Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Engineered AAV capsids mediate transduction of murine neurofibroma and sciatic nerve

Gene Therapy volume 32, pages 385–397 (2025)Cite this article

Subjects

Abstract

Genetic diseases such as Neurofibromatosis type 1 (NF1) and Charcot-Marie Tooth disease involve Schwann cells (SCs) associated with peripheral nerves. Gene therapy using adeno-associated virus (AAV) vector mediated gene delivery is a promising strategy to treat these diseases. However, AAV-mediated transduction of SCs in vivo after intravascular delivery is relatively inefficient, with a lack of extensive characterization of different capsids to date. Here, we performed an in vivo selection with an AAV9 capsid peptide display library in a mouse model of NF1. We chose one capsid variant, AAV-SC3, which was present in NF1 nerves for comparison to two benchmark capsids after systemic injection. AAV-SC3 significantly outperformed one of the two benchmark capsids at levels of transgene mRNA in the neurofibroma. Immunofluorescence microscopy revealed transgene expressing Sox10-positive SCs throughout the neurofibroma with AAV-SC3 injection. Next, we performed a pooled screen with four of the top capsids from our initial selection and AAV9 and identified one capsid, AAV-SC4, with enhanced biodistribution to and transduction of normal sciatic nerve in mice. This capsid displayed a peptide with a known laminin-binding motif, which may provide a conduit for future laminin-targeting strategies. Our results provide a baseline for future AAV-based gene therapies developed for NF1 or other diseases that affect SCs.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: NF1flox/flox/Flucflox induced tumor mouse model and characterization of neurofibroma.
Fig. 2: In vivo selection with an AAV peptide display library allows enrichment of variants recovered from the second round in the sciatic nerve of Nf1flox/flox/Flucflox induced tumor model.
Fig. 3: AAV-SC3 displays similar biodistribution to liver and brain as AAV9, but increased transgene expression in sciatic nerve of neurofibroma-bearing mice.
Fig. 4: Identification of an AAV capsid variant with enhanced biodistribution to sciatic nerve after systemic injection.
Fig. 5: Side by side comparison of AAV9 and AAV-SC4 biodistribution to and transduction of sciatic nerve in C57BL/6 mice after systemic administration.
Fig. 6: AAV-SC4 capsid packaging a self-complementary (sc) AAV-CAG-GFP genome transduces sciatic nerve after systemic injection.

Similar content being viewed by others

Data availability

Data are available upon request. Plasmid expressing the capsid gene for AAV-SC4 is available at Addgene (Plasmid ID 236254).

Code availability

All code written and used in this study can be accessed by emailing the authors, who will provide scripts and methods of use.

References

  1. Mendell JR, Al-Zaidy SA, Rodino-Klapac LR, Goodspeed K, Gray SJ, Kay CN, et al. Current clinical applications of in vivo gene therapy with AAVs. Mol Ther. 2021;29:464–88.

    Article  CAS  Google Scholar 

  2. Keeler AM, Flotte TR. Recombinant adeno-associated virus gene therapy in light of luxturna (and zolgensma and glybera): where are we, and how did we get here? Annu Rev Virol. 2019;6:601–21.

    Article  CAS  Google Scholar 

  3. Ozelo MC, Mahlangu J, Pasi KJ, Giermasz A, Leavitt AD, Laffan M, et al. Valoctocogene roxaparvovec gene therapy for hemophilia A. N. Engl J Med. 2022;386:1013–25.

    Article  CAS  Google Scholar 

  4. Pipe SW, Leebeek FWG, Recht M, Key NS, Castaman G, Miesbach W, et al. Gene therapy with etranacogene dezaparvovec for hemophilia B. N. Engl J Med. 2023;388:706–18.

    Article  CAS  Google Scholar 

  5. Tai CH, Lee NC, Chien YH, Byrne BJ, Muramatsu SI, Tseng SH, et al. Long-term efficacy and safety of eladocagene exuparvovec in patients with AADC deficiency. Mol Ther. 2022;30:509–18.

    Article  CAS  Google Scholar 

  6. Mendell JR, Sahenk Z, Lehman K, Nease C, Lowes LP, Miller NF, et al. Assessment of systemic delivery of rAAVrh74.MHCK7.micro-dystrophin in children with duchenne muscular dystrophy: a nonrandomized controlled trial. JAMA Neurol. 2020;77:1122–31.

