EP2329024A1

EP2329024A1 - An endogenous short hairpin rna and the use of the same

Info

Publication number: EP2329024A1
Application number: EP08784123A
Authority: EP
Inventors: Robert C. Zhao; James Q. Yin; Chunjing Bian
Original assignee: Institute of Basic Medical Sciences of AMMS; Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of AMMS; Institute of Basic Medical Sciences of CAMS
Priority date: 2008-08-25
Filing date: 2008-08-25
Publication date: 2011-06-08
Also published as: WO2010022553A1; AU2008361210A1; CN102131929A; EP2329024A4

Abstract

An endogenous short hairpin RNA having a sequence of SEQ ID NO: 1 or a complement thereof that induces the formation of hematopoietic cells particularly by repressing EID1 and the use thereof are provided. An expression construct, vector and polynucleotide comprising the sequence of SEQ ID NO: 1 are also provided. The SEQ ID NO: 1 shows 5'-CAA AUA CUC ACC GUG UUA CA -3'.

Description

An endogenous short hairpin RNA and the use of the same

Field of the Invention

The present invention relates to a RNA molecule, particularly to an endogenous short hairpin RNA as well as the application of the invented RNA to induce the formation of hematopoietic cells by repressing ElA-like inhibitor of differentiation- 1 (referred to hereinafter as EIDl). This application was supported by the program of Ministry of Science and Technology of P.R. China; National Natural Science Foundation of China; Beijing Ministry of Science and Technology as well as Cheung Kong Scholars programme.

Background of the Invention

Both bone marrow mesenchymal stem cells (referred to herein as BMSCs) and hematopoietic stem cells (referred to herein as HSCs) are characterized by their capacity to self-renew to supply differentiated cells during the lifetime of the organism. Said BMSCs expanded in culture can be distinguished from HSCs by the lack of expression of CD45 and CD34 (G. Chamberlain, J. Fox, B. Ashton, J. Middleton, Stem Cells. 25, 2739-49 (2007).). Since it is a great challenge to obtain a large number of HSCs for clinical application (B.P. Sorrentino, Nat Rev Immunol. 4(l l):878-88 (2004).), BMSCs would serve as an alternate source for HSCs if BMSCs can be induced to differentiate into hematopoietic cells. It is also known that mesenchymal stem cells (BMSCs) are multipotent stem cells that can differentiate into cells of bone, endothelium, adipose tissue, cartilage, muscle, and brain. However, whether or not BMSCs can become hematopoietic stem cells (HSCs) in vitro and in vivo remains controversial. (Y. Jiang et al., Nature 418, 41-49 (2002); M. Serafϊni et al., J Exp Med. 204,129-39 (2007); M.F. Pittenger, Science. 284, 143-7 (1999); K. W. Liechty et al., Nat Med. 6, 1282-6 (2000), and M. Angelopoulou et al., Exp. Hematol 31, 413-420 (2003)). In the present invention, it has been demonstrated that when BMSCs are cultured in the medium containing a cocktail of cytokines and growth factors, BMSCs expressing FIk-I (fetal liver kinase- 1) but negative for CD34 and CD31 appear and also can be readily expanded. It seems that expression of FIk-I renders BMSCs greater differentiation potential (L.Liao, L Li, R.C. Zhao, Philos Trans R Soc Lond B Biol Sci. 362:1107-12 (2007)). These FIk-I⁺ cells could even be induced to differentiate into hematopoietic cells although at a very low frequency (H. Guo et al., Exp. Hematol.31 : 650-658 (2003)). In addition, it is also very important for the person skilled in the art to understand what molecular mechanisms are controlling the self-renewal and differentiation of BMSCs in order to make BMSCs an alternate source for HSCs.

It has been shown by the prior art that miRNAs are differentially expressed in different hematopoietic cell types and play important regulatory roles. For example, miR-181 is associated with B lymphoid development (CZ. Chen et al., Science 303, 83-86 (2004)), miRNA-142 and -223 with T lymphopoiesis (S. H. Ramkissoon et al., Leukemia Research, 30, 643-647 (2005).), miRNA-221 and -222 with erythropoiesis (N. Felli et al., Proc Natl Acad Sci U S A. 102, 18081-18086 (2005).), miRNA-223 with granulocytic differentiation (F. Fazi et al., Cell, 123, 819-831 (2005).) and miR-10, -126 and -17 with megakaryocytopoiesis (R. Garzon et al., Proc Natl Acad Sci U S A.103, 5078-5083 (2006). ). Moreover, some miRNAs (miR-128 and -181) have been shown to prevent the differentiation of HSCs (III, R. W. Georgantas et al., Proc Natl Acad Sci U S A.104, 2750-2755 (2007).). Although some miRNAs such as miR-130a and miR-10a have been found to target the transcription factor genes HOXAl and MAFB, which are important for cellular differentiation (R. Garzon et al., Proc Natl Acad Sci U S A.103, 5078-5083 (2006)), it remains unclear whether any miRNAs or other small RNAs are involved in the determination of the self-renewal and differentiation of BMSCs and HSCs at early stages of development. In fact, little is known about intrinsic effectors of BMSC fate decisions.

