Adeno-Associated Virus (AAV) Packaging
Recombinant adeno-associated virus (AAV) is a versatile and popular viral vector used for in vitro and in vivo gene delivery. AAVs have emerged as one of the most effective vehicles for gene therapy due to their ability to transduce a wide variety of mammalian cell types and their non-pathogenicity and low immunogenicity in human.
VectorBuilder offers superior quality AAV packaging services to support your AAV-based gene therapy experiments. We have developed a series of proprietary technologies and reagents that have greatly improved recombinant AAV production protocols in terms of titer, purity, potency and consistency, especially for the AAV vector systems used in our vector cloning services. As a result, we have a growing base of highly satisfied customers who come back to us time after time for their AAV vector cloning and AAV packaging needs. In addition to research-grade AAV, we also offer GMP-grade AAV manufacturing for clinical applications.
Types of AAV offered
- Single-stranded AAV (ssAAV) and self-complementary AAV (scAAV)
- Various serotypes: 1, 2, 3, 4, 5, 6, 6.2, 7, 8, 9, rh10, DJ, DJ/8, PHP.eB, PHP.S, AAV2-retro, AAV2-QuadYF, AAV2.7m8, etc.
- AAV empty capsids or virus-like particles (VLPs)
Service Details
Research grade AAV packaging
Our research grade AAV packaging services meet the vast majority of AAV-based gene delivery needs in basic research. Both triple transfection and baculovirus-based packaging methods can be selected based on your needs.
Triple Transfection-Based Approach
Baculovirus-Based Approach
Price and turnaround Price Match
| Scale | Application | Typical Titer | Minimum Titer | Volume | Price (USD) | Turnaround |
|---|---|---|---|---|---|---|
| Pilot | Non-purified, suitable for most cell culture experiments | >1012 GC/ml | >2x1011 GC/ml | 250 ul (10x25 ul) | $449 |
6-12 days
|
| Medium | 1ml (10x100 ul) | $649 | ||||
| Large | >5x1012 GC/ml | >2x1012 GC/ml | 1ml (10x100 ul) | $1,099 | ||
| Ultra-purified pilot | Cell culture & in vivo | >2x1013 GC/ml | >1013 GC/ml | 100 ul (4x25 ul) | $1,399 |
7-14 days
|
| Ultra-purified medium | 500 ul (10x50 ul) | $1,999 | ||||
| Ultra-purified large | 1 ml (10x100 ul) | $3,099 | ||||
| Ultra-purified large 5 | 5 ml (10×500 ul) | From $9,899 | ||||
| Ultra-purified large 10 | 10 ml (10×1 ml) | From $15,899 | 14-21 days | |||
| Other scales | Please inquire | |||||
Note:
1. GC = Genome copies.
2. AAV serotype 4 tends to have lower yields than other serotypes. As such, we can only guarantee 50% of the minimum titers specified in the table for this serotype.
3. For ultra-purified scales, viral particles are purified by cesium chloride (CsCl) density gradient.
Deliverables
| For non-purified scales | For ultra-purified scales |
|---|---|
| Your custom AAV | Your custom AAV |
|
Free: non-purified control virus
|
Add-on purchase (optional): ultra-purified control virus
|
Control virus
The control AAV is designed to match the biological application of the custom virus and to be used for testing AAV transduction. For example, if the custom virus overexpresses a gene, then the control virus provided will be EGFP control AAV (AAV overexpressing EGFP), and if the custom virus expresses an shRNA against a gene, then the control virus provided will express a scramble shRNA. Detailed information on the control virus is shown below:
Research plus grade AAV packaging
Our research plus AAV packaging services are suitable for applications that are sensitive to impurities (e.g. host cell protein, endotoxin, etc.), or applications with special requirements on purification method, titer, or formulation. They are also optimal choices for your preclinical animal experiments.
Price and turnaround Price Match
| Scale | Application | Total Yield (GC) | Price (USD) | Turnaround |
|---|---|---|---|---|
| Research-plus 1 | Various in vitro & in vivo experiments | 1x1013 | From $4,699 | 10-16 days |
| Research-plus 5 | 5x1013 | From $14,899 | 14-21 days | |
| Research-plus 10 | 1x1014 | From $23,899 | ||
| Other scales | Up to 1x1016 | Please inquire | ||
Note:
1. GC = Genome copies.
2. The above listed price and turnaround are based on purification by CsCl density gradient centrifugation. When other purification methods (e.g. iodixanol density gradient, affinity chromatography, ion-exchange chromatography, etc.) are required, please send us a design request to get the quote.
