# US 10,500,232 B2 · Small molecule cellular reprogramming to generate neuronal cells

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- Number: US 10,500,232 B2
- Document ID: US10500232B2
- Status: Granted
- Kind: B2
- Application number: 14/913,549
- Filed: 2014-08-26
- Published: 2016-09-01
- Granted: 2019-12-10
- Earliest claimed: 2013-08-26
- Assignees: The J. David Gladstone Ins., a testamentary trust established under the Will of J. David Gladstone
- Applicants: The J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Gladstone
- Inventors: Changsheng Lin; Sheng Ding
- Web page: https://patentgrep.com/patent/US10500232B2

## 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 31/165: having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K 31/167: having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
- A61K 31/192: having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid {(cannabinoids A61K31/658)}
- A61K 31/196: the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
- A61K 31/198: Alpha-amino acids, e.g. alanine or edetic acid [EDTA] (betaine A61K31/205; proline A61K31/401; tryptophan A61K31/405; histidine A61K31/4172; peptides not degraded to individual amino acids A61K38/00)
- A61K 31/222: with compounds having aromatic groups, e.g. dipivefrine, ibopamine
- A61K 31/352: condensed with carbocyclic rings, e.g. methantheline {(cannabinoids A61K31/658)}
- A61K 31/4015: having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
- A61K 31/4402: only substituted in position 2, e.g. pheniramine, bisacodyl
- A61K 31/4406: only substituted in position 3, e.g. zimeldine (nicotinic acid A61K31/455)
- A61K 31/4439: containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole (nicotine A61K31/465)
- A61K 31/46: 8-Azabicyclo [3.2.1] octane; Derivatives thereof, e.g. atropine, cocaine
- A61K 31/506: not condensed and containing further heterocyclic rings
- A61K 31/688: both hydroxy compounds having nitrogen atoms, e.g. sphingomyelins
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- C12N 5/0619: {Neurons}
- C12N 2501/01: Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N 2501/395: Thyroid hormones
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/1307: from adult fibroblasts
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Compositions and methods are described herein for chemically inducing cells to change their differentiation state and become neuronal cells.

## Claims

### Claim 1

A method of generating a neuronal cell comprising incubating a selected population of non-pluripotent, non-neuronal cells with the composition comprising CHIR99021, an ALK5 and ALK4 inhibitor, a histone deacetylase inhibitor, and a p300 activator, to thereby generate neuronal cells, neuronal progenitor cells, or a combination thereof without de-differentiating the population of non-pluripotent, non-neuronal cells into a population of cells exhibiting pluripotent stem cell characteristics.

### Claim 2 (depends on 1)

The method of claim 1, wherein the selected population of non-pluripotent, non-neuronal cells contacted with the composition comprises a somatic cell, a differentiated cell, a heterogeneous mixture of cells, or a combination thereof.

### Claim 3 (depends on 2)

The method of claim 2, wherein the population of cells comprises cells from the spinal cord or brain.

### Claim 4 (depends on 1)

The method of claim 1, wherein the selected population of non-pluripotent, non-neuronal cells comprises newborn cord blood cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein the composition comprises about 0.01 micromolar to about 20 millimolar CHIR99021, about 0.01 micromolar to about 20 millimolar ALK5 and ALK4 inhibitor, about 0.001 micromolar to about 20 millimolar histone deacetylase inhibitor, and about 0.001 micromolar to about 20 millimolar p300 activator.

### Claim 6 (depends on 1)

The method of claim 1, further comprising administering the neuronal cells, neuronal progenitor cells, or a combination thereof to a subject.

### Claim 7 (depends on 6)

The method of claim 6, wherein the population of non-pluripotent, non-neuronal cells comprises allogenic or autologous cells.

### Claim 8 (depends on 6)

The method of claim 6, wherein the subject suffers or is suspected of suffering from Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, multiple sclerosis, Primary lateral sclerosis (PLS), Progressive bulbar palsy, Pseudobulbar palsy, Primary lateral sclerosis (PLS), Progressive muscular atrophy, Spinal muscular atrophy (SMA), Werdnig-Hoffmann disease, Kugelberg-Welander disease, Fazio-Londe disease, Huntington's disease, Kennedy's disease also known as progressive spinobulbar muscular atrophy, hereditary spastic paraplegia (HSP), congenital SMA with arthrogryposis, Post-polio syndrome (PPS), traumatic spinal cord injury, progressive pseudobulbar palsy, progressive muscular atrophy, stroke, head trauma, spinal cord injury, or a combination thereof.

### Claim 9 (depends on 1)

The method of claim 1, wherein the composition further comprises a PDE4 inhibitor, an Adenylyl cyclase agonist, a retinoic acid receptor γ agonist, a 5-HT3 antagonist, and a metabotropic glutamate (mGlu)receptor agonist.

### Claim 10 (depends on 1)

The method of claim 1, wherein the composition further comprises one or more of the following compounds: a ROCK inhibitor, a neuronal differentiation enhancer, an omega-3 fatty acid, an A3 adenosine receptor agonist or an L-type calcium channel blocker.

### Claim 11 (depends on 1)

The method of claim 1, wherein the composition comprises the following compounds: CHIR99021, SB431542, MS275, N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxybenzamide (CTB), Rolipram, Forskolin, CD1530, Tropanyl-3,5-dimethylbenzoate, and (1S,3R)-1-Aminocyclopentane-1,3-dic-arboxylic acid (ACPD).

### Claim 12 (depends on 1)

The method of claim 1, wherein the composition comprises the following compounds: CHIR99021, SB431542, MS275, N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxybenzamide (CTB), Rolipram, Forskolin, CD1530, Tropanyl-3,5-dimethylbenzoate, and (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (ACPD).

### Claim 13 (depends on 1)

The method of claim 1, wherein the composition comprises the following compounds: SB431542, Trichostatin A, Rolipram, CHIR99021, and N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB).