# US 2017/0283477 A1 · Animal model of longevity and related methods for increasing longevity and inhibiting tumorigenesis

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- Number: US 2017/0283477 A1
- Document ID: US20170283477A1
- Status: Published application
- Kind: A1
- Application number: 15/507,650
- Filed: 2015-09-01
- Published: 2017-10-05
- Earliest claimed: 2014-09-01
- Assignees: Academia Sinica
- Applicants: Academia Sinica
- Inventors: Che-Kun James SHEN; Yu-Chiau SHYU
- Web page: https://patentgrep.com/patent/US20170283477A1

## CPC

- C07K 14/4702: {Regulators; Modulating activity}
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A61K 38/1709: {from mammals}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/4703: {Inhibitors; Suppressors}
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A01K 2227/105: Murine
- A01K 2267/0331: Animal model for proliferative diseases
- 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
- 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)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention includes a genetically-modified non-human animal model of longevity and increased health span, which is associated with reduced tumorigenesis and tumor metastasis, as well as related methods for increasing longevity and health span, reducing tumorigenesis and tumor metastasis, and identifying active agents that confer increased longevity or health span, or reduced tumorigenesis or tumor metastasis.

## Claims

### Claim 1

A non-human transgenic animal comprising one or more modified Erythroid Kruppel-like factor (EKLF) genes encoding a modified EKLF polypeptide comprising one or more amino acid modifications as compared to a wild-type EKLF polypeptide, wherein the one or more amino acid modifications comprises a modification at an amino acid position that is sumoylated or phosphorylated in the wild-type EKLF polypeptide.

### Claim at position 2

2-4. (canceled)

### Claim 5 (depends on 1)

The non-human transgenic animal of claim 1, wherein the one or more amino acid modifications comprises a modification of an amino acid corresponding to position 74 of the full length wild-type mouse EKLF polypeptide.

### Claim 6 (depends on 5)

The non-human transgenic animal of claim 5, wherein the modification of the amino acid corresponding to position 74 is a substitution of Lys with Arg (K74R).

### Claim 7 (depends on 1)

The non-human transgenic animal of claim 1, wherein the one or more amino acid modifications comprises a modification of an amino acid corresponding to position 68 of the full length wild-type mouse EKLF polypeptide.

### Claim 8 (depends on 1)

The non-human transgenic animal of claim 1, wherein the modified EKLF polypeptide has reduced sumoylation or reduced phosphorylation as compared to the wild-type EKLF polypeptide.

### Claim 9 (depends on 1)

The non-human transgenic animal of claim 1, wherein the modified EKLF polypeptide has reduced translocation from the cytoplasm to the nucleus as compared to the wild-type EKLF polypeptide, and/or a modified transactivator activity or a modified repressor activity as compared to the wild-type EKLF polypeptide.

### Claim 10 (depends on 1)

The non-human transgenic animal of claim 1, wherein the expression of the modified EKLF polypeptide is associated with an increased lifespan or increased health span of the non-human transgenic animal, or wherein the expression of the modified EKLF polypeptide is associated with reduced tumor occurrence or reduced tumor metastasis in the non-human transgenic animal.

### Claim 11 (depends on 1)

The non-human transgenic animal of claim 1, wherein the wild-type EKLF polypeptide is from the same genus or species of animal as the non-human transgenic animal.

### Claim 12 (depends on 1)

The non-human transgenic animal of claim 1, which is a rodent, optionally a mouse or a rat.

### Claim at position 11

13-14. (canceled)

### Claim 15

A method of treating or preventing a cellular proliferative disorder, or inhibiting or reducing the incidence of tumor occurrence or tumor metastasis, in a subject in need thereof, comprising administering to the subject an effective amount of:
a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide is a modified EKLF polypeptide comprising one or more amino acid modifications that confer decreased sumoylation of the modified EKLF polypeptide as compared to a wild-type EKLF polypeptide, optionally a modified human EKLF polypeptide; or
b) a first active agent that alters one or more activities of an endogenous EKLF polypeptide, optionally an endogenous human EKLF polypeptide.

### Claim 16 (depends on 15)

The method of claim 15, wherein the polypeptide or the nucleic acid is administered, and the modified EKLF polypeptide has reduced translocation from the cytoplasm to the nucleus as compared to the wild-type EKLF polypeptide.

