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Downregulation of snca expression by targeted editing of dna-methylation

A targeted, gene-based technology for DNA/RNA fragmentation, recombinant DNA technology, stable introduction of foreign DNA into chromosomes, etc.

Pending Publication Date: 2021-01-01
DUKE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is an unmet need for the development of new therapeutic strategies targeting the regulation of SNCA expression

Method used

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  • Downregulation of snca expression by targeted editing of dna-methylation
  • Downregulation of snca expression by targeted editing of dna-methylation
  • Downregulation of snca expression by targeted editing of dna-methylation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0182] Materials and methods

[0183] Plasmid design and construction. The dCas9-DNMT3A transfer gene was derived from pdCas9-DNMT3A-EGFP (Addgene plasmid number 71666), and cloned into pBK301 (production-optimized lentiviral vector) as follows: by cloning the dCas9 fragment digested with AgeI-BamHI restriction enzymes Into pBK301, the pBK456 plasmid was generated. Next, the DNMT3A catalytic domain was transferred from pdCas9-DNMT3A-EGFP into pBK456 by amplifying a DNMT3A fragment from a plasmid using primers containing a BamHI restriction site: BamHI-429 / R 5'-GAGCGGATCCCCCTCCCG-3' (SEQ ID NO: 15), BamHI-429 / L 5'-CTCTCCACTGCCGGATCCGG-3' (SEQ ID NO: 16). The pBK456 was then digested with BamHI restriction enzyme for cloning, resulting in pBK492 plasmid (no gRNA plasmid). Next, the extra BsmBI site located in the DNMT3A fragment was eliminated by site-directed mutagenesis to generate pBK546 (SEQ ID NO:39; see Figure 12B ). This plasmid contains dCas9-DNMT3A-p2a-puromycin exp...

Embodiment 2

[0214] Development of a novel lentiviral vector system for efficient delivery of CRISPR / Cas9-based epigenetic tools

[0215] One disadvantage of the all-in-one integrative lentiviral vector system for the delivery of CRISPR / Cas9-based materials is low production titers. Approaches to overcome these issues include the development of binary plasmid vector systems that deliver the Cas9 and gRNA components separately. This approach has improved yield but is not suitable for gene editing applications including in vivo screening and disease modeling. Recently developed second-generation all-in-one vectors have shown increased production titers and transduction efficiencies compared to first-generation systems, but they still yield ~25-fold lower yields compared to traditional vectors. The ability to simultaneously deliver Cas9 and sgRNA via a single vector enables easy and robust gene editing in vivo, which is particularly advantageous for the development of translatable gene thera...

Embodiment 3

[0218] Results - Targeted methylation of SNCA-intron 1 using the all-in-one lentiviral vector-dCas9-DNMT3A system

[0219] SNCA intron 1 contains a CpG island (CGI) region containing 23 CpGs [Chr4: 89,836,150-89,836,593 (GRCh38 / hg38)] ( Figure 1A), where the methylation status changes with increasing SNCA expression. In addition, SNCA intron 1 subregions may be differentially methylated in disease states. CpG sites in this subregion of intron 1 may serve as candidate targets for epigenetic manipulations related to the fine regulation of SNCA transcription, and enhanced DNA methylation in these CpG sites may allow the regulation of SNCA expression. Stringent downregulation and reversal of PD-associated phenotypes. To assess this hypothesis, an all-in-one gRNA-dCas9-DNMT3A lentiviral vector was constructed using a production and expression-optimized backbone containing repeats of the transcription factor Sp1 binding site upstream of the human U6 (hU6) promoter and a present ...

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Abstract

Disclosed herein are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) 9-based epigenome modifier compositions for epigenomic modification of a SNCA gene and methods of use thereof.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Patent Application No. 62 / 661,134, filed April 23, 2018, U.S. Provisional Patent Application No. 62 / 676,149, filed May 24, 2018, U.S. Provisional Patent Application No. 6, filed January 8, 2019 62 / 789,932 and the priority of U.S. Provisional Patent Application No. 62 / 824,195, filed March 26, 2019, the contents of each of which are incorporated herein by reference. [0003] Statement of Government Interest [0004] This application was made with Government support under Federal Grant No. NS085011 awarded by the National Institutes of Neurological Disorders & Stroke (NIH / NINDS). The US Government has certain rights in this invention. technical field [0005] The present disclosure relates to a clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated protein (Cas) 9-based epigenome modifier composition for epigenome modification of SNCA genes and methods ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/62C12N15/86
CPCA61K48/00C12N15/90C12N2740/16043C12N9/1007C12N15/86C12N2310/20A61K38/00C07K2319/00C12N9/22C12N15/11C12N2740/15043C12N2800/80
Inventor 奥尼·基巴-法莱克鲍里斯·坎特
Owner DUKE UNIV
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