Design parameters of AAV serotype mixing.
The feasibility of mixing capsid proteins from different AAV serotypes was investigated by using the previously characterized AAV2 helper plasmid mutants H2634 and H/N3761 (31
). Both helper plasmids produce noninfectious virions as a result of unique mutations in their capsid regions. Virus produced from the H/N3761 mutant contains all three capsid subunits (VP1, VP2, and VP3); however, this virus does not bind heparin, nor will it compete with wild-type AAV2 for transduction into cells. The H2634 mutant produces virus with only VP3 subunits that will bind heparin and has the ability to compete with wild-type AAV2 for transduction into cells but is not infectious. When these two helper plasmids are mixed at different ratios during the transfection stage of recombinant AAV2 production, infectious virus was obtained as judged by the ability to transduce HeLa cells (Table ). These results indicated that the viable virions were not assembled homogeneously from the capsid subunits of one mutant but were instead composed of mixtures of subunits from each mutant. Importantly, these initial experiments indicated that advantageous phenotypes of each mutant could be built into a mosaic virus (Fig. ) and further suggested that properties of different AAV serotypes might be able to be mixed in an analogous manner.
Complementation of AAV2 capsid mutant viruses via helper plasmid mixing
FIG. 1. Plasmid mixing approach used to generate transcapsidated rAAV. Helper plasmid DNA containing the capsid gene from any two AAV serotypes (one is represented by a red capsid gene, and the other is represented by a blue capsid gene) was cotransfected at (more ...)
To investigate the possibility of serotype capsid mixing, helper plasmids containing the five currently available serotype capsid genes (AAV1 to AAV5) (29
) were mixed pairwise at specific ratios of 19:1, 3:1, 1:1, 1:3, and 1:19 (Fig. ). These helper plasmids have the same genetic backbone, efficiently replicate transgenes, produce replication and capsid proteins at nearly the same level, and package transgenes with AAV2 ITRs. As a result of these similarities and the number of DNA molecules used in the transfection (1.12 × 1012
in 10 μg), the number of molecules of each helper plasmid entering each cell should vary only slightly around the specific ratio. The resulting virions should be composed of subunits from each serotype used in the pairwise transfection mixture, and the ratio would determine the general composition of these virions (Fig. ). For nomenclature purposes, the mixtures are reported as fractions in which the numerator represents the first parent plasmid and the denominator represents the second parent. Accordingly, the first number in the ratio corresponds to the first parent, and the second number in the ratio corresponds to second parent. For example, a mixed-virus preparation consisting of 25% AAV1 plasmid and 75% AAV2 plasmid would be referred to as AAV1/2 with a ratio of 1:3.
Physical characterization of mixed virions.
Control experiments were performed to examine the potential for recombination of the helper plasmid pairs. Hirt DNA was isolated at 48 h posttransfection and transformed into Escherichia coli, and individual plasmids were assessed to determine whether recombination between serotype plasmids occurred. After numerous colonies from separate transfections were screened by restriction digestion, no evidence was found for recombination (data not shown).
Dot blot analyses were performed on cesium gradient purified samples from the 50 AAV mixing combinations, as well as the five serotype control samples, to determine whether the resultant viruses could package the GFP transgene. A sample of the peak fraction from each mixture is shown (Fig. ), and the corresponding physical titers are presented in Table . Three general classes of capsid interactions were identified from this type of analyses. Class A mixtures consist of serotypes that mix well at all ratios examined and generate titers almost equivalent to their respective 100% controls. In these experiments, class A mixtures consisted of any AAV1, AAV2, or AAV3 combination. Class B mixtures consist of interactions of AAV5 with serotypes 1, 2, 3, and 4. The physical titers from all of these mixtures were of intermediate numbers and were similar to the AAV5 parental 100% control (see Table ). Class C mixtures were composed of the serotypes (AAV1, AAV2, and AAV3) that do not mix very well with AAV4, as evidenced by the drastic reduction in physical titer of these mixtures outside of the 19:1 and 1:19 ratios.