    Article  Google Scholar 

  7. Kagiava A, Richter J, Tryfonos C, Leal-Julia M, Sargiannidou I, Christodoulou C, et al. Efficacy of AAV serotypes to target Schwann cells after intrathecal and intravenous delivery. Sci Rep. 2021;11:23358.

    Article  CAS  Google Scholar 

  8. Kagiava A, Karaiskos C, Richter J, Tryfonos C, Jennings MJ, Heslegrave AJ, et al. AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy. Gene Ther. 2021;28:659–75.

    Article  CAS  Google Scholar 

  9. Tanguy Y, Biferi MG, Besse A, Astord S, Cohen-Tannoudji M, Marais T, et al. Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice. Front Mol Neurosci. 2015;8:36.

    Article  Google Scholar 

  10. Homs J, Ariza L, Pages G, Udina E, Navarro X, Chillon M, et al. Schwann cell targeting via intrasciatic injection of AAV8 as gene therapy strategy for peripheral nerve regeneration. Gene Ther. 2011;18:622–30.

    Article  CAS  Google Scholar 

  11. Hanlon KS, Meltzer JC, Buzhdygan T, Cheng MJ, Sena-Esteves M, Bennett RE, et al. Selection of an Efficient AAV Vector for Robust CNS Transgene Expression. Mol Ther Methods Clin Dev. 2019;15:320–32.

    Article  CAS  Google Scholar 

  12. Ortonne N, Carroll SL, Rodriguez FJ, Miller DC, Nazarian RM, Blakeley JO, et al. Assessing interobserver variability and accuracy in the histological diagnosis and classification of cutaneous neurofibromass. Neurooncol Adv. 2020;2:i117–i23.

    Google Scholar 

  13. Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF. Neurofibromas in NF1: Schwann cell origin and role of tumor environment. Science. 2002;296:920–2.

    Article  CAS  Google Scholar 

  14. Woolfenden S, Zhu H, Charest A. A Cre/LoxP conditional luciferase reporter transgenic mouse for bioluminescence monitoring of tumorigenesis. Genesis. 2009;47:659–66.

    Article  CAS  Google Scholar 

  15. Prabhakar S, Taherian M, Gianni D, Conlon TJ, Fulci G, Brockmann J, et al. Regression of schwannomas induced by adeno-associated virus-mediated delivery of caspase-1. Hum Gene Ther. 2013;24:152–62.

    Article  CAS  Google Scholar 

  16. Ivanchenko MV, Hanlon KS, Devine MK, Tenneson K, Emond F, Lafond JF, et al. Preclinical testing of AAV9-PHP.B for transgene expression in the non-human primate cochlea. Hear Res. 2020;394:107930.

    Article  Google Scholar 

  17. Hanlon KS, Cheng M, Ferrer RM, Ryu JR, Lee B, De La Cruz D, et al. In vivo selection in non-human primates identifies AAV capsids for on-target CSF delivery to spinal cord. Mol Ther. 2024;32:2584–603.

  18. Morrison BM, Tsingalia A, Vidensky S, Lee Y, Jin L, Farah MH, et al. Deficiency in monocarboxylate transporter 1 (MCT1) in mice delays regeneration of peripheral nerves following sciatic nerve crush. Exp Neurol. 2015;263:325–38.

    Article  CAS  Google Scholar 

  19. Wu J, Williams JP, Rizvi TA, Kordich JJ, Witte D, Meijer D, et al. Plexiform and dermal neurofibromas and pigmentation are caused by Nf1 loss in desert hedgehog-expressing cells. Cancer Cell. 2008;13:105–16.

    Article  CAS  Google Scholar 

  20. Ribeiro S, Napoli I, White IJ, Parrinello S, Flanagan AM, Suter U, et al. Injury signals cooperate with Nf1 loss to relieve the tumor-suppressive environment of adult peripheral nerve. Cell Rep. 2013;5:126–36.

