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Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

ABSTRACT: Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage.

From the clinical editor

In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.

SUBMITTER: Gallego-Perez D

PROVIDER: S-EPMC5161095 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

Gallego-Perez Daniel D Otero Jose J JJ Czeisler Catherine C Ma Junyu J Ortiz Cristina C Gygli Patrick P Catacutan Fay Patsy FP Gokozan Hamza Numan HN Cowgill Aaron A Sherwood Thomas T Ghatak Subhadip S Malkoc Veysi V Zhao Xi X Liao Wei-Ching WC Gnyawali Surya S Gnyawali Surya S Wang Xinmei X Adler Andrew F AF Leong Kam K Wulff Brian B Wilgus Traci A TA Askwith Candice C Khanna Savita S Rink Cameron C Sen Chandan K CK Lee L James LJ

Nanomedicine : nanotechnology, biology, and medicine 20151219 2

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Rep ...[more]

PMID: 26711960

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Project description:BackgroundIntervertebral disc (IVD) degeneration is a major contributor to low back pain (LBP), yet there are no clinical therapies targeting the underlying pathology. The annulus fibrosus (AF) plays a critical role in maintaining IVD structure/function and undergoes degenerative changes such as matrix catabolism and inflammation. Thus, therapies targeting the AF are crucial to fully restore IVD function. Previously, we have shown nonviral delivery of transcription factors to push diseased nucleus pulposus cells to a healthy phenotype. As a next step in a proof-of-concept study, we report the use of Scleraxis (SCX) and Mohawk (MKX), which are critical for the development, maintenance, and regeneration of the AF and may have therapeutic potential to induce a healthy, pro-anabolic phenotype in diseased AF cells.MethodsMKX and SCX plasmids were delivered via electroporation into diseased human AF cells from autopsy specimens and patients undergoing surgery for LBP. Transfected cells were cultured over 14 days and assessed for cell morphology, viability, density, gene expression of key phenotypic, inflammatory, matrix, pain markers, and collagen accumulation.ResultsAF cells demonstrated a fibroblastic phenotype posttreatment. Moreover, transfection of SCX and MKX resulted in significant upregulation of the respective genes, as well as SOX9. Transfected autopsy cells demonstrated upregulation of core extracellular matrix markers; however, this was observed to a lesser effect in surgical cells. Matrix-degrading enzymes and inflammatory cytokines were downregulated, suggesting a push toward a pro-anabolic, anti-inflammatory phenotype. Similarly, pain markers were downregulated over time in autopsy cells. At the protein level, collagen content was increased in both MKX and SCX transfected cells compared to controls.ConclusionsThis exploratory study demonstrates the potential of MKX or SCX to drive reprogramming in mild to moderately degenerate AF cells from autopsy and severely degenerate AF cells from surgical patients toward a healthy phenotype and may be a potential nonviral gene therapy for LBP.

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Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

From the clinical editor

Publications

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

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