Cationic nanocapsules as a non-viral vector for CRISPR-Cas9–based genome editing for myocilin associated glaucoma therapy

Karthikeyan Kesavan; Virendra Kumar; Jothimani Rajeswari; Charles Searby; Val C. Sheffield; Karthikeyan Kesavan; Virendra Kumar; Jothimani Rajeswari; Charles Searby; Val C. Sheffield

doi:10.3934/molsci.2025020

AIMS Molecular Science

2025, Volume 12, Issue 4: 341-356. doi: 10.3934/molsci.2025020

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Cationic nanocapsules as a non-viral vector for CRISPR-Cas9–based genome editing for myocilin associated glaucoma therapy

1.
Department of Pharmacy, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, C.G., 495009, India
2.
Department of Pediatrics, Division of Medical Genetics and Genomics, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA

Received: 30 April 2025 Revised: 24 September 2025 Accepted: 14 October 2025 Published: 21 October 2025

Our aim of this work was the development of positively charged D-α-tocopheryl glycol succinate 1000 (TPGS)-chitosan nanocapsules (TCNs) as a non-viral vector for effective genome editing. A two-step process was used to create cationic TCNs. TPGS was initially esterified using succinic anhydride to add a carboxylic acid group. Then, using a complex coacervation technique, ionic cross-linking was used to join the activated TPGS with chitosan. Fourier transform infrared (FTIR) spectroscopy was used to validate the synthesis of TCNs. A gel retardation experiment was performed to evaluate TCNs/pDNA complex entrapments at various N/P ratios (2:1, 5:1, and 10:1). The physicochemical properties of the TCNs/pDNA complex were identified. Plasmid DNA was protected from DNase I activity through TCN complexation. HEK293 and GTM3 cell lines were used to test in vitro transfection efficiency. At N/P 5:1, TCNs/pDNA exhibited the maximum transfection efficiency, which was comparable to Lipofectamine 3000™. Cellular uptake and localization studies confirmed effective delivery of Cas9-GFP plasmids using TCNs. A T7 endonuclease mismatch detection assay further demonstrated effective targeting and editing of the MYOC gene in HEK293 cells using our TCN delivery system. Based on these findings, cationic TCNs represent a promising non-viral gene delivery platform for potential treatment of myocilin-associated glaucoma.
- CRISPR,
- Glaucoma,
- Non-viral,
- Myocilin,
- Chitosan,
- TPGS
Citation: Karthikeyan Kesavan, Virendra Kumar, Jothimani Rajeswari, Charles Searby, Val C. Sheffield. Cationic nanocapsules as a non-viral vector for CRISPR-Cas9–based genome editing for myocilin associated glaucoma therapy[J]. AIMS Molecular Science, 2025, 12(4): 341-356. doi: 10.3934/molsci.2025020

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Abstract

Our aim of this work was the development of positively charged D-α-tocopheryl glycol succinate 1000 (TPGS)-chitosan nanocapsules (TCNs) as a non-viral vector for effective genome editing. A two-step process was used to create cationic TCNs. TPGS was initially esterified using succinic anhydride to add a carboxylic acid group. Then, using a complex coacervation technique, ionic cross-linking was used to join the activated TPGS with chitosan. Fourier transform infrared (FTIR) spectroscopy was used to validate the synthesis of TCNs. A gel retardation experiment was performed to evaluate TCNs/pDNA complex entrapments at various N/P ratios (2:1, 5:1, and 10:1). The physicochemical properties of the TCNs/pDNA complex were identified. Plasmid DNA was protected from DNase I activity through TCN complexation. HEK293 and GTM3 cell lines were used to test in vitro transfection efficiency. At N/P 5:1, TCNs/pDNA exhibited the maximum transfection efficiency, which was comparable to Lipofectamine 3000™. Cellular uptake and localization studies confirmed effective delivery of Cas9-GFP plasmids using TCNs. A T7 endonuclease mismatch detection assay further demonstrated effective targeting and editing of the MYOC gene in HEK293 cells using our TCN delivery system. Based on these findings, cationic TCNs represent a promising non-viral gene delivery platform for potential treatment of myocilin-associated glaucoma.

Acknowledgments

Research was supported by Department of Biotechnology (DBT) along with Indo U.S. Science & Technology Forum (IUSSTF), New Delhi, India, in terms of Overseas Fellowship of Genome Engineering/Editing Technology Initiative Program. This research was funded by DBT and IUSSTF, Award Letter No Indo-US GET in Overseas Fellowship 2019-0109/Kesavan Kathikeyan dated 19^th June 2019 (KK). This work was also supported by NIH P30EY025580 (VCS).

Conflict of interest

The authors declare no conflicts of interest in this paper.

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