We attempted to transduce NK-92 cells using lentivirus. The virus was produced in HEK293T cells, yielding a biological titer of 3×10⁶ particles per mL. We concentrated the virus using two different methods: PEG precipitation and Amicon (100 kDa) filtration, maintaining similar final concentrations.
For transduction, we used:
- Filter-concentrated virus at MOI 10, 20, 30, 40, and 50 (no positive results).
- PEG-concentrated virus at MOI 10 and 15 (11% positivity at MOI 10).
However, with PEG-concentrated virus, we observed a 50% cell loss due to cell death. Additionally, after changing the medium, all NK-92 cells died within 24 hours.
Has anyone encountered similar issues? What strategies could improve transduction efficiency while minimizing toxicity? Any recommendations would be highly appreciated!
What method did you use to obtain the titer?
It sounds like you are getting inefficient packaging of the viral genome into the virus particle. In other words you have a lot of virus particles without a genome in them. Which would explain why you are seeing no to very low positivity.
Too high a titer/too much virus will cause cell death whether there is a viral genome present or not.
I don't think you have a transduction problem. You have a packaging problem which occurs when you are producing the virus in the HEK cells. You may want to alter the ratios of the plasmids you use to make the virus.
Dear Masoud,
It is true that NK cells, primary as well as cell lines, are difficult to genetically modify, even with a lentiviral vector. One problem may be the MOI to use, and especially if the viral particles are “only” concentrated and not purified. A high dose of virus can induce a massive non expected innate immune response from the target cells that will lower the overall efficiency.
This is why it may be interesting to lower the MOI while increasing the efficiency, and to this end, I would like to propose you to try our LentiBlast Premium.
LentiBlast Premium is a patented modified poloxamer that we developed a couple of years ago and that acts by simultaneously neutralizing electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Due to a favorable “membrane permeable effect” limiting the transmembrane potential changes, it is non-toxic and totally compatible with cell viability.
It is a real asset to viral infection because it allows to increase the effectiveness of the viral vector, thus reducing the MOI, and consequently the immune response of the cell. Secondarily, it reduces the cost of experience.
LentiBlast Premium was demonstrated to be efficient of multiple cell types from Hematopoietic Stem Cells, to T lymphocytes and I am sure it would be worth give it a try for NK cell type.
Some examples of publications with LentiBlast Premium:
- Bone marrow derived macrophages: Fonseca GJ. Nat Commun. 2019 Jan 24;10(1):414.
- Hematopoietic Stem Cells: Burton OT. Immunity. 2024 Jun 11:S1074-7613(24)00277-2.
- HMEC: Nassour J. Nature . 2023 Feb;614(7949):767-773.
- Myeloid progenitor: Verdys P. EMBO J. 2024 Jul 18. doi: 10.1038/s44318-024-00173-7.
- T lymphocytes CD4+: Claireaux M. Nature Commun 2022 Jan 26;13(1):521.
- THP-1: Chirayath TW. Nat Commun. 2024 Sep 18;15(1):8179.
you can have more infos at: https://ozbiosciences.com/lentivirus-retrovirus/113-418-lentiblast-premium-transduction-enhancer.html#/12-capacity-500_l
Would you like to give LentiBlast a try, please do not hesitate to contact me via ResearchGate or directly at: [email protected]; I will be glad to send you a sample !
best regards,
Cedric
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