# US 2021/0277419 A1 · Genome Editing without Nucleases

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- Number: US 2021/0277419 A1
- Document ID: US20210277419A1
- Status: Published application
- Kind: A1
- Application number: 17/322,561
- Filed: 2021-05-17
- Published: 2021-09-09
- Earliest claimed: 2014-03-21
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Adi Barzel; Mark A. Kay
- Web page: https://patentgrep.com/patent/US20210277419A1

## CPC

- C12N 15/86: Viral vectors
- A61K 38/1866: {Vascular endothelial growth factor [VEGF]}
- 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}
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/08: for glucose homeostasis (pancreatic hormones A61P5/48)
- A61P 5/06: of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 7/06: Antianaemics
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 19/06: Antigout agents, e.g. antihyperuricemic or uricosuric agents
- A61P 25/16: Anti-Parkinson drugs
- A61P 31/04: Antibacterial agents
- A61P 31/10: Antimycotics
- A61P 31/14: for RNA viruses
- A61P 31/16: for influenza or rhinoviruses
- A61P 31/18: for HIV
- A61P 33/06: Antimalarials
- A61P 37/04: Immunostimulants
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 16/114: Lentivirus (G), e.g. human immunodeficiency virus [HIV], feline immunodeficiency virus [FIV] or simian immunodeficiency virus [SIV]
- C12N 9/644: {Coagulation factor IXa (3.4.21.22)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/864: Parvoviral vectors {, e.g. parvovirus, densovirus}
- C12N 15/907: {in mammalian cells}
- C12Y 304/21022: Coagulation factor IXa (3.4.21.22)
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/92: containing an intein ("protein splicing")domain
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2799/025: where the vector is derived from a parvovirus
- C12N 2800/24: Vectors characterised by the absence of particular element, e.g. selectable marker, viral origin of replication
- C12N 2840/20: translation of more than one cistron
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- C12Y: ENZYMES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Methods and compositions are provided for editing the genome of a cell without the use of an exogenously supplied nuclease. Aspects of the methods include contacting a cell with a targeting vector comprising nucleic acid sequence to be integrated into the target locus, where the cell is not also contacted with a nuclease. In addition, reagents, devices and kits thereof that find use in practicing the subject methods are provided.

## Claims

### Claim 1

A method for the targeted integration of a transgene into the genome of a cell in the absence of an exogenously provided nuclease, the method comprising:
contacting a cell with a recombinant viral vector, the recombinant viral vector comprising:
i. a polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes the transgene; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into the target integration site in the genome of the cell;
ii. a third nucleic acid sequence positioned 5′ to the polynucleotide and comprising sequence that is substantially homologous to genomic sequence 5′ of a target integration site in the genome of the cell; and
iii. a fourth nucleic acid sequence positioned 3′ of the polynucleotide and comprising sequence that is substantially homologous to genomic sequence 3′ of a target integration site in the genome of the cell;
wherein the cell is not contacted with a nuclease or nucleic acid encoding a nuclease.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the cell is a non-dividing cell.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the contacting occurs in vivo.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the method finds use in treating a medical condition associated with a gene deficiency.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the medical condition is selected from the group consisting of hemophilia, hemophilia A, hemophilia B, a branched-chain organic aciduria, maple syrup urine disease (MSUD), isovaleric acidaemia (IVA), propionic aciduria (PA), methylmalonic aciduria (MMA), 3 methylcrotonyl glycinuria, 3-methylglutaconic Aciduria Type I, short/branched-chain Acyl-CoA dehydrogenase deficiency, 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, isobutyryl-CoA dehydrogenase deficiency, 3-Hydroxyisobutyric aciduria, malonic aciduria, a long chained fatty acid oxidation disorder, a glycogen storage disease, Glycogen storage disease type I (GSD1), A carnitine cycle disorder, a urea cycle disorder, Crigler-Najjar syndrome, heraditary tyrosinemia, epidermolysis bullosa, Wilson disease, adenosine deaminase deficiency, sickle cell disease, X-Linked Severe Combined Immunodeficiency (SCID-X1), thalassemia, cystic fibrosis, alpha-1 anti-trypsin deficiency, diamond-blackfan anemia, Gaucher's disease, growth hormone deficiency, and Parkinson's Disease.

