Adeno-associated Virus Particles Research Articles

SLMAP-3 is downregulated in human dilated ventricles and its overexpression promotes cardiomyocyte response to adrenergic stimuli by increasing intracellular calcium.

Structural dilation of cardiomyocytes (CMs) imposes a decline in cardiac performance that precipitates cardiac failure and sudden death. Since membrane proteins are implicated in dilated cardiomyopathy and heart failure, we evaluated the expression of the sarcolemmal membrane-associated protein (SLMAP) in dilated cardiomyopathy and its effect on CM contraction. We found that all 3 SLMAP isoforms (SLMAP-1, -2, and -3) are expressed in CMs and are downregulated in human dilated ventricles. Knockdown of SLMAPs in cultured CMs transduced with recombinant adeno-associated viral particles releasing SLMAP-shRNA precipitated reduced spontaneous contractile rate that was not fully recovered in SLMAP-depleted CMs challenged with isoproterenol (ISO), thus phenotypically mimicking heart failure performance. Interestingly, the overexpression of the SLMAP-3 full-length isoform induced a positive chronotropic effect in CMs that was more pronounced in response to ISO insult (vs. ISO-treated naïve CMs). Confocal live imaging showed that H9c2 cardiac myoblasts overexpressing SLMAP-3 exhibit a higher intracellular calcium transient peak when treated with ISO (vs. ISO-treated cells carrying a control adeno-associated viral particle). Proteomics revealed that SLMAP-3 interacts with the regulator of CM contraction, striatin. Collectively, our data demonstrate that SLMAP-3 is a novel regulator of CM contraction rate and their response to adrenergic stimuli. Loss of SLMAPs phenotypically mimics cardiac failure and crystallizes SLMAPs as predictive of dilated cardiomyopathy and heart failure.

Multimodal Production of Adeno-Associated Virus.

Adeno-associated virus (AAV) is an increasingly popular tool in the research laboratory, and use of this viral vector clinically is occurring at an accelerated pace. Nevertheless, despite its popularity, AAV is a relatively cumbersome virus to produce; however, significant efforts have been invested to develop, optimize, and simplify methodology that allows the generation of high-quality AAV with significantly increased production yields. Here we describe multiple modalities for production and purification of AAV particles produced in HEK293 cell cultures using an iodixanol density gradient. We include two methods adapted for harvesting virus from the culture media: tangential flow filtration (TFF) and polyethylene glycol precipitation (PEGylation). Moreover, we also describe the protocol for anion exchange chromatography, which can be used after the iodixanol gradient as an additional purification step. Last, we provide various protocols for determining virus titer.

A Library-Based Screening Strategy for the Identification of DARPins as Ligands for Receptor-Targeted AAV and Lentiviral Vectors

Delivering genes selectively to the therapeutically relevant cell type is among the prime goals of vector development. Here, we present a high-throughput selection and screening process that identifies designed ankyrin repeat proteins (DARPins) optimally suited for receptor-targeted gene delivery using adeno-associated viral (AAV) and lentiviral (LV) vectors. In particular, the process includes expression, purification, and in situ biotinylation of the extracellular domains of target receptors as Fc fusion proteins in mammalian cells and the selection of high-affinity binders by ribosome display from DARPin libraries each covering more than 1012 variants. This way, DARPins specific for the glutamate receptor subunit GluA4, the endothelial surface marker CD105, and the natural killer cell marker NKp46 were generated. The identification of DARPins best suited for gene delivery was achieved by screening small-scale vector productions. Both LV and AAV particles displaying the selected DARPins transduced only cells expressing the corresponding target receptor. The data confirm that a straightforward process for the generation of receptor-targeted viral vectors has been established. Moreover, biochemical analysis of a panel of DARPins revealed that their functional cell-surface expression as fusion proteins is more relevant for efficient gene delivery by LV particles than functional binding affinity.

Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation

Adeno-associated virus (AAV) is one of the most promising gene therapy vectors and is widely used as a gene delivery vehicle for basic research. As AAV continues to become the vector of choice, it is increasingly important for new researchers to have access to a simplified production and purification protocol for laboratory grade recombinant AAV. Here we report a detailed protocol for serotype independent production of AAV using a helper-free HEK293 cell system followed by iodixanol gradient purification, a method described earlier.1 While the core principals of this mammalian AAV production system are unchanged, there have been significant advancements in the production and purification procedure that serve to boost yield, maximize efficiency, and increase the purity of AAV preps. Using this protocol, we are able to constantly obtain high quantities of laboratory grade AAV particles (>5 × 1012 vg) in a week’s time, largely independent of serotype.

Near‐Infrared Light‐Controlled Gene Expression and Protein Targeting in Neurons and Non‐neuronal Cells

Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner, QPAS1, is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested the functionality of two BphP1-QPAS1-based optogenetic tools-an NIR- and blue-light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA)-in several cell types, including cortical neurons. We found that the performance of these optogenetic tools often relied on physiological properties of a specific cell type, such as nuclear transport, which could limit the applicability of the blue-light-sensitive component of iRIS. In contrast, the NIR-light-sensing component of iRIS performed well in all tested cell types. The TA system showed the best performance in cervical cancer (HeLa), bone cancer (U-2 OS), and human embryonic kidney (HEK-293) cells. The small size of the QPAS1 component allowed the design of adeno-associated virus (AAV) particles, which were applied to deliver the TA system to neurons.

Anti‐Methamphetamine Antibody Gene Therapy Protects Against Some METH‐Induced Inflammation

Methamphetamine (METH) is a highly abused, addictive drug that can adversely affect the peripheral immune response, causing immune dysregulation in the user. This can lead to an overproduction of cytokines, chemokines, and cellular adhesion molecules causing potential physiologic damage. Thus novel, sustained treatments that focus on mitigating these effects should be evaluated.Our laboratory is combining an anti‐METH single chain variable antibody‐fusion fragment (7F9‐Fc) with adeno‐associated virus (AAV) particles as a potential gene therapy. AAV‐7F9‐Fc works as a pharmacokinetic antagonist to slow and reduce entry of METH into the brain and can achieve lasting serum concentrations in mice (>7 months) from a single treatment. Thus, this therapy has the clinical potential to attenuate the effects of periodic METH relapse during rehabilitation and possibly decrease associated physiological complications such as inflammation.We hypothesized that an AAV‐delivered anti‐METH antibody fragment (AAV‐7F9‐Fc) will provide protection from METH‐induced inflammation. To test this hypothesis, we established a mouse model of METH‐induced early withdrawal inflammation in our laboratory. Male BALB/c mice were divided into six groups (n=5/group); 1) Saline + Saline, 2) Saline + METH, 3) LPS, 4) AAV‐7F9‐Fc + Saline, 5) AAV‐7F9‐Fc + METH, 6) AAV‐empty vector + saline. METH administration followed a dosing regimen of 1 mg/kg i.p. for seven consecutive days with sacrifice after 72 hrs. We performed a MSD Proinflammatory Panel multiplex analysis to measure important serum cytokine and chemokine levels (e.g., IL‐10, IL‐6, IL‐1®). In brain tissues, we measured markers of glial cell activation, glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule (Iba1) and proinflammatory cytokines via qPCR.Our results indicate that this dosing regimen lead to a significant (p < 0.05) increase in proinflammatory mediators (e.g., IL‐1® and IL‐6) in the striatum of Saline + METH mice, but not AAV‐7F9‐Fc + METH mice. In the hippocampus and frontal cortex, the Saline + METH mice showed a significant increase in the mRNA transcripts of IL‐6 & IL‐1® vs those mice treated with AAV‐7F9‐Fc + METH. Noticeably, GFAP was also increased in all observed brain regions of Saline + METH vs those treated with AAV‐7F9‐Fc + METH. Our results also indicate that this dosing regimen lead to a significant increase in serum proinflammatory cytokines (e.g., IL‐1® and IL‐6) vs the AAV‐7F9‐Fc + METH mice. Taken together, these data indicate that a chronic, low dose METH regimen can induce proinflammatory cytokine elevations in serum and brain, increase activation of glial cell markers in brain tissue, and that AAV‐7F9‐Fc treatment has the potential to mitigate some of these proinflammatory effects.Support or Funding InformationFunded by NIH R01‐DA036600, R01 DA036600‐03S1, T32‐GM106999, and R25GM083297This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oligonucleotide conjugated multi-functional adeno-associated viruses

