Adeno-associated Virus Production Research Articles

AAVone: A Cost-Effective, Single-Plasmid Solution for Efficient AAV Production with Reduced DNA Impurities.

Currently, the most common approach for manufacturing GMP-grade adeno-associated virus (AAV) vectors involves transiently transfecting mammalian cells with three plasmids that carry the essential components for production. The requirement for all three plasmids to be transfected into a single cell and the necessity for high quantities of input plasmid DNA, limits AAV production efficiency, introduces variability between production batches, and increases time and labor costs. Here, we developed an all-in-one, single-plasmid AAV production system, called AAVone. In this system, the adenovirus helper genes ( E2A , E4orf6 , and VA RNA ), packaging genes ( rep and cap ), and the vector transgene cassette are consolidated into a single compact plasmid with a 13-kb backbone. The AAVone system achieves a two- to four-fold increase in yields compared to the traditional triple-plasmid system. Furthermore, the AAVone system exhibits low batch-to-batch variation and eliminates the need for fine-tuning the ratios of the three plasmids, simplifying the production process. In terms of vector quality, AAVs generated by the AAVone system show similar in vitro and in vivo transduction efficiency, but a substantial reduction in sequences attributed to plasmid backbones and a marked reduction in non-functional snap-back genomes. In Summary, the AAVone platform is a straightforward, cost-effective, and highly consistent AAV production system - making it particularly suitable for GMP-grade AAV vectors.

Knockout of Pro-Apoptotic BAX and BAK1 Genes in HEK293T Cells Enhances Adeno-Associated Virus (AAV) Production: AAV2 and AAV9.

Increasing demand for adeno-associated virus (AAV) used in gene therapy highlights the need to enhance AAV production. When intracellular AAV2 and extracellular AAV9 were produced in HEK293T cells using the triple transfection method, apoptosis occurred during the AAV production. To mitigate apoptosis induced by AAV production, the pro-apoptotic BAX/BAK1 genes were knocked out in HEK293T cells. BAX/BAK1 knockout (BBKO) in HEK293T cells significantly increased the production of both AAV2 and AAV9. For AAV2, BBKO increased the genome titer of AAV2 by 55% without negatively affecting the proportion of unwanted empty capsids generated during AAV production. Empty capsid ratios were determined based on viral genome and capsid titers and confirmed via transmission electron microscopy (TEM). Likewise, for AAV9, BBKO increased the genome titer of AAV9 by 66% without negatively affecting the proportion of empty capsids. Additionally, as assessed using a transduction assay, BBKO increased the functional titers of AAV2 and AAV9 by 30% and 46%, respectively. Therefore, BBKO increased AAV production, while maintaining full capsid ratio and infectivity. Taken together, BBKO proved to be an efficient method for enhancing AAV production in HEK293T cells for both AAV2 and AAV9.

Towards a Platform Chromatography Purification Process for Adeno-Associated Virus (AAV).

Adeno-associated virus (AAV) is a versatile viral vector technology that can be engineered for specific functionality in vaccine and gene therapy applications. One of the major challenges in AAV production is the need for a GMP-ready platform-based approach to downstream processing, as this would lead to a standardized method for multiple products. Chromatography has huge potential in AAV purification, as it is a scalable method that would enable manufacturing to a high degree of purity, potency, and consistency. Multiple factors need to be considered when developing a chromatography platform, including the chromatography type, format, and mode of operation, along with other commercial considerations that have not been comprehensively reviewed until now. In addition to chromatography factors, this review will also consider the current understanding of AAV characteristics: this will include net surface charge, structural properties, and size, as well as their interactions with metal ions or receptors, and how this impacts the development of a chromatography platform.

Virus Filtration Development for Adeno-Associated Virus-Based Gene Therapy Products.