### Claim 6 (depends on 3)

The method according to claim 3, wherein the method finds use in promoting immunoprophylaxis.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the transgene encodes an agent that promotes immunoprophylaxis.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the agent that promotes immunoprophylaxis is an antibody or a chimeric polypeptide, and is specific for a pathogen selected from: human immunodeficiency virus (HIV), influenza virus, respiratory syncytial virus (RSV), hepatitis C virus (HCV), a plasmodium, Plasmodium falciparum, Plasmodium malariae, a fungus, and a bacterium.

### Claim 9 (depends on 3)

The method according to claim 3, wherein the method finds use in promoting wound healing.

### Claim 10

A recombinant viral vector for integrating a transgene into a target integration site in the genome of the cell, comprising:
a polynucleotide cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises the transgene; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into the target integration site in the genome of the cell;
a third nucleic acid sequence positioned 5′ to the polynucleotide cassette and comprising sequence that is substantially homologous to genomic sequence 5′ of the target integration site in the genome of the cell; and
a fourth nucleic acid sequence positioned 3′ of the polynucleotide cassette and comprising sequence that is substantially homologous to genomic sequence 3′ of the target integration site in the genome of the cell.

### Claim 11 (depends on 10)

The recombinant viral vector according to claim 10, wherein the viral vector is an rAAV vector.

### Claim 12 (depends on 10)

The recombinant viral vector according to claim 10, wherein the nucleic acid sequence that promotes the production of two independent gene products at the target integration site is selected from: a sequence that encodes a 2A peptide; an IRES; an intein; a recognition sequence for a site specific protease; a sequence that encodes a cleavable linker that is cleaved as part of the coagulation cascade; a sequence that encodes a factor XI cleavage site; and an intronic splice donor/splice acceptor sequence.

### Claim 13 (depends on 12)

The recombinant viral vector according to claim 12, wherein the expression and activity of an endogenous gene comprising the target integration site is not disrupted by the integration of the transgene.

### Claim 14 (depends on 13)

The recombinant viral vector according to claim 13, wherein:
the 3′ end of the endogenous gene comprises the target integration site,
the sequence of the third nucleic acid sequence is substantially homologous to the DNA sequence upstream of the stop codon of the endogenous gene; and
the sequence of the fourth nucleic acid sequences is substantially homologous to the DNA sequence downstream of the stop codon of the endogenous gene.

### Claim 15 (depends on 13)

The recombinant viral vector according to claim 13, wherein:
the 5′ end of the endogenous gene comprises the target integration site,
the sequence of the third nucleic acid sequence is substantially homologous to the DNA sequence upstream of the start codon of the endogenous gene; and
the sequence of the fourth nucleic acid sequences is substantially homologous to the DNA sequence downstream of the start codon of the endogenous gene.

### Claim 16 (depends on 13)

The recombinant viral vector according to claim 13, wherein the endogenous gene is selected from the group consisting of the albumin gene, a collagen gene, and an actin gene.

### Claim 17 (depends on 10)

The recombinant viral vector according to claim 10, wherein the transgene complements a gene deficiency.

### Claim 18 (depends on 10)

The recombinant viral vector according to claim 10, wherein the transgene encodes an agent that promotes immunoprophylaxis.

### Claim 19 (depends on 18)

The recombinant viral vector according to claim 18, wherein the agent that promotes immunoprophylaxis is a polypeptide comprising an immunoglobulin domain and an effector domain.

### Claim 20 (depends on 18)

The recombinant viral vector according to claim 18, wherein the agent that promotes immunoprophylaxis is an antibody.

### Claim 21 (depends on 18)

The recombinant viral vector according to claim 18, wherein the agent that promotes immunoprophylaxis is an antibody or a chimeric polypeptide, and is specific for a pathogen selected from: human immunodeficiency virus (HIV), influenza virus, respiratory syncytial virus (RSV), hepatitis C virus (HCV), a plasmodium, Plasmodium falciparum, Plasmodium malariae, a fungus, and a bacterium.

### Claim 22 (depends on 10)

The recombinant viral vector according to claim 10, wherein the transgene promotes vasculogenesis and/or angiogenesis.

### Claim 23 (depends on 22)

The recombinant viral vector according to claim 22, wherein the transgene is VEGF.