAV-2 capsid proteins can elicit a humoral host immune response, methods to determine exact numbers of total AAV-2 capsids in a given rAAV preparation are urgently needed.
In this report, we present a novel AAV-2 capsid ELISA that fits in this gap by allowing the rapid and reliable quantification of assembled, empty or full, AAV-2 particles. Given this property, the ELISA not only permits a better characterization of rAAV preparations, but also a more profound investigation of the AAV-2 genome packaging process in general. Indeed, we obtained and present first evidence that encapsidation of recombinant AAV-2 genomes occurs rather inefficiently and can be a limiting step for rAAV vector production.
A sandwich ELISA allowing the quantification of assembled AAV-2 particles was developed based on a previously described monoclonal antibody, A20, that specifically binds to assembled AAV-2 capsids, but not to single capsid proteins.26 A scheme depicting further details is shown in Figure 1a. To calibrate the ELISA, stocks of empty AAV-2 capsids or full AAV-2 virions were used as particle standards. The empty capsids were prepared from 293 cells infected with a recombinant adenovirus which expressed the AAV-2 cap gene under the control of a human CMV promoter (rAdVP) and purified as described.27 To determine the maximum number of AAV-2 capsids in this empty particle standard, the protein concentration of an aliquot was determined using a commercial protein quantification assay (BioRad, München, Germany). Assuming a stoichiometry of the three VP proteins of 1:1:10 in the assembled capsid (Figure 1b), an approximate number of 1.2 × 1012 capsids/ml was deduced from the total protein concentration of 7.8 ng/μl measured for the empty particle standard. In addition, the capsid number was determined by mixing equal amounts of the empty particle stock and an adenovirus 5 (Ad-5) stock of known titer (5 × 1010 pfu/ml) and by counting the AAV-2 and Ad-5 particles in 40 randomly chosen electron microscopy pictures of this mixture (Figure 1c). A 34.6-fold excess of AAV-2 capsids relative to Ad-5 capsids was counted, and since the Ad-5 stock contained 2.3 × 1011 capsids/ml (determined according to Ref. 28), the particle standard was calculated to have 8 × 1012 empty AAV-2 capsids/ml (assuming that AAV-2 and Ad-5 particles bind equally well to the EM grids). Likewise, a preparation of CsCl-purified, UV-inactivated wild-type AAV-2 particles was established as full particle standard, having a similar titer of 7.9 × 1012 capsids/ml.
Serial two-fold dilutions of the empty or full particle standard were used to obtain ELISA titration curves (examples are shown in Figure 1d). In the experiments reported below, only the linear part of the standard curves, ie the part between optical densities of 0.4 and 1.6 (corresponding to 107 to 108 particles per 100 μl), was used to derive values for unknown samples. The coefficient of variation in this part of the curves was 3–25%, but increased up to 67% at lower optical densities (calculated from 10 standard curves). Therefore, we consider the optical density of 0.4, which corresponds to approximately 107 assembled particles per sample, as the quantification limit of the AAV-2 capsid ELISA, while the actual detection limit is somewhat lower (approximately 106 particles). This renders the AAV-2 ELISA sensitive enough to be used for titration of any rAAV preparation, assuming an average generation of at least 104 assembled particles per cell during rAAV production.
The finding that the curves derived from empty or full particle standard samples with similar titers were nearly identical proved that the ELISA can be applied to the titration of both types of AAV-2 particles. This was expected since the ELISA relies on antibody(A20)-mediated recognition of an epitope which is supposed to be present on any assembled AAV-2 capsid, irrespective of being empty or full. Furthermore, earlier experiments have already shown that the monoclonal antibody A20 recognizes both wild-type and recombinant particles.20,26 Thus, the AAV-2 capsid ELISA not only provides a high grade of versatility, but is also highly suitable for standardization between laboratories, as it allows the titration of any type of AAV-2 particle via one invariant protocol.
Finally, spiking experiments were performed to address the question if sample matrix effects would influence the outcome of the ELISA measurement. Aliquots of a stock of purified empty capsids were mixed with different aliquots of cell culture medium, CsCl or MgCl2 and titrated via ELISA. Except for MgCl2 at concentrations above 20 mM, particle titers were found to vary less than 20%, which was within the range that could be expected from the variation coefficient of the standard curves (see above). From these findings, it was concluded that ELISA-based titration is not influenced by sample matrix effects.
