# US 12,180,546 B2 · Methods for identifying and modulating co-occurant cellular phenotypes

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- Number: US 12,180,546 B2
- Document ID: US12180546B2
- Status: Granted
- Kind: B2
- Application number: 16/494,911
- Filed: 2018-03-19
- Published: 2020-04-16
- Granted: 2024-12-31
- Earliest claimed: 2017-03-17
- Assignees: Massachusetts Institute of Technology
- Applicants: Massachusetts Institute of Technology
- Inventors: Alexander K. Shalek; Alexander S. Genshaft; Carly G. K. Ziegler; Jeffrey F. Van Humbeck; Ryan Philip King
- Web page: https://patentgrep.com/patent/US12180546B2

## CPC

- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C07D 405/12: linked by a chain containing hetero atoms as chain links
- C07D 495/04: Ortho-condensed systems
- C12Q 1/6869: Methods for sequencing
- G01N 1/28: Preparing specimens for investigation {including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q}(mounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 {; laboratory gas handling apparatus B01L5/00})
- G16B 45/00: ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
- 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
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

The present invention provides tools and methods for the systematic analysis of genetic interactions between cells. The present invention provides tools and methods for modulating cell phenotypes and compositions, combinatorial probing of cellular circuits, for dissecting cellular circuitry, for delineating molecular pathways, and/or for identifying relevant targets for therapeutics development.

## Claims

### Claim 1

A cell functionalizing probe comprising a polyadorned molecule adorned with at least 3 and up to 5 groups, the polyadorned molecule comprising,
a core selected from the group consisting of —NH—C═, —NH—C═CH2, —NH—C═S, and —NH—C≡N, wherein the at least 3 groups comprise of:
(a) a cell-surface reactive group comprising a photoactivated cell-surface reactive group of the formula
(b) a bio-orthogonal reactive group of the formula
attached to the core of the polyadorned molecule opposite the cell-surface reactive group; and
(c) a label.

### Claim 2 (depends on 1)

The cell functionalizing probe of claim 1, wherein the core is —NH—C═O.

### Claim 3 (depends on 1)

The cell functionalizing probe of claim 1 further comprising a second reactive group.

### Claim 4 (depends on 1)

The cell functionalizing probe of claim 1, wherein the label is a fluorophore, a peptide-based tag, biotin, affinity reagent, hapten, lanthanide heavy metal(s) or combination thereof or an oligonucleotide, optionally wherein when the label is a fluorophore, the fluorophore comprises a compound of Formula (II):
wherein R3 is selected from the group consisting of —H, C1-C6 alkyl, —(CH2)a—NR8R9, —NHC(O)—Y—R8, —NHC(O)CHR8R9, —CHR8R9; —(CH2)aNR8C(O)R9,
R4 is selected from the group consisting of —H, —OH, and —OR8;
R5 and R6 are selected from the group consisting of —H, —OH, —X, —NO2, —CN, —NH2, —NHR8; —C(O)R8, —C1-C3 perfluoro alkyl; R7 is selected from the group consisting of —H, —OH, —X, —NO2, —CN, —C(O)NH(CH2)a —O(C1-C6 alkyl), —NH2, —NHR8, —NHC(O)—Y—R8, —(CH2)a—NR68C(O)R9, —NHC(O)CHR8R9, —C1-C3 perfluoro alkyl;
R8 and R9 are independently selected from the group consisting of —H, NH2, —(CH2)a—C(O)NH(CH2)bCH3, —(CH2)aC(O)NH(CH2)bC(O)NHPG3, —(CH2)a—CO2NHPG3, —C1-C8 straight chain alkyl, —C1-C8 branched alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, —(C1-C6 alkyl)-O—(C1-C6 alkyl) each of which is optionally substituted by a halogen, ether, vinyl group, allylic group, —NH2, or —CN, —(CH2)aNR88R89, —(CH2)a—C(O)NR88R89, an aromatic group, heteroaromatic group, C3-C7 cycloalkyl, a three to twelve membered heterocyclic having up to 3 heteroatoms each of which preceding cyclic group is optionally substituted from 1 to 3 substituents independently selected from a halogen, —C1-C6 alkyl, —C2-C6 alkenyl, —O(C1-C6 alkyl), —C(O)—, —OH, —NH2, —CN, and —C1-C3 perfluoro alkyl;
R88 and R89 are independently selected from the group consisting of —H, —O(C1-C6 alkyl), —C1-C8 straight chain alkyl, —C1-C6-branched alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, —(C1-C6alkyl)-O—(C1-C6 alkyl);
Y is selected from a covalent bond, —O—, —NH—, and —C1-C6 alkyl;
X is selected from the group consisting of Cl, Br, I, F;
PG3 is any photolabile protecting group;
a is independently any integer between 0 and 6;
b is independently any integer between 0 and 6.