    Article  CAS  Google Scholar 

  21. Rizvi TA, Akunuru S, de Courten-Myers G, Switzer RC 3rd, Nordlund ML, et al. Region-specific astrogliosis in brains of mice heterozygous for mutations in the neurofibromatosis type 1 (Nf1) tumor suppressor. Brain Res. 1999;816:111–23.

    Article  CAS  Google Scholar 

  22. Hinderer C, Katz N, Buza EL, Dyer C, Goode T, Bell P, et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum Gene Ther. 2018;29:285–98.

    Article  CAS  Google Scholar 

  23. Huang Q, Chen AT, Chan KY, Sorensen H, Barry AJ, Azari B, et al. Targeting AAV vectors to the central nervous system by engineering capsid-receptor interactions that enable crossing of the blood-brain barrier. PLoS Biol. 2023;21:e3002112.

    Article  CAS  Google Scholar 

  24. Kawabata H, Konno A, Matsuzaki Y, Sato Y, Kawachi M, Aoki R, et al. Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization. Mol Ther Methods Clin Dev. 2024;32:101185.

    Article  CAS  Google Scholar 

  25. Beharry A, Gong Y, Kim JC, Hanlon KS, Nammour J, Hieber K, et al. The AAV9 variant capsid AAV-F mediates widespread transgene expression in nonhuman primate spinal cord after intrathecal administration. Hum Gene Ther. 2022;33:61–75.

    Article  CAS  Google Scholar 

  26. Huang Q, Chen AT, Chan KY, Sorensen H, Barry AJ, Azari B, et al. Targeting AAV vectors to the CNS via <em>de novo</em> engineered capsid-receptor interactions. bioRxiv. 2022:2022.10.31.514553.

  27. Akalu A, Roth JM, Caunt M, Policarpio D, Liebes L, Brooks PC. Inhibition of angiogenesis and tumor metastasis by targeting a matrix immobilized cryptic extracellular matrix epitope in laminin. Cancer Res. 2007;67:4353–63.

    Article  CAS  Google Scholar 

  28. Jiang M, Chen M, Liu N. Interactions between Schwann cell and extracellular matrix in peripheral nerve regeneration. Front Neurol. 2024;15:1372168.

    Article  Google Scholar 

  29. Dodd RD, Mito JK, Eward WC, Chitalia R, Sachdeva M, Ma Y, et al. NF1 deletion generates multiple subtypes of soft-tissue sarcoma that respond to MEK inhibition. Mol Cancer Ther. 2013;12:1906–17.

    Article  CAS  Google Scholar 

  30. Stemmer-Rachamimov AO, Louis DN, Nielsen GP, Antonescu CR, Borowsky AD, Bronson RT, et al. Comparative pathology of nerve sheath tumors in mouse models and humans. Cancer Res. 2004;64:3718–24.

    Article  CAS  Google Scholar 

  31. Kershner LJ, Choi K, Wu J, Zhang X, Perrino M, Salomonis N, et al. Multiple Nf1 Schwann cell populations reprogram the plexiform neurofibroma tumor microenvironment. JCI Insight. 2022;7:e154513.

  32. Bohlen MO, McCown TJ, Powell SK, El-Nahal HG, Daw T, Basso MA, et al. Adeno-associated virus capsid-promoter interactions in the brain translate from rat to the nonhuman primate. Hum Gene Ther. 2020;31:1155–68.

    Article  CAS  Google Scholar 

  33. Powell SK, Samulski RJ, McCown TJ. AAV capsid-promoter interactions determine CNS cell-selective gene expression in vivo. Mol Ther. 2020;28:1373–80.

    Article  CAS  Google Scholar 

  34. Gonzalez-Sandoval A, Pekrun K, Tsuji S, Zhang F, Hung KL, Chang HY, et al. The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner. Nat Commun. 2023;14:2448.

    Article  CAS  Google Scholar 

  35. Okada Y, Hosoi N, Matsuzaki Y, Fukai Y, Hiraga A, Nakai J, et al. Development of microglia-targeting adeno-associated viral vectors as tools to study microglial behavior in vivo. Commun Biol. 2022;5:1224.

    Article  CAS  Google Scholar 

  36. McKee KK, Yang DH, Patel R, Chen ZL, Strickland S, Takagi J, et al. Schwann cell myelination requires integration of laminin activities. J Cell Sci. 2012;125:4609–19.