### Claim 17 (depends on 16)

The method of claim 16, wherein the polypeptide or the nucleic acid is administered, and the modified EKLF polypeptide has a modified transactivator activity or a modified repressor activity as compared to the wild-type EKLF polypeptide.

### Claim 18 (depends on 15)

The method of claim 15, wherein the nucleic acid is viral expression vector.

### Claim 19

(canceled)

### Claim 20 (depends on 18)

The method of claim 18, wherein the viral vector is derived from a herpes virus, a retrovirus, a vaccinia virus, an attenuated vaccinia virus, a canary pox virus, an adenovirus, or an adeno-associated virus.

### Claim 21 (depends on 15)

The method of claim 15, wherein the one or more amino acid modifications comprises a modification at an amino acid position that is sumoylated or phosphorylated in the wild-type EKLF polypeptide.

### Claim 22 (depends on 21)

The method of claim 21, wherein the one or more amino acid modifications comprises a modification of an amino acid corresponding to position 54 of the full length wild-type human EKLF polypeptide.

### Claim 23 (depends on 22)

The method of claim 22, wherein the modification of the amino acid at position 54 is a substitution of Lys with Arg (K54R).

### Claim 24 (depends on 20)

The method of claim 20, wherein the one or more amino acid modifications comprises a modification of an amino acid corresponding to position 68 of the full length wild-type mouse EKLF polypeptide.

### Claim 25 (depends on 15)

The method of claim 15, wherein the first active agent is administered, and the first active agent reduces translocation of the endogenous EKLF polypeptide from the cytoplasm to the nucleus.

### Claim 26 (depends on 15)

The method of claim 15, wherein the first active agent is administered, and the first active agent modifies a transactivator activity or modifies a repressor activity of the endogenous EKLF polypeptide.

### Claim 27 (depends on 15)

The method of claim 15, wherein the first active agent binds to the endogenous EKLF polypeptide.

### Claim 28 (depends on 27)

The method of claim 27, wherein the first active agent is a small organic molecule or a polypeptide, optionally an antibody or functional fragment thereof.

### Claim 29 (depends on 27)

The method of claim 27, wherein binding of the first active agent to the endogenous EKLF polypeptide inhibits its translocation from the cytoplasm to the nucleus.

### Claim 30 (depends on 15)

The method of claim 15, wherein the polypeptide or the nucleic acid is administered, and wherein the method further comprises administering a second active agent that inhibits expression of the endogenous EKLF polypeptide.

### Claim 31 (depends on 30)

The method of claim 30, wherein the second active agent is a nucleic acid molecule, optionally an antisense RNA, siRNA, shRNA or miRNA that binds an mRNA or complement thereof that encodes the endogenous EKLF polypeptide.

### Claim 32 (depends on 30)

The method of claim 30, wherein the second active agent inhibits transcription or translation of the endogenous EKLF polypeptide.

### Claim 33 (depends on 15)

The method of claim 15, wherein the cellular proliferative disorder, tumor or tumor metastasis is a liver cancer, a colon cancer, a breast cancer, a prostate cancer, a hepatocellular carcinoma, a melanoma, a lung cancer, a glioblastoma, a brain tumor, a hematopoetic malignancy, a cholangiocarcinoma, a retinoblastoma, a renal cell carcinoma, a head and neck cancer, a cervical cancer, a pancreatic cancer, an esophageal cancer, or a squamous cell carcinoma.

### Claim 34 (depends on 15)

The method of claim 15, further comprising administering to the subject an effective amount of an anti-proliferation agent.

### Claim 35 (depends on 34)

The method of claim 34, wherein the anti-proliferation agent is an alkylating agent, a topoisomerase inhibitor, an anti-metabolite, or a cytotoxicity antibiotic.

### Claim 36 (depends on 35)

The method of claim 35, wherein the alkylating agent is selected from the group consisting of: cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea (MNU), carmustine, lomustine, semustine, fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mytomycin, and diaziquone.

### Claim 37 (depends on 35)

The method of claim 35, wherein the topoisomerase inhibitor is selected from the group consisting of: camptothecin, irinotecan, topotecan, etoposide, doxorubicin, teniposide, novobiocin, merbarone, and aclarubicin.

### Claim 38 (depends on 35)

The method of claim 35, wherein the anti-metabolite is selected from the group consisting of: fluoropymidine, deoxynucleoside analogue, thiopurine, methotrexate, and pemetrexed.

