Adeno-Associated Virus Capsids Displaying Immunoglobulin-Binding Domains Permit Antibody-Mediated Vector Retargeting to Specific Cell Surface Receptors

Abstract

Recombinant adeno-associated virus type 2 (rAAV2) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for some applications, because it reduces the specificity of the gene transfer. To overcome this limitation, we sought to create a versatile rAAV vector targeting system which would allow us to redirect rAAV binding to specific cell surface receptors by simple coupling of different ligands to its capsid. For this purpose, an immunoglobulin G (IgG) binding domain of protein A, Z34C, was inserted into the AAV2 capsid at amino acid position 587. The resulting AAV2-Z34C mutants could be packaged and purified to high titers and bound to IgG molecules. rAAV2-Z34C vectors coupled to antibodies against CD29 (␤ 1 -integrin), CD117 (c-kit receptor), and CXCR4 specifically transduced distinct human hematopoietic cell lines. In marked contrast, no transduction was seen in the absence of antibodies or in the presence of specific blocking reagents. These results demonstrate for the first time that an immunoglobulin binding domain can be inserted into the AAV2 capsid and coupled to various antibodies, which mediate the retargeting of rAAV vectors to specific cell surface receptors.The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for human somatic gene therapy (9, 11). However, its broad host range might represent a limitation for some applications, because recombinant AAV (rAAV)-mediated gene transfer would not be specific for the tissue or cell type of interest. The host range is determined by the interaction of the AAV2 capsid with specific cellular receptors and coreceptors (18,26,27).Recently, a hypothetical model of the AAV capsid was generated, and several regions which were exposed on the viral capsid accepted the insertion of an integrin-specific 14-aminoacid (aa) RGD ligand (L14) and bound to target cells expressing the corresponding receptor (6). Moreover, AAV2 vectors with a ligand insertion at site 587 infected wild-type AAVresistant B16F10 melanoma cells with infectious targeting titers of 5 ϫ 10 4 LacZ expression-forming units (EFU) per ml (multiplicity of infection, 1), indicating that the susceptibility of these cells to AAV2 infection was increased by at least 4 orders of magnitude (6).However, with this approach it remained difficult and laborious to generate targeting vectors, because the design and optimization of new AAV capsid mutants were required for each specific receptor and cell type. Thus, it seemed desirable to generate a universal AAV targeting capsid on which different ligands could bind and redirect the virus to specific cell surface receptors (Fig. 1A). Such a vector would allow rapid screening of appropriate receptors mediating virus binding, uptake, and correct intracellular processing, which are all prerequisites for successful retargeting of AAV-based vectors.For this purpose, an immunoglobulin G (IgG) binding domain was introduced into the capsid to enable AAV to bind different ...

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…Conjugation of ligands to the capsid [12][13][14][15][16] offers a high degree of versatility. However, such conjugates may show reduced stability in vivo, higher immunogenicity and reduced infectivity.…”

“…Genetic modification of the AAV2 capsid by oligonucleotide insertions, random mutagenesis or DNA shuffling is able to circumvent some of these limitations. [8][9][10][17][18][19] Whereas larger peptides up to the size of GFP (238 amino acids) could be accommodated at the VP2 N-terminus 15,[20][21][22][23][24][25] the addition of small peptides up to 34 amino acids is tolerated at several positions of the AAV2 capsid. 1 Among the tolerated insertion sites for small peptides, insertions in the AAV2 heparin binding domain 26,27 adjacent to amino acids 587 or 588 was most successful.…”

Receptor targeting of adeno-associated virus vectors

“…Taken together, all studies underline the potential value of the 587 site of the AAV-2 capsid, which is positioned at the tip of the GH loop 32 for the generation of cell-specific AAV-2 vectors by the direct targeting approach ( Figure 2C). 25,76,77 This strategy was successfully repeated by Grifman et al 72 They also used a sequence alignment approach to identify potential targeting sites of the AAV-2 capsid by expanding their comparisons to parvoviruses other than CPV and to the other AAV serotypes (AAV-1, AAV-3, AAV-4 and AAV-5). They identified identical regions to Girod et al, 25 and finally used sites 448 and 587 for their studies.…”

“…77 Protein A recognizes and binds the Fc part of immunoglobulins (Ig), but not the variable Ig domain, which therefore remains free to bind the antigen. Z34C is derived from the protein A subunit B, which encompasses 56 amino acids and binds the Fc portion with a dissociation constant of about 10-50 nM.…”

Adeno-associated virus vectors: potential applications for cancer gene therapy

“…Recently, Reid et al 99 combined the two approaches of chemically crosslinked bifunctional antibodies and genetic manipulation of the capsid gene to change the tropism of AAV2. In this study, a peptide (Z34C) from Staphylococcus aureus protein A was inserted at position 587 of the AAV2 capsid.…”