rAAV Particles Research Articles

Assessment of cell-binding capacity of shed rAAV particles after gene therapy vector administration: implications for environmental risk and hygiene recommendations

ABSTRACT Background Currently, adeno-associated viruses (AAVs) are the most commonly used in vivo gene therapy (GT) vector platform. Risks posed to the environment, including the public, have not been well studied in the past. There is uncertainty concerning the necessary level of biocontainment and appropriate hygiene behavior for the handling of secreta/excreta of GT patients during the shedding phase. Research design and methods Here, feces and urine samples from non-human primates, treated with an AAV9-based vector at 2 × 1013 vector genomes per kilogram body weight (vg/kg), were analyzed for vector presence and subsequently analyzed for their capacity to bind to cells. Results Both sample types contained particles which bound to cells at concentrations in the range of ~104 (and higher) vg/mL of culture medium. Novel control rAAV vector displayed a ~2-3 orders of magnitude higher affinity to cells than shed particles. Conclusions The lower binding capacity of the shed vector particles speaks in favor of a more relaxed containment and hygiene approach in the context of GT. It is recommended that current hygiene and contact-avoidance-based containment measures after GT administration are reduced. The results also support the efforts to achieve a simplification of the regulatory review process of medicinal genetically modified organisms.

Assessing the environmental fate of rAAV in activated sludge and water: Implications for environmental risk assessments and GMO regulatory frameworks

During the past +20 years, recombinant adeno-associated virus (rAAV) vectors have emerged as the primary vehicle of choice for in vivo gene therapy. rAAV vectors are classified as genetically modified organisms (GMOs), therefore specific biosafety laws apply regarding their use. Environmental agencies participating in the review of clinical trial applications involving viral-based gene therapies (eg based on AAV) focus among other phenomena especially on shedding, a mechanism by which rAAV vectors exit a patient's body and enter the natural environment. For example, following patient use, shed viral particles excreted in urine and feces enter the wastewater treatment facility (WWTF) and subsequently may be released into the natural environment through wastewater effluent discharges. Based on basic molecular biology, it is generally assumed by the scientific community that shed rAAV particles will undergo degradation during the wastewater treatment process. However, despite their importance and increase in use during the last few decades, actual data to support our understanding of the environmental fate of shed rAAV vector particles is unavailable. Data to support this assumption would greatly enhance our knowledge and understanding of degradation kinetics associated with rAAV in the environment. Such data would also provide strong scientific support for changes in current legislation regarding the medicinal use of GMOs. Therefore, the goal of this research was to conduct laboratory experiments to assess the actual environmental fate of rAAV virions.In this study the stability of 4 different rAAV vectors (based on wildtype (wt) AAV serotypes 2,3,6,9) was assessed during incubation in activated sludge (containing live microorganisms). This setting corresponds to conditions as encountered in WWTFs, and has been used in order to assess rAAV fate under environmentally relevant conditions, to gain a better understanding of the general environmental risk posed by shed rAAV particles. The amount of detectable virions in the supernatant, as measured by sensitive and specific qPCR, rapidly decreased within hours and continued to decline, reaching the lower limit of quantitation prior to or by study termination on day 7. Furthermore, a half-life of approximately 7 days for rAAV virions was determined under abiotic conditions, during a room temperature incubation experiment of rAAV vectors in water in the absence of any microbiota or sludge.The findings from this study provide the first insight of its kind into the actual environmental fate of shed rAAV particles, and help the community to better understand the potential impact of rAAVs on the environment. It has become evident now that shed particles are not equipped to remain stable and/or soluble once entering a typical WWTF and therefore do not pose a threat to the natural environment. These findings support a data-driven approach towards a simplified, risk-based regulation of medicinal GMOs in the EU and other regions.

MiR133b-mediated inhibition of EGFR-PTK pathway promotes rAAV2 transduction by facilitating intracellular trafficking and augmenting second-strand synthesis.

