Patent Description:
Blood vessel formation processes are largely classified into two types, namely vasculogenesis, in which angioblasts or vascular progenitor cells differentiate to form a primitive vascular network, and angiogenesis, in which new blood vessels are formed from existing vessels. During vasculogenesis, vascular progenitor cells differentiate into vascular endothelial cells and the like, yielding principal blood vessels. Vasculogenesis may include, depending on the pattern of differentiation of vascular progenitor cells, Type <NUM> vasculogenesis, in which vascular endothelial cells differentiate in situ, as in the production of blood vessels in the body, and Type <NUM> vasculogenesis, which occurs when vascular progenitor cells migrate over some significant distance and then differentiate, as in the formation of blood vessels in the endocardium or the cranial region. This acts as an important mechanism in various pathological states of inflammation, tumors and the like, as well as physiological states such as wound healing, ovulation, and pregnancy, including the fetal development process, and is thus under study.

Vascular injury causes a variety of ischemic diseases, and may be fundamentally treated through restoration of endogenous cells or transplantation of functional vascular cells for forming blood vessels. In this regard, treatment through the transplantation of functional vascular cells is problematic because effective methods of differentiating vascular cells are not readily available and because it is difficult to obtain large amounts of cells.

Also, methods of inducing the differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells into vascular cells have been proposed, but are disadvantageous because the efficiency of induction into cells of interest is low and there is the risk of activating tumor genes from the embryonic stem cells or pluripotent stem cells upon differentiation into specific cells. Furthermore, although induced pluripotent stem cells are able to avoid ethical issues associated with embryonic destruction and are resistant to immune system rejection when transplanted, they are undesirably liable to form teratoma, as in the embryonic stem cells.

Also, methods of preparing vascular progenitor cells from somatic cells through direct transdifferentiation have not yet been reported. In particular, the establishment of multi potency of vascular cells by direct transdifferentiation of somatic cells through transduction of ETV2 (ETS variant gene <NUM>) or FLI1 (Friend leukemia virus integration <NUM>) is not known.

It is known from patent application <CIT>, a method of providing endothelial cells from pluripotent stem cells or somatic cells by increasing the expression level of one or more endothelial programming factor genes.

It is also known from patent application <CIT> and from document <NPL>") methods for generating (induced vascular) endothelial cells by culturing amniotic cells under conditions where transcription factors are expressed, in the presence of a TGFβ signaling inhibitor.

It is finally known from document <NPL>") a method of transdifferentiation of fat skeletal muscle cells into endothelial cells, based on transient overexpression of ETV2.

Accordingly, an object of the present invention is to provide a use of a composition for inducing a direct transdifferentiation of a fibroblast into a vascular progenitor cell in vitro, wherein the composition comprises a vector comprising the nucleic acid molecule encoding, as an active ingredient, the proteins FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), wherein the vascular progenitor cell is induced through direct transdifferentiation by introducing the composition to the fibroblast, wherein the composition further comprising a culture medium to induce direct transdifferentiation of fibroblasts.

Another obj ect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of ischemic vascular disease, wherein the composition comprises a vascular progenitor cell induced through direct transdifferentiation, by introducing a lentivirus vector comprising the nucleic acid molecule encoding the proteins FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), to a dermal fibroblast.

Still another object of the present invention is to provide a method for in vitro direct transdifferentiation of a fibroblast into a vascular progenitor cell, comprising introducing a vector comprising the nucleic acid molecule encoding FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), to the fibroblast.

In order to accomplish the above objects, the present invention provides a use of a composition for inducing a direct transdifferentiation of a fibroblast into a vascular progenitor cell in vitro, wherein the composition comprises a vector comprising the nucleic acid molecule encoding, as an active ingredient, the proteins FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), wherein the vascular progenitor cell is induced through direct transdifferentiation by introducing the composition to the fibroblast, wherein the composition further comprising a culture medium to induce direct transdifferentiation of fibroblasts.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of ischemic vascular disease, wherein the composition comprises a vascular progenitor cell induced through direct transdifferentiation, by introducing a lentivirus vector comprising the nucleic acid molecule encoding the proteins FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), to a dermal fibroblast.

In addition, the present invention provides a method for in vitro direct transdifferentiation of a fibroblast into a vascular progenitor cell, comprising introducing a vector comprising the nucleic acid molecule encoding FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), to the fibroblast.

According to the unclaimed embodiment of the invention, vascular progenitor cells can be prepared from somatic cells through direct transdifferentiation, thereby decreasing the period of time required to prepare vascular progenitor cells and avoiding the formation of teratoma, which is a dysfunction of induced pluripotent stem cells, ultimately minimizing the side effects of stem cell therapy agents.

The present invention addresses a use of a composition for inducing a direct transdifferentiation of a fibroblast into a vascular progenitor cell in vitro, wherein the composition comprises a vector comprising the nucleic acid molecule encoding, as an active ingredient, the proteins FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), wherein the vascular progenitor cell is induced through direct transdifferentiation by introducing the composition to the fibroblast, wherein the composition further comprising a culture medium to induce direct transdifferentiation of fibroblasts.

In the present invention, ETV2 (ETS variant gene <NUM>) is a member of the ETS (E26 transformation-specific or E-twenty-six) family, and is registered with NCBI Registration No. NM_014209. The ETS factor is known to be associated with embryonic blood vessel development. In particular, ETV2 is known to perform an essential regulatory function in vascular endothelial differentiation, but the function thereof for inducing the direct transdifferentiation of somatic cells into vascular progenitor cells is not known at all.

Also, FLI1 (Friend leukemia virus integration <NUM>) is a member of the ETS family, and is registered with NCBI Registration No. NM_002017. It is known to inhibit the differentiation of red blood cells through constitutive activation in the erythroblasts. In particular, the function of FLI1 for inducing the direct transdifferentiation into vascular progenitor cells is not known at all, like ETV2.

In the present invention, ETV2, FLI1, or a combination thereof may be provided in the form of a protein or a nucleic acid encoding the protein, and the protein may include any ETV2 or FLI1 protein derived from humans or animals, such as mice, horses, sheep, pigs, goats, camels, antelopes, and dogs. Furthermore, the ETV2 or FLI1 protein used in the present invention includes not only a protein having a wild-type amino acid sequence but also a protein variant of the ETV2 or FLI1 protein.

The term "protein variant" refers to a protein, at least one amino acid residue of which differs from the native amino acid sequence of an ETV2 or FLI1 protein, resulting from deletion, insertion, non-conservative or conservative substitution, or combinations thereof. The variant may be a functional equivalent that shows the same biological activity as a native protein, may be a variant in which the physical and chemical properties of a protein are modified as necessary, or may be a variant the structural stability of which is increased under certain physical or chemical conditions, or the physiological activity of which is increased.

In the present invention, a nucleic acid encoding ETV2 or FLI1 may have a base sequence encoding the wild-type or variant-type ETV2 or FLI1 protein, and may be mutated by subjecting at least one base to substitution, deletion, insertion, or combinations thereof. Also, it may be prepared via extraction from nature or using a chemical synthesis method. The nucleic acid having the base sequence encoding the ETV2 or FLI1 protein may be a single chain or a double chain, and may be a DNA molecule (genomic DNA, cDNA) or an RNA molecule.

In the present invention, the vector may include a signal sequence or a reader sequence for membrane targeting or secretion, in addition to an expression regulatory element, such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, or an enhancer, and may be variously manufactured so as to be adapted for some purpose. The promoter of the vector may be constructive or inductive. Furthermore, the expression vector includes a selective marker for selecting a host cell containing the vector, and a replicable expression vector includes a replication origin. The vector may be self-replicating, or may be integrated into the host DNA.

