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)
  • 18 serotypes: 1, 2, 3, 4, 5, 6, 6.2, 7, 8, 9, rh10, DJ, DJ/8, PHP.eB, PHP.S, AAV2-retro, AAV2-QuadYF and AAV2.7m8

Service Details

Triple Transfection-Based Approach
Baculovirus-Based Approach
Price and turnaround
Scale Application Typical Titer Minimum Titer Volume Price (USD) Turnaround
Pilot Cell culture >1012 GC/ml >2x1011 GC/ml 250 ul (10x25 ul) $399 10-18 days
Medium 1ml (10x100 ul) $599
Large >5x1012 GC/ml >2x1012 GC/ml 1ml (10x100 ul) $999
Ultra-purified pilot Cell culture & in vivo >2x1013 GC/ml >1013 GC/ml 100 ul (4x25 ul) $1,199 16-26 days
Ultra-purified medium 500 ul (10x50 ul) $1,799
Ultra-purified large 1 ml (10x100 ul) $2,799

Note:

1. GC = Genome copies.

2. Triple transfection-based approach is available for the following serotypes: 1, 2, 3, 4, 5, 6, 6.2, 7, 8, 9, rh10, DJ, DJ/8, PHP.eB, PHP.S, AAV2-retro, AAV2-QuadYF and AAV2.7m8.

Deliverables
For non-ultra-purified scales For ultra-purified scales
Your custom AAV Your custom AAV

Free: control virus

  • Pilot: >2x1011 GC/ml, 100 ul
  • Medium: >2x1011 GC/ml, 2x100 ul
  • Large: >2x1011 GC/ml, 2x100 ul

  Add-on purchase (optional): ultra-purified control virus

  • Ultra-purified pilot: >1013 GC/ml, 4x25 ul, at $400
  • Ultra-purified medium: >1013 GC/ml, 10x50 ul, at $600
  • Ultra-purified large: >1013 GC/ml, 10x100 ul, at $940
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:

Vector System Control Virus Vector Name Control Virus Vector ID
AAV gene expression system pAAV[Exp]-CMV>EGFP:WPRE VB010000-9394npt
AAV U6-based shRNA knockdown system pAAV[shRNA]-EGFP-U6>Scramble_shRNA VB010000-0023jze
AAV miR30-based shRNA knockdown system pAAV[miR30]-CMV>EGFP:Scramble_miR30-shRNA:WPRE VB010000-9397wgw
AAV CRISPR system pAAV[Exp]-CMV>EGFP:WPRE VB010000-9394npt
Scale Application Minimum Titer Volume Price (USD) Turnaround
Ultra-purified pilot Cell culture & in vivo >5x1013 GC/ml 1 ml (10x100 ul) $5,599 42-56 days
Ultra-purified medium 5 ml (25x200 ul) $20,199 49-63 days
Ultra-purified large 10 ml (50x200 ul) $38,199 56-70 days

Note:

1. GC = Genome copies.

2. Baculovirus-based approach is available for the following serotypes: 1, 2, 5, 6, 8, 9.

Deliverables
Your custom AAV

  Add-on purchase (optional): ultra-purified control virus

  • Ultra-purified pilot: >1013 GC/ml, 10x100 ul, at $1,870
  • Ultra-purified medium: >1013 GC/ml, 25x200 ul, at $6,740
  • Ultra-purified large: >1013 GC/ml, 50x200 ul, at $12,740
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 AAV is stored in PBS buffer and is 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

Triple Transfection-Based Approach
Baculovirus-Based Approach
AAV production and QC

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.

AAV production and QC

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.

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. 293T cells were transduced with EGFP expressing, baculovirus-based AAV1 and AAV2 at MOI 10000. Magnification: 100x. Left: bright field. Right: GFP.

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, retina, inner ear, testes, kidney, adipose
AAVrh10 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
AAV2.7m8 Retina, inner ear
Tissue type Recommended AAV serotypes
Smooth muscle AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10
Skeletal muscle AAV1, AAV9
CNS AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB
PNS AAV-PHP.S
Brain AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8
Retina AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8
Inner ear AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8
Lung AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10
Liver AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
Pancreas AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10
Heart AAV1, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ
Kidney AAV2, AAV4, AAV8, AAV9, AAVrh10, 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 >>

How to Order

Order both vector cloning and virus packaging
Order virus packaging of your own vector

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.

FAQ

What features does AAV have comparing to other viral vectors?
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 Very High 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
How is viral titer determined in VectorBuilder?

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.

What is VectorBuilder's guarantee for virus titer and turnaround?

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:

  • Serotypes 4, 6 and DJ/8 which can only achieve 30-50% of the titer of the other serotypes.
  • 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).
  • Customer-supplied transfer plasmid containing undisclosed sequences or atypical ITRs that may introduce uncertainties to packaging efficiency.
  • Customer-supplied rep-cap plasmid and/or helper plasmid.

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.