AAVone 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 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 triple plasmid transfection system. AAV9 yield in AAVone system reaches as high as 3.7×10^15 VG/L in HEK 293T cells and 2.0x10^15 in HEK 293 cells.
Advantages of AAVone system
1. Increase AAV yield up to 400%.
2. Reduce plasmid number from 3 to 1.
3.Reduce plasmid amount from 1 to 0.25 ug/1e6 cells.
4.Reduce batch variations.
5.Without need the plasmid ratio optimization steps.
6.Compact plasmid size(16±2kb) and comparable yields.
7.Comparable full-to-empty ratio.
8.Similar viral activity, capsid and genomic components.
9.Similar levels of rcAAV, hcDNAs and producer plasmid contaminations.
10.The same upstream and downstream processes, analytical release or characterization assays.
| Plasmid Backbone | Ad Genes | Rep/Cap | Genome | Selection Marker | Available AAV Serotypes |
| pAAVone | Yes | Yes | ssAAV | Kana | AAV1, AAV2, AAV2-Retro, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, AAVrh.10, and AAVrh74 |
| pAAVone-sc | Yes | Yes | scAAV | Kana |
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. 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 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.
After successful development of the mini-pHelper, we finally cooperated all the necessary elements into one plasmid (pAAVone) and developed the AAVone system. 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 . 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 2.8 kb genome and the corresponding pAAVone plasmid is 15.8 kb.
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 triple plasmid transfection system. AAV9 yield in AAVone system reaches as high as 3.7×10^15 VG/L in HEK 293T cells and 2.0×10^15 in HEK 293 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.
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 regent/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 0.2-0.4 ug/1e6 cells with PEI/DNA ratio of 2-3. At this condition, we can achieve>1.2e 15 VG/ml for AAV2-CMV-EGFP. For the high yield AAV serotypes, such as AAV1, 5, 8, 9, and AAVrh.10, AAVone system can easily achieved>1e 15 VG/ml without any optimizations.
AAV Yield In Suspension Cultured HEK 293T Cells
Serotypes | 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 | 4.90E+14 | 1.59E+14 |
160 | 6.92E+14 | 1.46E+14 | ||
160 | 7.91E+14 | 1.61E+14 | ||
400 | 8.90E+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 |
Optimization of AAVone system In Suspension Cultured HEK 293T Cells
Notes:
All titers are measured with dPCR using primers targeting EGFP.
Cell lines: AAVhigh-HEK 293T, a subclone cell lines of HEK 293 developed by AAVnerGene.
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.
On the other hand, HEK 293 cells are a more established and widely accepted cell line for GMP AAV production. They have been used in numerous clinical trials and have a history of regulatory acceptance. These cells have been extensively characterized and optimized for GMP production processes, making them a reliable choice for therapeutic AAV vector production.
AAVone also achieved high AAV yield in suspension cultured HEK 293 cells.
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 for both AAV8 and AAV9 serotypes. By reducing the input plasmid to 0.25ug/1e6 cells in the AAVone system, researchers can still achieve high AAV yields, which demonstrates the efficiency and effectiveness of this approach.
The reduced input plasmid in the AAVone system has additional benefits. It can lead to increased full-to-empty capsid ratios in both AAV8 and AAV9 vectors, 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.
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 triple plasmid systems in producing AAV2 vectors. All systems demonstrate two prominent peaks at 66S and 92S, which correspond to empty and full AAV particles, respectively. Approximately 30% of the AAV particles in each system are full capsids.
When AAVone system use less input plasmid amount(0.25ug/1e6 cells), it can lead to increased full-to-empty capsid ratios in both AAV8 and AAV9 vectors.
AAVone did not change AAV capsid ratio of different VPs, and genomic component and integrity, as well as viral potency.
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:
- Replication-Competent AAV (rcAAV): 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.
Helper Plasmid Contamination: 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.
Host Cell DNA: DNA from the host cell line used for AAV production can also be present as impurities. Host cell DNA contamination can occur if the purification process is not effective in removing host cell components.
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.
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.
Systems | ITR Titer (gc/ml) | EGFP titer (gc/ml) | EGFP/ITR(%) | rcAAV/ITR (%) | Ori/ITR (%) | Cap/ITR (%) | Rep/ITR (%) |
Triple-mini-pHelper | 5.95E+13 | 7.35E+12 | 12.36 | 0.0046 | 0.3590 | 0.1065 | 0.0363 |
Dual-V2#1 | 5.14E+13 | 6.02E+12 | 12.52 | 0.0060 | 0.2679 | 0.1260 | 0.0491 |
Dual-V2#2 | 5.25E+13 | 6.58E+12 | 16.34 | 0.0040 | 0.3189 | 0.1010 | 0.0482 |
AAVone-100% | 9.67E+13 | 1.20E+13 | 16.05 | 0.0048 | 0.2463 | 0.1835 | 0.0607 |
AAVone-66.7% | 9.47E+13 | 1.55E+13 | 19.11 | 0.0030 | 0.1952 | 0.1159 | 0.0466 |
AAVone-33.3% | 7.47E+13 | 1.81E+13 | 22.81 | 0.0026 | 0.1529 | 0.1018 | 0.0313 |
Notes:
1. AAVone-100%, AAVone-66.7%,AAVone-33.3%, means the total plasmid amounts used in transfection is 100%, 66.7% and 33.3% of the total plasmid amounts used in triple transfection
AAVnerGene offers pre-constructed pAAVone plasmids with different AAV serotypes, promoters, transgenes, as well as scAAVs. AAVnerGene also provide custom clone services to help clone any AAV capsid into our pAAVone backbones.
