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Journal of Virology, July 2006, p. 7265-7269, Vol. 80, No. 14
0022-538X/06/$08.00+0     doi:10.1128/JVI.00076-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Heparan Sulfate Proteoglycan Binding Properties of Adeno-Associated Virus Retargeting Mutants and Consequences for Their In Vivo Tropism

Luca Perabo,1,2,{dagger} Daniela Goldnau,1,2,{dagger} Kathryn White,3,{dagger} Jan Endell,1,2,{dagger} Jorge Boucas,2 Sibille Humme,2,4 Lorraine M. Work,3 Hanna Janicki,2 Michael Hallek,1,2,4,5 Andrew H. Baker,3 and Hildegard Büning1,2,4*

Genzentrum, Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, Munich, Germany,1 Klinik I für Innere Medizin, Universität zu Köln,Joseph-Stelzmann-Str. 9, Cologne, Germany,2 British Heart Foundation Glasgow Cardiovascular Research Centre, Division of Cardiovascular and Medical Sciences, University of Glasgow, 126 University Place, Glasgow, United Kingdom,3 Center for Molecular Medicine, Universität zu Köln, Joseph-Stelzmann-Str. 52, Cologne, Germany,4 Gesellschaft für Strahlenforschung-National Center for Research and Environment, Marchioninistr. 25, Munich, Germany5

Received 11 January 2006/ Accepted 15 April 2006


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ABSTRACT
 
Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype. Interestingly, in vivo studies correlated the inability to bind to HSPG with liver and spleen detargeting in mice after systemic application, suggesting several strategies to improve efficiency of AAV-2 retargeting to alternative tissues.


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TEXT
 
Adeno-associated virus type 2 (AAV-2) is gaining increasing attention as a gene therapy vector. However, the wide distribution of its primary receptor, heparan sulfate proteoglycan (HSPG) (11), hampers selective transduction of target tissue. Vectors aiming to redirect AAV-2's tropism have been generated by insertion of ligands at position 587/588 of the capsid (1, 2). This is likely to interfere with the HSPG binding of at least two (R585 and R588) of the five positively charged amino acids of the recently identified HSPG binding motif (5, 7), explaining the ablation of HSPG binding of some targeting vectors (3, 4, 6, 8, 10). In some cases, however, binding was only partially affected (12) or was even restored (4, 8, 14). To investigate molecular mechanisms responsible for these differences, we applied a library of AAV-2 capsids carrying insertions of seven randomized amino acids at position 587 (8) to a heparin affinity column (13) to separate binding from nonbinding mutants. We sequenced and statistically analyzed (Table 1) at least 80 clones from (i) the original DNA-library, (ii) the viral AAV-2 display library, (iii) the flowthrough fraction (nonbinders were designated the NB-AAV-pool), and (iv) the 1 M NaCl eluted fraction (binders were designated the B-AAV-pool).


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  		TABLE 1. Peptides detected in B-AAV-pool and NB-AAV-pool and statistical analysesa

The DNA library showed a higher than expected presence of alanines, which originates from the oligonucleotide synthesis procedure. Occurrence of every other amino acid met statistical expectations for an unselected library.

The AAV-2 display library showed an excess of the amino acids P, G, and A and a defect of C, L, F, W, and Y (Table 1B). Since P, G, and A are three of the four smallest amino acids, whereas F, Y, and W are three of the four biggest, this bias suggests that the packaging process selects against bulky inserts which would introduce dramatic structural rearrangements and have a deleterious effect on capsid structure. In addition, prolines could favor spatial accommodation of the peptide by introducing kinks and reducing its bulkiness.

The B-AAV-pool showed a significant increase of arginine residues (Table 1B). Strikingly, arginines were particularly frequent at the seventh position of the peptide (30%). In contrast, in the AAV-2 display library and the NB-AAV-pool, the frequency of R at this position (15% and 9%, respectively) was equal to or lower than the expectation for a randomized distribution among the seven amino acid positions (14.3%).

Interestingly, B-AAV-pool insertions carrying no positive amino acids displayed an exceptionally large amount of A, G, and S (data not shown), the three smallest amino acids, suggesting a reduced impact on the wild-type capsid structure, which is less likely to interfere with binding of heparin.