Recombinant adeno-associated virus (rAAV) is an extremely attractive vector in the in vivo delivery of gene therapy as it is safe and its genome is simple. However, challenges including low permissiveness to specific cells and restricted tissue specificity have hindered its clinical application. Based on the previous studies, epidermal growth factor receptor-protein tyrosine kinase (EGFR-PTK) negatively regulated rAAV transduction, and EGFR-positive cells were hardly permissive to rAAV transduction. We constructed a novel rAAV-miRNA133b vector, which co-expressed miRNA133b and transgene, and investigated its in vivo and in vitro transduction efficiency. Confocal microscopy, live-cell imaging, pharmacological reagents and labelled virion tracking were used to analyse the effect of miRNA133b on rAAV2 transduction and the underlying mechanisms. The results demonstrated that miRNA133b could promote rAAV2 transduction and the effects were limited to EGFR-positive cells. The increased transduction was found to be a direct result of decreased rAAV particles degradation in the cytoplasm and enhanced second-strand synthesis. ss-rAAV2-miRNA133b vector specifically increased rAAV2 transduction in EGFR-positive cells or tissues, while ss-rAAV2-Fluc-miRNA133b exerted an antitumor effect. rAAV-miRNA133b vector might emerge as a promising platform for delivering various transgene to treat EGFR-positive cell-related diseases, such as non-small-cell lung cancer.

Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations

Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles.

An efficient rAAV vector for protein expression in cortical parvalbumin expressing interneurons

Recombinant adeno—associated viruses (rAAV) are extensively used in both research and clinical applications. Despite significant advances, there is a lack of short promoters able to drive the expression of virus delivered genes in specific classes of neurons. We designed an efficient rAAV vector suitable for the rAAV-mediated gene expression in cortical interneurons, mainly in the parvalbumin expressing cells. The vector includes a short parvalbumin promoter and a specialized poly(A) sequence. The degree of conservation of the parvalbumin gene adjoining non-coding regions was used in both the promoter design and the selection of the poly(A) sequence. The specificity was established by co-localizing the fluorescence of the virus delivered eGFP and the antibody for a neuronal marker. rAAV particles were injected in the visual cortex area V1/V2 of adult rats (2–4 months old). Neurons expressing the virus delivered eGFP were mainly positive for interneuronal markers: 66.5 ± 2.8% for parvalbumin, 14.6 ± 2.4% for somatostatin, 7.1 ± 1.2% for vasoactive intestinal peptide, 2.8 ± 0.6% for cholecystokinin. Meanwhile, only 2.1 ± 0.5% were positive for CaMKII, a marker for principal cells in the cortex. The efficiency of the construct was verified by optogenetic experiments: the expression of the virus delivered ChR2 channels was sufficient to evoke by blue light laser high frequency bursts of action potentials in putative fast spiking neurons. We conclude that our promoter allows highly specific expression of the rAAV delivered cDNAs in cortical interneurons with a strong preference for the parvalbumin positive cells.

Adeno-associated viral vector process development challenges in early research& preclinical study

Significant advances have been made in gene therapy using recombinant adeno-associated virus (rAAV) in recent years. Given the interest in and success of rAAV as a novel therapeutic modality, there has been an increase in preclinical proof of concept studies and clinical trials. However, with the momentum and competition in gene therapy, many research groups are coming up with novel AAV variants, new combinations of rAAV constructs, combined with targeted genes of interest to outperform traditional and competitor therapies. Also, screening and generation of rAAV libraries have helped identify novel rAAV serotypes and variants. The preclinical research studies require the production of highly pure rAAV particles with multiple serotypes and different gene of interest, thus creating a major challenge for the production and purification of rAAV. It is worth mentioning that the preclinical phase often overlooks the process development aspect and manufacturability of targeted rAAV and thus undermines the challenges that may arise as the rAAV-based gene therapy program progresses. This article outlines some of the major challenges, quality and quantity requirements, and scalability considerations during process development in the research preclinical phase. The process development and research scale production can be challenging considering volume, serotypes, and ever-changing GOI whilst maintaining the high-speed delivery of vectors. Thus, there is an urgent need to create a platform for the production and purification systems that can generate a high quantity of pure rAAV.

Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors.

Recombinant adeno-associated virus (rAAV) vectors have evolved as one of the most promising technologies for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in nondividing cells. rAAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. There is a high demand for efficient and scalable production and purification methods for rAAVs. This is particularly true for the downstream purification methods. The current standard methods are based on multiple steps of gradient ultracentrifugation, which allow for the purification and enrichment of full rAAV particles, but the scale up of this method is challenging. Here, we explored fast, scalable, and universal liquid chromatography-based strategies for the purification of rAAVs. In contrast to the hydrophobic interaction chromatography (HIC), where a substantial amount of AAV was lost, the cation exchange chromatography (CEX) was performed robustly for multiple tested serotypes and resulted in a mixture of full and empty rAAVs with a good purity profile. For the used affinity chromatography (AC), a serotype dependence was observed. Anion exchange chromatography (AEX) worked well for the AAV8 serotype and achieved high levels of purification and a baseline separation of full and empty rAAVs. Depending on the AAV serotype, a combination of CEX and AEX or AC and AEX is recommended and holds promise for future translational projects that require highly pure and full particle-enriched rAAVs.

Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques.

Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

A Simple, Two-Step, Small-Scale Purification of Recombinant Adeno-Associated Viruses

Recombinant adeno-associated viruses (rAAVs) are robust and versatile tools for in vivo gene delivery. Natural and designer capsid variations in rAAVs allow for targeted gene delivery to specific cell types. Low immunogenicity and lack of pathogenesis also add to the popularity of this virus as an innocuous gene delivery vector for gene therapy. rAAVs are routinely used to express recombinases, sensors, detectors, CRISPR-Cas9 components, or to simply overexpress a gene of interest for functional studies. High production demand has given rise to multiple platforms for the production and purification of rAAVs. However, most platforms rely heavily on large amounts of starting material and multiple purification steps to produce highly purified viral particles. Often, researchers require several small-scale purified rAAVs. Here, we describe a simple and efficient technique for purification of recombinant rAAVs from small amounts of starting material in a two-step purification method. In this method, rAAVs are released into the packaging cell medium using high salt concentration, pelleted by ultracentrifugation to remove soluble impurities. Then, the resuspended pellet is purified using a protein spin-concentrator. In this protocol, we modify the conventional rAAV purification methods to eliminate the need for fraction collection and the labor-intensive steps for evaluating the titer and purity of individual fractions. The resulting rAAV preparations are comparable in titer and purity to commercially available samples. This simplified process can be used to generate highly purified rAAV particles on a small scale, thereby saving resources, generating less waste, and reducing a laboratory’s environmental footprint.

Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls

Although the clinical use of recombinant adeno-associated virus (rAAV) vectors is constantly increasing, the development of suitable quality control methods is still needed for accurate vector characterization. Among the quality criteria, the titration of infectious particles is critical to determine vector efficacy. Different methods have been developed for the measurement of rAAV infectivity in vitro, based on detection of vector genome replication in trans-complementing cells infected with adenovirus, detection of transgene expression in permissive cells, or simply detection of intracellular vector genomes following the infection of indicator cells. In the present study, we have compared these methods for the titration of infectious rAAV8 vector particles, and, to assess their ability to discriminate infectious and non-infectious rAAV serotype 8 particles, we have generated a VP1-defective AAV8-GFP vector. Since VP1 is required to enter the cell nucleus, the lack of VP1 should drastically reduce the infectivity of rAAV particles. The AAV8 reference standard material was used as a positive control. Our results demonstrated that methods based on measurement of rAAV biological activity (i.e., vector genome replication or transgene expression) were able to accurately discriminate infectious versus non-infectious particles, whereas methods simply measuring intracellular vector genomes were not. Several cell fractionation protocols were tested in an attempt to specifically measure vector genomes that had reached the nucleus, but genomes from wild-type and VP1-defective AAV8 particles were equally detected in the nuclear fraction by qPCR. These data highlight the importance of using suitable controls, including a negative control, for the development of biological assays such as infectious unit titration.

Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses

Recent advances using CRISPR-Cas9 approaches have dramatically enhanced the ease for genetic manipulation in rodents. Notwithstanding, the methods to deliver nucleic acids into pre-implantation embryos have hardly changed since the original description of mouse transgenesis more than 30 years ago. Here we report a novel strategy to generate genetically modified mice by transduction of CRISPR-Cas9 components into pre-implantation mouse embryos via recombinant adeno-associated viruses (rAAVs). Using this approach, we efficiently generated a variety of targeted mutations in explanted embryos, including indel events produced by non-homologous end joining and tailored mutations using homology-directed repair. We also achieved gene modification in vivo by direct delivery of rAAV particles into the oviduct of pregnant females. Our approach greatly simplifies the generation of genetically modified mice and, more importantly, opens the door for streamlined gene editing in other mammalian species.

Viral Vector-Based Transduction of Slice Cultures

Transgenes can be introduced into the cells of organotypic slice cultures using different delivery methods, such as biolistic transfection, electroporation, and viral vector-based transduction. These methods produce different patterns of transgene expression. Local injection of recombinant adeno-associated virus (rAAV) produces a small cluster of transgene-expressing neurons around the injection site. Expression in individual cells varies with the distance from the injection site, indicating that many neurons take up several rAAV particles. The serotype and promoter also play a role in transgene expression. Here, we present a protocol for the transduction of previously prepared hippocampal slice cultures with rAVV.

