GI Partners Acquires CDMO and Cell Solutions
As Rose BioSolutions, the established CDMO and Cell Solutions businesses continue to support the biotechnology ecosystem with cell sourcing capabilities and CDMO services to accelerate your advanced therapy from development to delivery.
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AAV Packaging Services
Below is a list of the AAV packaging services that we provide. We are also happy to customize your order if a different volume, titer, or purification is needed (not including plasmid and cloning - additional two weeks for plasmid, and two weeks for cloning.) Inquire about synthesis.
| AAV Packaging Services | Titer | Standard Timeline* | |
|---|---|---|---|
| Crude (for in vitro) | Small-scale AAV packaging | >5x1012 GC/mL, 500 µL, crude sample (1 x 500 µL) | 2-3 Weeks |
| Routine packaging -500ul | >1.00x 1013 GC/mL, purified samples (2 x 250 µL) >5x1012 GC/mL for AAV2, AAV6** | ||
| Purified (for in vivo) | Routine packaging -1 mL | >1.00x1013 GC/mL, purified samples (4 x 250 µL) 5x1012 GC/mL for AAV2, AAV6** | |
| Large-scale AAV packaging - > 1 mL | >1.00x1013 GC/mL, purified samples (custom aliquoting) 5x1012 GC/mL for AAV2, AAV6** | ||
| Preclinical AAV packaging | >2x1013 GC/mL | ||
| Clinical-grade | CGMP AAV manufacturing | Total yield up to 1017 GC | View price and estimated timeline information |
GC = genome copies (physical titer)
Charles River will make commercially reasonable efforts to achieve the requested titer in a single production run.
* Timelines do not include cloning and synthesis
** AA2 and AAV6 are considered low producers
Please see the FAQ section at the bottom of this page for more information.
Quality Control
We use a validated assay for measuring physical titer (genome copies) for all AAV packaging services using qPCR with primers targeting the ITRs present in the viral genome and quantified by comparison to a standard curve of a plasmid sample of known concentration.
Custom AAV Vector Design
Charles River offers a broad range of custom AAV cloning services at a reasonable cost and with a quick turnaround time. Our highly experienced scientists are ready to tackle difficult cloning projects, including clones with high GC content or highly repetitive sequences. Please contact us with your project details and our team will get in touch as soon as possible with an estimated price and projected turnaround time. You can also visit our Molecular Cloning page for more information.
Recombinant AAV Packaging
AAV belongs to the Dependovirus genus, meaning that it depends on a co-infecting helper virus (usually adenovirus) for productive infection to occur. Additionally, the recombinant AAV genome has two essential genes removed to prevent integration and replication to make AAV a safe and effective tool for gene delivery. Therefore, in order to generate more AAV particles, essential genes must be provided in trans.
The triple transfection method involves co-transfecting the packaging cell line (usually HEK293T) with the recombinant AAV plasmid containing the gene of interest (GOI), a plasmid containing the essential rep and cap genes, and a third adenovirus-derived helper plasmid supplying genes needed for replication. For our large-scale and preclinical AAV packaging services, the AAV particles are purified using IDX gradient ultracentrifugation to remove impurities and empty capsids. Chromatography purification is also available depending on your needs. Viral titer is then determined using qPCR with quantification based on the inverted terminal repeats (ITRs) present in the viral genome. The AAV particles are then shipped overnight and can be used immediately for in vitro or in vivo research.
Frequently Asked Questions (FAQs) About AAV Packaging Services
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Why choose AAV?
Although AAV has a high prevalence in humans, it is non-pathogenic and elicits a very mild immune response. Wild-type AAV is able to integrate into the host genome at the AAVS1 loci, however, recombinant AAV lacks two essential genes for viral integration and replication. As a result, recombinant AAV remains primarily episomal and can persist in non-dividing cells for long periods of time. Due to these characteristics, along with the ability to target specific tissue types, recombinant AAV has become one of the main viral vectors used for research and gene therapy applications. Note: We offer only recombinant AAV products and services. We do not work with or sell wild-type AAV.
Advantages of using AAV:
- Very low immunogenicity
- Tissue specific targeting by choice of serotype
- Non-integrating
- Potential for long-term gene expression
Comparison to Other VirusesCharacteristic AAV Adenovirus Lentivirus Genome 4.8 kb (ssDNA) 36 kb (dsDNA) 9 kb (ssRNA) Packaging capacity 4.7 kb 7.5 kb 9 kb Infection Most dividing and non-dividing cells Most dividing and non-dividing cells Most dividing and non-dividing cells Transduction efficiency Moderate High Moderate Integration Non-integrating Non-integrating Integrating Expression Transient or stable Transient Stable Immunogenicity Very low High Low Biosafety BSL-1 BSL-2 BSL-2 NOTE: Please see our Lentivirus Packaging page for more information.
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Which AAV serotype should I use?
The tissue specificity of AAV is determined by the viral capsid serotype. This specificity allows you to target your gene of interest to certain tissues and cells, so it is critical to select the right AAV serotype to ensure optimal gene delivery. Below is our list of recommended AAV serotypes for different tissue types.
Tissue type Recommended AAV Serotypes Muscle AAV1, AAV6, AAV8, AAV9 Liver AAV8, AAV9, AAV-DJ Lung AAV5, AAV6, AAV9 Central Nervous System AAV1, AAV5, AAV8, AAV9, AAV-DJ Retina AAV1, AAV2, AAV5, AAV8 Pancreas AAV8 Kidney AAV2, AAV9 Heart AAV1, AAV8, AAV9 -
How much plasmid DNA do I need to provide for AAV packaging?
If you will be ordering a clone that you want packaged in AAV, please provide us with the RefSeq number or Charles River catalog number. If you will be sending us a plasmid, please refer to the table below. When possible, we recommend sending DNA obtained from an endo-free maxiprep. If you do not have the recommended amount of plasmid for the desired AAV service, we can prepare endotoxin-free DNA from your sample for an additional fee.
AAV services Amount of plasmid to send Cloning 5-10 µg at conc. of >0.5 µg/µL Transformation (DNA preps) >1 µg Small-scale AAV packaging
Titer: 5×1012 GC/mL>150 µg Prior to sending us a transfer plasmid for AAV packaging, we strongly recommend that you check the integrity of your plasmid by restriction digestion to ensure there are no issues during AAV packaging. This can be done by performing a restriction enzyme digest with SmaI or XmaI, which will cut at a site within each ITR to remove the insert and linearize your plasmid. If you notice excessive amounts of linearized full-length plasmid, recombination has occurred and this plasmid is unlikely to package successfully into AAV. If you are unable to check the integrity of your plasmid, we can perform the digestion on your plasmids for an additional fee.
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What is the AAV packaging capacity?
The primary limitation of AAV is its relatively small packaging capacity (~4.7 kb from ITR to ITR). AAV packaging capacity is further reduced if you are using self-complementary AAV (~2.4 kb). This can be especially limiting when working with a large gene of interest. To overcome this packaging constraint, Charles River has developed a Cre-dependent trans-splicing approach that allows for reconstituted gene expression from two independent recombinant AAV vectors. However, trans-splicing has an expression efficiency of ~20% when compared to use of a single recombinant AAV vector.