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. 2009 Nov 10;18(1):87–92. doi: 10.1038/mt.2009.258

Characterization of Genome Integrity for Oversized Recombinant AAV Vector

Biao Dong 1, Hiroyuki Nakai 2, Weidong Xiao 1,3
PMCID: PMC2803017  NIHMSID: NIHMS161703  PMID: 19904236

Abstract

Application of recombinant adeno-associated virus (rAAV) in gene therapy has been limited by its packaging capacity. Recent studies suggested that rAAV could achieve persistent transgene expression beyond 4.7-kb packaging limit. To clarify the mechanism leading to transgene expression from oversized rAAV vector, we constructed a series of rAAV vectors with genomes ranging from 2.9 to 7.2 kb. A plasmid replication origin and an ampicillin-resistant marker were included in the vector to facilitate the recovery of circularized, post-transduction AAV genome. Southern dot-blot analysis and silver staining confirmed that rAAVs could be produced at varying vector size. However, the vector yields decreased approximately tenfold for oversized vectors as compared to regular ones. Alkaline Southern blot hybridization suggested that the packaged genomes for oversized vectors were truncated. In the cells transduced by the above vectors, circularized rAAV monomers could be rescued at 24 hours after infection. Few recovered AAV genomes were >5 kb regardless of the initial vector size. In mice receiving the above vectors, larger circularized rAAV genomes could be recovered for oversized vectors at day 21 after vector administration. Our studies suggested that the partially packaged rAAV sequences may complement each other to restore full expression cassette.

Introduction

Adeno-associated virus (AAV) is a nonpathogenic and replication-defective human parvovirus. In addition to two open reading frames encoding the rep gene and cap gene, the 4.7-kb single-stranded DNA genome is flanked by two inverted terminal repeats (ITRs). There are 145 nucleotides in each ITR and the palindromic sequences within the ITR can form a T-shape structure, which facilitates AAV DNA replication in a self-priming manner.1 These two ITRs are the only cis elements in recombinant AAV (rAAV) genome essential for efficient packaging.

With a vector plasmid carrying the transgene cassette between two ITRs, rAAV can be produced by supplying necessary AAV trans elements, and helper virus functions, such as adenovirus or herpes virus.2 Extensive studies have documented rAAV as one of the most promising vector for human gene therapy.3,4,5 Many new AAV serotypes have been isolated or generated through directed evolution, which broadened the cell types and tissues that rAAV can efficiently transduce and direct long-term transgene expression.6,7,8,9,10 AAV vectors are currently being evaluated in human clinical trials for a variety of diseases and the preliminary results are very encouraging.11,12,13,14,15,16,17

Wild-type AAV virus is able to integrate site-specifically into a specific locus of human chromosome 19 at a high frequency in a latent infection.18,19 However, cellular junctions between rAAV vectors and host chromosomes are rare to detect using various approaches.20,21 On the other hand, episomal AAV genomes, linear or circular, can be isolated readily in both tissue culture cells and transduced target tissues. As demonstrated by Duan et al. the circularized rAAV genomes can be formed right after rAAV vectors enter the host cells.22,23,24 The major transgene expression by rAAV transduction most likely resulted from episomal AAV genomes.25

The packaging capacity for rAAV is a critical issue that may determine how rAAV vectors can be applied for human gene therapy, as many target genes and regulatory elements are large in size. It was reported that an rAAV genome <4.9 kb could be completely packaged into a virus.26 By step-increasing the wild-type AAV genome, it was suggested that 5.3 kb of nucleotides should be the maximum for efficiently packaging AAV.27 Using the same constructs as Dong et al.,26 it was found that the full length of 6-kb rAAV genome could be packaged into virus, with or without VP2 protein.28 Another report showed that a 3.3 kb of double-stranded DNA could be packaged into rAAV, which suggested the rAAV packaging capacity could be enlarged to 6.6 kb (ref. 29). Recently, it was reported that large vectors up to 8.9 kb could be packaged into rAAVs by selection of suitable AAV serotypes.30 In our effort to package large vectors, it was shown that a 5.8-kb vector for the expression of factor VIII was only partially packaged, although this vector could functionally correct the phenotype of hemophilia A.31

