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Engraftment of a galactose receptor footprint onto adeno-associated viral capsids improves transduction efficiency.

ABSTRACT: New viral strains can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. However, such mutants generally harbor amino acid changes that affect viral binding to a single class of carbohydrate receptors. We describe the rational design and synthesis of novel, chimeric adeno-associated virus (AAV) strains that exploit an orthogonal glycan receptor for transduction. A dual glycan-binding AAV strain was first engineered as proof of concept by grafting a galactose (Gal)-binding footprint from AAV serotype 9 onto the heparan sulfate-binding AAV serotype 2. The resulting chimera, AAV2G9, continues to bind heparin affinity columns but interchangeably exploits Gal and heparan sulfate receptors for infection, as evidenced by competitive inhibition assays with lectins, glycans, and parental AAV strains. Although remaining hepatotropic like AAV2, the AAV2G9 chimera mediates rapid onset and higher transgene expression in mice. Similarly, engraftment of the Gal footprint onto the laboratory-derived strain AAV2i8 yielded an enhanced AAV2i8G9 chimera. This new strain remains liver-detargeted like AAV2i8 while selectively transducing muscle tissues at high efficiency, comparable with AAV9. The AAV2i8G9 chimera is a promising vector candidate for targeted gene therapy of cardiac and musculoskeletal diseases. In addition to demonstrating the modularity of glycan receptor footprints on viral capsids, our approach provides design strategies to expand the AAV vector toolkit.

SUBMITTER: Shen S

PROVIDER: S-EPMC3789977 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Engraftment of a galactose receptor footprint onto adeno-associated viral capsids improves transduction efficiency.

Shen Shen S Horowitz Eric D ED Troupes Andrew N AN Brown Sarah M SM Pulicherla Nagesh N Samulski Richard J RJ Agbandje-McKenna Mavis M Asokan Aravind A

The Journal of biological chemistry 20130812 40

New viral strains can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. However, such mutants generally harbor amino acid changes that affect viral binding to a single class of carbohydrate receptors. We describe the rational design and synthesis of novel, chimeric adeno-associated virus (AAV) strains that exploit an orthogonal glycan receptor for transduction. A dual glycan-binding AAV strain was first engineered as proof of concept by grafting a ga ...[more]

PMID: 23940044

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Project description:Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.IMPORTANCE Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.

Project description:The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 157 intraventricularly injected barcoded AAV variants profiled by transcriptome sequencing. Transcriptome analysis identified two synthetic capsids, AAV9_A2 and AAV1_P5 transducing active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of promoter and dose of injected viral genomes enabled targeting of 57% of the NSC compartment. Importantly, transduced NSC readily produced neurons. The present study identifies AAV variants with a high specificity and transduction efficiency of adult NSCs thereby paving the way for preclinical testing of regenerative gene therapy. We identified a synthetic Adeno-associated virus (AAV) capsid, AAV1_P5, that transduces active and quiescent neural stem cells (NSCs). In order to fully characterize the identity of AAV1_P5-transduced cells, as well as potential changes arising by the AAV-transduction itself, we profiled these cells and untransduced ones from the same mouse by single cell RNA sequencing. To this end, three months-old eYFP-reporter mice were injected with 109 vg/mouse AAV1_P5 harboring the CMV_Cre construct. Transduction induces expression of eYFP. 37 days post injection, we isolated cells from the v-SVZ and other brain regions. More precisely, we isolated labeled cells of the ventricular-subventricular zone and the striatum, rostral migratory stream (RMS) and olfactory bulb, here referred to as rest of the brain (RoB). To capture the remaining unlabeled cells of the NSC-lineage in the v-SVZ, we also isolated GLAST+ v-SVZ cells. Two samples of two pooled mice each were subjected to single cell RNA sequencing.

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Engraftment of a galactose receptor footprint onto adeno-associated viral capsids improves transduction efficiency.

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Engraftment of a galactose receptor footprint onto adeno-associated viral capsids improves transduction efficiency.

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