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1.  Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site 
Journal of Virology  2016;90(11):5219-5230.
ABSTRACT
The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wild-type AAV6. Furthermore, three of the AAV1 SIA binding site mutants—S472R, V473D, and N500E—escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface “hot spot” dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering.
IMPORTANCE The adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect to transduction efficiency and the detrimental effects of preexisting host antibodies. This study aimed to identify key capsid regions which can be engineered to overcome these limitations. A sialic glycan receptor recognition pocket was identified in AAV1 and its closely related AAV6, using X-ray crystallography. The site was confirmed by mutagenesis followed by cell binding and transduction assays. Significantly, residues controlling gene expression efficiency, as well as antibody escape variants, were also identified. This study thus provides, at the amino acid level, information for rational structural engineering of AAV vectors with improved therapeutic efficacy.
doi:10.1128/JVI.00161-16
PMCID: PMC4934739  PMID: 26962225
2.  Glymphatic fluid transport controls paravascular clearance of AAV vectors from the brain 
JCI insight  2016;1(14):e88034.
Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4−/− mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4−/− mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design.
doi:10.1172/jci.insight.88034
PMCID: PMC5033923  PMID: 27699236
3.  Glymphatic fluid transport controls paravascular clearance of AAV vectors from the brain 
JCI Insight  null;1(14):e88034.
Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design.
Glymphatic transport or glial-lymphatic transport driven by aquaporin 4 water channels controls CNS spread and systemic leakage of AAV vectors.
doi:10.1172/jci.insight.88034
PMCID: PMC5033923  PMID: 27699236
4.  Optical Control of CRISPR/Cas9 Gene Editing 
The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function.
doi:10.1021/ja512664v
PMCID: PMC4919123  PMID: 25905628
5.  rAAV-compatible MiniPromoters for restricted expression in the brain and eye 
Molecular Brain  2016;9:52.
Background
Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters–however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo.
Methods
For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were “cut down” to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP.
Results
The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia.
Conclusions
Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-016-0232-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-016-0232-4
PMCID: PMC4862195  PMID: 27164903
rAAV Gene therapy; Raphe nuclei; Purkinje cells; Retina; Cornea
6.  AAV Gene Therapy for MPS1-associated Corneal Blindness 
Scientific Reports  2016;6:22131.
Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness.
doi:10.1038/srep22131
PMCID: PMC4761992  PMID: 26899286
7.  Strategies to circumvent humoral immunity to adeno-associated viral vectors 
Introduction
Recent success in gene therapy of certain monogenic diseases in the clinic has infused enthusiasm into the continued development of recombinant adeno-associated viral (AAV) vectors as next-generation biologics. However, progress in clinical trials has also highlighted the challenges posed by the host humoral immune response to AAV vectors. Specifically, while pre-existing neutralizing antibodies (NAbs) limit the cohort of eligible patients, NAb generation following treatment prevents vector re-dosing.
Areas covered
In this review, we discuss a spectrum of complementary strategies that can help circumvent the host humoral immune response to AAV.
Expert opinion
Specifically, we present a dual perspective, that is, vector versus host, and highlight the clinical attributes, potential caveats and limitations as well as complementarity associated with the various approaches.
doi:10.1517/14712598.2015.1035645
PMCID: PMC4689135  PMID: 25985812
adeno-associated virus; capsid; humoral immune response; neutralizing antibody; recombinant adeno-associated viral vectors
8.  Unique Glycan Signatures Regulate Adeno-Associated Virus Tropism in the Developing Brain 
Journal of Virology  2015;89(7):3976-3987.
ABSTRACT
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.
IMPORTANCE Viruses 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.
doi:10.1128/JVI.02951-14
PMCID: PMC4403422  PMID: 25631075
9.  An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses 
PLoS Pathogens  2015;11(8):e1005082.
Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.
