# US 11,994,512 B2 · Single-cell genomic methods to generate ex vivo cell systems that recapitulate in vivo biology with improved fidelity

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- Number: US 11,994,512 B2
- Document ID: US11994512B2
- Status: Granted
- Kind: B2
- Application number: 16/240,361
- Filed: 2019-01-04
- Published: 2019-07-04
- Granted: 2024-05-28
- Earliest claimed: 2018-01-04
- Assignees: Massachusetts Institute of Technology; The Brigham and Women's Hospital, Inc.
- Applicants: Massachusetts Institute of Technology; The Brigham and Women's Hospital, Inc.
- Inventors: Benjamin E. Mead; Jose Ordovas-Montanes; Alexander K. Shalek; Jeffrey Karp; Robert Langer
- Web page: https://patentgrep.com/patent/US11994512B2

## CPC

- G01N 33/5023: {on expression patterns}
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- C12N 5/0679: {Cells of the gastro-intestinal tract}
- C12N 5/068: {Stem cells; Progenitors}
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C40B 30/06: by measuring effects on living organisms, tissues or cells
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/5011: {for testing antineoplastic activity}
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/39: Steroid hormones
- C12N 2501/392: Sexual steroids
- C12N 2501/40: Regulators of development
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2510/00: Genetically modified cells
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/158: Expression markers
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

Disclosed here is a generally applicable framework that utilizes massively-parallel single-cell RNA-seq to compare cell types/states found in vivo to those of in vitro models. Furthermore, Applicants leverage identified discrepancies to improve model fidelity. Applicants uncover fundamental gene expression differences in lineage-defining genes between in vivo systems and in vitro systems. Using this information, molecular interventions are identified for rationally improving the physiological fidelity of the in vitro system. Applicants demonstrated functional (antimicrobial activity, niche support) improvements in Paneth cell physiology using the methods.

## Claims

### Claim 1

A method of generating an in vitro cell-based system that faithfully recapitulates an in vivo phenotype of interest comprising:
a) determining, using single cell RNA sequencing, gene expression for single cells in an initial in vitro cell-based system to computationally identify cell clusters of enteroendocrine Paneth cell types, wherein the initial in vitro cell-based system is an in vitro intestinal organoid cell-based system comprising Paneth cells, wherein the organoid cell-based system is obtained from an intestinal stem cell enriched organoid produced by enriching for murine LGR5+ intestinal stem cells in a scaffold and medium containing growth factors EGF (E), Noggin (N), R-spondin I (R), CHIR99021 (C), and valproic acid (V);
b) identifying differences in the gene expression for single Paneth cells in the initial in vitro cell-based system by performing differential gene expression analysis for clusters of single Paneth enteroendocrine cell types and Paneth cells in the in vivo system having the phenotype of interest, wherein differential gene expression analysis comprises comparing a gene expression distribution as determined by single cell RNA sequencing of the initial in vitro cell-based system and a gene expression distribution as determined by single cell RNA sequencing in the Paneth cells in vivo system;
c) identifying differential gene expression for Wnt and Notch pathways for Paneth cells identified in the initial in vitro cell-based system and the Paneth cell in vivo system in step (b); and
d) modulating Wnt and/or Notch signaling in the initial in vitro cell-based system with one or more agents comprising a Wnt signaling activator and Notch signaling inhibitor to induce a shift that reduces the differences in gene expression for the Paneth cells between the initial in vitro cell-based system and the Paneth cell in vivo system, thereby generating the in vitro cell-based system that faithfully recapitulates the in vivo phenotype of interest,
wherein the differential gene expression analysis comprises measuring a Euclidean distance, Pearson coefficient, Spearman coefficient, or any combination thereof, and
wherein the differential gene expression analysis comprises 10 or more genes, 20 or more genes, 30 or more genes, 40 or more genes, 50 or more genes, 100 or more genes, 500 or more genes, or 1000 or more genes; or wherein the differential gene expression analysis comprises one or more cell pathways; or wherein the differential gene expression analysis comprises a transcriptome of the Paneth cell in vivo system.

### Claim 2 (depends on 1)

The method of claim 1, wherein the shift that reduces the differences in gene expression for the Paneth cells in the initial cell-based in vitro system as compared to the target in vivo system is a statistically significant shift as measured by a P value of 0.05 or less in the gene expression distribution of the initial in vitro cell-based system toward that of the in vivo system.

### Claim 3 (depends on 1)

The method of claim 1, wherein comparing a gene expression distribution comprises comparing only Paneth cells from the initial in vitro cell-based system with the lowest differences in gene expression as compared to the Paneth cells from the in vivo system and wherein the differences in gene expression are a statistically significant shift measured by a P value of 0.05 or less.

### Claim 4 (depends on 1)

The method of claim 1, further comprising modulating the initial in vitro cell-based system to induce
a gain of function by modulating expression of one or more genes, gene expression cassettes, or gene expression signatures associated with the gain of function, wherein the gain of function is in addition to and different from the in vivo phenotype of interest; or
a loss of function by modulating expression of one or more genes, gene expression cassettes, or gene expression signatures associated with the loss of function, wherein the loss of function is in addition to and different from the in vivo phenotype of interest.

### Claim 5 (depends on 1)

The method claim 1, wherein modulating comprises increasing or decreasing expression of one or more genes, gene expression cassettes, or gene expression signatures in the in vitro cell-based system; or
wherein modulating comprises activating or inhibiting one or more genes, gene expression cassettes, or gene expression signatures in the in vitro cell-based system.

### Claim 6 (depends on 1)

The method of claim 1, wherein modulating Wnt and/or Notch signaling in the initial in vitro cell-based system with one or more agents comprising a Wnt signaling activator and Notch signaling inhibitor comprises delivering one or more modulating agents wherein the one or more modulating agents comprise one or more cytokines, growth factors, hormones, transcription factors, metabolites, synthetic ligands, or small molecules; or wherein the one or more modulating agents are comprise a genetic modifying agent or an epigenetic modifying agent.

### Claim 7 (depends on 6)

The method of claim 6, wherein the genetic modifying agent comprises a CRISPR system, a zinc finger nuclease system, a TALEN, or a meganuclease; or
wherein the epigenetic modifying agent comprises a DNA methylation inhibitor, HDAC inhibitor, histone acetylation inhibitor, histone methylation inhibitor or histone demethylase inhibitor.

### Claim 8 (depends on 2)

The method of claim 2, wherein the statistically significant shift having a P value of 0.05 or less is a shift that reduces the differences in the gene expression between the initial cell-based in vitro system and the in vivo system by at least a 10%.

### Claim 9 (depends on 1)

The method of claim 1, further comprising step (e) performing single cell RNA sequencing on the modulated in vitro cell-based system to determine the shift in the gene expression distribution of the initial in vitro cell-based system toward that of the in vivo system, optionally, repeating step (d) to further modulate Wnt and/or Notch signaling.

### Claim 10 (depends on 1)

The method of claim 1, wherein the Wnt signaling activator comprises 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR99021).

### Claim 11 (depends on 1)

The method of claim 1, wherein the Notch signaling inhibitor comprises N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

### Claim 12 (depends on 1)

The method of claim 1, wherein modulating Wnt and/or Notch signaling in the initial in vitro cell-based system to induce a shift that reduces the differences in gene expression is temporally modulated to further reduce the differences in gene expression for the Paneth cells.