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Adeno-associated virus-mediated trastuzumab delivery to the central nervous system for human epidermal growth factor receptor 2+ brain metastasis

Cancer Gene Therapy (2024)Cite this article

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Abstract

Trastuzumab improves overall survival for HER2+ breast cancer, but its short half-life in the cerebrospinal fluid (~2–4 days) and delivery limitations restrict the ability to target HER2+ central nervous system (CNS) disease. We developed an adeno-associated virus (AAV) vector expressing a codon-optimized, ubiquitin C (UbC)-promoter-driven trastuzumab sequence (AAV9.UbC.trastuzumab) for intrathecal administration. Transgene expression was evaluated in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intra-cisterna magna (ICM) AAV9.UbC.trastuzumab injection, respectively, using real-time PCR, ELISA, Western blot, in situ hybridization, single-nucleus RNA sequencing, and liquid chromatography-mass spectrometry; antitumor efficacy was evaluated in brain xenografts using HER2+ breast cancer cell lines (BT-474, MDA-MB-453). Transgene expression was detected in brain homogenates of Rag1 knockout mice following a single ICV injection of AAV9.UbC.trastuzumab (1 × 1011 vector genome copies [GC]/mouse) and tumor progression was inhibited in xenograft models of breast-to-brain metastasis. In NHPs, ICM delivery of AAV9.UbC.trastuzumab (3 × 1013 GC/animal) was well tolerated (36–37 days in-life) and resulted in transgene expression in CNS tissues and cerebrospinal fluid at levels sufficient to induce complete tumor remission in MDA-MB-453 brain xenografts. With AAV9’s proven clinical safety record, this gene therapy may represent a viable approach for targeting HER2 + CNS malignancies.

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Fig. 1: Single ICV administration of AAV9 vector encoding a codon-optimized version of trastuzumab results in robust transgene expression in RAG1 KO mice.
Fig. 2: AAV9.UbC.trastuzumab-mediated trastuzumab expression is not affected by systemic pre-treatment with IVIg containing broad neutralizing antibodies against AAV9.
Fig. 3: Single-dose ICV administration of AAV9.UbC.trastuzumab leads to tumor regression in xenograft mouse models of HER2+ breast-to-brain metastases.
Fig. 4: Detection of trastuzumab in the CSF of female NHPs following ICM administration of AAV9 vectors encoding trastuzumab.
Fig. 5: CNS-wide gene delivery and transgene transcription following ICM treatment with 3 × 1013 GC/animal of AAV9.UbC.trastuzumab.
Fig. 6: ICM delivery of 3 × 1013 GC/animal AAV9.UbC.trastuzumab results in transgene protein expression in NHP CNS tissues at levels sufficient to induce complete anti-tumor responses in tumor-bearing mice.

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Data availability

All data discussed in the manuscript are available in the main text or supplementary materials. Sequencing data associated with the snRNA-seq experiments were uploaded to the NCBI Gene Expression Omnibus with accession number GSE255027.

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Acknowledgements

The authors thank Celensia Tolson for her assistance with the biodistribution studies, and Rucha Fadnavis for her assistance with single-nucleus RNA sequencing. We thank the Immunology Core, Histopathology Core, Nucleic Acid Technologies Core, Program for Comparative Medicine, and Vector Core of the Gene Therapy Program at the University of Pennsylvania for study support. All vectors were produced by the Penn Vector Core (RRID: SCR_022432).

Funding

This study was supported by G2 Oncology.

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Authors and Affiliations

  1. Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Marcela S. Werner, Shweta Aras, Ashleigh R. Morgan, Jillian Roamer, Nesteene J. Param, Kanyin Olagbegi, R. Jason Lamontagne, Jenny A. Greig & James M. Wilson

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Contributions

MSW—conceptualization, data curation, formal analysis, investigation, methodology, project administration, software, supervision, validation, visualization, writing-original draft preparation, writing-review and editing. SA—data curation, formal analysis, investigation, methodology, software, validation, visualization, writing-original draft preparation, writing-review and editing. ARM—data curation, investigation, methodology, validation, visualization, writing-original draft preparation. JR—data curation, formal analysis, investigation, methodology, software. NJP—data curation, investigation, methodology. KO—formal analysis. RJL—data curation, formal analysis, software, visualization. JAG—conceptualization, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, writing-review and editing. JMW—conceptualization, funding acquisition, resources, supervision, writing-review and editing.

Corresponding author

Correspondence to James M. Wilson.

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Competing interests

JMW is a paid advisor to and holds equity in iECURE, Scout Bio, Passage Bio, and the Center for Breakthrough Medicines (CBM). He also holds equity in the former G2 Bio asset companies. He has sponsored research agreements with Amicus Therapeutics, CBM, Elaaj Bio, FA212, Foundation for Angelman Syndrome Therapeutics, former G2 Bio asset companies, iECURE, Passage Bio, and Scout Bio, which are licensees of Penn technology. JAG and JMW are inventors on patents that have been licensed to various biopharmaceutical companies and for which they may receive payments. The other authors have no disclosures to declare.

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All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

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Werner, M.S., Aras, S., Morgan, A.R. et al. Adeno-associated virus-mediated trastuzumab delivery to the central nervous system for human epidermal growth factor receptor 2+ brain metastasis. Cancer Gene Ther (2024). https://doi.org/10.1038/s41417-024-00751-1

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