Recombinant adeno-associated viruses (AAVs) are among the most commonly used vehicles for in vivo gene delivery. However, their tropism is limited, and additionally their efficacy can be negatively affected by prevalence of neutralizing antibodies in sera. Methodologies to systematically engineer AAV capsid properties would thus be of great relevance. In this regard, we develop here multi-functional AAVs by engineering precision tethering of oligonucleotides onto the AAV surface, and thereby enabling a spectrum of nucleic-acid programmable functionalities. Towards this, we engineered genetically encoded incorporation of unnatural amino acids (UAA) bearing bio-orthogonal chemical handles onto capsid proteins. Via these we enabled site-specific coupling of oligonucleotides onto the AAV capsid surface using facile click chemistry. The resulting oligo-AAVs could be sequence specifically labeled, and also patterned in 2D using DNA array substrates. Additionally, we utilized these oligo conjugations to engineer viral shielding by lipid-based cloaks that efficaciously protected the AAV particles from neutralizing serum. We confirmed these ‘cloaked AAVs’ retained full functionality via their ability to transduce a range of cell types, and also enable robust delivery of CRISPR-Cas9 effectors. Taken together, we anticipate this programmable oligo-AAV system will have broad utility in synthetic biology and AAV engineering applications.

Influence of Pre-existing Anti-capsid Neutralizing and Binding Antibodies on AAV Vector Transduction

Pre-existing immunity to adeno-associated virus (AAV) is highly prevalent in humans and can profoundly impact transduction efficiency. Despite the relevance to AAV-mediated gene transfer, relatively little is known about the fate of AAV vectors in the presence of neutralizing antibodies (NAbs). Similarly, the effect of binding antibodies (BAbs), with no detectable neutralizing activity, on AAV transduction is ill defined. Here, we delivered AAV8 vectors to mice carrying NAbs and demonstrated that AAV particles are taken up by both liver parenchymal and non-parenchymal cells; viral particles are then rapidly cleared, without resulting in transgene expression. In vitro, imaging of hepatocytes exposed to AAV vectors pre-incubated with either NAbs or BAbs revealed that virus is taken up by cells in both cases. Whereas no successful transduction was observed when AAV was pre-incubated with NAbs, an increased capsid internalization and transgene expression was observed in the presence of BAbs. Accordingly, AAV8 vectors administered to mice passively immunized with anti-AAV8 BAbs showed a more efficient liver transduction and a unique vector biodistribution profile compared to mice immunized with NAbs. These results highlight a virtually opposite effect of neutralizing and binding antibodies on AAV vectors transduction.

Gene Therapy Using a miniCEP290 Fragment Delays Photoreceptor Degeneration in a Mouse Model of Leber Congenital Amaurosis.

