Frequently Asked Questions:

VirusGEN® AAV Transfection Kit

The VirusGEN® AAV Transfection Kit, available in Research Use Only (RUO) and GMP formats, enables high titer AAV production in suspension 293-derived cell types. The components of the VirusGEN® AAV Transfection Kit and VirusGEN® GMP AAV Transfection Kit are identical in formulation, which equips researchers with the same reliable and scalable performance and streamlined viral vector production workflow as they progress from discovery and development to clinical trials and commercial manufacturing. The VirusGEN® GMP AAV Transfection Kit undergoes release testing for sterility, formulation identity, mycoplasma and endotoxin. Building on our commitment to product quality, the VirusGEN® GMP AAV Transfection Kit is manufactured under Current Good Manufacturing Practices (cGMP), which follow the regulations defined in U.S. 21 CFR 820 from the FDA.

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

Q13. Which serum-free medium should I use for preparing TransIT-VirusGEN®:DNA transfection complexes?
Q14. Can I filter TransIT-VirusGEN® Reagent?
Q15. For AAV generation, how much TransIT-VirusGEN® Reagent, total DNA (GOI containing transfer + rep/cap + pHelper) and VirusGEN® Complex Formation Solution and Enhancer should be used for transfection?
Q16. What is the recommended plasmid ratio for optimal AAV generation?
Q17. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Q18. Will antibiotics interfere with my transfection?
Q19. Is a media change required post-transfection?
Q20. Are cell density and passage number important factors for successful transfection?
Q21. At what time point do you recommend harvesting AAV post-transfection?
Q22. How can I titer my AAV stock?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q23. What can I do to further improve my AAV titers when using VirusGEN® AAV Transfection Kit?
Q24. Should I be concerned about cellular toxicity when using VirusGEN® AAV Transfection Kit?

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in VirusGEN® AAV Transfection Kit?
The VirusGEN® AAV Transfection Kit consists of two components: TransIT-VirusGEN® Transfection Reagent and VirusGEN® AAV Complex Formation Solution and Enhancer.

Q2. What is the difference between VirusGEN® AAV Transfection Kit and VirusGEN® GMP AAV Transfection Kit?
The components of the three products have the same formulation, but each product has different quality control release requirements:

The TransIT-VirusGEN® Transfection Reagent is a research-grade product which is released based on appearance and a functional AAV titer assay for all volume configurations; additional release testing (sterility, endotoxin, and mycoplasma) is performed for configurations that are 1.5 ml or greater in volume. The 100-ml VirusGEN® AAV Complex Formation Solution and Enhancer is a research-grade product which is released based on appearance, sterility, identity, endotoxin, osmolality and pH. The 1-L VirusGEN® AAV Complex Formation Solution and Enhancer is a research-grade product which is released based on appearance, sterility, identity, mycoplasma, endotoxin, osmolality and pH.
The TransIT-VirusGEN® GMP Transfection Reagent and VirusGEN® GMP AAV Complex Formation Solution and Enhancer are manufactured in accordance with the Mirus Bio Quality Management System, which incorporates the appropriate cGMP product and production requirements. The products are manufactured in audited facilities that are certified to ISO 13485 and comply with 21 CFR 820. Release testing for TransIT-VirusGEN® GMP Transfection Reagent consists of appearance, sterility, identity, mycoplasma and endotoxin assays. In addition to these tests, the VirusGEN® GMP AAV Complex Formation Solution and Enhancer is assayed for pH and osmolality.

Q3. Is a functional AAV titer assay performed for quality release testing of the VirusGEN® GMP AAV Transfection Kit?
A functional AAV titer assay is not performed for quality release testing of the VirusGEN® GMP AAV Transfection Kit. The VirusGEN® GMP AAV Transfection Kit is formulated through a validated manufacturing process which was established to ensure consistent functional AAV titer thereby removing the need to perform a functional AAV titer assay on each lot.

