# US 10,246,498 B2 · Genetically encoded histone reporter allele constructs

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- Number: US 10,246,498 B2
- Document ID: US10246498B2
- Status: Granted
- Kind: B2
- Application number: 15/315,137
- Filed: 2015-05-29
- Published: 2017-04-27
- Granted: 2019-04-02
- Earliest claimed: 2014-05-30
- Assignees: The Johns Hopkins University
- Applicants: The Johns Hopkins University
- Inventors: Hans Tomas Bjornsson; Harry Dietz
- Web page: https://patentgrep.com/patent/US10246498B2

## CPC

- C07K 14/47: from mammals
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A01K 67/0276: Knock-out vertebrates
- A61K 31/165: having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K 31/167: having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- G01N 33/582: {with fluorescent label}
- G01N 33/6893: {related to diseases not provided for elsewhere}
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2227/105: Murine
- A01K 2267/0356: Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
- C07K 2319/09: containing a nuclear localisation signal
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- C07K 2319/61: containing an enzyme fusion for detection (lacZ, luciferase)
- C07K 2319/90: containing a motif for post-translational modification
- C07K 2319/91: containing a motif for glycosylation
- C07K 2319/915: containing a motif for acylation
- C07K 2319/95: containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
- G01N 2500/04: Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
- G01N 2500/10: involving cells
- G01N 2800/50: Determining the risk of developing a disease
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Non-FRET-based fusion protein reporter molecules are provided that can be used to monitor histone modifications in living cells. Transgenic animals, particularly non-human mammals, whose genomes comprise an expression cassette encoding a non-FRET-based fusion protein reporter, are also provided. Methods of using the fusion reporter molecules for diagnosing histone-modification-associated disorders and to identify candidate pharmaceutical agents that effect histone modification in cells and tissues are also provided.

## Claims

### Claim 1

A fusion protein reporter comprising:
a) a histone-modification-specific binding domain that is configured to bind to said histone polypeptide substrate when said histone polypeptide substrate is modified;
b) a histone polypeptide substrate; and
c) a flexible linker region, wherein the flexible linker region is flanked on one side by a region encoding a C-terminal portion of a circularly permutated fluorescent protein and on the other side by a region encoding the N-terminal portion of the circularly permutated fluorescent protein, and wherein said C-terminal portion of said circularly permutated fluorescent protein and said N-terminal portion of said circularly permutated fluorescent protein form a functional fluorescent protein when said histone-modification-specific binding domain binds to said histone polypeptide substrate.

### Claim 2 (depends on 1)

The fusion protein reporter of claim 1, wherein the histone polypeptide is selected from the group consisting of H3 and H4.

### Claim 3 (depends on 1)

The fusion protein reporter of claim 1, wherein the histone polypeptide is selected from the group consisting of the N-terminus of H3 and the N-terminus of H4, wherein said N-terminus retains the ability to bind with said histone-modification specific binding domain.

### Claim 4 (depends on 1)

The fusion protein reporter of claim 1, wherein the histone modification is selected from the group consisting of acetylation, methylation, and phosphorylation.

### Claim 5 (depends on 1)

The fusion protein reporter of claim 1, wherein the histone modification-specific binding domain is selected from the group consisting of a TBP associated factor II (TAFII) bromodomain and a TBP associated factor III (TAFIII) homeodomain.

### Claim 6 (depends on 1)

The fusion protein reporter of claim 1, wherein the histone polypeptide is a polypeptide substrate for the histone-modification-specific binding domain.

### Claim 7 (depends on 1)

The fusion protein reporter of claim 1, further comprising a targeting polypeptide associated with the fusion protein.

### Claim 8 (depends on 7)

The fusion protein reporter of claim 7, wherein the targeting polypeptide comprises at least one nuclear localization sequence (NLS).

### Claim 9 (depends on 8)

The fusion protein reporter of claim 8, wherein the targeting polypeptide comprises a repeated NLS.

### Claim 10 (depends on 1)

The fusion protein reporter of claim 1, wherein the circularly permutated fluorescent protein comprises a circularly permutated green fluorescent protein (GFP).

