# US 10,865,419 B2 · Orally administered plastid expressed cholera toxin B subunit-exendin 4 as treatment for type 2 diabetes

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- Number: US 10,865,419 B2
- Document ID: US10865419B2
- Status: Granted
- Kind: B2
- Application number: 14/353,980
- Filed: 2012-10-24
- Published: 2015-01-29
- Granted: 2020-12-15
- Earliest claimed: 2011-10-24
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: University of Central Florida Research Foundation, Inc.
- Inventors: Henry Daniell
- Web page: https://patentgrep.com/patent/US10865419B2

## CPC

- C12N 15/8214: {Plastid transformation}
- A61K 36/28: Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K 36/81: Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
- A61K 38/164: {from bacteria}
- A61K 38/26: Glucagons
- A61K 38/51: Lyases (4)
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/04: Anorexiants; Antiobesity agents
- C07K 14/605: Glucagons
- A61K 38/162: {from virus}
- A61K 38/2278: {Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)}
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Disclosed herein are compositions and methods for treating Type II diabetes. The compositions comprise plant expressed Exendin 4. Particularly exemplified are plant derived compositions that include a CTB-EX4 conjugate that is bioencapsulated in chloroplasts.

## Claims

### Claim 1

A plant plastid comprising a plastid genome transformed with a heterologous expression cassette consisting of, as operably linked components in the 5′ to the 3′ direction of translation, a flanking sequence having homology to a trnl region in the plastid genome, a Prrn promoter operative in said plastid, an aadA selectable marker sequence or an antibiotic free selectable marker sequence, a psbA 5′ untranslated region sequence, a cholera toxin B subunit(CTB)-Exendin fusion gene sequence, a psbA 3′ untranslated region sequence, and a flanking sequence having homology to a trnA region in the plastid genome, said expression cassette being integrated into said plastid genome such that said CTB-Exendin is expressed in and present in said plastid, wherein the CTB-Exendin 4 fusion protein has a hinge region, and a furin cleavage site between the CTB and the Exendin 4, and wherein the hinge-furin cleavage site-exendin portion consists of SEQ ID NO: 7, said plastid being suitable for oral administration of said CTB-Exendin 4 to a subject in need thereof.

### Claim 2 (depends on 1)

An edible plant cell comprising the plastid of claim 1.

### Claim 3 (depends on 2)

A lettuce, apple, tomato or carrot plant comprising the plant cell of claim 2.

### Claim 4 (depends on 2)

A method of retarding the development of or treating Type II diabetes in a subject in need thereof, said method comprising orally administering to said subject a composition comprising plastid transformed plant cells of claim 2, said composition comprising a plant remnant.

### Claim 5 (depends on 4)

The method of claim 4, wherein said plant remnant comprises rubisco.

### Claim 6 (depends on 2)

The plant cell of claim 2, wherein said plant cell is homoplasmic and comprises an antibiotic free selectable marker.

### Claim 7 (depends on 3)

Transplastomic progeny or seeds of the plant of claim 3, said progeny or seeds having a plastid genome transformed with said CTB-exendin 4 expression cassette.

### Claim 8 (depends on 3)

The plant of claim 3, wherein said plant is homoplasmic and stably transformed.

### Claim 9 (depends on 3)

A process for producing a CTB-Exendin 4 polypeptide, said process comprising:
a) generating the lettuce plant of claim 3, which is Lactuca sativa;
b) growing said stably transformed Lactuca sativa plant under conditions suitable for expression and production of said CTB-Exendin polypeptide; and
c) optionally homogenizing material of said stably transformed Lactuca sativa plant to produce homogenized material.

### Claim 10 (depends on 9)

The method of claim 9, further comprising purifying CTB-Exendin 4 from said homogenized material.

### Claim 11 (depends on 9)

The method of claim 9, wherein said homogenized material is dried to produce a powder.

### Claim 12 (depends on 11)

The method of claim 11, further comprising encapsulating said powder.

### Claim 13 (depends on 2)

The plant cell of claim 2, which is freeze dried and retains therapeutic effectiveness when stored at room temperature for at least 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 weeks.

### Claim 14 (depends on 4)

The method of claim 4, wherein said subject in need exhibits at least one symptom comprising ketoacidosis, Kussmaul breathing, nausea, vomiting, abdominal pain, polyuria, polydipsia, polyphagia, increased or decreased insulin levels, and elevated serum glucose.

### Claim 15 (depends on 4)

The method of claim 4, wherein said patient in need exhibits impaired glucose tolerance.

### Claim 16 (depends on 4)

The method of claim 4, wherein said patient in need exhibits fasting glucose levels from 100 to 125 mg/dL (5.6 to 6.9 mmol/L).

### Claim 17 (depends on 4)

The method of claim 4, wherein said patient in need exhibits plasma glucose at or above 140 mg/dL (7.8 mmol/L), but not over 200 mg/dL (11.1 mmol/L), two hours after a 75 g oral glucose load.