Q4. What analytical assays are available for VirusGEN® AAV Transfection Kit?
Residual reagent and identity tests are available for VirusGEN® products. Please contact techsupport@mirusbio.com for more information.

Q5. What configurations are available for VirusGEN® AAV Transfection Kit and VirusGEN® GMP AAV Transfection Kit?

VirusGEN® AAV Transfection Kit is available in three formats (MIR 6750, MIR 6745 and MIR 6755). MIR 6750 includes 2×1.5 ml TransIT-VirusGEN® Transfection Reagent and 1×100 ml VirusGEN® AAV Complex Formation Solution and Enhancer. MIR 6745 includes 1×30 ml TransIT-VirusGEN® Transfection Reagent and 1×1 L VirusGEN® AAV Complex Formation Solution and Enhancer. MIR 6755 includes 1×150 ml TransIT-VirusGEN® Transfection Reagent and 5×1 L VirusGEN® AAV Complex Formation Solution and Enhancer.
VirusGEN® GMP AAV Transfection Kit (MIR 6815-GMP) includes 1×150 ml TransIT-VirusGEN® GMP Transfection Reagent and 5×1 L VirusGEN® GMP AAV Complex Formation Solution and Enhancer.

Q6. What is the composition of TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® Reagent is a proprietary, animal origin free formulation composed of a lipid and polymer mixture.

Q7. What is the composition of VirusGEN® AAV Complex Formation Solution and Enhancer?
VirusGEN® AAV Complex Formation Solution and Enhancer is a proprietary, chemically defined and animal origin free formulation.

Q8. What is the mechanism of action of each of the components of VirusGEN® AAV Transfection Kit?
TransIT-VirusGEN® Reagent complexes with negatively charged DNA to form lipopolyplexes that associate with the negatively charged cell membrane via electrostatic interactions. These lipopolyplexes are taken into the cell via endocytosis. We believe the enhancer component increases production of certain viral proteins when added between 0-24 hours post-transfection. The enhancer component does not directly impact transfection efficiency because improved AAV titers are observed even when the enhancer component is added several hours post-transfection.

Q9. What is the shelf life of VirusGEN® AAV Transfection Kit?
For the VirusGEN® AAV Transfection Kit: the TransIT-VirusGEN® Transfection Reagent component is guaranteed for 1 year from the date of purchase, and the VirusGEN® AAV Complex Formation Solution and Enhancer is guaranteed for 6 months from the date of purchase. All components of the VirusGEN® GMP AAV Transfection Kit are guaranteed for a minimum of three months from the date of purchase with recertification of Retest Dates based on real-time stability data. If the Retest Date is updated it will be revised on the product Certificate of Analysis (CoA). Please contact techsupport@mirusbio.com for the CoA.

Q10. How should VirusGEN® AAV Transfection Kit be stored?
Store TransIT-VirusGEN® Reagent from -10 to -30°C. Store VirusGEN® AAV Complex Formation Solution and Enhancer from 4 to 10°C.

Q11. Can VirusGEN® AAV Transfection Kit be used to generate AAV in both adherent and suspension cells?
VirusGEN® AAV Complex Formation Solution and Enhancer is only recommended for suspension HEK 293 cell lines. Generally, use of VirusGEN® AAV Complex Formation Solution and Enhancer in adherent HEK 293 cell lines is ineffective or even detrimental to AAV production. However, TransIT-VirusGEN® Reagent component is compatible for high titer AAV production in both adherent and suspension 293-derived cell types and can be purchased separately from the enhancer component of VirusGEN® AAV Transfection Kit.

Q12. What titers are expected when using VirusGEN® AAV Transfection Kit?
Virus titers vary widely and ultimately depend on the HEK 293 cell subtype, packaging system and transfer vector and serotype used for AAV generation. For more information, see VirusGEN® AAV Transfection Kit product page.