The vector includes a plasmid vector, a cosmid vector, a viral vector, and an episomal vector. Preferably useful is a viral vector. An example of the viral vector may include, but is not limited to, a vector derived from Retrovirus, for example, HIV (Human Immunodeficiency Virus), MLV (Murine Leukemia Virus), ASLV (Avian Sarcoma/Leukosis), SNV (Spleen Necrosis Virus), RSV (Rous Sarcoma Virus), MMTV (Mouse Mammary Tumor Virus), Adenovirus, Adeno-associated virus, Herpes simplex virus, etc. In particular, the vector is used to increase the efficiency of direct transdifferentiation. Any vector that exhibits the effects of the present invention may be used, so long as it causes genes associated with cells to be converted to be over-expressed in typical somatic cells. Specifically, the vector may be exemplified by a lentiviral vector that expresses ETV2 or FLI1, and particularly an SF-based lentiviral vector as an SFFV promoter.

Also, the nucleic acid encoding the ETV2 or FLI1 protein may be transferred or introduced into cells using any process known in the art, for example, using a vector-type naked DNA, or using a liposome, cationic polymer, etc..

The liposome is a phospholipid membrane obtained through mixing with cationic phospholipids such as DOTMA or DOTAP for gene delivery. When a cationic liposome and an anionic nucleic acid are mixed at a predetermined ratio, a nucleic acid-liposome complex may be formed, and may thereby be introduced into the cells.

Specifically in the present invention, the nucleic acid molecule encoding the ETV2 or FLI1 protein is contained in a viral vector, and thereby the viral vector, which includes the nucleic acid encoding the ETV2 or FLI1 protein, may be introduced into somatic cells together with a packaging-defective helper plasmid. Examples of the virus may include, but are not limited to, Retrovirus, Adenovirus, Adeno-associated virus, Herpes simplex virus, etc..

As used herein, the term "somatic cell" refers to any cell other than a germ cell. Examples of somatic cells may include fibroblasts, muscle cells, nerve cells, gastric mucosal cells, goblet cells, G cells, pericytes, astrocytes, B cells, blood cells, epithelial cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and cord blood stem cells. However, direct transdifferentiation may be applied regardless of the specific kind of tissue cell, so long as it starts from somatic cells, and the present invention is not limited to the above examples of somatic cells. In the present invention, direct transdifferentiation is induced using fibroblasts.

As used herein, the term "vascular progenitor cell" refers to a progenitor cell having the ability to differentiate into a vascular endothelial cell, a vascular smooth muscle cell, a pericyte, or a vascular cell, which is a constituent of the blood vessel in vivo. Also, in the present invention, "iVPC" designates an induced vascular progenitor cell, for example, an induced vascular progenitor cell obtained from a somatic cell through direct differentiation according to the method of the present invention.

In addition, the present invention addresses a pharmaceutical composition for the prevention or treatment of ischemic vascular disease, wherein the composition comprises a vascular progenitor cell induced through direct transdifferentiation, by introducing a lentivirus vector comprising the nucleic acid molecule encoding the proteins FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), to a dermal fibroblast.

Ischemic vascular disease refers to any disease that causes blood flow disorder due to blood vessel impairment by external or internal causes, and is not limited to generation at specific portions in vivo. Specific examples of the ischemic vascular disease may include, but are not limited to, cerebrovascular disease, cardiovascular disease, limb ischemia, peripheral vascular disease, and ischemic muscle necrosis. More specifically, the cerebrovascular disease may be cerebral infarction, stroke, or brain hemorrhage, and may include any blood flow disorder attributable to cerebrovascular damage, but the present invention is not limited to the examples of the above disease. Also, the cardiovascular disease may be arteriosclerosis, ischemic reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary micro embolism, tachycardia, bradycardia, pressure overload, coronary artery ligation, arrhythmias, stroke, angina, myocardial infarction, heart failure, or hypertension, but may include any blood flow disorder attributable to cardiovascular damage, and the present invention is not limited to the examples of the above disease.

As used herein, the term "cell therapy agent" refers to a medicine (US FDA-regulated) used for the purpose of treatment, diagnosis or prevention of diseases with cells and tissues manufactured through culturing and specialized tasks after separation from human beings, especially a medicine used for the purpose of treatment, diagnosis or prevention of diseases by proliferating or screening autologous, allogeneic or xenogeneic living cells in vitro or otherwise changing the biological characteristics of cells in order to restore the functions of cells or tissues.

In addition, the present invention addresses a method for in vitro direct transdifferentiation of a fibroblast into a vascular progenitor cell, comprising introducing a vector comprising the nucleic acid molecule encoding FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), to the fibroblast.

More specifically, the method includes: culturing fibroblasts in a culture medium, transducing the fibroblasts with a vector containing ETV2, FLI1 or combined genes thereof, and culturing the infected fibroblasts under culture conditions for inducing direct transdifferentiation.

The culture medium used for culturing the fibroblasts includes any medium typically useful in the culture not only of somatic cells but also of stem cells and progenitor cells in the art. The culture medium usually contains a carbon source, a nitrogen source, and small amounts of elements. In a specific embodiment of the present invention, transduced fibroblasts are cultured in a culture medium supplemented with protamine sulfate (Sigma), but elements necessary for culturing the cells may be contained without limitation, in addition to the protamine sulfate.

Also, the culture conditions for inducing the direct transdifferentiation of somatic cells may include any culture medium typically used to induce direct transdifferentiation of somatic cells in the art. In a specific embodiment of the present invention, useful is a culture medium for growing vascular progenitor cells, comprising <NUM>% FBS-containing minimal essential medium (MEM), <NUM> L-glutamine, β-mercaptoethanol, penicillin/streptomycin, and <NUM> ng/ml VEGF<NUM>.

A better understanding of the present invention may be obtained through the following examples, which are set forth to illustrate, but are not to be construed as limiting the scope of the present invention. The examples of the present invention are provided to fully describe the present invention to those having ordinary knowledge in the art to which the present invention pertains.

Dermal fibroblasts were cultured in a fibroblast medium (<NUM>% FBS-containing DMEM high glucose, <NUM> L-glutamine, 1x MEM nonessential amino acid, β-mercaptoethanol, 1x penicillin/streptomycin).

<NUM> cells were infected with an SFFV promoter, that is, an SF-based lentiviral vector encoding complementary DNA of ETV2 and FLI1, and with a packaging-defective helper plasmid using a Fugene <NUM> transfection reagent (Roche). After <NUM> hr, a viral supernatant was obtained according to the method disclosed in <NPL>.

The dermal fibroblasts were aliquoted at a density of <NUM>×<NUM><NUM> cells into a <NUM>% gelatin-coated <NUM>-well plate and cultured for <NUM> hr, together with the viral supernatant that contained ETV2 and FLI1 (<NUM>:<NUM>) and was supplemented with <NUM>µg/ml protamine sulfate (Sigma). The transduction efficiency was calculated using SF-GFP control virus.

Two days after injection, the cells were aliquoted again into a new fibroblast medium, and the medium was replaced with a vascular progenitor cell growth medium (<NUM>% FBS-containing minimal essential medium (MEM, Sigma), <NUM> L-glutamine, β-mercaptoethanol, penicillin/streptomycin, <NUM> ng/ml VEGF<NUM> (Peprotech)). Thereafter, the medium was replaced with a new one at an interval of three days, and the colony was physically separated for proliferation.

Five days after infection, the expression of ETV2 and FLI1 was observed using RT-PCR.

Specifically, total RNA was extracted from each cell using an RNeasy kit (Qiagen), five days after the infection, and cDNA was synthesized using Omniscript RT (Qiagen). PCT was performed using a Taq DNA polymerase recombinant (Invitrogen).

After RT-PCR, expression was confirmed through loading on agarose gel, using GAPDH as a control (<FIG>).

As illustrated in <FIG>, using phase-contrast microscopy, colonies began to appear in the cell populations infected with ETV2 and ETV2/FLI1 within <NUM> to <NUM> days after the infection, and the number of colonies increased over time.

The present inventors observed colonies in the cell populations infected with FLI1 within <NUM> days after the infection. In order to proliferate the colonies, the cell populations were physically separated and cultured in a gelatin-coated dish.

In order to perform immunocytochemistry, the cells were immobilized for <NUM> in <NUM>% para-formaldehyde and treated with <NUM>% Triton X-<NUM> for <NUM> so as to be permeable. The cells were cultured for <NUM> in a <NUM>% FBS/PBS blocking solution and then reacted for <NUM> hr with a primary antibody diluted with the blocking solution at room temperature. The primary antibody used was as follows: vWF (<NUM>:<NUM>, Abeam), CD31 (<NUM>:<NUM>, Chemicon) and α-SMA (<NUM>:<NUM>, Abcam).

After reaction with the primary antibody, the cells were washed three times with <NUM>% PBST (tween20/PBS). Thereafter, a secondary fluorescent antibody was diluted with PBS and then reacted with the cells for <NUM> hr (Alexa Fluor <NUM> and <NUM>; <NUM>:<NUM>, Molecular Probes). The cells were washed three times with <NUM>% PBST, and the nuclei were counterstained for <NUM> sec using Hoechst <NUM> (Thermo Scientific). After the staining, the cells were observed using an Olympus Cell^TIRF (UOBC center, UNIST) microscope.

The vascular progenitor cells had properties such that they were capable of differentiation into vascular endothelial cells and smooth muscle cells, and CD31 and vWF were used as markers for the vascular endothelial cells, and α-SMA was used as a marker for the smooth muscle cells. Through immunocytochemical analysis, induced vascular progenitor cells (iVPCs) obtained through transformation by each of ETV2, FLI1 and ETV2/FLI1 were confirmed (<FIG>).

In the ischemic limb model, whether induced vascular progenitor cells (induced-VPCs, iVPCs), obtained through transformation with ETV2, FLI1 and ETV2/FLI1, were effective at restoring blood flow was evaluated. To this end, the induced vascular progenitor cells were transplanted into the model. After a predetermined period of time, the blood flow was measured.

Specifically, thymus-free nude mice (male, <NUM> to <NUM>-week-old, weight <NUM> to <NUM>) were paralyzed with <NUM>/kg of pentobarbital for incision of the femoral artery and laser Doppler perfusion imaging. The femoral artery was incised to the distal point at which it is divided into the saphenous vein and the popliteal vein from the proximal tissue as branches of the external iliac artery. After arterial ligation, the mice were divided into the following test groups: ETV2, FLI1, and ETV2/FLI1 iVPC and a control (HBSS; saline injection) (n=<NUM> per group).

Before the transplantation, the cells were labeled with CM-Dil (Invitrogen). Thereafter, four points of the gracilis muscle in the central thigh of each mouse were intramuscularly injected with <NUM>×<NUM><NUM> cells (<NUM>µL) or HBSS. <NUM>, <NUM> and <NUM> days after transplantation of the cells into ischemic and normal limbs, as well as on the day of transplantation, measurement was performed using a laser Doppler perfusion imaging (LDPI) analyzer (Moor instruments, Devon, UK) (<FIG>).

The blood flows of the ischemic and nonischemic limbs were calculated on the basis of colored histogram pixels. The red and the blue indicated high blood flow and low blood flow, respectively. The blood flow was represented by the LDPI index, corresponding to the ischemic/nonischemic limb blood flow ratio. In <FIG>, the ratio before surgery, <NUM>, indicates the same blood flow in two limbs.

Claim 1:
Use of a composition for inducing a direct transdifferentiation of a fibroblast into a vascular progenitor cell in vitro, wherein the composition comprises a vector comprising the nucleic acid molecule encoding, as an active ingredient, the proteins FLI1 (Friend leukemia virus integration <NUM>) and ETV2 (ETS variant gene <NUM>), wherein the vascular progenitor cell is induced through direct transdifferentiation by introducing the composition to the fibroblast, wherein the composition further comprises a culture medium to induce direct transdifferentiation of fibroblasts.