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Genome editing by directed non-homologous DNA insertion using a retroviral integrase-cas9 fusion protein

A technology of retrovirus and fusion protein, applied in the field of genome editing through directional non-homologous DNA insertion using retroviral integrase-Cas9 fusion protein, which can solve the problems of preventing clinical application, off-target mutation, disease, etc.

Pending Publication Date: 2021-08-24
UNIVERSITY OF ROCHESTER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the random nature of current lentiviral integration has the potential to lead to off-target mutations and disease, which prevents its use in clinical applications (Milone et al., 2018, Leukemia 23:1529-41)

Method used

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  • Genome editing by directed non-homologous DNA insertion using a retroviral integrase-cas9 fusion protein
  • Genome editing by directed non-homologous DNA insertion using a retroviral integrase-cas9 fusion protein
  • Genome editing by directed non-homologous DNA insertion using a retroviral integrase-cas9 fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0325] Example 1: Amplification of Retroviral Integrase-dCas9 Fusion Protein for Editing Mammalian Genomic DNA strong nuclear localization

[0326] Efficient CRISPR-Cas9 editing of mammalian genomic DNA requires nuclear localization of Cas9, a large bacterial RNA-guided endonuclease that normally functions in prokaryotic cells lacking a nuclear membrane. Studies have shown that effective nuclear localization of Cas9 in mammalian cells requires the addition of at least two mammalian nuclear localization signals, one at the N-terminus and one at the C-terminus (Cong et al., 2013, Science 339:819-23) .

[0327] To facilitate nuclear localization of the retroviral integrase-dCas9 fusion protein for editing, an N-terminal SV40 NLS was included on the integrase in addition to the C-terminal SV40 NLS on dCas9 ( figure 1A). Surprisingly, only a small fraction of the IN-dCas9 fusion protein was nuclear localized when expressed in mammalian cells, as detected using a FLAG antibody...

Embodiment 2

[0331] Example 2: Integrative gene editing approach for correction of muscular dystrophy

[0332] As demonstrated elsewhere herein, the fusion of lentiviral integrases to CRISPR-Cas9 allows sequence-specific integration of large DNA sequences into genomic DNA. This approach can be used to deliver therapeutically beneficial genes to non-pathogenic genomic locations (safe harbor) for permanent correction of human genetic diseases ( figure 2 ). This technique allows sequence-specific integration of large DNA donor sequences containing short viral terminal motifs.

[0333] A major advantage of the gene therapy methods of the present invention is the ability to deliver donor DNA sequences to targeted genomic locations. Furthermore, this approach eliminates the need for homology arms and relies on targeting by guide RNAs, greatly simplifying genome editing. Thus, once a specific reporter donor sequence is generated, it can be directed to any location (or locations) for a varie...

Embodiment 3

[0345] Example 3: Genome Editing - Directed Non-Homologous DNA Integration

[0346] The data presented here demonstrate that optimized integrase-Cas enables efficient editing of mammalian genomes.

[0347] optimized editing

[0348] To optimize IN-mediated integration, determine whether amino acid mutations that enhance integrase catalytic activity, solubility, or interaction with host cell cofactors enhance editing. In addition, the efficiency and fidelity of IN proteins isolated from seven unique classes of retroviruses were assessed.

[0349] To quantify and characterize IN-dCas9-mediated integration in mammalian cells, a plasmid-based reporter system utilizing the blue chromoprotein (amilCP) from Acropora polyporia, when expressed in Escherichia coli, was used. Chromoproteins produce dark blue colonies. Disruption of the amilCP open reading frame abolishes blue protein expression, which can be used as a direct readout for targeting fidelity. In addition, a donor temp...

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Abstract

The present invention provides fusion proteins comprising a retroviral integrase and a Cas protein, and related nucleic acids, systems and methods for editing genomic material.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 62 / 748,703, filed October 22, 2018, which is incorporated herein by reference in its entirety. [0003] Background of the invention [0004] CRISPR-Cas9 has greatly enhanced the ability to rapidly alter mammalian genomes for basic research and clinical applications. CRISPR-Cas9 uses a guide RNA to direct Cas9 to a specific DNA target sequence, where it induces double-stranded DNA cleavage and triggers cellular repair pathways to introduce frameshift mutations or insert donor sequences through homology-directed repair (HDR). Despite these significant advances, targeted delivery of large DNA sequences for genome editing using CRISPR-Cas9-mediated HDR remains inefficient, requires donor templates containing significant flanking regions of homology and induces the p53 DNA damage pathway ( Byrne et al., 2015, NAR 43:e21; Happaniemi et al., 2018, Nat Med...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/52C12N15/867
CPCC12N9/22C07K2319/09C07K2319/00C07K2319/80C12N2740/16043C12N9/12C12N15/62C12N15/63C12N15/113C12N15/102C12N2310/20C12N15/11C12N15/111
Inventor 道格拉斯·马修·安德松
Owner UNIVERSITY OF ROCHESTER
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