Price and turnaround Price Match
| Scale | Application | Minimum Titer | Volume | Price (USD) | Turnaround |
|---|---|---|---|---|---|
| Ultra-purified pilot | Cell culture & in vivo | >5x1013 GC/ml | 1 ml (10x100 ul) | $5,599 | 35-49 days |
| Ultra-purified medium | 5 ml (25x200 ul) | $20,199 | 35-49 days | ||
| Ultra-purified large | 10 ml (50x200 ul) | $38,199 | 35-49 days |
Note:
1. GC = Genome copies.
2. Baculovirus-based approach is available for the following serotypes: 1, 2, 5, 6, 8, 9.
3. For ultra-purified scales, viral particles are purified by cesium chloride (CsCl) density gradient.
Deliverables
| Your custom AAV |
|
Add-on purchase (optional): ultra-purified control virus
|
Control virus
The control AAV is designed to match the biological application of the custom virus and to be used for testing AAV transduction. For example, if the custom virus overexpresses a gene, then the control virus provided will be EGFP control AAV (AAV overexpressing EGFP), Detailed information on the control virus is shown below:
| Vector System | Control Virus Vector Name | Control Virus Vector ID |
|---|---|---|
| Chimeric baculovirus-AAV gene expression system | pBV-ITR-CMV>EGFP-ITR | VB010000-9306ded |
Shipping and storage
Our non-purified AAV is stored in a Tris-based buffer and our ultra-purified AAV is stored in a PBS-based buffer. All our AAVs are shipped on dry ice. Upon receiving, it should be stored at -80°C for long term (stable for at least 1 year), or -20°C for short term, e.g. 2-3 weeks. Thawed AAV virus can be stored at 4°C for 1-2 weeks. Although AAV is more stable than many other virus (e.g. lentivirus) and can be frozen and thawed several times with minimal loss of virus activity, it is best to avoid repeated freeze-thaw cycles in practice.
Technical Information
Comparison of different AAV grades
The table below is an overview comparison of different grades of AAV we offer for research use.
| Non-purified research grade | Ultra-purified research grade | Research plus grade | |
|---|---|---|---|
| Available purification method | - | CsCl density gradient | CsCl density gradient (default), iodixanol density gradient, affinity chromatography, ion-exchange chromatography |
| Titer | >1012 GC/ml | >1013 GC/ml | 1x1013 - 5x1013 GC/ml |
| Achievable purity (assessed by SDS-PAGE) | - | >80% | >90% |
| Achievable endotoxin level | <30 EU/ml | <10 EU/ml | <2 EU/ml |
| Typical full capsid ratio | - | >70% | >80% |
AAV production and quality control (QC)
Triple Transfection-Based Approach
Baculovirus-Based Approach
For our recombinant AAV manufacturing, the transfer plasmid carrying the gene of interest (GOI) is co-transfected with our proprietary Rep-cap plasmid and helper plasmid encoding adenovirus genes (E4, E2A and VA) that mediate AAV replication into HEK293T packaging cells. After a short incubation period, viral particles are harvested from cell lysate or supernatant depending on serotype and concentrated by PEG precipitation. For ultra-purified AAV (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We use a qPCR-based approach to measure AAV titer.
Figure 1. Typical workflow of triple transfection-based AAV packaging
For each AAV produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the transfer vector encodes a fluorescent protein, we would perform transduction test to detect corresponding fluorescence. Additionally, for ultra-purified AAV, we routinely sample virus quality by SDS-PAGE analysis and endotoxin assay. To include the endotoxin results in your COA, an extra cost is required. Additional QC services as below can be provided upon request.
| Additional QC service | Method |
|---|---|
| Endotoxin testing | LAL |
| Titer determination | ddPCR |
| TCID50 | |
| Replication-competent AAV testing | qPCR |
| Full/empty capsid analysis | TEM |
| CDMS | |
| SV-AUC |
Our baculovirus-based AAV packaging workflow consists of two main steps as shown in Figure 1 below. Step 1 involves the generation of two recombinant baculoviruses, specifically the first one expressing the gene of interest (GOI) flanked by the AAV inverted terminal repeats (ITRs) and a second helper baculovirus expressing the AAV rep and cap genes. In step 2 recombinant AAV particles are generated by co-infecting insect cells with the two recombinant baculoviruses produced in step 1.
