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AAV Packaging Protocol for AAV Transfection

BioInnovatise Viral Vector Team

Updated December 9, 2024

What Is An AAV Packaging Protocol and Which Is Right for My Research?

Producing viral vectors capable of efficiently transferring the desired genetic material into target cells requires the development a reliable and reproducible method, this what is known as an AAV packaging protocol.

AAV packaging protocols differ for several technical reasons including materials required and actions taken. protocols vary mainly because of their differing desired outcomes. 

AAV Diagram

Here is a breakdown of the different reasons for variations between transfection protocol:

  • Serotype: Different AAV, such as AAV8 serotypes, have unique capsid proteins, which influence production yield and tropism. Protocols may adjust to optimize packaging for a specific serotype. Learn more about AAV serotype choices.
  • Scale of Production: Small scale research applications use transient transfection in HEK293T cells, while large scale production often relies on stable producer cell lines such as Sf9 for greater yield and scalability.
  • Target Tissue or Disease: Specific tissues or diseases might require optimized ratios of genome-containing particles versus empty capsids, purity levels, and capsid design to enhance transduction efficiency and minimize immune response.
  • Vector Genome: The size and sequence of the plasmid DNA transgene can impact production efficiency, necessitating adjustments to vector-to-helper plasmid ratios or process timing. If you are unsure your transgene is too large to be packaged in AAV vectors, reach out to our molecular cloning team for assistance.
  • Downstream Applications: Research grade AAV packaging prioritizes rapid production, while AAV vector manufacturing  focus on purity, stability, and endotoxin levels, leading to different purification and quality assurance processes.
  • Regulatory and Purity Requirements: Clinical and commercial grade AAV vector manufacturing requires cGMP production which requires more strict protocol details and adherence.

Our AAV packaging team will always use the specific transfection protocol requested by the customer. If you are unsure which AAV transfection protocol is right for you, contact our team. The below transfection protocol is a general protocol for educational purposes and should not be used outside of BioInnovatise productions.

Learn about our quick turnaround AAV packaging services. 

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The above diagram illustrates the AAV packaging process at BioInnovatise, where our team uses HEK293T cells for AAV packaging.

Materials Required

  1. Plasmids: Learn more about AAV packaging plasmids.
    1. Transfer plasmid: Contains the transgene flanked by AAV ITRs
    2. Helper plasmid: Provides adenoviral helper functions
    3. Packaging plasmid: Encodes AAV rep and cap genes for the desired serotype
  2. Cell line (our team uses a HEK293T cell line for high transfection efficiency)
  3. Transfection reagents
  4. Media
  5. Purification reagents
  6.  Equipment:
    1. 37°C incubator with 5% CO₂.
    2. 15 cm culture dishes.
    3. RT-qPCR machinery for titer determination.

Procedure

  1. Prepare cells
    1. Seed HEK293T cells in 15 cm dishes the day before transfection.
      1. Aim for ~70–80% confluency on the day of transfection.
  2. Transfection
    1. Prepare the DNA mixture:
      1. Use a 1:1:1 molar ratio of the transfer, helper, and packaging plasmids.
      2. Example: For one 15 cm dish, use 10 µg total DNA (3.3 µg each plasmid).
    2. Prepare transfection reagent:
      1. Mix the DNA with PEI in a 1:3 ratio (e.g., 10 µg DNA + 30 µL PEI).
      2. Dilute the DNA and PEI separately in 1 mL of serum-free DMEM, then mix and incubate for 10–15 minutes at room temperature.
    3. Add the transfection complex to the cells:
      1. Gently add the mixture dropwise to the culture dish.
      2. Swirl the plate to ensure even distribution.
    4. Incubate at 37°C, 5% CO₂.
  3. Media Change
    1. Replace the media with fresh DMEM (with 10% FBS) to enhance viral production.
  4. Harvest AAV
  5. Collect supernatant:
    1. Remove and centrifuge the supernatant at 3,000 rpm for 10 minutes to remove debris.
    2. Harvest cell pellet:
      1. Detach cells with PBS, centrifuge, and resuspend in lysis buffer (e.g., 50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl₂, pH 8.0).
      2. Perform freeze-thaw cycles (3 times) to release the virus.
    3. Combine lysate and supernatant for downstream processing.
  6. Purification and Titering
    1. Iodixanol gradient ultracentrifugation:
      1. Prepare a 4-layer iodixanol gradient (15%, 25%, 40%, and 60%).
      2. Load the viral lysate on top and centrifuge at 350,000 × g for 2 hours at 18°C.
      3. Extract the 40% layer containing AAV particles.
    2. Titer Determination: Use RT-qPCR to measure the genome copy titer.
    3. (Optional) Additional QC assays.

Packaging Helper Plasmids In AAV Packaging

Certain packaging protocols involve the use of helper plasmids. Ultimately the usage of helper plasmids occurs where cells lack endogenous helper genes (for small scale AAV manufacturing when certain cell lines like HEK293T require the usage of packaging helper plasmids) or if a particular serotype requires helper plasmids. Learn more about AAV packaging plasmids and pHelper.

Notes:

  • Titer measuring: RT-qPCR is an ideal assay technique to measure AAV titer because of its high sensitivity, vector genome specificity, and very scalable characteristics. 
  • Safety: Follow appropriate safety guidelines for working with adeno-associated viral vectors, including the use of biosafety cabinets and personal protective equipment.

Notes from the BioInnovatise laboratory:

  • Our team uses a HEK293T cell line for a highly efficient AAV packaging cell line
  • We use RT-qPCR for all AAV packaging productions. 
  • Our recommended QC assays options may be ELISA, infectious titer assay, SDS-PAGE / western blot, transmission electron microscopy, analytical ultracentrifugation, and sterility test / endotoxin assays.

Want to learn more about the latest in AAV based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News are always collecting and publishing the latest information on AAV based research.

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