Author Summary
Mammalian cells have developed diverse innate/intrinsic immune strategies to counteract viral infections. Post-entry infection steps of a single-strand DNA virus, adeno-associated virus (AAV), are subject to such restrictions. Here, we screened an siRNA library to identify a novel cellular factor involved in AAV restriction. We found PHF5A, a component of the U2 snRNP mRNA splicing factor, blocks expression from recombinant AAV vectors. Disruption of PHF5A expression specifically enhanced AAV vector performance. Moreover, genetic and pharmacological inhibition of other U2 snRNP proteins, but not spliceosome proteins involved in other splicing steps, strongly increased transgene expression from AAV vectors. Further study demonstrated that U2 snRNP proteins recognize incoming AAV capsids to mediate this cellular restriction at the step after second-strand synthesis. In summary, we identify the U2 snRNP spliceosome complex as novel host factors that effectively restrict recombinant AAV vectors. Considering frequent reorganization of host splicing machinery in DNA virus infections, it is conceivable that U2 snRNP plays a role as a broad spectrum antiviral factor and helper viruses have evolved to counteract this restriction through sequestration of snRNP proteins.
doi:10.1371/journal.ppat.1005082
PMCID: PMC4526370  PMID: 26244496
10.  Gene Therapy: Charting a Future Course—Summary of a National Institutes of Health Workshop, April 12, 2013 
Human Gene Therapy  2014;25(6):488-497.
Abstract
Recently, the gene therapy field has begun to experience clinical successes in a number of different diseases using various approaches and vectors. The workshop Gene Therapy: Charting a Future Course, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities, brought together early and mid-career researchers to discuss the key scientific challenges and opportunities, ethical and communication issues, and NIH and foundation resources available to facilitate further clinical advances.
doi:10.1089/hum.2014.045
PMCID: PMC4064731  PMID: 24773122
11.  An Emerging Adeno-Associated Viral Vector Pipeline for Cardiac Gene Therapy 
Human Gene Therapy  2013;24(11):906-913.
Abstract
The naturally occurring adeno-associated virus (AAV) isolates display diverse tissue tropisms in different hosts. Robust cardiac transduction in particular has been reported for certain AAV strains. Successful applications of these AAV strains in preclinical and clinical settings with a focus on treating cardiovascular disease continue to be reported. At the same time, these studies have highlighted challenges such as cross-species variability in AAV tropism, transduction efficiency, and immunity. Continued progress in our understanding of AAV capsid structure and biology has provided the rationale for designing improved vectors that can possibly address these concerns. The current report provides an overview of cardiotropic AAV, existing gaps in our knowledge, and newly engineered AAV strains that are viable candidates for the cardiac gene therapy clinic.
doi:10.1089/hum.2013.2515
PMCID: PMC3815036  PMID: 24164238
12.  Preclinical toxicity evaluation of AAV for pain: evidence from human AAV studies and from the pharmacology of analgesic drugs 
Molecular Pain  2014;10:54.
Gene therapy with adeno-associated virus (AAV) has advanced in the last few years from promising results in animal models to >100 clinical trials (reported or under way). While vector availability was a substantial hurdle a decade ago, innovative new production methods now routinely match the scale of AAV doses required for clinical testing. These advances may become relevant to translational research in the chronic pain field. AAV for pain targeting the peripheral nervous system was proven to be efficacious in rodent models several years ago, but has not yet been tested in humans. The present review addresses the steps needed for translation of AAV for pain from the bench to the bedside focusing on pre-clinical toxicology. We break the potential toxicities into three conceptual categories of risk: First, risks related to the delivery procedure used to administer the vector. Second, risks related to AAV biology, i.e., effects of the vector itself that may occur independently of the transgene. Third, risks related to the effects of the therapeutic transgene. To identify potential toxicities, we consulted the existing evidence from AAV gene therapy for other nervous system disorders (animal toxicology and human studies) and from the clinical pharmacology of conventional analgesic drugs. Thereby, we identified required preclinical studies and charted a hypothetical path towards a future phase I/II clinical trial in the oncology-palliative care setting.
doi:10.1186/1744-8069-10-54
PMCID: PMC4237902  PMID: 25183392
Adeno-associated virus; Pain; Gene therapy; Toxicology; Interleukin-10; Beta-endorphin
13.  Multiple Roles for Sialylated Glycans in Determining the Cardiopulmonary Tropism of Adeno-Associated Virus 4 
Journal of Virology  2013;87(24):13206-13213.
