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AAV2 vector optimization for retinal ganglion cell-targeted delivery of therapeutic genes

Gene Therapy volume 31, pages 175–186 (2024)Cite this article

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Abstract

Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken β-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5’ and 3’ ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.

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Fig. 1: AAV2 and AAV8BP2 transduction potential.
Fig. 2: Time- and dose-dependent transduction potential of the AAV2 vector.
Fig. 3: Optimization of the AAV2 vector for high efficiency delivery of transgenes to RGCs in vivo.
Fig. 4: RGC selectivity of AAV2 vectors.
Fig. 5: AAV2-mediated expression of hSIRT1 in RGCs.

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Data availability

The datasets generated and analyzed during the course of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

AAV vectors used in this study were produced and packaged by the Research Vector Core at the Center for Advanced Retinal and Ocular Therapeutics (University of Pennsylvania).

Funding

This study is supported, at least in part, by grants from Gyroscope Therapeutics Limited, a Novartis Company; National Institutes of Health Grants (EY019014, EY030163); Department of Defense (USAMRAA) award number HT9425-23-1-0725; RWJ-Harold Amos Faculty Development; Linda Pechenik Montague Investigator Award; Research to Prevent Blindness; Paul and Evanina Mackall Foundation Trust; Center for Advanced Retinal and Ocular Therapeutics; and the F. M. Kirby Foundation.

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Author notes

  1. Thu T. Duong

    Present address: GlaxoSmithKline, Collegeville, PA, 19426, USA

  2. Tehui Liu

    Present address: Spark Therapeutics, Philadelphia, PA, 19104, USA

Authors and Affiliations

  1. Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Brahim Chaqour, Thu T. Duong, Jipeng Yue, Tehui Liu, David Camacho, Kimberly E. Dine, Jean Bennett, Kenneth S. Shindler & Ahmara G. Ross

  2. F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA

    Brahim Chaqour, Thu T. Duong, Jipeng Yue, Tehui Liu, David Camacho, Kimberly E. Dine, Jean Bennett, Kenneth S. Shindler & Ahmara G. Ross

  3. Gyroscope Therapeutics Limited, a Novartis Company, London, N7 9AS, UK

    Julian Esteve-Rudd & Scott Ellis

  4. Department of Neurology, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Kenneth S. Shindler & Ahmara G. Ross

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Contributions

BC: data curation, formal analysis, supervision, writing original draft, review and editing. TTD: investigation, methodology. JPY: methodology, data extraction, formal data analysis. TL: methodology, validation. DC: data curation. KED: methodology, formal analysis, investigation. JER: conceptualization, review. SE: conceptualization, review. JB: conceptualization, review. KSS: conceptualization, data curation, formal analysis, funding acquisition, investigation, project administration, resources, supervision, review and editing. AGR: project administration, validation, visualization, investigation, methodology, funding acquisition, resources, review and editing.

Corresponding authors

Correspondence to Kenneth S. Shindler or Ahmara G. Ross.

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Competing interests

AGR and KSS hold intellectual property relevant to this study and received research funding from Gyroscope Therapeutics Limited, a Novartis Company. JE-R and SE are full time employees of Gyroscope. Other authors declare no conflict of interests.

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All experiments were performed in accordance with relevant guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, as well as with institutional and federal regulations.

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Chaqour, B., Duong, T.T., Yue, J. et al. AAV2 vector optimization for retinal ganglion cell-targeted delivery of therapeutic genes. Gene Ther 31, 175–186 (2024). https://doi.org/10.1038/s41434-023-00436-8

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