Methods for Nuclear Reprogramming Using Synthetic Transcription Factors

Abstract

The current disclosure provides methods for reprogramming mammalian somatic cells by regulating the expression of endogenous cellular genes. Cellular reprogramming of somatic cells can be induced by activating the transcription of embryonic stem cell-associated genes (e.g., oct3 / 4) and suppressing the transcription of somatic cell-specific and / or cell death-associated genes. The endogenous transcription machinery can be modulated using synthetic transcription factors (activators and suppressors), to allow for faster, and more efficient nuclear reprogramming under conditions amenable for clinical and commercial applications. The current disclosure further provides cells obtained from such methods, along with therapeutic methods for using such cells for the treatment of diseases amendable to stem cell therapy, as well as kits for such uses.

Description

FIELD OF THE INVENTION[0001]The invention relates to methods of nuclear reprogramming of mammalian somatic cells to produce induced pluripotent stem cells (iPSCs).BACKGROUND OF THE INVENTION[0002]Cellular reprogramming, also referred to as nuclear reprogramming, is the process of generating stem cells, e.g., iPSCs from somatic cells. The derivation of iPSCs from numerous normal and diseased cell sources has revolutionized stem cell biology, and has enabled the generation of stem cells for eventual use in cell therapy and regenerative medicine.[0003]iPSCs can be differentiated to many cell types, obviating the need to use discarded embryos from in vitro fertilization procedures to generate embryonic stem cells (ESCs) and minimizes the ethical issues involved. In addition, while ESCs can be only used for allogeneic cell therapy applications, iPSCs can be applied to both allogeneic and autologous cell therapy applications[0004]Seminal studies by Yamanaka and colleagues revealed that ec...

AI Technical Summary

Benefits of technology

This patent describes methods for reprogramming mammalian somatic cells by regulating the expression of endogenous cellular genes using synthetic transcription factors. The CRISPR / Cas9 gene editing system has been used successfully in a wide range of organisms and cell lines to induce DSB formation with the wild type Cas9 protein or to nick a single DNA strand using a mutant protein termed Cas9n / Cas9 D10A. The system has advantages such as speed and ease of use, and it allows for faster and more efficient nuclear reprogramming under conditions amenable for clinical and commercial applications. The patent also discusses the use of multiplex gene regulation using the CRISPR system to construct complex regulatory networks and comprehensive interrogation of gene pathway function. The direct modulation of endogenous gene transcription has advantages such as shortening the period of time from somatic cell transfection to iPSC colony appearance, ensuring self-renewal and pluripotency, minimizing possible side-effects, and reducing somatic cell-type dependent variability of reprogramming efficiency.

Problems solved by technology

However, much remains to be understood about the underlying mechanisms of reprogramming of somatic cells to iPSCs, and there is concern regarding potential clinical applications in the absence of mechanistic insights.

As an alternative to genetic modification, mRNA, episomal DNA plasmids, and cell permeant proteins (CPP) have been shown to be effective in reprogramming. mRNA reprogramming has high reprogramming efficiency rate, but method robustness (reproducibility) is low.

Persistent expression of exogenous reprogramming factors may limit the cell's differentiation potential.

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