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.
doi:10.1128/JVI.02109-13
PMCID: PMC3838263  PMID: 24067974
14.  Biology of adeno-associated viral vectors in the central nervous system 
Gene therapy is a promising approach for treating a spectrum of neurological and neurodegenerative disorders by delivering corrective genes to the central nervous system (CNS). In particular, adeno-associated viruses (AAVs) have emerged as promising tools for clinical gene transfer in a broad range of genetic disorders with neurological manifestations. In the current review, we have attempted to bridge our understanding of the biology of different AAV strains with their transduction profiles, cellular tropisms, and transport mechanisms within the CNS. Continued efforts to dissect AAV-host interactions within the brain are likely to aid in the development of improved vectors for CNS-directed gene transfer applications in the clinic.
doi:10.3389/fnmol.2014.00076
PMCID: PMC4168676  PMID: 25285067
adeno-associated virus (AAV); viral vectors; gene therapy; neurological disorders; neurodegenerative diseases
15.  Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release 
Journal of Virology  2013;87(6):2994-3002.
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.
doi:10.1128/JVI.03017-12
PMCID: PMC3592113  PMID: 23269804
16.  Tyrosine crosslinking reveals interfacial dynamics in adeno-associated viral capsids during infection 
ACS Chemical Biology  2012;7(6):1059-1066.
Viral capsid dynamics are often observed during infectious events such as cell surface attachment, entry and genome release. Structural analysis of adeno-associated virus (AAV), a helper-dependent parvovirus, revealed a cluster of surface-exposed tyrosine residues at the icosahedral two-fold symmetry axis. We exploited the latter observation to carry out selective oxidation of Tyr residues, which yielded crosslinked viral protein (VP) subunit dimers, effectively “stitching” together the AAV capsid two-fold interface. Characterization of different Tyr-to-Phe mutants confirmed that the formation of crosslinked VP dimers is mediated by dityrosine adducts and requires the Tyr704 residue, which crosses over from one neighboring VP subunit to the other. When compared to unmodified capsids, Tyr-crosslinked AAV displayed decreased transduction efficiency in cell culture. Surprisingly, further biochemical and quantitative microscopy studies revealed that restraining the two-fold interface hinders externalization of buried VP N-termini, which contain a phospholipase A2 domain and nuclear localization sequences critical for infection. These adverse effects caused by tyrosine oxidation support the notion that interfacial dynamics at the AAV capsid two-fold symmetry axis play a role in externalization of VP N-termini during infection.
doi:10.1021/cb3000265
PMCID: PMC3376196  PMID: 22458529
17.  Glycan Binding Avidity Determines the Systemic Fate of Adeno-Associated Virus Type 9 
Journal of Virology  2012;86(19):10408-10417.
Glycans are key determinants of host range and transmissibility in several pathogens. In the case of adeno-associated viruses (AAV), different carbohydrates serve as cellular receptors in vitro; however, their contributions in vivo are less clear. A particularly interesting example is adeno-associated virus serotype 9 (AAV9), which displays systemic tropism in mice despite low endogenous levels of its primary receptor (galactose) in murine tissues. To understand this further, we studied the effect of modulating glycan binding avidity on the systemic fate of AAV9 in mice. Intravenous administration of recombinant sialidase increased tissue levels of terminally galactosylated glycans in several murine tissues. These conditions altered the systemic tropism of AAV9 into a hepatotropic phenotype, characterized by markedly increased sequestration within the liver sinusoidal endothelium and Kupffer cells. In contrast, an AAV9 mutant with decreased glycan binding avidity displayed a liver-detargeted phenotype. Altering glycan binding avidity also profoundly affected AAV9 persistence in blood circulation. Our results support the notion that high glycan receptor binding avidity appears to impart increased liver tropism, while decreased avidity favors systemic spread of AAV vectors. These findings may not only help predict species-specific differences in tropism for AAV9 on the basis of tissue glycosylation profiles, but also provide a general approach to tailor AAV vectors for systemic or hepatic gene transfer by reengineering capsid-glycan interactions.
doi:10.1128/JVI.01155-12
PMCID: PMC3457279  PMID: 22787229
18.  Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles 
Journal of Virology  2012;86(15):7752-7759.
Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration.
doi:10.1128/JVI.00675-12
PMCID: PMC3421647  PMID: 22593151
19.  Human Galectin 3 Binding Protein Interacts with Recombinant Adeno-Associated Virus Type 6 
Journal of Virology  2012;86(12):6620-6631.
Recombinant adeno-associated viruses (rAAVs) hold enormous potential for human gene therapy. Despite the well-established safety and efficacy of rAAVs for in vivo gene transfer, there is still little information concerning the fate of vectors in blood following systemic delivery. We screened for serum proteins interacting with different AAV serotypes in humans, macaques, dogs, and mice. We report that serotypes rAAV-1, -5, and -6 but not serotypes rAAV-2, -7, -8, -9, and -10 interact in human sera with galectin 3 binding protein (hu-G3BP), a soluble scavenger receptor. Among the three serotypes, rAAV-6 has the most important capacities for binding to G3BP. rAAV-6 also bound G3BP in dog sera but not in macaque and mouse sera. In mice, rAAV-6 interacted with another protein of the innate immune system, C-reactive protein (CRP). Furthermore, interaction of hu-G3BP with rAAV-6 led to the formation of aggregates and hampered transduction when the two were codelivered into the mouse. Based on these data, we propose that species-specific interactions of AAVs with blood proteins may differentially impact vector distribution and efficacy in different animal models.
doi:10.1128/JVI.00297-12
PMCID: PMC3393578  PMID: 22496229
20.  Production of Recombinant Adeno-Associated Viral Vectors and Use in In Vitro and In Vivo Administration 
Adeno-associated virus is a nonpathogenic human virus that has been developed into a gene-delivery vector due to its high efficiency of infection for many different cell types and its ability to persist and lead to long-term gene expression. This unit describes efficient methods to generate high-titer, research-grade, adenovirus-free recombinant single-stranded and self-complementary adeno-associated virus in various serotypes, along with methods to quantify the viral vectors. Two detailed methods are provided for viral vector delivery into the rodent brain and spinal cord, and for histological detection of transgene expression of GFP.
doi:10.1002/0471142301.ns0417s57
PMCID: PMC3209619  PMID: 21971848
Gene Therapy; Viral Vectors; Adeno Associated Virus; Purification; Serotypes; Transfection; Brain; Spinal Cord; Transduction; Gene Delivery
21.  Glycated AAV Vectors: Chemical Redirection of Viral Tissue Tropism 
Bioconjugate chemistry  2011;22(4):529-532.
A chemical approach for selective masking of arginine residues on viral capsids, featuring an exogenous glycation reaction has been developed. Reaction of adeno-associated viral (AAV) capsids with the α-dicarbonyl compound, methylglyoxal resulted in formation of arginine adducts. Specifically, surface exposed guanidinium side chains were modified into charge neutral hydroimidazolones, thereby disrupting a continuous cluster of basic amino acid residues implicated in heparan sulfate binding. Consequent loss in heparin binding ability and decrease in infectivity were observed. Strikingly, glycated AAV retained ability to infect neurons in the mouse brain and were redirected from liver to skeletal and cardiac muscle following systemic administration in mice. Further, glycated AAV displayed altered antigenicity demonstrating potential for evading antibody neutralization. Generation of unnatural amino acid side chains through capsid glycation might serve as an orthogonal strategy to engineer AAV vectors displaying novel tissue tropisms for gene therapy applications.
doi:10.1021/bc100477g
PMCID: PMC3089942  PMID: 21388193
22.  Intra- and Inter-Subunit Disulfide Bond Formation Is Nonessential in Adeno-Associated Viral Capsids 
PLoS ONE  2012;7(2):e32163.