Adeno-associated virus (AAV) vectors have become a leading platform for gene delivery. A major portion of gene therapy currently in clinical trials are AAV-based for a wide range of diseases. A commonly used method for AAV production is by mammalian or insect cell culture, with or without added viruses to introduce needed genetic elements for AAV production. There are potential risks of virus contamination from the production cell line, process residuals, or adventitious contamination in the production of biotherapeutics, including AAV-based gene therapy products; therefore, it is imperative to demonstrate that the drug substance manufacturing process has sufficient capability to clear process-related or adventitious viruses. In the AAV-based gene therapy manufacturing process, cell lysis, affinity chromatography, and ion exchange chromatography steps are often effective to inactivate or remove viruses. To increase the viral clearance robustness, virus filtration (VF) is increasingly recommended by regulatory agencies for gene therapy products as a dedicated viral clearance step in the downstream purification process. In the current study, two commercially available virus filters were evaluated in the context of AAV manufacturing. The filter throughput and process yield were assessed under different operational modes. Virus clearance performance was evaluated by spiking in Adenovirus type 5 (Adv-5) and Simian virus 40 (SV-40). The viral filters assessed in this study demonstrated manufacturable throughputs, acceptable process yields, and robust virus clearance capabilities for viruses greater than 40nm.

An Optimized Triple-Plasmid System with Enhanced Viral and Helper Gene Expression for Improved Recombinant Adeno-Associated Virus Production

An Optimized Triple-Plasmid System with Enhanced Viral and Helper Gene Expression for Improved Recombinant Adeno-Associated Virus Production

E2A, VA RNA I, and L4-22k adenoviral helper genes are sufficient for AAV production in HEK293 cells

The replication-defective adeno-associated virus (AAV) is extensively utilized as a research tool or vector for gene therapy. The production process of AAV remains intricate, expensive, and mechanistically underexplored. With the aim of enhancing AAV manufacturing efficiencies in mammalian cells, we revisited the questions and optimization surrounding the requirement of the various adenoviral helper genes in enabling AAV production. First, we refined the minimal set of adenoviral genes in HEK293 AAV production to E2A, L4-22 K /33 K, and VA RNA I. These findings challenge the previously accepted necessity of adenoviral E4orf6 in AAV production. In addition, we identified L4-22 K genes as crucial helpers for AAV production. Next, a revised minimal adenoviral helper plasmid comprising E2A, L4-22 K, and VA RNA I genes was designed and demonstrated to yield high titer and potent AAV preps in HEK293 transient transfection. Lastly, stable packaging cells harboring inducible E2A and L4-22 K genes were shown to maintain AAV production yields comparable to transient transfection over a culture period of ∼10weeks. Combined, these findings further our understanding of adenoviral helper function in mammalian AAV production and provide novel plasmid and cell-line reagents with an improved safety profile for potential broad applicability in the research and gene therapy community.

The Unveiled Novel regulator of Adeno-associated virus production in HEK293 cells

The field of gene therapy using Adeno-associated viral (AAV) vector delivery is rapidly advancing in the biotherapeutics industry. Despite its successes, AAV manufacturing remains a challenge due to limited production yields. The triple plasmid transfection of HEK293 cells represents the most extensively utilized system for AAV production. The regulatory factors and mechanisms underlying viral production in HEK293 cells are largely unknown. In this study, we isolated high-titer AAV production clones from a parental HEK293 population using a single limiting dilution step, and subsequently elucidating their underlying molecular mechanisms through whole transcriptome analysis. LncRNA TCONS_00160397 was upregulated in clones and shown to promoted HEK293 cells proliferation and improved the titer of AAV production. Mechanistically, results from proteomics and metabolomics indicated that TCONS_00160397 regulated the ABC transporters pathway. These findings furnish a rich repository of knowledge and actionable targets for the rational optimization of HEK293-based producer lines, thereby paving the way for tangible improvements in AAV vector output and expediting the broad implementation of gene therapies.

An Improved Helper Plasmid Containing Deletions Within the E4 and E2a Genes Results in Increased Adeno-Associated Virus Productivity.

The use of a helper plasmid to replace adenovirus infection for adeno-associated virus (AAV) manufacturing has been common practice for decades. Adenovirus E4, E2a, and VA RNA genes are sufficient to support efficient AAV replication. In an effort to ensure that all transfected DNA has a functional role in AAV production, deletions were introduced to the E4 and E2a genes to determine if any portions were dispensable. Although a 900 bp deletion in the E2a intron did not have an impact, the removal of open reading frames (orf) 1-4 from the E4 gene resulted in a doubling of AAV productivity. The E4Δorf1-4 deletion was associated with a reduction in E4orf6 transcripts, along with an increase in Rep and Cap transcripts and protein levels, which corresponded to increased AAV productivity in crude lysate. The final product of these studies was a helper plasmid, termed OXB-Helper_3, that is >3.4 kb smaller than the original control plasmid and resulted in ∼2× improvement in vector genome productivity across multiple capsid serotypes, genome designs, and transfection platforms.

