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Crispr/cas-related methods and compositions for treating beta hemoglobinopathies

A nucleic acid composition and molecular technology, applied in the combined application field of β-hemoglobinopathies, can solve problems such as unclear long-term efficacy and safety, inability to identify allogeneic donors, etc.

Pending Publication Date: 2019-01-04
EDITAS MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the long-term efficacy and safety of this pathway is unknown
Hematopoietic stem cell transplantation from HLA-matched allogeneic stem cell donors has been demonstrated to treat SCD and β-Thal, but the approach involves risks, including those associated with excisional therapy, preparation of transplant subjects and post-transplant graft resistance. host disease risk
Additionally, matching allogeneic donors are often not identified

Method used

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  • Crispr/cas-related methods and compositions for treating beta hemoglobinopathies
  • Crispr/cas-related methods and compositions for treating beta hemoglobinopathies
  • Crispr/cas-related methods and compositions for treating beta hemoglobinopathies

Examples

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example

[0976] The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

example 1

[0977] Example 1: Streptococcus pyogenes for insertion of 13bp del c. -114 to -102 into HBG1 and HBG2 regulatory regions gRNA screening

[0978] The S. pyogenes gRNA designed as described herein targets a 26nt fragment spanning and including 13 nucleotides at c.-114 to -102 of HBG1 (e.g., nucleotides of SEQ ID NO: 902 (HBG1) 2824-2836, resulting in a change of HBG113bp del c.-114 to -102) and HBG2 (for example, nucleotides 2748-2760 of SEQ ID NO:903 (HBG2), resulting in a change of HBG1 13bp del c.-114 to -102 Change). After designing gRNAs after in silico simulation and fractionation, a portion of gRNAs were selected and screened for activity and specificity in human K562 cells. The gRNAs selected for screening contained the targeting domain sequences set forth in Table 8. DNA encoding the U6 promoter and each S. pyogenes gRNA was co-electroporated (Amaxa nucleofection apparatus) into human K562 cells with plasmid DNA encoding S. pyogenes Cas9. Experimental conditions ...

example 2

[0983] Example 2: Cas9RNP containing gRNA targeting HPFH mutation supports gene coding in human hematopoietic stem / progenitor cells edit

[0984] Human cord blood (CB) was prestimulated with human cytokines (stem cell factor (SCF), thrombopoietin (TPO), Flt3 ligand (FL)) and small molecules (prostaglandin E2 (PGE2), StemRegenin 1 (SR1)) CD34 + cells for two days. Experimental conditions were generally according to the method provided in Gori 2016 pp. 240-241, which is hereby incorporated by reference. CB CD34 + Cells were electroporated (Amaxa Nucleofector) with S. pyogenes Cas9 RNP containing (eg, 5' ARCA cap and 3' polyA(20A) tail) sgRNAs (Table 8) targeting HBG1 and HBG2 regulatory regions. 3 days after electroporation, CD34 from RNP-treated CB + gDNA was extracted from cells and analyzed for gene editing by T7E1 assay and DNA sequencing.

[0985] in CB CD34 + Of the RNPs containing different gRNAs tested in cells, only the Sp37 gRNA (comprising SEQ ID NO:333) res...

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Abstract

CRISPR / CAS-related compositions and methods for treatment of beta hemoglobinopathies are disclosed.

Description

[0001] References to related applications [0002] This application claims the benefit of U.S. Provisional Application No. 62 / 308,190, filed March 14, 2016, and U.S. Provisional Application No. 62 / 456,615, filed February 8, 2017, the contents of each of which are hereby incorporated by reference in their entirety. [0003] sequence listing [0004] This application contains a Sequence Listing submitted via EFS-Web in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 14, 2017, is named 8009WO00_SequenceListing.txt and is 335KB in size. technical field [0005] The present invention relates to CRISPR / Cas-related methods and components for editing target nucleic acid sequences or regulating the expression of target nucleic acid sequences, and their combined application with β-hemoglobinopathies including sickle cell disease and β-thalassemia. Background technique [0006] Hemoglobin (Hb) carries oxygen from the lungs to ti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713C12N15/09C12N15/63A61P7/06
CPCA61K31/713C12N15/113C12N2310/10C12N2310/20A61P43/00A61P7/06C12N9/22C12N15/102C12N15/907C12N5/0602C12N15/85C12N2310/315C12N2310/322C12N2510/00C12N2800/80
Inventor J·L·戈里L·A·巴雷拉
Owner EDITAS MEDICINE
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