570. Transient Reconstitution of Cas9 Protein for Safer Gene Editing In Vivo - A Ferry Tale Starring rAAV Capsid

Transforming the gene editing technology into therapeutic uses encounters several obstacles including the concern over safety. These gene editing platforms, such as the Cas9/sgRNA system, have been shown to induce off-target DNA double-stranded breaks (DSBs) throughout genomes, which is associated with toxicity. Such off-target effects not only stem from the intrinsic ambiguity of DNA sequence recognition by nucleases, but also attribute to the prolonged presence of an active gene editing system in a given cell. As a result, off-target DSBs accumulate over time, and ultimately lead to genotoxicity. To mitigate the potential toxicity due to prolonged expression of a gene editing system in vivo, we sought to transiently deliver the key component, i.e. the endonuclease protein, which will fulfill the task of inducing permanent gene editing followed by natural degradation. Specifically, we use the VP2 protein of AAV capsid as a protein delivery vehicle to ferry the Cas9 protein in vivo. We first constructed a sensitive gene editing reporter plasmid, and demonstrated that co-transfection of the reporter plasmid and a plasmid expressing the SpCas9-VP2 fusion protein induced gene editing in HEK293 cells. We then modified our rAAV packaging system to include a plasmid expressing VP1 and VP3, and another plasmid expressing either the SpCas9-VP2 fusion protein or the EGFP-VP2 fusion protein. We successfully produced EGFP-AAV2 (EGFP protein grafted on the AAV2 capsid). However, we were not able to produce rAAV particles carrying SpCas9 protein, likely because the large size of SpCas9 protein interfered with the AAV packaging process. Therefore, we split SpCas9 into halves aiming to utilize split intein-mediated protein trans-splicing (PTS) to transiently reconstitute the full-length SpCas9 (Figure)(Figure). When the two parts of a split intein (termed IntN and IntC, respectively) fused with two proteins, the split intein is able to mediate PTS, resulting in the generation of a fusion protein with the intein being spliced out. We generated plasmids expressing the fusion proteins SpCas9N-IntN and IntC-SpCas9C-VP2, respectively, and demonstrated productive intein-mediated reconstitution of SpCas9-VP2 protein in HEK293 cells by co-transfection. Importantly, co-transfection of plasmids expressing SpCas9N-IntN and IntC-SpCas9C-VP2 in HEK293 cells led to gene editing based on the reporter assay. Guided by structural analysis and prediction, we strategically screened and identified the SpCas9 split sites close to the C-term of SpCas9. Therefore, the IntC-SpCas9C protein to be grafted on VP2 is equal or smaller than EGFP. Because we were able to produce EGFP-AAV2, we reasoned that the IntC-SpCas9C-VP2 will likely be amenable to the rAAV packaging process, which is underway. If successful, our novel off-target mitigating strategy described here will greatly facilitate the translational application of gene editing technology in vivo. In addition, this transient in vivo protein reconstitution approach is applicable to the delivery of therapeutic but highly toxic proteins for the treatment of cancer and other diseases.FigureReconstituting SpCas9 by intein-mediated protein trans-splicing (PTS). In the first AAV vector, the AAV genome encodes the N-terminal portion of SpCas9 (SpCas9N) fused with IntN. The second AAV vector carries Intc and the C-terminal portion of SpCas9 fused to VP2. In vivo transduction of the first AAV vector produces the fusion protein SpCas9N-lntN, which is followed by delivery of the second vector. PTS occurs to reconstitute the full-length SpCas9 protein.View Large Image | Download PowerPoint Slide

Molecular imaging reveals epileptogenic Ca2+-channel promoter activation in hippocampi of living mice.

Focal epilepsies often originate in the hippocampal formation of the temporal lobe (temporal lobe epilepsy) and are generally acquired after transient brain insults. Such insults induce cellular and structural reorganization processes of the hippocampus, referred to as epileptogenesis that finally convert the brain spontaneous epileptic. Here, we developed a new molecular imaging strategy in a state-of-the-art animal model to provide insights into key epileptogenic mechanisms. Our new approach combines recombinant adeno-associated virus (rAAV) gene delivery with in vivo bioluminescence imaging. rAAV particles harboring the luciferase reporter gene under control of the minimal T type Ca(2+)-channel subunit Ca V 3.2-promoter were generated and injected stereotaxically in the hippocampal region of mice. Bioluminescent signals, corresponding to Ca V 3.2 promoter activation, were imaged in vivo in the pilocarpine model of status epilepticus (SE). We detected activation of key Ca V 3.2 promoter motifs at 3 and 10 days after SE but not after the onset of chronic seizures. These data suggest Ca V 3.2 promoter activation as novel anti-epileptogenic target. In more general terms, we have established an experimental approach that allows to follow cerebral gene promoter dynamics longitudinally and to correlate this activity to behavioral parameters in the same mice.