Shuttle vectors have been used as a tool to study rAAV transduction mechanisms for conventional vectors with rAAV genomes <4.7 kb (refs. 22,32,33). In an rAAV shuttle vector, the ampicillin-resistant gene and plasmid replication region are presented in the rAAV genomes. Upon transduction, duplex rAAV genome may become a circular molecule via the two ITRs. The circularized rAAV genome could be rescued by transforming competent Escherichia coli. Here, we took advantage of AAV genome circularization and characterized the genome integrity of rAAV vectors produced from oversized rAAV genome vectors. The rescued rAAV genomes were analyzed by restriction enzyme digestion mapping and DNA sequencing. Our detailed analysis of encapsidated AAV genomes demonstrated that the packaging of oversized vectors was inefficient and only partial AAV genomes can be packaged. Complementation of incomplete AAV genomes may have led to the regeneration of full AAV gene expression cassette.

Results

Analysis of the effects of vector genome size on rAAV packaging

In order to characterize the vector genome integrity, we choose to employ a rescue strategy as previously published.22,32,33 The key elements in this approach are the plasmid replication origin (ori) and ampicillin resistant (amp) flanked by the two regular AAV ITRs. The detailed illustration for these plasmids is shown in Figure 1. The sequences flanked by AAV ITRs based on these vector plasmids ranged from 2.9 to 7.2 kb. The stuffer sequences for these vector plasmids were nonfunctional human factor VIII codons.

Figure 1.

Figure 1

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Illustration of AAV vector genomes. The stuffer fragments were partial sequences of factor VIII gene. The vectors were named according to their rAAV genome size. The DNA fragment used for the probe for Southern blot hybridization was identified, which corresponds to the sequences in the ampicillin-resistant gene. AAV, adeno-associated virus; ampR, an ampicillin-resistant gene, β-lactamase; ITR, inverted terminal repeat sequence; ori, a plasmid replication region of pUC18; ↓, AleI site; *, BamHI site; Δ, SmaI site.

The packaging efficacy using these vector plasmids were then analyzed following our standard, triple plasmid transfection, AAV production protocol.34 Under cesium chloride–gradient ultracentrifugation, we noticed that the usual distinctive AAV vector bands could no longer be observed from preparations that used the oversized vector plasmids DB6.4 and DB7.2 for rAAV production. Individual gradient fractions from these vector preparations were then collected and analyzed by both Southern dot-blot and silver staining. The packaged vector DNA as revealed by Southern blot (Figure 2a) suggested that vector size >4.7 kb (DB5.4, DB6.4, and DB7.2) caused a considerable decrease in vector yields. Despite that there were some variations in the yields of packaged of 7.2, 6.4, and 5.4 kb vectors, the vector yields from these vector plasmids were comparable to each other based on our quantitative analysis. Overall vector yields were approximately tenfold less than their smaller counter parts (DB2.9 and DB 3.9). On the other hand, AAV capsids revealed by silver staining showed that the ratio of vector capsid proteins VP1, VP2, and VP3 were similar among all vector preparations, which is independent of vector genome size. Therefore, capsid formation, either empty particles or full particles, was not affected by the size of AAV vector genomes.

Figure 2.

Figure 2

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Oversized AAV vectors are inefficient in packaging. rAAV vectors were packaged using AAV5 capsids using triple plasmid transfection method as described in the Materials and Methods section. The resulting rAAV vectors were purified by two rounds of cesium chloride–gradient ultracentrifugation. (a) rAAV vectors in each gradient fraction were quantified by use of Southern dot-blotting. Each dot represented 2 µl of purified vectors from each gradient fraction. The membrane was hybridized with a probe to a region that is identical in all vectors. (b) Detection of capsid proteins by silver staining. A volume of 1 × 1010 viral particles for each vector were used for the silver staining. rAAV, recombinant adeno-associated virus; Std, the standard for quantification of the purified viral genomes; VP1, VP2, VP3, are AAV capsid proteins.

