DELIVERY, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION AND THERAPEUTIC APPLICATIONS

• US 20170152528A1
• Filed: 02/10/2017
• Published: 06/01/2017
• Est. Priority Date: 12/12/2012
• Status: Active Application

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: ZHANG, Feng
• Application Number:
  US15/430,260
• Publication Number:
  US 20170152528A1
• Time in Patent Office: Days
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/07 Animals genetically altered by homologous recombination
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: 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
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 : Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 : Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/06 : Antihyperlipidemics
• CPC Class Codes: A61P 31/12 : Antivirals
• CPC Class Codes: A61P 31/14 : for RNA viruses
• CPC Class Codes: A61P 31/18 : for HIV
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 15/01 : Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/102 : Mutagenizing nucleic acids
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/63 : Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/8213 : Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/85 : for animal cells
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/86 : Viral vectors
• CPC Class Codes: C12N 15/90 : Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 : in mammalian cells
• CPC Class Codes: C12N 2310/10 : Type of nucleic acid
• CPC Class Codes: C12N 2310/20 : involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2320/11 : for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/30 : Special therapeutic applications
• CPC Class Codes: C12N 2750/14143 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/22 : Vectors comprising a coding region that has been codon optimised for expression in a respective host
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 : Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: G16B 20/00 : ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 : Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 : Detection of binding sites or motifs
• CPC Class Codes: G16B 20/50 : Mutagenesis
• CPC Class Codes: G16B 30/00 : ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 : Sequence alignment; Homology search

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

1. A method of altering expression of at least one gene product comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding the gene product an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:

a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and

b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,

wherein components (a) and (b) are located on same or different vectors of the system,

whereby the guide RNA targets the target sequence and the Cas9 protein generates at least one site specific break in the DNA molecule, wherein the at least one site specific break is repaired through a cellular repair mechanism; and

, wherein the Cas9 protein and the guide RNA do not naturally occur together.

2. The method of claim 1, wherein the cellular repair mechanism is Non-homologous end joining (NHEJ).

3. The method of claim 1, wherein the cellular repair mechanism is Homology-directed repair (HDR).

4. The method of claim 1, wherein the Cas9 protein is a nickase that generates a site specific break in only one strand of the DNA molecule.

5. The method of claim 1, wherein the method further comprises the insertion of a recombination template into the site specific break in the DNA molecule.

6. The method of claim 1, wherein the expression of two or more gene products is altered.

7. The method of claim 1, wherein the CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)).

8. The method of claim 1, wherein the CRISPR-Cas system comprises a trans-activating cr (tracr) sequence.

9. The method of claim 1, wherein the guide RNAs comprise a guide sequence fused to a tracr sequence.

10. The method of claim 1, wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.

11. The method of claim 1, wherein the eukaryotic cell is a mammalian or human cell.

12. The method of claim 1, wherein the expression of one or more gene products is increased.

13. The method of claim 1, wherein the expression of one or more gene products is decreased.

14. The method of claim 1, wherein the one or more vectors are viral vectors.

15. The method of claim 1, wherein the one or more viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

16. A CRISPR-Cas system-mediated genome editing method comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding at least one gene product an engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:

a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and

b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,

wherein components (a) and (b) are located on same or different vectors of the system,

whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system, whereby the Cas9 protein generates at least one site specific break in the DNA molecule, whereby there is genome editing, and, wherein the Cas9 protein and the guide RNA do not naturally occur together.

17. The method of claim 16, wherein the at least one site specific break is repaired through a NHEJ cellular repair mechanism.

18. The method of claim 16, wherein the at least one site specific break is repaired through a HDR cellular repair mechanism.

19. The method of claim 16, wherein the Cas9 protein is a nickase that generates a site specific break in only one strand of the DNA molecule.

20. The method of claim 16, wherein the method further comprises the insertion of a recombination template into the site specific break in the DNA molecule.

21. The method of claim 16, wherein the expression of two or more gene products is altered.

22. The method of claim 16, wherein the CRISPR-Cas system further comprises one or more NLS(s).

23. The method of claim 16, wherein the CRISPR-Cas system comprises a tracr sequence.

24. The method of claim 16, wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.

25. The method of claim 16, wherein the eukaryotic cell is a mammalian or human cell.

26. The method of claim 16, wherein the expression of one or more gene products is increased.

27. The method of claim 16, wherein the expression of one or more gene products is decreased.

28. An engineered, programmable, non-naturally occurring Type II CRISPR-Cas system comprising a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a eukaryotic cell, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product and the Cas9 protein generates at least one site specific break in the DNA molecule, wherein the at least one site specific break is repaired through a cellular repair mechanism;

and, wherein the Cas9 protein and the guide RNA do not naturally occur together.

29. The CRISPR-Cas system of claim 28, wherein the cellular repair mechanism is NHEJ.

30. The CRISPR-Cas system of claim 28, wherein the cellular repair mechanism is HDR.