AAV Vector Production

Gene delivery using AAV vectors have emerged as safe and effective therapeutic platform for numerous diseases. Hundreds of identified natural AAV serotypes and increasing number of capsid engineered AAV variants allow us to find better AAV vectors for each cell type and tissue, which is vital for effective gene therapies.

During the experiments, it is very common to compare the activity of different AAV vectors. However, it is very difficult  to generate, control and qualify the AAV vectors, especially for those who are new in AAV field.

AAVnerGene provides you consistent, high quality, affordable AAV vectors. Customers can compare different batch AAV vectors which are ordered from our company.  

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Scale, Price and Time
 

1. All the prices are just for AAV packaging servicies. Customer needs to provide enough amount of transgene plasmids (0.5mg/10e13 GC). We can help do Midiprep or Maxiprep with additional charge. 

2.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.

3. Our regular purification method is CsCl. We can also use Iodixanol and AAVx resin to purify the AAV vectors if customer prefers.

4. If the size of transgene is over 5.0 kb. The price would be doubled. We reserve the option to cancel the order if the efficiency is too low.

5. GC means Genome Copy. The titer is measured using primers targeting ITRs with SYBR method.

6. Our buffer is 0.001% F188l/DPBS with additional 150mM NaCl. Please let us know if you have your own buffer system.

Checklist for AAV packaging
 
  1. ITRs: Digestion by SmaI to make sure your transgene plasmids has two ITRs.

  2. AAV size: Confirm your AAV vectors size between two ITRS(including ITRs) is less than 5.0kb. AAV size less than 2.5 would package both monomer and dimmer genomes.

  3. 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.

  4. Serotypes: Check the AAV variants you want to use in your experiments.

  5. Empty AAV vectors: Do you need the empty AAV vectors? 

  6. Purification method: Our regular purification method is CsCl. We can also use Iodixanol and AAVx resin to purify the AAV vectors if customer prefers.

  7. Buffer: Our buffer is 0.001% F188l/DPBS with additional 150mM NaCl. Please let us know if you have your own buffer system.

  8. 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 Packaging Capacity

The main point of consideration in the rational design of an AAV vector is the packaging size of the expression cassette that will be placed between the two ITRs. wtAAV has a single strand DNA genome about 4.7kb. Regular single strand AAV(ssAAV) has a packaging capacity of ~5.0 Kb. Thus, as a starting point, it is generally accepted that anything under 5 kb (including ITRs) is sufficient. Since the two ITRs of AAV are about 0.2-0.3Kb total, the foreign sequence between two ITRs should be smaller than 4.7 Kb. When the length of inserted DNA  is close to the maximum, the packaging efficiency decreases significantly. If the insert DNA is over 4.7 Kb, only partial of DNA is packaged into AAV vectors. For large coding sequences, the use of dual, overlapping vector strategies may be a good  choice for you.

AAV Capsids for Packaging
 

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. We can package most of the AAV serotypes and can help you choose the best one. We can also help package AAV with your own capsid, although the titer can not be guaranteed. Contact us, our AAV experts will help you choose the potential AAV capsid for your projects. 

scAAV is a viral vector engineered from the naturally occurring AAV to be used as a tool for gene therapy. This lab-made progeny of AAV is termed "self-complementary" because the coding region has been designed to form an intra-molecular double-stranded DNA template. A rate-limiting step for the standard AAV genome involves the second-strand synthesis since the typical AAV genome is a single-stranded DNA template. However, this is not the case for scAAV genomes. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and  transcription. The caveat of this construct is that instead of the full coding capacity found in rAAV (5.0 kb).  scAAV can only hold about half of that amount (≈2.5kb).

  • Native AAV Serotypes: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13.

  • Y/F mutants: single-, double-, triple-, quad-, penta-, hexa-,  mutation of any native serotypes.

  • Engineered AAV Capsids: AAV-PHP.B, AAV-PHP.eB, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.S, AAV-retro, AAV-7M8, AAV-F, AAV-Vec, AAV-Nec, AAV-SCH9, AAV-SCH2, AAV-SHH10, AAV6.1, AAV6.2, AAV6.2FF, AAV-B1, AAV-BP2, AAVv66, AAV-Anc110, AAV-Anc113, AAV-Anc126, AAV-Anc127, AAV-Anc80L65, AAV-Anc81, AAV-anc82, AAV-anc83, AAV-Anc84, etc.