FIG. 2. (A) Dot blot of peak fractions from cesium gradient-purified AAV mixtures. 293 cells were transfected with AAV1 to AAV5 plasmids (AAV column) or combinations of the five serotypes (AAV serotype mixtures 1/2, 1/3, 1/4, 1/5, 2/3, 2/4, 2/5, 3/4, 3/5, and (more ...)
Particle numbers for AAV pseudotypes and controlsa
To eliminate the possibility that problems other than aberrant packaging of the transgene were responsible for the poor performance of the class C mixtures, additional physical properties of the mixed viruses or its corresponding transfection lysate were examined. The presence of replicating transgene DNA in all mixed and parental rAAV preparations was confirmed by analyses of Hirt DNA (Fig. ; AAV1/2, AAV1/3, AAV1/4, and AAV1/5 are shown). These results clearly demonstrate that replicating genomes were present at similar levels in transfection lysates of all mixed virus preparations, including those that produced low particle numbers.
Western blot analyses were performed on equivalent protein amounts from transfected cell lysates from each helper plasmid mixture ratio by using monoclonal antibodies against the AAV structural proteins or the GFP protein as a control (Fig. , AAV1/4 mixtures are shown). The B1 monoclonal antibody recognizes the capsid proteins of all serotypes with the exception of AAV4 (29
). Western blot analyses with this antibody on cell lysates from the AAV1/4 mixing experiments indicated that, as the contribution of AAV4 increases to 1:19, the ability of the antibody to recognize the capsid is lost. This absence in antibody recognition does not correlate with the loss of dot blot signal, which decreases as the contribution of AAV4 subunits increases toward the 1:1 ratio (Fig. , see 1/4, 2/4, and 3/4 mixtures).
Electron microscopy was used to examine the physical particles of the three classes of mixed virions. Class A virions were indistinguishable from preparations of AAV2 virions at the resolution viewed, whereas no intact particles were observed from preparations of class C virions of intermediate ratio of 1:1 (data not shown). This further suggests that the lack of a dot blot signal in class C virus preparations does not represent an absence of subunits or packageable genomes but rather an inability to assemble capsids with the type and proportion of the capsid subunits present. Class B mixed viruses (AAV1/5 [1:3] shown as an example) were indistinguishable from AAV2 (Fig. ); however, smaller components were evident in these preparations (Fig. , arrows) and may represent incompletely assembled or unassembled capsid components. These smaller entities were not observed in preparations of AAV2 (Fig. , AAV2).
In vitro binding properties of mixed virus.
It has been shown that AAV2 and AAV3 bind heparin agarose (29
) and that both AAV4 and AAV5 bind mucin (1
). To determine whether the mixed virions exhibit binding properties similar to the parental virions or whether the binding to both heparin and mucin can be coupled through mixing of capsid components, the binding properties of each ratio of all mixtures except the 1/4, 2/4, and 3/4 mixed viruses that were deficient in generating sufficient test material were examined.
Equivalent particles of parental and all ratios of mixed virions were applied to either a heparin agarose or a mucin column, washed, and eluted in three steps of increasing salt. Samples from each column fraction were analyzed by dot blot analyses, and the corresponding percentages of bound and unbound virus are presented as pie charts (Fig. and ).
FIG. 3. Heparin batch binding profiles of rAAV mixed virions. Totals of 1010 vg of each ratio (19:1, 3:1, 1:1, 1:3, or 1:19) of the rAAV1/2, rAAV1/3, rAAV2/3, rAAV2/5, and rAAV3/5 mixed virions or the rAAV1, rAAV2, or rAAV3 viral controls were applied to 50 μl (more ...)
FIG. 4. Mucin batch binding profiles of rAAV mixed virions. Totals of 109 vg of each ratio (19:1, 3:1, 1:1, 1:3, or 1:19) of the rAAV1/5, rAAV2/5, rAAV3/5, and rAAV4/5 mixed virions or the rAAV1 to rAAV5 viral controls were applied to 50 μl of mucin agarose, (more ...)
