# US 10,946,108 B2 · Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for targeting disorders and diseases using viral components

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- Number: US 10,946,108 B2
- Document ID: US10946108B2
- Status: Granted
- Kind: B2
- Application number: 14/970,967
- Filed: 2015-12-16
- Published: 2016-06-23
- Granted: 2021-03-16
- Earliest claimed: 2013-06-17
- Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology; President and Fellows of Havard College
- Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology; President and Fellows of Harvard College
- Inventors: Feng Zhang; Le Cong; Fei Ran; Matthias Heidenreich; Lukasz Swiech
- Web page: https://patentgrep.com/patent/US10946108B2

## CPC

- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- 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 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/86: Viral vectors
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## 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 CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing 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

An AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV1/2, or AAV2/8 viral vector that contains heterologous nucleic acid molecule(s) engineered for expression in a eukaryotic cell, wherein the heterologous nucleic acid molecule(s) encodes a Cas9 and a CRISPR-Cas system guide, wherein the Cas9 and the system guide are capable of forming a CRISPR-Cas complex after expression in the eukaryotic cell, wherein the system guide is capable of targeting the complex to a DNA sequence in the genome of the eukaryotic cell,
wherein the Cas9 and the system guide are encoded in a single AAV 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV1/2, or AAV2/8 viral vector, and wherein the Cas9 is Staphylococcus aureus Cas9 (SaCas9).

### Claim 2 (depends on 1)

The viral vector of claim 1, wherein one or more of the system guides comprises
(a) a guide sequence that hybridizes to the target sequence,
(b) a tracr mate sequence, and
(c) a tracr sequence,
wherein (a), (b), and (c) are arranged in a 5′ to 3′ orientation.

### Claim 3 (depends on 2)

The viral vector of claim 2, wherein expression of the guide sequence is under the control of a T7 promoter and is driven by the expression of T7 polymerase.

### Claim 4 (depends on 1)

The viral vector of claim 1, wherein the viral vector encodes two or more system guides.

### Claim 5 (depends on 1)

The viral vector of claim 1, wherein one or more of the system guides comprises a chimeric RNA (chiRNA).

### Claim 6 (depends on 1)

The viral vector of claim 1, wherein the heterologous nucleic acid molecule further encodes a homologous recombination (FIR) template.

### Claim 7 (depends on 1)

The viral vector of claim 1, wherein the Cas9 is a nuclease directing cleavage of both strands of the targeted DNA sequence.

### Claim 8 (depends on 1)

The viral vector of claim 1, wherein the Cas9 comprises one or more mutations.

### Claim 9 (depends on 8)

The viral vector of claim 8, wherein the Cas9 comprises one or more of D10A, E762A, H840A, N854A, N863A and D986A.

### Claim 10 (depends on 8)

The viral vector of claim 8, wherein the one or more mutations is in a RuvC1 domain of the Cas9.

### Claim 11 (depends on 8)

The viral vector of claim 8, wherein the Cas9 is a nickase directing cleavage of one strand of the targeted DNA sequence.

### Claim 12 (depends on 11)

The viral vector of claim 11, wherein the nickase is a double nickase.

### Claim 13 (depends on 1)

The viral vector of claim 1, wherein the nucleic acid molecule encoding the Cas9 comprises at least one or more NLS.

### Claim 14 (depends on 1)

The viral vector of claim 1, wherein the nucleic acid molecule encoding the Cas9 comprises at least two or more NLS.

### Claim 15 (depends on 1)

The viral vector of claim 1, wherein the Cas9 has one or more mutations in a catalytic domain, wherein the Cas9 further comprises a functional domain.

### Claim 16 (depends on 15)

The viral vector of claim 15, wherein the functional domain is a transcriptional activation domain.

### Claim 17 (depends on 15)

The viral vector of claim 15, wherein the functional domain is VP64.

### Claim 18 (depends on 1)

The viral vector of claim 1, wherein the targeted DNA sequence is associated with a condition or defect of a neural cell.

### Claim 19 (depends on 18)

The viral vector of claim 18, wherein the targeted DNA sequence is associated with a neurological or neuronal disease or disorder in Table 7, 8, or 9.

### Claim 20 (depends on 18)

The viral vector of claim 18, wherein the serotype of the viral vector is AAV1, AAV2, AAV4, AAV5, AAV8, AAV9 or a combination thereof.

### Claim 21 (depends on 1)

The viral vector of claim 1, wherein the targeted DNA sequence is associated with a condition or defect of a muscle cell.

### Claim 22 (depends on 21)

The viral vector of claim 21, wherein the muscle cell is a cardiac muscle cell.

### Claim 23 (depends on 21)

The viral vector of claim 21, wherein the targeted DNA sequence is associated with a muscular/skeletal disease or disorder in Table 7, 8, or 9.

