Delivery, use and therapeutic applications of the crispr-cas systems and compositions for modeling mutations in leukocytes

Patent metadata

Field	Value
Publication number	US-2018255751-A1
Application number	US-201715468652-A
Country	US
Kind code	A1
Filing date	Mar 24, 2017
Priority date	Sep 24, 2014
Publication date	Sep 13, 2018
Grant date	—

Abstract

The invention involves a method for modulating leukocyte activity, comprising delivering to a leukocyte a vector containing nucleic acid molecule(s), whereby the leukocyte contains Cas9 and the vector expresses one or more RNAs to guide the Cas9 to introduce mutations in one or more target genetic loci in the leukocyte, thereby modulating expression of one or more genes expressed in the leukocyte. The invention also involves identifying genes associated with leukocyte responses and experimental modeling of aberrant leukocyte activation and diseases associated with leukocytes by introducing mutations into leukocytes. The invention comprehends testing putative treatments with such models, e.g., testing putative chemical compounds that may be pharmaceutically relevant for treatment or gene therapy that may be relevant for treatment, or combinations thereof. The invention allows for the study of genetic diseases and putative treatments to better understand and alleviate leukocyte associated diseases.

First claim

1 - 79 . (canceled)

80 . A method for modeling an aberrant leukocyte response or a disease associated with leukocytes, comprising delivering to leukocytes a vector containing nucleic acid molecule(s), whereby the leukocytes contain or express or are able to be induced to express or conditionally express Cas9 and the vector expresses a plurality of guides (i) to guide the Cas9 to introduce mutations in target genetic loci or (ii) to guide the Cas9 to activate or inhibit expression of target genes in the leukocytes, thereby modulating expression of one or more genes expressed in the leukocytes, wherein expression of the one or more genes is associated with the response or disease and optionally deliver donor templates and plurality of leukocyte-specific mutations or precise sequence substitutions.

81 . The method of claim 80 , wherein the leukocytes are derived from a non-human transgenic mammal having cells that contain or express or are able to be induced to express or conditionally express Cas9, and the vector is delivered to the leukocyte ex vivo.

82 . The method of claim 80 , wherein the specific mutations or precise sequence substitutions are or have been correlated to the aberrant leukocyte response or disease associated with leukocytes.

83 . The method of claim 80 , wherein each of the guides targets a different genetic locus associated with an aberrant leukocyte response or disease associated with leukocytes.

84 . The method of claim 80 , wherein the leukocytes comprise T cells and the response comprises differentiation, exhaustion, activation, or inhibition.

85 . The method of claim 84 , wherein the T cells are stimulated with interleukin-12 and the response comprises differentiation to a Th1 phenotype.

86 . The method of claim 84 , wherein the T cells are stimulated with interleukin-4 and the response comprises differentiation to a Th2 phenotype.

87 . The method of claim 84 , wherein the T cells are stimulated with TGF-β and the response comprises differentiation to a Treg phenotype.

88 . The method of claim 84 , wherein the T cells are stimulated with TGF-β and interleukin-6 and the response comprises differentiation to a Th17 phenotype.

89 . The method of claim 84 , wherein the T cells are from a transgenic mouse having cells that express Cas9 and an ovalbumin-specific αβ-T cell receptor.

90 . The method of claim 80 , wherein the guides target one or more leukocyte genes so as to introduce a loss-of-function or a gain-of-function mutation.

91 . The method of claim 80 , wherein the guides target one or more loci which are transcriptional control elements.

92 . The method of claim 81 , wherein the transgenic non-human mammal comprises a non-human primate, a rodent, a canine, a bovine, an ovine, a mouse, a dog, a goat, a pig, a rabbit, or a fowl or poultry.

93 . A method for modulating leukocyte activity, comprising delivering to a leukocyte a vector containing nucleic acid molecule(s), whereby the leukocyte contains or expresses or is able to be induced to express or conditionally expresses Cas9 and the vector expresses a plurality of guides to guide the Cas9 to a plurality of leukocyte-specific target loci and optionally delivers donor template(s) to introduce mutations in one or more target genetic loci in the leukocyte, thereby modulating expression of one or more genes in the leukocyte.

