# US 12,215,320 B2 · Compositions and methods for gene editing by targeting transferrin

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,215,320 B2
- Document ID: US12215320B2
- Status: Granted
- Kind: B2
- Application number: 16/961,314
- Filed: 2019-01-11
- Published: 2021-08-19
- Granted: 2025-02-04
- Earliest claimed: 2018-01-12
- Assignees: Crispr Therapeutics AG; Bayer HealthCare LLC
- Applicants: CRISPR Therapeutics AG; Bayer Healthcare LLC
- Inventors: Alan Richard Brooks; Karen Vo
- Web page: https://patentgrep.com/patent/US12215320B2

## CPC

- 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)}
- A61K 35/407: Liver; Hepatocytes
- A61K 48/0008: {characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- C12Y 304/21: Serine endopeptidases (3.4.21)
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2510/00: Genetically modified cells
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2830/42: being an intron or intervening sequence for splicing and/or stability of RNA
- C12N 2830/50: regulating RNA stability, not being an intron, e.g. poly A signal
- 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)
- C12Y: ENZYMES

## Abstract

Provided include compositions, methods, and systems for modulating the expression, function, and/or activity of a target gene, for example a blood-clotting protein such as Factor VIII (FVIII), in a cell by genome editing. Also provided include compositions, methods, and systems for treating a subject having or suspected of having a disorder or health condition, e.g., Hemophilia A, employing ex vivo and/or in vivo genome editing.

## Claims

### Claim 1

A system comprising:
a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding the DNA endonuclease;
a guide RNA (gRNA) comprising a spacer sequence that is complementary to a sequence within intron 1 of an endogenous transferrin gene in a cell; and
a donor template comprising a nucleic acid sequence encoding a protein-of-interest (POI), wherein the POI is Factor VIII (FVIII).

### Claim 2 (depends on 1)

The system of claim 1, wherein the gRNA comprises:
i) a spacer sequence from any one of SEQ ID NOs: 96, 5, 6, 9, 8, 11, 15, 16, 12, 7, 10, 17, 18, 29, 76, 50, 54, 81, 64, 51, 1-4, 13, 14, 19-28, 30-49, 52, 53, 55-63, 65-75, 77-80, 82-95, and 97-190 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 96, 5, 6, 9, 8, 11, 15, 16, 12, 7, 10, 17, 18, 29, 76, 50, 54, 81, 64, 51, 1-4, 13, 14, 19-28, 30-49, 52, 53, 55-63, 65-75, 77-80, 82-95, and 97-190;
ii) a spacer sequence from any one of SEQ ID NOs: 5, 6, 9, 8, 11, 15, 16, 12, 7, and 10 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 5, 6, 9, 8, 11, 15, 16, 12, 7, and 10;
iii) a spacer sequence from any one of SEQ ID NOs: 17, 18, 29, 76, 50, 54, 81, 96, 64, and 51 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 17, 18, 29, 76, 50, 54, 81, 96, 64, and 51; or
iv) a spacer sequence from any one of SEQ ID NOs: 5, 6, 9, 8, and 11 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 5, 6, 9, 8, and 11.

### Claim 3 (depends on 2)

The system of claim 2, wherein the spacer sequence is 19 nucleotides in length and does not include the nucleotide at position 1 of the sequence from which it is selected.

### Claim 4 (depends on 1)

The system of claim 1, wherein the DNA endonuclease is selected from the group consisting of a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease, or a functional derivative thereof.

### Claim 5 (depends on 1)

The system of claim 1, wherein the DNA endonuclease is Cas9.

### Claim 6 (depends on 1)

The system of claim 1, wherein I) the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in a host cell; II) the nucleic acid sequence encoding the POI is codon-optimized for expression in a host cell; and/or III) the nucleic acid sequence encoding the POI comprises a reduced content of CpG di-nucleotides than a nucleic acid sequence encoding the wild-type POI.

### Claim 7 (depends on 1)

The system of claim 1, wherein the nucleic acid sequence encoding the POI:
A) comprises about or less than 20 CpG di-nucleotides;
B) comprises about or less than 10 CpG di-nucleotides;
C) comprises about or less than 5 CpG di-nucleotides; or
D) does not comprise CpG di-nucleotides.

### Claim 8 (depends on 1)

The system of claim 1, wherein the nucleic acid encoding the DNA endonuclease is a ribonucleic acid (RNA).

### Claim 9 (depends on 1)

The system of claim 1, wherein the donor template is encoded in an Adeno Associated Virus (AAV) vector.

### Claim 10 (depends on 1)

The system of claim 1, wherein the donor template comprises a donor cassette comprising the nucleic acid sequence encoding the POI, and wherein the donor cassette is flanked on one or both sides by a gRNA target site.

### Claim 11 (depends on 10)

The system of claim 10, wherein the gRNA target site is a target site for a gRNA in the system.

### Claim 12 (depends on 11)

The system of claim 11, wherein the gRNA target site of the donor template is the reverse complement of a genomic gRNA target site for a gRNA in the system.

### Claim 13 (depends on 1)

The system of claim 1, wherein the DNA endonuclease or nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.

### Claim 14 (depends on 13)

The system of claim 13, wherein the liposome or lipid nanoparticle also comprises the gRNA.

### Claim 15 (depends on 1)

The system of claim 1, comprising the DNA endonuclease precomplexed with the gRNA, forming a Ribonucleoprotein (RNP) complex.