WO2014139539A1 - Inhibitor of dpysl3/crmp4 for treatment of bone loss - Google Patents

Abstract

There is provided an antibody, fragments hereof, or derivatives hereof, which specifically blocks CRMP4 for use in treating or preventing bone loss in a patient. The antibody may be selected from isolated polyclonal antiserum, a preparation of purified polyclonal antibodies, and a preparation containing one or more monoclonal antibodies. The invention further provides the use of siRNA to block CRMP4 gene expression.

Description

INHIBITOR OF DPYSL3/CRMP4 FOR TREATMENT OF BONE

LOSS

FIELD OF THE INVENTION

The present invention relates to the inhibition of CRMP4 thereby increasing osteogenic differentiation. Specifically the present invention provides an antibody and siRNA for use in treating or preventing bone loss in a patient.

BACKGROUND OF THE INVENTION

A variety of disorders in humans and other mammals involve or are associated with abnormal bone remodelling. Such disorders include, but are not limited to, senile and postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, Paget's disease, periodontitis, tooth loss, osteoarthritis, bone fractures, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma. One of the most common of these disorders is postmenopausal and senile osteoporosis. Osteoporosis is a systemic skeletal disease characterized by a low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Osteoporotic fractures are a major cause of morbidity and mortality in the elderly population. As many as 50% of women and a third of men will experience an osteoporotic fracture. A large segment of the older population already has low bone density and a high risk of fractures. There is a significant need to both prevent and treat osteoporosis and other conditions associated with bone loss.

Bone marrow-derived human mesenchymal (stromal, skeletal) stem cells (hMSC) are a population of self-renewing, multipotent cells that have significant clinical potential in cellular therapies for skeletal tissue and other tissue regeneration. hMSC can differentiate along several mesodermal lineages, including the osteogenic lineage, in response to stimulation by multiple exogenous factors. Dihydropyrimidinase-like protein 3 (DPYSL3), a member of TUC (TOAD-64/Ulip/CRMP), is also known as dihydropyrimidinase related protein 3 (DRP-3), collapsing response- mediated protein-4 (CRMP-4), TUC-4 and Ulip-1 (unc-33-like phosphoprotein) (1). CRMP4 is strongly expressed throughout the developing central and peripheral nervous systems and was shown to mediate the signaling pathway of Sema 3A/collapsin-induced growth cone collapse and to play an important role in neuronal differentiation and axonal outgrowth (2,3). Furthermore, CRMP4 was shown to have a regulatory function in the cytoskeletal dynamics of neuronal cells via interaction with GAP-43 (4) and regulating F- actin bundling (5). CRMP4 contains a number of consensus phosphorylation sites and serves as physiological substrates for the signaling molecule glycogen synthase kinase 3β (Θ8Κ3β) after priming phosphorylation by Dual specificity tyrosine-phosphorylation- regulated kinase (DYRK2) and Cyclin-dependent kinase 5 (Cdk5) (6,7). Despite these extensive studies on the role of CRMP4 in neurogenesis, nothing is known regarding its function in bone marrow-derived MSC differentiation and bone remodelling.

WO 2007/098198 A2 discloses agents for increasing osteoblast differentiation and for treating or preventing osteoporosis. The agents may be antibodies against the Ror-2 protein resulting in dimerization and thereby activation of the Ror-2 protein, which then phosphorylates and inactivates the 14-3-3β protein. This inactivation results in an increase in osteoblast differentiation at about 85% (osteoblast formation) and an increase in bone mass at about 50%. The agent may also be a shRNA or a siRNA against the 14-3-3β gene for the purpose of inhibiting the expression of said gene. It is shown that shRNA against the 14-3-3β gene results in an increase in osteoblast differentiation at about 60% (osteoblast formation) and increase in bone mass at about 50%. The effects of said antibody and said shRNA were not additive or synergistic indicating the same pathway of differentiation.

