AAVnerGene offers a valuable service by providing consistent, high-quality, and affordable AAV vectors for gene delivery experiments. Gene therapy using AAV vectors has indeed shown great promise for treating various diseases, and the ability to compare different AAV vectors is crucial for optimizing their therapeutic potential.
The challenges associated with generating, controlling, and qualifying AAV vectors are well-recognized in the field. The variability in production methods, purification processes, and capsid characteristics can lead to differences in vector performance. Having a reliable source for AAV vectors can significantly aid researchers, especially those who are new to the AAV field, in obtaining consistent and trustworthy tools for their experiments.
By offering batch AAV vectors for comparison, AAVnerGene can help researchers save time and resources that would otherwise be spent on vector production and validation. This approach also ensures that researchers can focus more on the experimental aspects of their work rather than grappling with vector-related challenges.
However, it’s important for AAVnerGene to maintain high standards of quality control to ensure the vectors they provide are reliable and accurately characterized. Consistency across batches is critical, as any variations in vector quality could introduce confounding factors into experimental results.
AAVnerGene offers a variety of packaging systems for AAV vectors, catering to different experimental needs and preferences. Each of these systems likely has its own advantages and considerations, providing researchers with options to choose the most suitable method for their specific gene delivery experiments.
Traditional Triple Plasmid System: This is a well-established method for AAV vector production. It typically involves using three separate plasmids: one containing the gene of interest (pGOI), one with the AAV rep and cap genes(pRep-Cap) required for packaging, and a third with the adenoviral helper(pHelper) genes to facilitate AAV replication and packaging.
Mini-pHelper Based Triple Plasmid System: This system streamlines the traditional triple plasmid approach. The “mini-pHelper” refers to a modified smaller version of the adenoviral helper genes that’s more efficient and easier to work with.
Dual V2 Plasmid System: The dual V2 plasmid system combine the mini-pHelper and pGOI in one plasmid, and remain the pRep-Cap alone. This system offers a flexible and efficient approach to generate AAV vectors, as users only need put the GOI into our mini-pHelper-AAV backbone and package AAV with different serotypes.
AAVone Single Plasmid System: In this 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 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.
Each of these systems comes with its own set of protocols, advantages, and potential limitations. Researchers would need to consider factors like vector yield, ease of use, and compatibility with their experimental setup when choosing the most suitable system.
Sum of plasmid Genome Size(kb)
Number of plasmids
Dual-plasmid system V2
AAVone (All-in-one system)
AAVnerGene offers a range of cell lines for AAV vector production, both adherent and suspension, which allows researchers to choose the most suitable system for their production needs. Here’s a summary of the cell lines you provide:
HEK 293 Cells: These are a widely used cell line that originated from human embryonic kidney cells. They are commonly used for AAV vector production due to their ability to express the adenoviral gene E1, which aids in the packaging of AAV vectors.
HEK 293T Cells: HEK 293T cells are a derivative of HEK 293 cells that express the SV40 T antigen. This results in increased cell cycling and higher plasmid amplification upon transfection, leading to improved transfection efficiency and higher vector production.
- HEK 293 Cells (Suspension): These are suspension-adapted HEK 293 cells derived from adherent HEK 293 cells. Suspension culture systems offer advantages for large-scale production due to ease of scaling and better mixing compared to adherent cultures. HEK 293 cells in suspension culture have become a widely used platform for the production of GMP (Good Manufacturing Practice) grade AAV vectors.
- HEK 293T Cells (Suspension): These are suspension-adapted HEK 293T cells derived from adherent HEK 293T cells. The benefits of HEK 293T cells, including their high transfection efficiency, are likely retained in the suspension-adapted version.
By offering both adherent and suspension cell lines, AAVnerGene addresses the diverse requirements of researchers who may have different preferences and needs for AAV vector production. Adherent cells are often suitable for smaller-scale experiments or when adherent culture methods are preferred, while suspension cells are advantageous for larger-scale production intended for research or potential clinical applications.
The purification of AAV vectors is a critical step in their production for both research and clinical applications. Different methods are employed to achieve high-quality and pure AAV vector preparations.
