AAVone system-A simple, Scalable Solution for AAV Production

AAVone System

Introduction of AAVone system

AAVone^® system is designed to streamline AAV production process, increase AAV yield, improve product consistency and reduce the cost and labor, especially for GMP grade AAV production. In the AAVone system, all the Ad helper genes (E2A, E4orf6 and VA RNA), AAV helper genes (Rep and Cap), and AAV vector genome are assembled into one plasmid and AAV vectors are simply generated by transfection one plasmid into host cells. AAVone has demonstrated impressive productivity, which is 2~4 fold higher than original triple plasmid transfection system. AAV9 yield in AAVone system reaches as high as 5.0 x 10^15 VG/L in HEK 293 cells.

Recent breakthrough-AAVone2.0

We recently developed the second-generation AAVone system (AAVone 2.0), which significantly improves the full-to-empty capsid ratio. For AAV9, AAVone 2.0 achieves approximately 70% full particles in both our HEK293one cell line and VPC2.0 cells.

AAVone2.0 Performance in Full-To-Empty Ratio:

AAV9: ~70% full particles
Most other AAVs: >50% full particles

AAVone2.0 Performance in AAV Productivity (vs AAVone1.0)

AAV9: ~50% increase
AAV1/2/6/8: up to 2× increase
AAV5: up to 4 × increase (Yield is comparable to AAV9)

AAVone2.0 is now available for testing. To request access, please contact customer@aavnergene.com.

AAVone1.0 system data

pAAVone is a compact plasmid with 13 kb oversized backbone. The AAV Rep-Cap expression cassette was positioned between E4orf6 and VA RNA, while the AAV genome was situated between the VA RNA and the bacterial backbone (bacterial origins of replication, Ori, and antibiotic resistant gene, Kana). In this configuration, the 3’ end of cap and VA RNA are closer to the left (L)-ITR and bacterial backbone is located after the right (R)-ITR. With pAAVone plasmid, AAV vectors can be produced by transfecting single-plasmid into host cells. For example, AAV2-CMV-EGFP viral particles are generated by transfecting of pAAVone-AAV2-CMV-EGFP plasmid into HEK-293T cells. Its relatively compact size make it easier to handle and produce during the manufacturing process and would not significantly reduce the plasmid yield. The compact size of the pAAVone plasmid also offers practical benefits and simplifies cloning procedures. We were able to quickly make pAAVone plasmids with different AAV serotypes, different transgenes, different AAV sizes as well as scAAV vectors, by using of standard molecular biology techniques.

pAAVone backbones

Plasmid Backbone	Ad Genes	Rep/Cap	Genome	Selection Marker	Available AAV Serotypes
pAAVone	Yes	Yes	ssAAV	Kana	AAV1, AAV2, AAV2-Retro, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAV12, AAV13, AAV-DJ, AAVrh.10, AAVrh.74, AAV-PHP.eB
pAAVone-sc	Yes	Yes	scAAV	Kana

pAAVone plasmids at AAVnerGene

AAVnerGene offers pre-constructed pAAVone plasmids with different AAV serotypes, promoters, reporters, as well as scAAVs. MTA is required for academic use, and a license agreement is required for industry use.

pAAVone-AAVs-CMV-EGFP Plasmids:

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pAAVone-AAVs-CMV-mCherry Plasmids:

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pAAVone-sc-AAVs-CMV-EGFP Plasmids:

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pAAVone plasmid yields

The compact pAAVone plasmids are easy to clone and amplify without reducing yields.

To create an efficiency single-plasmid system, it is critical to control the plasmid size. Among the three plasmids for used in traditional triple-plasmid AAV production system, the Ad Helper (pHelper) is the largest one. The common used pHelper is developed based Ad2, which has a 11.6 kb total genome with 9.3 kb of Ad genome. This plasmid is still too big for generating AAVone AAV production system. Therefore, we created the mini-pHelper, which only remains 6.1 kb of Ad2 genome and with a total plasmid size 8.4 kb. The mini-pHelper and its parental pHelper have been shown to generate similar AAV vectors in terms of the ratio of empty to full particles, as well as the AAV potency, capsid and genome components. We then created AAVdual system, in which the GOI cassette was integrated into mini-pHelper, resulting in a plasmid with a 8.4-kb backbone (pAAVdual). The AAVdual system exhibited AAV yields that were comparable to or surpassed those of the AAVtri system.