Recently, we have reported that a novel class of endogenous shRNAs is generated from the introns of protein-coding genes in human cells (Yin JQ and Zhao RC. Methods. 43(2): 123-30 (2007). T. Gu et al. (Accompanying manuscript) 2008. See an accompanying manuscript).

Summary of the Invention

The first object of the invention is to provide an endogenous short hairpin RNA or a complement thereof having a sequence of SEQ. ID. NO.: 1 that induces the formation of hematopoietic cells particularly by repressing EIDl. The said SEQ. ID. NO.: 1 shows:

5'-CAA AUA CUC ACC AUU GUG UUA CA-3'. The said sequence of SEQ. ID. NO.: 1 essentially includes the nucleic acid of 5'-CAA AUA CUC A-3'.

The second object of the invention is to provide an expression construct including the sequence of SEQ. ID. NO.: 1.

The third object of the invention is to provide a vector comprising an expression construct having an expression construct including the sequence of SEQ. ID. NO.: 1.

All vectors known by the person skilled in the art can be used in the present invention. In an embodiment of the invention, the vector preferably is a retrovirus plasmid (retrovirus vector pMSCV ) that expresses the sequence of SEQ. ID. NO.: 1.

The fourth object of the invention is to provide an application of the invented RNA or a complement thereof having a sequence of SEQ. ID. NO.: 1 to induce the formation of hematopoietic cells or a method of treatment for a subject suffering from dysfunction of formation of blood cells or a method of treatment for hematopoiesis in mammalian including human.

In other word, the invention also provides a use of an endogenous short hairpin RNA having SEQ. ID. NO.: 1 or SEQ. ID. NO.: 2 in manufacture of medicament for treatment of a subject suffering from dysfunction of hematopoiesis in mammalian including human. The said SEQ. ID. NO.: 2 shows 5'-UGU AAC ACA AUG GUG AGU

AUU UG-3'.

The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, the query sequence is at most 23 nucleotides in length. Preferably, the sequence is at least 10 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at most 23 nucleotides. And the GAP analysis aligns the two sequences over a region of at least 10 nucleotides.

With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that a polynucleotide of the invention comprises a sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ. ID. NO.: 1.

Polynucleotides of the present invention are natural ones, and may possess one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site- directed mutagenesis on the nucleic acid).

Oligonucleotides and/or polynucleotides of the invention hybridize to a sill- gene of the present invention, or a region flanking said gene, under stringent conditions.

The term "stringent hybridization conditions" and the like as used herein refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an oligonucleotide. Nucleic acid hybridization parameters may be found in references which compile such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra). For example, stringent hybridization conditions, as used herein, can refer to hybridization at 65°C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH2PO4 (pH7), 0.5% SDS, 2 niM EDTA), followed by one or more washes in 0.2.xSSC, 0.01% BSA at 500C. Alternatively, the nucleic acid and/or oligonucleotides (which may also be referred to as "primers" or "probes") hybridize to the region of the an insect genome of interest, such as the genome of a honeybee, under conditions used in nucleic acid amplification techniques such as PCR. BMSCs transduced with a newly identified short hairpin RNA (shRNA) of the present invention can differentiate into hematopoietic stem cells (HSCs) and their descendant multipotent progenitor cells that have the capacity of further differentiating into blood cells in vitro, according to the invention, on transplantation into sublethally irradiated NOD/SCID mice, transduced human BMSCs engrafted and differentiated into all hematopoietic lineages including lymphocytes and myelocytes. Furthermore this new shRNA alters BMSC fate by repressing the translation of EIDl. Thus a model has been established that the endogenous shRNA can confer definitive tropism to BMSCs and unveil an intrinsic roadmap for programming hematopoiesis-related genes in HSCs.

Like artificial shRNAs, the two strands of their stems are perfectly complementary with a length of at least 21 nucleotides. To identify shRNAs important in regulating the self-renewal and directional differentiation of stem cells, we employed a custom microarray for a high-throughput screen (see. table 1).

Table.1 The different expression patterns of sRNA between BM

Name Intensity Intensity Ratio

(sRNA) (BMSC) (HSC) (HSC/BMSC)

ZK-693 2943 9 0.0031

ZK-871 1853 9 0.0049

ZK- 125 1846 12 0.0063

ZK-633 5281 62 0.0117

ZK-313 2360 39 0.0167

ZK-428 2582 48 0.0186

ZK-358 3800 178 0.0468

ZK-381 2505 160 0.0639

ZK-290 2147 138 0.0641

ZK-401 3000 197 0.0658

ZK-574 1598 106 0.0661

ZK-IOl 1 3176 253 0.0797

ZK-266 2641 216 0.0818

ZK-921 2225 248 0.1116

ZK-826 11330 1388 0.1225 ZK-960 11452 1643 0.1434

ZK- 1040 9095 1454 0.1598

ZK-768 4641 939 0.2023

ZK-296 4722 1011 0.2141

ZK-630 3109 766 0.2463

ZK-588 4896 1264 0.2582

ZK-798 3122 854 0.2735

ZK-548 2367 694 0.2932

ZK-908 1055 5329 5.0533

ZK-249 309 1571 5.0852

ZK-907 298 4168 14. 0090

*ZK-249 is referred as shR-CH in this paper.

