# US 11,306,309 B2 · Chemically modified guide RNAs for CRISPR/CAS-mediated gene regulation

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- Number: US 11,306,309 B2
- Document ID: US11306309B2
- Status: Granted
- Kind: B2
- Application number: 15/724,073
- Filed: 2017-10-03
- Published: 2018-05-03
- Granted: 2022-04-19
- Earliest claimed: 2015-04-06
- Assignees: The Board of Trustees of the Leland Stanford Junior University; Agilent Technologies, Inc.
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Matthew H. Porteus; Ayal Hendel; Joe Clark; Rasmus O. Bak; Daniel E. Ryan; Douglas J. Dellinger; Robert Kaiser; Joel Myerson
- Web page: https://patentgrep.com/patent/US11306309B2

## CPC

- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- 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}
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 7/06: Antianaemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 13/12: of the kidneys
- A61P 19/00: Drugs for skeletal disorders
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/18: Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 25/34: Tobacco-abuse
- A61P 25/36: Opioid-abuse
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- A61P 37/04: Immunostimulants
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/907: {in mammalian cells}
- C12N 2310/10: Type of nucleic acid
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2320/51: modulating the chemical stability, e.g. nuclease-resistance
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are methods for inducing CRISPR/Cas-based gene regulation (e.g., genome editing or gene expression) of a target nucleic acid (e.g., target DNA or target RNA) in a cell. The methods include using modified single guide RNAs (sgRNAs) that enhance gene regulation of the target nucleic acid in a primary cell for use in ex vivo therapy or in a cell in a subject for use in in vivo therapy. Additionally, provided herein are methods for preventing or treating a genetic disease in a subject by administering a sufficient amount of a modified sgRNA to correct a mutation in a target gene associated with the genetic disease.

## Claims

### Claim 1

A method for inducing gene regulation of a target nucleic acid in a primary cell, the method comprising:
introducing into the primary cell:
(a) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to the target nucleic acid and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein:
(i) about 10% to about 30% of nucleotides in the first nucleotide sequence and/or about 1% to about 10% of nucleotides in the second nucleotide sequence are modified nucleotides;
(ii) the modified sgRNA comprises a modified nucleotide within five nucleotides from the 5′ end of the first nucleotide sequence and/or within five nucleotides from the 3′ end of the second nucleotide sequence; and
(iii) the modified nucleotides are selected from the group consisting of a 2′-O-methyl 3′-phosphorothioate (MS) nucleotide, a 2′-O-methyl 3′-thioPACE (MSP) nucleotide, and a combination thereof; and
(b) a Cas polypeptide, an mRNA encoding the Cas polypeptide, or a recombinant expression vector comprising a nucleotide sequence encoding the Cas polypeptide,
wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid, and wherein the modified sgRNA induces a gene regulation of the target nucleic acid with an enhanced activity relative to a corresponding unmodified sgRNA.

### Claim 2 (depends on 1)

The method of claim 1, wherein the enhanced activity comprises increased stability of the modified sgRNA and/or increased specificity of the modified sgRNA for the target nucleic acid.

### Claim 3 (depends on 1)

The method of claim 1, wherein the target nucleic acid comprises a target DNA or a target RNA.

### Claim 4 (depends on 3)

The method of claim 3, wherein the gene regulation comprises genome editing of the target DNA.

### Claim 5 (depends on 4)

The method of claim 4, wherein the genome editing comprises homologous-directed repair (HDR) or nonhomologous end joining (NHEJ) of the target DNA.

### Claim 6 (depends on 4)

The method of claim 4, further comprising introducing a recombinant donor repair template into the primary cell.

### Claim 7 (depends on 6)

The method of claim 6, wherein the recombinant donor repair template comprises two nucleotide sequences comprising two non-overlapping, homologous portions of the target DNA, wherein one of the two homologous portions is located at the 5′ end and the other homologous portion is located at the 3′ end of the recombinant donor template, wherein each homologous portion is homologous to a corresponding region of the target DNA to undergo genome editing.

### Claim 8 (depends on 6)

The method of claim 6, wherein the recombinant donor repair template comprises a synthetic single-stranded oligodeoxynucleotide (ssODN) template comprising a nucleotide sequence encoding a mutation to correct a single nucleotide polymorphism (SNP) and two nucleotide sequences comprising two non-overlapping, homologous portions that are homologous to corresponding regions of the target DNA, wherein one of the two homologous portions is located at the 5′ end and the other homologous portion is located at the 3′ end of the recombinant donor template.

### Claim 9 (depends on 3)

The method of claim 3, wherein the gene regulation comprises inhibition or activation of gene expression of the target DNA or the target RNA using a Cas polypeptide that is endonuclease-deficient.