    CAS  Google Scholar 

  37. Skoumal M, Seidlits S, Shin S, Shea L. Localized lentivirus delivery via peptide interactions. Biotechnol Bioeng. 2016;113:2033–40.

    Article  CAS  Google Scholar 

  38. Chan KY, Jang MJ, Yoo BB, Greenbaum A, Ravi N, Wu WL, et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci. 2017;20:1172–9.

    Article  CAS  Google Scholar 

  39. Drouyer M, Chu TH, Labit E, Haase F, Navarro RG, Nazareth D, et al. Novel AAV variants with improved tropism for human Schwann cells. Mol Ther Methods Clin Dev. 2024;32:101234.

    Article  CAS  Google Scholar 

  40. Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther. 2008;16:1073–80.

    Article  CAS  Google Scholar 

  41. Romo CG, Piotrowski AF, Campian JL, Diarte J, Rodriguez FJ, Bale TA, et al. Clinical, histological and molecular features of gliomas in adults with neurofibromatosis type 1. Neuro Oncol. 2023;25:1474–86.

  42. Wilson JM, Flotte TR. Moving Forward After Two Deaths in a Gene Therapy Trial of Myotubular Myopathy. Hum Gene Ther. 2020;31:695–6.

    Article  CAS  Google Scholar 

  43. Hordeaux J, Lamontagne RJ, Song C, Buchlis G, Dyer C, Buza EL, et al. High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury. Mol Ther. 2024;32:952–68.

    Article  CAS  Google Scholar 

  44. Xie J, Xie Q, Zhang H, Ameres SL, Hung JH, Su Q, et al. MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression. Mol Ther. 2011;19:526–35.

    Article  CAS  Google Scholar 

  45. Krolak T, Chan KY, Kaplan L, Huang Q, Wu J, Zheng Q, et al. A high-efficiency AAV for endothelial cell transduction throughout the central nervous system. Nat Cardiovasc Res. 2022;1:389–400.

    Article  Google Scholar 

Download references

Acknowledgements

We thank Dr. Akiko Yoshinaga for help with genotyping of the neurofibroma model and professional insights into NF1, Ms Suzanne McDavitt for skilled editorial assistance and Ms Diane M. Nguyen for assistance with figure artwork. We thank Dr. Judith S. Kempfle for the recommendation of the anti-Sox10 antibody for the immunofluorescence experiment. We thank Dr. Nancy Ratner for providing Nf1flox/flox mice and helpful review of the manuscript. We thank Dr. K.A. Kleopa for helpful discussion around the timing of the in-life mouse AAV gene transfer experiments.

Funding

This work was supported by NIH R01 grant DC017117 (C.A.M.), the Gilbert Family Foundation Award #521015 (C.A.M.) and the Gilbert Family Foundation Award #521013 (X.O.B.). I.C.H is supported in part by the National Institute of Biomedical Imaging and Bioengineering under award number 1K25EB032864-01A1.

Author information

Author notes

  1. These authors contributed equally: Edwina Abou Haidar, Shilpa Prabhakar.

Authors and Affiliations

  1. Molecular Neurogenetics Unit, Department of Neurology, The Massachusetts General Hospital, Charlestown MA and Program in Neuroscience, Harvard Medical School, Boston, MA, USA

    Edwina Abou Haidar, Shilpa Prabhakar, Pike See Cheah, Killian S. Hanlon, Paula Espinoza, Adam V. Crain, Nikita Patel, Greta W. Radcliff, Ming Cheng, Demitri de la Cruz, Carrie Ng, Cintia Carla da Hora, Xandra O. Breakefield & Casey A. Maguire

