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Gene regulation via conditional nuclear localization of gene modulating polypeptides

A technology for gene and target regulation, which can be applied to polypeptides containing positioning/targeting motifs, activity regulation, genetic engineering, etc., and can solve problems such as reduced immune cell response and reduced effectiveness of immunotherapy.

Pending Publication Date: 2021-03-16
REFUGE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insufficient signaling from co-stimulatory receptors may result in reduced immune cell responses and less effective immunotherapy

Method used

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  • Gene regulation via conditional nuclear localization of gene modulating polypeptides
  • Gene regulation via conditional nuclear localization of gene modulating polypeptides
  • Gene regulation via conditional nuclear localization of gene modulating polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0479] Nuclear factor of activated T cells (NFAT) is a family of T lymphocyte activation-specific transcription factors consisting of five members: NFATc1, NFATc2, NFATc3, NFATc4 and NFAT5. The NFAT response element (recognition sequence) is a transcriptional element in the IL-2 enhancer that, in the absence of physiological activation of T lymphocytes by antigen receptors or by combined treatment of T cells with ionomycin and PMA, Usually has no stimulatory effect on transcription.

[0480] In resting T cells, NFAT can exist in the cytoplasm in a phosphorylated state. In this state, nuclear localization signals (NLS) can be masked by phosphorylated serine residues or other inhibitory NFAT-binding proteins. Upon cellular activation, NFAT can be directly dephosphorylated by calcineurin (CN), and the NFAT NLS can then be exposed due to a conformational change or dissociation of an inhibitory binding partner, allowing NFAT nuclear translocation (Shibasaki, F. et al., 1996, Natu...

Embodiment 2

[0481] Example 2: CAR activation-dependent GFP protein expression mediated by N-NFATc2-dCas9-VP64 fusion protein

[0482] Upregulation of the GFP reporter gene using a catalytically inactive (dCas9) system. T cell activation-induced activity was observed when dCas9 was fused to the NFATc2 N-terminal domain and transfected into Jurkat T cells containing a GFP-targeting sgRNA and a GFP reporter gene ( Figure 3A and Figure 3B ).

[0483] Two stable Jurkat-derived cell lines (2sg and 2sg-CAR) were transfected with the indicated DNA constructs and BFP expression constructs, respectively, and then stimulated with CD19+ Raji cells. Two days later, cells were harvested for flow cytometry analysis. Raji-stimulated cells were stained with anti-CD3-APC780 and CD22-PE before sampling in the flow cytometer. CD3+CD22- cells were gated as Jurkat-derived cells. Transfected cells were gated for data analysis using BFP expression. The 2sg cell line contains both a GFP reporter gene and ...

Embodiment 3

[0486] Example 3: Down-regulation of target genes

[0487] A catalytically inactive (dCas9) system as described in Example 2 was used to downregulate target genes. In this example, dCas9 is fused to a transcriptional repressor (eg, KRAB) rather than a transcriptional activator (eg, VP64). When dCas9-KRAB was fused to the N-terminal domain of NFATc2 (N-NFATc2-dCas9-KRAB) (SEQ ID NO:36) and transfected with sgRNA targeting the PD1 gene into the Jurkat T cell line expressing the CD19 CAR, Downregulation of PD1 was observed in activated T cells ( Figure 5 ).

[0488] Jurkat cells and Jurkat-derived cell lines constitutively expressing the CD19 CAR were transfected with N-NFATc2-dCas9-KRAB constructs and Gal4 control or PD1 sgRNA constructs, and then stimulated with CD19+ Raji cells one day later. After three days, cells were harvested for flow cytometry analysis. Raji-stimulated cells were stained with anti-CD22-APC and PD1-PE prior to sample collection in the flow cytometer....

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Abstract

The present disclosure provides a system for regulating expression of a target polynucleotide in a cell. The system may comprise a chimeric polypeptide comprising a gene modulating polypeptide fused in-frame with a heterologous nuclear localization domain. The heterologous nuclear localization domain may be operable to translocate the chimeric polypeptide to a cell nucleus upon activation by an active cellular signaling pathway. The cellular signaling pathway may be inducible in response to an extracellular signal. In response to the extracellular signal, the chimeric polypeptide may localizeto the cell nucleus and the gene modulating polypeptide may regulate expression of a target polynucleotide in the cell nucleus.

Description

[0001] cross reference [0002] This application claims the benefit of U.S. Provisional Application No. 62 / 647,543, filed March 23, 2018, and U.S. Provisional Application No. 62 / 675,134, filed May 22, 2018, each of which is incorporated by reference Incorporated into this article as a whole. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been filed electronically in ASCII format, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 22, 2019, is named 50489-711_601_SL.txt and is 137,422 bytes in size. Background technique [0005] Modulation of cellular activity may involve ligand binding to a membrane-bound receptor comprising an extracellular ligand-binding domain and an intracellular (eg, cytoplasmic) signaling domain. The formation of a complex between the ligand and the ligand-binding domain can result in a conformational change and / or chemical modification of the receptor, which can re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00
CPCC12N15/63C12N2830/002C12N2830/005C12N15/111C12N2310/20C12N2320/50C07K14/4702A61K2039/5158A61K2039/5156C07K14/7051C07K2319/03A61K39/0011C12N5/0636C07K14/705C07K2319/09C12N9/22C12N2529/00
Inventor 刘佩琪汪建斌
Owner REFUGE BIOTECH INC
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