AAV Production Research Articles

Advancing AAV production with high-throughput screening and transcriptomics

Advancing AAV production with high-throughput screening and transcriptomics

Navigating variability and scalability challenges in AAV production

Navigating variability and scalability challenges in AAV production

Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production

Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production.

Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production.

Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production.

Advanced biomanufacturing and evaluation of adeno-associated virus

Recombinant adeno-associated virus (rAAV) has been developed as a safe and effective gene delivery vehicle to treat rare genetic diseases. This study aimed to establish a novel biomanufacturing process to achieve high production and purification of various AAV serotypes (AAV2, 5, DJ, DJ8). First, a robust suspensive production process was developed and optimized using Gibco Viral Production Cell 2.0 in 30–60 mL shaker flask cultures by evaluating host cells, cell density at the time of transfection and plasmid amount, adapted to 60–100 mL spinner flask production, and scaled up to 1.2–2.0-L stirred-tank bioreactor production at 37 °C, pH 7.0, 210 rpm and DO 40%. The optimal process generated AAV titer of 7.52–8.14 × 1010 vg/mL. Second, a new AAV purification using liquid chromatography was developed and optimized to reach recovery rate of 85–95% of all four serotypes. Post-purification desalting and concentration procedures were also investigated. Then the generated AAVs were evaluated in vitro using Western blotting, transmission electron microscope, confocal microscope and bioluminescence detection. Finally, the in vivo infection and functional gene expression of AAV were confirmed in tumor xenografted mouse model. In conclusion, this study reported a robust, scalable, and universal biomanufacturing platform of AAV production, clarification and purification.

Advancing AAV production scalability: enhancing purity, productivity, and yield

Advancing AAV production scalability: enhancing purity, productivity, and yield

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

AAV production in stable packaging cells requires expression of adenovirus 22/33K protein to allow episomal amplification of integrated rep/cap genes

Efficient manufacture of recombinant adeno-associated virus (rAAV) vectors for gene therapy remains challenging. Packaging cell lines containing stable integration of the AAV rep/cap genes have been explored, however rAAV production needs to be induced using wild-type adenoviruses to promote episomal amplification of the integrated rep/cap genes by mobilizing a cis-acting replication element (CARE). The adenovirus proteins responsible are not fully defined, and using adenovirus during rAAV manufacture leads to contamination of the rAAV preparation. ‘TESSA’ is a helper adenovirus with a self-repressing Major Late Promoter (MLP). Its helper functions enable efficient rAAV manufacture when the rep and cap genes are provided in trans but is unable to support rAAV production from stable packaging cells. Using rAAV-packaging cell line HeLaRC32, we show that expression of the adenovirus L4 22/33K unit is essential for rep/cap amplification but the proteins are titrated away by binding to replicating adenovirus genomes. siRNA-knockdown of the adenovirus DNA polymerase or the use of a thermosensitive TESSA mutant decreased adenovirus genome replication whilst maintaining MLP repression, thereby recovering rep/cap amplification and efficient rAAV manufacture. Our findings have direct implications for engineering more efficient adenovirus helpers and superior rAAV packaging/producer cells.

Engineered CHO cells as a novel AAV production platform for gene therapy delivery

The Herpes simplex virus (HSV)-based platform for production of recombinant adeno-associated viral vectors (rAAVs) yields higher titers and increased percentage of full capsids when compared to the triple transient transfection (TTT) method. However, this platform currently faces two major challenges. The first challenge is the reliance on commercial media, sometimes supplemented with serum, leading to costly manufacturing and a high risk for introduction of adventitious agents. The second challenge is that the production of HSV-1 relies on adherent complementing Vero cells (V27), making it difficult to scale up. We engineered serum-free-adapted CHO cells expressing key HSV-1 entry receptors, HVEM and/or Nectin-1 to address the first challenge. Using high-throughput cloning methods, we successfully selected a HVEM receptor-expressing clone (CHO–HV–C1) that yields 1.62 × 109, 2.51 × 109, and 4.07 × 109 viral genome copies/mL with rAAV6.2-GFP, rAAV8-GFP, and rAAV9-GFP vectors respectively, within 24 h post rHSV-1 co-infection. Moreover, CHO–HV–C1-derived rAAVs had comparable in vitro transduction, infectivity, and biodistribution titers to those produced by TTT. The second challenge was addressed via engineering CHO–HV–C1 cells to express HSV-1 CP27. These cells successfully produced rHSV-1 vectors, but with significantly lower titers than V27 cells. Taken together, the CHO/HSV system provides a novel, scalable, reduced cost, serum-free AAV manufacturing platform.

