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THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

a genome editing and genome technology, applied in the field of therapeutic use of genome editing with crispr/cas systems, can solve the problems of only 2%-4% efficiency of allele targeting in human stem cells, and achieve the effect of effectively deleting target polynucleotide sequences

Inactive Publication Date: 2015-06-25
PRESIDENT & FELLOWS OF HARVARD COLLEGE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes methods for using CRISPR / Cas systems to efficiently target and modify genes in primary somatic cells, such as blood cells and T cells. This is possible by using multiple guide strategies that use ribonucleic acids to direct the Cas protein to specific sequences and delete or modify them. The methods also allow for the delivery of CRISPR / Cas systems to human stem cells. Additionally, the patent provides specific RNA guide sequences for targeting specific genes, such as B2M, HPRT, CCR5, and CXCR4. Finally, the patent provides methods for using the CRISPR / Cas systems to treat HIV infection. The efficiency of altering cells that express Cas protein can range from about 50% to about 80%.

Problems solved by technology

Thus, CRISPR / Cas systems could be useful tools for therapeutic applications, but unfortunately prior published reports have demonstrated an efficiency of allele targeting of only 2%-4% in human stem cells (Mali et al., Science 339:823-826 (2013)).

Method used

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  • THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

Examples

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Effect test

example 1

[0322]Transcription activator-like effector nucleases (TALENs) bind as a pair around a genomic site, in which a double-strand break (DSB) is introduced by a dimer of FokI nuclease domains. The use of a TALEN genome-editing system to rapidly and efficiently generate mutant alleles of 15 different genes in human pluripotent stem cells (hPSCs) as a means of performing rigorous disease modeling was recently reported (Ding et al., Cell Stem Cell 12:238-251 (2013)); the proportions of clones bearing at least one mutant allele ranged from 2%-34%.

[0323]As described below, the relative efficacies of CRISPRs and TALENs targeting the same genomic sites in the same hPSC lines was assessed with the use of the same delivery platform described previously (Ding et al., Cell Stem Cell 12:238-251 (2013)). In the TALEN genome-editing system, the CAG promoter was used to co-translate (via a viral 2A peptide) each TALEN with green fluorescent protein (GFP) or red fluorescent protein (RFP). For CRISPRs, ...

example 2

Efficient Targeting of Clinically Relevant Genes in Primary Somatic Cells

[0329]Work described herein shows for the first time that the CRISPR / Cas9 system can be used to edit the genome of somatic cells (e.g., primary) with high efficiency by using a double guide strategy. The inventors posit that this work will help bring genome editing in clinically relevant primary cells into reality.

[0330]The advent of genome editing tools that allow one to target any desired genomic site has greatly advanced the investigation of human biology and disease. In particular, the CRISPR / Cas9 system has become the gold standard in targeted genome editing technology, due to its flexibility and high efficacy. This system is constituted by the Cas9 nuclease from the microbial type II CRISPR / Cas system, which is targeted to specific genomic loci by a 20-nucleotide region in a synthetic guide RNA molecule. Similar to other targeted nucleases (ZFNs and TALENs), Cas9 induces double strand breaks (DSBs) that a...

example 3

Modified Cas9 mRNA Functions to Efficiently Introduce On-Target Mutations

[0356]The inventors generated Figment (Fgm) knockout mice by CRIPSR / Cas9 gene editing utilizing a modified Cas9 mRNA. Fgm is a coding gene within the long non-coding RNA Lnc-Rap-5 (referred to herein as Fgm (Lnc-Rap-5; see Sun et al., “Long noncoding RNAs regulate adipogenesis,” PNAS; 2013; 110(9):3387-3392, incorporated herein by reference in its entirety). The guide RNA (gRNA) sequence employed in this example was: 5′ gaggegaaagccactagcac 3′ (SEQ ID NO: 599). The modified Cas9 mRNA used in this example was made using an in vitro transcription reaction in which pseudouridine and 5-methyl-cytosine are reacted with unmodified nucleotides and randomly integrated into the resulting modified Cas9 mRNA. An exemplary protocol for generating Fgm knockout mice using CRISPR / Cas9 gene editing utilizing a modified Cas9mRNA is shown in FIG. 11A. As shown in FIG. 11A, 100 ng / μl of the resulting modified Cas9 mRNA and 50 ng / ...

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Abstract

Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells.

Description

RELATED APPLICATIONS[0001]This application is a continuation-in-part of PCT Application No. PCT / US2014 / 033082, filed Apr. 4, 2014, which claims the benefit of U.S. Provisional Application Ser. No. 61 / 808,594, filed Apr. 4, 2013, the teachings of which are incorporated herein by reference in their entirety.GOVERNMENT SUPPORT[0002]This invention was made with government support under R01-HL118744, R00-HL098364 and R01-DK095384 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems are a new class of genome-editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR / Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/63
CPCC12N15/63A61K48/00C12N15/907A61P31/00A61P31/18A61P35/00A61P43/00C12N5/0606
Inventor MUSUNURU, KIRANCOWAN, CHAD A.ROSSI, DERRICK J.
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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