  2. Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia

    Pike See Cheah

  3. Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    Killian S. Hanlon

  4. EGA-Institute for Women’s Health, University College London, London, UK

    Killian S. Hanlon

  5. Transduction Consulting, London, UK

    Killian S. Hanlon

  6. Surgical Photonics & Engineering Laboratory, Mass Eye and Ear, Harvard Medical School, Boston, MA, 02114, USA

    Iván Coto Hernández, Steven Minderler & Nate Jowett

  7. Mayo Clinic College of Medicine and Science, Jacksonville, FL, 32224, USA

    Cintia Carla da Hora

  8. Department of Medicine, Harvard Medical School, Boston, MA, 02215, USA

    Alain Charest

  9. Cancer Research Institute, Beth Israel Deaconess Medical Center, Boston, MA, 02215, USA

    Alain Charest

  10. Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Anat Stemmer-Rachamimov

Authors
  1. Edwina Abou Haidar
    View author publications

    Search author on:PubMed Google Scholar

  2. Shilpa Prabhakar
    View author publications

    Search author on:PubMed Google Scholar

  3. Pike See Cheah
    View author publications

    Search author on:PubMed Google Scholar

  4. Killian S. Hanlon
    View author publications

    Search author on:PubMed Google Scholar

  5. Paula Espinoza
    View author publications

    Search author on:PubMed Google Scholar

  6. Adam V. Crain
    View author publications

    Search author on:PubMed Google Scholar

  7. Nikita Patel
    View author publications

    Search author on:PubMed Google Scholar

  8. Greta W. Radcliff
    View author publications

    Search author on:PubMed Google Scholar

  9. Ming Cheng
    View author publications

    Search author on:PubMed Google Scholar

  10. Iván Coto Hernández
    View author publications

    Search author on:PubMed Google Scholar

  11. Steven Minderler
    View author publications

    Search author on:PubMed Google Scholar

  12. Demitri de la Cruz
    View author publications

    Search author on:PubMed Google Scholar

  13. Carrie Ng
    View author publications

    Search author on:PubMed Google Scholar

  14. Cintia Carla da Hora
    View author publications

    Search author on:PubMed Google Scholar

  15. Alain Charest
    View author publications

    Search author on:PubMed Google Scholar

  16. Anat Stemmer-Rachamimov
    View author publications

    Search author on:PubMed Google Scholar

  17. Nate Jowett
    View author publications

    Search author on:PubMed Google Scholar

  18. Xandra O. Breakefield
    View author publications

    Search author on:PubMed Google Scholar

  19. Casey A. Maguire
    View author publications

    Search author on:PubMed Google Scholar

Contributions

C.A.M., K.S.H. and X.O.B. conceived of the study, analyzed data, and wrote the manuscript. C.A.M. performed all of the tail vein injections of mice and assisted in tissue harvesting. S.P., P.S.C., C.C.D.H. and E.A.H. maintained the mice colony, breeding, genotyping, sciatic nerve injections, perfusion, and sacrifice of the mice for further analysis. A.S.R. carried out the pathological analyses in collaboration with S.P. and E.A.H. E.A.H. performed the RT-ddPCR analysis on neurofibroma and confocal microscopy. P.E. performed confocal microscopy and image analysis on the neurofibroma from mice injected with the different AAV vectors. N.P. cloned the AAV-P0-tdTomato construct. D.D.L.C. and A.V.C. produced, purified, and titered all AAV vectors used in the study, C.N. and G.W.R. performed cryosectioning of nerves and the immunofluorescence staining of neurofibroma sections. A.C. provided the luciferase floxed mouse model and advised on the study. M.C. assisted with image analysis. I.C.H, S.M. and N.J. performed microscopy analyses. All authors helped edit, revise, and approve of the manuscript.

Corresponding authors

Correspondence to Xandra O. Breakefield or Casey A. Maguire.

Ethics declarations

Competing interests

CAM has a financial interest in Sphere Gene Therapeutics, Inc., Chameleon Biosciences, Inc., and Skylark Bio, Inc., companies developing gene therapy platforms. CAM’s interests were reviewed and are managed by MGH and Mass General Brigham in accordance with their conflict-of-interest policies.

Ethical approval

All animal procedures were performed in accordance with Massachusetts General Hospital’s (MGH’s) recommendations for the care and use of animals and were maintained and handled under protocols approved by the Institutional Animal Care and Use Committee (IACUC).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Abou Haidar, E., Prabhakar, S., Cheah, P.S. et al. Engineered AAV capsids mediate transduction of murine neurofibroma and sciatic nerve. Gene Ther 32, 385–397 (2025). https://doi.org/10.1038/s41434-025-00542-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41434-025-00542-9

Search

Advanced search

Quick links