Purification of Adeno-Associated Viral Vector Serotype 9 Using Ceramic Hydroxyapatite Chromatography and its Analysis.

Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of AAV9 vectors in 293EB cells Basic Protocol 2: Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration Basic Protocol 3: Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography Basic Protocol 4: Analysis of the purified AAV9 vectors.

Hybrid modeling of an ultracentrifugation process for separation of full and empty adeno-associated virus particles.

Ultracentrifugation is an attractive method for separating full and empty capsids, exploiting their density difference. Changes of the serotype/capsid, density of loading material, or the genetic information contained in the adeno-associated viruses (AAVs) require the adaptation of the harvesting parameters and the density gradient loaded onto the centrifuge. To streamline these adaptations, a mathematical model could support the design and testing of operating conditions.Here, hybrid models, which combine empirical functions with artificial neural networks, are proposed to describe the separation of full and empty capsids as a function of material and operational parameters, i.e., the harvest model. In addition, critical quality attributes are estimated by a quality model which is operating on top of the harvest model. The performance of these models was evaluated using test data and two additional blind runs. Also, a "what-if" analysis was conducted to investigate whether the models' predictions align with expectations.It is concluded that the models are sufficiently accurate to support the design of operating conditions, though the accuracy and applicability of the models can further be increased by training them on more specific data with higher variability.

Isolation of Adeno-associated Viral Vectors Through a Single-step and Semi-automated Heparin Affinity Chromatography Protocol.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Adeno-associated virus type 2 in US children with acute severe hepatitis.

As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA1,2. Previous studies have found human adenoviruses (HAdVs) in the blood from patients in Europe and the USA3-7, although it is unclear whether this virus is causative. Here we used PCR testing, viral enrichment-based sequencing and agnostic metagenomic sequencing to analyse samples from 16 HAdV-positive cases from 1 October 2021 to 22 May 2022, in parallel with 113 controls. In blood from 14 cases, adeno-associated virus type 2 (AAV2) sequences were detected in 93% (13 of 14), compared to 4 (3.5%) of 113 controls (P < 0.001) and to 0 of 30 patients with hepatitis of defined aetiology (P < 0.001). In controls, HAdV type 41 was detected in blood from 9 (39.1%) of the 23 patients with acute gastroenteritis (without hepatitis), including 8 of 9 patients with positive stool HAdV testing, but co-infection with AAV2 was observed in only 3 (13.0%) of these 23 patients versus 93% of cases (P < 0.001). Co-infections by Epstein-Barr virus, human herpesvirus 6 and/or enterovirus A71 were also detected in 12 (85.7%) of 14 cases, with higher herpesvirus detection in cases versus controls (P < 0.001). Our findings suggest that the severity of the disease is related to co-infections involving AAV2 and one or more helper viruses.

Adeno-associated virus 2 infection in children with non-A-E hepatitis.

An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland1 in April 2022 and has now been identified in 35 countries2. Several recent studies have suggested an association with human adenovirus with this outbreak, a virus not commonly associated with hepatitis. Here we report a detailed case-control investigation and find an association between adeno-associated virus 2 (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing, PCR with reverse transcription, serology and in situ hybridization, we detected recent infection with AAV2 in plasma and liver samples in 26 out of 32 (81%) cases of hepatitis compared with 5 out of 74 (7%) of samples from unaffected individuals. Furthermore, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsy samples. In keeping with a CD4+ T-cell-mediated immune pathology, the human leukocyte antigen (HLA) class II HLA-DRB1*04:01 allele was identified in 25 out of 27 cases (93%) compared with a background frequency of 10 out of 64 (16%; P = 5.49 × 10-12). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a co-infection with human adenovirus that is usually required as a 'helper virus' to support AAV2 replication) and disease susceptibility related to HLA class II status.

Surprise virus tied to pediatric hepatitis cases.

Two viruses plus a child's genetic background may explain a recent surge in the United Kingdom.

Comprehensive Preclinical Assessment of ADVM-022, an Intravitreal Anti-VEGF Gene Therapy for the Treatment of Neovascular AMD and Diabetic Macular Edema.

Inhibition of vascular endothelial growth factor is the mode of action for several approved therapies, including aflibercept, for the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME). Lack of compliance due to the frequent intravitreal dosing requirements may result in inadequately treated disease, leading to irreversible vision impairment. To date, the majority of gene therapy clinical trials providing sustained anti-VEGF levels in the retina have been limited to subretinal injections requiring a vitrectomy. A single intravitreal injection of a gene therapy product could drastically reduce the treatment burden and improve visual outcomes. ADVM-022, an adeno-associated virus vector encoding aflibercept, has been optimized for intravitreal delivery and strong protein expression. Long-term expression and efficacy of ADVM-022-derived aflibercept were evaluated in a laser-induced choroidal neovascularization (CNV) model in non-human primates. Ocular safety was evaluated following long-term suppression of VEGF by clinical scoring (inflammatory parameters) as well as optical coherence tomography (OCT) and electroretinography (ERG). Intravitreal administration of ADVM-022 was well tolerated and resulted in sustained aflibercept levels in ocular tissues. In addition, ADVM-022 administration 13 months before laser-induced CNV prevented the occurrence of clinically relevant CNV lesions, to the same degree as a bolus of aflibercept delivered at the time of laser. These results demonstrate that a single intravitreal administration of ADVM-022 may provide a safe and effective long-term treatment option for nAMD and DME, and may ultimately improve patients' visual outcomes. Clinical trials are currently underway, evaluating safety and efficacy following a single intravitreal injection of ADVM-022.

