Engineering Liver-detargeted AAV9 Vectors for Cardiac and Musculoskeletal Gene Transfer

Pulicherla, Nagesh; Shen, Shen; Yadav, Swati; Debbink, Kari; Govindasamy, Lakshmanan; Agbandje‐McKenna, Mavis; Asokan, Aravind

doi:10.1038/mt.2011.22

Cited by 184 publications

(157 citation statements)

References 47 publications

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“…Despite this, our transgene was not delivered to the liver, transduced muscle, and was highly expressed at 7 months with significant therapeutic efficacy. Recently, novel-engineered AAV2/9 variants were tested in mice and displayed 10-to 25-fold lower gene transfer efficacy in liver, while transducing cardiac and skeletal muscle as effectively as AAV2/9 (Pulicherla et al, 2011). As previously reported (Vitiello et al, 2009), the results obtained in our laboratory using AAV2/9 were unsatisfactory in BIO14.6 and were in contrast with reported observations on the ability of AAV2/9 in mice to transcend vasculature and transduce myocardium (Pacak et al, 2006).…”

Section: Discussioncontrasting

confidence: 56%

Use of a Lower Dosage Liver-Detargeted AAV Vector to Prevent Hamster Muscular Dystrophy

et al. 2013

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The BIO14.6 hamster carries a mutation in the delta sarcoglycan gene causing muscular dystrophy and cardiomyopathy. The disease can be prevented by systemic delivery of delta sarcoglycan cDNA using adeno-associated viruses (AAVs). However, all AAVs also target the liver, raising concerns about their therapeutic efficacy in human applications. We compared the AAV2/8 with the chimeric AAV2/2i8, in which the 585-QQNTAP-590 motif of the AAV8 serotype was added to the heparan sulfate receptor footprint of the AAV2 strain. Both vectors carrying the human delta sarcoglycan cDNA were delivered into 24 14-day-old BIO14.6 hamsters. We followed transgene expression in muscle and liver for 7 months. We detected a sustained ectopic expression of delta sarcoglycan in the liver when using AAV2/8 but not AAV2/2i8. Genomic copies of AAV2/2i8 were not detectable in the liver, while at least 100-fold more copies of AAV2/8 were counted. In contrast, the hamster skeletal muscle expressed more delta sarcoglycan using AAV2/2i8 and were still healthy after 7 months at the lower dosage. We conclude that this chimeric vector is a robust option for safer and longer-term diseased muscle targeting.

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Section: Discussioncontrasting

confidence: 56%

Use of a Lower Dosage Liver-Detargeted AAV Vector to Prevent Hamster Muscular Dystrophy

et al. 2013

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“…All viral vectors were generated at the UNC Vector Core by iodixanol gradient ultracentrifugation followed by ion-exchange chromatography and vector genome titers determined by dot blot assay as well as verified by quantitative PCR (qPCR) as described previously (23). Single-stranded AAV serotype 2 vectors packaging different transgene cassettes were as follows: wild-type AAV genome containing rep and cap genes (4.7 kb) (24), chicken ␤-actin (CBA) promoter-driven firefly luciferase (4.1 kb) (25), CBA promoter-driven tdTomato (3.8 kb) (25), cytomegalovirus (CMV) promoter-driven firefly luciferase (3.6 kb) (26), CMV promoter-driven green fluorescent protein (GFP) (3.4 kb) (23), and an EF1a promoter-driven mCherry with an internal ribosome entry site (IRES) signal followed by WGA-Cre (5.3 kb) (27). Self-complementary AAV2 vectors utilized in the study were as follows: CMV promoter-driven green fluorescent protein with lambda phage genome stuffer DNA (5.0 kb) (23), CMV promoter-driven green fluorescent protein (4.6 kb) (28), and CBA hybrid promoter-driven green fluorescent protein (4.1 kb) (28).…”

Section: Methodsmentioning

confidence: 99%

Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release

Horowitz

Rahman

Bower

et al. 2013

J Virol

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We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release.A deno-associated virus (AAV) is a small (25 nm) nonenveloped virus belonging to the family Parvoviridae and genus Dependovirus. The AAV capsid packages a single-stranded (ssDNA) genome approximately 4.7 kb in length (1). The wildtype genome consists of two open reading frames flanked by two inverted terminal hairpin repeats (ITRs). The ITRs, which are 145 nucleotides each, are the only cis element in the AAV genome required for successful packaging (2, 3). The AAV capsid is composed of 60 (T ϭ 1) viral protein subunits VP1, VP2, and VP3, in approximately the ratio 1:1:10. The three different subunits are generated from overlapping reading frames and interact within the capsid through the common VP3 subunit region. The largest VP1 subunit is known to possess a phospholipase A2 domain required for infectivity (4). Because of its broad tropism, lack of pathogenicity, and flexibility in genome content, AAV has become a promising candidate for therapeutic gene transfer applications. In the past 2 decades, AAV has been utilized as a gene transfer vector in a number of phase I and phase II clinical trials treating various genetic diseases (5).Different AAV serotypes infect cells by engaging a variety of cell surface glycans and coreceptors, followed by endocytic uptake (4, 6, 7). Viral particles are then thought to escape from the endosome and translocate to th...

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“…The AAV capsid surface features that are unique to AAV5-shorter 3-fold protrusions, extended VR-VII, and smaller HI loop-contain amino acid residues or are proximate to capsid regions reported to play essential roles in the AAV life cycle. For example, residues within VR-IV, VR-V, and VR-VIII which make up the 3-fold protrusions have been reported to control glycan receptor attachment (VR-V and VR-VIII for AAV2 and VR-V for AAV9) (90)(91)(92)(93)122), transduction (VR-IV, VR-V, and VR-VIII in AAV2, VR-VIII in AAV8, and VR-IV, VR-V, and VR-VIII in AAV9) (94)(95)(96)(97)(98)(99) and antigenic phenotypes (VR-IV, VR-V, and VR-VIII in AAV2 and VR-VIII in AAV8 (97,100,101). These regions have similar roles in AAV5.…”

Section: Fig 2 Aav Capsid Surfaces (A Cmentioning

confidence: 99%

Structural Insights into Adeno-Associated Virus Serotype 5

Govindasamy

DiMattia

Gurda

et al. 2013

J Virol

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The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between ␤H and ␤I of the core ␤-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity. R ecombinant adeno-associated viruses (rAAVs) are promising viral vectors for gene delivery applications (1, 2). These viruses belong to the single-stranded DNA (ssDNA)-packaging Parvoviridae and genus Dependovirus. Hundreds of AAV genotypes have been sequenced from several mammalian species, and to date 12 serotypes (AAV1 to AAV12) have been defined for the human and nonhuman primate isolates (3-15). The 12 serotypes are classified into eight genetic groups, clades A to F and clonal isolates AAV4 and AAV5, based on antigenic reactivity and sequence similarity (15). The groups are represented by AAV1 to AAV9, with AAV1 and AAV6 belonging to the same clade A because of their high sequence similarity and antigenic cross-reactivity. The representative members share ϳ55 to 99% sequence identity, with AAV4 and AAV5 being the most divergent from each other and from the other members.The linear ssDNA AAV genome, ϳ4.7 kb in length, contains two genes: cap, which encodes the capsid viral proteins (VPs; VP1, ϳ87 kDa; VP2, ϳ73 kDa; VP3, ϳ62 kDa) and rep, which encodes the replication proteins necessary for genome replication and genome packaging. Inverted terminal repeats (ITRs) at the end of the AAV genome, 145 bp in length, are the only essential active sequence required to function as (i) the origin for DNA replication, (ii) the packaging signal, and (iii) integration sites (16)(17)(18)(19). Recombina...

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Engineering Liver-detargeted AAV9 Vectors for Cardiac and Musculoskeletal Gene Transfer

Cited by 184 publications

References 47 publications

Use of a Lower Dosage Liver-Detargeted AAV Vector to Prevent Hamster Muscular Dystrophy

Use of a Lower Dosage Liver-Detargeted AAV Vector to Prevent Hamster Muscular Dystrophy

Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release

Structural Insights into Adeno-Associated Virus Serotype 5

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