CsCl Gradients (Cesium Chloride Density Gradient Ultracentrifugation): CsCl gradients have been a conventional method for purifying AAV vectors. This technique relies on the differences in density among AAV particles, empty capsids, and contaminants. By subjecting the AAV mixture to ultracentrifugation in a CsCl density gradient, the components separate into distinct bands based on their density. This visual distinction of distinct bands aids in identifying and collecting the desired AAV particles and separating them from contaminants.
Iodixanol Gradients: Iodixanol is used to create a density gradient similar to CsCl gradients. The gradient composition and steps are carefully designed to separate contaminants from impure AAV preparations. Different iodixanol concentrations help separate empty capsids and contaminants from genome-containing virions. The 15% iodixanol step has 1M NaCl to destabilize ionic interactions between macromolecules. The 40% and 25% steps are used to remove contaminants with lower densities, including empty capsids. The 60% step serves as a cushion for genome-containing virions. Phenol red is often added to visualize the distinct steps in the gradient. One of the limitations of using iodixanol gradients for AAV purification is the inability to visually observe distinct bands in the gradient as you would with CsCl gradients. The lack of visible bands can make it more challenging to precisely identify and collect specific fractions during the purification process.
AAVx Resin: AAVx resin is a purification technology that exploits the binding reactivity of certain AAV serotypes to the resin. This resin facilitates one-step purification of AAV vectors from crude material. It offers several advantages:
- High Purity and Yield: AAVx resin allows for efficient purification, resulting in high purity and yield of AAV vectors.
- High Specificity and Capacity: The resin’s specificity ensures that only AAV vectors bind, reducing the need for additional purification steps.
- Scalability: AAVx resin can be scaled up easily, making it suitable for both research and production applications.
- Non-Animal Derived: The resin being non-animal derived is significant, especially for processes intended for clinical applications.
Choosing the right purification method depends on various factors, including the specific AAV serotype, the desired purity level, the intended application (research or clinical), and the available resources. The CsCl and iodixanol gradient methods are traditional but effective approaches, while the AAVx resin offers the advantages of simplicity and scalability. Overall, the purification methods you’ve described highlight the ongoing efforts in the field of AAV vector production to optimize purity, yield, and safety while streamlining the process for various applications.
Scale, Price and Time
AAV packaging and production: We will package your vector into one of our AAV serotypes, followed by two rounds of CsCl gradient or one round of iodixanol gradient ultracentrifugation purification and tittering with SYBR Green method, using primers targeting the ITRs.
1 X100 ul
2 X 250 ul
2 X 500 ul
2 X 1000 ul
5 X 1000 ul
10 X 1000 ul
20 X 1000 ul
50 X 1000 ul
100 X 1000 ul
- All the prices are just for AAV packaging services. Customer needs to provide enough amount of transgene plasmids (0.5mg/10e13 GC). We can help do Midiprep or Maxiprep with additional charge(See Gene synthesis and Clone).
- GC means Genome Copy. The titer is measured using primers targeting ITRs with SYBR method. Pricing may vary for the viral tittering with gene specific primers and ddPCR, as larger production scales are needed to achieve viral titers with these methods. The titers are based on our typical productions of common AAV serotypes and transfer vectors. Usually, our delivery titers are higher. However, for some serotypes or transfer vectors in some rare cases, the titer can’t be reached, we reserve the right to discuss with the customers and cancel the orders. For the customer special capsids, we can’t grantee the titer is over 1e13 gc/ml.
- Our regular purification method is CsCl. We can also use Iodixanol and AAVx resin to purify the AAV vectors if customer prefers.
- If the size of transgene is over 5.0 kb, additional 50%-100% services fee may charge depending on the yield of each vector. We reserve the option to cancel the order if the efficiency is too low.
- Our buffer is 0.001% F-68/DPBS with additional 150mM NaCl. Please let us know if you have your own buffer system.
There are hundreds of AAV capsids published, including well known AAV serotypes (AAV1-13), engineered capsids and other native capsids isolated from human, monkey or other species. These versatile AAV serotypes provide us different tropism for specific cells in vitro and in vivo. For your specific purpose, the researcher needs to choose the best AAV capsids to performer for your projects.
AAVnerGene has such an extensive collection of over 1000 different AAV capsids available for research and applications. This diversity provides researchers with a remarkable resource for selecting the most suitable AAV vectors for their specific projects.
- For researchers who have identified specific capsid names or sequences, AAVnerGene offers capsid cloning services and packaged into AAV vectors.
- For researchers who are unsure about which capsid to choose for their specific research goals, AAVnerGene offers AAV serotype testing kits and ATHENA I capsid platform, presumably tools that help researchers assess the transduction efficiency and tropism of different capsids.
- For researchers who want to discovery novel AAV capsids, AAVnerGen offers premade ATHENA II capsid libraries and ATHENA II capsid platform to help researchers evolute their our capsids.
AAVnerGene is committed to providing comprehensive support to researchers at every stage of their projects involving AAV capsids. Whether researchers are utilizing established capsids, exploring new ones, or seeking assistance with various AAV-related services, AAVnerGene is well-equipped to cater to your needs.
Here is a list of AAV capsids, including native serotypes, engineered variants, and species-specific capsids.
1. Native AAV Serotypes:
- AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13
2. AAV Isolated from Human:
- Isolation AAV-Hu.10, AAV-Hu.37, AAV-Hu.52, AAV-Hu.58, etc.
- Isolation AAVhu68, AAVhu69, AAVhu70, AAVhu71.74 ,AAVhu74,etc.
- Isolation hu.T17, hu.T32, hu.T40, hu.T41 etc.
- Isolation Hu.S17, PAK56, Hu.LG15,etc.
- Isolation hu.Lvr01, hu.Lvr02, hu.Lvr03, hu.Lvr04, hu.Lvr05, hu.Lvr06 etc.
- Isolation CVR_1, CVR_2, CVR_3, CVR_4, CVR_5, CVR_6, CVR_7, etc.
- Isolation JBL1,JBL2, JBL3, JBL4,JBL5, etc.
- Isolation JBB1, JBB2, JBB3, JBB4, JBB6, JBB7, JBB8, JBB9, JBB11, JBB12, JBB13, etc
- Isolation CONB23, CONB36, CONB37, CONB39, CONS3, CONS6 etc.
3. AAV Isolated from non-human primate:
- AAVbb.1,AAVbb.2, AAVcy.2,AAVcy.3,AAVcy.4,AAVcy.5,AAVcy.6.
- AAVpi.1, AAVpi.2, AAVpi.3.
- AAVrh.2,AAVrh.8, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.14, AAVrh.16, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.22, AAVrh.37, etc
AAVrh74, AAVrh75, AAVrh76, AAVrh77, AAVrh78, AAVrh79, AAVrh81, etc.
- KN01_S1, KN02_S2, KN03_S3, KN04_S4, KN05_S5, KN06_S6, etc.
4. AAV Isolated from other species:
- AAV Isolated from Rat: AAV-ra.1, stain YY.12, YY.25, YY.54, YY.78, YY.80, YY.93, XM.70, GZ.512, HD.16,HD.20, HD.43, HD.94, MLP.6, MLP.26
- AAV isolated from Porcine/pig: AAVpo1, AAVpo2.1, AAVpo4, AAVpo5, AAVpo6, AAVpo7, AAVpo8.
- AAV-Isolated from Goat: AAV-Go1
- AAV isolated from Avian: Strain VR-865, DA-1, YZ-1, ZN1,BR_DF12, RS/BR/15/1R, GA/1360/1994.
- AAV-isolated from Bat: Strain 09YN, 1285, 10HB, 07YN ,YNM.
