# US 12,595,462 B2 · High throughput genetic barcoding and analysis methods

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- Number: US 12,595,462 B2
- Document ID: US12595462B2
- Status: Granted
- Kind: B2
- Application number: 17/758,828
- Filed: 2021-01-14
- Published: 2023-02-23
- Granted: 2026-04-07
- Earliest claimed: 2020-01-14
- Assignees: William Marsh Rice University
- Applicants: William Marsh Rice University
- Inventors: David Yu Zhang; Ping Song; Omid Veiseh; Siavash Parkhideh; Sudip Mukherjee; Maria Isabel Ruocco; Boram Kim; Yuxuan Cheng
- Web page: https://patentgrep.com/patent/US12595462B2

## CPC

- C12N 5/0012: {Cell encapsulation}
- A61K 9/0024: {Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue (compositions for intravenous administration, normal injectable solutions or dispersions for, e.g. subcutaneous administration A61K9/0019; brain implants A61K9/0085; (coated) prostheses, catheters or stents A61L)}
- A61K 9/5036: {Polysaccharides, e.g. gums, alginate; Cyclodextrin}
- A61K 35/39: Pancreas; Islets of Langerhans (Langerhans cells of epidermis A61K35/36)
- A61K 49/0008: {Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure}
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 2503/02: Drug screening
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

Provided herein are high-throughput methods for genetic barcoding and analysis, e.g., for tagging each biomaterial capsule with a barcode cell. These barcode cells are derived from patient samples, and thus embody natural human genetic variation. Also provided are SNP panels that can be used as genetic barcodes to identify the identity of a cell.

## Claims

### Claim 1

A method for identifying a cell encapsulation material following in vivo screening of cell encapsulation materials, the method comprising (a) preparing capsules containing barcode cells using various encapsulation materials, wherein the barcode cells comprise a plurality of SNPs that serve as a genetic barcode for each cell encapsulation material; (b) implanting the capsules into a test subject; (c) explanting the capsules after a set period of time; and (d) determining the sequence of the plurality of SNPs in the barcode cells of each explanted capsule, thereby identifying the cell encapsulation material of each capsule.

### Claim 2 (depends on 1)

The method of claim 1, wherein all of the capsules made with the same encapsulation material comprise identical barcode cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein each type of barcode cell is used for only one encapsulation material.

### Claim 4 (depends on 1)

The method of claim 1, wherein the plurality of SNPs have variant allele frequencies of between 10% and 90%.

### Claim 5 (depends on 1)

The method of claim 1, wherein the plurality of SNPs are spaced across the 22 pairs of human autosomes.

### Claim 6 (depends on 1)

The method of claim 1, wherein the plurality of SNPs are present in non-coding regions of the genome.

### Claim 7 (depends on 1)

The method of claim 1, wherein the plurality of SNPs comprises at least 5 SNPs.

### Claim 8 (depends on 7)

The method of claim 7, wherein the SNPs are selected form the group consisting of rs10230708, rs2043583, rs955456, rs966516, rs11247921, rs10510620, rs3789806, rs1884444, rs16754, rs28932178, rs10741037, rs10805227, rs10833604, rs10964389, rs11045749, rs12213948, rs12259813, rs1516755, rs1937037, rs2616187, rs2710998, rs2874755, rs4665582, rs4712476, rs611628, rs7893462, rs7902135, rs9466930, rs2862909, and rs1338945.

### Claim 9 (depends on 1)

The method of claim 1, wherein determining the sequence of the plurality of SNPs in the barcode cells of each explanted capsule comprises (i) isolating each explanted capsule by distributing each capsule individually into a well of a multi-well plate; and (ii) amplifying each of the SNPs in the barcode cells of each isolated capsule using locus-specific forward and reverse primers.

### Claim 10 (depends on 9)

The method of claim 9, wherein the location-specific forward and location-specific reverse primers comprise unique Hamming barcodes.

### Claim 11 (depends on 10)

The method of claim 10, wherein each Hamming barcode is seven nucleotides long.

### Claim 12 (depends on 10)

The method of claim 10, wherein each of the Hamming barcodes lack sequence identity at at least 2 nucleotide positions as compared to any of the other Hamming barcodes.

### Claim 13 (depends on 10)

The method of claim 10, wherein each of the Hamming barcodes has a Hamming distance of at least two relative to every other Hamming barcode.

### Claim 14 (depends on 1)

The method of claim 1, wherein the cell encapsulation material is an alginate encapsulation material.

### Claim 15 (depends on 1)

The method of claim 1, wherein each capsule further comprises therapeutic cells.

### Claim 16

A method for identifying a cell encapsulation material following in vivo screening of cell encapsulation materials, the method comprising (a) preparing a plurality of barcode cells derived from one or more subjects, wherein each composition of barcode cells comprises a unique profile of a plurality of SNPs that serve as a genetic barcode for each cell encapsulation material; (b) fabricating capsules using various encapsulation materials and barcode cells; (c) implanting the capsules into a test subject; (d) explanting the capsules after a set period of time; and (d) determining the sequence of the plurality of SNPs in the barcode cells of each explanted capsule, thereby identifying the cell encapsulation material of each capsule.