PROTOCOL QUESTIONS AND ANSWERS

Q13. Which serum-free medium should I use for preparing TransIT-VirusGEN®:DNA transfection complexes?
For best results, we recommend using AAV Complex Formation Solution and Enhancer for preparing TransIT-VirusGEN® Reagent:DNA complexes. If you would like to evaluate transfection without the enhancer component (i.e. without AAV Complex Formation Solution and Enhancer), we recommend using Phosphate Buffered Saline (PBS) without calcium or magnesium. Serum-free media formulations such as Opti-MEM® I Reduced-Serum Medium can also be used. NOTE: Some serum-free media formulations contain polyanions such as heparin or polydextran sulfate that may inhibit formation of active transfection complexes and should be avoided.

Q14. Can I filter TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® Reagent can be filtered with no effect on reagent performance if it has not been diluted in aqueous solution. TransIT-VirusGEN® Reagent cannot be filtered after it has been diluted in an aqueous solution such as PBS as this results in loss of critical formulation components.

Q15. For AAV generation, how much TransIT-VirusGEN® Reagent, total DNA (GOI containing transfer + rep/cap + pHelper) and VirusGEN® Complex Formation Solution and Enhancer should be used for transfection?
The quantities of TransIT-VirusGEN® Reagent, total DNA and Complex Formation Solution and Enhancer needed for transfection will depend upon cell culture vessel and volume (see Table 1 in the VirusGEN® AAV Transfection Kit Full Protocol). Per 1 ml of complete growth medium, our starting recommendation is 3 µl TransIT-VirusGEN® Reagent and 2 µg total DNA in 100 µl of VirusGEN® AAV Complex Formation Solution and Enhancer, which represents a 1.5:1 ratio (µl of reagent/µg of DNA, vol:wt) in 10% culture volume.

Q16. What is the recommended plasmid ratio for optimal AAV generation?
The TransIT-VirusGEN® Reagent was optimized using a 1:1:1 weight ratio of pAAV-hrGFP, pAAV-RC and pHelper (AAV Helper-Free System, Agilent Technologies). The optimal ratio may differ by serotype and packaging system and should be empirically determined for each unique experimental system.

Q17. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Our starting recommendation is 15-60 min. However, users should empirically determine optimal complex formation time for each unique experimental system. Incubating complexes at 4°C may allow for longer, optimal complex formation times. Importantly, complexes should not be agitated after the initial mixing of reagent and DNA, which may disrupt complexes and result in decreased viral titers.

Q18. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q19. Is a media change required post-transfection?
No. A media change to remove TransIT-VirusGEN® Reagent:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to AAV titers.

Q20. Are cell density and passage number important factors for successful transfection?
Yes. For suspension 293 cell types, the recommended cell density at transfection is 2×10⁶ cells/ml. Passage cells 18-24 hours before transfection to ensure that cells are actively dividing and reach the appropriate density at time of transfection.

Q21. At what time point do you recommend harvesting AAV post-transfection?
The optimal incubation time for harvesting high-titer AAV is generally 48-72 hours post-transfection but should be determined empirically for each unique experimental system

Q22. How can I titer my AAV stock?
ELISA assays designed to measure intact AAV particles and qPCR/ddPCR assays measuring the viral nucleic acid content of purified viruses (genome copy/ml) are commonly used to titer AAV. These methods measure particles or nucleic acid present which does not equate to functional virus (i.e. virions capable of transducing host cells). The VirusGEN® AAV Transfection Kit Full Protocol describes a method to determine functional titer by transducing HT-1080 cells with a GFP reporter AAV2. In this method, cells are transduced at a low MOI and assayed for GFP expression after 72 hours of incubation. Transducing Units per milliliter (TU/ml) are calculated based on the following formula:

Titer (HT-1080 Transducing Units/ml) = Number of target cells (Count at day 2, transduction) x [% GFP positive cells/100]
(Volume of AAV2 Stock in ml)

Reference Card: Methods to Measure Lentivirus and AAV in Your Sample
The TransMISSION: Transfection 101 - Understand Viral Titer and Quality