In these experiments of the present invention, several hundreds of new short hairpin RNAs derived from human introns were identified already. The custom array analysis successfully revealed a differential expression of shRNAs between human BMSCs and HSCs. The majority of shRNAs were more highly expressed in BMSCs than in HSCs. According to the invention, two shRNAs, namely shR-CH having SEQ. ID. NO.:. 1 and a complementary sequence of shR-CH having SEQ. ID. NO.: 2, displayed stronger signals in HSCs than MSCs (Fig. IA). The shR-CH precursor was expected to have effect as same as the shR-CH. To confirm the differential expression pattern of shR-CH, quantitative real-time PCR was performed. The expression of shR-CH was found to be at least 10 times higher in HSCs than in MSCs. To determine the length of mature shR-CH, this endogenous shRNA has been cloned. As shown in Figure 2B, mature shR-CH was about 21nt in length and its precursor formed a perfectly complementary hairpin structure. The said shR-CH resides within the first intron of SH3PXD2B gene that is located on chromosome 5 and is not phylogenetically conserved as most endogenous shRNAs (17 Accompanying manuscript).

To identify potential target genes of shR-CH, similar bioinformatics based approaches as those used in prior art to predict potential targets of miRNAs was used. Among the predicted target genes of shR-CH (see the following Table2), Table.2 Prediction of shR-CH target genes No Gene Function 1 NIPAl Related with neural system development

2 FLJ35424 No detailed reports

3 FLJ21625 No detailed reports

4 EIDl CREBBP/EP300 inhibitory protein l;Rb binding protein

EIDl (ElA-like inhibitor of differentiation- I)(NM O 14335.2) was selected for validation by qRT-PCR and Western blot assay because it contains five naturally-occurring putative shR-CH binding sites at its 3'UTR region (see Fig 1C). More importantly, EIDl has been shown to have a close relationship with two essential hematopoiesis-related transcriptional coactivators (S. Miyake et al., MoI Cell Biol. 20, 8889-902 (2000) ; W. Xu et al., Blood. 107, 4407-16 (2006).). Several lines of studies have been reported that the transcriptional coactivators CBP (Cyclic adenosine monophosphate response element binding protein (CREB-binding protein) and its paralogue p300 interact with over 312 proteins, at least 65 of which are encoded by genes that are essential for hematopoiesis (D. N. Messina, J. Glasscock, W. Gish, M. Lovett. Genome Res. 14:2041-2047 (2004); L.H. Kasper, Nature. 419, 738-43 (2002)). CBP/p300 was thought to provide both an assembly platform as well as protein acetyltransferase functions with many transcription factors and histones that regulate gene expression (V. V. Ogryzko et al., Cell 87, 953-959 (1996) ; X. Liu et al., Nature. 451, 846-50 (2008)).The differential expression of shR-CH between human BMSCs and HSCs raised the possibility that it might be a key factor for HSCs to maintain their sternness and thus dictate the directional differentiation from BMSCs to HSCs. To investigate this possibility, vectors containing the murine stem-cell retrovirus backbone, a RNA polymerase III (pol III)-specific U6 gene promoter and shR-CH or mock shRNA in BMSCs (Fig. 2A) have been constructed. To examine the biological effect of ectopic expression of shR-CH on BMSC differentiation, mesenchymal stem cells from human bone marrow are infected with those vectors. As shown in Fig. 2B, the levels of shR-CH were significantly elevated by transduction of shR-CH vectors, compared with those in control or mock infected cells. Elevated levels of shR-CH can be sustained for a long time in BMSCs infected with shR-CH (data not shown). Next whether increased levels of shR-CH affected the expression of its target mRNA was determined. Levels of EIDl mRNA were analyzed via application of RT-PCR at day 2 following transduction of shR-CH vectors. As shown in Figure 2C and 2D, whereas there was only a slight change in the level of EIDl mRNA, a remarkably decreased level of EIDl protein was detected when assayed by Western blotting. Consistent with the potential involvement of EIDl in shR-CH-dependent hematopoietic differentiation, endogenous EIDl protein was down-regulated in HSCs, coupled with a concomitant occurrence in expression of CD45 marker.