For generating the two recombinant baculoviruses (one expressing the GOI flanked by AAV ITRs and the other expressing the AAV rep/cap genes), the expression cassette for each baculovirus is first cloned into a baculovirus transfer vector. The baculovirus transfer plasmid is then co-transformed with a helper plasmid expressing the Tn7 transposase into the bacterial host harboring the empty baculovirus shuttle vector (a.k.a. bacmid) to generate recombinant bacmid. The recombinant bacmid is then transfected into insect Sf9 cells. After a short incubation period, viral particles are harvested from medium and further concentrated by sucrose cushion centrifugation. We use a qPCR-based approach to measure baculovirus titer. Both recombinant baculoviruses are then co-infected into insect cells. After an incubation period of 72-96 hours, AAV particles are harvested from the cell lysate as well as the supernatant and concentrated by PEG precipitation. For ultra-purified AAV (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We use a qPCR-based approach to measure AAV titer.
Figure 1. Typical workflow of baculovirus-based AAV packaging
For each AAV produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the transfer vector encodes a fluorescent protein, we would perform transduction test to detect corresponding fluorescence. Additionally, for ultra-purified AAV, we routinely sample virus quality by SDS-PAGE analysis and endotoxin assay. Extra cost is required for including endotoxin result in your COA report. Additional QC services as below can be provided upon request.
| Other QC services | Methods |
|---|---|
| Titer determination | ddPCR |
| TCID50 | |
| Replication-competent AAV testing | qPCR |
| Empty capsid analysis | TEM |
| CDMS | |
| SEC-AUC |
Recommended AAV serotypes
List by Serotype
List by Tissue Type
| Serotype | Tissue tropism |
|---|---|
| AAV1 | Smooth muscle, skeletal muscle, CNS, brain, lung, retina, inner ear, pancreas, heart, liver |
| AAV2 | Smooth muscle, CNS, brain, liver, pancreas, kidney, retina, inner ear, testes |
| AAV3 | Smooth muscle, liver, lung |
| AAV4 | CNS, retina, lung, kidney, heart |
| AAV5 | Smooth muscle, CNS, brain, lung, retina, heart |
| AAV6 | Smooth muscle, heart, lung, pancreas, adipose, liver |
| AAV6.2 | Lung, liver, inner ear |
| AAV7 | Smooth muscle, retina, CNS, brain, liver |
| AAV8 | Smooth muscle, CNS, brain, retina, inner ear, liver, pancreas, heart, kidney, adipose |
| AAV9 | Smooth muscle, skeletal muscle, lung, liver, heart, pancreas, CNS, brain, retina, inner ear, testes, kidney, adipose |
| AAV-rh10 | Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney |
| AAV-DJ | Liver, heart, kidney, spleen |
| AAV-DJ/8 | Liver, brain, spleen, kidney |
| AAV-PHP.eB | CNS |
| AAV-PHP.S | PNS |
| AAV2-retro | Spinal nerves |
| AAV2-QuadYF | Endothelial cell, retina |
| AAV2.7m8 | Retina, inner ear |
| Tissue type | Recommended AAV serotypes |
|---|---|
| Smooth muscle | AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-rh10 |
| Skeletal muscle | AAV1, AAV9 |
| CNS | AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAV-rh10, AAV-PHP.eB |
| PNS | AAV-PHP.S |
| Brain | AAV1, AAV2, AAV5, AAV7, AAV8, AAV9, AAV-DJ/8 |
| Retina | AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAV-rh10, AAV2-QuadYF, AAV2.7m8 |
| Inner ear | AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8 |
| Lung | AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAV-rh10 |
| Liver | AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV-rh10, AAV-DJ, AAV-DJ/8 |
| Pancreas | AAV1, AAV2, AAV6, AAV8, AAV9, AAV-rh10 |
| Heart | AAV1, AAV4, AAV5, AAV6, AAV8, AAV9, AAV-rh10, AAV-DJ |
| Kidney | AAV2, AAV4, AAV8, AAV9, AAV-rh10, AAV-DJ, AAV-DJ/8 |
| Adipose | AAV6, AAV8, AAV9 |
| Testes | AAV2, AAV9 |
| Spleen | AAV-DJ, AAV-DJ/8 |
| Spinal nerves | AAV2-retro |
| Endothelial cells | AAV2-QuadYF |
* Please note that the ITRs carrying your gene of interest (GOI) is from the AAV2 genome. Different serotypes are distinguished by the capsid protein serotype.
Click here to view the amino acid sequence of the major viral capsid protein 1 (VP1) for different AAV serotypes >>Experimental validation
We have developed a number of proprietary techniques to optimize our baculovirus-based AAV packaging protocol and our virus has been validated to exhibit high transduction efficiency in mammalian cells.
Figure 2. HEK293T cells were transduced with EGFP expressing, baculovirus-based AAV1 and AAV2 at MOI 10000. Magnification: 100x. Left: bright field. Right: EGFP.
