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

• US 20140179770A1
• Filed: 12/12/2013
• Published: 06/26/2014
• Est. Priority Date: 12/12/2012
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: ZHANG, Feng, RAN, Fei
• Application Number:
  US14/104,837
• Publication Number:
  US 20140179770A1
• Time in Patent Office: Days
• US Class Current: 514/44.R
• 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 modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest comprising

delivering a non-naturally occurring or engineered composition comprising:

A)—

I. a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises;

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a polynucleotide sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,

or

(B) I. polynucleotides comprising;

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and

(b) at least one or more tracr mate sequences,

II. a polynucleotide sequence encoding a CRISPR enzyme, and

III. a polynucleotide sequence comprising a tracr sequence,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA.

2. The method of claim 1, wherein any or all of the polynucleotide sequence encoding a CRISPR enzyme, guide sequence, tracr mate sequence or tracr sequence, is/are RNA.

3. The method of claim 1, wherein the polynucleotides encoding the sequence encoding a CRISPR enzyme, the guide sequence, tracr mate sequence or tracr sequence is/are RNA and are delivered via liposomes or nanoparticles.

4. The method of claim 1 wherein the polynucleotides are comprised within a vector system comprising one or more vectors.

8. The method of claim 1, 5 or 7, wherein the method is carried out in vitro, and/or ex vivo.

9. The method of claim 1, 5 or 7 including inducing expression.

10. The method of claim 1, 5 or 7 wherein the organism or subject is a eukaryote.

11. The method of claim 10 wherein the organism or subject is a non-human eukaryote.

12. The method of claim 1, 5 or 7 wherein the organism or subject is a mammal or a non-human mammal.

14. The method of claim 1, 5 or 7 wherein the CRISPR enzyme is a Cas9.

15. The method of claim 1, 5 or 7 wherein expression of the guide sequence is under the control of the T7 promoter and is driven by the expression of T7 polymerase.

16. A method of delivering a CRISPR enzyme of claim 1, 5 or 7, comprising delivering to a cell mRNA encoding the CRISPR enzyme.

17. The method of claim 1, 5 or 7, wherein the polynucleotide or enzyme coding sequence encoding the CRISPR enzyme is delivered to the cell by delivering mRNA encoding the CRISPR enzyme to the cell.

22. The method of claim 1, 5 or 7 wherein the target sequence is flanked at its 3′

end or followed by 5′

-NRG (where N is any Nucleotide), or where the CRISPR enzyme is (or is derived from) a genus belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphyloccoccus, Nitratifractor, Mycoplasma and Campylobacer.

5. A method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest comprising

delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors operably encoding a composition for expression thereof, wherein the composition comprises:

(A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein components I and II are located on the same or different vectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

or

(B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and

(b) at least one or more tracr mate sequences,

II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and

III. a third regulatory element operably linked to a tracr sequence,

wherein components I, II and III are located on the same or different vectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence.

6. The method of claim 5, wherein one or more of the viral vectors are delivered via liposomes or nanoparticles.

13. The method of claim 5 wherein the viral vector is an AAV or lentiviral vector.

7. A method of treating or inhibiting a condition caused by a defect in a target sequence in a genomic locus of interest in a subject or a non-human subject in need thereof comprising modifying the subject or a non-human subject by manipulation of the target sequence and wherein the condition is susceptible to treatment or inhibition by manipulation of the target sequence comprising providing treatment comprising:

delivering a non-naturally occurring or engineered composition comprising an AAV or lentivirus vector system, comprising one or more AAV or lentivirus vectors operably encoding a composition for expression thereof, wherein the target sequence is manipulated by the composition when expressed, wherein the composition comprises;

(A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein components I and II are located on the same or different vectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

or

(B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and

(b) at least one or more tracr mate sequences,

II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and

III. a third regulatory element operably linked to a tracr sequence,

wherein components I, II and III are located on the same or different vectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence.

18. A method of preparing the AAV or lentivirus vector of claim 7 comprising transfecting plasmid(s) containing or consisting essentially of nucleic acid molecule(s) coding for the AAV or lentivirus into AAV-infected or lentivirus-infected cells, and supplying AAV AAV or lentivirus rep and/or cap and/or helper nucleic acid molecules obligatory for replication and packaging of the AAV or lentivirus.

19. A method of preparing an AAV or lentivirus vector for use in the method of claim 7, comprising transfecting plasmid(s) containing or consisting essentially of nucleic acid molecule(s) coding for the AAV or lentivirus into AAV-infected or lentivirus-infected cells, and supplying AAV AAV or lentivirus rep and/or cap and/or helper nucleic acid molecules obligatory for replication and packaging of the AAV or lentivirus.

20. The method of claim 18 wherein the cells are mammalian cells.

21. The method of claim 19 wherein the cells are insect cells and the helper virus (where present) is baculovirus.