Overcoming upstream & downstream process barriers for large-scale AAV production

Overcoming upstream & downstream process barriers for large-scale AAV production

AAV production by transient transfection: strategies & challenges

AAV production by transient transfection: strategies & challenges

Inducible HEK293 AAV packaging cell lines expressing Rep proteins

Packaging or producer cell lines for scalable recombinant adeno-associated virus (rAAV) production have been notoriously difficult to create due in part to the cytostatic nature of the Rep proteins required for AAV production. The most difficult challenge being creating AAV packaging cell lines using HEK293 parental cells, currently the best mammalian platform for rAAV production due to the constitutive expression of E1A in HEK293 cells, a key REP transcription activator. Using suspension and serum-free media adapted HEK293SF carrying a gene expression regulation system induced by addition of cumate and coumermycin, we were able to create REP-expressing AAV packaging cells. This was achieved by carefully choosing two of the AAV Rep proteins (Rep 40 and 68), using two inducible promoters with different expression levels and integrating into the cells through lentiviral vector transduction. Three of our best clones produced rAAV titers comparable to titers obtained by standard triple plasmid transfection of their parental cells. These clones were stable for up to 7weeks under continuous cultures condition. rAAV production from one clone was also validated at scale of 1L in a wave bioreactor using serum-free suspension culture.

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

A new T-antigen negative HEK293 cell line with improved AAV productivity.

Viral vectors for gene therapy, such as recombinant adeno-associated viruses,are produced in human embryonic kidney (HEK) 293 cells. However, the presence of the SV40 T-antigen-encoding CDS SV40GP6 and SV40GP7 in the HEK293T genome raises safety issues when these cells are used in manufacturing for clinical purposes. We developed a new T-antigen-negative HEK cell line from ExcellGene's proprietary HEKExpress,®using the CRISPR-Cas9 strategy. We obtained a high number of clonally-derived cell populations and all of them were demonstrated T-antigen negative. Stability study and AAV production evaluation showed that the deletion of the T-antigen-encoding locus did not impact neither cell growth nor viability nor productivity. The resulting CMC-compliant cell line, named HEKzeroT,®is able to produce high AAV titers, from small to large scale.

AAV- based vector improvements unrelated to capsid protein modification.

Recombinant adeno-associated virus (rAAV) is the leading platform for delivering genetic constructs in vivo. To date, three AAV-based gene therapeutic agents have been approved by the FDA and are used in clinical practice. Despite the distinct advantages of gene therapy development, it is clear that AAV vectors need to be improved. Enhancements in viral vectors are mainly associated with capsid protein modifications. However, there are other structures that significantly affect the AAV life cycle and transduction. The Rep proteins, in combination with inverted terminal repeats (ITRs), determine viral genome replication, encapsidation, etc. Moreover, transgene cassette expression in recombinant variants is directly related to AAV production and transduction efficiency. This review discusses the ways to improve AAV vectors by modifying ITRs, a transgene cassette, and the Rep proteins.

Exploring the potential of insect cell lines for efficient AAV production

Exploring the potential of insect cell lines for efficient AAV production

Cryptic resolution sites in the vector plasmid lead to the heterogeneities in the rAAV vectors.

Recombinant adeno-associated virus (rAAV) vectors carry a cassette of interest retaining only the inverted terminal repeats (ITRs) from the wild-type virus. Conventional rAAV production primarily uses a vector plasmid as well as helper genes essential for AAV replication and packaging. Nevertheless, plasmid backbone related contaminants have been a major source of vector heterogeneity. The mechanism driving the contamination phenomenon has yet to be elucidated. Here we identified cryptic resolution sites in the plasmid backbone as a key source for producing snapback genomes, which leads to the increase of vector genome heterogeneity in encapsidated virions. By using a single ITR plasmid as a model molecule and mapping subgenomic particles, we found that there exist a few typical DNA break hotspots in the vector DNA plasmid backbone, for example, on the ampicillin DNA element, called aberrant rescue sites. DNA around these specific breakage sites may assume some typical secondary structures. Similar to normal AAV vectors, plasmid DNA with a single ITR was able to rescue and replicate efficiently. These subgenomic DNA species significantly compete for trans factors required for rAAV rescue, replication, and packaging. The replication of single ITR contaminants during AAV production is independent of size. Packaging of these species is greatly affected by its size. A single ITR and a cryptic resolution site in the plasmid work synergistically, likely causing a source of plasmid backbone contamination.