pAAVone-CMV-EGFP Series
| Plamsid Name | AAV Serotype | Reporter | Transgene | Genome |
| pAAVone-AAV1-CMV-EGFP | AAV1 | CMV | EGFP | ssAAV |
| pAAVone-AAV2-CMV-EGFP | AAV2 | CMV | EGFP | ssAAV |
| pAAVone-AAV3B-CMV-EGFP | AAV3B | CMV | EGFP | ssAAV |
| pAAVone-AAV4-CMV-EGFP | AAV4 | CMV | EGFP | ssAAV |
| pAAVone-AAV5-CMV-EGFP | AAV5 | CMV | EGFP | ssAAV |
| pAAVone-AAV6-CMV-EGFP | AAV6 | CMV | EGFP | ssAAV |
| pAAVone-AAV7-CMV-EGFP | AAV7 | CMV | EGFP | ssAAV |
| pAAVone-AAV8-CMV-EGFP | AAV8 | CMV | EGFP | ssAAV |
| pAAVone-AAV9-CMV-EGFP | AAV9 | CMV | EGFP | ssAAV |
| pAAVone-AAV10-CMV-EGFP | AAV10 | CMV | EGFP | ssAAV |
| pAAVone-AAV11-CMV-EGFP | AAV11 | CMV | EGFP | ssAAV |
| pAAVone-AAV12-CMV-EGFP | AAV12 | CMV | EGFP | ssAAV |
| pAAVone-AAV13-CMV-EGFP | AAV13 | CMV | EGFP | ssAAV |
| pAAVone-AAVrh.10-CMV-EGFP | AAVrh.10 | CMV | EGFP | ssAAV |
| pAAVone-AAVrh74-CMV-EGFP | AAVrh74 | CMV | EGFP | ssAAV |
| pAAVone-AAV2-Retro-CMV-EGFP | AAV2-Retro | CMV | EGFP | ssAAV |
| pAAVone-AAV-PHP.eB-CMV-EGFP | AAV-PHP.eB | CMV | EGFP | ssAAV |
pAAVone-CMV-mCherry Series
| Plamsid Name | AAV Serotype | Reporter | Transgene | Genome |
| pAAVone-AAV1-CMV-mCherry | AAV1 | CMV | mCherry | ssAAV |
| pAAVone-AAV2-CMV-mCherry | AAV2 | CMV | mCherry | ssAAV |
| pAAVone-AAV3B-CMV-mCherry | AAV3B | CMV | mCherry | ssAAV |
| pAAVone-AAV4-CMV-mCherry | AAV4 | CMV | mCherry | ssAAV |
| pAAVone-AAV5-CMV-mCherry | AAV5 | CMV | mCherry | ssAAV |
| pAAVone-AAV6-CMV-mCherry | AAV6 | CMV | mCherry | ssAAV |
| pAAVone-AAV7-CMV-mCherry | AAV7 | CMV | mCherry | ssAAV |
| pAAVone-AAV8-CMV-mCherry | AAV8 | CMV | mCherry | ssAAV |
| pAAVone-AAV9-CMV-mCherry | AAV9 | CMV | mCherry | ssAAV |
| pAAVone-AAV10-CMV-mCherry | AAV10 | CMV | mCherry | ssAAV |
| pAAVone-AAV11-CMV-mCherry | AAV11 | CMV | mCherry | ssAAV |
| pAAVone-AAV12-CMV-mCherry | AAV12 | CMV | mCherry | ssAAV |
| pAAVone-AAV13-CMV-mCherry | AAV13 | CMV | mCherry | ssAAV |
| pAAVone-AAVrh.10-CMV–mCherry | AAVrh.10 | CMV | mCherry | ssAAV |
| pAAVone-AAVrh74-CMV-mCherry | AAVrh74 | CMV | mCherry | ssAAV |
| pAAVone-AAV2-Retro-CMV-mCherry | AAV-Retro | CMV | mCherry | ssAAV |
| pAAVone-AAV-PHP.eB-CMV-mCherry | AAV-PHP.eB | CMV | mCherry | ssAAV |
pAAVone-sc-CMV-EGFP Series
| Plamsid Name | AAV Serotype | Reporter | Transgene | Genome Type |
| pAAVone-sc-AAV1-CMV-EGFP | AAV1 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV2-CMV-EGFP | AAV2 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV3B-CMV-EGFP | AAV3B | CMV | EGFP | scAAV |
| pAAVone-sc-AAV4-CMV-EGFP | AAV4 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV5-CMV-EGFP | AAV5 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV6-CMV-EGFP | AAV6 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV7-CMV-EGFP | AAV7 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV8-CMV-EGFP | AAV8 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV9-CMV-EGFP | AAV9 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV10-CMV-EGFP | AAV10 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV11-CMV-EGFP | AAV11 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV12-CMV-EGFP | AAV12 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV13-CMV-EGFP | AAV13 | CMV | EGFP | scAAV |
| pAAVone-sc-AAVrh.10-CMV-EGFP | AAVrh.10 | CMV | EGFP | scAAV |
| pAAVone-sc-AAVrh74-CMV-EGFP | AAVrh74 | CMV | EGFP | scAAV |
| pAAVone-sc-AAV2-Retro-CMV-EGFP | AAV-DJ | CMV | EGFP | scAAV |
| pAAVone-sc-AAV-PHP.eB-CMV-EGFP | AAV-PHP.eB | CMV | EGFP | scAAV |
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Its relatively compact size(16±2kb) can 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
Notes:
1.AAVnerGene provides different templates for customers to clone their transgenes into our backbones.
2.AAVnerGene also provides DNA synthesis and clone services. Please contact us if you need a custom clone service.
3. All the plasmids are kanamycin resistance(KanR).
4. A material transfer agreement(MTA) is required to use any of mini-pHelper based plasmids, including pAAVone.