Conjugate-Based Targeting of Recombinant Adeno-Associated Virus Type 2 Vectors by Using Avidin-Linked Ligands

Abstract

The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1␣ receptor (FGFR1␣), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1␣-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.Adeno-associated virus (AAV)-based vectors are becoming increasingly popular for gene therapy of human diseases (3,16,22,33,34). Despite the potential for long-term expression, genomic integration, and low immunogenicity, the transduction efficiency of AAV type 2 (AAV-2) vectors varies significantly among cell types. For example, the cells of muscle and brain are highly permissive for AAV-2 infection. However, certain human megakaryocytic cell lines and primary cells of hematopoietic origin show inconsistencies in infection (8,15,19,20,23), which may be due to either insufficient receptor numbers, an absence of optimal amounts of intracellular factors necessary for AAV second-strand synthesis, or a combination of both (2, 6, 7, 10, 30). Thus, improvements to overcome these limitations will have a positive impact on the application of AAV vectors in gene therapy. Whereas recent studies on intracellular events conducive for AAV transgene expression have identified the roles of several host cell factors (10,14,30), in order to overcome the limitations of receptormediated AAV infection, both genetic and nongenetic approaches are currently being developed (19,24,26,37,38). Although genetic modification of AAV capsid may be a preferred means of achieving vector r...

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.…”

Intracellular trafficking of adeno-associated viral vectors

“…The repertoire of rAAV vectors has also been greatly expanded by the development of technologies to pseudo-package rAAV genomes, [17][18][19][20] package AAV genomes with two different ITR serotypes, 21 generate mosaic rAAV particles with more than one capsid serotype, [22][23][24] retarget AAV by generating rAAV capsid modification [25][26][27][28][29] and generate rAAV with chemically modified capsids. 30 These technologies have greatly expanded the ability to tailor rAAV for specific applications in gene therapy. 31 Despite the great number of rAAV serotypes and rAAV variants available, several biologic barriers appear to limit the effectiveness of rAAVs for gene therapy.…”

Library selection and directed evolution approaches to engineering targeted viral vectors

“…For example, numerous studies have attempted to enhance selective AAV vector gene delivery to specific cell types via altering binding affinity to receptors expressed on the target cells. Several such strategies to modify AAV tropism include: (i) association with ''adaptor'' bispecific antibodies, with one arm directed against AAV and the other against a target receptor (Bartlett et al, 1999), (ii) chemical or metabolic conjugation of biotin to the virus, followed by the addition of avidin and a biotinylated-targeting molecule (Arnold et al, 2006;Ponnazhagan et al, 2002), and (iii) genetic modification of the viral capsid structures through the insertion of defined peptide sequences (Girod et al, 1999;Grifman et al, 2001;Nicklin et al, 2001;Work et al, 2004). These rational design approaches have resulted in moderately selective receptormediated gene delivery and demonstrated the strong potential for engineering the AAV capsid for targeting.…”