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UF Health University of Florida
Powell Gene Therapy Center Department of Pediatrics | College of Medicine
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Frequently Asked Questions (FAQs)

What serotype should I select?

  • Selection of AAV serotype can be driven by several factors including In vitro and In vivo application, tissue tropism, serotype productivity and transgene construct.  Refer to the following link for a general tropism chart.    

How much plasmid is required for plasmid production?

  • The minimum amount we will need is 5-10ug, to allow for upfront QC work and transformation into our preferred bacterial line.  We will require a detailed electronic plasmid map for each plasmid you provide.  Maps are used for QC analysis as well as primer selection for final titering.

How much plasmid is required to produce AAV vector?

  • We generally request 700ug of transgene plasmid for each 10-layer cell stack being transfected. If you cannot provide sufficient plasmid for transfection, please request plasmid production. We will require a detailed electronic plasmid map for each plasmid you provide.  Maps are used for QC analysis as well as primer selection for final titering.

How does the Vector Core manufacture AAV?

  • Our standard vector production platform uses adherent HEK293 cells grown in a Corning 10-layer cell stack (6,360cm2 cell growth area). HEK293 cells are co-transfected with Ad helper, AAV rep–cap and rAAV vector plasmids. Cell lysates are purified using Iodixanol, then concentrated, and buffer exchanged into the excipient buffer.

What total vector genome (VG) can I expect from one cell stack?

  • The yield from one Corning 10-layer cell stack production is typically 1E+12 to 1E+13 total vector genomes (VG). The yield can be influenced by the specific serotype requested as well as the transgene being packaged.  Please contact us to discuss specifics of your project.

What are empty capsids?

  • Some clients request production of true empty capsids for experimentation. Empty capsids are produced using our standard production protocol with the omission of the transgene plasmid resulting in production of complete capsid lacking a genetic payload.

What is an enhanced for full capsids vector?

  • Enhanced for full capsid vector preparations were created for use as a standard for comparing capsid protein to viral genome titer. Enhanced for full capsid preparations are processed using additional purification steps beyond standard Iodixanol. The additional steps include ultracentrifugation using cesium chloride gradients and FPLC affinity chromatography. Titers are determined by ddPCR and the full to empty capsid ratio is determined by cryogenic electron microscopy and found to be greater than 98.6% full capsids.

Do I need to request additional purification?

  • Affinity purification is usually requested for studies involving non-human primates (NHP) and when the highest level of purity is required. Our standard production platform purifies via Iodixanol resulting in 50-90% full capsids. The purity of our vector product is sufficient for use in most animal research and in vitro studies. Additionally, large production scales will require FPLC affinity purification due to the large volume of material being processed. 

What is the difference between qPCR and ddPCR?

  • Both qPCR and ddPCR are very sensitive and reliable means of quantifying AAV viral titers. Our standard method of vector titering is assessed using qPCR which is suitable for most client needs. Alternatively, ddPCR will yield viral titers that represent the absolute copy number and regarded as the highest level of titering accuracy. Titering by ddPCR is a requirement if your plasmid construct is self-complementary.  

Can I request a specific final viral titer (VG/ml)

  • Requested final titers (VG/ml) can usually be achieved. To do this, we titer the midstream product and concentrate using the appropriate buffer volumes to achieve the desired titer target. The preliminary titer will require an additional cost.  Some capsid serotypes will not support high concentrations without loss to aggregation.   In these specific instances, we will advise our clients of the safe upper level of concentration.

What excipient buffer should I choose?

  • Buffer choices are usually based on route of administration, targeted tissues and capsid serotype.  Some capsids require specific buffer formulations at higher concentrations.  Most capsids have multiple suitable excipient buffer choices.  If you are uncertain which buffer would be best for your specific application, please feel free to reach out for assistance.

What are the proper storage conditions for my AAV vector?   

  • Vector products should be held at -80°C until used. Once thawed, the product can be stored at 4°C for one week without significant loss of biological activity.
  • We recommend aliquoting your vectors into low protein binding tubes upon receipt. This helps avoid repeated freeze-thaw cycles and prevent loss of virus. To maintain accurate titer, aliquot a minimum of 20ul per tube.
  • Alternatively, vector aliquots can be made at the completion of the production for a nominal charge.

What timeline can be expected for my custom AAV preparation?

  • Production proceeds on a first come first serve basis with the timeline depending on the length of our vector production queue. Smaller (1-3CS) production projects can usually be completed in 5-6 weeks. Larger scale production runs require additional time. Please contact us for an up-to-date production timeline.

What is your shipping address?

  • Please do not ship anything to us without email confirmation by our lab.
  • University of Florida
    Attention: Sanford Boye
    1200 Newell Drive ARB, room RG-171
    Gainesville FL, 32610

What is your billing address?

  • University of Florida
    Powell Gene Therapy Center
    Attention: Deena Sanders
    PO Box 100292
    Gainesville, FL 32610