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Materials and methods for engineering cells and uses thereof in immuno-oncology

An engineering, cellular technology, applied in other methods of inserting foreign genetic materials, animal cells, anti-tumor drugs, etc.

Pending Publication Date: 2020-03-24
CRISPR THERAPEUTICS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These platforms offer a higher degree of repeatability, but limitations remain

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  • Materials and methods for engineering cells and uses thereof in immuno-oncology
  • Materials and methods for engineering cells and uses thereof in immuno-oncology
  • Materials and methods for engineering cells and uses thereof in immuno-oncology

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example

[0832] The present invention will be more fully understood by reference to the following examples, which provide illustrative, non-limiting aspects of the invention.

[0833] The examples describe the use of the CRISPR system as an illustrative genome editing technology to create defined therapeutic genomic deletions, insertions, or substitutions (referred to herein as "genomic modifications") in or near target genes, which result in mutations in genomic loci permanent correction, or restore expression of target protein activity at heterologous loci. As described and shown herein, the introduction of defined therapeutic modifications represents novel therapeutic strategies that have the potential to ameliorate various medical conditions.

example 1

[0834] Example 1 - Screening gRNA

[0835] To identify a wide range of gRNAs capable of editing cognate DNA target regions, an in vitro transcribed (IVT) gRNA screen was performed. A spacer sequence is incorporated into the backbone sequence to generate a full-length sgRNA. Examples of backbone sequences are shown in Table 1. To generate a series of spacer sequences for gene disruption, each target gene (especially those containing the initiation ATG initiation codon and / or encoding key protein domains (e.g., DNA binding domain, Target genes for extracellular domains, etc.) select protein-coding exons. Submit relevant genomic sequences for analysis using gRNA design software. The resulting list of gRNAs was narrowed down to a list of ~200 gRNAs based on sequence uniqueness (screening only gRNAs that did not have a perfect match elsewhere in the genome) and minimal expected off-target effects. This set of gRNAs was transcribed in vitro and transfected into HEK293T cells con...

example 2

[0899] Example 2 - Gene knockout at the genotype and phenotype level in cells

[0900] This example demonstrates efficient knockdown by CRISPR / Cas9 of graft-versus-host (GVH) or host-versus-graft (HVG) genes or immune checkpoint genes at the genotype and phenotype level in primary human T cells remove.

[0901] Primary human T cells were isolated from peripheral blood (AllCells Inc., Alameda, CA) using the EasySep Direct Human T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada). cells at 0.5 x 10 6 cells / mL were plated in large flasks. Human T-activator CD3 / CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA) were resuspended and washed with PBS before adding to the cells. Cells were incubated with human T-activator CD3 / CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA) at a 1:1 bead-to-cell ratio in X-vivo15 hematopoietic serum-free medium (Thermo Fisher Shier Technology Company, Waltham, Massachusetts) supplemented with 5% human serum (Sigma-Aldrich (Si...

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PUM

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Abstract

The present invention disclsoes materials and methods for producing genome-edited cells engineered to express a chimeric antigen receptor (CAR) construct on the cell surface, and materials and methodsfor genome editing to modulate the expression, function, or activity of one or more immuno-oncology related genes in a cell, and materials and methods for treating a patient using the genome-edited engineered cells.

Description

[0001] Cross References to Related Applications [0002] This application requires U.S. Provisional Application No. 62 / 505,649, filed May 12, 2017, U.S. Provisional Application No. 62 / 508,862, filed May 19, 2017, filed July 2017, pursuant to 35 U.S.C. § 119(e) U.S. Provisional Application No. 62 / 538,138 filed on October 28, U.S. Provisional Application No. 62 / 567,012 filed on October 2, 2017, U.S. Provisional Application No. 62 / 567,008 filed on October 2, 2017, filed in 2017 U.S. Provisional Application No. 62 / 583,793, filed November 9, U.S. Provisional Application No. 62 / 639,332, filed March 6, 2018, U.S. Provisional Application No. 62 / 648,138, filed March 26, 2018, and Priority to U.S. Provisional Application No. 62 / 655,510, filed April 10, 2018, each of which is incorporated herein by reference in its entirety. technical field [0003] In some aspects, the application provides materials and methods for generating genome-edited cells engineered to express a chimeric antigen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/60A61K35/17C07K14/725C07K14/705C12N15/10C12N15/63C12N15/90A61K39/00C12N15/62
CPCA61K35/17A61K39/001112A61K39/001138A61K2039/5156A61K2039/5158A61P35/00C07K14/7051C07K14/70517C07K14/70521C07K14/70539C07K14/70578C07K2319/02C07K2319/03C07K2319/33C12N15/102C12N15/62C12N15/63C12N15/907A61K38/00A61K48/005C07K2319/00C07K2319/30C12N15/1138C12N2310/20C12N2750/14143A61K39/4611A61K2239/38A61K2239/31C12N5/0634A61K2239/26A61K39/464414A61K39/464411C12N5/0636A61K39/4631A61K39/464438A61K39/464417A61K2239/56A61K2239/48A61K39/464412C12Q2521/301C12N15/113C07K16/2803C12N2510/00C07K2317/24C07K2317/622A61K2121/00A61K2300/00A61K48/0066C12N9/22C12N15/86
Inventor J.A.特瑞特D.卡莱齐迪斯L.克莱因
Owner CRISPR THERAPEUTICS AG
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