AAVone1.0

After successful development of the mini-pHelper and AAVdual, we finally cooperated all the necessary elements into one plasmid (pAAVone) and developed the AAVone system. In the AAVone1.0 system, all the Ad helper genes (E2A, E4orf6 and VA RNA), AAV helper genes (Rep and Cap), and AAV vector genome are assembled into one plasmid, which has a 13 kb backbone. The original pAAVone plasmid (pAAVone1.0) features the following structure: E4-E2-Bacterial Backbone-(L)ITR-GOI-(R)ITR-VARNA-P5-Cap-Rep-P5, with an additional P5 promoter located between VA RNA II and Cap PolyA. AAV vectors can by simply generated by transfection one plasmid into host cells. For example, AAV2-CMV-EGFP vectors can be generated by transfecting of pAAVone-AAV2-CMV-EGFP plasmid into HEK 293 cells. This AAV vector has 2.2 kb genome and the corresponding pAAVone plasmid is 15.2 kb.

We found that removing the additional P5 and VA RNA II elements in pAAVone1.0, resulted into pAAVone1.1 significantly increased AAV2 productivity by 36-50% in both HEK 293T and HEK 293 cells. Additionally, this modification improved the AAV2 full particle ratios (increasing from 28.7% to 34.4%), as determined by mass photometry analysis. Most importantly, pAAVone1.1 significantly reduced VA RNA-related impurities, which are located outside of R-ITR, compared to pAAVone.

AAVone System vs Triple Plasmid System

AAVone has demonstrated impressive results, achieving unpurified yields of over 1×10^15 viral genomes (VGs) per liter in suspension-cultured HEK-293T cells for most AAV serotypes, which is 2~4 fold higher than original pHelper based triple plasmid transfection system. AAV9 yield in AAVone system reaches as high as 5 x10^15 VG/L in HEK 293 cells

AAVone Productivity with Different Payloads

pAAVone names	AAV Genome	Payload size(Kb)	Plasmid Size(Kb)	AAV Serotype	Productivity (10^15 VG/L)
pAAVone-AAV9-CMV-EGFP	AAV-CMV-EGFP	2.2	15.2	AAV9	2.87 ± 0.18
pAAVone-AAV9-CMV-EGFP-4.0kb	AAV-CMV-EGFP-4.0K	4.0	17.0	AAV9	2.36 ± 0.28
pAAVone-AAV9-CMV-EGFP-4.6kb	AAV-CMV-EGFP-4.6K	4.6	17.6	AAV9	1.76 ± 0.65
pAAVone-AAV9-CMV-EGFP-4.7kb	AAV-CMV-EGFP-4.7K	4.7	17.7	AAV9	1.94 ± 0.02

DNA amount: 0.30-0.35 ug pDNA/1e6 cells; Cell: Suspension cultured HEK 293T Cells; Transfection Reagent: PEImax; PEI/DNA ratio=2:1

AAVone Productivity in HEK 293T Cells

AAV production in suspension culture offers scalability, cost-effectiveness, higher cell densities, efficient transfection, enhanced AAV yield, process control, and simplified downstream processing. These advantages make suspension culture an attractive option for large-scale AAV vector production to meet the increasing demand for AAV-based gene therapies and research applications.