De Jong et al (9) disclose osteoblast differentiation induced by BMP-2 and/or ΤΘΡβ, of which the CRMP4 ("DPYSL3") gene among 26 genes is demonstrated to be up- regulates by BMP-2 during said differentiation (tablel). The CRMP4 gene is among 7 genes selected for further investigation, where it is shown that 5 of said selected genes were specifically regulated by BMP-2, of which 5 selected genes the CRMP4 gene is the only gene not previously disclosed to play a role in osteoblast differentiation. De Jong, et al (10) disclose BMP2-induced osteoblast differentiation, where the CRMP4 gene is one gene among several genes which are up-regulated during said differentiation. Thus, said up-regulated genes are sorted in 6 clusters depending on their expression pattern during said differentiation. CRMP4 is one gene among 17 genes in cluster 1 showing a linear expression level when plot against time.

Elucidating the mechanisms regulating human MSC (hMSC) differentiation into osteogenic lineage is of importance to improve therapeutic treatments of bone loss diseases, such as osteoporosis.

SUMMARY OF THE INVENTION

The present inventors have found that inhibition of CRMP4 by e.g. an antibody markedly increases osteogenic differentiation.

Accordingly, the present invention provides an antibody, fragments hereof, or derivatives hereof, which specifically blocks CRMP4 phosphorylation sites at the c-terminal end (including Phosphothreonine, Phosphoserine) by targeting the epitope having the sequence: VFDLTTTPKGGTPAGSARGSPTRPNPPVRNLHSGFSLSGTQVDEGVRSA (SEQ ID 1) for use in treating or preventing bone loss in a patient.

Preferably, the antibody is selected from the group consisting of an isolated polyclonal antiserum, a preparation of purified polyclonal antibodies, and a preparation containing one or more monoclonal antibodies.

The bone loss may be associated with ankylosing spondylitis, renal osteodystrophy, osteoporosis, glucocorticoid-induced osteoporosis, Paget's disease, abnormally increased bone turnover due to other conditions e.g. periodontitis, bone fractures, rheumatoid arthritis, osteoarthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, multiple myeloma, bone loss associated with microgravity, Langerhan's Cell Histiocytosis (LHC), or bone loss associated with renal tubular disorders, or bone loss associated with prolonged immobilization. In a particularly preferred embodiment the bone loss is associated with osteoporosis.

In another aspect the present invention is directed at the use of an siRNA to block the transcription of all CRMP4 variants mRNA by targeting the following DNA sequence of the gene GTGTTGATGACGTACGTTA (SEQ ID 2).

Further the present invention provides a method for treating or preventing bone loss in a patient comprising the steps of providing an antibody of the present invention and administering said one or more nucleic acids to the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of the microarray approach used to select for DPYSL3/CRMP4.

Figure 2 shows exclusive expression and regulation of CRMP4 during osteoblast differentiation of MSC.

Figure 3 shows tissue distribution of CRMP4 mRNA expression in adult mouse organs and bone related cells.

Figure 4 shows that CRMP4 expression and phosphorylation is regulated during osteoblast differentiation of MSC.

Figure 5 shows paraffin embedded sections of mouse embryos at different gestations and new born pups.

Figure 6 shows expression of CRMP4 by bone lining osteoblasts during endochondral bone development.

Figure 7 shows expression of CRMP4 by osteoblast lining cells in adult mouse bone.

Figure 8 shows expression of CRMP4 by proliferating chondrocyte and osteoblast during mouse bone fracture healing. Figure 9 shows increased bone mineral density in CRMP4 knockout mice.

Figure 10 shows increased trabecular and cortical bone volume/total volume (BV/TV) in CRMP4-/- mice.

Figure 11 shows results from the bone cellular phenotype of CRMP4-/- mice.

Figure 12 shows the effect of different signalling molecules on osteoblast differentiation of CRMP^^'O versus WT OB.

Figure 13 shows WT and CRMP4-/- OB induced to osteoblast differentiation in the presence of different BMPs. Figure 14 shows WT and CRMP4^'1' OB induced with different concentration of BMP-2.

Figure 15 shows WT and CRMP4-/- MSC isolated from the bone marrow of 2 months old mice and cultured in osteogenic media. Figure 16 shows WT and CRMP4-/- OB induced with BMP-2 and western blot was performed for the indicated proteins of canonical and non-canonical BMP2 signals.