### Claim 39 (depends on 35)

The method of claim 35, wherein the cytotoxicity antibiotic is selected from the group consisting of: actinomycin, bleomycin, plicamycin, mitomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, piraubicin, alcarubicin, and mitoxantrone.

### Claim 40

A method of extending the lifespan or health span of a subject, comprising administering to the subject an effective amount of:
a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide is a modified EKLF polypeptide comprising one or more amino acid modifications that confer reduced sumoylation of the modified EKLF polypeptide as compared to a wild-type EKLF polypeptide, optionally a modified human EKLF polypeptide; or
b) a first active agent that alters one or more activities of an endogenous EKLF polypeptide, optionally an endogenous human EKLF polypeptide.

### Claim 41 (depends on 40)

The method of claim 40, wherein the polypeptide or the nucleic acid is administered, and the modified EKLF polypeptide has reduced translocation from the cytoplasm to the nucleus as compared to the wild-type EKLF polypeptide.

### Claim 42 (depends on 40)

The method of claim 40, wherein the polypeptide or nucleic acid is administered, and the modified EKLF polypeptide has a modified transactivator activity or a modified repressor activity as compared to the wild-type EKLF polypeptide.

### Claim 43 (depends on 40)

The method of claim 40, wherein the nucleic acid is a viral expression vector.

### Claim 44

(canceled)

### Claim 45 (depends on 43)

The method of claim 43, wherein the viral vector is derived from a herpes virus, a retrovirus, a vaccinia virus, an attenuated vaccinia virus, a canary pox virus, an adenovirus, or an adeno-associated virus.

### Claim 46 (depends on 40)

The method of claim 40, wherein the one or more amino acid modifications comprises a modification at an amino acid position that is sumoylated or phosphorylated in the wild-type EKLF polypeptide.

### Claim 47 (depends on 46)

The method of claim 46, wherein the one or more amino acid modifications comprises a modification of an amino acid corresponding to position 54 of the full length wild-type human EKLF polypeptide.

### Claim 48 (depends on 47)

The method of claim 47, wherein the modification of the amino acid corresponding to position 54 is a substitution of Lys with Arg (K54R).

### Claim 49 (depends on 45)

The method of claim 45, wherein the one or more amino acid modifications comprises a modification of an amino acid corresponding to position 68 of the full length wild-type mouse EKLF polypeptide.

### Claim 50 (depends on 40)

The method of claim 40, wherein the first active agent is administered, and the one or more activities comprises reduced translocation from the cytoplasm to the nucleus.

### Claim 51 (depends on 40)

The method of claim 40, wherein the first active agent is administered, and the one or more activities comprises a modified transactivator activity or a modified repressor activity.

### Claim 52 (depends on 40)

The method of claim 40, wherein the first active agent binds to the endogenous EKLF polypeptide.

### Claim 53 (depends on 52)

The method of claim 52, wherein the first active agent is a small organic molecule or a polypeptide, optionally an antibody or functional fragment thereof.

### Claim 54 (depends on 52)

The method of claim 52, wherein binding of the first active agent to the endogenous EKLF polypeptide inhibits its translocation from the cytoplasm to the nucleus.

### Claim 55 (depends on 52)

The method of claim 52, wherein binding of the first active agent to the endogenous EKLF polypeptide modifies its transactivator activity or modifies its repressor activity.

### Claim 56 (depends on 40)

The method of claim 40, wherein the polypeptide or the nucleic acid is administered, and the method further comprises administering a second active agent that inhibits expression of the endogenous EKLF polypeptide.

### Claim 57 (depends on 56)

The method of claim 56, wherein the second active agent is a nucleic acid molecule, optionally an antisense RNA, siRNA, shRNA or miRNA that binds an mRNA or complement thereof that encodes the endogenous EKLF polypeptide.

### Claim 58 (depends on 40)

The method of claim 40, wherein the method results in reduced graying of the hair, increased motor coordination, increased muscle strength, reduced muscle weakness, increased motor coordination, reduced osteoporosis, greater bone volume, greater bone density, greater trabecular number, reduced trabecular spacing, or reduced loss of balance in the subject.

### Claim 59 (depends on 40)

The method of claim 40, wherein the subject is a mammal, optionally a human.

### Claim at position 57

60-72. (canceled)