# US 10,865,381 B2 · Multi-lineage hematopoietic precursor cell production by genetic programming

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- Number: US 10,865,381 B2
- Document ID: US10865381B2
- Status: Granted
- Kind: B2
- Application number: 15/298,931
- Filed: 2016-10-20
- Published: 2017-04-20
- Granted: 2020-12-15
- Earliest claimed: 2015-10-20
- Assignees: FUJIFILM Cellular Dynamics, Inc.
- Applicants: FUJIFILM Cellular Dynamics, Inc.
- Inventors: Junying Yu; Maksym A. Vodyanyk; Jeffrey Sasaki; Deepika Rajesh; Sarah A. Burton
- Web page: https://patentgrep.com/patent/US10865381B2

## CPC

- C12N 5/0639: {Dendritic cells, e.g. Langherhans cells in the epidermis}
- A61K 40/10: characterised by the cell type used
- A61K 40/22: Immunosuppressive or immunotolerising
- A61K 40/416: {Antigens related to auto-immune diseases; Preparations to induce self-tolerance}
- C12N 5/0634: {Cells from the blood or the immune system}
- C12N 5/0635: {B lymphocytes}
- C12N 5/0636: {T lymphocytes}
- C12N 5/0637: {Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg}
- C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- C12N 15/85: for animal cells
- C12N 15/86: Viral vectors
- A61K 2239/31: characterized by the route of administration
- C12N 2500/38: Vitamins
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL]
- C12N 2501/145: Thrombopoietin [TPO]
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/165: Vascular endothelial growth factor [VEGF]
- C12N 2501/22: Colony stimulating factors (G-CSF, GM-CSF)
- C12N 2501/2303: Interleukin-3 (IL-3)
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/2307: Interleukin-7 (IL-7)
- C12N 2501/25: Tumour necrosing factors [TNF]
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N 2501/60: Transcription factors
- C12N 2501/602: Sox-2
- C12N 2501/603: Oct-3/4
- C12N 2501/604: Klf-4
- C12N 2501/605: Nanog
- C12N 2501/606: c-Myc
- C12N 2501/608: Lin28
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/025: from extra-embryonic cells, e.g. trophoblast, placenta
- C12N 2506/03: from non-embryonic pluripotent stem cells
- C12N 2506/09: from epidermal cells, from skin cells, from oral mucosa cells
- C12N 2506/11: from blood or immune system cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2510/00: Genetically modified cells
- C12N 2533/50: Proteins
- C12N 2840/20: translation of more than one cistron
- 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)

## Abstract

The present disclosure generally regards methods and compositions for providing multi-lineage hematopoietic precursor cells from pluripotent stem cells (PSCs). The PSCs comprise an expression construct encoding an ETS/ERG gene, GATA2 and HOXA9. Also provided are methods for providing hematopoietic stem cells capable of long-term engraftment in mammals, such as humans. Further provided are therapeutic compositions including the provided hematopoietic stem cells and precursors of hematopoietic cells, and methods of using such for the treatment of subjects.

## Claims

### Claim 1

An in vitro method for producing hematopoietic precursor cells from pluripotent stem cells comprising:
(a) providing pluripotent stem cells (PSCs) comprising at least one expression construct encoding hematopoietic precursor programming genes, wherein the hematopoietic precursor programming genes comprise ETS/ERG, GATA2, and HOXA9 genes co-expressed;
(b) culturing the PSCs wherein the PSCs are induced to express the hematopoietic precursor programming genes, thereby producing hematopoietic precursor cells (HPCs); and
(c) further culturing the HPCs, wherein expression of the hematopoietic precursor programming genes is no longer induced, to produce a population of cells comprising at least 70% multi-lineage HPCs with expression of CD34 and CD43.

### Claim 2 (depends on 1)

The method of claim 1, wherein the multi-lineage HPCs are capable of differentiating into myeloid and lymphoid lineages.

### Claim 3 (depends on 1)

The method of claim 1, wherein the expression construct is a transposon- or episomal-based expression construct.

### Claim 4 (depends on 1)

The method of claim 1, wherein the hematopoietic precursor programming genes are under the control of a single promoter.

### Claim 5 (depends on 4)

The method of claim 4, wherein the single promoter is an inducible promoter.

### Claim 6 (depends on 5)

The method of claim 5, wherein the inducible promoter is a tetracycline-inducible promoter.

### Claim 7 (depends on 1)

The method of claim 1, wherein the culturing of step (b) is about four to about ten days.

### Claim 8 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

### Claim 9 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells are human pluripotent stem cells.

### Claim 10 (depends on 1)

The method of claim 1, wherein the multi-lineage HPCs further express one or more hematopoietic precursor markers in addition to the CD34 and the CD43.

### Claim 11 (depends on 10)

The method of claim 10, wherein the one or more hematopoietic precursor markers are selected from the group consisting CD33, CD45, CD235a, and CD41a.

### Claim 12 (depends on 10)

The method of claim 10, wherein the one or more hematopoietic precursor markers is CD45.