The size of vector DNA packaged into AAV capsids was further analyzed by alkaline Southern blot analysis. The peak fraction of AAV vectors from the above preparations were digested by proteinase K, separated in agarose gel, and probed with DNA sequence specific to the ampR region. This result is presented in Figure 3. The major vector size for DB2.9 and DB 3.9 were close to 2.9 and 3.9 kb, which was nearly identical to their original size. However, the vector DNA from preparation from vector plasmids DB5.4, DB6.4, and DB7.2 were not >4.7 kb. It suggested that only partial DNA sequences can be encapsidated if vector DNA is >4.7 kb.

Figure 3.

Figure 3

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Analysis of the rAAV genome integrity. The size of the single-stranded DNAs packaged in rAAV capsids were analyzed using alkaline agarose gel electrophoresis. After extensive DNaseI treatment, the packaged DNAs from different vectors were resolved on 1% alkaline agarose gel. The membrane was hybridized with a probe specific to the ampicillin-resistant gene. rAAV, recombinant adeno-associated virus.

Rescue of rAAV DNA in vitro

As AAV genomes in circular configuration can be formed in the host cells after rAAV transduction, the inclusion of amp and ori in the AAV vector DNA could therefore facilitate the rescue of circularized AAV genomes. We transduced 293 cells with equal genome titers of AAV vectors (multiplicity of infection 10,000) made from vector plasmids that varied in their genome size. Small-molecular weight DNA was then extracted from these cells at 24 hours postinfection. After electro-transformation into competent cells, the number of amp-resistant colonies are counted and graphed in Figure 4. On average, AAV vector with small/regular genomes such as DB2.9 and DB3.9 gave rise to 4,000–6,000 colonies. In contrast, vectors with oversized genomes, DB5.4, DB6.4, and DB7.2, resulted in substantial fewer colonies, which were in the range of 500–1,000.

Figure 4.

Figure 4

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Rescued rAAV genomes from HeLa cells. HeLa cells were transduced with similar amounts of rAAVs at a multiplicity of infection of 10,000. After 24-hour infection, low-molecular-weight DNAs were extracted and transformed into competent cells. The ampicillin-resistant colonies growing on Luria–Bertani plates were counted for each vector. The experiment was repeated twice. rAAV, recombinant adeno-associated virus.

Analysis of AAV genome regeneration from large AAV vectors in vivo and in vitro

The circularized AAV genomes recovered from transduced 293 cells by DB7.2 were then mapped by both restriction digestions and DNA sequencing. The missing AAV fragments are summarized in Figure 5. Of all clones recovered, 80% of clones had deletions >5 kb, 7% had deletions between 3 and 4 kb, and only 13% had deletions between 2 and 3 kb. There were no clones with deletions <1 kb.

Figure 5.

Figure 5

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Summary of the deleted regions in the rescued rAAV genomes for DB7.2. All plasmids rescued from HeLa cells and mouse muscle tissues were analyzed by restriction enzyme digestion and DNA sequencing. The percentage was calculated as dividing the total number of rescued plasmids by the number of classified plasmids. The semi-filled box illustrates missing region from recovered rAAV genome. ampR, an ampicillin-resistant gene, β-lactamase; ITR, inverted terminal repeat sequence; ori, a plasmid replication region of pUC18; rAAV, recombinant adeno-associated virus.

Similar experiments were carried out in vivo. Vectors made from DB7.2 were injected into the muscle of C57BL6 mice. The small-molecular weight DNA was extracted from the muscle tissue at the injection site on day 21. Upon transformation in competent E. coli, the clones containing AAV genomes are shown in the bottom panel of Figure 5. In contrast to the in vitro results, the clones with large deletions (>4 kb) decreased to 17%. Clones with deletion in the range of 3, 2, and 1 kb are 26, 35, and 15%, respectively. Seven percent of the clones had deletions <1 kb. It suggested that AAV vector genomes undergo significant changes in vivo.

Discussion

Traditionally, it has been documented that a virus can generally package no more than 105% nucleotides of normal virus genome. Analysis of the wild-type AAV virus packaging revealed that increasing AAV genome size drastically reduces virus encapsidation.26,35 An oversized wild-type AAV virus has also been explored as a helper for rAAV production due to its deficiency in packaging itself.36 Despite the outstanding performance of rAAV vectors in transduction, the packaging capacity of AAV vectors has been a limiting factor, preventing broader application of rAAV vectors for human gene therapy. Recently, several studies explored oversized AAV vectors in a variety of settings.26,27,28,29,30,31 All these studies suggested that oversized AAV vectors may be useful for human gene therapy. On the other hand, these results also raised a critical question of how AAV vectors could package more than the normal size of AAV genomes.

