Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

Shen, Shen; Horowitz, Eric D.; Troupes, Andrew N.; Brown, Sarah M.; Pulicherla, Nagesh; Samulski, R. Jude; Agbandje‐McKenna, Mavis; Asokan, Aravind

doi:10.1074/jbc.m113.482380

Cited by 82 publications

(77 citation statements)

References 38 publications

(39 reference statements)

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“…Self-complementary rAAV carrying the GFP gene under the control of the ubiquitous CBh promoter (34) were produced by the triple-transfection method using polyethylenimine (35). Viruses were harvested as previously described (36). Lysate was clarified by centrifugation at 6,200 ϫ g and purified by iodixanol gradient ultracentrifugation at 402,000 ϫ g for 1 h. Viruses were pulled from the 40% to 60% interface, purified by ion-exchange chromatography on a 1-ml Q HyperD F column (Pall), and eluted with 200 mM NaCl, 25 mM Tris (pH 9.0).…”

Section: Methodsmentioning

confidence: 99%

Heparan Sulfate Binding Promotes Accumulation of Intravitreally Delivered Adeno-associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism

Woodard

Liang

Bennett

et al. 2016

J Virol

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Many adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV serotype 2 (AAV2) and its heparan sulfate (HS)-binding-deficient capsid led to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated recombinant AAV2 (rAAV2) led to a 300-fold decrease in transduction compared to AAV2. Fluorescence in situ hybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed that HS binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on human ex vivo retinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhance their transduction, AAV1 and AAV8 were modified to bind HS with a single-amino-acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction than their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual-glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated with a change in tropism. Taken together, these data indicate that HS binding serves to sequester AAV capsids from the vitreous to the ILM but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery. A deno-associated virus (AAV) is a small (25-nm), nonpathogenic virus that has been extensively studied as a vector for gene transfer applications (1-3). The virus consists of two parts: the viral genome and the protein capsid. The viral genome can be largely replaced with a desired transgene to create recombinant AAV (rAAV) vectors used for gene delivery. The protein capsid is responsible for cell attachment and entry via a variety of glycans and cell surface receptors. There are 11 naturally occurring serotypes of AAV, denoted AAV1 to AAV11. Glycans and receptors have been elucidated for several AAV serotypes. Heparan sulfate proteoglycan (HSPG) has been shown to be used for both rAAV2 and rAAV3 cell entry (4). rAAV6 displays dual-glycan interaction with HSPG and sialic acid; however, HSPG binding alone is insufficient for cellular entry (5). Various linkages of sialic acid are important for the transduction of the rAAV1, rAAV4, and rAAV5 serotypes (6,7). N-linked galactose is used for the transduction of the rAAV9 serotype (8). Glycans expressed on the cell surface dictate the tissue and cellular tropism observed with the various AAV capsids. In addition to attachment to these glycans, AAV serotypes interact with cell receptors, including human growth factor rec...

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Section: Methodsmentioning

confidence: 99%

Heparan Sulfate Binding Promotes Accumulation of Intravitreally Delivered Adeno-associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism

Woodard

Liang

Bennett

et al. 2016

J Virol

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“…Several techniques have been proposed to engineer new AAV variants, including rationale or random capsid mutagenesis, 162,163 DNA shuffling, 164 in vitro and in vivo molecular evolution, and direct selection. 165,166 Thanks to rational receptor footprint engineering, new AAV2 chimera, such as AAV2i8, 167 AAV2i8G9, 168 and AAV-SASTG, 169 are now available, which transduce cardiac and skeletal muscle with high efficiency but are detargeted from liver tissue. Similarly, new AAV9 variants have been generated by random mutagenesis and selected for strong cardiac tropism and significantly less transgene expression in off-target by guest on http://circres.ahajournals.org/ Downloaded from organs (liver, kidney, pancreas).…”

Section: Capsid Engineering To Achieve Improved and Selective Tropismmentioning

confidence: 99%

Adeno-Associated Virus Vectors as Therapeutic and Investigational Tools in the Cardiovascular System

Zacchigna

Zentilin

Giacca

2014

Circulation Research

111

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Abstract:The use of vectors based on the small parvovirus adeno-associated virus has gained significant momentum during the past decade. Their high efficiency of transduction of postmitotic tissues in vivo, such as heart, brain, and retina, renders these vectors extremely attractive for several gene therapy applications affecting these organs. Besides functional correction of different monogenic diseases, the possibility to drive efficient and persistent transgene expression in the heart offers the possibility to develop innovative therapies for prevalent conditions, such as ischemic cardiomyopathy and heart failure. Therapeutic genes are not only restricted to protein-coding complementary DNAs but also include short hairpin RNAs and microRNA genes, thus broadening the spectrum of possible applications. In addition, several spontaneous or engineered variants in the virus capsid have recently improved vector efficiency and expanded their tropism. Apart from their therapeutic potential, adeno-associated virus vectors also represent outstanding investigational tools to explore the function of individual genes or gene combinations in vivo, thus providing information that is conceptually similar to that obtained from genetically modified animals. Finally, their single-stranded DNA genome can drive homology-directed gene repair at high efficiency. Here, we review the main molecular characteristics of adeno-associated virus vectors, with a particular view to their applications in the cardiovascular field.

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“…Recombinant AAV4 and mutant AAV4.18 vectors were generated using an updated triple-plasmid transfection method (25). Briefly, this involved transfection of (i) the pXR4 helper plasmid (26) or the mutant pXR4.18 helper plasmid (21), (b) the adenoviral helper plasmid pXX6-80, and (c) pTR-CBA-tdTom or pTR-CBA-Luc plasmids encoding the tdTomato (tdTom) or luciferase (Luc) reporter genes driven by the chicken beta actin (CBA) promoter and flanked by inverted terminal repeats (ITRs) derived from the AAV2 genome.…”

Section: Methodsmentioning

confidence: 99%

Unique Glycan Signatures Regulate Adeno-Associated Virus Tropism in the Developing Brain

Murlidharan

Corriher

Ghashghaei

et al. 2015

J Virol

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Adeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 (AAV4) as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain. Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans in AAV4 transduction. Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low-affinity AAV4 mutant (AAV4.18) displayed a striking shift in cellular tropism from 2,3-linked SA ؉ ependymal lining to 2,8-linked PSA ؉ migrating progenitors in the rostral migratory stream and olfactory bulb. In addition, this gain-of-function phenotype correlated with robust CNS spread of AAV4.18 through paravascular transport pathways. Consistent with these observations, altering glycan dynamics within the brain by coadministering SA-and PSA-specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors. We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain. IMPORTANCEViruses invade the CNS through various mechanisms. In the current study, we utilized AAV as a model to study the dynamics of virus-carbohydrate interactions in the developing brain and their impact on viral tropism. Our findings suggest that carbohydrate content can be exploited to regulate viral transport and tropism in the brain.

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Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

Cited by 82 publications

References 38 publications

Heparan Sulfate Binding Promotes Accumulation of Intravitreally Delivered Adeno-associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism

Heparan Sulfate Binding Promotes Accumulation of Intravitreally Delivered Adeno-associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism

Adeno-Associated Virus Vectors as Therapeutic and Investigational Tools in the Cardiovascular System

Unique Glycan Signatures Regulate Adeno-Associated Virus Tropism in the Developing Brain

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