# US 2021/0171902 A1 · Methods and compositions for the production of oligodendrocyte progenitor cells

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- Number: US 2021/0171902 A1
- Document ID: US20210171902A1
- Status: Published application
- Kind: A1
- Application number: 16/768,348
- Filed: 2018-11-30
- Published: 2021-06-10
- Earliest claimed: 2017-12-01
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Parastoo Khoshakhlagh; Hon Man Alex Ng; George M. Church
- Web page: https://patentgrep.com/patent/US20210171902A1

## CPC

- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 5/0622: {Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells}
- C12N 15/86: Viral vectors
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 2501/60: Transcription factors
- C12N 2501/602: Sox-2
- C12N 2501/603: Oct-3/4
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2800/90: Vectors containing a transposable element
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are pluripotent stem cells comprising particular combinations of transcription factor for the production of oligodendrocyte progenitor cells (OPCs). Also provided herein are methods of producing the pluripotent stem cells and methods of using the pluripotent stem cells to produce (OPCs) and oligodendrocytes.

## Claims

### Claim 1

A pluripotent stem cell comprising at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1), (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4).

### Claim 2 (depends on 1)

The pluripotent stem cell of claim 1 comprising OCT4, SOX9, NKX6.1, and NKX6.2.

### Claim 3 (depends on 1)

The pluripotent stem cell of claim 1 comprising OLIG2, OCT4, SOX9, NKX6.1, NKX6.2.

### Claim 4 (depends on 1, 2, 3)

The pluripotent stem cell of any one of claims 1-3 further comprising at least one cytokine selected from IL-10 and IFNβ.

### Claim 5 (depends on 1, 2, 3, 4)

The pluripotent stem cell of any one of claims 1-4 further comprising IL-10 and IFNβ.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The pluripotent stem cell of any one of claims 1-5, wherein at least one of the transcription factors and/or at least one of the cytokines is encoded by a nucleotide sequence of an engineered nucleic acid.

### Claim 7 (depends on 6)

The pluripotent stem cell of claim 6, comprising 5 to 50 copies of the nucleotide sequence encoding at least one transcription factor.

### Claim 8 (depends on 7)

The pluripotent stem cell of claim 7, wherein the engineered nucleic acid is a modified engineered nucleic acid.

### Claim 9 (depends on 6, 7, 8)

The pluripotent stem cell of any one of claims 6-8, wherein the engineered nucleic acid comprises DNA or RNA.

### Claim 10 (depends on 9)

The pluripotent stem cell of claim 9, wherein the RNA is messenger (mRNA).

### Claim 11 (depends on 6, 7, 8, 9, 10)

The pluripotent stem cell of any one of claims 6-10, wherein the engineered nucleic acid is operably linked to a promoter.

### Claim 12 (depends on 11)

The pluripotent stem cell of claim 11, wherein the promoter is an inducible promoter.

### Claim 13 (depends on 6, 7, 8, 9, 10, 11, 12)

The pluripotent stem cell of any one of claims 6-12, wherein the engineered nucleic acid is present on a PIGGYBAC™ transposon vector.

### Claim 14 (depends on 13)

The pluripotent stem cell of claim 13, wherein the PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeat sequences flanking at least one nucleotide sequence encoding at least one of the transcription factors.

### Claim 15 (depends on 13, 14)

The pluripotent stem cell of claim 13 or 14 further comprising a PIGGYBAC™ transposase or an engineered nucleic acid encoding a PIGGYBAC™ transposase.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The pluripotent stem cell of any one of claims 1-15, wherein the engineered nucleic acid further encodes a selection marker protein.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The pluripotent stem cell of any one of claims 1-16, wherein the pluripotent stem cell is an induced pluripotent stem cell.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

The pluripotent stem cell of any one of claims 1-17, wherein the pluripotent stem cell is a human pluripotent stem cell.

### Claim 19

A method comprising:
introducing into pluripotent stem cells at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4), optionally wherein each transcription factor is introduced on an engineered nucleic acid comprising a nucleotide sequence encoding the transcription factor, and optionally wherein at least two transcription factors are encoded by the same engineered nucleic acid; and
culturing the pluripotent stem cells to produce a population of cells comprising oligodendrocyte progenitor cells (OPCs).