Class A mixed viruses interacted on the heparin column in two manners: either as an abrupt shift (Fig. ) or as a gradual change (Fig. ) in the heparin-binding profile upon increase of the second capsid component. Less than 10% of the AAV1/2 (19:1) mixture bound to heparin (Fig. ). Heparin binding abruptly changed to >75% at the 3:1 ratio (Fig. ). This finding is in contrast to AAV1/3 mixed viruses, with which there was a gradual change in heparin binding: <25% binding at the 3:1 ratio increasing to 50% at 1:1 ratio to >90% at the 1:3 ratio (Fig. ). As expected, all ratios of the AAV2/3 mixtures bound heparin (Fig. ).
Since neither AAV4 nor AAV5 bound heparin, the heparin-binding profiles were not determined for the AAV1/5 and AAV4/5 mixtures. In the case of the other class B interactions, the heparin-binding profiles of the AAV2/5 and AAV3/5 mixtures were essentially identical (Fig. ); the 19:1 ratios were the only mixtures that significantly bound to heparin (Fig. ). Heparin-binding analyses of the class C mixes could not be determined because of the low particle numbers.
Mucin-binding experiments with class A parental or class A mixed virus did not result in significant mucin binding, as expected (data not shown). Interactions of class C mixtures on the mucin column could not be determined since these mixtures did not contain sufficient viral particles.
The mucin-binding profiles for the AAV1/5 and AAV3/5 mixtures resemble their respective parents in the extreme ratios (19:1 and 1:19; Fig. ). These mixtures at the 1:1 ratio bind to mucin more efficiently than AAV5 (Fig. ). AAV4/5 mixed viruses bound mucin as expected, since both parental serotypes individually bound mucin. Intermediate ratios of AAV4/5 (3:1, 1:1, and 1:3) appear to bind mucin better than either parent or the more extreme ratios 19:1 and 1:19 (Fig. ). Interestingly, only AAV3/5 (3:1) was able to bind both mucin and heparin agarose (Fig. and ). To determine whether individual particles bound both mucin and heparin agarose, a preparation of AAV3/5 (3:1) was used to first bind mucin. Virus eluted from this column was then used to bind heparin. The results from this experiment indicate that the AAV3/5 (3:1) preparation was able to bind both matrices at levels similar to those shown in Fig. and . The control viruses (AAV3 and AAV5) were not able to successively bind the different matrices, indicating that the individual mosaic particles of AAV3/5 (3:1) were dual binders due to assembly of virus particles composed of mixed parental (types 3 and 5, respectively) capsid components (data not shown).
Both heparin competition and neuraminidase treatment of cells were used to assess whether this binding duality correlated with cell infection. Heparin pretreatment was unable to inhibit AAV3b (Fig. ). This was unexpected since Handa et al. (16
) previously demonstrated that AAV3a is inhibited by soluble heparin at these concentrations. The same concentration of heparin was able to inhibit the ability of rAAV2 to transduce C2C12 cells (see Fig. ). As expected, the ability of rAAV5 to transduce HeLa cells was greatly reduced upon pretreatment with neuraminidase, whereas the ability of rAAV3 to transduce these cells was unaffected (Fig. ). More importantly, the AAV3/5 (3:1) mixture was reliant only on sialic acid for infectivity, as judged by the decrease in transduction of cells pretreated with neuraminidase (Fig. ).
FIG. 5. Neuraminidase and soluble heparan inhibition. HeLa cells, either untreated (virus) or pretreated with neuraminidase (N) and/or soluble heparin (H or H+N) for 1 h, were transduced with 3,000 vg of AAV3, AAV5, or AAV3/5 (3:1)/cell. The cells were (more ...)
FIG. 7. (A) Transduction of C2C12 cells with mixtures of purified rAAV1 and rAAV2 virus. C2C12 cells were transduced with 300 vg of rAAV1, rAAV2, or both viruses mixed at the following ratios (19:1, 3:1, 1:1, 1:3, and 1:19)/cell. At 24 h posttransduction, cells (more ...) Transduction of different cell lines with AAV mixed virus.
