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Methods for Nuclear Reprogramming Using Synthetic Transcription Factors

Inactive Publication Date: 2016-12-15
LONZA WALKERSVILLE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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.

Method used

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  • Methods for Nuclear Reprogramming Using Synthetic Transcription Factors
  • Methods for Nuclear Reprogramming Using Synthetic Transcription Factors
  • Methods for Nuclear Reprogramming Using Synthetic Transcription Factors

Examples

Experimental program
Comparison scheme
Effect test

example 1

Reprogramming Rescue by Endogenous Activation of the Human POU5F1 / OCT4 Gene Transcription (CRISPR-Based Reprogramming)

Vector Sequences

[0174]DNA sequences for dCas9, dCas9-VP64 and guide RNA constructs were prepared as described in Mali, P. et al. “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.”Nat Biolechnol 2013, 31(9): 833-8, the disclosure of which is incorporated herein in its entirety. Additional sequences for gRNAs containing MS2 binding loops and the MS2-transcriptional regulator fusion proteins (e.g. MS2-VP64) were prepared as described in Konermann et al., Nature 2015, 517: 583-588 (and supporting material), the disclosure of which is incorporated herein in its entirety. Sequences were synthesized by GeneART and cloned into episomal cloning vectors. These vectors were used directly in the following experiments.

[0175]Selection of gRNA for Human POU5F1 / OCT4 Transcription Activation

[0176]Human peripheral...

example 2

Reprogramming Rescue by Endogenous Activation of the Human OCT4 Gene Transcription (CRISPR-Based Reprogramming)

Vector Sequences

[0187]DNA sequences for dCas9-VPR consisting of VP64-p65-Rta activation domains fused to the C-terminus of dCas9 protein and guide RNA constructs were prepared as described in Mali, P. et al. (Mali, P. et al. “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.”Nat Biotechnol 2013, 31(9): 833-8) and Chavez, A. et al. (Chavez, A. et al. “Highly efficient Cas9-mediated transcriptional programming.”Nat Methods. 2015 April; 12(4):326-8). Sequences were synthesized by GeneART and cloned into standard cloning vectors. These vectors were used directly in the following experiments.

Determining gRNA Combination for Human OCT4 Transcription Activation

[0188]HEK293T cells were transfected with various combinations of transient Cas9-VPR and gRNA encoding vectors (see Table 1). The plasmids were co-transf...

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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...

Claims

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Application Information

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IPC IPC(8): C12N15/85A61K35/12C12N15/11
CPCA61K35/12C12N15/85A61K35/15A61K35/33A61K35/51C12N5/0696C12N2501/40C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2501/608C12N2506/11C12N2510/00G01N33/5073A61P43/00
Inventor ABRAHAM, EYTANPAYNE, THOMASYOUNG, ROBERT J.FRIEDRICH BEN NUN, INBAR
Owner LONZA WALKERSVILLE INC
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