An in vivo Cell-Based Delivery Platform for Zinc Finger Artificial Transcription Factors in Pre-clinical Animal Models.

Peter Deng ,Ulrika Beitnere,Michele L Martinez,Anna Adhikari,Stela P Petkova,Jill L Silverman,Kyle D Fink,Miranda Palomares,Fernando A Fierro,Krista Thongphanh,Jan A Nolta,David J Segal,Alice F Tarantal,Nycole A Copping ,Charles C Lee ,Henriette O’geen ,Jennifer J Waldo ,Julian Halmai ,Fiona K B Buchanan ,Samantha Lock ,Casiana E Gonzalez ,David L Cameron ,Jasmine Carter ,Jonathan D Anderson ,Ruth D Lee

doi:10.3389/fnmol.2021.789913

Abstract

Zinc finger (ZF), transcription activator-like effectors (TALE), and CRISPR/Cas9 therapies to regulate gene expression are becoming viable strategies to treat genetic disorders, although effective in vivo delivery systems for these proteins remain a major translational hurdle. We describe the use of a mesenchymal stem/stromal cell (MSC)-based delivery system for the secretion of a ZF protein (ZF-MSC) in transgenic mouse models and young rhesus monkeys. Secreted ZF protein from mouse ZF-MSC was detectable within the hippocampus 1 week following intracranial or cisterna magna (CM) injection. Secreted ZF activated the imprinted paternal Ube3a in a transgenic reporter mouse and ameliorated motor deficits in a Ube3a deletion Angelman Syndrome (AS) mouse. Intrathecally administered autologous rhesus MSCs were well-tolerated for 3 weeks following administration and secreted ZF protein was detectable within the cerebrospinal fluid (CSF), midbrain, and spinal cord. This approach is less invasive when compared to direct intracranial injection which requires a surgical procedure.

Highlights

Engineered stem cell therapies have been utilized as a gene replacement and crosscorrection approach by producing proteins at supraphysiologic levels in vivo (Kohn et al, 1995, 1998, 2021; Visigalli et al, 2010; Pollock et al, 2016; Adhikari et al, 2019, 2021; Beegle et al, 2020)
We report the use of mesenchymal stem/stromal cell (MSC) to secrete a bioactive zinc finger (ZF) to alter Ube3a expression in Angelman Syndrome (AS) mouse models and demonstrate detectable distribution in midbrain and spinal cord of rhesus monkeys
One-Way ANOVA [F(2,9) = 6.041] followed by Fisher’s LSD, ∗p < 0.05 to non-transduced MSC (NT-MSC). (C) A significant increase in midbrain UBE3A-YFP protein expression was observed in both ZF-MSC IC and ZF-MSC cisterna magna (CM) treated mice

Summary

Introduction

Engineered stem cell therapies have been utilized as a gene replacement and crosscorrection approach by producing proteins at supraphysiologic levels in vivo (Kohn et al, 1995, 1998, 2021; Visigalli et al, 2010; Pollock et al, 2016; Adhikari et al, 2019, 2021; Beegle et al, 2020). Cell-Based Delivery of ATF cytokines, and demonstrate a clinically favorable safety profile (Thompson et al, 2020). These unique characteristics of MSCs make them an attractive potential delivery vehicle for gene modifying artificial transcription factors (ATF) such as zinc finger (ZF), transcription activator-like effectors (TALE), and CRISPR/Cas (Russ and Lampel, 2005; Thakore et al, 2015; Bailus et al, 2016; Fink et al, 2016; Halmai et al, 2020). Direct administration of purified protein (Bailus et al, 2016; Perdigão et al, 2020), lipid nanoparticle (Wang et al, 2016; Cheng et al, 2020), and viral-mediated approaches such as adeno-associated virus (Mendell et al, 2017; Russell et al, 2017) have been explored as putative delivery systems for ATF in vivo; a cell-based system such as MSCs has not been reported to date

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Conclusion