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Multiple roles for sialylated glycans in determining the cardiopulmonary tropism of adeno-associated virus 4.

ABSTRACT: Adeno-associated virus 4 (AAV4) is one of the most divergent serotypes among known AAV isolates. Mucins or O-linked sialoglycans have been identified as the primary attachment receptors for AAV4 in vitro. However, little is known about the role(s) played by sialic acid interactions in determining AAV4 tissue tropism in vivo. In the current study, we first characterized two loss-of-function mutants obtained by screening a randomly mutated AAV4 capsid library. Both mutants harbored several amino acid residue changes localized to the 3-fold icosahedral symmetry axes on the AAV4 capsid and displayed low transduction efficiency in vitro. This defect was attributed to decreased cell surface binding as well as uptake of mutant virions. These results were further corroborated by low transgene expression and recovery of mutant viral genomes in cardiac and lung tissue following intravenous administration in mice. Pharmacokinetic analysis revealed rapid clearance of AAV4 mutants from the blood circulation in conjunction with low hemagglutination potential ex vivo. These results were recapitulated with mice pretreated intravenously with sialidase, directly confirming the role of sialic acids in determining AAV4 tissue tropism. Taken together, our results support the notion that blood-borne AAV4 particles interact sequentially with O-linked sialoglycans expressed abundantly on erythrocytes followed by cardiopulmonary tissues and subsequently for viral cell entry.

SUBMITTER: Shen S

PROVIDER: S-EPMC3838263 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Multiple roles for sialylated glycans in determining the cardiopulmonary tropism of adeno-associated virus 4.

Shen Shen S Troupes Andrew N AN Pulicherla Nagesh N Asokan Aravind A

Journal of virology 20130925 24

Adeno-associated virus 4 (AAV4) is one of the most divergent serotypes among known AAV isolates. Mucins or O-linked sialoglycans have been identified as the primary attachment receptors for AAV4 in vitro. However, little is known about the role(s) played by sialic acid interactions in determining AAV4 tissue tropism in vivo. In the current study, we first characterized two loss-of-function mutants obtained by screening a randomly mutated AAV4 capsid library. Both mutants harbored several amino a ...[more]

PMID: 24067974

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Project description:Adeno-associated viruses (AAV) are composed of nonenveloped, icosahedral protein shells that can be adapted to package and deliver recombinant therapeutic DNA. Approaches to engineer recombinant capsids for gene therapy applications have focused on rational design or library-based approaches that can address one or two desirable attributes; however, there is an unmet need to comprehensively improve AAV vector properties. Such cannot be achieved by utilizing sequence data alone but requires harnessing the three-dimensional (3D) structural properties of AAV capsids. Here, we solve the structures of a natural AAV isolate complexed with antibodies using cryo-electron microscopy and harness this structural information to engineer AAV capsid libraries through saturation mutagenesis of different antigenic footprints. Each surface loop was evolved by infectious cycling in the presence of a helper adenovirus to yield a new AAV variant that then serves as a template for evolving the next surface loop. This stepwise process yielded a humanized AAV8 capsid (AAVhum.8) displaying nonnatural surface loops that simultaneously display tropism for human hepatocytes, increased gene transfer efficiency, and neutralizing antibody evasion. Specifically, AAVhum.8 can better evade neutralizing antisera from multiple species than AAV8. Further, AAVhum.8 displays robust transduction in a human liver xenograft mouse model with expanded tropism for both murine and human hepatocytes. This work supports the hypothesis that critical properties, such as AAV capsid antibody evasion and tropism, can be coevolved by combining rational design and library-based evolution for clinical gene therapy.IMPORTANCE Clinical gene therapy with recombinant AAV vectors has largely relied on natural capsid isolates. There is an unmet need to comprehensively improve AAV tissue tropism, transduction efficiency, and antibody evasion. Such cannot be achieved by utilizing capsid sequence data alone but requires harnessing the 3D structural properties of AAV capsids. Here, we combine rational design and library-based evolution to coevolve multiple, desirable properties onto AAV by harnessing 3D structural information.

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Multiple roles for sialylated glycans in determining the cardiopulmonary tropism of adeno-associated virus 4.

Publications

Multiple roles for sialylated glycans in determining the cardiopulmonary tropism of adeno-associated virus 4.

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