Unlock instant, AI-driven research and patent intelligence for your innovation.

Regulated gene editing system

A gene and editing technology, applied in the field of regulating gene editing compositions, can solve off-target and other problems

Pending Publication Date: 2021-07-23
THE UNIV OF NORTH CAROLINA AT CHAPEL HILL
View PDF66 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite advances in genome editing technology, it has been found that a large number of off-targets (e.g., unintentional mutations) can occur during gene editing, which limits the approach as a therapeutic option

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Regulated gene editing system
  • Regulated gene editing system
  • Regulated gene editing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0404] Example 1. Differential regulation of multiple transgenes in AAV vectors by alternative splicing

[0405] introduce

[0406] Wild-type AAV is a non-pathogenic, non-enveloped, small single-stranded DNA virus with a genome of 4.7 kb in length. Recombinant AAVs have been developed and used as gene therapy vectors for decades. The ability to regulate transgene expression is critical to ensuring the safety of many gene therapy strategies. Several strategies for controlling transgene expression (eg tet-on or rapamycin-inducible systems) have been tested for AAV vector-mediated gene transfer. Each regulatory system has advantages and disadvantages depending on the target to be treated. As a strategy to develop a transgenic regulatory system that simplifies the gene delivery system, eliminates immune responses against transactivators, and induces the induction of AAV-mediated gene delivery by adapting the splicing-switching mechanism of the IVS2-654 intron Multiple transgen...

Embodiment 2

[0438] Example 2. Generation of saCas9 comprising regulatory nucleic acid sequences

[0439] As described in Example 1, saCas9 containing regulatory sequences (beta globin intron regions) was generated. The regulatory sequence intron region (eg, SEQ ID NO: 53 (IVS2-654 intron with a 200 bp deletion) was subcloned into an AAV vector plasmid carrying saCas 9 using restriction digestion.

Embodiment 3

[0440] Example 3. Assay for off-target effects of gene editing

[0441] Sequencing of digested genomes (Digenome-seqover), an in vitro Cas9 digested whole-genome sequencing, is a robust, sensitive, unbiased, and cost-effective method for analyzing programmable nucleases (e.g., Cas9) in mammals ( Genome-wide off-target effects in human cells).

[0442] HeLa, HEK, and CHO cells expressing Nav 1.8-directed gRNAs were transfected using lipofectamine 2000 (Life Technologies) with: (1) nuclease-free (eg, untransfected population); (2) constitutively active Casp 9; (3) a gene editing system described herein that does not contain an oligonucleotide that binds a regulatory sequence, such as a nuclease in an "OFF" position; and (4) a gene editing system described herein and Oligonucleotides that bind regulatory sequences, eg, nucleases in the "ON" position. HeLa cells were cultured in DMEM medium containing 10% FBS. Cells were cultured for 48 hours.

[0443] In vitro lysis of genomi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides a gene editing system having reduced off target effects comprising (a) a vector comprising a nucleic acid sequence encoding a nuclease, wherein the nucleic acid encoding the nuclease contains within its sequence a regulatory nucleic acid sequence having a first and second set of splice elements defining a first and second intron, wherein the first and second intron flank a sequence encoding a non-naturally occurring exon sequence containing an in-frame stop codon sequence, and wherein the first and second intron are spliced from the mRNA message to produce an mRNA encoding a non-functional nuclease that contains an amino acid sequence encoded by the non-naturally occurring exon; and (b) an oligonucleotide that binds to the regulatory sequence. Further provided are methods of using the gene editing system of this invention to regulate transgene expression.

Description

[0001] claim of priority [0002] This application claims the benefit of US Provisional Application No. 62 / 743,317, filed October 9, 2018, and US Provisional Application No. 62 / 870,427, filed July 3, 2019, under 35 U.S.C. §119(e), Its entire content is incorporated herein by reference in its entirety. [0003] Statement Regarding Electronic Submission of Sequence Listings [0004] A Sequence Listing in ASCII text format, named 5470-858WO_ST25.txt, 371,885 bytes in size, was submitted in accordance with 37 C.F.R. §1.821, generated on October 8, 2019, and submitted via EFS-Web in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification herein as its disclosure. technical field [0005] The present invention relates to compositions and methods of use for modulating gene editing. [0006] Background of the Invention [0007] Recent advances in genome sequencing technologies and analytical methods have significantly accelerated the ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N9/22C12N15/10C12N15/113C12N15/90A61K31/7088
CPCC12N9/22C12N15/113C12N15/86C12N15/907C12N2310/20C12N2310/11C12N2310/3231C12N2320/33C12N2750/14143
Inventor 理查·J·萨谬斯基
Owner THE UNIV OF NORTH CAROLINA AT CHAPEL HILL