Membrane chromatography for AAV full capsid enrichment: Process development to scale up

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing “empty” capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.

<i>In vivo</i> evaluation of tropism and biodistribution of synthetic and natural adeno-associated viral vectors by next-generation sequencing

INTRODUCTION. The creation of synthetic adeno-associated virus (AAV) vectors during gene therapy development is a labour-intensive and expensive process. The optimal solution to minimise the time and costs associated with gene therapy development lies in the improvement of methods aimed at assessing AAV vector biodistribution and transduction efficiency in vivo.AIM. This study aimed to develop a new bioinformatics-based assessment method for synthetic AAV vector libraries to analyse AAV vector biodistribution and transduction efficiency in vivo.MATERIALS AND METHODS. The production of synthetic AAV vectors involved assigning AAV serotype-specific barcodes (12-nucleotide tags flanked at the 5' end with a sequence encoding the green fluorescent reporter protein). Plasmids carrying unique barcodes were propagated in competent Escherichia coli XL10-Gold cells and used to create two AAV libraries: L1 with a viral genome count of 1010 and L2 with a viral genome count of 1011. AAV production involved HEK293T cell transfection. L1 and L2 library vectors were administered to C57Bl/6N mice by intravenous injection. DNA and RNA were isolated from transduced organs for analysis by next-generation sequencing. The obtained data on DNA and RNA barcode quantities in different murine organs were analysed to assess the biodistribution and transduction efficiency of synthetic AAVs. Barcodes were identified by aligning them to the expected sequences and counted. The resulting values were normalised to the quantity of barcodes in the original library.RESULTS. Seven viral constructs based on different AAV serotypes were created as part of two AAV libraries. Six of the AAV serotypes were synthetic (sAAV1, sAAV2, sAAV3, sAAV4, sAAV5, and sAAV6). Sequencing of murine organ samples revealed significant quantities of DNA barcodes from both AAV libraries in all organs except the brain. For the L1 library, RNA barcodes were detected at a sufficient level in 4 organs, including the skeletal muscles, the heart, the liver, and the adrenal glands. For the L2 library, in addition to the listed organs, sufficient RNA-barcode levels were observed in the gonads and the kidneys. According to transduction efficiency analysis based on RNA barcode levels adjusted for DNA barcodes, sAAV5 was considered the most promising variant for gene therapy of liver-related diseases, whereas sAAV2 and sAAV6 were recognised as holding the most promise for adrenal diseases.CONCLUSIONS. The developed bioinformatics-based assessment method for synthetic AAV vector libraries can analyse AAV vector biodistribution and transduction efficiency in the body. The presented approach has the potential for selecting optimal AAV vectors for specific organs and tissues in further gene therapy development.

Suppression of toxic transgene expression by optimized artificial miRNAs increases AAV vector yields in HEK-293 cells

Adeno-associated virus (AAV) vectors have become the leading platform for gene delivery in both preclinical research and therapeutic applications, making the production of high-titer AAV preparations essential. To date, most AAV-based studies use constitutive promoters (e.g., CMV, CAG), which are also active in human embryonic kidney (HEK)-293 producer cells, thus leading to the expression of the transgene already during production. Depending on the transgene's function, this might negatively impact producer cell performance and result in decreased AAV vector yields. Here, we evaluated a panel of diverse microRNA (miRNA)-based shRNA designs to identify a highly potent artificial miRNA for the transient suppression of transgenes during AAV production. Our results demonstrate that insertion of miRNA target sites into the 3' UTR of the transgene and simultaneous expression of the corresponding miRNA from the 3' UTR of conventional AAV production plasmids (rep/cap, pHelper) enabled efficient silencing of toxic transgene expression, thereby increasing AAV vector yields up to 240-fold. This strategy not only allows to maintain the traditional triple-transfection protocol, but also represents a universally applicable approach to suppress toxic transgenes, thereby boosting vector yields with so far unprecedented efficiency.

