Modalities for the treatment of degenerative diseases of the retina

• US 9,040,770 B2
• Filed: 05/18/2010
• Issued: 05/26/2015
• Est. Priority Date: 01/23/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Ocata Therapeutics, Inc. (Astellas Pharma Incorporated)
• Inventors: Klimanskaya, Irina, Lanza, Robert
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US12/781,929
• Publication Number:
  US 20100299765A1
• Time in Patent Office: 1,834 Days
• US Class Current: 800/3
• CPC Class Codes: 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
• CPC Class Codes: 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
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61K 9/0048 Eye, e.g. artificial tears
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 27/00 Drugs for disorders of the senses
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/062 Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062

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

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.

2. 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.

3. 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.

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.

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

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.

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

8. 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.

9. 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.

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

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

12. 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.

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

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

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

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