# US 2014/0193341 A1 · Human induced neuronal cells

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2014/0193341 A1
- Document ID: US20140193341A1
- Status: Published application
- Kind: A1
- Application number: 13/980,832
- Filed: 2012-01-19
- Published: 2014-07-10
- Earliest claimed: 2011-01-19
- Inventors: Asa Abeliovich; Liang Qiang
- Web page: https://patentgrep.com/patent/US20140193341A1

## CPC

- C12N 5/0619: {Neurons}
- A61K 49/0008: {Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure}
- G01N 33/5044: {involving specific cell types}
- G01N 33/5058: {Neurological cells}
- 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 2501/13: Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
- C12N 2501/60: Transcription factors
- C12N 2506/1307: from adult fibroblasts
- C12N 2510/00: Genetically modified cells
- C12N 2533/32: Polylysine, polyornithine
- G01N 2800/28: Neurological disorders
- G01N 2800/2821: Alzheimer
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A method for reprogramming a fibroblast into a human induced neuronal cell (hIN) is described. The method comprises expressing heterologous reprogramming factors Bm2, Myt11, Zic1, Olig2, Asc11 or any combination thereof, in said fibroblast, and culturing the fibroblast in a medium comprising BDNF, NT3, GeM or any combination thereof. Biomarkers describing the obtained hiN cells are also presented. In another aspect, methods for screening compounds using the hiN cells is described.

## Claims

### Claim 1

A method for reprogramming a human fibroblast into a human induced neuronal (hiN) cell, the method comprising:
a) expressing heterologous Brn2, Myt1l, Zic1, Olig2 and Ascl1 in the fibroblast,
b) culturing the fibroblast in a medium comprising BDNF, NT3, and GCM such that the fibroblast is reprogrammed into a hiN.

### Claim 2

An induced human neuron obtained by reprogramming a human fibroblast to express heterologous Brn2, Myt1l, Zic1, Olig2 and Ascl1 and culturing said fibroblast in a medium comprising BDNF, NT3, and GCM.

### Claim 3 (depends on 1, 2)

The hiN cell of claim 1 or 2, wherein the hiN cell has increased expression of synaptophysin, Tau, Map2, NeuN, Tuj1, NCAM, neurofilament 160 kd, or any combination thereof as compared to a fibroblast that has not been reprogrammed.

### Claim 4 (depends on 1, 2)

The hiN cell of claim 1 or 2, wherein the hiN cell has increased expression of vGLUT1, GAD65, TBR1, or any combination thereof as compared to a fibroblast that has not been reprogrammed.

### Claim 5 (depends on 1, 2)

The hiN cell of claim 1 or 2, wherein the hiN cell does not have increased expression of GFP, FSP1, Pax 6, Nestin, Otx2, En2, FoxG1, or any combination thereof as compared to a fibroblast that has not been reprogrammed.

### Claim 6 (depends on 1, 2)

The method of claim 1, 2, or 28 wherein the skin fibroblast is reprogrammed into a hiN cell without undergoing reprogramming into a neural progenitor intermediate cell.

### Claim 7 (depends on 1, 2)

The method of claim 1, 2, or 28 wherein the fibroblast is from a biological sample.

### Claim 8 (depends on 1, 2, 28)

The method of claim 1, 2 or 28, wherein the fibroblast is from a subject having a neurodegenerative disorder.

### Claim 9 (depends on 8)

The method of claim 8, wherein the neurodegenerative disorder is Alzheimer's disease.

### Claim 10 (depends on 8)

The method of claim 8, wherein the neurodegenerative disorder is Familial Alzheimer's disease.

### Claim 11 (depends on 8)

The method of claim 8, wherein the neurodegenerative disorder is Sporadic Alzheimer's disease.

### Claim 12 (depends on 1, 2, 28)

The method of claim 1, 2 or 28, wherein the fibroblast is a skin fibroblast.

### Claim 13 (depends on 1, 2, 28)

The method of claim 1, 2 or 28, wherein the fibroblast comprises a PS1 A246E allele.

### Claim 14 (depends on 1, 2, 28)

The method of claim 1, 2 or 28, wherein the fibroblast comprises a PS2 N141I allele.

### Claim 15 (depends on 1, 2, 28)

The method of claim 1, 2 or 28, wherein the fibroblast is from a subject having a neurodegenerative disorder and wherein the hiN cell has increased Aβ40, increased Aβ42, increased sAPPβ accumulation, increased APP-positive puncta within cell soma, enlarged APP positive puncta, enlarged early endosomes, enlarged late endosomes, or any combination thereof as compared to a fibroblast from a subject having a neurodegenerative disorder that has not been reprogrammed.

### Claim 16 (depends on 1, 2, 28)

The method of claim 1, 2 or 28, wherein the medium further comprises drosomorphin.

### Claim 17

A method for determining whether a test compound can ameliorate a condition associated with a neurodegenerative disorder, the method comprising:
a) isolating a fibroblast cell from a subject having a neurodegenerative disorder;
b) expressing heterologous Brn2, Myt1l, Zic1, Olig2 and Ascl1 in the fibroblast,
c) culturing the fibroblast in a medium comprising BDNF, NT3, and GCM such that the fibroblast is reprogrammed into a hiN cell derived from a subject having a neurodegenerative disorder,
d) contacting the hiN cell with a test compound,
e) measuring an indicator of the neurodegenerative disorder in the hiN, and
f) comparing the measured indicator of the neurodegenerative disorder in the hiN cell contacted with a test compound, with a second hiN cell of step (c) wherein a reduction in the indicator of the neurodegenerative disorder in hiN cell contact with the test compound indicates that the test compound can ameliorate a condition associated with a neurodegenerative disorder.

