Modalities for the treatment of degenerative diseases of the retina

• US 9,730,962 B2
• Filed: 04/21/2015
• Issued: 08/15/2017
• Est. Priority Date: 01/23/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Inventors: Klimanskaya, Irina V., Lanza, Robert P.
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US14/692,191
• Publication Number:
  US 20150328261A1
• Time in Patent Office: 847 Days
• 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 generating a retinal pigment epithelium (RPE) cell preparation suitable for transplantation therapy comprising:

(a) obtaining RPE cells that have a cobblestone polygonal epithelial-like cellular morphology and pigmentation, wherein the RPE cells include cells that express RPE65 and bestrophin;

(b) dispersing the RPE cells and culturing the dispersed RPE cells under adherent conditions in the presence of basic FGF, wherein the cultured RPE cells dedifferentiate and proliferate, losing pigmentation and epithelial-like morphology;

(c) culturing the dedifferentiated cells in the absence of basic FGF, wherein the cells form an RPE cell monolayer, become quiescent, and regain pigmentation and epithelial-like morphology, and wherein the RPE cell monolayer includes cells that express RPE65 and bestrophin; and

(d) expanding the RPE cells by repeating steps (b) and (c) through multiple passages.

2. The method of claim 1, wherein steps (b) and (c) are repeated to provide for three passages.

3. The method of claim 1, wherein the RPE cell preparation is a human RPE cell preparation.

4. The method of claim 1, wherein the dedifferentiated cells that have lost pigmentation and epithelial morphology are positive for mitf, Pax6, tubulin beta III and nestin.

5. The method of claim 1, wherein the RPE cell monolayer includes cells that are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65.

6. The method of claim 1, wherein the RPE cell monolayer is characterized by the absence of any cells expressing at least one ES cell marker selected from the group consisting of Oct4, Sox2, and TDGF-1.

7. The method of claim 1, further comprising

(e) isolating a pure preparation of human RPE cells suitable for transplantation therapy after the multiple passages of (d).

8. The method of claim 7, wherein the preparation of RPE cells suitable for transplantation therapy is provided with a matrix or substrate.

9. The method of claim 7, wherein the preparation of RPE cells suitable for transplantation therapy is provided in a suspension.