### Claim 13 (depends on 1)

The method of claim 1, wherein the multi-lineage HPCs are immature HPCs.

### Claim 14 (depends on 13)

The method of claim 13, wherein at least 90 percent of the multi-lineage HPCs are immature HPCs.

### Claim 15 (depends on 1)

The method of claim 1, wherein the HPCs of step (c) are cultured in the absence of stromal cells.

### Claim 16 (depends on 1)

The method of claim 1, wherein the HPCs of step (c) are cultured in serum free or defined medium.

### Claim 17 (depends on 15)

The method of claim 15, wherein the multi-lineage HPCs can be differentiated into two or more cell types selected from the group consisting of plasma cell, natural killer cell, macrophage, mast cell, megakaryocyte, erythrocyte, granulocyte, lymphocyte, monocyte, leukocyte, and thrombocyte.

### Claim 18 (depends on 17)

The method of claim 17, wherein the lymphocyte is a B lymphocyte and/or a T lymphocyte.

### Claim 19 (depends on 1)

The method of claim 1, wherein the ETS/ERG gene is ERG (v-ets erythroblastosis virus E26 oncogene homolog), ETV2 (ets variant 2), FLI-1 (Friend leukemia virus integration 1), ELK3 (ETS domain-containing protein), ETS1 (C-ets-1), or ETS2 (C-ets-2).

### Claim 20 (depends on 1)

The method of claim 1, wherein the ETS/ERG gene is ERG or ETV2.

### Claim 21 (depends on 1)

The method of claim 1, wherein the hematopoietic precursor programming genes are fused to a targeting sequence.

### Claim 22 (depends on 21)

The method of claim 21, wherein the targeting sequence is NUP98 or a homeodomain thereof.

### Claim 23 (depends on 1)

The method of claim 1, wherein the hematopoietic precursor programming genes comprise ERG, GATA2, HOXA9, NUP98-HOXA9 and NUP98-HOXA10.

### Claim 24 (depends on 1)

The method of claim 1, wherein the hematopoietic precursor programming genes comprise ETV2, GATA2, HOXA9, NUP98-HOXA9 and NUP98-HOXA10.

### Claim 25 (depends on 1)

The method of claim 1, wherein the PSCs of step (a) further comprise at least one additional expression construct encoding one or more hematopoietic stem cell programming genes.

### Claim 26 (depends on 25)

The method of claim 25, wherein the one or more hematopoietic stem cell programming genes are selected from the group consisting of BCL2, BEND4, BMI1, CIITA, EGR3, ETV6, EZH1, EZH2, FOXL1, HIF3A, HLF, HMGA2, HOXA10, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXB3, HOXB6, HSF5, KLF2, KLF4, MECOM, MEIS1, MIR29A, MIR29B1, MSI2, MYB, MYCN, NKX2-3, NR4A2, PEG3, PRDM12, PRDM16, RBAK, RUNX1, RUNX3, SETBP1, SOX17, SOX8, TFEC, ZBTB14, ZBTB20, ZMAT1, ZNF131, ZNF134, ZNF136, ZNF256, ZNF26, ZNF300, ZNF337, ZNF350, ZNF414, ZNF662, ZNF667 and ZNF682.

### Claim 27 (depends on 25)

The method of claim 25, wherein expression of the one or more hematopoietic stem cell programming genes is constitutive in the HPCs of step (b).

### Claim 28 (depends on 25)

The method of claim 25, wherein expression of the one or more hematopoietic stem cell programming genes is essentially silenced in the pluripotent stem cells.

### Claim 29 (depends on 25)

The method of claim 25, wherein the hematopoietic stem cell programming genes are fused to a targeting sequence.

### Claim 30 (depends on 29)

The method of claim 29, wherein the targeting sequence is NUP98 or a homeodomain thereof.

### Claim 31

An in vitro method for producing hematopoietic precursor cells from pluripotent stem cells comprising:
(a) providing pluripotent stem cells (PSCs) comprising an expression construct encoding ERG, GATA2, and HOXA9 under the control of a single promoter; and
(b) culturing the pluripotent stem cells with coexpression of ERG, GATA2, and HOXA9, thereby producing hematopoietic precursor cells (HPCs); and
(c) further culturing the HPCs without coexpression of ERG, GATA2, and HOXA9, to produce a population of cells comprising at least 70% multi-lineage HPCs with expression of CD34 and CD43.

### Claim 32 (depends on 5)

The method of claim 5, wherein the inducible promoter is a bi-directional Tight promoter.

### Claim 33 (depends on 1)

The method of claim 1, wherein the ETS/ERG, GATA2, and/or HOXA9 genes are linked through a 2A-cleavage peptide.

### Claim 34 (depends on 1)

The method of claim 1, further comprising step (d) differentiating the HPCs to myeloid lineage cells or lymphoid lineage cells.

### Claim 35 (depends on 6)

The method of claim 6, wherein the PSCs are cultured in the presence of 0.25 μg/mL doxycycline for induction of gene expression in step (b).