In BMSCs and HSCs , it was noted that a reciprocal relationship between the expression of EIDl and the expression of the CD45 marker that is characteristic for hematopoietic cells. Moreover, CD45 was upregulated after shR-CH induced repression of EIDl, raising the possibility that EIDl is involved in suppressing the hematopoietic program in BMSCs. According to the present invention a single BMSC was cloned by the single cell cloning method to reach this end and rule out the possibility of HSC contamination. The results from a fluorescence activated cell sorter illustrated that the cloned BMSCs were characterized with the presence of specific CD29, CD 105 and CD44 surface antigens and FIk-I nuclear antigen, and without the CD34, CD45 and CD133 specific for HSCs (Suppl. Fig. 1). Then these cloned cells were infected with vectors expressing shR-CH or mock shRNA and seeded onto the 24-well plate coated with fϊbronectin and collagen, and supplemented with a cocktail of hematopoietic cytokines and growth factors. Cells originating from transduced BMSCs were identified on the basis of the green fluorescent protein (GFP) marker carried by the vector, and differentiation of BMSCs to hematopoietic cells was characterized by expression of specific CD45 surface antigen (Fig. 3A). Ex vivo expansion of infected BMSCs was determined by using a modified CFU assay. 5000 seed cells from each group were selected and cultured for an additional 14 days. shR-CH overexpression rendered a substantial growth advantage during the 14-day culture as determined by in vitro colony-forming capacity (CFU-GM). This was also reflected by the predominance of GFP- and CD45- positive cells in the shR-CH culture (Fig. 3B). Although the same number of cells (-5,000) was initially seeded in three cases, shR-CH-transduced cells proliferated and differentiated significantly (Fig. 3C) whereas very few mock-infected BMSCs could change their phenotypes by day 14, suggesting that non-infected BMSCs and mock-infected BMSCs fail to thrive under these conditions. In contrast, many cells infected with shR-CH vectors could form CD45-positive colonies. This significant increase in hematopoietic cells indicated that increased levels of shR-CH induced the hematopoietic differentiation of BMSCs. The induction of hematopoietic differentiation was shR-CH specific since transduction with mock vectors had no effects on CD45 expression and the formation of CFU. These results strongly suggest that the enforced expression of shR-CH can overcome the blockade of hematopoietic differentiation and set out the program for the directional differentiation of hematopoiesis in BMSCs.

If an increase in shR-CH level is required for definitive differentiation of BMSCs, the direct inhibition of its target gene, EIDl, should have similar effects on BMSC differentiation. Therefore, we tested whether the EIDl gene was of functional importance in inducing the directional differentiation of BMSCs. Quantitative real time RT-PCR analysis illustrated that EIDl-siR effectively reduced endogenous EIDl expression to 40% of that observed with a mock siRNA in the infected BMSCs (Fig. 2C). As expected, BMSCs treated with EIDl-siRNA were indeed induced to differentiate into hematopoietic cells, as determined by the specific expression of CD45 antigen (Fig. 3). This si-EIDl is highly specific for its target mRNA since mock siRNAs showed no effects on the differentiation of BMSCs. Therefore, like the ectopic expression of shR-CH, the knockdown of EIDl expression in BMSCs can result in the differentiation of these stem cells toward hematopoietic cells. Together, the findings described above provide the direct evidence that shR-CH dictates the differentiation of MSCs via the repression of EIDl.

To directly assess whether the ectopic expression of shR-CH induced the directional differentiation of human BMSCs in vivo, the capacity of infected BMSCs for multilineage hematopoietic repopulation in mice was examined. In initial experiments, human BMSCs were infected with either the retrovirus that expressed shR-CH or a control vector that expressed unrelated shRNA and were then transplanted into sublethally irradiated recipient mice. BMSC-derived GFP-expressing hematopoietic cells were detected through 60 days post infected-BMSC transplantation. By using antibodies against the corresponding antigens specific for human cells, immunochemical staining of mouse bone marrow indicated that these BMSC-derived cells could differentiate into multiple hematopoietic lineages as shown by expression of markers for hematopoietic progenitors and then further into myeloid cells marked by CD33 and lymphoid cells expressing the CD3 marker (Fig. 4A). In contrast, very few human CD45-, CD33- or CD3-positive cells were seen in the bone marrow sections of control and mock-transduced groups. The few human CD45-, CD33- or CD3-positive cells seen in the control and mock-transduced groups may reflect the intrinsic capacity of FIk-I⁺ BMSCs to differentiate into HSC. Several organs were also collected and determined the presence of human BMSC-derived cells by GFP illumination. However, no positive results were obtained. These findings strongly suggest that shR-CH induce the directional differentiation of BMSCs into hematopoietic cells but not to other types of cells.

To quantify the phenotype of the BMSC-derived cells in bone marrow, the cells were analyzed by flow cytometry by using three monoclonal antibodies against human leukocyte and stem cell markers. Consistent with the observation on the bone marrow sections, FACS profiles of bone marrow cells from recipients also showed robust engraftment of the BMSC-derived cells in medullary canal (Fig. 4B). After 8 weeks, the lineage composition of medullary canal cells descending from infected human BMSC (GFP⁺ cells) was examined. The shR-CH expression in human BMSCs led to a significant increase in lymphoid (CD3) cells in medullary canal (2.8% vs 0.5% in the control). Similarly, there was also a substantial elevation in CD33-positive myeloid lineage cells (Fig. 4B). The presence of human BMSC-derived hematopoietic cells in bone marrow of the mouse recipients indicated that the ectopic expression of shR-CH in human BMSCs initiated a program for the hematopoietic lineage differentiation.

