Cytoplasmic Trafficking, Endosomal Escape, and Perinuclear Accumulation of Adeno-Associated Virus Type 2 Particles Are Facilitated by Microtubule Network

Understanding adeno-associated virus (AAV) trafficking is critical to advance our knowledge of AAV biology and exploit novel aspects of vector development. Similar to the case for most DNA viruses, after receptor binding and entry, AAV traverses the cytoplasm and deposits the viral genome in the cell nucleus. In this study, we examined the role of the microtubule (MT) network in productive AAV infection. Using pharmacological reagents (e.g., nocodazole), live-cell imaging, and flow cytometry analysis, we demonstrated that AAV type 2 (AAV2) transduction was reduced by at least 2-fold in the absence of the MT network. Cell surface attachment and viral internalization were not dependent on an intact MT network. In treated cells at 2 h postinfection, quantitative three-dimensional (3D) microscopy determined a reproducible difference in number of intracellular particles associated with the nuclear membrane or the nucleus compared to that for controls (6 to 7% versus 26 to 30%, respectively). Confocal microscopy analysis demonstrated a direct association of virions with MTs, further supporting a critical role in AAV infection. To investigate the underling mechanisms, we employed single-particle tracking (SPT) to monitor the viral movement in real time. Surprisingly, unlike other DNA viruses (e.g., adenovirus [Ad] and herpes simplex virus [HSV]) that display bidirectional motion on MTs, AAV2 displays only unidirectional movement on MTs toward the nuclei, with peak instantaneous velocities at 1.5 to 3.5 μm/s. This rapid and unidirectional motion on MTs lasts for about 5 to 10 s and results in AAV particles migrating more than 10 μm in the cytoplasm reaching the nucleus very efficiently. Furthermore, electron microscopy analysis determined that, unlike Ad and HSV, AAV2 particles were transported on MTs within membranous compartments, and surprisingly, the acidification of AAV2-containing endosomes was delayed by the disruption of MTs. These findings together suggest an as-yet-undescribed model in which after internalization, AAV2 exploits MTs for rapid cytoplasmic trafficking in endosomal compartments unidirectionally toward the perinuclear region, where most acidification events for viral escape take place

Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus

Recombinant adeno-associated viral (rAAV) vectors have garnered much promise in gene therapy applications. However, widespread clinical use has been limited by transduction efficiency. Previous studies suggested that the majority of rAAV accumulates in the perinuclear region of cells, presumably unable to traffic into the nucleus. rAAV nuclear translocation remains ill-defined; therefore, we performed microscopy, genetic, and biochemical analyses in vitro in order to understand this mechanism. Lectin blockade of the nuclear pore complex (NPC) resulted in inhibition of nuclear rAAV2. Visualization of fluorescently labeled particles revealed that rAAV2 localized to importin-β-dense regions of cells in late trafficking steps. Additionally, small interfering RNA (siRNA) knockdown of importin-β partially inhibited rAAV2 nuclear translocation and inhibited transduction by 50 to 70%. Furthermore, coimmunopreciptation (co-IP) analysis revealed that capsid proteins from rAAV2 could interact with importin-β and that this interaction was sensitive to the small GTPase Ran. More importantly, mutations to key basic regions in the rAAV2 capsid severely inhibited interactions with importin-β. We tested several other serotypes and found that the extent of importin-β interaction varied, suggesting that different serotypes may utilize alternative import proteins for nuclear translocation. Co-IP and siRNA analyses were used to investigate the role of other karyopherins, and the results suggested that rAAV2 may utilize multiple import proteins for nuclear entry. Taken together, our results suggest that rAAV2 interacts with importin-β alone or in complex with other karyopherins and enters the nucleus via the NPC. These results may lend insight into the design of novel AAV vectors that have an enhanced nuclear entry capability and transduction potential