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q23. What can I do to further improve my AAV titers when using VirusGEN® AAV Transfection Kit?
Multiple factors can contribute to AAV titers; refer to the following critical troubleshooting tips to improve your transfection results:

Optimize cell density at time of transfection - The recommended cell density for suspension 293 cells at the time of transfection is 2×10⁶ cells/ml.
Optimize TransIT-VirusGEN® Reagent to DNA ratio - For AAV generation, we recommend using 3 µl TransIT-VirusGEN® Reagent per 2 µg total DNA.
Use high quality DNA - DNA used for transfection should be highly purified, sterile and free from contaminants such as endotoxin. Remove any traces of endotoxin (e.g. bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit (MIR 5910 or 5900).
Do NOT incubate TransIT-VirusGEN® Reagent without DNA - Diluting TransIT-VirusGEN® Reagent can be performed, but diluted reagent should be mixed with DNA within 5 minutes for optimal transfection complex formation. NOTE: TransIT-VirusGEN® Reagent cannot be filtered after it has been diluted in an aqueous solution such as PBS as this results in loss of critical formulation components.
Do NOT disturb TransIT-VirusGEN® Reagent:DNA complexes after formation - After the initial mixing of TransIT-VirusGEN® Reagent with DNA, do not agitate or mix the complexes further. Agitation of the complexes will likely result in suboptimal functional virus production.
Form complexes in appropriate complex formation solution volume – We recommend forming transfection complexes in 10% of total culture volume, e.g. form complexes in 2.5 ml VirusGEN® AAV Complex Formation Solution and Enhancer (or PBS) to transfect 25 ml culture volume. Increasing or decreasing the complex formation solution volume will require optimization of complex formation time.
Cells which are normally cultured in serum-containing medium should remain in serum-containing medium during and post-transfection – Though complex formation steps should occur in serum-free medium (e.g. VirusGEN® AAV Complex Formation Solution and Enhancer or PBS), the cells themselves should be maintained in their normal complete (serum-containing) medium. A cell culture medium change is not necessary.
Optimize quantity of DNA used for transfection - Refer to Table 1 in the VirusGEN® AAV Transfection Kit Full Protocol for recommended total DNA per reaction. Using amounts out of range of the recommended amounts of total DNA will likely result in suboptimal functional virus production.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

Q24. Should I be concerned about cellular toxicity when using VirusGEN® AAV Transfection Kit?
When generating AAV with VirusGEN® AAV Complex Formation Solution and Enhancer, cell growth may decrease. This is normal and does not adversely affect virus titers. However, cellular toxicity that impacts viral titers may be observed if the transfection protocol is not followed properly. The following are considerations for improving cell health post-transfection:

Cell density too low at time of transfection - If cytotoxicity is observed, increase the cell density to maintain cell health.
Plasmid DNA contains high level of endotoxin - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit (MIR 5910 or 5900).
Expressed target gene is toxic to cells - Compare toxicity levels against a cells alone control and cells transfected with an empty vector to assess the cytotoxic effects of the target protein being expressed. If lower levels of target gene expression are desired, consider reducing the amount of target plasmid. Maintain the optimal TransIT-VirusGEN® Reagent:DNA ratio by using carrier DNA such as an empty cloning vector. For example, use 0.5 µg transfer DNA, 0.5 µg pUC18 and 1 ug packaging plasmids for complexes with a reduced amount of target plasmid.
Cell morphology has changed – If the passage number of the cells is too high or too low, they can be more sensitive to transfection reagents. Maintain a similar passage number between experiments to ensure reproducibility.
Mycoplasma contamination affecting cell health - Check your cells for mycoplasma contamination. Use a freshly thawed stock of cells or use appropriate antibiotics to eliminate mycoplasma.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

Frequently Asked Questions:

VirusGEN® AAV Transfection Kit

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS AND ANSWERS

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS AND ANSWERS

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