  • Peptide Insert AAV2 vectors:

  • AAVs isolated  from Human: AAV-Hu.1, AAV-Hu.2, AAV-Hu.3, AAV-Hu.6, AAV-Hu.10, AAV-Hu.11, AAV-Hu.13, AAV-Hu.15, AAV-Hu.16, AAV-Hu.17, AAV-Hu.18, AAV-Hu.19, AAV-Hu.20,  AAV-Hu.37, AAV-Hu.45, AAV-Hu.47, AAV-Hu.48, AAV-Hu.49, AAV-Hu.52, AAV-Hu.58, etc.

  • AAV  isolated  from Rh: AAV-Rh10, AAV-Rh.22, AAV-Rh.23, AAV-Rh.24, AAV-Rh.32, AAV-Rh.32.33, AAV-Rh.33, AAV-Rh.34, AAV-Rh.35, AAV-Rh.39, AAV-Rh.43, AAV-Rh.47, AAV-Rh.49, AAV-Rh.52, AAV-Rh.74, etc.

  • AAVs isolated  from Rat: AAV-Rat-YY.12, AAV-Rat-YY.25, AAV-Rat-YY.78, AAV-Rat-YY.80, AAV-Rat-YY.90, AAV-Rat-YY.93, AAV-Rat-TT.54,AAV-Rat-HD.16, AAV-Rat-HD-20, AAV-Rat-HD-43, AAV-Rat-HD-94, AAV-Rat-MLP.26, AAV-Rat-MLP-6, AAV-Rat-XM.70,  etc.

  • AAVs isolated  from Other Specials: AAV-Po.1,AAV-Po.2,AAV-Po.3, AAV-Po.4, AAV-Po.5, AAV-Po.6, AAV-Po.7, AAV-Po.8, AAV-Go.1, AAV-VR355,AAV-Cy.2,AAV-Cy.3, AAV-Cy.4,AAV-Cy.5 AAV-Cy.6, AAV-Avian-DA, AAV-Avian-VR865, AAV-Bat-YNM, AAV-bovine,​ AAV-Pi.1,AAV-Pi.2,AAV-Pi.3, AAV-BB.1, AAV-BB.2,AAV-ch.5.

  • Peptide Insert AAV2 vectors: AAV2-588NGR, AAV2-MO7A, AAV2-MO7T, AAV2-MecA, AAV2-MecB, AAV2-rRGD587, AAV2-C4, AAV2-D10, AAV2-SIG, AAV2-MTP, AAV2-QPE, AAV2-VNT, AAV2-CNH, AAV2-CAP, AAV2-EYH, AAV2-587MTP, AAV2-r3.45, AAV2-LSS, AAV2-PFG, AAV2-PPS, AAV2-TLH, AAV2-GMN, AAV2-Kera1, AAV2-Kera2, AAV2-Kera3, AAV2-588Myc, AAV2-BAP, AAV2-Ald13, AAV2-DMD4, AAV2-DMD6, AAV2-588-RGD4C, AAV2-VTAGRAP, AAV2-APVTRPA, AAV2-DLSNLTR, AAV2-NQVGSWS, AAV2-EARVRPP, AAV2-NSVSLYT, AAV2-LS1, AAV2-LS2, AAV2-LS3, AAV2-LS4, AAV2-RGDLGLS, AAV2-RGDMSRE, AAV2-ESGLSQS, AAV2-EYRDSSG, AAV2-DLGSARA, AAV2-GPQGKNS, AAV2-NSSRDLG, AAV2-NDVRAVS, AAV2-PRSTSDP, AAV2-DIIRA, AAV2-SYENV, AAV2-PENSV, AAV2-LSLAS, AAV2-NDVWN, AAV2-NRTYS, AAV2-ESGHGYF, AAV2-GQHPRPG, AAV2-PSVSPRP, AAV2-VNSTRLP, AAV2-GQHPR, AAV2-LSPVR, AAV2-MSSDP, AAV2-GARPS, AAV2-GNEVL, AAV2-KMRPG, AAV2-588MTP, AAV2-RGDLGLS, AAV2-RGDAVGV, AAV2-SPGARAF, AAV2-DGPWRKM, AAV2-FGQKASS, AAV2-MPLGAAG, AAV2-NYSRGVD, AAV2-GSVPRLG, AAV2-PVVPRPA, AAV2-GERAKPA, AAV2-NEARVRE, AAV2-MVNNFEW, AAV2-SEGLKNL, AAV2-PSVPRPP, AAV2-SLRSPPS, AAV2-NFTRLSA, AAV2-GDVGPPG, AAV2-NDVRPER, AAV2-NRVEEKL, AAV2-TTSVRPA, AAV2-HLHGRPA, AAV2-588MTP-R587G, AAV2-MecA-N584H/586A, AAV2-TTSVRPA, AAV2-HLHGRPA,AAV2-588MTP. etc