### Claim 24 (depends on 21)

The viral vector of claim 21, wherein the condition is Duchenne Muscular Dystrophy (DIV)).

### Claim 25 (depends on 21)

The viral vector of claim 21, wherein the serotype of the viral vector is AAV1, AAV6, AAV7, AAV8, or AAV9.

### Claim 26 (depends on 1)

The viral vector of claim 1, wherein the targeted DNA sequence is associated with a condition or defect of a lung cell.

### Claim 27 (depends on 26)

The viral vector of claim 26, wherein the targeted DNA sequence is associated with a lung cell gene or disorder in Table 7, 8, or 9.

### Claim 28 (depends on 26)

The viral vector of claim 26, wherein the condition is cystic fibrosis or alpha-1 antitrypsin deficiency (AAD).

### Claim 29 (depends on 26)

The viral vector of claim 26, wherein the serotype of the viral vector is AAV1, AAV2, AAV5, AAV6, or AAV9.

### Claim 30 (depends on 1)

The viral vector of claim 1, wherein the targeted DNA sequence is associated with a condition or defect of a hepatic cell.

### Claim 31 (depends on 30)

The viral vector of claim 30, wherein the targeted DNA sequence is associated with a hepatic cell gene or disorder in Table 7, 8, or 9.

### Claim 32 (depends on 30)

The viral vector of claim 30, wherein the condition is a glycogen storage disease or alpha-1 antitrypsin deficiency (AAD).

### Claim 33 (depends on 30)

The viral vector of claim 30, wherein the serotype of the viral vector is AAV1, AAV5, AAV7, AAV8, AAV2/8, or AAV9.

### Claim 34 (depends on 1)

The viral vector of claim 1, wherein the targeted DNA sequence is associated with a condition or defect of a pancreatic cell.

### Claim 35 (depends on 34)

The viral vector of claim 34, wherein the targeted DNA sequence is associated with a pancreatic cell gene or disorder in Table 7, 8, or 9.

### Claim 36 (depends on 34)

The viral vector of claim 34, wherein the serotype of the viral vector is AAV6 or AAV8.

### Claim 37 (depends on 1)

The viral vector of claim 1, wherein the targeted DNA sequence is associated with a condition or defect of an epithelial cell.

### Claim 38 (depends on 37)

The viral vector of claim 37, wherein the targeted DNA sequence is associated with an epithelial gene or disorder in Table 7, 8, or 9.

### Claim 39 (depends on 37)

The viral vector of claim 37, wherein the serotype of the viral vector is AAV1, AAV2, AAV5, AAV6, or AAV9.

### Claim 40 (depends on 1)

The viral vector of claim 1, wherein the targeted DNA sequence is associated with a condition or defect of a cell of the eye.

### Claim 41 (depends on 40)

The viral vector of claim 40, wherein the targeted DNA sequence is associated with an ocular gene or disorder in Table 7, 8, or 9.

### Claim 42 (depends on 40)

The viral vector of claim 40, wherein the ocular condition is Leber's Congenital Amaurosis (LCA) or Usher Syndrome.

### Claim 43 (depends on 40)

The viral vector of claim 40, wherein the serotype of the viral vector is AAV1, AAV2, AAV4, AAV5, or AAV8.

### Claim 44 (depends on 1)

The viral vector of claim 1, wherein the targeted DNA sequence is associated with an immune-related disease or disorder.

### Claim 45 (depends on 44)

The viral vector of claim 44, wherein the targeted DNA sequence is associated with an immune-related gene or disorder in Table 7, 8, or 9.

### Claim 46 (depends on 44)

The viral vector of claim 44, wherein the immune-related disease or disorder is severe combined immunodeficiency (SCID).

### Claim 47 (depends on 1)

The viral vector of claim 1, wherein the targeted DNA sequence is associated with a blood disease or disorder.

### Claim 48 (depends on 47)

The viral vector of claim 47, wherein the targeted DNA sequence is associated with a blood gene or disorder in Table 7, 8, or 9.

### Claim 49 (depends on 47)

The viral vector of claim 47, wherein the blood disease or disorder is hemophilia, sickle cell disease, or beta-thalassemia.

### Claim 50 (depends on 1)

An isolated cell comprising a viral vector of claim 1.

### Claim 51 (depends on 50)

A non-human organism comprising a cell of claim 50.

### Claim 52 (depends on 51)

The non-human organism of claim 51, wherein the organism is an eukaryote.

### Claim 53 (depends on 51)

The non-human organism of claim 51, wherein the organism is a mammal.

### Claim 54 (depends on 50)

An isolated tissue comprising the cell of claim 50.

### Claim 55 (depends on 54)

An non-human organism comprising the tissue of claim 54.