# US 10,767,174 B2 · Method for RGEN RNP delivery using 5′-phosphate removed RNA

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- Number: US 10,767,174 B2
- Document ID: US10767174B2
- Status: Granted
- Kind: B2
- Application number: 15/347,871
- Filed: 2016-11-10
- Published: 2017-11-02
- Granted: 2020-09-08
- Earliest claimed: 2015-11-13
- Assignees: Institute for Basic Science
- Applicants: Institute for Basic Science
- Inventors: Jin-Soo Kim; So Jung Kim
- Web page: https://patentgrep.com/patent/US10767174B2

## CPC

- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/224: {Class 1 CAS enzyme complex, e.g. multi-enzyme}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/1024: {In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/31: of the backbone
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2320/53: reducing unwanted side-effects
- C12N 2330/30: chemically synthesised
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Provided are a composition for ribonucleoprotein delivery, comprising a guide RNA free of 5′-terminal phosphates, and a method for ribonucleoprotein delivery, using the same.

## Claims

### Claim 1

A method of decreasing expression of a gene involved in a Type I interferon response following delivery of an RNA-guided endonuclease ribonucleoprotein into a cell wherein the cell comprises the gene involved in the Type I interferon response, the method comprising
preparing a guide RNA having a 5′ end free of both a diphosphate residue and a triphosphate residue by in vitro transcription using a prokaryote RNA polymerase followed by removing the diphosphate and the triphosphate residues of the guide RNA,
forming the RNA-guided endonuclease ribonucleoprotein by combining a Cpf1 protein with the guide RNA having the 5′ end free of both the diphosphate residue and the triphosphate residue, administering the RNA-guided endonuclease ribonucleoprotein directly to the cell, and
wherein the gene involved in the Type I interferon response comprises a gene selected from the group consisting of IFN-β (Interferon-beta), RIG-1 (retinoic acid-inducible gene 1), and OAS2 (2′-5′-oligoadenylate synthetase 2) genes, and the expression level of the gene involved in the type I interferon response is decreased compared to a control in which a RNA-guided endonuclease ribonucleoprotein comprising a Cpf1 protein and a guide RNA having the diphosphate residue or the triphosphate residue at the 5′ end is administered.

### Claim 2 (depends on 1)

The method of claim 1, wherein the Cpf1 protein is obtained from Parcubacteria bacterium, Peregrinibacteria bacterium, Acidaminococcus sp., Porphyromonas macacae, Lachnospiraceae bacterium, Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi, Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida, Candidatus methanoplasma termitum, or Eubacterium eligens.

### Claim 3 (depends on 1)

The method of claim 1, wherein the prokaryotic RNA polymerase is a bacteriophage RNA polymerase.

### Claim 4 (depends on 3)

The method of claim 3, wherein the bacteriophage RNA polymerase is at least one selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.

### Claim 5

A method of decreasing expression of a gene involved in a Type I interferon response following delivery of an RNA-guided endonuclease ribonucleoprotein into a cell wherein the cell comprises the gene involved in the Type I interferon response, the method comprising
preparing a guide RNA by in vitro transcription using a prokaryote RNA polymerase followed by removing the diphosphate and the triphosphate residues of the guide RNA,
forming the RNA-guided endonuclease ribonucleoprotein by combining a Cas9 protein with the guide RNA having the 5′ end free of both the diphosphate residue and the triphosphate residue, administering the RNA-guided endonuclease ribonucleoprotein directly to the cell,
wherein the guide RNA comprises CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), and wherein the gene involved in the Type I interferon response is comprises a gene selected from the group consisting of IFN-β, RIG 1, and OAS2 genes, and the expression level of the gene involved in the type I interferon response is decreased compared to a control in which a RNA-guided endonuclease ribonucleoprotein comprising a Cas9 protein and a guide RNA having the diphosphate residue or the triphosphate residue at the 5′ end is administered.

### Claim 6 (depends on 5)

The method of claim 5, wherein the prokaryotic RNA polymerase is a bacteriophage RNA polymerase.