94 . The method of any claim 93 , wherein the guides target one or more genes expressed in the leukocyte so as to introduce loss-of-function or gain-of-function mutations.

95 . An isolated leukocyte comprising a vector containing a nucleic acid molecule(s), wherein the leukocyte contains or expresses or is able to be induced to express or conditionally express Cas9, (i) one or more target genetic loci in the leukocyte comprise mutations derived from Cas9 activity guided by a plurality of guides encoded by the vector or (ii) expression of one or more target genes in the leukocytes is activated or inhibited by Cas9 guided by a plurality of guides encoded by the vector, and wherein expression of one or more genes in the leukocyte is modulated thereby.

96 . The isolated leukocyte of claim 94 , wherein the one or more target genetic loci or one or more target genes comprise a plurality pf target genetic loci or target genes associated with an aberrant leukocyte response or a disease associated with leukocytes.

97 . The isolated leukocyte of claim 94 , wherein the leukocyte is derived from a non-human transgenic animal having cells that contain or express or are able to be induced to express or conditionally express Cas9.

98 . The isolated leukocyte of claim 97 , wherein the non-human transgenic animal comprises a non-human primate, a rodent, a canine, a bovine, an ovine, a mouse, a dog, a goat, a pig, a rabbit, or a fowl or poultry.

99 . A non-human transgenic mammal comprising cells that express or that are able to be induced to express or that conditionally express Cas9 and an aberrant leukocyte response or a disease associated with leukocytes, obtained or obtainable by the method of claim 80 .

100 . An individualized or personalized treatment of a leukocyte-associated disease in a subject in need of such treatment comprising:
(a) introducing multiple mutations ex vivo in a tissue, organ, or cell line comprising Cas9-expressing leukocytes that are able to be induced to express or conditionally express Cas9, or in vivo in a transgenic non-human mammal comprising cells that express or that are able to be induced to express or that conditionally express Cas9, comprising delivering to the cell(s) of the tissue, organ, cell or mammal a vector which expresses a plurality of guides (i) to guide the Cas9 and optionally delivers one or more donor template(s) to introduce mutations in one or more target genetic loci or (ii) to guide the Cas9 to activate or inhibit expression of a target gene in the leukocytes, thereby modulating expression of one or more genes in the leukocytes; (b) testing treatment(s) for the disease on the leukocytes to which the vector has been delivered; and (c) treating the subject based on results from the testing of treatment(s) of step (b).

101 . The method of claim 100 , wherein the leukocytes are derived from a non-human transgenic animal having cells that contain or express or are able to be induced to express or conditionally express Cas9, and the vector is delivered to the leukocyte ex vivo.

102 . The method of claim 100 , wherein the disease is an immune system disorder.

103 . The method of claim 100 , wherein the disease is cancer.

104 . A method of identifying a gene associated with a leukocyte response, comprising:
(a) delivering to a plurality of leukocytes a plurality of different vectors containing nucleic acid molecule(s), whereby the leukocytes contain or express or are able to be induced to express or conditionally express Cas9 and each vector expresses a plurality of guides (i) to guide the Cas9 and optionally delivers donor template(s) to introduce mutations in different target genetic loci or (ii) to guide the Cas9 to activate or inhibit expression of target gene(s) in the leukocytes, thereby modulating expression of different genes expressed in the leukocytes; (b) measuring a response in the leukocytes; and (c) isolating leukocyte(s) in which the response is modulated following delivery of the vector, thereby identifying gene(s) or genetic elements as

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Massachusetts Gen Hospital

Inventors

• Regev Aviv
• Parnas Oren
• Jovanovic Marko
• Hacohen Nir
• Eisenhaure Thomas

Classifications

• A01K2267/0312

  Animal model for Alzheimer's disease · CPC title

• A01K2227/105

  Murine · CPC title

• A01K67/0271 Primary

  Chimeric vertebrates, e.g. comprising exogenous cells · CPC title

• C12N15/907

  in mammalian cells · CPC title

• A01K2267/0331

  Animal model for proliferative diseases · CPC title