Insertional Mutagenesis at Positions 520 and 584 of Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Generation of AAV2 Vectors with Eliminated Heparin-Binding Ability and Introduced Novel Tropism

Shi, Xiangqun; Fang, Guangguang; Shi, Wenfang

doi:10.1089/hum.2006.17.ft-177

Cited by 11 publications

(18 citation statements)

References 26 publications

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“…The RGD motif has been displayed previously on the surfaces of many viruses (measles virus [45], parvovirus [27], adenovirus [46][47][48], and adeno-associated virus [49]) with the goal of enhancing their oncolytic specificities and/or transduction efficiencies. In selecting potential insertion sites for the VSV G protein, we relied heavily on the crystal structure of the protein and sequence comparisons with the G proteins of various related vesiculoviruses.…”

Section: Discussionmentioning

confidence: 99%

Characteristics of Oncolytic Vesicular Stomatitis Virus Displaying Tumor-Targeting Ligands

Ammayappan

Peng

Russell

2013

J Virol

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bWe sought proof of principle that tumor-targeting ligands can be displayed on the surface of vesicular stomatitis virus (VSV) by engineering its glycoprotein. Here, we successfully rescued VSVs displaying tumor vasculature-targeting ligands. By using a rational approach, we investigated various feasible insertion sites on the G protein of VSV (VSV-G) for display of tumor vasculature-targeting ligands, cyclic RGD (cRGD) and echistatin. We found seven sites on VSV-G that tolerated insertion of the 9-residue cRGD peptide, two of which could tolerate insertion of the 49-amino acid echistatin domain. All of the ligand-displaying viruses replicated as well as the parental virus. In vitro studies demonstrated that the VSVechistatin viruses specifically bound to targeted integrins. Since the low-density lipoprotein receptor (LDLR) was recently identified as a major receptor for VSV, we investigated the entry of ligand-displaying viruses after masking LDLR. The experiment showed that the modified viruses can enter the cell independently of LDLR, whereas entry of unmodified virus is significantly blocked by a specific monoclonal antibody against LDLR. Both parental and ligand-displaying viruses displayed equal oncolytic efficacies in a syngeneic mouse myeloma model. We further demonstrated that single-chain antibody fragments against tumor-specific antigens can be inserted at the N terminus of the G protein and that corresponding replication-competent VSVs can be rescued efficiently. Overall, we demonstrated that functional tumor-targeting ligands can be displayed on replication-competent VSVs without perturbing viral growth and oncolytic efficacy. This study provides a rational foundation for the future development of fully retargeted oncolytic VSVs. V esicular stomatitis virus (VSV) is an enveloped, negativestrand RNA virus that belongs to the Vesiculovirus genus of the Rhabdoviridae family. VSV has the ability to infect and kill cancer cells while sparing normal cells (1-4). Exploitation of this oncolytic property provides a promising alternative approach for the treatment of cancer. For disseminated cancer, virotherapy should ideally be administered systemically (2, 5-7), but this route of delivery brings its own set of problems. The major concerns for VSV virotherapy are neurotoxicity, antibody neutralization, and sequestration in off-target organs, especially the liver and spleen. Many attempts have been made to address these drawbacks. To reduce the neurotoxicity, the matrix protein of VSV was mutated (8, 9), and microRNA targets (10) or picornaviral internal ribosome entry sites (11) were engineered into the VSV genome. Serum neutralization has been avoided by PEGylating the virus (12, 13) or loading onto antigen-specific T cells (14), which ultimately improved virotherapy outcomes.To circumvent all of the above hurdles in a single step, pseudotyping VSV with other viral envelope glycoproteins was considered a potentially feasible approach. Recent studies demonstrated that VSV neurotoxicity can be circumvented by...

