# US 9,040,770 B2 · Modalities for the treatment of degenerative diseases of the retina

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- Number: US 9,040,770 B2
- Document ID: US9040770B2
- Status: Granted
- Kind: B2
- Application number: 12/781,929
- Filed: 2010-05-18
- Published: 2010-11-25
- Granted: 2015-05-26
- Earliest claimed: 2004-01-23
- Assignees: Ocata Therapeutics, Inc.
- Applicants: Irina Klimanskaya; Robert Lanza
- Inventors: Irina Klimanskaya; Robert Lanza
- Web page: https://patentgrep.com/patent/US9040770B2

## CPC

- A61K 35/30: Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
- A61K 9/0048: {Eye, e.g. artificial tears}
- A61K 35/44: Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 9/14: Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
- A61P 25/16: Anti-Parkinson drugs
- A61P 27/00: Drugs for disorders of the senses
- A61P 27/02: Ophthalmic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 5/062: {Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain}
- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- C12N 2501/01: Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/33: Insulin
- C12N 2506/02: from embryonic cells
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This invention relates to methods for improved cell-based therapies for retinal degeneration and for differentiating human embryonic stem cells and human embryo-derived into retinal pigment epithelium (RPE) cells and other retinal progenitor cells.

## Claims

### Claim 1

A method of producing isolated human RPE cells, comprising:
(a) culturing a multilayer culture of human pluripotent cells that express Oct-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-I-60, and TRA-I-81 under adherent conditions for a sufficient time for the appearance of a pigmented cell population having a cobblestone, polygonal, epithelial-like appearance and comprising cells that express RPE65 and bestrophin; and
(b) isolating the pigmented cell population from the resultant cell culture, thereby obtaining human RPE cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the multilayer culture of step (a) is produced by allowing pluripotent cells to overgrow and form a thick multilayer of cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein the multilayer culture of step (a) is produced by allowing pluripotent cells to overgrow and form a thick multilayer of cells on a substrate comprising laminin, fibronectin, collagen I, collagen IV, or MATRIGEL (TM) a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.

### Claim 4

A method of producing isolated human RPE cells, comprising:
(a) culturing embryoid bodies comprising human pluripotent cells that express Oct-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-I-60, and TRA-I-81for a sufficient time for the appearance of a pigmented cell population having a cobblestone polygonal epithelial-like appearance and comprising cells that express RPE65 and bestrophin; and
(b) isolating the pigmented cell population from the resultant cell culture, thereby obtaining human RPE cells.

### Claim 5 (depends on 4)

The method of claim 4, wherein said embryoid bodies are produced by culturing pluripotent cells under low adherent conditions.

### Claim 6

A method of producing isolated human RPE cells, comprising:
(a) differentiating in vitro human pluripotent cells that express Oct-4, alkaline phosphatase,SSEA-3, SSEA-4, TRA-I-60, and TRA-I-81to obtain a culture of a pigmented cell population having a cobblestone polygonal epithelial-like appearance and comprising cells that express RPE65 and bestrophin; and
(b) isolating the pigmented cell population from the resultant cell culture, thereby obtaining human RPE cells.

### Claim 7 (depends on 6)

The method of claim 6, further comprising cryopreserving said human RPE cells.

### Claim 8 (depends on 7)

The method of claim 7, wherein said cryopreserved human RPE cells are suitable for prevention or treatment of a retinal disease or condition in a human subject in need thereof.

### Claim 9 (depends on 6)

The method of claim 6, wherein said human RPE cells are suitable for prevention or treatment of a retinal disease or condition in a human subject in need thereof.

### Claim 10 (depends on 9)

The method of claim 9, wherein said human RPE cells are disposed on a substrate or matrix.

### Claim 11 (depends on 10)

The method of claim 10, wherein said substrate or matrix comprises at least one of gelatin, fibronectin, laminin, collagen, or extracellular matrix.

### Claim 12 (depends on 6)

The method of claim 6, wherein said human RPE cells are suitable for prevention or treatment of a retinal disease or condition comprising retinitis pigmentosa, RPE detachment, retinal dysplasia, retinal atrophy, retinopathy, macular dystrophy, cone dystrophy, cone-rod dystrophy, Malattia Leventinese, Doyne honeycomb dystrophy, Sorsby's dystrophy, Stargardt disease, pattern/butterfly dystrophies, Best vitelliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid streaks, or a toxic maculopathy in a human subject in need thereof.

### Claim 13 (depends on 6)

The method of claim 6, wherein RPE65 expression is detected by RT-PCR.

### Claim 14 (depends on 6)

The method of claim 6, wherein bestrophin expression is detected by Western blot or immunostaining.

### Claim 15 (depends on 6)

The method of claim 6, wherein said human RPE cells are dispersed in a suspension.

### Claim 16 (depends on 6)

The method of claim 6, wherein said human RPE cells are passaged one or more times.