### Claim 11 (depends on 1)

An expression vector comprising an expression cassette encoding a fusion protein reporter of claim 1.

### Claim 12 (depends on 11)

A host cell transformed or transfected with the expression vector of claim 11.

### Claim 13 (depends on 1)

A non-human transgenic animal, wherein the genome of the transgenic animal comprises an expression cassette encoding a fusion protein reporter of claim 1.

### Claim 14 (depends on 1, 13)

A method for producing a cell specific screening assay for test agents, comprising:
a) providing a transgenic animal of claim 13;
b) crossing the transgenic animal with a compatible animal, wherein the compatible animal is an animal model of a disease, thereby producing a hybrid;
c) isolating a disease relevant cell type that comprises the fusion protein reporter of claim 1 from the hybrid; and
d) utilizing the isolated disease relevant cell types in a high throughput screening assay for test agents that inhibit a histone modification activity in said disease relevant cell types.

### Claim 15 (depends on 1)

A method of determining the level of histone modification activity in a biological sample comprising:
a) contacting a biological sample with a fusion protein reporter of claim 1; and
b) monitoring the level of in the biological sample as a measure of the level of histone modification activity in the biological sample.

### Claim 16 (depends on 15)

The method of claim 15, wherein the biological sample is selected from the group consisting of cells and tissues.

### Claim 17 (depends on 1)

A method of monitoring the onset, progression, or regression of a histone-modification disorder in a subject comprising:
a) contacting a first biological sample obtained from a subject with a fusion protein reporter of claim 1;
b) determining the level of fluorescence in the first biological sample;
c) contacting a subsequent second biological sample obtained from the subject with the fusion protein reporter;
d) determining the amount of fluorescence in the second biological sample; and
e) comparing the level of fluorescence in the first biological sample to the level of fluorescence in the second biological sample as a measure of the onset, regression or progression of a histone modification disorder in the subject.

### Claim 18 (depends on 17)

The method of claim 17, further comprising administering after the first biological sample is obtained from the subject and before the second biological sample is obtained from the subject, a candidate pharmacological agent to the subject, wherein the measure of the onset, progression, or regression of a histone modification disorder in the subject is an indication of the effect of the candidate pharmacological agent on histone modification in the subject.

### Claim 19 (depends on 17)

The method of claim 17, wherein the biological sample is selected from the group consisting of cells and tissues.

### Claim 20 (depends on 1)

A method for evaluating the effect of candidate pharmacological agents on histone modification activity in a biological sample comprising:
a) contacting a biological sample with a fusion protein reporter fusion protein reporter of claim 1;
b) determining a first level of fluorescence in the biological sample;
c) contacting the biological sample with a candidate pharmacological agent;
d) determining a second level of fluorescence in the biological sample; and
e) comparing the first level of fluorescence in the cell with the second level of fluorescence in the cell, wherein a relative increase or relative decrease in fluorescence indicates an effect of the candidate pharmacological agent on histone modification activity in the biological sample.

### Claim 21 (depends on 20)

The method of claim 20, wherein the biological sample is selected from the group consisting of cells and tissues.

### Claim 22 (depends on 20)

The method of claim 20, wherein the biological sample is a cell.

### Claim 23 (depends on 22)

The method of claim 22, wherein the cell is undergoing cell division.

### Claim 24 (depends on 22)

The method of claim 22, wherein the cell is a living cell.

### Claim 25 (depends on 1)

A kit for diagnosing and/or monitoring treatment efficacy of a histone modification disorder, comprising:
a) a container containing a fusion protein reporter fusion protein reporter of claim 1; and
b) instructions for the use of the fusion protein reporter in the diagnosis and/or monitoring treatment efficacy of a histone-modification disorder.

### Claim 26 (depends on 25)

The kit of claim 25, wherein the kit comprises reagents and/or instructions for chromatin immunoprecipitation (ChiP).

### Claim 27 (depends on 26)

The kit of claim 26, wherein the kit comprises reagents and/or instructions for chromatin immunoprecipitation-sequencing (ChiP-seq).