Engineering a synthetic gene circuit for high-performance inducible expression in mammalian systems

Inducible gene expression systems can be used to control the expression of a gene of interest by means of a small-molecule. One of the most common designs involves engineering a small-molecule responsive transcription factor (TF) and its cognate promoter, which often results in a compromise between minimal uninduced background expression (leakiness) and maximal induced expression. Here, we focus on an alternative strategy using quantitative synthetic biology to mitigate leakiness while maintaining high expression, without modifying neither the TF nor the promoter. Through mathematical modelling and experimental validations, we design the CASwitch, a mammalian synthetic gene circuit based on combining two well-known network motifs: the Coherent Feed-Forward Loop (CFFL) and the Mutual Inhibition (MI). The CASwitch combines the CRISPR-Cas endoribonuclease CasRx with the state-of-the-art Tet-On3G inducible gene system to achieve high performances. To demonstrate the potentialities of the CASwitch, we apply it to three different scenarios: enhancing a whole-cell biosensor, controlling expression of a toxic gene and inducible production of Adeno-Associated Virus (AAV) vectors.

Enhancing the production of adeno-associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design-of-experiment optimization of triple plasmid ratio.

The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI=1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI=1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.

Production of adeno-associated virus in insect cells using multiple gene deleted baculovirus

Adeno-associated virus (AAV) is one of the most frequently used viral vectors in the field of gene therapy. However, the industrial production of AAV is facing key bottlenecks such as low yield and high-cost. The aim of this study was to establish a technology system for production of AAV in the double virus infected insects by using multiple-gene deleted baculovirus. First, a multiple gene deleted baculovirus for AAV production was constructed, and the baculovirus titer and its effect on infected cells was examined. Subsequently, the insect cells were co-infected with the double baculovirus and the infection conditions were optimized. At the final stage, we performed AAV production based on optimized conditions, and evaluated relevant parameters including production titer and quality. The results showed that the titer of AAV produced in the multiple gene deleted baculovirus was not different from that of the wild type, but the rate of cell death was significantly slower upon infection. Using the double virus route for optimized production of AAV, the genome titers were 1.63×1011 VG/mL for Bac4.0-1 and 1.02×1011 VG/mL for Bac5.0-2, which were elevated 240% and 110%, respectively, compared with that of the wild-type. Electron microscopy observations revealed that all three groups exhibited normal AAV viral morphology and they showed similar transduction activity. Taken together, we developed an AAV production system based on the infection of insect cells using multiple-gene deleted baculovirus, which significantly improved the virus yield and showed application potential.

A Comprehensive Study of the Effects by Sequence Truncation within Inverted Terminal Repeats (ITRs) on the Productivity, Genome Packaging, and Potency of AAV Vectors.

One of the primary challenges in working with adeno-associated virus (AAV) lies in the inherent instability of its inverted terminal repeats (ITRs), which play vital roles in AAV replication, encapsidation, and genome integration. ITRs contain a high GC content and palindromic structure, which occasionally results in truncations and mutations during plasmid amplification in bacterial cells. However, there is no thorough study on how these alterations in ITRs impact the ultimate AAV vector characteristics. To close this gap, we designed ITRs with common variations, including a single B, C, or D region deletion at one end, and dual deletions at both ends of the vector genome. These engineered ITR-carrying plasmids were utilized to generate AAV vectors in HEK293 cells. The crude and purified AAV samples were collected and analyzed for yield, capsid DNA-filled percentage, potency, and ITR integrity. The results show that a single deletion had minor impact on AAV productivity, packaging efficiency, and in vivo potency. However, deletions on both ends, except A, showed significant negative effects on the above characteristics. Our work revealed the role of ITR regions, A, B, C, and D for AAV production and DNA replication, and proposes a new strategy for the quality control of ITR-bearing plasmids and final AAV products.

Multi-omics kinetic analysis of recombinant adeno-associated virus production by plasmid transfection of HEK293 cells.

Recombinant adeno-associated virus (rAAV) is among the most commonly used vectors for gene therapy. It is commonly produced by transfection of HEK293 cells with three plasmids each containing the vector genome including gene of interest (GOI), helper functions, and rep and cap genes for genome replication and capsid formation. To meet the potential clinical needs, the productivity of the production system needs to be enhanced. A better process characterization of the production system will further advance our insights into ways to enhance productivity. Here, we employed transcriptomic analysis to quantify the dynamics of different isoforms of viral transcripts and to assess the shift of cellular physiology, and deployed targeted proteomic analysis for absolute quantification of viral proteins and tandem mass tags (TMTs) for assessing cellular responses at the protein level. Functional analysis at transcriptome and proteome levels identified defense and immune response, unfolded protein response, p53 signaling as enriched. The small molecule additive intervention study based on functional analysis showed the potential of such omics-guided productivity enhancement. Together, multi-omics analysis advanced understanding of rAAV production and provided insight into enhancing rAAV production by plasmid transfection.

Recombinant AAV genome size effect on viral vector production, purification, and thermostability

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. This work studied the effect of the genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension adapted HEK293 cells via triple transfection using AAV plasmids containing the same green fluorescent protein (GFP) transgene with DNA stuffers for variable size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography and the results suggest that the triple transfection condition significantly impacts the AEX retention time and resolution between the full and empty capsid peaks. Charge detection mass spectrometry (CD-MS) was performed on all AEX full capsid peak samples showing a wide distribution of empty, partial, full length, and co-packaged DNA in the capsids. The AEX purified samples were then analyzed by differential scanning fluorimetry (DSF) and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.

Streamlined Adeno-Associated Virus Production Using Suspension HEK293T Cells.

Recombinant adeno-associated viruses (rAAVs) are valuable viral vectors for in vivo gene transfer, also having significant ex vivo therapeutic potential. Continued efforts have focused on various gene therapy applications, capsid engineering, and scalable manufacturing processes. Adherent cells are commonly used for virus production in most basic science laboratories because of their efficiency and cost. Although suspension cells are easier to handle and scale up compared to adherent cells, their use in virus production is hampered by poor transfection efficiency. In this protocol, we developed a simple scalable AAV production protocol using serum-free-media-adapted HEK293T suspension cells and VirusGEN transfection reagent. The established protocol allows AAV production from transfection to quality analysis of purified AAV within two weeks. Typical vector yields for the described suspension system followed by iodixanol purification range from a total of 1 × 1013 to 1.5 × 1013 vg (vector genome) using 90 mL of cell suspension vs. 1 × 1013 to 2 × 1013 vg using a regular adherent cell protocol (10 × 15 cm dishes). Key features • Adeno-associated virus (AAV) production using serum-free-media-adapted HEK293T suspension cells. • Efficient transfection with VirusGEN. • High AAV yield from small-volume cell culture. Graphical overview.

Evolving membrane-associated accessory protein variants for improved adeno-associated virus production

Manufacturing sufficient adeno-associated virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, although the underlying mechanisms of MAAP function remain unknown. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 open reading frame (ORF) (AAV2-ΔMAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1×106 MAAP protein variants to five rounds of packaging selection into the AAV2-ΔMAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk-selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a frameshifted C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

A Novel Role for the Adenovirus L4 Region 22K and 33K Proteins in Adeno-Associated Virus Production.

Despite decades of research in adeno-associated virus (AAV) and the role of adenovirus in production, the interplay of AAV and adenovirus is not fully understood. Specific regions of the adenoviral genome containing E1, E2a, E4 open reading frame (ORF), and VA RNA have been demonstrated as necessary for AAV production; however, incorporating these regions into either a producer cell line or subcloning into an Ad helper plasmid may lead to inclusion of neighboring adenoviral sequence or ORFs with unknown function. Because AAV is frequently used in gene therapies, removing excessive adenovirus sequences improves the Ad helper plasmid size and manufacturability, and may lead to safer vectors for patients. Furthermore, deepening our understanding of the helper virus genes required for recombinant AAV (rAAV) production has the potential to increase yields and manufacturability of rAAV for clinical and commercial applications. One region continuously included in various Ad helper plasmid iterations is the adenoviral E2a promoter region that appears to be necessary for E2a expression. Due to the compact nature of viral genomes, the E2a promoter region overlaps with the Hexon Assembly/100K protein and the L4 region. The L4 region, which contains the coding sequences for 22K and 33K proteins, had not been thought to be necessary for AAV production. Through molecular techniques, this study demonstrates that the adenoviral 22K protein is essential for rAAV production in HEK293 cells by triple transfection and that the 33K protein synergistically increases rAAV yield.