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Use of RNA for reprogramming somatic cells

a somatic cell and rna technology, applied in the field of rna for reprogramming somatic cells, can solve the problems of limited number of types, restricted differentiation potential, poor growth, etc., and achieve the effect of facilitating reprogramming of somatic cells, controlling the amount of rna, and relatively high transfection ra

Inactive Publication Date: 2011-03-17
BIONTECH AG +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for reprogramming somatic cells to have stem cell characteristics. This is achieved by introducing RNA into somatic cells, which can then be directed to differentiate into various types of stem cells. The use of RNA instead of DNA reduces the risk of mutations and allows for greater control over the expression of certain factors. The amount of RNA introduced can be adjusted, and the RNA is degraded quickly, resulting in a safer profile for stem cells. The use of RNA instead of DNA also avoids gene therapy and allows for the development of cells with non-discriminable genetic markers. The method can be carried out using electroporation and specific embryonic stem cell morphologies and markers. Overall, the invention provides a way to produce stem cells with greater control and safety for use in regenerative medicine.

Problems solved by technology

At the next stage, cells become multipotent, meaning they can give rise to several other cell types, but those types are limited in number.
However, scientific and ethical considerations have slowed the progress of research using embryonic stem cells recovered from aborted embryos or embryos formed using in vitro fertilization techniques.
Adult stem cells are present only at low frequencies and exhibit restricted differentiation potential and poor growth.
A further problem associated with using adult stems cells is that these cells are not immunologically privileged, or can lose their immunological privilege after transplant, wherein the term “immunologically privileged” is used to denote a state where the recipient's immune system does not recognize the cells as foreign.
Thus, only autologous transplants are possible in most cases when adult stem cells are used.
Most presently envisioned forms of stem cell therapy are essentially customized medical procedures and therefore economic factors associated with such procedures limit their wide ranging potential.
However, the resulting cells are hybrids, often with a tetraploid genotype, and therefore not suited as normal or histocompatible cells for transplant purposes.
The stresses placed on both the egg cell and the introduced nucleus are enormous, leading to a high loss in resulting cells.
Furthermore, the procedure has to be performed manually under a microscope, and therefore, somatic cell nuclear transfer is very resource intensive.
As a consequence, clones are not perfect copies of the donor of the nucleus.
A major disadvantage of viral delivery is the stochastic reactivation of integrated retroviruses encoding potent oncogenes, which in the case of c-MYC led to the induction of tumors in chimeric mice (Okita et al., 2007, Nature 448, 313-317).
Genomic integration has not been detected in this study, however, stable genomic integration in a small fraction of the cells of transfected plasmid DNA cannot be completely excluded.

Method used

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  • Use of RNA for reprogramming somatic cells
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  • Use of RNA for reprogramming somatic cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of IVT RNA

[0206]The first step in the production of IVT RNA comprises linearization of a plasmid containing the coding sequence for a particular factor and having an SP6 promoter or T7 promoter before the start codon, starting from which an in vitro transcription is possible. To this end, restriction enzymes are used, for example. Following linearization, the enzyme is inactivated by phenol-chloroform precipitation and removed. For this, an isovolume of a mixture of phenol and chloroform is added and mixed thoroughly.

[0207]Brief centrifugation at 10 000×g provides separation into a lower organic phase and an upper aqueous phase, which contains the DNA. The latter is transferred to a new reaction vessel. Then the aqueous phase is mixed with an isovolume of pure chloroform, to remove any phenol residues. After centrifugation, the aqueous phase is removed and precipitated for 2 h by adding two isovolumes of ethanol and 10% v / v 3M sodium acetate pH 4.5 at −20° C. The DNA is s...

example 2

Electroporation of Cells

[0211]The principle of electroporation is based on disturbing the transmembrane potential of the cells by a brief current pulse. The alteration of the transmembrane potential by an external stimulus is described by the following equation:

ΔVm=fEextr cos φ

[0212]Vm is the transmembrane potential and f is a form factor, which describes the influence of the cell on the extracellular field distribution. fEext describes the applied electric field, r the cell radius and φ the angle to the externally applied electric field. Factor f is often given as 1.5, though it depends on many other factors. The electroporation of the cells is successful if the applied electric field exceeds the capacity of the cell membrane, i.e. ΔVm is greater than a threshold value ΔVs, given as 1V (Kinosita, K., Jr. and Tsong, T. Y. (1977) Nature 268, 438-441). Since construction of the cell membrane as a bilayer is a feature that is common to eukaryotic cells, this value shows little variatio...

example 3

Stability of Proteins Expressed by Transfected RNA

[0220]We next examined for how long proteins with different halftimes following transfer of IVT RNA can be stably detected in cells. 786-0 cells were transfected with 20 μg eGFP IVT RNA and 2dGFP IVT RNA, respectively, and the fluorescence intensity was measured in the time course of 3 h to 120 h. The eGFP protein has a halftime of 16 h, while the halftime of the destabilized variant 2dGFP due to the integration of a PEST amino acid sequence effecting protein degradation is reduced to 2 h (Clontech, 1998). The experiment showed that already after 4 h a substantial amount of translated protein was detectable which further increased until 24 h after transfection. The amount of eGFP protein remains relatively constant for more than 120 h. Even the destabilized 2dGFP shows stable protein expression for 48 h (FIG. 1). In order to induce protein expression of 2dGFP which is stable over a long period of time RNA can be transfected every 48 ...

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Abstract

The present invention provides methods for de-differentiating somatic cells into stem-like cells without generating embryos or fetuses. More specifically, the present invention provides methods for effecting the de-differentiation of somatic cells to cells having stem cell characteristics, in particular pluripotency, by introducing RNA encoding factors inducing the de-differentiation of somatic cells into the somatic cells and culturing the somatic cells allowing the cells to de-differentiate.

Description

FIELD OF THE INVENTION[0001]The present invention provides methods for de-differentiating somatic cells into stem-like cells without generating embryos or fetuses. More specifically, the present invention provides methods for effecting the de-differentiation of somatic cells to cells having stem cell characteristics, in particular pluripotency, by introducing RNA encoding factors inducing the de-differentiation of somatic cells into the somatic cells and culturing the somatic cells allowing the cells to de-differentiate. After being de-differentiated, the cells can be induced to re-differentiate into the same or a different somatic cell type such as neuronal, hematopoietic, muscle, epithelial, and other cell types. The stem-like cells derived by the present invention have medical applications for treatment of degenerative diseases by “cell therapy” and may be utilized in novel therapeutic strategies in the treatment of cardiac, neurological, endocrinological, vascular, retinal, derm...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N5/0789C12N13/00C12N15/12
CPCC12N2510/00C12N5/0696A61P43/00C12N2501/602C12N2501/603C12N2501/604C12N2501/606
Inventor SAHIN, UGURPOLEGANOV, MARCOBEISSERT, TIM
Owner BIONTECH AG
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