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Section: Discussionmentioning

confidence: 99%

Characteristics of Oncolytic Vesicular Stomatitis Virus Displaying Tumor-Targeting Ligands

Ammayappan

Peng

Russell

2013

J Virol

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“…Many groups have successfully inserted peptides, specifically at the threefold loops, on the capsid surface as a means to retarget the virus for specific tissue types (14,38,39,53). In this study, we used peptides not as insertions but as substitutions in a novel region of the capsid.…”

Section: Discussionmentioning

confidence: 99%

“…The threefold axis has the largest amount of buried surface area and the highest contact energy, being the most interdigitated region of the capsid (60). The surface loops at the threefold axis of symmetry are thought to be involved in host cell receptor binding (4,23) and have been the target of several mutagenesis studies (27,31,39,55,56). In addition, recent data have shown that a single amino acid change (K531E) located at the base of the threefold loops has the ability to alter the phenotypes of multiple AAV serotypes (56), suggesting an incomplete understanding of this critical region.…”

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confidence: 99%

Surface Loop Dynamics in Adeno-Associated Virus Capsid Assembly

DiPrimio

Asokan

Govindasamy

et al. 2008

J Virol

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The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.Adeno-associated virus (AAV), a 26-nm nonpathogenic human parvovirus, is distinct from most viruses due to the dependence on a helper virus for productive infection (adenovirus or herpes simplex virus) (6). In light of the rapidly growing applications of AAV as a gene therapy vector (51, 57), several efforts to understand events in the infectious pathway including host cell recognition (3,11,23,33,49), intracellular trafficking (5, 10), and u...

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“…Among the all applied insertion peptide and molecules, RGD4C is the most popular peptide for viral capsid modification to target the intergrin on the cellular surface majorly expressed on the tumor cells [32,34,54,[87][88][89][90] . AAV mosaics revealed the selective and efficient transduction in targeted tumor cell [19,32,91] .…”

Section: Chemical Conjugation Modified Viral Capsidmentioning

confidence: 99%

“…Replicating adenovirus with mutated capsid proteins, in which the promiscuous adenovirus native tropism was abolished and a bi-specific adapter molecule to target the virus to the Epidermal Growth Factor Receptor (EGFR) was encoded [11] . Adenoassociated virus 2 natural tropism has been abrogated via double mutation in capsid at site 520 and 584 and a novel tropism was achieved via inserted RGD4C peptide proved by in vitro RGD-integrin mediated specific infectivity and heparin non-dependant infectivity [90] . Metabolically biotinylated AAV was produced via biotin peptide inserted into AAV capsid gene, such as serotype 1, 2, 3, 4 and 5.…”

Section: Chemical Conjugation Modified Viral Capsidmentioning

confidence: 99%

AAV-Based Targeting Gene Therapy

Shi¹,

Mu²,

Qian³

2008

American J. of Immunology

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Abstract:Since the first parvovirus serotype AAV2 was isolated from human and used as a vector for gene therapy application, there have been significant progresses in AAV vector development. AAV vectors have been extensively investigated in gene therapy for a broad application. AAV vectors have been considered as the first choice of vector due to efficient infectivity, stable expression and nonpathogenicity. However, the untoward events in AAV mediated in vivo gene therapy studies proposed the new challenges for their further applications. Deep understanding of the viral life cycle, viral structure and replication, infection mechanism and efficiency of AAV DNA integration, in terms of contributing viral, host-cell factors and circumstances would promote to evaluate the advantages and disadvantages and provide more insightful information for the possible clinical applications. In this review, main effort will be focused on the recent progresses in gene delivery to the target cells via receptor-ligand interaction and DNA specific integration regulation. Furthermore AAV receptor and virus particle intracellular trafficking are also discussed.

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Insertional Mutagenesis at Positions 520 and 584 of Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Generation of AAV2 Vectors with Eliminated Heparin-Binding Ability and Introduced Novel Tropism

Cited by 11 publications

References 26 publications

Characteristics of Oncolytic Vesicular Stomatitis Virus Displaying Tumor-Targeting Ligands

Characteristics of Oncolytic Vesicular Stomatitis Virus Displaying Tumor-Targeting Ligands

Surface Loop Dynamics in Adeno-Associated Virus Capsid Assembly

AAV-Based Targeting Gene Therapy

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