# US 2021/0277370 A1 · Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders

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- Number: US 2021/0277370 A1
- Document ID: US20210277370A1
- Status: Published application
- Kind: A1
- Application number: 17/108,771
- Filed: 2020-12-01
- Published: 2021-09-09
- Earliest claimed: 2013-12-12
- Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology; University of Iowa Research Foundation
- Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology; University of Iowa Research Foundation
- Inventors: Beverly Davidson; Chie-Yu Lin; Edgardo Rodriguez; Feng Zhang
- Web page: https://patentgrep.com/patent/US20210277370A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- A01K 67/0276: Knock-out vertebrates
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 21/02: Muscle relaxants, e.g. for tetanus or cramps
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- 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
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- A01K 2217/075: inducing loss of function, i.e. knock out
- A01K 2227/105: Murine
- A01K 2267/0318: Animal model for neurodegenerative disease, e.g. non- Alzheimer's
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences especially for use as to nucleotide repeat disorders. Provided are delivery systems and tissues or organ which are targeted as sites for delivery especially for use as to nucleotide repeat disorders. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex or system especially for use as to nucleotide repeat disorders, as well as methods for the design and of such. Also provided are methods of directing CRISPR complex or system formation in eukaryotic cells especially for use as to nucleotide repeat disorders including with consideration of specificity for target recognition and avoidance of toxicity and editing or modifying a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

## Claims

### Claim at position 1

1-18. (canceled)

### Claim 19

A method of in vivo genome editing in a multicellular organism, comprising delivering a CRISPR-Cas system to at least one eukaryotic cell in the multicellular organism, wherein the CRISPR-Cas system comprises a Cas9 protein and a first and a second CRISPR-Cas system guides, or one or more vectors encoding the Cas9 protein and the first and second CRISPR-Cas system guides; wherein the Cas9 is linked to at least one nuclear localization signal (NLS); wherein the first and second CRISPR-Cas system guides are engineered to target the Cas9 protein to a first and a second genomic loci in nucleus of the eukaryotic cell that together flank a defective nucleotide element, repeat or expansion; and wherein the Cas9 protein excises the defective nucleotide element, repeat or expansion.

### Claim 20 (depends on 19)

The method of claim 19, wherein the CRISPR-Cas system comprises one or more viral vectors encoding the Cas9 protein and the first and second CRISPR-Cas system guides.

### Claim 21 (depends on 20)

The method of claim 20, wherein the viral vectors are adeno-associate viral (AAV) vectors.

### Claim 22 (depends on 21)

The method of claim 21, wherein the Cas9 protein and the first and second CRISPR-Cas system guides are encoded on the same vector.

### Claim 23 (depends on 21)

The method of claim 21, wherein the Cas9 protein is encoded on a first vector, and the first and second CRISPR-Cas system guides are encoded on a second vector.

### Claim 24 (depends on 19)

The method of claim 19, wherein the one or more vectors further comprise a repair template for homology-directed repair.

### Claim 25 (depends on 19)

The method of claim 19, wherein the first and second CRISPR-Cas system guides are each a chimeric RNA (chiRNA) comprising (a) a guide sequence that hybridizes to a target sequence at the genomic locus, (b) a tracr mate sequence, and (c) a tracr sequence, wherein (a), (b), and (c) are arranged in a 5′ to 3′ orientation.

### Claim 26 (depends on 19)

The method of claim 19, wherein the Cas9 is a Cas9 ortholog of a genus selected from the group consisting of Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Streptococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

### Claim 27 (depends on 19)

The method of claim 19, wherein the Cas9 is S. pyogenes Cas9.

### Claim 28 (depends on 19)

The method of claim 19, wherein the Cas9 is S. aureus Cas9.

### Claim 29 (depends on 19)

The method of claim 19, wherein the Cas9 is linked to at least two NLSs.

### Claim 30 (depends on 19)

The method of claim 19, wherein the Cas9 comprises one or more mutations in a catalytic domain and is fused to a heterologous protein domain.

### Claim 31 (depends on 19)

The method of claim 19, wherein the multicellular organism is a mammalian organism, and the eukaryotic cell is a brain cell, a neuronal cell, or a central nervous tissue cell.

### Claim 32 (depends on 19)

The method of claim 19, wherein the defective nucleotide element, repeat or expansion is selected from the group consisting of: a trinucleotide repeat comprising CTG, CAG, CGG, CCG, GAA, or TTC; a tetranucleotide repeat comprising CCTG, a pentanucleotide repeat comprising ATTCT or AGAAT; a hexanucleotide repeat comprising GGGGCC; and a dodecanucleotide repeat comprising CCCCGCCCCGCG (SEQ ID NO:1) or CGCGGGGCGGGG (SEQ ID NO:2).

### Claim 32 (depends on 19)

The method of claim 19, wherein the defective nucleotide element, repeat or expansion is a trinucleotide repeat comprising CAG or CTG.

### Claim 33 (depends on 19)

The method of claim 19, wherein the defective nucleotide element, repeat or expansion is in the coding sequence of HTT gene.

### Claim 34 (depends on 19)

The method of claim 19, wherein the defective nucleotide element, repeat or expansion is associated with a brain or central nervous system disease or disorder selected from the group consisting of: a Fragile X (FXS); Spinocerebellar ataxia type-12 (SCA12); Friedreich Ataxia; Myotonic Dystrophy type-1 (DM1); Spinocerebellar ataxia type-8 (SCAB); Spinocerebellar ataxia type-10 (SCA10); Spinocerebellar ataxia type-31 (SCA31); Spinocerebellar ataxia type-1 (SCA1); Spinocerebellar ataxia type-2 (SCA2); Spinocerebellar ataxia type-3 (SCA3); Spinocerebellar ataxia type-6 (SCA6); Spinocerebellar ataxia type-7 (SCAT); Spinocerebellar ataxia type-17 (SCA17); Huntington's Disease (HD); Fragile X Tremor Ataxia (FXTAS); Unverricht-Lundborg disease (EPM1); Amyotrophic Lateral Sclerosis (ALS); Fronto Temporal Dementia (FTD); Myotonic Dystrophy type-2 (DM2); Oculopharyngeal muscular dystrophy (OPMD); Dentatorubral-pallidoluysian atrophy (DRPLA); Spinobulbar muscular atrophy (SBMA); and Huntington's disease like type-2 (HDL2).

### Claim 35 (depends on 19)

The method of claim 19, wherein excision of the defective nucleotide element, repeat or expansion produces a phenotypic change in the multicellular organism.

### Claim 36 (depends on 19)

The method of claim 19, wherein the CRISPR-Cas system is delivered via injection.

### Claim 37 (depends on 19)

The method of claim 19, wherein the CRISPR-Cas system is delivered via a liposome, a nanoparticle, an exosome, or a microvesicle.

### Claim 38 (depends on 19)

The method of claim 19, further comprising delivering to the multicellular organism a third CRISPR-Cas system guide capable of hybridizing to a nucleotide sequence encoding the Cas9 protein.