The preferential expression of shR-CH in HSCs implicates an important role of shR-CH in hematopoiesis. The findings in the invention show that ectopic expression of shR-CH is able to initiate and direct the human BMSCs to differentiate into hematopoietic cell lines in vitro and vivo (Fig. 3 and 4) substantiate that notion. Through the repression of EIDl, shR-CH probably up-regulates the activities of hematopoiesis-related genes such as CBP and p300 (Fig 4C). CBP and p300 knockout or point-mutant mice were reported to have dramatically reduced numbers of definitive erythroid, myeloid, and B-lymphocytic progenitors in bone marrow (L. H. Kasper, Nature. 419, 738-43 (2002); Y. Chen, P. Haviernik, K.D. Bunting, Y.C. Yang, Blood. 110, 2889-98 (2007); A.L. Kung et al, Genes Dev. 14, 272-7 (2000)). Other studies further showed that CBP and p300 were fate decision factors for HSCs, responsible for HSC self-renewal and hematopoietic differentiation, respectively (V.I. Rebel et al., Proc Natl Acad Sci U S A. 99, 14789-94 (2002); MX. Sandberg et al., Dev Cell. 8, 153-66 (2005)). To test whether the expression of genes downstream of CBP/p300 is affected by the change of shR-CH and EIDl, the expression of RUNXl, an important hematopoiesis-related transcription factor, in BMSCs expressing shR-CH and in MSCs treated with si-EIDl was also measured. As shown in Fig.4D, RUNXl was up-regulated significantly in both cases, indicating that the transcriptional activity of CBP/p300 was increased.

The capacity of BMSCs to contribute to progeny of all three germ layers makes them a potential resource for regenerative medicine (Y. Jiang et al., Nature 418, 41-49 (2002).3, Y.S. Yoon et al., J. Clin. Invest 115, 326-338 (2005); M. T. Reyes et al., Blood 98, 2615-2625 (2001).). However, before the clinical trials, it is important to develop methods that allow reproducible differentiation of BMSCs. According to the study of the present invention, it showed for the first time that transplantation of shR-CH-infected BMSCs can give rise to high levels of BMSC-derived hematopoietic cells in bone marrow of the recipients. These findings also provide another explanation for the enhanced myelopoiesis and megakaryocytopoiesis caused by co-transplantation of human BMSCs and HSCs (K. W. Liechty et al., Nat Med. 6, 1282-6 (2000)). Moreover, it has been shown that BMSCs can prevent from lethal graft- versus-host disease, leading to a speedy recovery of hematopoiesis (X. Chen, H. Xu, C. M. Wan, McCaigue, G. Li, .Stem Cells. 24, 2052-9 (2006); G. Ren et al., Cell Stem Cell 2, 141-150 (2008)). In other words, shR-CH-infected BMSCs could serve as an alternate source of hematopoietic stem cells for transplantation.

In summary, these studies characterized a new endogenous shRNA in structure and function, and identified a regulatory way in which endogenous shRNAs participate to control the directional differentiation of mesenchymal stem cells.

Brief description of the drawings

Figure 1 shows an identification and characterization of a novel shR-CH in human stem cells, wherein Figure IA indicates the differential expression of newly identified shR-CH. Data are averages of at least three independent determinations. Error bars indicate standard deviations. *P<0.01 and **P<0.001, expression of shR-CH detected by microarrays or qRT-PCR assay in BMSCs compared with HSCs, respectively. Figure IB indicates the cloning and sequencing of mature shR-CH and the secondary structure of the same. Solid red line stands for the sequence of mature shR-CH. Figure 1C indicates the alignment of the sequences of mature shR-CH and its three putative binding sites at the 3'UTR region of EIDl mRNA. Figure 2 shows that the ectopic expression of shR-CH can effectively inhibit the expression of endogenous EIDl gene at the translational level. Wherein Fig 2A indicates the construction of the mock-sRNA /GFP and shR-CH/GFP retroviral vectors used in this study. Fig 2B indicates the differential expression of newly identified shR-CH in different cases. Data are averages of at least three independent determinations. Error bars indicate standard deviations. *p < 0.001. And Fig 2C indicates Real Time-PCR analysis on the expression levels of EIDl mRNA under various conditions were carried out and normalized to that of GAPDH, and the resultant expression levels in different cases are normalized to their levels in the control. Data are averages of at least three independent determinations. Error bars indicate standard deviations. Fig 2D indicates the levels of EIDl protein from different cases were analyzed by Western blotting. The Western blot was stripped and re-probed with actin antibody to check for equal loading of total protein.

Figure 3 shows that the enforced expression of shR-CH can induce the proliferation and differentiation of human BMSCs in vitro. Cells from a single clone were used. Fig 3A indicates the morphological alterations of normal BMSC, mock-, shR-CH- and siEIDl -infected BMSCs illustrated by different staining methods. Fig 3B indicates CFC assay of CD45⁺ hematopoietic cells. Cells were cultured in growth medium for 24 hours after transduction and then transferred into differentiation medium for 12 hours before immunostaining for CD45 specific for hematopoietic cells. Observation under inverted microscope showed that both the shR-CH-infected BMSCs and the siEIDl -infected BMSCs generated the colonies effectively. Fig 3C indicates quantitative analysis of colonies from three different cases. At Day 14, the colony number from the BMSCs infected by shR-CH, siEIDl or mock vectors gave the result of n>6/case; *p < 0.001. Value inside bars represents fold increase.

