# US 2026/0085331 A1 · Biallelic Knockout of PDCD1

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- Number: US 2026/0085331 A1
- Document ID: US20260085331A1
- Status: Published application
- Kind: A1
- Application number: 19/113,031
- Filed: 2023-09-18
- Published: 2026-03-26
- Earliest claimed: 2022-09-19
- Assignees: EmendoBio Inc.
- Applicants: EmendoBio Inc.
- Inventors: Rafi EMMANUEL
- Web page: https://patentgrep.com/patent/US20260085331A1

## CPC

- A61K 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
- C12N 15/907: {in mammalian cells}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- 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)

## Abstract

Compositions comprising an RNA molecule comprising a guide sequence portion having 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-6220 and methods and uses thereof.

## Claims

### Claim 1

A method for inactivating alleles of the Programmed cell death protein 1 (PDCD1) gene in a cell, the method comprising
introducing to the cell a composition comprising:
at least one CRISPR nuclease, or a polynucleotide molecule encoding a CRISPR nuclease; and
an RNA molecule comprising a guide sequence portion, or a polynucleotide molecule encoding the RNA molecule,
wherein a complex of the CRISPR nuclease and the RNA molecule affects a double strand break in alleles of the PDCD1 gene, and
wherein the guide sequence portion of the RNA molecule comprises 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-6220.

### Claim 2

(canceled)

### Claim 3 (depends on 1)

The method of claim 1, wherein the cell is a lymphocyte, a T cell, a T regulatory cell, a B cell, a natural killer (NK) cell, a macrophage, a stem cell, or a fibroblast, blood cell, hepatocyte, keratinocyte, or any other cell type capable of being reprogrammed to an induced pluripotent stem cell (iPSC), or
wherein the cell is a hematopoietic stem cell (HSC), induced pluripotent stem cell (iPS cell), iPSc-derived cell, natural killer cell (NK), iPS-derived NK cell (iNK), T cell, innate-like T cell (iT), natural killer T cell (NKT), γδ T cell, iPSc-derived T cell, invariant NKT cells (iNKT), iPSc-derived NKT, monocyte, or macrophage.

### Claim 4

(canceled)

### Claim 5

(canceled)

### Claim 6 (depends on 1)

The method of claim 1, wherein alleles of the PDCD1 gene in the cell are subjected to an insertion or deletion mutation.

### Claim 7

(canceled)

### Claim 8

(canceled)

### Claim 9 (depends on 1)

The method of claim 1, wherein the composition introduced to the cell further comprises a second RNA molecule comprising a guide sequence portion, wherein the guide sequence portion of the second RNA molecule comprises 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-6220, wherein the guide sequence portion of the second RNA molecule differs from the guide sequence portion of the first RNA molecule.

### Claim 10

A method for inactivating alleles of the Programmed cell death protein 1 (PDCD1) gene in a cell, the method comprising
introducing to the cell a composition comprising:
at least one CRISPR nuclease, or a polynucleotide molecule encoding a CRISPR nuclease; and
an RNA molecule comprising a guide sequence portion, or a polynucleotide molecule encoding the RNA molecule,
wherein a complex of the CRISPR nuclease and the RNA molecule affects a double strand break in alleles of the PDCD1 gene, and
wherein the guide sequence portion of the RNA molecule comprises 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-6220 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to a fully-complementary target sequence of the guide sequence portion.

### Claim 11

(canceled)

### Claim 12 (depends on 10)

The method of claim 10, wherein the guide sequence portion provides higher targeting specificity to the complex of the CRISPR nuclease and the RNA molecule relative to a guide sequence portion that has higher complementarity to an allele of the PDCD1 gene.

### Claim 13 (depends on 10)

The method of claim 10, wherein the composition introduced to the cell further comprises a second RNA molecule comprising a guide sequence portion, wherein the guide sequence portion of the RNA molecule comprises 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-1-6220, or any one of SEQ ID NOs: 1-1-6220 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to a fully-complementary target sequence of the guide sequence portion, wherein the guide sequence portion of the second RNA molecule differs from the guide sequence portion of the first RNA molecule.

### Claim 14

A composition comprising:
at least one CRISPR nuclease, or a polynucleotide molecule encoding a CRISPR nuclease; and
an RNA molecule comprising a guide sequence portion, or a polynucleotide molecule encoding the RNA molecule,
wherein the guide sequence portion of the RNA molecule comprises 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-6220 or in any one of SEQ ID NOs: 1-6220, modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to a fully target sequence of the guide sequence portion.

### Claim 15 (depends on 14)

The composition of claim 14, further comprising a second RNA molecule which comprises a guide sequence portion comprising 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-6220, or any one of SEQ ID NOs: 1-6220 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to a fully-complementary target sequence of the guide sequence portion, wherein the guide sequence portion of the second RNA molecule differs from the guide sequence portion of the first RNA molecule.

### Claim 16

(canceled)

### Claim 17

(canceled)

### Claim 18

(canceled)

### Claim 19

(canceled)

### Claim 20 (depends on 14)

A cell modified using the composition of claim 14.

### Claim 21

(canceled)

### Claim 22

(canceled)

### Claim 23 (depends on 20)

The modified cell of claim 20, wherein the cell is a stem cell or any cell type capable of being reprogrammed to an induced pluripotent stem cell (iPSC).

### Claim 24

(canceled)

### Claim 25

(canceled)

### Claim 26

(canceled)

### Claim 27 (depends on 14)

Use of the composition of claim 14 for adoptive immunotherapy, comprising delivering the composition of claim 14, or a cell modified by the composition of claim 14, to a subject in need of adoptive immunotherapy.

### Claim 28

(canceled)

### Claim 29

(canceled)

### Claim 30

(canceled)

### Claim 31

(canceled)

### Claim 32

(canceled)

### Claim 33 (depends on 14)

A method of treating cancer, the method comprising delivering the composition of claim 14, or a cell modified by the composition of claim 14, to a subject in need of cancer treatment.

### Claim 34

(canceled)

### Claim 35 (depends on 1)

The method of claim 1, wherein the guide sequence portion of the RNA molecule further comprises 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 2226, 2573, 2757, 2887, or 6215.

### Claim 36 (depends on 35)

The method of claim 35, wherein at least one CRISPR nuclease is OMNI-103.

### Claim 37 (depends on 10)

The method of claim 10, wherein the guide sequence portion of the RNA molecule further comprises 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 2226, 2573, 2757, 2887, or 6215.

### Claim 38 (depends on 37)

The method of claim 37, wherein at least one CRISPR nuclease is OMNI-103.

### Claim 39 (depends on 14)

The composition of claim 14, wherein the guide sequence portion of the RNA molecule further comprises 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: SEQ ID NOs: 2226, 2573, 2757, 2887, or 6215.

### Claim 40 (depends on 39)

The composition of claim 39, wherein at least one CRISPR nuclease is OMNI-103.