Transduction of human B-lymphocytes using adeno-associated viruses of various serotypes
Abstract
Introduction. Adeno-associated virus (AAV) vectors are the leading platform for gene therapy in the treatment of various human diseases. Gene delivery to human B-lymphocytes using AAV vectors presents significant challenges, as they are resistant to AAV infection. Researches is being carried out to find effective methods to overcome this problem.
The aim of the study was to develop a method for delivering genes using AAV vectors to human B-lymphocytes in vitro.
Material and methods. B-lymphocytes were isolated from the peripheral blood of healthy donors by Ficoll-Verografin density gradient centrifugation with further enrichment by negative selection using magnetic beads. B-lymphocytes were stimulated in vitro with a lymphokine mixture containing IL-2, IL-4, BAFF, IL-21 and multimerized CD40L (mCD40L). After 24 hours, stimulated B-lymphocytes were infected with recombinant AAV carrying the green fluorescent protein reporter gene (GFP). 3 serotypes AAV2, AAV6 and AAV-DJ have been tested. 48 hours after infection, the proportion of transduced B-lymphocytes was determined. After 7 days, the number and phenotype of stimulated B-lymphocytes were determined by flow cytometry, and the secretion of Ig into the supernatant of cultured cells was assessed using the enzyme-linked immunosorbent assay. HEK293T cells were used as a transduction control.
Results. Serotypes AAV2, AAV6 and AAV-DJ efficiently and equally transduced HEK293T cells. Unstimulated B-lymphocytes were resistant to the action of AAV and did not show a noticeable fluorescence of the GFP reporter protein. B-lymphocytes stimulated with a mixture of lymphokines, which included IL-2, IL-4, BAFF, IL-21 and mCD40L, were efficiently transduced with AAV2, AAV6 and AAV-DJ. The highest expression of the transgene was provided by the AAV6 serotype. The transduced B-lymphocytes continued to actively proliferate, acquired the plasmablast phenotype and secreted Ig into the supernatant. In terms of their functional properties, the transduced B-lymphocytes did not differ from the control cells that were not exposed to infection. Thus, at moderate doses, AAV infection did not have a toxic effect on B-lymphocytes and did not affect the physiological parameters of B-cells. Efficient transduction was observed in both subpopulations of memory B-cells, both with switched (IgG+CD27+) and non-switched (IgM+CD27+) Ig synthesis.
Conclusion. Stimulation with a mixture of lymphokines is critical for efficient transgene transfer into human B-lymphocytes using rAAV vectors. In the transduction of B-lymphocytes the AAV6 serotype is the most effective.
Keywords:adeno-associated virus (rAAV); AAV serotypes; gene transfer; B-lymphocytes; transduction; GFP; B lymphocyte stimulation
For citation: Byazrova M.G., Mikhailov A.A., Sukhova M.M., Bardina M.V., Shmidt A.A., Prilipov A.G., Filatov A.V. Transduction of human B-lymphocytes using adeno-associated viruses of various serotypes. Immunologiya. 2023; 43 (4): 443–54. DOI: https://www.doi.org//10.33029/0206-4952-2023-44-4-443-454 (in Russian)
Funding. The study was supported by the grant of Russian Science Foundation (RSF) No. 23-15-00289 (https://rscf.ru/project/23-15-00289/).
Conflict of interests. The authors declare no conflict of interests.
Authors’ contribution. Collection and processing of material – Byazrova M.G., Mikhailov A.A., Sukhova M.M., Shmidt A.A.; text writing, editing – Bardina M.V., Filatov A.V.; the final version of the text – Prilipov A.G., Filatov A.V.
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