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A Combinatorial Library of Biodegradable Polyesters Enables Non-viral Gene Delivery to Post-Mitotic Human Stem Cell-Derived Polarized RPE Monolayers

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Abstract

Safe and effective delivery of DNA to post-mitotic cells, especially highly differentiated cells, remains a challenge despite significant progress in the development of gene delivery tools. Biodegradable polymeric nanoparticles (NPs) offer an array of advantages for gene delivery over viral vectors due to improved safety, carrying capacity, ease of manufacture, and cell-type specificity. Here we demonstrate the use of a high-throughput screening (HTS) platform to synthesize and screen a library of 148 biodegradable polymeric nanoparticles, successfully identifying structures that enable efficient transfection of human pluripotent stem cell differentiated human retinal pigment epithelial (RPE) cells with minimal toxicity. These NPs can deliver plasmid DNA (pDNA) to RPE monolayers more efficiently than leading commercially available transfection reagents. Novel synthetic polymers are described that enable high efficacy non-viral gene delivery to hard-to-transfect polarized human RPE monolayers, enabling gene loss- and gain-of-function studies of cell signaling, developmental, and disease-related pathways. One new synthetic polymer in particular, 3,3′-iminobis(N,N-dimethylpropylamine)-end terminated poly(1,5-pentanediol diacrylate-co-3 amino-1-propanol) (5–3-J12), was found to form self-assembled nanoparticles when mixed with plasmid DNA that transfect a majority of these human post-mitotic cells with minimal cytotoxicity. The platform described here can be utilized as an enabling technology for gene transfer to human primary and stem cell-derived cells, which are often fragile and resistant to conventional gene transfer approaches.

Lay Summary

Many retinal diseases are attributable to dysregulation in gene expression or lack of expression of specific genes, allowing for the possibility of prevention or cure of these diseases by effective delivery of nucleic acids coding for the necessary gene to the retina. Delivery of nucleic acids to cells of the retina is challenging due to the non-dividing nature of most retinal cells, preventing DNA from reaching the nucleus. To overcome this barrier, we engineered and tested a library of nanoparticle formulations to identify polymers that enabled safe and effective delivery of nucleic acid cargoes to retinal pigment epithelial cells. The nanoparticle technology explored here has the potential to be utilized for therapeutic delivery of nucleic acids to retinal cells, possibly enabling treatment for otherwise untreatable retinal diseases for which a specific genetic deficit is known but no drugs are available.

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Acknowledgements

We are grateful to Baranda S. Hansen for technical assistance. The authors thank the Wilmer Microscopy and Imaging Core Facility (EY001765) at Johns Hopkins for use of their confocal microscopy.

Funding

This research was supported by grants from the Maryland Stem Cell Research Fund to D.J.Z. (EY027266), NIH grant (EY024249) to D.J.Z, BrightFocus Foundation to D.J.Z., RPB Nelson Trust Award for Retinitis Pigmentosa and unrestricted funds from Research to Prevent Blindness, Inc. to D.J.Z., generous gifts from the Guerrieri Family Foundation and from Mr. and Mrs. Robert and Clarice Smith to D.J.Z., Foundation Fighting Blindness to D.J.Z., Thome Foundation to D.J.Z., Beckman Foundation to D.J.Z., NSF Graduate Research Fellowships DGE-0707427 to DRW and DGE-1232825 to YR; the Bloomberg~Kimmel Institute for Cancer Immunotherapy to JJG; the NIH (R21EY026148, R01EB022148, R01CA228133, and the Wilmer Core Grant P30 EY001765); and a Research to Prevent Blindness / Dr. H. James and Carole Free Catalyst Award for Innovative Research Approaches for Age-Related Macular Degeneration to JJG.

Author information

Author notes

  1. Karl J. Wahlin

    Present address: Shiley Eye Institute, University of California San Diego, La Jolla, CA, USA

  2. Bibhudatta Mishra and David R. Wilson contributed equally to this work.

Authors and Affiliations

  1. Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    Bibhudatta Mishra, Srinivas R. Sripathi, Karl J. Wahlin, Cynthia A. Berlinicke, Jordan J. Green & Donald J. Zack

  2. Biomedical Engineering, Johns Hopkins University, Baltimore, MD, 21231, USA

    David R. Wilson, Mark P. Suprenant, Yuan Rui & Jordan J. Green

  3. Translational Tissue Engineering Center, Johns Hopkins of Medicine, Baltimore, MD, 21231, USA

    David R. Wilson, Mark P. Suprenant, Yuan Rui & Jordan J. Green

  4. Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD, 21231, USA

    David R. Wilson, Yuan Rui & Jordan J. Green

  5. Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    Jordan J. Green

  6. Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, 21231, USA

    Jordan J. Green

  7. Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    Jordan J. Green

  8. Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    Donald J. Zack

  9. Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    Donald J. Zack

  10. Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    Donald J. Zack

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  1. Bibhudatta Mishra
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Contributions

Overall conceptualization, B.M., D.R.W., D.J.Z and J.J.G; Methodology and Investigation, B.M., D.R.W., S.S.R, M.P.S., C.B., and Y.R.; Resource Generation (plasmid DNA designing and production), K.J.W., (stem cell differentiation and maintenance), S.S.R.; Writing—Original Draft, B.M., D.R.W., C.B., D.J.Z and J.J.G; Writing—Review & Editing, B.M., D.R.W., C.B., D.J.Z and J.J.G.; Funding Acquisition, D.J.Z., J.J.G., S.S.R., D.R.W., and Y.R.; Supervision and Project Administration, D.J.Z and J.J.G.

Corresponding authors

Correspondence to Jordan J. Green or Donald J. Zack.

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Conflict of Interest

DJZ is on the scientific advisory board of Spark Therapeutics, which is interested in developing optimized approaches for retinal gene delivery.

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Mishra, B., Wilson, D.R., Sripathi, S.R. et al. A Combinatorial Library of Biodegradable Polyesters Enables Non-viral Gene Delivery to Post-Mitotic Human Stem Cell-Derived Polarized RPE Monolayers. Regen. Eng. Transl. Med. 6, 273–285 (2020). https://doi.org/10.1007/s40883-019-00118-1

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