### Claim 20

A method comprising:
introducing into pluripotent stem cells at least one engineered nucleic acid encoding IL-10, IFNβ, or a combination thereof;
introducing into the pluripotent stem cells at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4), optionally wherein each transcription factor is introduced on an engineered nucleic acid comprising a nucleotide sequence encoding the transcription factor, and optionally wherein at least two transcription factors are encoded by the same engineered nucleic acid; and
optionally culturing the pluripotent stem cells to produce a population of cells comprising oligodendrocyte progenitor cells (OPCs).

### Claim 21 (depends on 19, 20)

The method of claim 19 or 20, comprising introducing 5 to 50 copies of at least one nucleotide sequence encoding at least one transcription factor.

### Claim 22 (depends on 19, 20, 21)

The method of any one of claims 19-21, wherein OCT4, SOX9, NKX6.1, and NKX6.2 are introduced into the pluripotent stem cells.

### Claim 23 (depends on 19, 20, 21, 22)

The method of any one of claims 19-22, wherein at least 40% of the cells of the population express O4.

### Claim 24 (depends on 19, 20, 21, 23)

The method of any one of claim 19-21 or 23, wherein OLIG2, OCT4, SOX9, NKX6.1, NKX6.2 are introduced into the pluripotent stem cells.

### Claim 25 (depends on 19, 20, 21, 22, 24)

The method of any one of claim 19-22 or 24, wherein at least 25% of the cells of the population express O4.

### Claim 26 (depends on 19, 20, 21, 22, 23, 24, 25)

The method of any one of claims 19-25, wherein the cells are cultured for 1 to 10 days.

### Claim 27 (depends on 19, 20, 21, 22, 23, 24, 25, 26)

The method of any one of claims 19-26, wherein at least 25% of the cells of the population express O4 following 4 days of culture.

### Claim 28 (depends on 19, 21, 22, 23, 24, 25, 26, 27)

The method of any one of claim 19 or 21-27, wherein at least one engineered nucleic acid encoding a cytokine selected from IL-10 and IFNβ is introduced into the pluripotent stem cells.

### Claim 29 (depends on 28)

The method of claim 28, wherein at least one engineered nucleic acid encoding IL-10 and at least one engineered nucleic acid encoding IFNβ are introduced into the pluripotent stem cells, optionally wherein IL-10 and IFNβ are encoded by the same engineered nucleic acid.

### Claim 30 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)

The method of any one of claims 20-29, wherein the OPCs secrete IL-10.

### Claim 31 (depends on 30)

The method of claim 30, wherein the OPCs secrete IL-10 following 3, 4, 5, or 7 days of culturing.

### Claim 32 (depends on 31)

The method of claim 31, wherein the level of IL-10 secreted by the OPCs is at least 50-fold higher, at least 100-fold, or at least 1000-fold higher than control cells, wherein the control cells are (a) naturally-occurring OPCs, (b) OPCs that are not engineered to express IL-10, and/or (c) OPCs that are engineered to express IL-10 under an inducible promoter and are cultured in the absence of an inducing agent.

### Claim 33 (depends on 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)

The method of any one of claims 19-32, further comprising culturing the OPCs to produce oligodendrocytes.

### Claim 34 (depends on 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)

The method of any one of claims 19-33, wherein at least one engineered nucleic acid is a modified engineered nucleic acid.

### Claim 35 (depends on 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)

The method of any one of claims 19-34, wherein at least one engineered nucleic acid comprises DNA or RNA.

### Claim 36 (depends on 35)

The method of claim 35, wherein the RNA is messenger (mRNA).

### Claim 37 (depends on 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)

The method of any one of claims 19-36, wherein at least one engineered nucleic acid is operably linked to a promoter.

### Claim 38 (depends on 37)

The method of claim 37, wherein the promoter is an inducible promoter.

### Claim 39 (depends on 38)

The method of claim 38, wherein the pluripotent stem cells are cultured in the presence of an agent that induces the inducible promoter to activate transcription of the transcription factors and/or cytokines.

### Claim 40 (depends on 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The method of any one of claims 19-39, wherein at least one engineered nucleic acid is present on a PIGGYBAC™ transposon vector.

### Claim 41 (depends on 40)

The method of claim 40, wherein the PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeat sequences flanking at least one nucleotide sequence encoding at least one of the transcription factors.

### Claim 42 (depends on 40, 41)

The method of claim 40 or 41 further comprising a PIGGYBAC™ transposase or an engineered nucleic acid encoding a PIGGYBAC™ transposase.

### Claim 43 (depends on 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)

The method of any one of claims 19-42, wherein at least one engineered nucleic acid further encodes a selection marker protein.