The NB-AAV-pool clearly showed a greater presence of negatively charged amino acids (D and E) (Table 1A and B). Moreover, although the number of negative residues observed in the B-AAV-pool matched statistical expectations, only 2% of the clones carried a net negative charge in its insertion, while 76% carried a positive charge and 21.5% a neutral charge. In clear contrast, the NB-AAV-pool consisted of 53% negative, 8% positive, and 39% neutral net charged inserts. This bias becomes even clearer if histidine is considered neutral due to its low pKa: B-AAV-pool insertions would then be 2% negative, 64% positive, and 34% neutral, while the NB-AAV-pool consisted of 64% negative, 2% positive, and 34% neutral. These observations strongly suggest that the presence of negative charges is deleterious for functional binding of AAV-2 targeting vectors to negatively charged heparin/HSPG, while positive charges facilitate this interaction. Capsid binding to heparin/HSPG is driven by electrostatic interactions (5). Since R has the highest pKa (12.48) and therefore the strongest interaction, the high frequency with which this amino acid was selected in the B-AAV-pool (Table 1B) can be explained. Accordingly, due to their lower pKa values (10.79 and 6.04, respectively), K and H seem to play a less important role compared to that of R.

Taken together, these data suggest a model in which peptide insertions at 587 can either disrupt or conserve the capsid ability to bind heparin by different mechanisms. Insertion of a peptide between R585 and R588 (Fig. 1A and B) could cause their spatial separation or sterically block the heparin binding ability. In either case, bulky amino acids are prone to lead to one or both of these results (Fig. 1C and D). If the peptide consists of small residues, the insertion could be less invasive and the structure of the HSPG binding motif maintained functional (Fig. 1E). Insertion of positively charged peptides could lead to an HSPG binding phenotype by reconstituting a binding motif in combination with one of the original arginines (Fig. 1F) or independently from them (Fig. 1G). The proximity of R588 to the last position of the inserted peptide could facilitate reconstitution of a functional motif if an arginine is present at the latter position (Fig. 1H). It should be noted that this behavior could be due to the particular sequence of the construct we used, where the seventh position of the randomized peptide and R588 are separated by two residues, resembling the wild-type situation.


Figure 1
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  		FIG. 1. Proposed model for the influence of several peptide classes on capsid stability and on binding to heparin. A) Three-dimensional atomic structure of the 587 region. R585 and R588 are depicted in blue. B) The two arginines are part of the binding motif. C) A bulky peptide disrupts the heparin binding motif, taking the arginines apart. D) A bulky peptide obstructs the HSPG binding motif. E) Small peptides could preserve the original structure of the loop and heparin binding motif. F) and G) The presence of one or more arginines in the inserted peptide restores the heparin binding ability. H) Due to its proximity to R588, an arginine in the last amino acid position of the insertion is prone to restore heparin binding. In all panels, a functional heparin binding site is indicated by a red pattern. A loop conformation that confers capsid stability is indicated by the blue arrow. wt, wild type.

To prove that lack of heparin affinity column binding reflected lack of cellular HSPG binding, binding and nonbinding pools and AAV with wild-type capsid (RC) were produced as recombinant AAV vectors (rAAV) as previously described (9), and heparin competition studies on the cervix carcinoma cell line HeLa were performed (Fig. 2A). The addition of heparin inhibited transduction by rAAV-RC and the B-rAAV-pool, while the effect on the NB-rAAV-pool was negligible, demonstrating the initial hypothesis.


Figure 2
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  		FIG. 2. Transduction efficiency of rAAV-RC, the B-rAAV-pool, the NB-rAAV-pool, and rAAV-A3 on HeLa and HepG2 cells. A) HeLa cells were transduced with rAAV-RC (RC; 500 genomic particles per cell [GOI]), the B-rAAV-pool (B; 1,000 GOI), and the NB-rAAV-pool (NB; 5,000 GOI), coding for the green fluorescent protein (GFP) in the absence (gray bars) or presence (black bars) of 425 IU of heparin/ml medium. Transduction levels were determined by fluorescence-activated cell sorter analysis. Each experiment was repeated independently twice, and the mean values and standard deviations (indicated by error bars) are shown. B) HepG2 cells were transduced in the presence of adenovirus (1 PFU/cell) with a 1,000 GOI of rAAV-RC, the B-rAAV-pool, the NB-rAAV-pool, or rAAV-A3 (coding for beta-galactosidase) and were analyzed by Galactolight Plus beta galactosidase assay according to manufacturer's instructions (Tropix). Gene expression was normalized for total protein using bicinchoninic acid (Perbio, United Kingdom) and expressed as relative light units (RLU) per milligram of protein. Each experiment was repeated independently twice, and the mean values and standard deviations (indicated by error bars) are shown. rAAV-RC, dotted bars; B-rAAV-pool, black bars; NB-rAAV-pool, striped bars; rAAV-A3, gray bars.