Figure 17 shows the effect of specific BMP-type I receptor inhibitor LDN-193189 on BMP2 -induced osteoblast differentiation.

Figure 18 shows BMP2-induced migration of CRMP4-/- OB in response to BMP2.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors used a comparative microarray analysis to screen for non- canonical osteogenic factors controlling the osteoblast differentiation of mesenchymal stem cells (MSC) and identified CRMP4, as a novel neuro-protein to be expressed and control the early commitment of MSC into osteoblast lineage. The results demonstrated a novel function of CRMP4 in regulating osteoblast differentiation of MSC and osteoprogenitors. In addition, preliminary skeletal analysis of CRMP4 knockout mice revealed significant increased bone mass in CRMP4-/- mice over wild type controls.

In the following a detailed description on the identification of the CRMP4 gene as the only member of CRMP osteoblast differentiation is provided. The inventors screened for non-canonical osteogenic factors by comparing the transcriptome of previously established bone marrow-derived pre-osteogenic (MSC-bone) and pre-adipogenic (MSC-adipo) cell lines (8) during their differentiation course into osteoblast and adipocyte lineages respectively.

Genes up-regulated during early osteogenesis versus adipogenesis were subjected to several rounds of selection to exclude all previously studied osteogenic factors. This screening resulted in the selection of DPYSL3/CRMP4 as a novel gene that exclusively expressed among CRMP1-5 family during the early commitment of MSC into osteogenic differentiation (Figure 1).

Figure 1 shows a schematic diagram of the microarray approach used to select for DPYSL3/CRMP4. Two clonal mouse bone marrow-derived mesenchymal stem cells (MSC) with opposite differentiation potential were used to screen for novel factor involved in regulating bone formation, using comparative microarray analysis. -mMSC- bone denotes mMSC cells only differentiated into osteoblast and chondrocyte lineages, whereas -mMSC-Adipo corresponds to mMSC cells only differentiated into adipocyte lineage. Microarray analysis was performed at different time points (day 0, 3, 6 and 10) during osteoblast differentiation of MSC-Bone and adipocyte differentiation of MSC- Adipo.

As shown in Figure 2, CRMP4 mRNA was shown to be expressed by BM-MSC during their early osteoblast differentiation, while its expression was down-regulated afterwards. Referring to Figure 2 there is shown exclusive expression and regulation of CRMP4 during osteoblast differentiation of MSC as shown by (A) microarray analysis and fluorescent immune staining for CRMP4. (B) Real time PCR analysis during the time course of differentiation. For quantitative RT-PCR, CRMP4 mRNA expression was represented as relative expression to b-actin. Values are mean ±SD of three replicates in three independent experiments. Referring to Figure 3 there is shown tissue distribution of CRMP4 mRNA expression in adult mouse organs and bone related cells. (A) Different tissues were dissected from 2 months old mice and homogenized to isolate RNA. (B) RNA was isolated from different cell cultures at baseline. Calvarial cells were isolated from calvarias of 3 days old pups; MEF cells (mouse embryonic fibroblasts) E11.5 mouse embryos; primary MSC were isolated from 2 month old mouse bone marrow. CRMP4 mRNA expression was analysed by quantitative RT-PCR and presented as relative expression to b-actin. Values are means ± SD of three replicates in three independent experiments.

Referring to Figure 4 there is shown that CRMP4 expression and phosphorylation is regulated during osteoblast differentiation of MSC. To study the regulation of CRMP4 expression and phosphorylation, the inventors used ST2 cell line as a model of mMSC. ST2 cells were induced to osteoblast differentiation for 12-20 days and both protein extract and RNA were purified at the indicated time points. A) shows quantitative PCR for CRMP4 mRNA expression. The expression level of CRMP4 is up-regulated during early OB diff and then down-regulated during matrix mineralization. B) shows protein extracts were subjected to Western blot analysis. The phosphorylation of CRMP4 appears to increase at day6 during OB diff and then decline after day 9. The regulation of CRMP4-p indicates the role of CRMP4 phosphorylation in OB diff.