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

ABSTRACT 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. IMPORTANCE Adeno-associated virus (AAV) has become the vector of choice for viral gene transfer and has shown great promise in clinical trials. The need for development of an easy, less invasive injection route for ocular gene therapy is met by intravitreal delivery, but delivery of AAV by this route results in poor transduction outcomes. The inner limiting membrane (ILM) creates a barrier separating the vitreous and the retina. Binding of AAV to heparan sulfate proteoglycan (HSPG) at the ILM may allow the virus to traverse this barrier for better retinal transduction. We show that HSPG binding is correlated with greater accumulation and penetration of AAV in the retina. We demonstrated that this accumulation is conserved across mouse and human retinas and that the addition of HSPG binding to other AAV capsids can increase the number of vectors accumulating at the ILM without dictating tropism

Novel Transcriptional Regulatory Signals in the Adeno-Associated Virus Terminal Repeat A/D Junction Element

Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604–1611, 1998) demonstrated the use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the “off” state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other cis-acting elements (AAV terminal repeats [TR]) were contributing to this activity. In the present study, we identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using deletion analysis and specific PCR-derived TR reporter gene templates, we mapped this activity to a 37-nucleotide stretch in the A/D elements of the TR. Although the mRNA derived from the TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green fluorescent protein expression both in vitro and in vivo in brain by using recombinant virus carrying only the TR element. Since the AAV terminal repeat is a necessary component of all recombinant AAV vectors, this TR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel TR split gene vectors currently being considered for genes too large to be packaged

Prediction of adeno-associated virus neutralizing antibody activity for clinical application

Patients with neutralizing antibodies (Nab) against adeno-associated virus (AAV) are usually excluded from treatment with AAV vectors. To develop a standard assay for detecting Nab inhibition activity, we systematically studied current AAV Nab assays in vitro and in vivo. Several factors were found that influence the Nab titers based on the in vitro assay, including: sera volume, AAV dose/cell, cell number and choice of transgenes. When the Nab titer assay was performed in vivo via intramuscular (IM) or systemic administration, a 4-fold increase in sensitivity for measurement of Nab titers was observed compared to an identical in vitro test. To better mimic the clinical setting, after passively transferring human Nabs into mice, blood was collected before systemic injection of AAV vector and used for Nab titer analysis in vitro or via IM injection. The results showed that AAV delivered via IM injection had a similar inhibition pattern to systemic administration. These studies indicate critical parameters necessary for optimizing Nab sensitivity and that an in vivo Nab assay is more sensitive than an in vitro assay for inclusion/exclusion criteria. The variables identified by this study may explain some of the compounding clinical data seen to date with respect to efficiency of AAV transduction in various Phase I clinical trials

Delivering multiple gene products in the brain from a single adeno-associated virus vector

For certain gene therapy applications, the simultaneous delivery of multiple genes would allow for novel therapies. In the case of adeno-associated virus (AAV) vectors, the limited packaging capacity greatly restricts current methods of carrying multiple transgene cassettes. To address this issue, a recombinant AAV (rAAV) vector was designed such that a furin proteolytic cleavage site (RKRRKR) was placed between the coding sequences of two genes (green fluorescent protein (GFP) and galanin), to allow cleavage of the chimeric protein into two fragments. In addition, these constructs contained the fibronectin secretory signal sequence that causes the gene products to be constitutively secreted from transduced cells. In vitro studies show that after transfection of HEK293 cells, the appropriate cleavage and constitutive secretion occurred regardless of the order of the genes in the transgene cassette. In vivo, infusion of rAAV vectors into the piriform cortex resulted in both GFP expression and significant galanin attenuation of kainic acid-induced seizure activity. Thus, the present results establish the utility of a proteolytic approach for the expression and secretion of multiple gene products from a single AAV vector transgene cassette

Adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 suppresses seizure activity in vivo