We previously described an AAV targeting vector, rAAV-MTP, that allowed systemic vascular targeting (12). Simultaneous detargeting of this vector from liver and spleen was observed. This vector showed a reduced ability to bind to heparin (12). Here, we analyzed whether the inability of AAV insertion mutants to bind heparin directly correlates with detargeting from liver and spleen. First, the ability to transduce the hepatocellular carcinoma cell line HepG2 was analyzed (Fig. 2B). In addition to rAAV-RC, the B-rAAV-pool, and the NB-rAAV-pool, a known HSPG knockout mutant (rAAV-A3) was generated by three amino acid substitutions (R585A, N587A, and R588A) (15) and was used as a control. rAAV-RC and the B-rAAV-pool showed a comparable transduction efficiency, whereas the NB-rAAV-pool showed an eightfold reduction in infectivity. In contrast, a reduction of ~500-fold was determined for rAAV-A3. Higher transduction levels of the NB-rAAV-pool in comparison to rAAV-A3 could be due to new ligand-receptor interactions of some of the inserts displayed within the pool.

Thereafter, 4 x 109 genomic particles were injected intravenously into C57/B6 mice (n = 4/group), and biodistribution studies were performed as described previously (12). Animals injected with rAAV-RC and the B-rAAV-pool showed a comparable biodistribution, with the highest vector DNA level in spleen and liver (Fig. 3). In contrast, the NB-rAAV-pool showed a 102- and 31.8-fold reduction in vector DNA level in the spleen in comparison to rAAV-RC and the B-rAAV-pool, respectively, whereas in the liver an 8.8- and 6.7-fold reduction was detected. In addition, elevated levels of viral DNA in the blood were measured for the NB-rAAV-pool consistently with the level of liver and spleen detargeting. rAAV-A3 also showed a detargeting from liver and spleen as well as the highest amount of viral DNA in the blood. Results obtained with rAAV-A3 and the NB-rAAV-pool are in agreement with previously published data obtained with other mutants impaired in HSPG binding, which showed a detargeting from liver (5, 12) and spleen (12).


Figure 3
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  		FIG. 3. Bioistribution of rAAV-RC, the B-rAAV-pool, the NB-rAAV-pool, and rAAV-A3 in C57/B6 mice. A total of 4 x 109 genomic particles of the different vector preparations (coding for beta-galactosidase) were injected into the tail vein of 12-week-old C57/B6 mice. At 24 h postinfection mice were sacrificed. DNA was extracted from blood and tissues. Vector genomes per tissue were quantified by PCR (TaqMan). rAAV-RC, dotted bars; B-rAAV-pool, black bars; NB-rAAV-pool, striped bars; rAAV-A3, gray bars.

This suggests an unspecific HSPG-dependent retention of rAAV-2 and of HSPG-binding rAAV targeting vectors in liver and spleen, and it provides the rationale for using the NB-AAV-pool for AAV display selections of cell/tissue type-specific AAV targeting vectors to avoid the HSPG-dependent retention in liver and spleen, thereby increasing the in vivo targeting ability of the respective vectors. Furthermore, our studies revealed different ways by which an inserted peptide is able to confer HSPG binding abilities to AAV targeting vectors, and they may help to fine tune the peptide insertion in order to ablate HSPG binding and to obtain tissue-specific vectors. This knowledge could also improve targeting mutants where R585 and/or R588 are replaced by other amino acids, since even in this case, some peptides (Fig. 1G) would restore HSPG binding.


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ACKNOWLEDGMENTS
 
This work was supported by the Center for Molecular Medicine of the University of Cologne (S.H., M.H., and H.B.), Köln Fortune (M.H.), the Deutsche Forschungsgemeinschaft (Forschergruppe Xenotransplantation) (M.H. and L.P.), the European Union (J.B.), Deutsche Krebshilfe (J.E. and H.J.), the Medical Research Council (A.H.B.), and the Biotechnology and Biophysical Research Council (A.H.B.).

We thank Richard Jude Samulski (University of North Carolina at Chapel Hill) for kindly providing pXX6.


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FOOTNOTES
 
* Corresponding author. Mailing address: Clinic I for Internal Medicine, University of Cologne, LFI, Level 4, Room 052, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany. Phone: 49-221-478-4448. Fax: 49-221-478-87941. E-mail: buening{at}lmb.uni-muenchen.de. Back

{dagger} These authors contributed equally to this work. Back


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Journal of Virology, July 2006, p. 7265-7269, Vol. 80, No. 14
0022-538X/06/$08.00+0     doi:10.1128/JVI.00076-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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