AAVnerGene developed an high AAV titer suspension cell line AAVhigh-HEK 293T. This cell line is a subclone of the transformed human embryonic kidney cell line HEK 293T, which is highly transfectable. The cell line also constitutively expresses SV40 large T antigen. When combined with the AAVone system, these cells can produce high titers of AAV vectors. With AAVone system, you can achieve>5e 14 VG/L of crude titers for AAV2. For the high yield AAV serotypes, such as AAV1, 5, 8, 9, and AAVrh.10, AAVone system can easily achieved>1e 15 VG/L.

erotypes	pAAVone Plasmid	Scale (ml)	Crude titer (GC/L)	Purification yield(GC/L)
AAV1	pAAVone-AAV1-CMV-EGFP	130	1.83E+15	2.84E+14
AAV2	pAAVone-AAV2-CMV-EGFP	130	4.9E+14	1.59E+14
		160	6.92E+14	1.46E+14
		160	7.91E+14	1.61E+14
		400	8.9E+14	2.14E+14
AAV5	pAAVone-AAV5-CMV-EGFP	130	1.23E+15	1.65E+14
AAV6	pAAVone-AAV6-CMV-EGFP	130	2.01E+14	5.20E+13
AAV8	pAAVone-AAV8-CMV-EGFP	130	1.06E+15	1.66E+14
AAV9	pAAVone-AAV9-CMV-EGFP	130	3.70E+15	1.03E+15
		2100	2.04E+15	5.20E+14
AAVrh.10	pAAVone-AAVrh.10-CMV-EGFP	130	1.61E+15	3.42E+14

AAVone Productivity in HEK 293 Cells

In the AAVone system, optimization of several parameters is also necessary to achieve the best yield of AAV vectors. These parameters include cell density, transfection reagent/DNA ratio, and total DNA amount. By carefully adjusting these factors, researchers can enhance the efficiency and productivity of the AAVone system. In our experiences, the best conditions for AAV production is transfection at 2.5-3.5 e6 cell/ml, using DNA 0.3-0.4 ug/1e6 cells with PEIone/pDNA ratio of 0.6-0.7 in HEK 293 cells. The common used transfection reagents, such as PEImax, FactorVIR, AAVmax, are all worked well with AAVone system.

Advantages of using suspension cells

Scalability: Suspension culture allows for easy scale-up of AAV production. Bioreactors of various sizes can be employed to increase production volumes, making it feasible to meet the growing demand for AAV vectors. This scalability is particularly important for clinical applications and large-scale production needs.
Cost-effectiveness: Suspension culture systems, especially those utilizing disposable bioreactors, can be more cost-effective compared to adherent cell culture methods. They eliminate the need for expensive culture vessels, such as culture flasks or roller bottles, and reduce labor and cleaning requirements. This makes suspension culture a more economical choice for large-scale AAV production.
Higher Cell Density: Suspension culture allows for higher cell densities compared to adherent culture systems. Cells can be grown in a three-dimensional environment, allowing for better nutrient and oxygen availability. This promotes increased AAV vector production as the cells can reach higher densities, leading to improved productivity.
Efficient Transfection: Suspension culture provides a favorable environment for efficient transfection of cells with the AAV vector components. The use of transfection reagents or electroporation methods can result in high transfection efficiencies, leading to increased AAV production.
Enhanced AAV Yield: Suspension culture systems offer the potential for improved AAV vector yield. With optimized process parameters, including agitation, aeration, and nutrient supply, AAV production can be maximized, resulting in higher yields of AAV vectors per unit volume.
Process Control: Suspension culture allows for better control and monitoring of critical process parameters. Parameters such as pH, temperature, dissolved oxygen, and nutrient concentrations can be tightly regulated in bioreactors, ensuring optimal conditions for cell growth and AAV vector production. This facilitates consistent and reproducible AAV vector production.
Simplified Downstream Processing: AAV vectors produced in suspension culture can be readily harvested and processed downstream. The absence of adherent cells simplifies the separation and purification steps, reducing the complexity and time required for downstream processing.

The use of HEK 293T cells for GMP-level AAV production might face challenges related to regulatory acceptance due to the presence of the SV40 T antigen. Regulatory agencies have stringent requirements for cell lines used in GMP manufacturing to ensure safety, consistency, and quality of therapeutic products. In general, the use of cell lines with viral or oncogenic elements, such as the SV40 T antigen, can raise concerns about potential risks and regulatory approvals. While HEK 293T cells, which are derived from HEK 293 cells, do offer advantages in terms of transfection efficiency and plasmid replication, their use in GMP production could require careful scrutiny and validation to ensure that any potential risks associated with the T antigen are mitigated.