Driving the viral vector expressway: speeding through AAV manufacturing

Watch this FastFacts to discover:     How the VirusExpress® 293 AAV Production Platform can help speed your gene therapy to patients     How high-through put screening has enhanced efficiency of the platform     How improvements to transfection and cell culture media impact performance     How our platform performs with GFP model virus in serotypes AAV2, AAV5, AAV6, and a customer’s GOI and AAV9.Andi Ushijima is a Scientist in the Viral Vector Innovation and Technology group at MilliporeSigma. She has a variety of upstream bioprocessing experience working on bioreactor development and scale up of monoclonal antibodies, virus-like particles, and viral vectors. She received her BSc in Bioengineering at UCSD.Henry has worked since 1981 in both the biotechnology and pharmaceutical sectors, developing protein expression systems in bacterial, insect and mammalian hosts. He joined MilliporeSigma in 2001 as an R&D manager, and currently leads the Viral Vector Producer Cell Lines R&D group.

Tips and tricks for transfection, adaptation, and scale-up for AAV production

Watch the video or view the poster to learn about:How to adapt a cell line using the Gibco Viral Vector HEK Media PanelStrategies to transfect HEK293 cells in large-scale processesStrategies to implement a new media and process at the 50 L scaleAmanda Zunic is currently an R&D scientist for Thermo Fisher Scientific. She’s been a part of Thermo Fisher Scientific since 2020 and joined to enable product development to ensure customers have the necessary tools to help create a healthier world. She’s earned a Masters degree in Microbiology and Immunology from The State University of New York at Buffalo where she was investigating the interactions between HPV viral proteins and the eukaryotic replication machinery. Her current focus is on development of products and solutions to enhance therapeutic potential of gene therapy manufacturing.

Abstract P2104: Adeno-associated Virus Serotype 5 Is A Suitable Vector For S100a1-based Gene Therapy Of Post-ischemic Chronic Cardiac Dysfunction

Introduction: For S100A1-based heart failure gene therapies, AAV9 and 6 have shown efficacy in pre-clinical large animal studies. As AAV9 has shown concerning signs of toxicity in clinical studies and AAV6 displays poor production yields, there is need for a novel safe and cardiac-specific AAV serotype. Hypothesis: We hypothesized that in a pig model the safety proven and scalably manufacturable AAV5 may be a suitable vector for S100A1-based gene therapy of post-ischemic cardiac dysfunction. Methods: AAV production, 2h balloon-occlusion of the LCX, retrograde cardiac gene delivery, cardiac MRI, late gadolinium enhancement (LGE), global T1 relaxation, qPCR, RNA-Seq, WGCNA, KEGG, Reactome, LAD-ligation mouse model Results: In a comparative study of AAV5-, 6- and 9-luciferase (luc) in healthy farm pigs (n=5 each; 1x10 13 vgc/pig), AAV5 achieved a more homogeneous cardiac apical-basal transduction pattern than AAV6 with a higher luc activity than AAV9. In a clinically relevant endpoint driven study, we demonstrated a significant improvement in EF (+19 ± 5 %) 12 weeks after retrograde AAV5- S100A1 gene delivery compared to AAV5-luc in infarcted pigs (n=4 each; 1x10 13 vgc/pig). Moreover, S100A1 -treated pigs showed significantly less infarct extension (-0.5 ± 0.3 g vs. 5 ± 1.3 g (luc)) measured by cardiac MRI. There were no unfavorable alterations in blood chemistry or ECG. S100A1 expression was predominantly contained to the heart. The WGCNA unveiled a significant correlation between the improved EF and a suppression of inflammatory and immunological pathways (r=-0.96, p < 0.01) and between the absent infarct extension and enhanced activity of cardioprotective signaling (r=-0.82, p < 0.05). With injections of 2х10 11 vgc of AAV5- S100A1 or AAV5-gfp (n=4 each) into the remote myocardium in the mouse model, we confirmed a significant improvement in FS (+43.8 ± 8.8 %, vs. gfp) and suppression of inflammatory gene expression including i.e., IL1b or TNFa by S100A1. Conclusion: We conclude that AAV5 is suitable for S100A1-based gene therapy of post-ischemic cardiac dysfunction and that this vector/target combination can help accelerating the way towards a clinical trial. We also found novel signaling pathways that may be involved in S100A1’s therapeutic actions.