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Methods and compositions for nuclease-mediated genome engineering and correction in hematopoietic stem cells

A hematopoietic stem cell and genome technology, applied in the field of genome engineering of hematopoietic stem cells, can solve the problems of life expectancy patients' quality of life, unknown long-term side effects, unwanted side effects, etc.

Active Publication Date: 2017-05-31
桑格摩治疗股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the long-term side effects of long-term hydroxyurea therapy remain unknown, and the treatment causes undesired side effects and may have variable efficacy between different patients
Despite the increased efficacy of sickle cell therapy, the life expectancy of patients remains only 55 to 59 years and the associated morbidity of the disease has a profound impact on patients' quality of life

Method used

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  • Methods and compositions for nuclease-mediated genome engineering and correction in hematopoietic stem cells
  • Methods and compositions for nuclease-mediated genome engineering and correction in hematopoietic stem cells
  • Methods and compositions for nuclease-mediated genome engineering and correction in hematopoietic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0180] Example 1: Design, Construction and General Characterization of Zinc Finger Nucleases (ZFNs)

[0181] Zinc finger proteins were designed and incorporated into plasmid, AAV or adenoviral vectors essentially as Urnov et al. (2005) Nature 435(7042):646-651, Perez et al (2008) Nature Biotechnology 26(7) : 808-816, and as described in U.S. Patent No. 6,534,261 and tested for binding. See commonly owned US Patent No. 7,888,121 and US Patent Publication Nos. 20130137104 and 20130122591 for ZFNs and TALENs specific for the human beta globin locus.

Embodiment 2

[0182] Example 2: Activity of globin-specific ZFNs

[0183] Pairs of ZFNs targeting the human globin locus were used to test the ability of these ZFNs to induce DSBs at specific target sites. The amino acid sequences of the recognition helical regions of each finger of the indicated ZFNs are shown in Table 1 below. Target sites (DNA target sites are indicated in capital letters; non-contact nucleotides are indicated in lower case letters) are shown in Table 2.

[0184] Table 1: Human β-globin-specific zinc finger protein recognition helix design

[0185]

[0186]

[0187] Table 2: Target sites of human β-globin-specific zinc fingers

[0188]

[0189] Human CD34+ cells were obtained and processed as follows. All cord blood (CB) specimens were obtained according to UC-approved guidelines and were considered anonymous medical waste exempt from IRB review. Process cells within 48 hours of collection. Bone marrow (BM) aspirates from volunteer donors with SCD were obta...

Embodiment 4

[0201] Example 4: ZFN-driven correction at sickle cell bases

[0202] Following successful cleavage of the targeted β-globin locus, we sought to determine whether it was possible to correct sickle bases at this locus using a cognate donor template. To this end, two types of gene correction templates were simultaneously designed and tested: short DNA oligonucleotides and IDLV. The 1.1 kb human β-globin gene fragment donor template cloned into IDLV was designed to include correction changes for sickle mutations as well as silent restriction fragment length polymorphisms (RFLPs) to generate replacements for homologous recombination The analyzed HhaI restriction site ( Figure 1C ). Donor templates are delivered via IDLV (see Figure 2D ), enabling efficient transduction of CD34+ HSPCs with minimal cytotoxicity, resulting in transient generation of high template copy numbers with minimal genomic integration.

[0203] Levels of gene modification in CD34+ cells treated with ZFN ...

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PUM

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Abstract

The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic stem cell. Disclosed herein are methods and compositions for altering the expression or for correcting one or more genes encoding proteins involved in a genetic disease (e.g., producing proteins lacking, deficient or aberrant in the disease and / or proteins that regulate these proteins) such as sickle cell disease. The present invention describes compositions and methods for use in gene therapy and genome engineering.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 62 / 051,159, filed September 16, 2014, the disclosure of which is hereby incorporated by reference in its entirety. technical field [0003] The present disclosure belongs to the field of genome engineering of hematopoietic stem cells, especially the targeted modification of the genome of hematopoietic cells. Background technique [0004] One of the most promising approaches in gene therapy for a large number of diseases involves the use of genetic modification of stem cells in vitro, followed by transplantation and implantation of the modified cells in patients. It is particularly promising when the introduced stem cells display long-term persistence and multi-lineage differentiation. Hematopoietic stem cells (most commonly an enriched form of cells based on the expression of the CD34 cell surface marker) are the most particularly useful cell popula...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC07K14/805C12N5/0645C12N2510/00C12N15/111C12N2310/20C12N9/22A61K48/005A61P7/06A61K39/464A61K39/461A61K35/28C12N15/11C12N15/907C12N2800/80
Inventor 格雷戈里·J·科斯特杰弗里·C·米勒张雷
Owner 桑格摩治疗股份有限公司
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