Compositions and methods for re-programming cells without genetic modification for repairing cartilage damage

A technology of cells and substances applied in the field of compositions and methods of non-genetically modified reprogrammed cells for repairing cartilage damage

Inactive Publication Date: 2014-06-11
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Healing of cartilage injuries via hyaline cartilage rather than fibrocartilage remains a challenging clinical problem

Method used

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  • Compositions and methods for re-programming cells without genetic modification for repairing cartilage damage
  • Compositions and methods for re-programming cells without genetic modification for repairing cartilage damage
  • Compositions and methods for re-programming cells without genetic modification for repairing cartilage damage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1 Reprogramming somatic cells into induced pluripotent stem cells (iPSCs)

[0112] 1.a. Preparation of transduction substances Oct4-11R, Sox2-11R, Klf4-11R and cMyc-llR.

[0113] The polyarginine protein transduction domain is fused to the C-terminus of each reprogramming protein Oct4, Sox2, Klf4 and cMyc A through the linker SEQ ID NO.55 to form fusion proteins Oct4-11R, Sox2-11R, Klf4 respectively -11R and cMyc-l lR ( 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, Klf4-11R and cMyc-11R. Protein identity was confirmed by mass spectrometry and Western blot analysis ( figure 1 B).

[0114] 1.b. Cell permeability and stability of transduction substances Oct4-11R, Sox2-11R, Klf4-11R and cMyc-11R

[0115] Add transduction substances (Oct4-11R, Sox2-11R, Klf4-11R or cMyc-11R) at differ...

Embodiment 2

[0120] Example 2 Reprogramming of liver and pancreatic exocrine cells into insulin-producing beta cells in mice by transduction substances His6-Ngn3-11R, His6-PDX1-11R and His6-MafA-11R.

[0121] The polyarginine protein transduction domain was fused to the C-terminus of each reprogramming protein (Ngn3, PDX1 and MafA) through 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 poly-arginine fusion proteins were expressed in E. coli in the form of inclusion bodies, which were subsequently dissolved, refolded, and further purified to prepare transducible substances His6-Ngn3-11R, His6-PDX1-11R, and His6-MafA-11R. Protein identity was confirmed by mass spectrometry and Western blot analysis.

[0122] Six CD-1 mice (Charles River Laboratory) were divided into two groups: treatment group and control group. Transduce substances His6-Ngn3-11R (lmg / ...

Embodiment 3

[0123] Example 3 Reprogramming T cells with the transduction substance Foxp3 and programming them into Treg cells.

[0124] 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 polyarginine 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.

[0125] 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 embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), MSCs, fibroblasts, hematopoietic stem cells, endothelian stem cells, adipocytes, chondrocytes, osteoblasts, osteoclasts and endothelial cells) into chondrogenic cells 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 US Provisional Patent Application: US Provisional Patent Application 61 / 509,114, filed July 19, 2011, which is hereby incorporated by reference in its entirety. 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. Ectopic expression of four transcription factors, Oct4 (e.g., SEQ ID NO:1), Sox2 (e.g., SEQ ID NO:2), Klf4 (e.g., SEQ ID NO:3), and cMyc (e.g., SEQ ID NO:4) by viral transduction , somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) (Okita et al., Nature 448, 313-317 (2007); Takahashi and Yamanaka, Cell 126, 663-676 (2006)). A num...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C07K14/00C12N5/077A61K38/16A61L27/38C07K19/00
CPCC07K2319/60C12N2501/603C12N2506/14C12N5/0637C12N5/0655C12N2501/606C07K14/4702A61K47/48246C12N2501/604A61L27/3654C12N2501/602C07K17/06A61K47/48315A61K38/00C07K2319/21C12N5/0696C07K14/4703C12N2501/60C07K14/4716G01N33/5073A61L27/3612C12N5/0676A61K47/64A61K47/645
Inventor 朱勇吴时丽包骏初从秋
Owner VIVOSCRIPT
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