Separating Empty and Full Recombinant Adeno-Associated Virus Particles Using Isocratic Anion Exchange Chromatography.

The development of recombinant adeno-associated virus (rAAV) gene therapies is becoming an increasing priority in the biotherapeutic landscape. One of the challenges associated with the production of rAAV is the formation of empty AAV particles that do not contain a therapeutic gene. The concerns about the impact of empty particles on clinical safety and rAAV-mediated gene expression have necessitated the development of purification processes to remove these species. The development of a robust and scalable purification process to separate empty and full AAV particles at large scale remains a challenge. In this study, a novel anion exchange chromatography process based on isocratic wash and elution steps to enrich full rAAV2 particles is presented. An operating design space is identified to ensure the robustness of the process. The isocratic chromatography provides several advantages over the traditional shallow linear gradient elution, including lower buffer consumption, smaller intermediate pool volumes, and more robust manufacturing.

A comparison of AAV-vector production methods for gene therapy and preclinical assessment.

Adeno Associated Virus (AAV)-mediated gene expression in the brain is widely applied in the preclinical setting to investigate the therapeutic potential of specific molecular targets, characterize various cellular functions, and model central nervous system (CNS) diseases. In therapeutic applications in the clinical setting, gene therapy offers several advantages over traditional pharmacological based therapies, including the ability to directly manipulate disease mechanisms, selectively target disease-afflicted regions, and achieve long-term therapeutic protein expression in the absence of repeated administration of pharmacological agents. Next to the gold-standard iodixanol-based AAV vector production, we recently published a protocol for AAV production based on chloroform-precipitation, which allows for fast in-house production of small quantities of AAV vector without the need for specialized equipment. To validate our recent protocol, we present here a direct side-by-side comparison between vectors produced with either method in a series of in vitro and in vivo assays with a focus on transgene expression, cell loss, and neuroinflammatory responses in the brain. We do not find differences in transduction efficiency nor in any other parameter in our in vivo and in vitro panel of assessment. These results suggest that our novel protocol enables most standardly equipped laboratories to produce small batches of high quality and high titer AAV vectors for their experimental needs.

Rapid Determination of Full and Empty Adeno-Associated Virus Capsid Ratio by Capillary Isoelectric Focusing.

Adeno-associated virus (AAV) is one of the most promising gene transfer vector types featuring long-term gene expression and low toxicity. The lack of pathogenicity and the availability of many serotypes augmented the applicability of AAV virions in gene therapy applications. The recombinant AAV capsid includes the therapeutic protein-coding transgene as well as a promoter to initiate translation and a poly A sequence portion for stabilization. Current AAV manufacturing technologies, however, cannot guarantee the generation of only full capsids, i.e., including the entire required genome. Partially filled and empty capsids are also part of the product, decreasing in this way the efficacy and safety upon clinical translation. Therefore, rapid, accurate and QC friendly analysis of the full and empty capsid ratio is of high importance during AAV vector manufacturing and release testing. In this paper, an automated capillary isoelectric focusing technique is introduced, readily applicable in the biopharmaceutical industry for fast and efficient determination of the full and empty capsid ratio. The method also reveals information about the proportion of partially filled capsids. For higher resolution (<0.1 pI unit), mixtures of wide and narrow range ampholytes were utilized. The isoelectric point and peak area percentage reproducibility (RSD) of the mixed ampholyte assay were as low as 1.67% and 2.45 %, respectively, requiring only 65 nL of sample volume per injection.

Purification of Recombinant Adeno-Associated Viruses (rAAVs) by Cesium Chloride Gradient Sedimentation.

Centrifugation to equilibrium in cesium chloride gradients has been used for more than 40 yr to purify viruses. The application of high G-forces for a long period of time to a solution of CsCl generates a density gradient that allows separation of empty, partially packaged, and fully packaged viral particles from cellular debris, proteins, and nucleic acids in the crude viral lysate on the basis of their buoyant densities. This protocol describes the use of CsCl gradients to purify AAV vectors from crude viral lysates.

Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation.

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design.

Sensitive Determination of Infectious Titer of Recombinant Adeno-Associated Viruses (rAAVs) Using TCID50 End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR).

Adeno-associated virus (AAV) recombinants are currently the vector of choice for many gene therapy applications. As experimental therapies progress to clinical trials, the need to characterize recombinant adeno-associated viruses (rAAVs) accurately and reproducibly increases. Accurate determination of rAAV infectious titer is important for determining the activity of each vector lot and for ensuring lot-to-lot consistency. The following protocol developed in our laboratory uses a 96-well TCID50 format and quantitative polymerase chain reaction (qPCR) detection for the determination of rAAV infectious titer.

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.

Distribution and genetic diversity of adeno-associated viruses in bats from coastal areas of Southeast China.

Bats are associated with several important zoonotic viruses from different families. One example includes adeno-associated viruses (AAVs), that are extensively detected in several animals, especially primates. To understand AAVs distribution and genetic diversity in the coastal areas of Southeast China, a total of 415 intestine samples were mostly collected from two provinces of southeast China, i.e., Zhejiang and Fujian province. Intestine samples from five bat species were collected for AAVs detection. The average prevalence rate for AAV detection among these samples was 18.6% (77 positives out of 415 samples) and ranged from 11.8 to 28.9% between the five bat species. This suggests that AAVs are widely distributed in diverse bat populations in southeast coastal areas of China. Based on the genome sequence of bat adeno-associated virus-CXC1(BtAAV-CXC1) from one AAV-positive sample, the genetic diversity of the detected AAVs were assessed and analyzed. Phylogenetic analysis revealed that BtAAV-CXC1 was comparatively distant to other major AAVs from mammals and non-mammals, with only a 52.9~64.7% nucleotide identity. However, they were phylogenetically closer to Rhinolophus sinicus bat adeno-associated virus (Rs-BtAAV1), with a 74.5% nt similarity. Partial analysis of the rep and cap overlapping open reading frame (ORF) sequences from bat AAV samples revealed 48 partial rep sequences and 23 partial cap sequences from positive samples shared 86.9 to 100% and 72.3 to 98.8% nucleotide identities among themselves, respectively. This suggests that the detected AAVs had a distinctly high genetic diversity. These findings led us to conclude that diverse AAVs may be widely distributed in bat populations from the southeast regions of China.

Purification of Recombinant Adeno-Associated Viruses (rAAVs) by Iodixanol Gradient Centrifugation.

This is a simple method for rapid preparation of recombinant adeno-associated virus (rAAV) stocks, which can be used for in vivo gene delivery. The purity of these vectors is considerably lower than that obtained by either CsCl gradient centrifugation or by combination of iodixanol gradient ultracentrifugation followed by column chromatography.

Enrichment of Fully Packaged Virions in Column-Purified Recombinant Adeno-Associated Virus (rAAV) Preparations by Iodixanol Gradient Centrifugation Followed by Anion-Exchange Column Chromatography.

This rapid and efficient method to prepare highly purified recombinant adeno-associated viruses (rAAVs) is based on binding of negatively charged rAAV capsids to an anion-exchange resin that is pH dependent.

Adeno-associated virus in the liver: natural history and consequences in tumour development.

OBJECTIVE: Adeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35%-80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed on normal liver. This study aimed to characterise the natural history of AAV infection in the liver and its consequence in tumour development. DESIGN: Viral DNA was quantified in tumour and non-tumour liver tissues of 1461 patients. Presence of episomal form and viral mRNA expression were analysed using a DNAse/TaqMan-based assay and quantitative RT-PCR. In silico analyses using viral capture data explored viral variants and new clonal insertions. RESULTS: AAV DNA was detected in 21% of the patients, including 8% of the tumour tissues, equally distributed in two major viral subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Episomal viral forms were found in 4% of the non-tumour tissues, frequently associated with viral RNA expression and human herpesvirus type 6, the candidate natural AAV helper virus. In 30 HCC, clonal AAV insertions were recurrently identified in CCNA2, CCNE1, TERT, TNFSF10, KMT2B and GLI1/INHBE. AAV insertion triggered oncogenic overexpression through multiple mechanisms that differ according to the localisation of the integration site. CONCLUSION: We provided an integrated analysis of the wild-type AAV infection in the liver with the identification of viral genotypes, molecular forms, helper virus relationship and viral integrations. Clonal AAV insertions were positive selected during HCC development on non-cirrhotic liver challenging the notion of AAV as a non-pathogenic virus.

A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins.

Adeno-Associated Viruses (AAV) are widely used gene-therapy vectors for both clinical applications and laboratory investigations. The titering of different AAV preparations is important for quality control purposes, as well as in comparative studies. However, currently available methods are limited in their ability to detect various serotypes with sensitivity and convenience. Here, we took advantage of a newly discovered AAV receptor protein with high affinity to multiple AAV serotypes, and developed an ELISA-like method named "VIRELISA" (virus receptor-linked immunosorbent assay) by adopting fusion with a streptavidin-binding peptide (SBP). It was demonstrated that optimized VIRELISA assays exhibited satisfactory performance for the titering of AAV2. The linear range of AAV2 was 1 × 105 v.g. to 5 × 109 v.g., with an LOD (limit of detection) of 5 × 104 v.g. Testing of VIRELISA for the quantification of AAV1 was also successful. Our study indicated that a generic protocol for the quantification of different serotypes of AAVs was feasible, reliable and cost-efficient. The applications of VIRELISA will not only be of benefit to laboratory research due to its simplicity, but could also potentially be used for monitoring the circulation AAV loads both in clinical trials and in wild type infection of a given AAV serotype.

Production of adeno-associated virus vectors for in vitro and in vivo applications.

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010-1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.