Mutations in the cilia-centrosomal protein CEP290 are frequently observed in autosomal recessive childhood blindness disorder Leber congenital amaurosis (LCA). No treatment or cure currently exists for this disorder. The Cep290rd16 (retinal degeneration 16) mouse (a model of LCA) carries a mutation in the Cep290 gene. This mutation leads to shorter cilia formation and defective photoreceptor structure and function. A roadblock to developing a gene replacement strategy for CEP290 using conventional adeno-associated virus (AAV) vectors is its large size. The identification and characterization is reported of a miniCEP290 gene that is amenable to AAV2/8-mediated delivery and delaying retinal degeneration in the Cep290rd16 mice. Using the ability of Cep290rd16 mouse embryonic fibroblasts to from shorter cilia as a platform, a human CEP290 domain encoded by amino acids 580–1180 (miniCEP290580-1180) was identified that can recover the cilia length in vitro. Furthermore, subretinal injection of AAV particles carrying the cDNA expressing miniCEP290580-1180 into neonatal Cep290rd16 mice resulted in significantly improved photoreceptor survival, morphology, and function compared to control injected mice. These studies show the potential of using a truncated CEP290 to treat this fast progressing and devastating disease.

One-Step Fabrication of Bone Morphogenetic Protein-2 Gene-Activated Porous Poly-L-Lactide Scaffold for Bone Induction

Bone morphogenetic protein 2 (BMP2) is an efficacious inducer for the osteogenesis of mesenchymal stem cells (MSCs). Conventional applications of BMP2 have involved either the direct incorporation of BMP2 protein or ex vivo BMP2 gene transfer into stem cells prior to their transplantation. These approaches are able to promote bone formation to some extent; however, they are hampered by either the lack of stability and sustainability of BMP2 protein or the time-consuming and cost-prohibitive in vitro cell culture procedure. To overcome these limitations, we have developed a gene-activated poly-L-lactide acid (PLLA) scaffold with the encapsulation of recombinant adeno-associated viral (AAV) vector encoding a full-length cDNA of human BMP2 using an ice-based microparticle porogenization method that was recently developed. Results showed continuous release of AAV particles from the micropores of scaffolds for up to 1 week, subsequently transducing embedded human MSCs and producing functional BMP2. MSCs within scaffolds underwent efficacious osteogenesis, on the basis of osteoinductive gene expression and osteogenic differentiation, which resulted in robust new bone formation in vivo at 4 weeks. These findings show the potential of the technology toward developing clinical applications of a rapid, cost-effective, and potentially point-of-care approach for the repair of bone defects.

Adeno-Associated Virus (AAV) as a Vector for Gene Therapy.

There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies. The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations

Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

Sustained viral gene delivery from a micro-fibrous, elastomeric cardiac patch to the ischemic rat heart.

Biodegradable and elastomeric patches have been applied to the surface of infarcted hearts as temporary mechanical supports to effectively alter adverse left ventricular remodeling processes. In this report, recombinant adeno-associated virus (AAV), known for its persistent transgene expression and low pathogenicity, was incorporated into elastomeric polyester urethane urea (PEUU) and polyester ether urethane urea (PEEUU) and processed by electrospinning into two formats (solid fibers and core-sheath fibers) designed to influence the controlled release behavior. The extended release of AAV encoding green fluorescent protein (GFP) was assessed invitro. Sustained and localized viral particle delivery was achieved over 2 months invitro. The biodegradable cardiac patches with or without AAV-GFP were implanted over rat left ventricular lesions three days following myocardial infarction to evaluate the transduction effect of released viral vectors. AAV particles were directly injected into the infarcted hearts as a control. Cardiac function and remodeling were significantly improved for 12 weeks after patch implantation compared to AAV injection. More GFP genes was expressed in the AAV patch group than AAV injection group, with both α-SMA positive cells and cardiac troponin T positive cells transduced in the patch group. Overall, the extended release behavior, prolonged transgene expression, and elastomeric mechanical properties make the AAV-loaded scaffold an attractive option for cardiac tissue engineering where both gene delivery and appropriate mechanical support are desired.

Viral delivered gene therapy to treat catecholaminergic polymorphic ventricular tachycardia (CPVT2) in mouse models

The recessive form of catecholaminergic polymorphic ventricular tachycardia 2 (CPVT2) is caused by mutations in cardiac calsequestrin (CASQ2), leading to protein deficiency. The aims of this study were to develop a viral-delivered gene therapy for CPVT2 and to determine the relationship between CASQ2 expression and antiarrhythmic efficacy in a murine model. We used a murine model of CPVT2 caused by the D307H human mutation (CASQ2D307H) or CASQ2 knockout (CASQ2Δ/Δ). Adeno-associated virus (AAV) particles containing the CASQ2 gene (AAVCASQ2) were injected into the heart or intraperitoneally to 12-week-old mice. A telemetry device was implanted, and mice underwent provocation testing 7-8 weeks after gene therapy. CASQ2Δ/Δ mice injected intracardiacally with AAVCASQ2 expressed 40% ± 25% of the normal CASQ2 protein level, which was increased compared to untreated CASQ2Δ/Δ mice (n = 10; P< .05). Intraperitoneal therapy led to a significantly elevated expression of the CASQ2 protein, which was comparable in CASQ2D307H (n = 12) and CASQ2Δ/Δ (n = 4) mice. All control mice with CPVT2 had nonsustained ventricular tachycardia (VT) and 8 of 13 had sustained VT on provocation. Expressing ≥33% of the normal CASQ2 level was needed to protect from nonsustained VT as well as stress-induced premature ventricular contractions. Lower levels of expression prevented sustained VT in AAVCASQ2-treated mice (0 of 26; P < .001 vs controls). AAVCASQ2 displays a long-lasting capacity to attenuate and potentially cure CPVT2. Systemic delivery is feasible and convenient, reproducibly providing adequate levels of transgene expression. Antiarrhythmic efficacy depends on the CASQ2 level: ≥33% of the normal CASQ2 level is needed to prevent arrhythmia. However, even lower levels of protein protect from sustained VT, thereby potentially reducing the risk of sudden death.

Striatal activation by optogenetics induces dyskinesias in the 6-hydroxydopamine rat model of Parkinson disease.

Long-term levodopa (l-dopa) treatment is associated with the development of l-dopa-induced dyskinesias in the majority of patients with Parkinson disease (PD). The etiopathogonesis and mechanisms underlying l-dopa-induced dyskinesias are not well understood. We used striatal optogenetic stimulation to induce dyskinesias in a hemiparkinsonian model of PD in rats. Striatal dopamine depletion was induced unilaterally by 6-hydroxydopamine injection into the medial forebrain bundle. For the optogenetic manipulation, we injected adeno-associated virus particles expressing channelrhodopsin to stimulate striatal medium spiny neurons with a laser source. Simultaneous optical activation of medium spiny neurons of the direct and indirect striatal pathways in the 6-hydroxydopamine lesion but l-dopa naïve rats induced involuntary movements similar to l-dopa-induced dyskinesias, labeled here as optodyskinesias. Noticeably, optodyskinesias were facilitated by l-dopa in animals that did not respond initially to the laser stimulation. In general, optodyskinesias lasted while the laser stimulus was applied, but in some instances remained ongoing for a few seconds after the laser was off. Postmortem tissue analysis revealed increased FosB expression, a molecular marker of l-dopa-induced dyskinesias, primarily in medium spiny neurons of the direct pathway in the dopamine-depleted hemisphere. Selective optogenetic activation of the dorsolateral striatum elicits dyskinesias in the 6-hydroxydopamine rat model of PD. This effect was associated with a preferential activation of the direct striato-nigral pathway. These results potentially open new avenues in the understanding of mechanisms involved in l-dopa-induced dyskinesias. © 2017 International Parkinson and Movement Disorder Society.

Probing the Link among Genomic Cargo, Contact Mechanics, and Nanoindentation in Recombinant Adeno-Associated Virus 2.

Recombinant adeno-associated virus (AAV) is a promising gene therapy vector. To make progress in this direction, the relationship between the characteristics of the genomic cargo and the capsid stability must be understood in detail. The goal of this study is to determine the role of the packaged vector genome in the response of AAV particles to mechanical compression and adhesion to a substrate. Specifically, we used atomic force microscopy to compare the mechanical properties of empty AAV serotype 2 (AAV2) capsids and AAV2 vectors packaging single-stranded DNA or self-complementary DNA. We found that all species underwent partial deformation upon adsorption from buffer on an atomically flat graphite surface. Upon adsorption, a preferred orientation toward the twofold symmetry axis on the capsid, relative to the substrate, was observed. The magnitude of the bias depended on the cargo type, indicating that the interfacial properties may be influenced by cargo. All particles showed a significant relative strain before rupture. Different from interfacial interactions, which were clearly cargo-dependent, the elastic response to directional stress was largely dominated by the capsid properties. Nevertheless, small differences between particles laden with different cargo were measurable; scAAV vectors were the most resilient to external compression. We also show how elastic constant and rupture force data sets can be analyzed according a multivariate conditional probability approach to determine the genome content on the basis of a database of mechanical properties acquired from nanoindentation assays. Implications for understanding how recombinant AAV capsid-genome interactions can affect vector stability and effectiveness of gene therapy applications are discussed.

Regulation of Synaptic Amyloid-β Generation through BACE1 Retrograde Transport in a Mouse Model of Alzheimer's Disease.

Amyloid-β (Aβ) peptides play a key role in synaptic damage and memory deficits in the early pathogenesis of Alzheimer's disease (AD). Abnormal accumulation of Aβ at nerve terminals leads to synaptic pathology and ultimately to neurodegeneration. β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the major neuronal β-secretase for Aβ generation. However, the mechanisms regulating BACE1 distribution in axons and β cleavage of APP at synapses remain largely unknown. Here, we reveal that dynein-Snapin-mediated retrograde transport regulates BACE1 trafficking in axons and APP processing at presynaptic terminals. BACE1 is predominantly accumulated within late endosomes at the synapses of AD-related mutant human APP (hAPP) transgenic (Tg) mice and patient brains. Defective retrograde transport by genetic ablation of snapin in mice recapitulates late endocytic retention of BACE1 and increased APP processing at presynaptic sites. Conversely, overexpressing Snapin facilitates BACE1 trafficking and reduces synaptic BACE1 accumulation by enhancing the removal of BACE1 from distal AD axons and presynaptic terminals. Moreover, elevated Snapin expression via stereotactic hippocampal injections of adeno-associated virus particles in mutant hAPP Tg mouse brains decreases synaptic Aβ levels and ameliorates synapse loss, thus rescuing cognitive impairments associated with hAPP mice. Altogether, our study provides new mechanistic insights into the complex regulation of BACE1 trafficking and presynaptic localization through Snapin-mediated dynein-driven retrograde axonal transport, thereby suggesting a potential approach of modulating Aβ levels and attenuating synaptic deficits in AD.SIGNIFICANCE STATEMENT β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) trafficking and synaptic localization significantly influence its β secretase activity and amyloid-β (Aβ) production. In AD brains, BACE1 is accumulated within dystrophic neurites, which is thought to augment Aβ-induced synaptotoxicity by Aβ overproduction. However, it remains largely unknown whether axonal transport regulates synaptic APP processing. Here, we demonstrate that Snapin-mediated retrograde transport plays a critical role in removing BACE1 from presynaptic terminals toward the soma, thus reducing synaptic Aβ production. Adeno-associated virus-mediated Snapin overexpression in the hippocampus of mutant hAPP mice significantly decreases synaptic Aβ levels, attenuates synapse loss, and thus rescues cognitive deficits. Our study uncovers a new pathway that controls synaptic APP processing by enhancing axonal BACE1 trafficking, thereby advancing our fundamental knowledge critical for ameliorating Aβ-linked synaptic pathology.