### Claim 5 (depends on 1)

A method of single-cell profiling in a subject in need thereof wherein the single cells are spatially resolved, the method comprising:
(a) saturating a tissue in the subject in need thereof with a cell functionalizing probe according to claim 1;
(b) activating the cell functionalizing probe;
(c) labelling the tissue with a cell functionalizing barcoded tag comprising a click-enabled moiety; and an oligonucleotide barcode comprising a spatial barcode or a space barcode;
(d) washing the tissue with an aqueous solution to remove unbound functionalizing probe;
(e) repeating steps (a) through (d) from 1 to about 100 times;
(f) separating labeled cells into a suspension of single cells or small cell aggregates;
(g) profiling single cell sequences, whole cell populations, or cell subpopulations; and
(h) analyzing cellular phenotypes using categorical spatial information.

### Claim 6 (depends on 5)

The method of claim 5, further comprising sorting and enriching cells comprising a cell functionalizing tag and a cell functionalizing probe via a cell separation method between step (f) and step (g).

### Claim 7 (depends on 5)

The method of claim 5, further comprising assembling the single cell sequences into a visual representation between step (g) and step (h), wherein the relationship between amplified shared spatial barcodes of single cell sequences is obtained by a computational method.

### Claim 8 (depends on 1)

A method of single-cell profiling in a subject in need thereof wherein the single cells are spatially resolved, the method comprising:
(a) conjugating a cell functionalizing probe of claim 1 to a cell functionalizing barcoded tag comprising a click-enabled moiety; and an oligonucleotide barcode comprising a spatial barcode or a space barcode, whereby an active complex is formed;
(b) saturating a tissue in the subject in need thereof with the active complex;
(c) activating the cell functionalizing probe;
(d) washing the tissue with an aqueous solution to remove excess active complex;
(e) repeating steps (a) through (d) from 1 to 100 times;
(f) separating labeled cells into a suspension of single cells or small cell aggregates;
(g) profiling single cell sequences.

### Claim 9 (depends on 8)

The method of claim 8, further comprising sorting and enriching cells comprising a cell functionalizing tag and a cell functionalizing probe via a cell separation method between step (f) and step (g).

### Claim 10 (depends on 8)

The method of claim 8, further comprising assembling the single cell sequences into a visual representation, wherein the relationship between amplified shared spatial barcodes of single cell sequences is obtained.

### Claim 11 (depends on 8)

The method of claim 8, further comprising using spatial information as a categorical variable for downstream computational analysis.

### Claim 12 (depends on 1)

A method of spatially patterning cells on surfaces wherein single cells are spatially localized, the method comprising:
(a) conjugating a cell functionalized barcode of claim 1 to cell(s);
(b) streaming cells conjugated with a cell-functionalized barcode over a surface patterned with the cell-functionalized probe, whereby cells are attached to the cell-functionalized probe via complementary pairing chemistry.

### Claim 13 (depends on 12)

The method of claim 12,
wherein the cell functionalized probe is labelled with an oligonucleotide; whereby cells are conjugated non-specifically; or streaming cells conjugated with a cell functionalized barcode over a surface patterned with the cell functionalized probe, wherein the cell functionalized probe is labelled with complementary oligonucleotides; whereby cells are conjugated specifically.