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Compositions and methods for re-programming cells without genetic modification for treatment of cardiovascular diseases

A cell and progenitor cell technology, applied in cardiovascular system diseases, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as insufficient control of transgene expression levels

Inactive Publication Date: 2013-02-27
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, all of these existing methods still involve the use of genetic material, which has the drawback of requiring the introduction of unknown, unwanted, or even deleterious genome modifications into target cells through exogenous sequences, and of not expressing sufficient levels of transgenes. control

Method used

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  • Compositions and methods for re-programming cells without genetic modification for treatment of cardiovascular diseases
  • Compositions and methods for re-programming cells without genetic modification for treatment of cardiovascular diseases
  • Compositions and methods for re-programming cells without genetic modification for treatment of cardiovascular diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1 Reprogramming somatic cells into induced pluripotent stem cells (iPSCs)

[0107] 1.a. Preparation of transduction substances Oct4-11R, Sox2-11R, K1f4-11R and cMyc-11R.

[0108] The polyarginine protein transduction domain was fused to the C-terminus of each reprogramming protein Oct4, Sox2, K1f4 and cMyc A through the linker SEQ ID NO.55 to form fusion proteins Oct4-11R, Sox2-11R, K1f4 respectively -11R and cMyc-11R ( figure 1 A). These poly-arginine fusion proteins were expressed in E. coli in the form of inclusion bodies, which were subsequently dissolved, refolded and further purified to obtain transducible substances Oct4-11R, Sox2-11R, K1f4-11R and cMyc-11R. Protein identity was confirmed by mass spectrometry and Western blot analysis ( figure 1 B).

[0109] 1.b. Cell permeability and stability of transduction substances Oct4-11R, Sox2-11R, K1f4-11R and cMyc-11R

[0110] Transduced substances (Oct4-11R, Sox2-11R, K1f4-11R or cMyc-11R) were added to m...

Embodiment 2

[0115] Example 2 Reprogramming of liver and pancreatic exocrine cells into insulin-producing beta cells by transduction substances His6-Ngn3-11R, His6-PDX1-11R and His6-MafA-11R in mice.

[0116] The polyarginine protein transduction domain was fused to the C-terminus of each reprogramming protein (Ngn3, PDX1 and MafA) via a linker (SEQ ID NO:55) to form His6-Ngn3-11R, His6-PDX1, respectively -11R and His6-MafA-11R ( Figure 7 ). The addition of His6 (SEQ ID NO:59) facilitates protein purification. These polyarginine fusion proteins were expressed in E. coli in the form of inclusion bodies, which were subsequently dissolved, refolded and further purified to prepare the transductants His6-Ngn3-11R, His6-PDX1-11R and His6-MafA-11R. Protein identity was confirmed by mass spectrometry and Western blot analysis.

[0117] Six CD-1 mice (Charles River Laboratory) were divided into two groups: treatment group and control group. Transduce substances His6-Ngn3-11R (1 mg / kg), His6-PD...

Embodiment 3

[0118] Example 3 Reprogramming T cells with the transduction substance Foxp3 and programming them into Treg cells.

[0119] The polyarginine protein transduction domain was fused to the C-terminus of each reprogramming protein Foxp3 through a linker (SEQ ID NO:55) to form His6-Foxp3-11R ( Figure 7 ). The addition of His6 (SEQ ID NO:59) facilitates protein purification. The poly-arginine fusion protein was expressed in Escherichia coli in the form of inclusion body, and then dissolved, refolded and further purified to prepare the transduction material His6-Foxp3-11R. Protein identity was confirmed by Western blot analysis.

[0120]100 ml of healthy human blood was collected from donors, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St Louis, MO). CD14+ monocytes were depleted by magnetic bead selection (Miltenyi Biotec, Auburn, CA). Briefly, 108 PBMCs were incubated with 200 μL of anti-C...

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Abstract

The present inventions are directed to compositions and methods regarding the reprogramming of other cells (such as fibroblast cells) into cardiomyocytes without introducing exogenous genes to the samples. In particular, the present inventions are directed to transducible materials that are capable of transducing into the biological samples but are not genes or causing genetic modifications. The present inventions also are directed to methods of reprogramming the path of biological samples or treating diseases using the tranducible compositions thereof.

Description

[0001] priority statement [0002] This application claims priority to the following U.S. Provisional Patent Applications: U.S. Provisional Patent Application 61 / 398,279, filed June 23, 2010, and U.S. Provisional Patent Application 61 / 360,852, filed July 1, 2010, which are incorporated by reference in their entirety into this article. Background of the invention [0003] Embryonic stem cells are capable of differentiating into many types of human cells. Most somatic cells are terminally differentiated cells and are thought to lack the ability to transform into other types of somatic cells. Recent advances in the field of induced pluripotent stem cells (iPSCs) and transdifferentiation have changed this paradigm. Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopically expressing four transcription factors, Oct4 (e.g. SEQ ID NO:1), Sox2 (e.g. SEQ ID NO:2) through viral transduction , K1f4 (SEQ ID NO:3) and cMyc (eg SEQ ID NO:4) (Okita et al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N5/07C12N15/12C07K14/47A61K38/17A61P9/00
CPCC12N2501/603C12N5/0696A61K38/16C12N2501/604C12N2501/60A61K38/00B82Y5/00C12N2501/602Y10S977/773C12N5/0637C12N2501/606A61P43/00A61P9/00
Inventor 朱勇吴时丽包骏
Owner VIVOSCRIPT
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