- AAV Isolated from Bovine: BAAV, isolate BSRI1
5.Engineering AAV capsids
- Tyrosine (Y) to phenylalanine (F):
- AAV2: Y252F, Y272F, Y444F, Y500F, Y700F, Y704F,Y730F
- AAV3: Y701F, Y705F and Y731F
- AAV5: Y263F, Y719F
- AAV6: Y445F, F731F
- AAV8: Y447, 733F
- AAV9: Y446F, Y731F
- Tissue specific AAV capsids
- AAV8Muscle: AAVmyo, AAVmyo2, AAVmyo3, myoAAV-1A, myoAAV-2A, myoAAV-3A, myoAAV-4A, AAV587MTP, AAVM41, AAV9.45, AAV9.61
- Lung: AAV2-ESGHGYF, AAV2H22, AAV6.2FF, AAV2.5T, AAV9.452sub.LUNG1, AAV5-PK2
- Retina: AAV2-HBKO, AAV2-7M8, AAV2.GL, AAV2.NN, AAV44.9, AAV44.9(E531D), AAV8BP2, AAV6-K531E-R576Q-K493S-K459S, AAV9.GL, AAV9.NN ,ShH10
- Liver: AAVhum.8, AAV-L2-10, AAV3B-DE5, AAV-KP1, AAV-DJ, AAV-DJ-K137R/T251A/S503A, AAV3-S663V-T492V, AAVrh10, Spark100, AAVrh74, AAV-SL65
- Crossing blood brain barrier(BBB): AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV.CAP-B10, AAV-F, AAV-S, AAV-PHP.Cs, AAV-PHP.C2, AAV 9P31, AAV-MaCPNS1 ,AAV-MaCPNS2 ,AAV.CPP.16, AAV.CPP.21, AAV9.HR, AAV-B1, AAV/Olig001 ,AAV-AS , AAV-BR1, AAV-ShH10, AAV-ShH10Y445F, AAV Retrograde, AAV1/2 hybrid, AAV.GTX, AAV-Cap-B22, PHP.B1, PHP.B2, PHP.B3, AAV1RX, AAV-True Type (AAV-TT), AAV-PHP.V ,AAV-PHP.C , AAV-PHP.N, AAV9_A2, AAV1_P5, VCAP-101, d VCAP-102, AAV8-THR.
Checklist for AAV packaging
ITRs: Digestion by SmaI to make sure your transgene plasmids has two ITRs.
AAV size: Confirm your AAV vectors size between two ITRS(including ITRs) is less than 4.7kb. AAV size between 4.7-5.2kb are still packageable, but have low yield and high partial genomic containing particles. AAV size less than 2.5 would package both monomer and dimmer genomes.
Amount of AAV vectors: Plan according you experiment design and produce at least 2X AAV vectors. Usually, our titer is measured using primers targeting ITRs with SYBR method. Titer with ddPCR method may be lower than SYBR method.
Serotypes: Select the AAV variants you want to use in your experiments.
Control AAV vectors: Control AAV vectors play a crucial role in experimental design and data interpretation. Control vectors are used as references to assess the effects of the experimental AAV vectors and to distinguish between specific effects and background noise. They provide a baseline for comparison and help ensure the accuracy and reliability of experimental results.
Empty AAV vectors: Do you need the by-products empty AAV or real empty AAV vectors without any genetic materials as control?
Purification method: Our regular purification method is CsCl. We can also use Iodixanol and AAVx resin to purify the AAV vectors if customer prefers.
Buffer: Our buffer is 0.001% F-68/DPBS with additional 150mM NaCl. Please let us know if you have your own buffer system.
Additional Tests: Check whether you need additional tests besides titers, such as purity, endotoxin, sterility, Mycoplasma, AAV Capsid quantification with Elisa, Empty/Full Ratio, etc.
AAV QC tests
|Catalog||QC||Method||Turnaround Time||Price||Sample Requirement|
|AQC001||Genome Titration||ddPCR/qPCR||3-5 day||$200/sample||Primer/probe sequences; ~20μl of samples|
|AQC002||Capsid Titration||ELISA||3-5 day||$400/sample||Serotypes and buffers; ~20μl of samples|
|AQC003||Purity Analysis||SDS-PAGE||3-5 day||$200/sample||~20μl of sample with titer >1E+12vg/ml|
|AQC004||Full/Empty Ratio||AUC||3-5 day||$2000/sample||~400μl of sample with titer >1E+12vg/ml|
|AQC005||Endotoxin||LAL assay||3-5 day||$200/sample||~20μl of samples|
|AQC006||Endotoxin Removal||Resin-PMB||3-5 day||$200/sample||~20μl of samples|
|AQC007||Bioburden Testing||Direct plating||3-5 day||$200/sample||~20μl of samples|
|AQC008||Vector Genome Identity||PacBio Seq||4 weeks||Request||~100μl of sample with titer >1E+12vg/ml|
|PCR||3-5 day||$200/sample||~20μl of samples|