# US 2016/0354487 A1 · Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders

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- Number: US 2016/0354487 A1
- Document ID: US20160354487A1
- Status: Published application
- Kind: A1
- Application number: 15/179,941
- Filed: 2016-06-10
- Published: 2016-12-08
- 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: Feng Zhang; Beverly Davidson; Chie-yu Lin; Edgardo Rodriguez
- Web page: https://patentgrep.com/patent/US20160354487A1

## 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. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a SIN CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing SIN CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

## Claims

### Claim 1

A non-naturally occurring or engineered self-inactivating CRISPR-Cas composition comprising:
I. a first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises:
(a) at least one first guide sequence capable of hybridizing to a target DNA,
(b) at least one tracr mate sequence, and
(c) at least one tracr sequence, and
wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,
II. a second regulatory element operably linked to a polynucleotide sequence encoding a CRISPR enzyme,
wherein parts I and II comprise a first CRISPR-Cas system, and wherein,
III. the composition further comprises
(a) at least one second guide sequence capable of hybridizing to a sequence in or of the CRISPR-Cas system,
(b) at least one tracr mate sequence, and
(c) at least one tracr sequence, and
wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, wherein parts I and III comprise a second CRISPR-Cas system,
and said composition when transcribed comprises
a first CRISPR complex comprising the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized or is hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
a second CRISPR complex comprising a CRISPR enzyme complexed with (1) the second guide sequence that is hybridized or hybridizable to a sequence of a polynucleotide comprising or encoding the CRISPR-Cas system, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
wherein the first guide sequence directs sequence-specific binding of a first CRISPR complex to the target DNA, and
wherein the second guide sequence directs sequence-specific binding of a second CRISPR complex to a sequence comprising a polynucleotide comprising or encoding a component of the CRISPR-Cas system and whereby there is diminished activity of the first CRISPR-Cas system over a period of time, and the CRISPR-Cas composition is self-inactivating (“SIN CRISPR-Cas composition”).

### Claim 2 (depends on 1)

The composition of claim 1, wherein the target DNA sequence is within a cell.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the cell is a eukaryotic cell.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the first CRISPR-Cas system and/or the second CRISPR-Cas system is codon optimized.

### Claim 5 (depends on 4)

The composition of claim 4, wherein part II includes coding for one or more nuclear localization signals (NLSs).

### Claim 6 (depends on 5)

The composition of claim 5, wherein the CRISPR enzyme comprises a C-terminal NLS and an N-terminal NLS.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the RNA of part I is chimeric RNA (chiRNA).

### Claim 8 (depends on 1)

The composition of claim 1, wherein part I is encoded by a first viral vector and part II is encoded by a second viral vector.

### Claim 9 (depends on 8)

The composition of claim 8, wherein the first and second viral vectors are lentiviral vectors or recombinant AAV.

### Claim 10 (depends on 9)

The composition of claim 9, wherein the recombinant AAV genome comprises inverted terminal repeats (iTRs).

### Claim 11 (depends on 10)

The composition of claim 10, wherein expression of the CRISPR enzyme is driven by the inverted terminal repeat (iTR) in the AAV genome.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the first regulatory element is a RNA polymerase type III promoter and the second regulatory element is a RNA polymerase type III promoter.

### Claim 13 (depends on 1)

The composition of claim 1, wherein the first regulatory element is a U6 promoter or a H1 promoter.

### Claim 14 (depends on 1)

The composition of claim 1, wherein the second regulatory element is a ubiquitous expression promoter or a cell-type specific promoter.

### Claim 15 (depends on 1)

The composition of claim 1, wherein there is a selection marker comprising a FLAG-tag.

### Claim 16 (depends on 1)

The composition of claim 1, which is delivered via injection.

### Claim 17 (depends on 1)

The composition of claim 1, wherein the composition or a part thereof is delivered via a liposome, a nanoparticle, an exosome, or a microvesicle.

### Claim 18 (depends on 2)

The composition of claim 2, wherein the first guide sequence directs sequence-specific binding of the first CRISPR complex to the target DNA sequence and alters expression of a genomic locus in the cell.

### Claim 19 (depends on 2)

The composition of claim 2, wherein the first CRISPR complex mediates binding to or a double or single stranded DNA break, thereby editing a genomic locus in the cell.

