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Quinoxalinone compounds, compositions, methods, and kits for increasing genome editing efficiency

A compound, alkyl technology, applied to quinoxalinone compounds used to improve genome editing efficiency, can solve problems such as ratios less than 1%

Pending Publication Date: 2020-10-13
VERTEX PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although the efficiency of achieving insertions or deletions (indels) from NHEJ is as high as 70% in some reports, the efficiency of HDR is still challenging, where the ratio is less than 1%

Method used

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  • Quinoxalinone compounds, compositions, methods, and kits for increasing genome editing efficiency
  • Quinoxalinone compounds, compositions, methods, and kits for increasing genome editing efficiency
  • Quinoxalinone compounds, compositions, methods, and kits for increasing genome editing efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0691] Example 1: Materials and methods

[0692] method:

[0693] Cells and culture

[0694] Bronchial epithelial cells (BEC) are derived from a human donor diagnosed with cystic fibrosis of the CFTR dF508 / dF508 genotype.

[0695] Induced pluripotent stem cells (iPSC) are derived from human dermal fibroblasts after viral transduction using Yamanaka's reprogramming factors Oct4, Sox2, KLF4 and c-Myc. The derived iPSC can differentiate into 3 germ layers and contains a normal karyotype with 23 pairs of chromosomes.

[0696] Primary human mobilization of peripheral blood (mPB) CD34 + Hematopoietic stem cells and progenitor cells (HSPC) were purchased from Hemacare or AllCells. The cells were thawed, washed and resuspended in a complete medium consisting of serum-free medium CellGro SCGM (CellGenix) and supplemented with a cytokine mixture (300ng / mL SCF, 300ng / mLFlt3L, 100ng / mL TPO, 60ng / mL). ml IL-3), the density is 1-3x 10 5 Cells / mL, and at 37°C / 5% CO before electroporation 2 Incubate...

Embodiment 2

[0730] Example 2: DNA-PK inhibitors improve the ratio of HDR gene editing in BEC

[0731] figure 1 The design of the gene editing assay used in the following examples is exemplified. In order to study the effect of DNA-PK inhibitors on HDR gene editing rate, BEC was electroporated with spCAS9 mRNA, NAV1.7 sgRNA and NAV1.7 non-PAM ssODN, and then incubated with different concentrations of compound 1 or untreated ( Control). The gene editing rate was determined 72 hours after electroporation by using the TIDE assay. The gene editing rate is expressed as a percentage and classified into HDR and NHEJ. The cell survival rate is expressed as a percentage in which the control cell is set to 100%.

[0732] Such as figure 2 As shown in, the DNA-PK inhibitor of compound 1 improves the gene editing rate in BEC. For compound 1, NHEJ IC50 is 0.4450 μM, HDR EC50 is 0.4448 μM, and HDR TOP% is 69.37.

Embodiment 3

[0733] Example 3: DNA-PK inhibitors improve CD34 + HDR gene editing ratio in cells

[0734] In order to study the effect of DNA-PK inhibitors on HDR gene editing rate, mPB CD34 was electroporated with RNP (spCAS9 protein + NAV1.7sgRNA) and NAV1.7 non-PAM ssODN + cell. The cells were then incubated with compound 1 at different concentrations. 48h after electroporation, the gene editing rate was determined by using the TIDE assay. The gene editing rate is expressed as a percentage, and such as Figure 3A (Donor B) and Figure 3B (Donor C) is classified as HDR and NHEJ. The cell survival rate was expressed as a percentage in which the control cells were set to 100%.

[0735] Such as Figure 3A with 3B Shown that the DNA-PK inhibitor of compound 1 improves CD34 + The rate of gene editing in the cell. The EC50 values ​​formed by the HDR and Indel of donor B were 0.29 μM and 0.35 μM, respectively.

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Abstract

Compounds, methods of editing a target genomic region(s), methods of repairing of a DNA break via a HDR pathway, methods of inhibiting or suppressing repair of a DNA break via a NHEJ pathway, and methods of modifying expression of a gene(s) or protein(s) comprise administering to one or more cells that include one or more target genomic regions, a genome editing system and a DNA protein-kinase (DNA-PK) inhibitor disclosed herein. Kits and compositions for editing a target gene comprise a genome editing system and a DNA-PK inhibitor disclosed herein.

Description

[0001] Cross references to related applications [0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 618,385 filed on January 17, 2018, the entire content of which is incorporated herein by reference. [0003] Sequence Listing [0004] This application contains a sequence listing, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on January 16, 2019 is called 14390-687Sequence listing_ST25.txt and is 4KB in size. [0005] Invention field [0006] The present invention generally relates to compounds, compositions, methods and kits for improving the efficiency of genome editing by applying DNA protein kinase (DNA-PK) inhibitors and genome editing systems to cells. [0007] Background of the invention [0008] Accurate genome targeting technology is needed to realize the systematic engineering of genetic variation. In the past few years, the application of genome editi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D403/12C07D405/14C07D241/44C07D491/048C07D498/08C12N15/00
CPCC07D403/12C07D405/14C07D241/44C07D491/048C07D498/08C12N15/87C12N15/907C12N15/102A61K31/5377C12N15/11C12N2310/20A61K31/5386C07D491/08C12N15/113C12N2800/80C12N9/22
Inventor S·马哈詹M·S·韦恩伯格D·S·达斯托尔佛K·M·寇特瑞尔M·A·莫里斯J·P·麦克斯威尔
Owner VERTEX PHARMA INC
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