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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: 2017-08-03
BIONTECH AG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for reprograming somatic cells to cells with stem cell characteristics by introducing RNA capable of expressing factors that induce reprogramming. The amount and time of expression of certain factors expressed by the RNA can be adjusted as necessary. The RNA can be derived from an undifferentiated cell or specific for it, and can be obtained by in vitro transcription. The factors capable of being expressed by the RNA include OCT4, SOX2, NANOG, and one or both of KLF4 and c-MYC. The method can involve culturing the somatic cell under embryonic stem cell culture conditions to allow the development of cells with stem cell characteristics.

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

Examples

Experimental program
Comparison scheme
Effect test

example 1

n 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 sedimented...

example 2

ration 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 variation for dif...

example 3

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 h.

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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

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 14 / 933,840, filed on Nov. 5, 2015, which is a continuation of U.S. patent application Ser. No. 12 / 735,060, filed on Nov. 24, 2010, now abandoned, which is the National Stage of International Patent Application No. PCT / EP2008 / 010593, filed on Dec. 12, 2008, which claims priority of European Patent Application No. 07024312.6, filed on Dec. 14, 2007, each of which is incorporated herein by reference.INCORPORATION OF SEQUENCE LISTING[0002]This application includes biological sequence information, which is set forth in an ASCII text file having the file name “VOS-128-CON-1_SEQ.txt”, created on Nov. 4, 2015, and having a file size of 32,591 bytes, which is incorporated herein by reference.FIELD OF THE INVENTION[0003]The present invention provides methods for de-differentiating somatic cells into stem-like cells without generating embryos or fetuses. More specifically, the pr...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/074
CPCC12N2510/00C12N5/0696A61P43/00C12N2501/602C12N2501/603C12N2501/604C12N2501/606
Inventor SAHIN, UGURPOLEGANOV, MARCOBEISSERT, TIM
Owner BIONTECH AG
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