 
AAV Production Methods

We use the most common triple-plasmid system method to produce AAV vector in E1-expressing cells.

  • Transgene plasmid: This plasmid contain the vector genome, which is an expression cassette with promoter, gene of interest and polyA signal , flanked by two ITRs (mostly from AAV2).

  • AAV helper plamsid: This plasmid encodes Rep proteins, commonly from AAV2, and Cap proteins that are specific for the desired serotypes.

  • Ad helper plamsid:  The third plasmid carries the minimal adenoviral genes required to support AAV replication (E2a, E4 and VA). The E1 gene is integrated into HEK-293T cells. 

 

When the above three plasmids are co-transfected into HKE-293T cells, the AAV vector is produced as efficiently as when infection by adenovirus is used as a source of helper virus.

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Cell Lines for AAV Production

We provide both adherent cells and suspension cells to produce AAV vectors.

Adherent cells: 

  • HEK 293 cells: Human embryonic kidney 293 cells, also referred to as HEK293, HEK-293, 293 cells, or less precisely as HEK cells, are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture. HEK293 cells express the adenoviral gene E1, which permits adenoviruses lacking this gene to grow, thus contribute AAV package.

  • HEK 293T cellsHEK 293T is an inbreed of HEK 293 along with SV40 T antigen. The cells are forced entering into more number of cell cycles. This adds on to increase the amplification of the transfected plasmid thus increasing the efficiency of transfection, higher vector production and higher transduction efficiency.

Suspension cells: 

  • HEK 293T cells: Suspension HEK 293T cells are adapted from adherent HEK 293 T cells.

 
AAV Purification

We provide cesium chloride (CsCl) based density gradients , iodixanol-based density gradients and AAVx resin methods for AAV purification.We can see clear viral bands for all the  AAV vectors we deliver. 

CsCl gradients

Cesium chloride density gradient ultracentrifugation is the conventional method of purification for all kind of AAV vectors. 

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Iodixanol gradients

The iodixanol gradient in this protocol is composed of steps that separate out contaminants from an impure AAV preparation. 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 added to clearly distinguish the steps. This method will help enrich the prep with full (genome containing) particles. 

AAVx resin

AAVX resin has demonstrated binding reactivity towards a set of AAV serotypes that includes AAV1 to AAV8, and AAVrh10 as well as synthetic serotypes.
With AAVX resin, we can do:
• One-step AAV purification from crude material with high purity and yield
• High specificity and capacity, maximizing yield by significantly reducing process steps
• Superior capacity maintained at high flow rates
• Excellent scalability and easier handling and packing due to rigid polymeric bead
• Non-animal derived

 

Quality Control of AAV Vectors
 

One of the main challenges in the using of AAV vectors is how to accurate quantify your AAV vectors. The AAV titration can be used to determine the number of genome-containing particles of an AAV prep using SYBR green technology. Usually, the viral titer is quantified by comparison to a standard curve of a plasmid sample of known concentration. However, even one sample performing two individual measures will get 2-fold difference. At AAVnerGene, we take care of every steps from production to qualification to make sure the two AAV vectors you got  from us are  comparable. 

  • All AAV vectors are produced with the same protocol.

  • All AAV vectors from one customer are produced and qualified together.  

  • The DNA used as Standard has very similar structure with the AAV genomes.

  • Besides the stardard curve, we always have an internal reference  to normalize the qPCR results. 

  • The uncapsidated DNA was digested by DNase I before qPCR.

  • Proteinase K is used to digest the capsid and completely release the capsidated genomes. 

AAVnerGene also offer other quality tests if customer request:

  • Endotoxin testing

  • Protein purity testing

  • Sterility testing

  • Mycoplasma

  • AAV Capsid quantification with Elisa

  • Empty/Full Ratio

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