Following the general characterization of the ELISA, we analyzed how the assembled particle titers obtainable via the ELISA would relate to titers determined by common methods for AAV-2 titration. Therefore, we first prepared a series of wild-type AAV-2 stocks (Table 1) and titrated them via the ELISA to gain numbers of assembled particles. In parallel, these stocks were analyzed via commonly used methods to determine corresponding numbers of DNA-containing, infectious or transducing particles. For details and results see Table 1. As expected, the assembled particle titers determined via ELISA were always highest, reaching up to approximately 1012 capsids/ml for the AAV-2 stocks derived from infection, and being only slightly lower for those generated by transfection. Importantly, the titers of DNA-containing particles were generally two- to four-fold lower than the assembled particle titers. This was clearly in line with the assumption that only a portion of the total particles present in an AAV-2 stock has packaged DNA or is infectious. Assuming that the DNA quantification method yielded reliable DNA-containing particle titers, the data suggest that a minimum of 25 up to 50% of the AAV-2 capsids were packaged or somehow associated with viral genomes. The numbers of infectious or transducing AAV-2 particles were one to three orders of magnitude lower than the numbers of assembled particles and at least 10-fold lower than those of the DNA-containing particles (Table 1 and Figure 2a-c).
In the course of these first titration experiments, the AAV-2 ELISA proved to be very reliable. Repeated titration of one distinct AAV-2 stock under consistent conditions (as described in Figure 1a) gave capsid titers which were highly reproducible. The inter-assay variation was actually found to be less than 20%, which is most likely due to the fact that ELISA-based AAV-2 titration, in contrast to common methods, neither involves steps which are prone to influence the particle measurement, such as prior enzymatic treatment of the virions, nor any biological components, eg cells or a helper virus.
Subsequent experiments aimed at confirming that the ELISA could also be used for the titration of recombinant AAV-2 particles. Table 2 lists a variety of rAAV stocks that were generated and then analyzed in a manner identical to the wild-type AAV-2 stocks. In general, most of the various titers measured for the rAAV stocks prepared using the pDG helper plasmid were several-fold higher than those obtained via transfection and infection of the cells with the pΔTR helper construct and Ad-5, respectively. This confirms and extends our own previous findings that rAAV production using pDG is more efficient than using a standard Rep-/VP-expressing packaging plasmid.20
Similar to wild-type AAV-2, the assembled particle titers obtained via the ELISA were highest in each of the rAAV stocks analyzed, being on average only slightly below those calculated for the wild-type virus particles (Tables 1 and 2). The numbers of DNA-containing rAAV particles, however, were markedly lower than the particular wild-type AAV-2 titers, resulting in higher average ratios of physical to DNA-containing rAAV particles of approximately 5 up to 60:1. These ratios depended mainly on the vector plasmid used but seemed not to be influenced by the choice of cell line or helper plasmid (Table 2 and Figure 2a–c). This indicated that, as compared with wild-type AAV-2, fewer capsids were loaded with recombinant genomes, namely a maximum of only 20% (eg pTRUFlacZ) down to 1.7% (pAVJE). The numbers of infectious or transducing rAAV showed an even stronger decrease not only compared with wild-type AAV-2, but in particular to the rAAV capsid titers, indicating that only a very small portion of less than 0.1% of the assembled rAAV particles were actually infectious or capable of transduction in these assays (Table 2 and Figure 2a–c).
Caution was taken, however, when interpreting these initial findings which were based on comparisons of capsid and genome numbers. This was because the DNA quantification method used is highly dependent upon a number of parameters and thus possibly yielded DNA-containing particle titers that were not fully reliable. In particular, it can not be excluded that numbers of DNA-containing particles were overestimated due to replicated genomes outside the capsids that were protected against DNaseI digestion (eg by Rep or cellular proteins). The preponderance of empty capsids in rAAV stocks thus needed additional confirmation. Therefore, a wild-type AAV-2 and two rAAV(lacZ) stocks were subjected to CsCl density gradient centrifugation and gradient fractions having densities of 1.30 to 1.50 g/cm3 were analyzed using the same titration methods as before (Figure 2d-f). The quantification of either DNA- containing or transducing wild-type AAV-2 particles revealed one distinct peak at a density of 1.40–1.43 g/cm3, where full and infectious particles were expected to be found.18,37,38 Importantly, ELISA-based measurement of assembled particles resulted in a peak which was not only the strongest one, but also matched the peaks of the DNA-containing or transducing particles. A second, but smaller culmination of physical particles was found at a density of around 1.33 g/cm3, where empty particles were supposed to accumulate.18,37,38 Two conclusions are drawn: firstly, the fact that measurement of capsid concentrations gave two peaks at the expected densities which complemented the data obtained using common titration methods strongly proved that the ELISA is dependable. Second, the appearance of a distinct peak of assembled particles being paralleled by culminations of DNA-containing and transducing particles reinforced the initial finding that a large portion of wild-type AAV-2 virions is actually packaged and capable of transduction.
