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Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical.

ABSTRACT: Adeno-associated viral (AAV) vectors 2 and 8 have been used in clinical trials for patients with hemophilia, and data suggest that the capsid-specific CD8? T cell response has had a negative impact on therapeutic success. To date the pattern of capsid cross-presentation from AAV2 and AAV8 transduction in vivo has not been elucidated. Previously, we have demonstrated that an engineered AAV2 virus carrying the immune-dominant SIINFEKL peptide in the capsid backbone was indistinguishable from wild type with respect to titer, tropism, and the ability to induce capsid-specific CD8? T cell responses in vivo. In this study, we used the same strategy to engineer an AAV8 vector and demonstrated that antigen from SIINFEKL peptide-integrated AAV8 capsid was effectively presented via either plasmid transfection or AAV8 transduction in vitro. The tissue tropism and transgene expression kinetics of the engineered AAV8 vector in vivo were identical to that of wild-type AAV8. Animal studies show that capsid antigen presentation from AAV transduction was dose dependent, and more importantly, the proliferation of capsid-specific CD8? T cells had similar kinetics (detectable before 30 days and undetectable after 40 days) for both AAV2 and AAV8 vectors. Elucidation of the kinetics of capsid antigen presentation from AAV transduction by various serotypes provides new insight into the potential impact CD8? T cells can have during clinical trials and may help with rational design of effective strategies to prevent capsid-specific CD8? T cell-mediated elimination of AAV-transduced target cells.

SUBMITTER: He Y

PROVIDER: S-EPMC3655625 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical.

He Yi Y Weinberg Marc S MS Hirsch Matt M Johnson Mark C MC Tisch Roland R Samulski R Jude RJ Li Chengwen C

Human gene therapy 20130502 5

Adeno-associated viral (AAV) vectors 2 and 8 have been used in clinical trials for patients with hemophilia, and data suggest that the capsid-specific CD8⁺ T cell response has had a negative impact on therapeutic success. To date the pattern of capsid cross-presentation from AAV2 and AAV8 transduction in vivo has not been elucidated. Previously, we have demonstrated that an engineered AAV2 virus carrying the immune-dominant SIINFEKL peptide in the capsid backbone was indistinguishable from wild ...[more]

PMID: 23534873

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Project description:Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP-/-) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty virions may allow us to rationally design effective strategies to prevent elimination of AAV transduced target cells by capsid specific CD8+ T cells.

Project description:Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be beta-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Project description:Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T?Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S?A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (~9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h.FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h.FIX:Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.

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Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical.

Publications

Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical.

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