In contrast, HEK293 cells are a well-established and widely accepted platform for cGMP AAV production, having been used in numerous clinical trials with a strong history of regulatory acceptance. These cells are extensively characterized and optimized for GMP manufacturing processes, making them a reliable choice for therapeutic AAV vector production. Notably, the AAVone system achieves high AAV yields in suspension-cultured HEK293 cells. In addition, AAVnerGene has developed a proprietary HEK293 cell line, HEK293one, specifically optimized for AAVone-based production.

Fully adapted to FreeStyle F17/Viral Production Medium, PEAK/PRIME, and Pro293 media
High AAV productivity across multiple production platforms and compatibility with various AAV serotypes
- AAV9: up to 5e12 vg/ml in AAVone system
- AAV8: over 2e12 vg/ml in AAVone system
- AAV2: over 1e12 vg/ml in AAVone system
- AAV6: over 1e12 vg/ml in AAVone system
Scalable for large-volume AAV production
Compatible with multiple transfection reagents, especially our PEIone transfection reagent
Available for research use under license, cGMP cell banks are currently in preparation.

Furthermore, commercially available HEK293 cell lines, such as Thermo Fisher’s VPC2.0, also demonstrate high productivity when combined with the AAVone system.

Here, we report that the productivity of the AAVone system can be further increased to reach 5E+15 vg/L of culture in commonly used HEK 293 cells, such as VPC2.0. To optimize production using the AAVone system, we initially tested the productivity of four different transfection reagents with varying transfection viable cell density (VCD) and plasmid DNA (pDNA) amounts in three different HEK293-based cell lines. Consistent with our previous report, the optimal total pDNA amount needed is significantly lower than that used in the triple plasmid system. We then selected potential combinations of transfection reagents with different culture media, enhancers, and boosters to conduct production at the same transfection VCD and pDNA amount. We identified one specific medium in combination with a specific transfection reagent and two booster reagents from different sources that consistently yielded higher productivity. The titer in the crude cell lysate can reach up to 5.8E+12 vg/mL of culture using a pAAVone-AAV9-CMV-EGFP construct in small shake flasks. Further productivity evaluation with other AAV serotypes and transgenes, as well as scale-up production and characterization of the AAV vectors produced by this protocol, is ongoing. Taken together, process optimization is crucial for new AAV production systems, including AAVone system. The high productivity of AAVone system could significantly reduce the cost of high-quality AAV production, especially for large-scale clinical use, potentially increasing patient accessibility to AAV gene therapy drugs.

In the triple-plasmid systems, a relatively high amount of input total plasmids (1 ug/1e6 cells) is typically needed to achieve high AAV yields. Surprising, the AAVone system allows for a substantial reduction in the amount of input plasmid while still increasing AAV yield. By reducing the input plasmid to 0.25 ug/1e6 cells in the AAVone system, researchers can still achieve high AAV yields, which demonstrates the efficiency and effectiveness of this approach. Overall, AAVone system enables the production of AAV vectors using only about one-quarter to one-half of the pDNA normally required in conventional approaches.

The reduced input plasmid in the AAVone system has additional benefits. It can lead to increased full-to-empty capsid ratios, which are desirable for obtaining higher-quality AAV vectors with a higher proportion of functional, transgene-carrying capsids. Moreover, reduced input plasmid can help minimize potential DNA contaminations in the AAVone system.

AAVone has similar full/empty with tri-plasmid systems at high pDNA conditions

When utilizing the same quantity of input plasmids (0.75ug/1e6 cells) to produce AAV vectors, the AAVone system exhibits a similar full-to-empty ratio compared to the tri-plasmid systems in producing AAV2 vectors. All systems demonstrate two prominent peaks at 66S and 92S, which correspond to empty and full AAV particles of 2.8 kb AAV-CMV-EGFP genomes, respectively. Approximately 30% of the AAV particles in each system are full capsids.