Differential effects of two MRI contrast agents on the integrity and distribution of rAAV2 and rAAV5 in the rat striatum

Intraoperative magnetic resonance imaging (MRI) has been proposed as a method to optimize intracerebral targeting and for tracking infusate distribution in gene therapy trials for nervous system disorders. We thus investigated possible effects of two MRI contrast agents, gadoteridol (Gd) and galbumin (Gab), on the distribution and levels of transgene expression in the rat striatum and their effect on integrity and stability of recombinant adeno-associated virus (rAAV) particles. MRI studies showed that contrast agent distribution did not predict rAAV distribution. However, green fluorescent protein (GFP) immunoreactivity revealed an increase in distribution of rAAV5-GFP, but not rAAV2-GFP, in the presence of Gd when compared with viral vector injected alone. In contrast, Gab increased the distribution of rAAV2-GFP not rAAV5-GFP. These observations pointed to a direct effect of infused contrast agent on the rAAV particles. Negative-stain electron microscopy (EM), DNAase treatment, and differential scanning calorimetry (DSC) were used to monitor rAAV2 and rAAV5 particle integrity and stability following contrast agent incubation. EMs of rAAV2-GFP and rAAV5-GFP particles pretreated with Gd appear morphologically similar to the untreated sample; however, Gab treatment resulted in surface morphology changes and aggregation. A compromise of particle integrity was suggested by sensitivity of the packaged genome to DNAase treatment following Gab incubation but not Gd for both vectors. However, neither agent significantly affected particle stability when analyzed by DSC. An increase in Tm was observed for AAV2 in lactated Ringer’s buffer. These results thus highlight potential interactions between MRI contrast agents and AAV that might affect vector distribution and stability, as well as the stabilizing effect of lactated Ringer’s solution on AAV2.

Toll–Like Receptor 2–Mediated Innate Immune Response in Human Nonparenchymal Liver Cells Toward Adeno–Associated Viral Vectors

Adeno-associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen-associated molecular patterns by Toll-like receptor 2. In contrast to the Toll-like receptor 9-mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up-regulated inflammatory cytokines through activation of nuclear factor κB. Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV-mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV.

A Potential Role of Distinctively Delayed Blood Clearance of Recombinant Adeno-associated Virus Serotype 9 in Robust Cardiac Transduction

Recombinant adeno-associated virus serotype 9 (rAAV9) vectors show robust in vivo transduction by a systemic approach. It has been proposed that rAAV9 has enhanced ability to cross the vascular endothelial barriers. However, the scientific basis of systemic administration of rAAV9 and its transduction mechanisms have not been fully established. Here, we show indirect evidence suggesting that capillary walls still remain as a significant barrier to rAAV9 in cardiac transduction but not so in hepatic transduction in mice, and the distinctively delayed blood clearance of rAAV9 plays an important role in overcoming this barrier, contributing to robust cardiac transduction. We find that transvascular transport of rAAV9 in the heart is a capacity-limited slow process and occurs in the absence of caveolin-1, the major component of caveolae that mediate endothelial transcytosis. In addition, a reverse genetic study identifies the outer region of the icosahedral threefold capsid protrusions as a potential culprit for rAAV9's delayed blood clearance. These results support a model in which the delayed blood clearance of rAAV9 sustains the capacity-limited slow transvascular vector transport and plays a role in mediating robust cardiac transduction, and provide important implications in AAV capsid engineering to create new rAAV variants with more desirable properties.

Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02- to 200-Liter Cultures

The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02- to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 ± 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32 × 10(6) cells/ml, ≥ 10(16) purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned.

Establishment of an AAV Reverse Infection-Based Array

BackgroundThe development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited.Principal FindingsWe established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vector-mediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments.Conclusions/SignificanceOur study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays.

Myosin IIb Regulates Actin Dynamics during Synaptic Plasticity and Memory Formation

Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation.