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Method for treating autoimmune disease using cd4 t-cells with engineered stabilization of expression of endogennous foxp3 gene

A gene and heterologous technology, applied in gene therapy, allergic diseases, animal cells, etc., can solve the problems of exacerbation of autoimmune symptoms and lack of stability of pTregs

Active Publication Date: 2019-08-16
SEATTLE CHILDRENS HOSPITAL (DBA SEATTLE CHILDRENS RES INST)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

pT regs The lack of stability is an important issue because pT reverts to an inflammatory phenotype regs Use of infusions can lead to exacerbation of autoimmune symptoms

Method used

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  • Method for treating autoimmune disease using cd4 t-cells with engineered stabilization of expression of endogennous foxp3 gene
  • Method for treating autoimmune disease using cd4 t-cells with engineered stabilization of expression of endogennous foxp3 gene
  • Method for treating autoimmune disease using cd4 t-cells with engineered stabilization of expression of endogennous foxp3 gene

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Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0093] Alternative Embodiment 1: Gene editing effectively targets the FOXP3 gene and drives high expression of the transgene.

[0094] Such as Image 6 As shown, cells engineered to express GFP under the control of the MND promoter fused to the N-terminus of the endogenous FOXP3 gene showed high levels of GFP-FOXP3 fusion protein expression. Such as Figure 7 As shown, the edited cells display HR-mediated seamless integration at the target site. for Figure 7 In the experiments in , PCR was performed with the indicated primer set (ie corresponding to the position of the red arrow on the figure), and the obtained PCR products were analyzed by agarose gel electrophoresis. PCR using one primer targeting the outside of the template (primer 1 or primer 2) and one primer targeting the inside of the template (primer 3 or primer 4) will only show bands of the correct size if precise targeted integration occurs. The gel panels on the left illustrate the lack of targeted integratio...

Embodiment approach 2

[0099] Alternative embodiment 2: Forced FOXP3 expression produces reg Surface and cytokine phenotypes of T cells

[0100] Figure 10 T showing surface marker and cytokine phenotypes are shown reg Cell cartoons. It is desirable to see whether the edited cells have a specific surface phenotype. Such as Figure 11 As indicated, CD25, CD127, CTLA4 and LAG3 expression of engineered T cells stably expressing FOXP3 were analyzed by flow cytometry. Similar to natural regulatory T cells, the engineered cells were shown to express high CD25, low CD127, and high CTLA4 and Lag3.

[0101] Figure 12 Cytokine expression profiles of edited cells are also shown in . Such as Figure 12 As shown, engineered cells exhibited intracellular cytokine expression in T cells compared to mock-edited T cells. reg Like profile, with low expression of IL2, IL4 and IFN-γ. Such as Figure 13 As shown in the bar graph in , engineered T cells stably expressing FOXP3 showed high expression of IL-10 c...

Embodiment approach 3

[0108] Alternative Embodiment 3: Functional Activity of Edited Cells, Edited Cells Can Inhibit T eff proliferation.

[0109] Figure 16 A schematic diagram of the assay to read out the suppressive activity of regulatory T cells is shown. Combine CFSE-labeled responder cells with edited T regs Or mock-edited cells were mixed and subjected to bead stimulation. The assay is read off by the extent of CFSE dilution at 96 hours.

[0110] Figure 17 show that edited T cells suppress T eff proliferation. Responder cells incubated with mock-edited cells were able to substantially dilute CFSE labeling through their proliferation, whereas responder cells incubated with engineered GFP+ cells remained largely undivided (with a large amount of residual labeling).

[0111] Figure 18 Functional activity of edited cells is shown: using edited T cells from IPEX subjects lacking response to T eff Inhibition of proliferation. use with Figure 17 In the same assay, normal subject contr...

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Abstract

Disclosed are methods of making a genetically cell that expressed FOXP3 and methods of treatment. In some embodiments, the method can providing a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand, the coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof providing a nuclease and performing a gene editing process on thefirst nucleotide sequence, which edits said one or more regulatory elements, and optionally edits the FOXP3 gene or portion thereof. Methods of treating a subject suffering from an autoimmune diseaseand subjects suffering the effects of organ transplantation are also provided.

Description

technical field [0001] Methods for autoimmune disease therapy and for inducing tolerance to transplanted organs are described herein. The approach could also be used in cell therapy for severe autoimmune diseases and organ transplants. Background technique [0002] Immune self-tolerance in the periphery can be achieved through the negative regulation of the immune response that can be exerted by a variety of cells, the most characterized population of which are regulatory T cells (T regs ). T regs By suppressing effector T cells (T effs ) activation and exert anti-inflammatory activity to mediate self-tolerance and tolerance to alloantigens (alloantigens). For example, in the context of autoimmune disease and organ transplantation, CD4+CD25+Foxp3+T regs is one of the best studied and best characterized T cells. [0003] T regs Also known as "suppressor T cells". It has also been specifically described that CD4+, CD25+ regulatory T cells can exhibit regulatory functio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N5/0783C12N15/66
CPCA61K48/00C12N15/102C12N15/66C12N15/85A61P37/06A61K38/2013C12N15/52A61K35/545C12N5/0647
Inventor 安德鲁·M·沙尔博格戴维德·J·罗林斯特洛伊·托格森
Owner SEATTLE CHILDRENS HOSPITAL (DBA SEATTLE CHILDRENS RES INST)
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