The capsid proteins of adeno-associated viruses (AAV) have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s) of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.
doi:10.1371/journal.pone.0032163
PMCID: PMC3289628  PMID: 22389684
23.  Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors 
Gene therapy of musculoskeletal disorders warrants efficient gene transfer to a wide range of muscle groups. Reengineered adeno-associated viral (AAV) vectors that selectively transduce muscle tissue following systemic administration are attractive candidates for such applications. Here we provide examples of several lab-derived AAV vectors that display systemic tissue tropism in mice. Methods to evaluate the efficiency of gene transfer to skeletal muscle following intravenous or isolated limb infusion of AAV vectors in mice are discussed in detail.
doi:10.1007/978-1-61737-982-6_9
PMCID: PMC3253371  PMID: 21194026
AAV; Reengineering; Capsid; Tropism; Muscle; Gene transfer; Isolated limb infusion; Gene therapy
24.  Bocavirus Episome in Infected Human Tissue Contains Non-Identical Termini 
PLoS ONE  2011;6(6):e21362.
Human bocaviruses (HBoV) are highly prevalent human infections whose pathogenic potential remains unknown. Recent identification of the first non-human primate bocavirus [1] in captive gorillas raised the possibility of the persistent nature of bocavirus infection. To characterize bocavirus infection in humans, we tested intestinal biopsies from 22 children with gastrointestinal disease for the presence of HBoV DNA. Four HBoV-positive tissue samples were analyzed to determine whether viral DNA was present in the linear genomic, the episomal closed circular or the host genome-integrated form. Whereas one tissue sample positive for HBoV3 contained the episomal form (HBoV3-E1), none had the genome-integrated form. The complete genome sequence of HBoV3-E1 contains 5319 nucleotides of which 513 represent the non-coding terminal sequence. The secondary structure of HBoV3-E1 termini suggests several conserved and variable features among human and animal bocaviruses. Our observation that HBoV genome exists as head-to-tail monomer in infected tissue either reflects the likely evolution of alternative replication mechanism in primate bocaviruses or a mechanism of viral persistence in their host. Moreover, we identified the HBoV genomic terminal sequences that will be helpful in developing reverse genetic systems for these widely prevalent parvoviruses.
Significance
HBoV have been found in healthy human controls as well as individuals with respiratory or gastrointestinal disease. Our findings suggest that HBoV DNA can exist as episomes in infected human tissues and therefore can likely establish persistent infection in the host. Previous efforts to grow HBoV in cell culture and to develop reverse genetic systems have been unsuccessful. Complete genomic sequence of the HBoV3 episome and its genomic termini will improve our understanding of HBoV replication mechanism and its pathogenesis.
doi:10.1371/journal.pone.0021362
PMCID: PMC3125170  PMID: 21738642
25.  Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle 
Nature biotechnology  2009;28(1):79-82.
Reengineering the receptor footprints of adeno-associated virus (AAV) isolates may yield variants with improved properties for clinical applications. We generated a panel of synthetic AAV2 vectors by replacing a hexapeptide sequence in a previously identified heparan sulfate receptor footprint with corresponding residues from other AAV strains. This approach yielded several chimeric capsids displaying systemic tropism after intravenous administration in mice. Of particular interest, an AAV2/AAV8 chimera designated AAV2i8 displayed an altered antigenic profile, readily traversed the blood vasculature, and selectively transduced cardiac and whole-body skeletal muscle tissues with high efficiency. Unlike other AAV serotypes, which are preferentially sequestered in the liver, AAV2i8 showed markedly reduced hepatic tropism. These features of AAV2i8 suggest that it is well suited to translational studies in gene therapy of musculoskeletal disorders.
doi:10.1038/nbt.1599
PMCID: PMC2912150  PMID: 20037580

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