### Claim 18

A method for determining whether a test compound can ameliorate a condition associated with a neurodegenerative disorder, the method comprising:
a) isolating a fibroblast cell from a subject having a neurodegenerative disorder;
b) expressing heterologous Brn2, Myt1l, Zic1, Olig2 and Ascl1 in the fibroblast,
c) culturing the fibroblast in a medium comprising BDNF, NT3, and GCM such that the fibroblast is reprogrammed into a hiN cell derived from a subject having a neurodegenerative disorder,
d) implanting the hiN cell into a host organism,
d) administering a test compound to the host organism,
e) measuring an indicator of the neurodegenerative disorder in the host organism, and
f) comparing the measured indicator of the neurodegenerative disorder in the host organism administered with the test compound, with a second host organism which has not been implanted with a hiN cell derived from a subject having a neurodegenerative disorder, wherein a reduction in the indicator of the neurodegenerative disorder in the host organism administered with the test compound indicates that the test compound can ameliorate a condition associated with a neurodegenerative disorder.

### Claim 19 (depends on 17, 18, 36, 37)

The method of claim 17, 18, 36 or 37 wherein the indicator of the neurodegenerative disorder is increased Aβ40, increased Aβ42, increased sAPPβ accumulation, increased APP-positive puncta within cell soma, enlarged APP positive puncta, enlarged early endosomes, enlarged late endosomes, or any combination thereof as compared to a hiN cell obtained by reprogramming a fibroblast from a subject not having a neurodegenerative disorder.

### Claim 20 (depends on 17, 18, 36, 37)

The method of claim 17, 18, 36 or 37 wherein the neurodegenerative disorder is Alzheimer's disease.

### Claim 21 (depends on 18)

The method of claim 18, wherein the host organism is a mammal.

### Claim 22 (depends on 18)

The method of claim 18, wherein the host organism is a mouse.

### Claim 23 (depends on 22)

The method of claim 22, wherein the mouse is an immunocompromised mouse.

### Claim 24

A human cell comprising a nucleic acid vector encoding Brn2, Myt1l, Zic1, Olig2 and Ascl1.

### Claim 25

An isolated human cell comprising a nucleic acid vector encoding Brn2, Ascl1 and Zic1.

### Claim 26 (depends on 24, 25)

The human cell of claim 24 or 25, wherein the human cell is a fibroblast.

### Claim 27 (depends on 24, 25)

The human cell of claim 24 or 25, wherein the human cell is a hiN.

### Claim 28

A method for reprogramming a human fibroblast into a human induced neuronal (hiN) cell, the method comprising:
a) expressing heterologous Brn2 and Ascl1 in the fibroblast,
b) culturing the fibroblast in a medium comprising BDNF, NT3, and GCM such that the fibroblast is reprogrammed into a hiN.

### Claim 29 (depends on 28)

The method of claim 28, further comprising expressing heterologous Myt1l, Zic1 or Olig2.

### Claim 30 (depends on 28)

The method of claim 28, further comprising expressing heterologous Zic1.

### Claim 31

An induced human neuron obtained by reprogramming a human fibroblast to express heterologous Brn2 and Ascl1 and culturing said fibroblast in a medium comprising BDNF, NT3, and GCM.

### Claim 32 (depends on 31)

The induced human neuron of claim 31 obtained by reprogramming a human fibroblast to further express heterologous Myt1l, Zic1 or Olig2.

### Claim 33 (depends on 31)

The induced human neuron of claim 31 obtained by reprogramming a human fibroblast to further express heterologous Zic1.

### Claim 34

An expression vector comprising nucleic acids encoding Brn2, Ascl1 and Zic1.

### Claim 35 (depends on 34)

The expression vector of claim 34, wherein the vector is sufficient to reprogram a human fibroblast into a human induced neuronal (hiN).

### Claim 36

A method for determining whether a test compound can ameliorate a condition associated with a neurodegenerative disorder, the method comprising:
a) expressing heterologous Brn2 and Ascl1 in a fibroblast isolated from a subject having a neurodegenerative disorder,
b) culturing the fibroblast in a medium comprising BDNF, NT3, and GCM such that the fibroblast is reprogrammed into a hiN cell derived from a subject having a neurodegenerative disorder,
c) contacting the hiN cell with a test compound,
d) measuring an indicator of the neurodegenerative disorder in the hiN, and
e) comparing the measured indicator of the neurodegenerative disorder in the hiN cell contacted with a test compound, with a second hiN cell of step (b) wherein a reduction in the indicator of the neurodegenerative disorder in hiN cell contact with the test compound indicates that the test compound can ameliorate a condition associated with a neurodegenerative disorder.

### Claim 37

A method for determining whether a test compound can ameliorate a condition associated with a neurodegenerative disorder, the method comprising:
a) expressing heterologous Brn2 and Ascl1 in a fibroblast isolated from a subject having a neurodegenerative disorder,
b) culturing the fibroblast in a medium comprising BDNF, NT3, and GCM such that the fibroblast is reprogrammed into a hiN cell derived from a subject having a neurodegenerative disorder,
c) implanting the hiN cell into a host organism,
d) administering a test compound to the host organism,
e) measuring an indicator of the neurodegenerative disorder in the host organism, and
f) comparing the measured indicator of the neurodegenerative disorder in the host organism administered with the test compound, with a second host organism which has not been implanted with a hiN cell derived from a subject having a neurodegenerative disorder, wherein a reduction in the indicator of the neurodegenerative disorder in the host organism administered with the test compound indicates that the test compound can ameliorate a condition associated with a neurodegenerative disorder.

### Claim 38 (depends on 36, 37)

The method of claim 36 or 37 further comprising expressing heterologous Myt1l, Zic1 or Olig2.

### Claim 39 (depends on 36, 37)

The method of claim 36 or 37 further comprising expressing heterologous Zic1.