ONCOGENIC MODELS BASED ON DELIVERY AND USE OF THE CRISPR-CAS SYSTEMS, VECTORS AND COMPOSITIONS

• US 20160168594A1
• Filed: 12/16/2015
• Published: 06/16/2016
• Est. Priority Date: 06/17/2013
• Status: Abandoned Application

• Current Assignee: Brigham and Women's Hospital Incorporated, Massachusetts Institute of Technology
• Original Assignee: Brigham and Women's Hospital Incorporated, Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Heckl, Dirk, Zhang, Feng, Ebert, Benjamin Levine
• Application Number:
  US14/971,459
• Publication Number:
  US 20160168594A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01K 2207/05 Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
• CPC Class Codes: A01K 2207/12 Animals modified by administration of exogenous cells
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/077 heterozygous knock out animals displaying phenotype
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0312 Animal model for Alzheimer's disease
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A01K 2267/0362 Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

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 enable genome engineering in an organism to recapitulate the genetic complexity of disease or a condition and further interrogate gene function.

Claims

1. A method of inducing a proliferative condition in an organism comprising:

isolating a first population of cells from the organism,

transducing the first population of cells with 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) three or more guide sequences capable of hybridizing to three or more target sequences in genome of the organism,

(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 (NLSs),

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 direct sequence-specific binding of CRISPR complexes to the target sequence,

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

wherein the CRISPR enzyme alters the genome of the first population of cells to obtain a second population of cells, and

transplanting the second population of cells into the organism thereby inducing the proliferative condition.

3. The method of claim 1 or 2, wherein the proliferative condition is a cancer.

4. The method of claim 3, wherein the first population of cells is a population of stem cells.

5. The method of claim 1 or 2, wherein the proliferative condition is a myeloid malignancy.

6. The method of claim 5, wherein the first population of cells is a population of hematopoietic stem and progenitor cells.

7. The method of claim 1 or 2, wherein the vector system is a lentiviral vector system.

8. The method of claim 7, wherein the first regulatory element is a U6 promoter.

9. The method of claim 7, wherein the second regulatory element is an EFS promoter.

10. The method of claim 1 or 2, wherein the polynucleotide sequence comprises 4, 5, 6, 7, 8, 9 or 10 guide sequences.

11. The method of claim 1 or 2, wherein the target sequences are associated with tumor suppressor gene(s).

2. A method of interrogating function of one or more genes in a proliferative condition in an organism comprising:

inducing the proliferative condition by a method comprising

isolating a first population of cells from the organism,

transducing the first population of cells with 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 chiRNA polynucleotide sequence, wherein the polynucleotide sequence comprises

(a) three or more guide sequences capable of hybridizing to three or more target sequences in genome of the organism,

(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 zero or at least one or more NLSs,

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 direct sequence-specific binding of CRISPR complexes to the target sequence,

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

wherein the CRISPR enzyme alters the genome of the first population of cells to obtain a second population of cells,

transplanting the second population of cells into the organism thereby inducing the proliferative condition; and

determining changes in expression of the one or more genes in the proliferative condition thereby interrogating the function of one or more genes.

12. A non-naturally occurring or engineered composition for generating an in vivo, in vitro or ex vivo proliferative condition comprising multiple genetic lesions in a population of cells comprising a lentiviral vector system comprising one or more lentiviral vectors comprising

I. a U6 promoter operably linked to a CRISPR-Cas system chiRNA polynucleotide sequence, wherein the polynucleotide sequence comprises

(a) three or more guide sequences capable of hybridizing to three or more target sequences in genome of the organism,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a EFS promoter operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising zero or at least one or more NLSs,

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 direct sequence-specific binding of CRISPR complexes to the target sequence, and

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

13. The composition of claim 12, wherein the proliferative condition is a cancer.

14. The composition of claim 12, wherein the population of cells is a population of stem cells.

15. The composition of claim 12, wherein the proliferative condition is a myeloid malignancy.

16. The composition of claim 12, wherein the population of cells is a population of hematopoietic stem and progenitor cells.

17. The composition of claim 12, wherein the polynucleotide sequence comprises 4, 5, 6, 7, 8, 9 or 10 guide sequences.

18. The composition of claim 12, wherein the target sequences are associated with tumor suppressor gene(s).