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Unveiling the sex bias: higher preexisting and neutralizing titers against AAV in females and implications for gene therapy

Gene Therapy volume 32, pages 339–348 (2025)Cite this article

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Abstract

Gene therapy with AAV vectors is a promising approach for treating numerous genetic disorders but is often hindered by preexisting antibodies that neutralize the vectors. Given that females may exhibit stronger immune responses than males, this study hypothesizes that females may have higher preexisting antibody titers against AAV. Serum samples from two U.S. cohorts were analyzed for antibody titers, antibody subtypes, and transduction inhibition activity against AAV serotypes AAV1, AAV2, AAV5, AAV8, and AAV9. We found that among seropositive samples, females had higher preexisting antibody levels and neutralizing activities against AAV9 and other serotypes. Immunoglobulin subclass analysis showed IgG1 dominance in both sexes, but females had higher IgA levels, whereas males had higher levels of IgG2. We further evaluated the cellular level of this differential immune response to AAV by stimulation of male and female human PBMCs. We observed dose-dependent increase in cytokines and chemokines in female PBMCs which suggests a differential inflammatory response. Altogether, our findings suggest that the enhanced immune response in females could lead to neutralization and faster clearance of AAV vectors with potential to impact the efficacy of gene therapy.

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Fig. 1: Female serum has higher titers of preexisting antibodies against AAV9 than male.
Fig. 2: Variable AAV9 specific immunoglobulin subclass patterns in male and female samples.
Fig. 3: Female serum has higher levels or preexisting antibodies against other natural AAV serotypes.
Fig. 4: Female sera is more neutralizing.
Fig. 5: Correlation between binding and neutralization titer.
Fig. 6: Analysis of cytokine levels in human PBMCs from females and males.
Fig. 7: Analysis of chemokine levels in human PBMCs from females and males.

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Acknowledgements

We thank Dr. Abdul Mohin Sajib and Dr. Alan Baer for training and advice on AAV manufacturing and Dr. Fei Mo and Dr. Ross Marklein for helpful review of the manuscript draft.

Funding

This work was supported by the FDA Office of Women’s Health. This project was supported in part by an appointment to the ORISE Research Participation Program at the CBER, U.S. Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and FDA/Center.

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Author notes

  1. Sima Saleh

    Present address: Office of Nonprescription Drugs, Office of New Drugs, Center for Drug Evaluation and Research, U. S. Food and Drug Administration, Silver Spring, MD, 20993, USA

Authors and Affiliations

  1. Office of Gene Therapy, Office of Therapeutic Products, Center for Biologics Evaluation and Research, U. S. Food and Drug Administration, Silver Spring, MD, 20993, USA

    Stephanee Warrington, Leila Abdelhamid, Hrittal Saha, Sojin Bing, Sima Saleh & Ronit Mazor

  2. Promega Corporation, Madison, WI, USA

    Trish T. Hoang & Morten Seirup

  3. Facility for Biotechnology Resources, Center for Biologics Evaluation & Research, U. S. Food and Drug Administration, Silver Spring, MD, 20993, USA

    Je-Nie Phue

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Contributions

Conceptualization: SW, RM, LA. Methodology: SW, TH, MS, LA, SB, SH, SS, JNP Supervision: RM. Writing (original draft): SE, LA, RM. Writing (review and editing): TH, MS, SB.

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Correspondence to Ronit Mazor.

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Warrington, S., Hoang, T.T., Seirup, M. et al. Unveiling the sex bias: higher preexisting and neutralizing titers against AAV in females and implications for gene therapy. Gene Ther 32, 339–348 (2025). https://doi.org/10.1038/s41434-025-00528-7

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