Neuropeptide Y (NPY) is a 36-amino-acid peptide that attenuates seizure activity following direct infusion or adeno-associated virus (AAV)-mediated expression in the central nervous system. However, NPY activates all NPY receptor subtypes, potentially causing unwanted side effects. NPY13-36 is a C-terminal peptide fragment of NPY that primarily activates the NPY Y2 receptor, thought to mediate the antiseizure activity. Therefore, we investigated if recombinant adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 could alter limbic seizure sensitivity. Rats received bilateral piriform cortex infusions of AAV vectors that express and constitutively secrete full-length NPY (AAV-FIB-NPY) or NPY13-36 (AAV-FIB-NPY13-36). Control rats received no infusion, as we have previously shown that vectors expressing and secreting reporter genes like GFP (AAV-FIB-EGFP), as well as vectors expressing peptides that lack secretion sequences (AAV-GAL) have no effect on seizures. One week later, all animals received kainic acid (10 mg kg−1, intraperitoneally), and the latencies to wet dog shakes and limbic seizure behaviors were determined. Although both control and vector-treated rats developed wet dog shake behaviors with similar latencies, the latencies to class III and class IV limbic seizures were significantly prolonged in both NPY- and NPY13-36-treated groups. Thus, AAV-mediated expression and constitutive secretion of NPY and NPY13-36 is effective in attenuating limbic seizures, and provides a platform for delivering therapeutic peptide fragments with increased receptor selectivity

Arsenic Trioxide Stabilizes Accumulations of Adeno-Associated Virus Virions at the Perinuclear Region, Increasing Transduction In Vitro and In Vivo

Interactions with cellular stress pathways are central to the life cycle of many latent viruses. Here, we utilize adeno-associated virus (AAV) as a model to study these interactions, as previous studies have demonstrated that cellular stressors frequently increase transduction of recombinant AAV (rAAV) vectors and may even substitute for helper virus functions. Since several chemotherapeutic drugs are known to increase rAAV transduction, we investigated the effect of arsenic trioxide (As2O3), an FDA-approved chemotherapeutic agent with known effects on several other virus life cycles, on the transduction of rAAV. In vitro, As2O3 caused a dose-dependent increase in rAAV2 transduction over a broad range of cell lines from various cell types and species (e.g., HEK-293, HeLa, HFF hTERT, C-12, and Cos-1). Mechanistically, As2O3 treatment acted to prevent loss of virions from the perinuclear region, which correlated with increased cellular vector genome retention, and was distinguishable from proteasome inhibition. To extend our investigation of the cellular mechanism, we inhibited reactive oxygen species formation and determined that the As2O3-mediated increase in rAAV2 transduction was dependent upon production of reactive oxygen species. To further validate our in vitro data, we tested the effect of As2O3 on rAAV transduction in vivo and determined that treatment initiated transgene expression as early as 2 days posttransduction and increased reporter expression by up to 10-fold. Moreover, the transduction of several other serotypes of rAAV was also enhanced in vivo, suggesting that As2O3 affects a pathway used by several AAV serotypes. In summary, our data support a model wherein As2O3 increases rAAV transduction both in vitro and in vivo and maintains perinuclear accumulations of capsids, facilitating productive nuclear trafficking

Bioluminescent virion shells: new tools for quantitation of AAV vector dynamics in cells and live animals

Current technologies for visualizing infectious pathways of viruses rely on fluorescent labeling of capsid proteins by chemical conjugation or genetic manipulation. For non-invasive in vivo imaging of such agents in mammalian tissue, we engineered bioluminescent Gaussia luciferase-tagged Adeno-Associated Viral (gLuc/AAV) vectors. The enzyme was incorporated into recombinant AAV serotype 1, 2, and 8 capsids by fusion to the N-terminus of the VP2 capsid subunit to yield bioluminescent virion shells. The gLuc/AAV vectors were utilized to quantify kinetics of cell surface binding by AAV2 capsids in vitro. Bioluminescent virion shells displayed an exponential decrease in luminescent signal following cellular uptake in vitro. A similar trend was observed following intramuscular injection in vivo, although the rate of decline in bioluminescent signal varied markedly between AAV serotypes. While gLuc/AAV1 and gLuc/AAV8 vectors displayed rapid decrease in bioluminescent signal to background levels within 30 min, the signal from gLuc/AAV2 vectors persisted for over 2 hrs. Bioluminescent virion shells might be particularly useful in quantifying dynamics of viral vector uptake in cells and peripheral tissues in live animals

Viral vectors for gene delivery to the central nervous system

The potential benefits of gene therapy for neurological diseases such as Parkinson’s, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer’s are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features