Analyses of the two rAAV(lacZ) stocks gave distinct peaks for DNA-containing and transducing particles that matched each other, although as compared with wild-type AAV-2, a shift to a higher density of 1.43–1.46 g/cm3 was noticed. However, ELISA-based titration revealed a predominant peak of assembled rAAV particles at a low density of 1.33–1.36 g/cm3, indicating a large portion of empty capsids. Vice versa, an ELISA peak matching the majority of DNA-containing and transducing rAAV particles at a density of about 1.44–1.45 g/cm3 was barely detectable (Figure 2d–f). A larger portion of empty versus full particles was also observed after CsCl gradient fractionation of rAAV stocks that were generated using the pΔTR plasmid (data not shown). Taken together, all results clearly confirmed the preponderance of non- packaged and non-transducing particles in rAAV stocks. Moreover, this finding is also clearly in line with our recently published electron microscopy analysis of rAAV stocks in which the majority of the observed rAAV particles were found to be empty.20 Two reasons for this phenomenon can be speculated: recombinant AAV-2 genomes could either be replicated less efficiently in cells than wild-type genomes, yielding fewer single stranded DNA molecules ready for encapsidation. Alternatively, recombinant AAV-2 genomes might also be lacking some yet undefined elements required for efficient DNA encapsidation additional to the terminal repeats. However, discriminating between these possibilities would have required additional investigation which was beyond the range of this report.
Irrespective of the exact reasons, the pure finding that rAAV preparations are characterized by an excess of empty capsids is of particular importance with respect to rAAV production. Several reports have recently shown that an efficient rAAV production depends on a strong VP protein expression,39,40 which, as demonstrated by others, itself is a prerequisite for a high rate of AAV-2 capsid assembly.20,41 Based on these correlations, we finally investigated whether further increasing VP protein expression and thus the rate of AAV-2 capsid assembly during rAAV production would result in a concomitant increase in assembled and functional rAAV particle titers. Therefore, several rAAV stocks were generated via cotransfection of 293 cells with pDG and pTRUFlacZ and by additionally overinfecting the cells with the rAdVP virus (see above). Control rAAV stocks were prepared under identical conditions, but leaving out the rAdVP infection. Titration of the stocks derived from the rAdVP-infected cells revealed a two- to five-fold increase in DNA-containing and only marginal changes of transducing rAAV particle numbers as compared with the control stocks (Figure 3a). However, the capsid numbers were up to 25-fold increased, indicating the generation of a massive excess of non-packaged and non-transducing assembled particles as a result of the rAdVP infection (Figure 3a).
Similar observations were made when VP protein expression was elevated by transfection of 293T cells with increasing molar amounts of pDG relative to pTRUFlacZ (Figure 3b/c). A 1:1 molar ratio of helper and vector plasmid resulted in the production of a seven- to eight-fold excess of assembled to DNA-containing particles, while yielding the highest transducing particle titers. As expected, transfection of a five- or 10-fold molar excess of pDG was followed by an elevated production of Rep and VP proteins (in a constant stoichiometry, Figure 3b) and consequently an increased generation of assembled capsids (as detected via ELISA, Figure 3c). On the other hand, the numbers of DNA-containing or transducing particle titers slightly declined, which led to increased ratios of assembled to DNA-containing or to transducing particles. After transfection of up to 10-fold lower molar amounts of pDG than pTRUFlacZ, AAV-2 protein expression as well as titers of assembled or transducing particles decreased, and only the numbers of DNA- containing particles remained constant (Figure 3b/c). Thus, the excess of assembled to DNA-containing and to a lesser extent, transducing particles was significantly reduced under those conditions, however, the absolute transducing particle titers were also lowest. Similar results were obtained using the 293 cell line except that the absolute particle titers were all several-fold lower (data not shown).
In sum, these two experiments show that VP expression is only limiting for rAAV production until a certain threshold of assembled capsids, ready to get packaged with recombinant genomes, is reached. Rather than improving yields of DNA-containing or transducing particles, further overexpression of VP proteins only results in the excessive production of empty capsids. This is of course highly undesirable since under these conditions host immune responses to VP proteins become more likely. Together these findings again strongly indicate that as yet undefined elements involved in DNA replication or encapsidation, which are missing on common rAAV vectors, but are present on wild-type genomes, are generally limiting rAAV production. With the feasibility of measuring capsid numbers and ratios of empty to full particles given now by the AAV-2 ELISA, future studies should aim at elucidating this obvious block in replication or encapsidation of rAAV genomes. The identification of these elements or mechanisms required for an optimum yield of packaged AAV-2 particles would certainly have a significant impact on the production of rAAV vectors for human gene therapy.
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We are grateful to Dr Anna Salvetti for providing the HeLaRC32 cell line and to Dr Michael Chapman for supplying CsCl purified wild-type AAV-2. Andrea Hörster and Birgit Teichmann are thanked for their help with the FACS analyses. Thorsten Belz was involved in initial development of the ELISA. Dirk Grimm was supported by the BMBF grant 01KV9517/6.
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Grimm, D., Kern, A., Pawlita, M. et al. Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2. Gene Ther 6, 1322–1330 (1999). https://doi.org/10.1038/sj.gt.3300946
- recombinant AAV-2
- AAV-2 titration
- AAV-2 ELISA
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