Experimental validation
We have developed a number of proprietary techniques to optimize our triple transfection-based AAV packaging protocol, and our virus has been validated to exhibit high transduction efficiency both in vitro and in vivo in a broad range of cell and tissue types.
In vitro
In vivo
Figure 2. HEK293T cells were transduced with 18 serotypes of recombinant AAV packaged from the same CMV>EGFP vector (VB010000-9394npt). Representative EGFP expression at 48 h post transduction in different serotypes is shown as indicated. Scale bars: 100 μm.
Figure 3. HeLa cells were transduced with 18 serotypes of recombinant AAV packaged from the same CMV>EGFP vector (VB010000-9394npt). EGFP (A) Mean Fluorescence Intensity (MFI) and (B) positivity were quantified using flow cytometry 48 h post transduction. (C) Representative EGFP expression in different serotypes is shown as indicated. Scale bars: 100 μm.
Figure 4. Huh-7 cells were transduced with 18 serotypes of recombinant AAV packaged from the same CMV>EGFP vector (VB010000-9394npt). EGFP (A) Mean Fluorescence Intensity (MFI) and (B) positivity were quantified using flow cytometry 48 h post transduction. (C) Representative EGFP expression in different serotypes is shown as indicated. Scale bars: 100 μm.
Figure 5. Mouse lung transduced with AAV6.2 expressing ZsGreen driven by the SPB promoter. Administration route: intratracheal injection. Imaging: light sheet microscopy at 4 weeks post-injection.
Figure 6. AAV9 expressing EGFP, packaged from the CAG>EGFP vector (VB010000-9287ffw), was administered to mice via various routes. EGFP and DAPI fluorescence were analyzed in the following organs: (A) inner ear, 13 days post-intratympanic injection; (B) hippocampus and spinal cord anterior horn, 10 days post-facial vein injection; (C) retina, liver, pancreas, and skeletal muscle, 12 days post-tail vein injection.
Figure 7. (A) AAV8 expressing mCherry driven by the ProA1 promoter or (B) AAV8 expressing EGFP driven by the hRHO promoter was administered to mice via subretinal injection. mCherry, EGFP, and DAPI fluorescence were analyzed in the retina 20 days post-injection.
Figure 8. Transduction of newborn mouse brain with AAV9 and PHP.eB expressing EGFP, packaged from the same CAG>EGFP vector (VB010000-9287ffw). Administration route: intracerebroventricular injection. Imaging: 10X magnification, 10 ms exposure.
Figure 9. Transduction of newborn mouse spinal cord with AAV9 and PHP.eB expressing EGFP, packaged from the same CAG>EGFP vector (VB010000-9287ffw). Administration route: intracerebroventricular injection. Imaging: 4X magnification, 200 ms exposure.
How to Order
Customer-supplied vectors
If customer-supplied AAV plasmids are used in packaging, please send them to us following the Materials Submission Guidelines. Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur $100 surcharge for each item. Please note that production may not be initiated until customer-supplied materials pass QC.
Resources
Documents
Brochures & Flyers
Certificate of Analysis (COA)
Material Safety Data Sheet (MSDS)
FAQ
| Lentivirus | MMLV | Adenovirus | AAV | |
|---|---|---|---|---|
| Tropism | Broad | Broad | Ineffective for some cells | Depending on viral serotype |
| Can infect non-dividing cells? | Yes | No | Yes | Yes |
| Stable integration or transient | Stable integration | Stable integration | Transient, episomal | Transient, episomal |
| Maximum titer | High | Moderate | High | Very high |
| Promoter customization | Yes | No | Yes | Yes |
| Primary use | Cell culture and in vivo | Cell culture and in vivo | In vivo | In vivo |
| Immune response in vivo | Low | Low | High | Very low |
We measure the physical titer of AAV by directly extracting viral genome from lysed viral particles, and then using qPCR to accurately quantify the copy number of viral genome (using the copy number of ITR region as a proxy) in the stock. Titration services using other methods (e.g. ddPCR, TCID50) can be purchased separately.
Our titer guarantee applies to vectors for which the region being packaged into virus (from 5’ ITR to 3’ ITR) is below the AAV cargo limit (4.7 kb). For sizes above this limit, the region packaged into the virus may be truncated and result in loss-of-function. Additionally, we may not be able to guarantee titer in the following situations:
- AAV serotype 4 tends to have lower yields than other serotypes. As such, we can only guarantee 50% of the minimum titers specified in the table for this serotype.
- Vectors containing sequences that could adversely affect the packaging process such as toxic genes (e.g. proapoptotic genes), genes that compromise the integrity of packaging cells or virus (e.g. membrane proteins that cause cell aggregation), and sequences prone to rearrangements or secondary structures (e.g. repetitive or highly GC-rich sequences).