### Claim 20 (depends on 1)

The composition of claim 1, wherein the first and/or second CRISPR-Cas system is a multiplexed CRISPR enzyme system further comprising multiple chimeras and/or multiple multiguide sequences and a single tracr sequence.

### Claim 21 (depends on 1)

The composition of claim 1, wherein the first CRISPR-Cas system is a multiplexed CRISPR enzyme system to minimize off-target activity.

### Claim 22 (depends on 1)

The composition of claim 1, wherein the CRISPR enzyme is a nickase.

### Claim 23 (depends on 1)

The composition of claim 1, wherein the CRISPR enzyme comprises one or more mutations.

### Claim 24 (depends on 23)

The composition of claim 23, wherein the CRISPR enzyme comprises one or more mutations selected from D10A, E762A, H840A, N854A, N863A or D986A.

### Claim 25 (depends on 23)

The composition according to claim 23, wherein the one or more mutations is in a RuvC1 domain of the CRISPR enzyme.

### Claim 26 (depends on 1)

The composition of claim 1, wherein the CRISPR enzyme further comprises a functional domain.

### Claim 27 (depends on 1)

The composition of claim 1, wherein the CRISPR enzyme is a Cas9.

### Claim 28 (depends on 1)

The composition of claim 1, wherein the second complex binds to a sequence for CRISPR enzyme expression.

### Claim 29 (depends on 1)

The composition of claim 1, wherein the second guide sequence is capable of hybridizing to (a) a sequence encoding the RNA or (b) a sequence encoding the CRISPR enzyme, or (c) a non-coding sequence comprising
i) a sequence within a regulatory element driving expression of non-coding RNA elements,
ii) a sequence within a regulatory element driving expression of the CRISPR enzyme,
iii) a sequence within 100 by of the ATG translational start codon of the CRISPR enzyme coding sequence, and
iv) a sequence within an inverted terminal repeat of a viral vector.

### Claim 30 (depends on 1)

The composition of claim 1, wherein the second guide sequence is expressed singularly to achieve inactivation of the first CRISPR-Cas system.

### Claim 31 (depends on 30)

The composition of claim 30, wherein the second CRISPR complex induces a frame shift in CRISPR enzyme coding sequence causing a loss of protein expression.

### Claim 32 (depends on 30)

The composition of claim 30, wherein the second guide sequence targets an iTR, wherein expression will result in the excision of an entire CRISPR-Cas cassette.

### Claim 33 (depends on 28)

The composition of claim 28, wherein the second guide sequence is expressed in an array format to achieve inactivation of the first CRISPR-Cas9 system.

### Claim 34 (depends on 28)

The composition of claim 28, wherein the second guide sequences is expressed in array format and targets both regulatory elements, thereby excising intervening nucleotides from within the first CRISPR-Cas system, effectively leading to its inactivation.

### Claim 35 (depends on 34)

The composition of claim 34, wherein expression of the second guide sequences are driven by a U6 promoter.

### Claim 36 (depends on 1)

The composition of claim 1, whereby the self-inactivation of the first CRISPR-Cas system limits duration of its activity and/or expression in targeted cells.

### Claim 37 (depends on 36)

The composition of claim 36, wherein transient expression of the CRISPR enzyme is normally lost within 48 hours.

### Claim 38 (depends on 1)

A method of treating or inhibiting a condition in a cell or tissue having a nucleotide element or trinucleotide repeat or other nucleic acid repeat element that gives rise to an adverse or disease condition caused by a defect in a genomic locus of interest in a cell in a subject or a non-human subject in need thereof comprising modifying the subject or a non-human subject by editing the genomic locus and wherein the condition is susceptible to treatment or inhibition by editing the genomic locus comprising providing treatment comprising: delivering the non-naturally occurring or engineered composition of claim 1.

### Claim 39 (depends on 38)

The method of claim 38, wherein part III is introduced into the cell sequentially or at a time point after the introduction of parts I and II.

### Claim 40

A non-naturally occurring or engineered RNA that is a first CRISPR-Cas system or first CRISPR-Cas complex guide sequence capable of hybridizing to an RNA sequence of a second CRISPR-Cas system or a nucleic acid molecule for expression of a component of the second CRISPR-Cas complex, to diminish or eliminate functional expression of the second system or complex, whereby the first and/or second system or complex is Self-Inactivating.