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

• US 20170107536A1
• Filed: 11/11/2016
• Published: 04/20/2017
• Est. Priority Date: 12/12/2012
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Zhang, Feng, Platt, Randall Jeffrey, Feng, Guoping, Zhou, Yang
• Granted Patent: US 11,041,173 B2
• 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 vector containing nucleic acid(s) encoding inducible Cas9 and at least one nuclear localization signal (NLS), whereby when the vector is delivered to a eukaryotic cell, when induced, Cas9 is expressed in vivo in the eukaryotic cell.

2. The vector of claim 1 wherein the nucleic acid encoding the Cas9 encodes two NLSs, whereby a first NLS is situated at or within 50 amino acids of the amino terminal of Cas9 and a second NLS is situated at or within 50 amino acids of the carboxy terminal of Cas9.

3. The vector of claim 1 wherein the inducible Cas9 is a Cre recombinase dependent Cas9.

4. The vector of claim 3 wherein the vector comprises a nucleic acid encoding a stop cassette element, wherein the stop cassette element is loxP-SV40 polyAx3-loxP.

5. The vector of claim 4 wherein the nucleic acid encoding the stop element is between a nucleic acid encoding a promoter and the Cas9.

6. The vector of claim 1 wherein the Cas9 is a Streptococcus pyogenes Cas9.

7. The vector of claim 1 further comprising a nucleic acid encoding a peptide cleavage sequence.

8. The vector of claim 1 further comprising a nucleic acid encoding a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

9. The vector of claim 1 further comprising a nucleic acid encoding a bovine growth hormone poly-A signal sequence (bGHpolyA).

10. The vector of claim 1 further comprising a nucleic acid encoding a peptide cleavage sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a bovine growth hormone poly-A signal sequence (bGHpolyA).

11. A cell comprising the vector of claim 1, wherein the cell gives rise to a Cas9 knock in transgenic non-human animal.

12. The cell of claim 11 wherein the cell is a mouse cell, the transgenic non-human animal is a mouse, and the vector includes nucleic acid(s) to target insertion of the nucleic acid(s) encoding inducible Cas9 and at least one nuclear localization signal (NLS) to a Rosa26 locus.

13. A cell comprising the vector of claim 4, wherein the cell gives rise to a Cas9 knock in transgenic non-human animal.

14. A method of making a transgenic non-human animal expressing Cas9 comprising crossing the animal from the cell of claim 13 with an animal that ubiquitously expresses Cre.

15. A transgenic animal expressing Cas9 produced by the method of claim 14.

16. The cell of claim 11 wherein the transgenic animal is a transgenic mammal.

17. The cell of claim 13 wherein the transgenic animal is a transgenic mammal.

18. The animal of claim 14 wherein the transgenic animal is a transgenic mammal.

19. The animal of claim 16 wherein the transgenic mammal is a transgenic mouse, rat, or rabbit.

20. The animal of claim 19 wherein the mammal is a transgenic mouse.

21. A cell comprising the vector of claim 1, wherein the cell gives rise to a Cas9 knock in transgenic plant.

22. A Cas9 knock in non-human eukaryote.

23. The Cas9 knock in transgenic non-human eukaryote of claim 22 wherein the eukaryote is a Cas9 knock in transgenic non-human animal or a Cas9 knock in transgenic plant.

24. The Cas9 knock in transgenic non-human eukaryote of claim 23 wherein the eukaryote is an animal.

25. The Cas9 knock in transgenic non-human eukaryote of claim 24 wherein the animal is a transgenic non-human mammal.

26. The Cas9 knock in transgenic non-human eukaryote of claim 25 wherein the mammal is a mouse, rat or rabbit.

27. A method for delivering RNA(s) to guide Cas9 of the mammal of claim 22 comprising delivering to cell(s) of the mammal vector(s) containing and/or expressing in vivo the RNA(s) to guide the Cas9, or nanoparticle(s) that carry(ies) the RNA(s) or the vector(s).

28. The method of claim 27 wherein the delivery is by vector or particle.

29. The method of claim 28 wherein vector is a viral vector.

30. The method of claim 28 wherein vector is AAV, Adenovirus or Lentivirus.