### Claim 10 (depends on 1)

The method of claim 1, wherein the primary cell is isolated from a multicellular organism prior to introducing the modified sgRNA and the Cas polypeptide into the primary cell.

### Claim 11 (depends on 10)

The method of claim 10, wherein the multicellular organism is a plant, a multicellular protist, a multicellular fungus, or an animal.

### Claim 12 (depends on 1)

The method of claim 1, wherein the primary cell is a stem cell or an immune cell.

### Claim 13 (depends on 12)

The method of claim 12, wherein the stem cell is a hematopoietic stem and progenitor cell (HSPC), a mesenchymal stem cell, a neural stem cell, or an organ stem cell.

### Claim 14 (depends on 12)

The method of claim 12, wherein the immune cell is a T cell, a natural killer cell, a monocyte, a peripheral blood mononuclear cell (PBMC), or a peripheral blood lymphocyte (PBL).

### Claim 15 (depends on 10)

The method of claim 10, wherein the primary cell or a progeny thereof is returned to the multicellular organism after introducing the modified sgRNA and the Cas polypeptide into the primary cell.

### Claim 16 (depends on 1)

The method of claim 1, comprising introducing (a) and (b) to a population of primary cells each comprising the target nucleic acid.

### Claim 17 (depends on 16)

The method of claim 16, wherein the modified sgRNA induces the gene regulation of the target nucleic acid in at least about 30% of the population of primary cells.

### Claim 18 (depends on 16)

The method of claim 16, wherein the modified sgRNA induces the gene regulation of the target nucleic acid in at least about 40% of the population of primary cells.

### Claim 19 (depends on 16)

The method of claim 16, wherein the modified sgRNA induces the gene regulation of the target nucleic acid in at least about 50% of the population of primary cells.

### Claim 20 (depends on 16)

The method of claim 16, wherein the modified sgRNA induces the gene regulation of the target nucleic acid in at least about 60% of the population of primary cells.

### Claim 21 (depends on 1)

The method of claim 1, wherein the first nucleotide sequence is about 20 nucleotides in length.

### Claim 22 (depends on 1)

The method of claim 1, wherein at least two nucleotides in the first nucleotide sequence are modified nucleotides.

### Claim 23 (depends on 1)

The method of claim 1, wherein one or more modified nucleotides are located within five nucleotides from the 5′-end of the first nucleotide sequence.

### Claim 24 (depends on 1)

The method of claim 1, wherein from about 10% to about 30% of the nucleotides in the first nucleotide sequence are modified nucleotides.

### Claim 25 (depends on 1)

The method of claim 1, wherein the second nucleotide sequence is about 80 nucleotides in length.

### Claim 26 (depends on 1)

The method of claim 1, wherein at least two nucleotides in the second nucleotide sequence are modified nucleotides.

### Claim 27 (depends on 1)

The method of claim 1, wherein one or more modified nucleotides are located within five nucleotides from the 3′-end of the second nucleotide sequence.

### Claim 28 (depends on 1)

The method of claim 1, wherein from about 1% to about 10% of the nucleotides in the second nucleotide sequence are modified nucleotides.

### Claim 29 (depends on 1)

The method of claim 1, wherein the modified sgRNA comprises one, two, or three consecutive or non-consecutive modified nucleotides at or near the 5′-end of the first nucleotide sequence and one, two, or three consecutive or non-consecutive modified nucleotides at or near the 3′-end of the second nucleotide sequence.

### Claim 30 (depends on 29)

The method of claim 29, wherein the modified sgRNA comprises three consecutive modified nucleotides at the 5′-end of the first nucleotide sequence and three consecutive modified nucleotides at the 3′-end of the second nucleotide sequence.

### Claim 31 (depends on 1)

The method of claim 1, wherein the modified sgRNA is chemically synthesized.

### Claim 32 (depends on 1)

The method of claim 1, further comprising introducing into the primary cell a second modified single guide RNA (sgRNA), wherein the second modified sgRNA guides the Cas polypeptide to a second target nucleic acid that is different from the target nucleic acid.

### Claim 33 (depends on 1)

The method of claim 1, comprising introducing into the primary cell the mRNA encoding the Cas polypeptide.

### Claim 34 (depends on 1)

The method of claim 1, wherein the Cas polypeptide is a Cas9 polypeptide, a variant thereof, or a fragment thereof.

### Claim 35 (depends on 1)

The method of claim 1, wherein the step of introducing into the primary cell comprises electroporating the primary cell.

### Claim 36 (depends on 1)

The method of claim 1,
wherein the modified sgRNA in (a) and the Cas polypeptide in (b) are introduced into the primary cell in a ribonucleoprotein (RNP) complex.

### Claim 37 (depends on 1)

The method of claim 1, wherein the gene regulation induced by the introduction of (a) and (b) is stable in the primary cell for at least 24 hours.