AAVone has improved full/empty when using low pDNA

When AAVone system use less input plasmid amount (0.25 ug/1e6 cells), it can lead to increased full-to-empty capsid ratios in both AAV8 and AAV9 vectors, which exhibited a ~10% increase in crude samples. Moreover, reduced pDNA can help minimize potential plasmid-related impurities in the AAVone system.

AAV vector quality

Compared with triple plasmid systems(mTri-plasmid and pTri-plasmid), AAVone did not change AAV capsid ratio of different VPs, and genomic component and integrity, as well as viral potency.

AAV in vivo potency

mTri-plsasmid: mini-pHelper based triple plasmid system; pTri-plasmid: pHelper based triple plasmid system

The majority of ITRs in the vectors produced by either systems are 140-150 bp in length at both the L-ITR and R-ITR

ITR Integrity

The majority of ITRs in the vectors produced by either systems are 140-150 bp in length at both the L-ITR and R-ITR

Genome Integrity

AAVone generated AAV vector with increased genomic integrity

Incomplete Genomes

AAVone generated AAV vectors with lower percentage of snap-back genomes

During AAV production, it is possible for various genomic impurities to be present in the final AAV DNA product. These impurities can arise from different sources and may impact the quality and safety of the AAV vector. Here are some common genomic impurities associated with AAV DNA production. In the AAVone system, where the Rep and Cap genes, as well as the inverted terminal repeats (ITRs), are present in the same plasmid, the issue of replication-competent AAV (rcAAV) becomes a major concern. However, by using the qPCR and PacBio Sequencing, we found comparable levels of rcAAV, Rep, Cap in the AAVone system and traditional triple plasmid system. Moreover, AAVone system has significantly lower plasmid backbone Ori genomes.

Producer Plasmid Related Contaminations

The presence of residual helper plasmid genomes used during the production process can be a genomic impurity. This can occur if the helper plasmid is not completely eliminated during downstream purification steps or the related genomes are packaged into AAV vectors

In the tri-plasmid systems, the dominant contaminants originate from the backbone of the pAAV-GOI plasmid, while trans plasmid sequences are packaged at lower levels. One major difference between AAVone and tri-plasmid system is the oversized 13 kb backbone, which not only includes the regular plasmid backbone (bacterial ORIs and selection maker genes), but also contains the rep, cap, E2A, E4orf6 and VA RNA genes

We employed SMRT sequencing to thoroughly examine the potential impurities originating from plasmids in the AAVone and tri-plasmid systems and found:.

The two ITRs are the primary cis elements responsible for AAV packaging.
The abundance of pDNA impurities is primarily determined by their proximity to the ITR regions;
AAVone system alter the prominent impurities from bacterial backbones to VA RNA.
Rep, Cap, E2, E4 impurities are comparable or slightly increased in AAVone system
No notable differences in host cell (hc) DNA contaminants among the AAV vectors produced by the AAVone and triple plasmid system

The overall total genomic contamination in the AAVone system does not exhibit a general increase in undesired genomic contaminants, and may even show a decrease in frequency.

Host Cell DNAs(hcDNAs)

DNA from the host cell line used for AAV production can also be present as impurities. hcDNA contamination can occur if the purification process is not effective in removing host cell components. Overall, there was no notable difference in hcDNA contamination among the AAV vectors produced by the three production systems, each showing approximately 3% of the total reads. The normalized densities of reads per chromosome suggested an overall random distribution among all three AAV samples.

Replication-Competent AAVs(rcAAVs)

One of the critical impurities in AAV production is the presence of rcAAV particles. These are AAV vectors that contain a functional Rep gene and can replicate in target cells, leading to potential adverse effects or interference with the intended gene delivery. In tri-plasmid systems, rcAAVs arise through the recombination of DNAs carrying ITRs with rep and cap sequences originating from the pRep-Cap plasmid. In AAVone system, rep, cap, and ITRs are coexist within the same plasmid.The FDA requires that AAV products be screened for the presence of possible rcAAVs. Current vector production systems in use have been optimized to reduce rcAAV to levels ≤1 rcAAV per 10^8 vector genomes. A cell-based rcAAV assay performed by an external contract research organization indicated that rcAAV levels are below one per 10^9 vector genomes in the AAV vectors generated by AAVone system. After three rounds of amplification with Ad, all three flasks in the 10^9 vector genome group showed no presence of rcAAV. And only one out of three samples tested positive for rcAAV in the group with 10^10 vector genomes. This underscores the AAVone system can effective produce safe AAV vectors with low rcAAV.