Figure 4 shows the effects of shR-CH forced expression of on hematopoietic lineage differentiation in vivo, wherein Fig 4A indicates the immunofluorescent and Hochest33342 staining of mouse bone marrow sections at 60 days post-trans. Enumeration of cells were stained with the human- specific antibodies against CD45, CD3 or CD33 in control, mock-shRNA and shR-CH sections of bone marrow obtained at 60 days post-transplantation. Fig 4B indicates hematopoietic reconstitution from human BMSCs grafted in NOD-SCID mice. 10⁵ GFP⁺CD45⁺ BMSCs were transplanted in sublethally irradiated NOD-SCID mice. Representative flow cytometry profiles of BM of NOD-SCID (human CD45) mice >8wk after transplant demonstrate multilineage (lymphoid and myeloid cells) reconstitution. For each quadrant, the fraction of cells relative to the total number of all the cells in mice bone marrow is given. Fig 4C and Fig 4D indicate that Real time -PCR analysis on the expression levels of RUNXl mRNA under various conditions were carried out and normalized to that of GAPDH, and the resultant expression levels in different cases are normalized to their levels in the control. Data are averages of at least three independent determinations. Error bars indicate standard deviations.

Detailed description of the invention

Example 1

Cells and Cell Culture

Human bone marrow (BM) was obtained from 30 healthy donors (ages 20 to 50 years) following informed consent according to guidelines from the Committee of Beijing Union Hospital on the Use of Human Subjects in Research. Isolation and culture of BM-derived FIkI⁺ CD31 CD34^" cells from healthy donors were performed as described previously with some modifications. Briefly, based on a known method in the art, mononuclear cells were separated by a Ficoll-Paque gradient centrifugation (specific gravity 1.077 g/mL; Nycomed Pharma AS, Oslo, Norway) and cultured in DF12 medium containing 5% FCS, 20 ng/niL EGF, 100 U/mL penicillin and 100 g/mL streptomycin (Gibco Life Technologies) at 37°C and a 5%CO₂ humidified atmosphere. The floating cells were discarded at 18 hours. Culture media were changed every 2 days. At day 6, cells were harvested and further depleted of hematopoietic cells with magnetic-activated cell separation (MACS) CD45, GIyA, and CD34 micromagnetic beads (Miltenyi Biotec, Auburn, CA). Cells were then replated and passaged. At passages 4 cells were harvested by trypsinization, transfected with retrovirus vectors and used in the transplantation assay. Cells derived from single clone were used in other assays. To ensure single-cell originality of each cell colony, sorted cells were plated in wells coated with fibronectin (Sigma, St Louis, MO) and collagen (Sigma) for each patient. Culture medium was Dulbecco modified Eagle medium and Ham F 12 medium (DF 12) containing 40% MCDB-201 medium complete with trace elements (MCDB) (Sigma), 2% fetal calf serum (FCS; Gibco Life Technologies, Paisley, United Kingdom), 1: insulin-transferrin-selenium (Gibco Life Technologies), 10 -^"9 M dexamethasone (Sigma), 10^~4 M ascorbic acid 2-phosphate (Sigma), 20 ng/mL interleukin-6 (Sigma), 10 ng/mL epidermal growth factor (Sigma), 10 ng/mL platelet-derived growth factor BB (Sigma), 50 ng/mL fetal liver tyrosine kinase 3 (Flt-3) ligand (Sigma), 30 ng/mL bone morphogenetic protein-4 (Sigma), and 100 LVmL penicillin and 100 g/mL streptomycin. Culture media were changed every 4 to 6 days. Wells with a single adherent cell were identified during the first 24 hours. The appearance of cell colonies was checked daily. Wells containing more than one colony were discarded. The cells deriving from a single cell colony usually reached 90% confluence after four weeks' culture. Then they were harvested by trypsinization and culture expanded. The phenotype of the cells was evaluated by FACS. CD133⁺ hematopoietic stem cells were isolated from cord blood (presented by Beijing Cord Blood Bank) by using the CD 133 positive immunomagnetic beads (MACS).

Example 2 Plasmid construction

A retrovirus vector pMSCV encoding shRNAs expressed from the U6 promoter was generated by the method in the art (Michael T Hemann 2003)., the short hairpin DNA sequence (SEQ. ID. NO.: 3 and SEQ. ID. NO.: 4, Table 4) coincident with the pre-shR-CH was chemically synthesized as a construct. The retroviruses containing the shR-CH-expressing cassette were packaged by H293T cells. The retroviral titers of the mock-GFP and shR-CH-GFP producer cells were 3 x 105/mL and 4 x 105/mL respectively, as assessed by transfer of GFP expression to H293T cells. (Fig.2 A)

Example 3 RNA Isolation and Purification

Total RNA was extracted from different cells by using TRIzol (Invitrogen), as described in the art, and then small RNA was isolated from large RNA by using mirVana miRNA Isolation kit (Ambion, Austin, TX) according to the manufacturer's instructions. The concentration of small RNA was measured by the UV absorbance at 260 nm.

