# US 11,998,567 B2 · Engineered chimeric guide RNA and uses thereof

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- Number: US 11,998,567 B2
- Document ID: US11998567B2
- Status: Granted
- Kind: B2
- Application number: 16/990,372
- Filed: 2020-08-11
- Published: 2020-11-19
- Granted: 2024-06-04
- Earliest claimed: 2018-03-12
- Assignees: Nanjing Bioheng Biotech Co., Ltd.
- Applicants: Nanjing Bioheng Biotech Co., Ltd.
- Inventors: Jiangtao Ren; Xuhua Zhang
- Web page: https://patentgrep.com/patent/US11998567B2

## CPC

- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/42: Cancer antigens
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 2319/03: containing a transmembrane segment
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2320/51: modulating the chemical stability, e.g. nuclease-resistance
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to engineered chimeric guide RNAs and their uses in systems and methods for gene expression perturbation in eukaryotic cells. Furthermore, the invention also relates to methods for generating a modified T cell using the engineered chimeric guide RNAs of the present application, and to the obtained modified T cell. Also included are methods and pharmaceutical compositions comprising the modified T cell for adoptive therapy and treating a condition, such as cancer, infections or autoimmune diseases.

## Claims

### Claim 1

An engineered chimeric guide RNA comprising:
(1) at least one crRNA sequences, and
(2) at least one additional direct repeat sequences and small RNA species,
wherein the crRNA sequence is capable of hybridizing with a target locus of interest, and the additional direct repeat sequence and/or small RNA species confers increased stability to the chimeric guide RNA;
wherein the small RNA species are a truncated form of pre-tRNA.

### Claim 2 (depends on 1)

The engineered chimeric guide RNA of claim 1, further comprises one or more elements selected from untranslated region (UTR), poly-A and 5′methylguanosine cap.

### Claim 3 (depends on 1)

The engineered chimeric guide RNA of claim 1, wherein the crRNA comprises a spacer sequence, which has a length from 10 to 30 bp.

### Claim 4 (depends on 1)

The engineered chimeric guide RNA of claim 1, wherein the engineered chimeric guide RNA is in the form of a pool or an array.