Other Contaminants

Additional impurities can arise from various sources, such as host cell proteins, media components, endotoxins, and process-related contaminants. These impurities can affect the safety, efficacy, and quality of the AAV vectors. Thorough purification steps and quality control measures are implemented to remove or minimize these contaminants.

This AAVone-ORF system is designed to use our AAVone single-plasmid AAV production system to efficiently produce AAV vectors. Take advantage of our innovative AAVone system and the comprehensive ORFoeme collections, which contains complete human or mouse ORFs. Researchers only need to provide us the information of gene of interest (GOI), AAV serotype and promoter. After a straightforward one-step reaction, the final AAVone packaging plasmid carrying the GOI will be constructed for AAV production, eliminating complex multi-step procedures. The AAVone-ORF system provides an efficient, one-step solution to advance your AAV research projects more quickly and effectively.

How to do it?

By combining our pAAVone-AAVs-Pro-CcdB destination plasmids and the ORFeome collections, we can efficiently generate pAAVone-AAVs-Pro-GOI packaging plasmids, which can be used to package corresponding AAV vectors directly. pAAVone-AAVs-Pro-CcdB destination plasmids carry CcdB gene flanked by attR1 and attR2, as well as AAV cap genes. The CcdB gene acts as a toxin to select for successful recombination events in bacterial cells, killing those that do not carry the desired recombinant plasmid. The ORFeome collections provides a comprehensive resource for studying genes, particularly focusing on the complete set of open reading frames (ORFs) within a genome. Each enter vector in ORFeome collections carrys an ORF flanked by attL1 and attL2. In the Gateway cloning, the entry clone is then recombined with the destination vector in an LR reaction between attL and attR sites. This results in the transfer of the gene of interest to the destination vector, replacing the ccdB gene.

Step1: Choose your AAV serotypes

We offer pAAVone-CcdB destination vectors with common used AAV serotypes, such as:

Step2: Choose your promoters

The premade pAAVone-CcdB destination vectors carry different promoters, such as CMV, hSyn, CaMKIIα, GFAP, EF1a, CAG. We can customize constructs destination vectors with promoters other promoters.

Step3: Determine the Entry clone

The Entry clone is how and where you start your experiment, as it contains your gene of interest or DNA fragment flanked by attL sequences, which are then used to recombine with attR sequences to create your desired pAAVOne packaging clone. Choose your gene of interest from the ORFeome collections:ORFeome Collaboration Complete Human collection ORFeome Collaboration Complete Mouse collection

Step4: Making the pAAVone-ORF packaging plasmids

The recombination reaction is mediated by LR Clonase enzyme mix, which contains the protein machinery necessary to excise the gene of interest from the Entry clone and integrate it into the Destination vector, which then becomes your packaging clone.

Step5: Producing AAV vectors with AAVone system

AAV vectors are simply produced by transfection of pAAVone packaging vectors into host cells, such as HEK 293T and HEK 293 cells. We offer AAVone packaging services at different production scale (1e12~1e16 VG), in both suspension and adherent cells and provide comprehensive AAV characterization services.

Step6: Deliver the plasmids and AAV vectors to customers.

After production and quality control are completed, the plasmids and AAV vectors are prepared for delivery to customers. Following delivery, technical support is provided to assist with any questions or troubleshooting related to vector handling, cloning, or transduction procedures.

Please contact AAVnerGene(customer@aavnergene.com) for more information and quote.

AAVone Paper

Rongze Yang et al., AAVone: A Cost-Effective, Single-Plasmid Solution for Efficient AAV Production with Reduced DNA Impurities. https://www.sciencedirect.com/science/article/pii/S2162253125001179