In our studies, we generated a series of AAV vectors with genomes ranging from 2.9 to 7.2 kb. In contrast to previous studies which focused on transgene expression, we included both an amp and ori sequence in the rAAV sequence to specifically facilitate the recovery of circularized AAV molecules. It is known that AAV vectors below the wild-type size generally performed similarly to wild-type-genome-sized vectors. Vectors DNA <4.7 kb (DB2.9 and DB 3.9) packaged and behaved similar to that standard-sized 4.7-kb vector. The packaging of oversized AAV genomes appeared to be inefficient with at least a tenfold decrease in packaging efficiency. This result is consistent with many previous studies27 showing that the optimal packaging requires the size of AAV vector, including AAV ITRs, not to exceed 4.7–5.3 kb. In addition, we also observed that vector >5.3 kb gave rise to similar low yield, suggesting a common packaging problem for the large vectors. The debatable issue is how much DNA AAV can encapsidate into AAV genomes when AAV vector genome size increases. In several recently published studies, significant transgene expression could be observed in vivo using an AAV vector with large genomes ranging from 5 to 9 kb (refs. 26,27,28,29,30,31). However, the reported size of packaged DNA varied. Our previous results27 suggested that only 3.9 kb could be packaged by alkaline gel analysis of AAV genomes, which confirms again that DNA inside the AAV capsid is <5 kb regardless of the size of the AAV genome (Figure 3). Analysis of different density fractions (higher up to 1.46–1.48 g/ml and lower to 1.35–1.38 g/ml) did not show any size difference as well (data not shown). Thus, we conclude that through physical limitation an AAV capsid cannot accommodate >5,000 nucleotides.

The conclusion that only limited nucleotides can be encapsidated was also supported by our data from rescuing AAV genomes in vitro and in vivo. It is clear that the larger the vector size, the fewer circularized AAV genomes that could be recovered (Figure 4). This result suggested that there were serious defects in encapsidated AAV genomes when rAAV genomes were over the normal range. The deletion of an ITR from the larger genomes during the truncation/deletion process of the larger genomes may have resulted in lower circularization efficiencies. In addition, deletion in elements controlling formation, amp gene, and plasmid ori would have reduced the clones, which could be recovered. Further analysis of recovered, circularized AAV genomes provided direct evidence supporting this hypothesis. The circular molecules recovered from AAV transduction of tissue cultures, at only 24 hours post-transduction, revealed that >80% of them contained large deletions. Of those with a 7.2-kb genome >80% had deletions >4 kb. There is nothing >6 kb, let alone full-size genome. This result is consistent with our Southern blot analysis of AAV genomes, that >5-kb genomes could not be packaged (Figure 3). We sequenced ~200 clones that were recovered (data not shown), which suggested that AAV ITR and non-AAV ITR sequences were joined together. Thus, we speculate that one ITR and non-ITR sequence junction could be formed by direct intramolecular ends joining or nonhomologous recombination. On the other hand, most of these clones were 2–3 kb, much <4.7 kb. The truncated genomes were probably being further processed in 293 cells after uncoating occurred to achieve circularization. Missing one ITR may also increase rAAV genome degradation/processing. In contrast, the circularized AAV molecules recovered from the muscle tissue that has been transduced by rAAV vectors exhibited a very different pattern (Figure 5). Not only did the number of clones with large deletions decrease dramatically, but also clones close to full AAV genomes could be recovered. These phenomena were observed with all other vectors >5.4 kb. Therefore, the above results suggested that the AAV vectors underwent significant changes in vivo. Longer time in the target tissue probably facilitated AAV genome conversion, which allowed larger AAV genomes containing the full-length transgene to be regenerated.

We wish to resolve the issue on AAV encapsidation of large genomes so that oversized AAV vectors can have broad applications in human gene therapy. Our results and other studies supported that AAV genomes packaged were <5 kb. However, through other molecular mechanism, most likely, AAV genome conversion from overlapping sequence, AAV vectors produced from oversized AAV genomes were able to give rise to meaningful transgene expression.37 The successful use of this approach will probably rest on the further improvement of AAV vector–packaging technology.

