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A method for engineering synthetic cis-regulatory DNA

A purposeful and integrated technology, applied in the field of reporting carriers, can solve problems such as limitations

Pending Publication Date: 2021-07-23
MAX DELBRUECK CENT FUER MOLEKULARE MEDIZIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is a common practice but limited in the sense that the corresponding markers must be known in advance and not all cell types have characteristic surface proteins
Furthermore, tracking cell types in vivo is impossible or very challenging using such methods

Method used

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  • A method for engineering synthetic cis-regulatory DNA
  • A method for engineering synthetic cis-regulatory DNA
  • A method for engineering synthetic cis-regulatory DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0347] Example 1: Design of an expression cassette comprising a subtype-specific synthetic locus control region (sLCR) of glioblastoma multiforme (GBM) tumor cells.

[0348] A high degree of cellular and molecular heterogeneity is thought to contribute to resistance to standard therapy in solid tumors, and this poses a barrier to the development of targeted approaches. Glioblastoma multiforme (GBM), the most common primary adult brain tumor, is unusually heterogeneous and resistant to therapy 13 . GBM is also one of the cancers with the highest degree of genomic and epigenomic characterization 14-16 . GBM tumors were recurrently classified into three subtypes based on the transcriptome, with mesenchymal and proneural more frequently cross-validated 52、53、54 . Several studies have debated the correlation between subtype-specific gene expression signatures and differential response to treatment and overall survival of patients. This suggests that GBM subtype identification ...

Embodiment 2

[0355] Example 2: Genetic tracking of mesenchymal fate in human glioma-initiating cells using lentiviral vectors including MGT#1 as sLCR

[0356] A typical lentiviral vector carrying an sLCR such as MGT#1 drives the expression of a fluorescent reporter subtype of mVenus or mCherry. To facilitate genetic tracing in vivo, drive mVenus to the plasma membrane (via Igk leader and platelet-derived growth factor receptor (PDGFR) transmembrane sequence tagging; figure 1 c) and shuttle mCherry to the nucleus via NLS. To enable fluorescence visualization and sorting of sLCRs independently of reporter expression, we also included a second cassette expressing the H2B-CFP fusion via the ubiquitous PGK promoter ( figure 1 c).

[0357] As a prototype test, we generated lentiviral particles in HEK293T cells with MGT#1-mVenus sLCR and used the viral particles to infect human glioma-initiating cells with the MES genotype (MES-hGIC). Membrane mVenus expression was observed both in transient...

Embodiment 3

[0360] Example 3: Use of MGT#1 and MGT#2 sLCR as readout for studying intrinsic and adaptive responses in GIC

[0361] Under the same experimental conditions, a second independent reporter (MGT#2) showed consistent results ( figure 2 a), which supports our ability to generate functional sLCRs starting from gene expression profiles. Interestingly, both MGT#1 and MGT#2 reporters indicated that FBS was able to induce mesenchymal differentiation, unlike the case of TNFα, which was accompanied by GIC differentiation as measured by visual inspection and flow cytometry (data not shown show). This finding can be explained only in part by the presence of TGFB1, which is indeed a known component of FBS. Indeed, TGFB1 is a mesenchymal inducer, but does not strongly induce MGT#1, and it does not promote differentiation when used as a purified cytokine in the same time frame ( figure 2 a). Perhaps more interestingly, this observation on FBS is highly consistent with the TCGA report t...

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Abstract

The invention relates to methods for generating cell-type specific expression cassettes and reporter vectors, as well as nucleic acid constructs that can be generated by such methods. The cell-type specific expression cassettes and reporter vectors are characterized synthetic cis-regulatory DNA, also termed synthetic locus regions (sLCRs). sLCRs allow for a cell-type specific expression of reporter or effector genes. The invention further relates to various uses of the reporter vectors, including the determination of a property of a cell, preferably a cell type, state or fate transition, in gene and viral therapy, drug discovery or validation.

Description

technical field [0001] The present invention relates to methods for producing cell type-specific expression cassettes and reporter vectors, as well as nucleic acid constructs that can be produced by such methods. Cell type-specific expression cassettes and reporter vectors are characterized by synthetic cis-regulatory DNA, also known as synthetic locus regions (sLCRs). sLCRs allow cell type-specific expression of reporter or effector genes. The invention further relates to various uses of reporter vectors, including the determination of cell properties, preferably cell type, state or fate transition, in gene and viral therapy, drug discovery or validation. Background technique [0002] Expression cassettes and reporter vectors have a wide range of applications in basic research, drug screening diagnostics, or gene therapy. [0003] Selective identification of cell type-specific signatures is critical to understanding biological processes in which diverse cell types contrib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12Q1/6897
CPCG16B20/30G16B25/10G16B40/00C12N15/63C12Q1/6809C12Q1/6897C12N15/64
Inventor 加埃塔诺·加尔朱洛
Owner MAX DELBRUECK CENT FUER MOLEKULARE MEDIZIN
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