Mixed virus preparations were analyzed for their ability to transduce human, mouse, and Chinese hamster cell lines. The CHO wild-type cells, K1, and derivatives with mutations in glycosaminoglycan biosynthesis (pgsD and pgsE cells) were selected since these cells demonstrate a biological phenomenon: heparin-dependent transduction of AAV2 (29
). HeLa cells are the standard line to titer rAAV, and C2C12 cells were used because they are known to bind AAV2 and yet are refractory to transduction (11
). Either 300 or 3,000 vg of the mixed virus/cell was used in the transduction assays. All transduction assays were performed in the presence of adenovirus and flow cytometry was used to determine the percentage of cells expressing GFP.
Four transduction profiles were apparent in these experiments: (i) a gradual change (titration), (ii) little or no change (plateau), (iii) abrupt changes (threshold), and (iv) synergistic changes as the amount of input plasmid increased from one parental serotype to the other (Fig. ). The titration effect is seen in HeLa cells for the AAV1/3 (Fig. ) and in CHO K1 and pgsE cells for the AAV 2/5 mixtures (Fig. ). The plateau effect was observed in C2C12 cells for both the AAV1/5 and the AAV2/5 mixtures (Fig. ). The threshold effect is clearly seen in HeLa cells infected with the AAV2/5 mixtures, for which the transduction efficiency decreased from 10 to 11% at the 19:1 ratio to 3% at 3:1 ratio (Fig. ). This pattern was also seen in HeLa cells with the AAV1/2 mixtures (Fig. ), in CHO K1 cells with the AAV1/3 (Fig. ) and 1/5 mixtures (Fig. ), in CHO pgsD cells with the AAV1/2 mixtures (Fig. ), and in CHO pgsE cells with the AAV1/5 mixtures (Fig. ). Finally, a synergistic pattern was observed in C2C12, CHOK1, and pgsE cells infected with vector from AAV1/2 mixtures (Fig. ) and in C2C12, pgsD, and pgsE cells infected with vector from AAV1/3 mixtures (Fig. ). This pattern resulted in some ratios of mosaic virus having a better transduction efficiency than either parental virus.
FIG. 6. Transduction efficiency of reference cell lines with mixed rAAV. Cells (cell types are indicated on top) were transduced with all ratios (19:1, 3:1, 1:1, 1:3, and 1:19) of the rAAV1/3, rAAV1/2, rAAV1/5, or rAAV2/5 mixed virus and their corresponding parental (more ...)
To test that the enhanced transduction observed from mosaic virus particles were not an artifact of mixing two parental serotypes virions, purified recombinant viruses from two serotypes (AAV1 and AAV2) were mixed at ratios identical to those used for the helper plasmids, and these mixtures were used to transduce C2C12 cells, with the same overall particle number (i.e., 300) that was used in the plasmid mixing experiments. A titration effect was observed in this instance as the ratio of the virus varied (Fig. ), in contrast to the synergistic effect observed (Fig. ) when rAAV1/2 mosaic viruses were used to transduce C2C12 cells. Based on in vitro binding studies and in vivo transduction assays, we probed known attributes of the parental virus to determine a mechanism for the synergism we observed from the mosaic virus particles.
Heparin inhibition of C2C12 cells transduced with AAV1/2 mixed viruses.
Heparin competition was used to determine whether the enhancement in transduction of C2C12 cells by AAV1/2 mixed viruses was due to HS-mediated binding. Recombinant AAV1/2 or the parental viruses were incubated in the presence or absence of soluble heparin for 1 h prior to transduction with 300 vg/cell. Soluble heparin reduced the enhanced transduction of 1:1, 1:3, and 1:19 ratios (Fig. ). However, the transduction of these mixtures was not completely abolished in the presence of HS-like AAV2 control (Fig. ). Instead, these mixtures plateau at levels identical to the 3:1 ratio, which was a modest enhancement over both parental viruses 1 and 2 in the presence of heparin. This suggests that not all of the enhancement was due to HS-mediated binding but rather related to other steps involved in vector transduction (e.g., coreceptor usage, trafficking, uncoating, or facilitation of second-strand synthesis [Fig. ]).
FIG. 8. (A) Model of potential interactions between AAV2 and AAV5 capsid subunits. The triangular shapes represent individual subunits of AAV2 (yellow) or AAV5 (light blue). The heparin-binding domain of AAV2 is represented by black circles near the threefold (more ...)