Materials and Methods

Cell lines. HEK 293 cells and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 µg of penicillin/ml, 100 U of streptomycin/ml as recommended by the manufacturer (Invitrogen, Carlsbad, CA). All cells were maintained in a humidified 37 °C incubator with 5% CO2.

Plasmid construction. The plasmid pAAV-IsceI.AO3 (accession number EU022316) has been described previously.33 Plasmids containing various rAAV genomes were made in the following procedures. First, pAAV-IsceI.AO3 was digested by NheI and EcoRV and subsequently filled-in by T4 DNA polymerase. The 2,855-bp fragment was then self-ligated, or dephosphorylated by calf intestinal alkaline phosphatase (NEB, Ipswich, MA) and used as the backbone for the construction of the other vectors. The self-ligated plasmid has around 2.9 kb of rAAV genome, so it was named as DB2.9. The above plasmid designation was also used for the other constructs DB3.9, DB5.4, DB6.4, and DB7.2, respectively. These plasmids were constructed by blunt-end ligation of the backbone and the filled-in DNA fragments digested from the plasmid pAAV-TTR-hF8. Specially, the filled-in DNA fragments were 1,046-bp fragment by ClaI and AccI, 2,564-bp fragment by KpnI and XhoI, 3,522-bp fragment by ClaI and ApaI, and 4,389-bp fragment by ClaI and XhoI. The constructs were confirmed by DNA sequencing.

rAAV production. A triple plasmid co-transfection method was used to produce rAAVs as described previously.38 One vector plasmid, one AAV5 helper plasmid, and one mini adenovirus function helper plasmid pFΔ6, were co-transfected into HEK293 cells cultured in roller bottles at a ratio of 1:2:1. The transfected cells were harvested 3 days later. rAAVs were then purified by two rounds of cesium chloride–gradient ultracentrifuge. After extensive buffer exchange against phosphate-buffered saline with 5% D-sorbitol, the peak fractions of purified virus were pooled and stored at −80 °C before administration.

rAAV titer determination. Genome titers were determined by dot-blot hybridization according to the standard protocol. Briefly, 2 µl of purified virus from each gradient fraction was used to release the genome in a reaction mix containing 50 mmol/l Tris–HCl, pH 8.0, 1 mmol/l EDTA, 0.5% sodium dodecyl sulfate (SDS), and 0.5 mg/ml proteinase K. The mixtures were extracted once using phenol:chloroform:isoamyl alcohol (25:24:1). After denaturing, the mixtures were dotted on a Hybond-XL nylon membrane (GE Healthcare, Little Chalfont, UK). After the membrane was rinsed with 2× sodium chloride and sodium citrate, it was dried at 65 °C. For hybridization, the probe was a DNA fragment located in the ampicillin-resistant gene that was shared by all vectors. This fragment was PCR amplified using a pair of primers 5′-GCCTTCCTGTTTTTGCTCAC-3′ and 5′-TTGCCGGGAAGCTAGAGTAA-3′. The purified PCR products were labeled by [α-32P]dCTP using Prime-It II Random Primer Labeling Kit (Stratagene, La Jolla, CA).

Silver staining. Purified virus (~1 × 1010 particles) was mixed with protein-loading dye and NuPAGE sample-reducing reagent (Invitrogen), the mixture was heated at 95 °C for 5 minutes. The denatured samples were then separated on 10% Tris–glycine gel (Invitrogen). After electrophoresis, the SDS–poly acrylamide gel was stained by SilverXpress silver staining kit (Invitrogen) according to the manufacturer's instruction.

rAAV DNA analysis. Alkaline agarose gel electrophoresis was used to analyze the size of the single-stranded DNA packaged in rAAV capsids as described previously.31 Briefly, viral genomes were treated with DNaseI and resolved on 1% alkaline agarose gel. After gel blotting, the membrane was hybridized with a 32P-labeled probe that was used for the titer determination. The membrane was washed once under low-stringent condition (2× salt-sodium citrate + 0.1% SDS, at room temperature), followed by washing for three times under high-stringent condition (0.2× salt-sodium citrate + 0.1% SDS, at 65 °C). The membrane was then analyzed by X-ray autoradiography.