- Packaging of customer-supplied vector because we have no control over the quality of the vector and the compatibility of the vector with our packaging system.
Our estimated turnaround is the time from production initiation to completion. It does not include waiting time for any customer-supplied materials (e.g. template DNA or viral vectors), QC of such materials, and transit time for shipping final deliverables to the customer.
Featured Citations
Trb3 Controls Mesenchymal Stem Cell Lineage Fate and Enhances Bone Regeneration by Scaffold-Mediated Local Gene Delivery
Jiabing Fan, et al.
Biomaterials, 2020, doi: 10.1016/j.biomaterials.2020.120445
Products used:
- Adeno-Associated Virus (AAV) Packaging (AAV1, 2, 5, 6, 8 & 9)
- Lentivirus Packaging
- Vector Cloning
Abstract: Aberrant lineage commitment of mesenchymal stem cells (MSCs) in marrow contributes to abnormal bone formation due to reduced osteogenic and increased adipogenic potency. While several major transcriptional factors associated with lineage differentiation have been found during the last few decades, the molecular switch for MSC fate determination and its role in skeletal regeneration remains largely unknown, limiting creation of effective therapeutic approaches. Tribbles homolog 3 (Trb3), a member of tribbles family pseudokinases, is known to exert diverse roles in cellular differentiation. Here, we investigated the reciprocal role of Trb3 in the regulation of osteogenic and adipogenic differentiation of MSCs in the context of bone formation, and examined the mechanisms by which Trb3 controls the adipo-osteogenic balance. Trb3 promoted osteoblastic commitment of MSCs at the expense of adipocyte differentiation. Mechanistically, Trb3 regulated cell-fate choice of MSCs through BMP/Smad and Wnt/β-catenin signals. Importantly, in vivo local delivery of Trb3 using a novel gelatin-conjugated caffeic acid-coated apatite/PLGA (GelCA-PLGA) scaffold stimulated robust bone regeneration and inhibited fat-filled cyst formation in rodent non-healing mandibular defect models. These findings demonstrate Trb3-based therapeutic strategies that favor osteoblastogenesis over adipogenesis for improved skeletal regeneration and future treatment of bone-loss disease. The distinctive approach implementing a scaffold-mediated local gene transfer may further broaden the translational use of targeting specific therapeutic gene related to lineage commitment for clinical bone treatment.
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Damaging mutations in liver X receptor-α are hepatotoxic and implicate cholesterol sensing in liver health
Lockhart, et al.
Nature Metabolism, 2024, doi: 10.1038/s42255-024-01126-4
Products used:
- AAV Packaging (AAV8)
- Vector Cloning
Abstract: Liver X receptor-α (LXRα) regulates cellular cholesterol abundance and potently activates hepatic lipogenesis. Here we show that at least 1 in 450 people in the UK Biobank carry functionally impaired mutations in LXRα, which is associated with biochemical evidence of hepatic dysfunction. On a western diet, male and female mice homozygous for a dominant negative mutation in LXRα have elevated liver cholesterol, difuse cholesterol crystal accumulation and develop severe hepatitis and fbrosis, despite reduced liver triglyceride and no steatosis. This phenotype does not occur on low-cholesterol diets and can be prevented by hepatocyte-specifc overexpression of LXRα. LXRα knockout mice exhibit a milder phenotype with regional variation in cholesterol crystal deposition and infammation inversely correlating with steatosis. In summary, LXRα is necessary for the maintenance of hepatocyte health, likely due to regulation of cellular cholesterol content. The inverse association between steatosis and both infammation and cholesterol crystallization may represent a protective action of hepatic lipogenesis in the context of excess hepatic cholesterol.
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Discovery and engineering of ChCas12b for precise genome editing
Wei, et al.
Science Bulletin, 2024, doi: 10.1016/j.scib.2024.06.012
Products used:
- AAV Packaging (AAV9)
Abstract: Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b (CRISPR-Cas12b) nucleases have been computationally identified, yet their potential for genome editing remains largely unexplored. In this study, we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing, identifying five active candidates. Candidatus hydrogenedentes Cas12b (ChCas12b) was found to recognize a straightforward WTN (W = T or A) proto-spacer adjacent motif (PAM), thereby dramatically expanding the targeting scope. Upon optimization of the single guide RNA (sgRNA) scaffold, ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endoge nous loci. Additionally, we identified nine mutations enhancing ChCas12b specificity. More importantly, we demonstrated that both ChCas12b and its high-fidelity variant, ChCas12b-D496A, enabled allele specific disruption of genes harboring single nucleotide polymorphisms (SNPs). These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.
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