Synthetically modified mRNA for efficient and fast human iPS cell generation and direct transdifferentiation to myoblasts

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Abstract

Synthetic mRNA transfection enables efficient and controlled gene expression in human cells, without genome integrations. Further, modifications to the mRNA and transfection protocol now allow for repeated transfection and long-term gene expression of an otherwise short-lived mRNA expression. This is mainly achieved through introducing modified nucleosides and interferon suppression. In this study we provide an overview and details of the advanced synthesis and modifications of mRNA originally developed for reprogramming. This mRNA allows for very efficient transfection of fibroblasts enabling the generation of high quality human iPS cells with a six-factor mRNA cocktail in 9 days. Furthermore, we synthesised and transfected modified MYOD1 mRNA to transdifferentiate human fibroblasts into myoblast-like cells without a transgene footprint. This efficient and integration-free mRNA technology opens the door for safe and controlled gene expression to reverse or redirect cell fate.

Introduction

To efficiently induce gene expression is a hallmark technology of molecular biology. It allows functional interrogation of genes as well as providing a tool to alter cell behaviour or even cell fate. Traditionally, DNA-based technologies have been successfully used to overexpress specific genes, but drawbacks significantly hinder their use in clinically relevant settings. Drawbacks include DNA integration into the host genome and poor control of gene expression often due to promoter choice, number of stable integrations or complex transgene vectors with induction systems. Furthermore, if DNA is transferred, there is always a chance of random integration, and such integrations come with the risk of causing insertional mutagenesis, inducing carcinogenic behaviour or other off-target effects.

In vitro transcription of mRNA has been made possible through discovery and cloning of SP6 and T7 RNA polymerases in the early 1980's [1], [2]. These bacteriophage RNA polymerases allowed for specific gene transcription from DNA templates and the production of large amounts of pre-mRNA. To improve stability and translation of synthetic mRNA, it is usually capped and polyadenylated [3], [4], mirroring the in-vivo processing of eukaryotic pre-mRNA to mature mRNA.

Transfection of synthetic RNA compared to DNA transfection has been shown to allow faster expression, and is able to transfect a higher number of cells in a more controlled fashion [5]. Additionally, mRNA translation does not require transit through the nuclear membrane and therefore also allows gene expression in non-dividing cells, such as neurons [6].

Until recently, applicability of such synthetic mRNA was limited to short-term gene expression, due to the short lifespan of mRNA and the high toxicity caused by innate anti-viral response mechanisms to long single stranded RNA [7]. However, the requirement for extended transgene expression in iPS cell generation brought forward the development of methods that can suppress this innate immune response [8]. Indeed, the combinatorial use of modified nucleoside bases [9] and addition of the soluble immunosuppressant, interferon receptor B18R, maintains transgene expression through repeated transfection without the normally associated toxicity of synthetic mRNA [10]. Not only have these modifications reduced toxicity, they also now provide the fastest, most efficient way to generate iPS cells from fibroblasts, which is integration and virus-free [11].

The ability to control expression of one or multiple genes over a defined time period through simple daily mRNA transfection provides more precise means to study the effect of gene expression levels without genome editing. At the same time, the ability to efficiently reprogram cells to iPS cells, clearly suggests that mRNA would also lend itself to directly convert cell types, with transcription factors specific for cell types of interest. Direct conversion of fibroblasts to neurons or cardiomyocytes can be induced with defined sets of transcription factors usually using integrating vectors [12], [13]. Furthermore, viral overexpression of the transcription factor MyoD1 alone has been shown to be sufficient to convert fibroblasts to myoblasts, identifying the transcription factor MyoD1 as a master regulator for the myoblast cell fate [14]. Myoblasts are clinically relevant cells that can be the optimal progenitor cells to regenerate and incorporate into cardiomyocytes to treat myocardial infarction [15]. Instead of integrating methods, mRNA would potentially allow the transdifferentiation to integration-free cells that could be relevant in clinical therapies.

In this study we provide an overview of the advanced synthesis and modifications of mRNA developed for reprogramming. We show the high efficiency of such modified synthetic mRNA transfection further allowing for very fast and high quality iPS cell generation. Finally, we synthesise modified MYOD1 mRNA to transdifferentiate human fibroblasts into myoblast-like cells with the potential for clinical application.

Section snippets

Cell culture

BJ fibroblasts were purchased from Stemgent (Lexington, MA, USA) and HFF human foreskin fibroblasts were obtained from ATCC (CRL-2429, LGC-standards, Middlesex, UK). Both fibroblast lines were cultured in DMEM (Invitrogen) plus 10% FBS (Hyclone) and passaged using Trypsin or TryPLE Express (Invitrogen).

Human myoblast control cells, KM155C25dist, were immortalised with hTert and Cdk4 as described previously [16].

Myoblast growth medium was made of DMEM/F12 (Sigma) base medium supplemented with

In vitro mRNA generation for efficient primary cell transfection

There are a number of practical approaches to generate mRNA in vitro and we will first summarise our method that we initially developed with the aim of reprogramming somatic cells. Since multiple transfections and high quality mRNA are required to be able to generate iPS cells, this method is useful as a base for producing mRNA for transfection and the precise overexpression of genes in cells. In this study we focussed on transfecting fibroblasts as a convenient and readily available starting

Conclusion

Synthetic mRNA transfection is an efficient and versatile methodology for controlled transgene expression in human cells. The in vitro modifications described, suppress the innate cellular immune response against synthetic long mRNA, and enables extended controlled transgene expression for highly efficient iPS cell generation without integrations. The same integration-free approach is also able to directly convert human fibroblasts into myoblast-like cells, providing a potentially safe source

Acknowledgement

We thank Duncan Baker for Karyotyping of the iPS cell lines. We thank the Allele Biotech team for help with their protocol and Stemgent for general advise on mRNA reprogramming. For the provision of the immortalised myoblast cell line we thank the platform for the immortalisation of human cells from the Insitut de Myologie in Paris, France. This project was funded by the Yorkshire Cancer Research.

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