Animal procedures. The 4- to 5-week-old C57BL/6 mice were housed in a pathogen-free environment with a normal diet. All surgical procedures involving mice were in accordance with institutional guidelines under approved protocols at the Children's Hospital of Philadelphia, PA. For each vector, three hindlimb muscles were injected independently and each injection contained about 5 × 1010 viral particles.

rAAV genome rescue. HeLa cells were grown to 95% confluence in a 6-well plate and transduced with rAAVs at an multiplicity of infection of 10,000. After 24-hour infection, cells were rinsed once with 1× phosphate-buffered saline, scraped into centrifuge tubes, and pelleted by centrifugation. To rescue rAAV genomes from HeLa cells, low-molecular-weight DNA was extracted using High Pure plasmid isolation kit (Roche Diagnostics, Mannheim, Germany) and eluted using 50 µl of elution solution (10 mmol/l Tris, pH 8.0).

To rescue rAAV genome from muscle tissue, transduced muscles were harvested on 21 day. Muscle tissue (100 mg) was chopped into small pieces and subsequently homogenized in 1 ml of buffer containing 10 mmol/l Tris (pH 8.0), 10 mmol/l EDTA, 1% SDS, and 100 µg/ml RNase (DNase free; Sigma, St Louis, MO). After incubation at 37 °C overnight, NaCl was added to a final concentration of 1.1 mol/l. The samples were further incubated at 4 °C overnight. The processed samples were then extracted twice with phenol–chloroform and once with chloroform, followed by ethanol precipitation. After washing with 70% ethanol, DNA pellet was resuspended in 20 µl of Tris–EDTA buffer.

For each vector, 3 µl of low-molecular-weight DNA prepared above was used to transform 40 µl of SURE electroporation-competent E. coli (Stratagene). Bacterial clones growing on Luria–Bertani plates containing 100 µg/ml ampicillin were randomly selected and cultured for plasmid extraction. At least 20 clones for each vector were analyzed by restriction enzyme digestion and DNA sequencing. The total colony number for each vector was also calculated according to the portion of DNA used for transformation.

Restriction enzyme digestion. To characterize the rescued rAAV genome, DNA fingerprinting was generated by AleI and BamHI double digestion. In a 20-µl reaction system, 300 ng of plasmid was digested with 2.5 units of AleI and 10 units of BamHI at 37 °C for 3 hours. To estimate the ITR integrity, 10 units of SmaI was used in a 20 µl reaction system that was incubated at 25 °C for 3 hours.

DNA sequencing. To assess rAAV genome accurately, a serial of primers were designed for DNA sequencing as following: DB7.2-2398F, 5′-ACC TCT GAC TTG AGC GTC GA-3′ DB7.2-2560F, 5′-AGA GCT CTC CAC CTG CTT CT-3′ DB7.2-3055F, 5′-TGA CCC ACT GTG CCT TAC CT-3′ DB7.2-3548F, 5′-TCC CAC CA ACA TGA TGG CAT-3′ DB7.2-4391F, 5′-CTC CCC AAT CCA GCT GGA GT-3′ DB7.2-4529F, 5′-AGC ATT GGA GCA CAG ACT GA-3′ DB7.2-5085F, 5′-ACA GGG CTC AGA GTG GCA GT-3′ DB7.2-5485F, 5′-ACC CCT TCT GGT CTG CCA CA-3′ DB7.2-6138F, 5′-TCA AGG TGG ATC TGT TGG CA-3′ DB7.2-6613F, 5′-GCT GCA AGT GGA CTT CCA GA-3′ DB7.2-6929F, 5′-AGC TTG CCA ATG CAT G-3′ DB7.2-321R, 5′-GTA GTT GTA CTC CAG CTT GT-3′ DB7.2-444R, 5′-GTG TTC TGC TGG TAG TGG T-3′ DB7.2-940R, 5′-CCA GTT CGA TGT AAC CCA CT-3′.

Acknowledgments

This work was supported by the National Institutes of Health (HL080789 and HL084381 to W.X.).

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