Example 4 Cloning and sequencing of shRNA

Short hairpin RNAs were polyadenylated at 37^°C for 30 min in a 50-ul reaction volume including 1.5 ug RNA and 5 U poly(A) polymerase (Takara). Poly(A)-tailed small RNA was recovered by phenol/chloroform extraction and ethanol precipitation. Reverse transcription was performed by applying 1.5 ug RNA and 1 ug of RT primer (SEQ. ID. NO.: 5, Table 4) with 200 U of Superscript III reverse-transcriptase (Invitrogen). The cDNA amplification was carried out for 40 cycles at a final annealing temperature of 60 ^°C by using primers SEQ. ID. NO.: 6 (Table 4) targeting specifically the novel shRNA shR-CH and SEQ. ID. NO.: 7 (Table 4). A 5' adapter (SEQ. ID. NO.: 8, Table 4) was ligated to small RNA by using T4 RNA ligase (TAKARA) and the ligation products were recovered by phenol/chloroform extraction followed by ethanol precipitation. Reverse transcription was performed by means of 1.5 ug RNA and 1 ug specific RT primer, SEQ. ID. NO.: 9 (Table 4). The cDNA amplification was carried out for 40 cycles at a final annealing temperature of 60 ^°C via primers, SEQ. ID. NO.: 10 and SEQ. ID. NO.: 11 (Table 4). The PCR products were separated on 2% agarose with Goldview staining. A gel fragment spanning 100 nt internal standards was excised and DNA was eluted into elution buffer (0.5M NH₄Ac, 10 mM Mg(Ac)₂, and 1 mM EDTA) at 37 ^°C and recovered by phenol/chloroform extraction followed by ethanol precipitation. PCR products spanning the insert were directly submitted for sequencing (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd). (Fig. IB)

Example 5 Real-time PCR

The expression levels of small RNA and related genes were determined by real-time RT-PCR. SYBR green assays with SYBR Premix Ex Taq™ (TAKARA) were run on the 7500HT real-time PCR instrument (Applied Biosystems). Reverse transcription of small RNA was performed by using 1.5 ug RNA and 1 ug shR-CH specific RT primer .

(SEQ. ID. NO.: 9, Table 4). Primers, SEQ. ID. NO.: 6 and SEQ. ID. NO.: 11 (Table 4) were used for the following real-time PCR. Reverse transcription of hematopoiesis-related genes were performed by using 1.5ug RNA and lug universe primer oligo-dT(18). The primers used for the cDNA amplication were listed in Table 3. The results refer to Fig. IA, Fig.2B, Fig.2C, Fig.4D.

Table 3 Primers used for the cDNA amplication

Table 4 Sequence of nucleotide acids referred in this paper

Example 6 Western blotting

Cells were washed twice with cold PBS and then extracted in 20 ul of RIPA lysis buffer (5OmM Tris-HCl pH7.5; 1% NP-40; 15OmM NaCl; lmg/ml aprotinin; lmg/ml leupeptin; ImM Na₃VO₄; ImM NaF; ImM PMSF) at 4^°C for 30 min. Total protein was resolved on 10% SDS-polyacrylamide gel eletrophoresis and bands of protein transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham). The membrane was blocked with 5% nonfat milk TBS buffer overnight at RT, and incubated for 2 hours with primary antibodies. The expression of beta-actin was used as loading control. The antibodies used included beta-actin (Santa Cruz Biotechnology), and EIDl (UPSTATE). The membranes then were incubated for one hour with HRP-conjugated rabbit anti-goat secondary antibody. Immunocomplexes were visualized with a commercial ECL kit.(Fig.2D) Example 7 Bioinformatic Analysis

The algorithm, miRanda 3.0 and targetscan 3.1( Miranda KC et al., Cell. 126, 1203-1217 (2006); Grimson A, Farh KK et al., MoI CeI. 27, 91-105 (2007)), were used in the invention for target prediction for shRNA. HSCs-overexpressed human-shR-CH was selected for target verification. Among hundreds of predicted targets, one potential targets for human shR-CH, EIDl was chosen for further experimental validation. The result was shown in Fig.1 C .

Eample 8 Small RNA MicroArray and Granulocyte-macrophage colony-forming unit (CFU-GM) assay

Small RNA MicroArray

1. The custom sRNA MicroArrays were provided by CapitoBio Corporation. The details were described in the above (Yin JQ and Zhao RC .Methods. 43(2): 123-30

(2007)).

Granulocyte-macrophage colony-forming unit (CFU-GM) assay The BM-derived mesenchymal stem cells stably expressing shR-CH were analyzed by CFU-GM assay to assess the hematopoietic colony forming ability. Cells from a single clone were used in this assay. The cells suspending in the supernatant were collected and seeded in the CFU mix. The cells were then incubated at 37 ⁰C under 5% CO₂ in a humidified atmosphere. At day 14, the numbers of colony-forming units (CFU) (CFU, defined as >50 cells/colony) were scored based on the morphology and size of colonies. (Fig.3B, Fig.3C)

Example 9 Immunostaining of cells and tissues

Cells from a single clone were fixed with paraformaldehyde 4% for 30 min at room temperature and then washed three times with PBS. The cells were permeabilized with Triton 0.2% in PBS for 5 min. After four washes in PBS, the cells were blocked with a blocking buffer (Dako) for 30 min and then incubated in PBS/BSA 0.1% with an anti-albumin (Dako) or anti-CD45, CD34, CD19, CD71, CD3 and CD33 antibody (PE; Becton Dickinson), or with rabbit IgG (Jackson ImmunoResearch) as a negative control for one hour at room temperature. After staining, the cells were washed three times in PBS and incubated with an antirabbit whole IgG-Cy3 (Jackson ImmunoResearch) or anti-rabbit whole IgG-Cy2 (Jackson ImmunoResearch) in PBS/BSA 0.1% for one hour at room temperature. The cells were washed again (four times in PBS) and the nuclei were stained with Hochest 33342 diluted in PBS for 5 min at room temperature. The stained cells were visualized by using a fluorescence microscope (Leica DMIRB) and images captured using Magnafϊre software. The results refer to Fig.3A, Fig.4A.

Example 10 Transplantation and FACS assay

Six to eight week-old male nonobese diabetic ( NOD)/ LtSz-scid/scid (SCID ) mice were bred and maintained under defined flora conditions in individually ventilated (high-efficiency particle-arresting filtered air) sterile micro-isolator cages (Techniplast, Milan, Italy). All animal handle and experiment procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences. Mice were sublethally irradiated (300 cGy) with a cesium source (MDS Nordion; Gammacell, Ottawa, QC, Canada) prior to transplantation. BMSCs (2.5 ^χl O⁵ cells/mice) transduced without or with shR-CH vectors or mock vectors in 0.4 ml of physiological saline (PS) were respectively injected via tail vain into the irradiated mice. The peripheral white blood cell count was done once a week. Mice were killed 2 months later by cervical dislocation. The BM from both femora and tibiae were collected. They were air dried and then stained with Jenner-Giemsa (BDH Ltd, Poole, United Kingdom). Conventional 4-um histologic sections of decalcified tibia were cut from formalin-fixed, paraffin-embedded material and stained. The BM of mice that received a transplant was assessed for presence of human cells by FACS. Mononuclear cells were harvested as described in the previous paragraph and red blood cells were lysed by adding 8.3% ammonium chloride. Single-cell suspensions were then prepared. After blocking of Fc receptors with human serum, cells were determined by labeling with anti-CD45, -CD33, or -CD3 phycoerythrin (PE; Becton Dickinson). Cells labeled with anti-immunoglobulin G (anti-IgG) monoclonal antibody (mAb) were used as control. In addition, cells were gated to include both lymphoid and myeloid fractions. (Fig.4B)

Statistical analysis

Data are described as means±SEM of the indicated number of separate experiments. A one-way analysis of variance was performed for multiple comparisons. If there was significant variation between treatment and control groups, the mean values were compared by using Student's two-tailed t-test. The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modification and improvements within the spirit and scope of the invention as set forth in the following claims.

Claims

What is claimed is:

1. An endogenous short hairpin RNA having a sequence of SEQ. ID. NO.: 1 that induces the formation of hematopoietic cells particularly by repressing EIDl.

2. A complement of endogenous short hairpin RNA according to claim 1 having a sequence of SEQ. ID. NO.: 2.

3. The endogenous short hairpin RNA according to claim 1, wherein said sequence of SEQ. ID. NO.: 1 essentially includes the nucleic acid of 5'-CAA AUA CUC A-3'.

4. An expression construct including the sequence of SEQ. ID. NO.: 1, wherein the expression construct has sequence of SEQ. ID. NO.: 3.

5. An expression construct including the sequence of SEQ. ID. NO.: 1, wherein the expression construct has a complement having sequence of SEQ. ID. NO.: 4.

6. A vector comprising an expression construct having an expression construct including the sequence of SEQ. ID. NO.: 1.

7. The vector according claim 6, wherein the vector is a retrovirus plasmid.

8. A polynucleotide comprising a sequence which is at least 70% identical to the relevant nominated SEQ. ID. NO.: 1.

9. A polynucleotide comprising a sequence which is at least 80% identical to the relevant nominated SEQ. ID. NO.: 1.

10. A polynucleotide comprising a sequence which is at least 90% identical to the relevant nominated SEQ. ID. NO.: 1.

11. A use of the RNA or a complement thereof having a sequence of SEQ. ID. NO. : 1 according to claim 1 or 2 in manufacture of a medicament for treatment for a subject suffering from dysfunction of formation of blood cells.

12. The use of claim 11, wherein the dysfunction of formation of blood cells includes hematopoiesis.

13. The use of claim 11 or 12, wherein the subject is mammalian animal including human.

14. A method of treatment for a subject suffering from dysfunction of formation of blood cells, wherein effective amount of a RNA or a complement thereof having a sequence of SEQ. ID. NO.: 1 or claim 2 is administrated enough to induce the formation of hematopoietic cells.