Automated Glycan Assembly of Oligo-N-Acetyllactosamine and Keratan Sulfate Probes to Study Virus-Glycan Interactions

Summary Oligo- N -acetyllactosamine (LacNAc) and keratan sulfate (KS) glycans exert crucial functions in disease-relevant processes, including cancer formation, inflammation, and viral infection. To facilitate structure-activity studies with these glycans, we established a universal strategy to synthesize linear and branched LacNAc as well as differentially sulfated KS oligosaccharides by automated glycan assembly. We synthesized oligosaccharides as long as hexamers by combining four monosaccharide building blocks. Key to the strategy was installing three orthogonal protection groups, 9-fluorenylmethoxycarbonyl (Fmoc), levulinoyl (Lev) ester, and 2-naphthylmethyl (Nap) ether, which were selectively removed from a common oligosaccharide precursor for differential sulfation. Microarrays presenting the synthetic oligosaccharides revealed a specific interaction between a disulfated KS tetrasaccharide and the adeno-associated virus AAVrh10 gene-therapy vector, which was further corroborated by surface plasmon resonance studies. Thus, KS represents a novel receptor candidate for AAVrh10. These insights could have implications for cell-type-specific gene-delivery approaches.

Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors

Adeno-associated virus (AAV) vectors have emerged as a safe and efficient gene therapy platform. One complication is that a significant amount of empty particles have always been generated as impurities during AAV vector production. However, the effects of such particles on AAV vector performance remain unclear. Here we systemically evaluated the biological properties of three types of “empty” AAV particles: syngeneic pseudo-vectors with partial AAV genomes derived from DNA of the corresponding full particles, allogeneic pseudo-vectors with partial genomes different from the corresponding full particles, and null pseudo-vectors with no DNA inside the capsids. The syngeneic particles in excess increased the corresponding full AAV vector transgene expression both in vivo and in vitro. However, such effects were not observed with null or allogeneic particles. The observed differences among these pseudo-AAV particles may be ascribed to the syngeneic pseudo-vector DNA facilitating the complementary DNA synthesis of the corresponding full AAV particles. Our study suggests that the DNA content in the pseudo-vectors plays a key role in dictating their effects on AAV transduction. The effects of residual “empty” particles should be adequately assessed when comparing AAV vector performance. The syngeneic AAV pseudo-vectors may be used to enhance the efficacy of gene therapy.

293. Discovery of an Essential Receptor for Adeno-Associated Virus Infection

Cellular entry of adeno-associated viruses (AAV) is poorly understood, despite the prominent use of AAV vectors in gene therapy for several monogenic diseases. Using an unbiased, haploid genetic screen, we identified critical players in AAV serotype 2 (AAV2) entry including members of distinct protein complexes involved in retrograde trafficking as well as genes involved in the biosynthesis of the AAV2 attachment factor, heparan sulphate. We focused on the single most significantly enriched gene of the screen, an uncharacterized type-I transmembrane protein. Based on the evidence below we renamed this gene AAV receptor (AAVR). We discovered AAVR as capable of rapidly endocytosing from the plasma membrane and trafficking to the trans-Golgi network, taking a similar path as AAV particles utilize. Genetic ablation of AAVR using CRISPR/Cas9 technology demonstrated a robust resistance to AAV2 infection in a wide range of mammalian cell types, which could be reversed upon AAVR complementation. This confirmed the essentiality of AAVR in AAV2 infection. Further investigation revealed that AAVR was also required for the infections of all tested human and simian-derived AAV serotypes including AAV1, 3B, 5, 6, 8 and 9. Deeper characterization of AAVR showed it to contain Ig-like domains, which are commonly found in many virus receptors including those for poliovirus and measles. We observed that these domains are capable of binding to AAV2 particles, and anti-AAVR antibodies efficiently block AAV2 infection. Moreover, the importance of AAVR for AAV infection in vivo was demonstrated by the strong resistance of AAVR−/− mice to AAV9 infection, recapitulating what we showed in vitro. Collectively, the data indicates that AAVR is a universal receptor involved in AAV infection. This has significant implications for the improvement of future AAV vector design.