### Claim 44 (depends on 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)

The method of any one of claims 19-43, wherein the pluripotent stem cell is an induced pluripotent stem cell.

### Claim 45 (depends on 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)

The method of any one of claims 19-44, wherein the pluripotent stem cell is a human pluripotent stem cell.

### Claim 46 (depends on 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)

A pharmaceutical composition comprising OPCs produced by the method of any one of claims 19-45.

### Claim 47 (depends on 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)

A pharmaceutical composition comprising oligodendrocytes produced by the method of any one of claims 33-45.

### Claim 48 (depends on 46, 47)

A method comprising administering to a subject having a demyelinating disorder the pharmaceutical composition of claim 46 or 47.

### Claim 49 (depends on 48)

The method of claim 48, wherein the demyelinating disorder is multiple sclerosis.

### Claim 50

A pluripotent stem cell or an oligodendrocyte progenitor cell comprising OCT4, OLIG2, NKX6.1, and SOX9.

### Claim 51

A pluripotent stem cell or an oligodendrocyte progenitor cell comprising OCT4, OLIG2, and SOX9.

### Claim 52

A pluripotent stem cell or an oligodendrocyte progenitor cell comprising OLIG2, NKX6.1, and SOX9.

### Claim 53 (depends on 50, 51, 52)

The pluripotent stem cell or an oligodendrocyte progenitor cell of any one of claims 50-52 further comprising IL-10.

### Claim 54 (depends on 53)

The pluripotent stem cell or an oligodendrocyte progenitor cell of claim 53 further comprising IFNβ.

### Claim 55

A pluripotent stem cell engineered to express IL-10 and/or IFNβ, optionally wherein the pluripotent stem cell is an induced pluripotent stem cell.

### Claim 56

An oligodendrocyte progenitor cell engineered to express IL-10 and/or IFNβ.

### Claim 57 (depends on 55, 56)

The cell of claim 55 or 56, wherein the cell is engineered to express IL-10 and IFNβ.

### Claim 58 (depends on 55, 56, 57)

The cell of any one of claims 55-57, wherein the IL-10 and/or IFNβ are encoded by at least one nucleic acid that is stably integrated into the genome of the cell.

### Claim 59 (depends on 55, 56, 57, 58)

The cell of any one of claims 55-58, wherein the cell secretes at least 50-fold, 100-fold, or 1000-fold more IL-10 than a control.

### Claim 60 (depends on 59)

The cell of claim 59, wherein the control cell is (a) a naturally-occurring pluripotent stem cell or naturally-occurring OPC, (b) a pluripotent stem cell or OPC that is not engineered to express IL-10, IFNβ, or a combination thereof, or (c) a pluripotent stem cell or OPC that is engineered to express IL-10, IFNβ, or a combination thereof under an inducible promoter and is cultured under conditions that lack an inducible agent.

### Claim 61

A method comprising:
introducing into pluripotent stem cells at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4), optionally wherein each transcription factor is introduced on an engineered nucleic acid comprising a nucleotide sequence encoding the transcription factor, and optionally wherein at least two transcription factors are encoded by the same engineered nucleic acid;
culturing the pluripotent stem cells to produce a population of cells comprising oligodendrocyte progenitor cells (OPCs); and
culturing the OPCs, optionally with another cell type, to produce a myelinated organoid.

### Claim 62 (depends on 61)

The method of claim 61, further comprising transplanting the myelinated organoid into a subject in need thereof.

### Claim 63

A method comprising:
introducing into pluripotent stem cells at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4), optionally wherein each transcription factor is introduced on an engineered nucleic acid comprising a nucleotide sequence encoding the transcription factor, and optionally wherein at least two transcription factors are encoded by the same engineered nucleic acid to produce engineered pluripotent stem cells capable of expressing at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1) (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4).

### Claim 64 (depends on 63)

The method of claim 63 further comprising administering the engineered pluripotent stem cells, or OPCs derived from the pluripotent stem cells, to a subject in need thereof.

### Claim 65 (depends on 63, 64)

The method of any one of claims 63-64 comprising inducing expression of the at least four transcription factors.

### Claim 66

A pluripotent stem cell comprising at least three transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1), (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4).

### Claim 67 (depends on 67)

The pluripotent stem cell of claim 67 comprising OCT4, SOX9, and OLIG2.

### Claim 68 (depends on 67)

The pluripotent stem cell of claim 67 comprising OCT4, SOX9, and NKX6.1.