The phosphorylation of CRMP4 is inhibited by blocking the up-stream signal of GSK3b and Cdk5 kinases activity in osteoprogenitor cells. Since CRMP4 was identified as a phosphorylated brain-specific substrate for GSK3 upon priming by DYRK2 and Cdk5 (6,7), the inventors aimed to investigate whether the CRMP4 phosphorylation mediates its effect on osteoblast differentiation. -Calvaria cells were isolated from 2-3 days pups and treated without (control) or with inhibitors of GSK3b kinase (CHIR-99021 , SB216763), or Cdk5 kinase inhibitor (purvalanol). -Western blots were performed after 2 days of induction. Results were simillar to what is known about the inhibition of CRMP4- p by inhibiting GSK3b kinase in neuronal cells, we found that the CRMP4-p is inhibited by different -The same results have been verified in mouse MSC, ST2 cell line.

Referring to Figure 5 there is shown paraffin embedded sections of mouse embryos at different gestations and new born pups (1 day) were immune-stained with mouse specific CRMP4 antibody. Immune-histochemical staining for CRMP4 showed the expression of CRMP4 protein by proliferating chondrocyte (red arrows) and osteoblast (blue arrows) during mouse ribs development (flat bone).

Referring to Figure 6 there is shown expression of CRMP4 by bone lining osteoblasts during endochondral bone development. Paraffin embedded sections of mouse embryonic femur derived from embryos at different gestations and new born pups (1 day) were stained with a CRMP4 specific antibody. Immunehistochemical staining for CRMP4 showed the expression of CRMP4 protein during endochondral ossification of mouse femur bone.

Referring to Figure 7 there is shown expression of CRMP4 by osteoblast lining cells in adult mouse bone. Immune-histochemical staining for CRMP4 in the paraffin embedded sections of 2 months old mouse distal femur. Almost all osteoblastic lining cells of cancellous and endostial cortical bone surface were staining positive for CRMP4.

Referring to Figure 8 there is shown expression of CRMP4 by proliferating chondrocyte and osteoblast during mouse bone fracture healing. The expression of CRMP4 was studied during bone union-fracture healing in mice at different time points. Results showed that CRMP4 is expressed early during callus formation by proliferating chondrocyte and later by osteoblasts. This expression pattern is similar to the expression of CRMP4 during embryonic bone formation.

The results indicate that loss function of CRMP4 in mice leads to significant increased bone mass phenotype. To investigate the in vivo physiological function of CRMP4 in bone homeostasis, the inventors performed bone analysis on tibiae and femurs derived from CRMP4 knockout mice (CRMP4-/-) and their wild type littermates (provided through a scientific collaboration with, Prof. Yoshio Goshima, Yokohama City University, Japan). Interestingly, CRMP4-/- mice exhibited significantly increased bone mass over WT controls as shown by 30% increase in the total bone mineral density (BMD) (measured by DEXA scan) and 100% increase in the trabecular bone volume/total volume (BV/TV)

(measured by micro-computed tomography analysis, microCT) (Figure 2). Taken together, the data identified CRMP4 as a novel negative regulator of bone mass.

Referring to Figure 9 there is shown increased bone mineral density in CRMP4 knockout mice. A) X-ray radiographs show the increasing bone mass in the 2 months old CRMP4- /- tibia vs wild type controls. B) DEXA scan analysis of the 2-months-old tibia of CRMP4- /- vs wild type. BMD=total bone mineral density; BMC= total bone mineral content and B area= total bone area.

Referring to Figure 10 there is shown increased trabecular and cortical bone volume/total volume (BV/TV) in CRMP4-/- mice. Micro-computed tomography analysis (uCT) was performed on the proximal tibia of 2-months-old tibia obtained from DPYSL3- /- and wild type control mice. A) uCT-3D reconstruction images of tibia from CRMP4-/- and wild type control mice. B) BV/TV measurements of cortical and trabecular bone of tibia bone samples.

Referring to Figure 11 there is shown results from the bone cellular phenotype of CRMP4-/- mice. Osteoprogenitor cells (OB) were isolated from both WT, heterozygote and CRMP4-/- neonatel calvaria (2-3 days old) and cultured in vitro. A) Western blot analysis and quantitative PCR confirmed the lacking of CRMP4 expression in CRMP4-/- OB. B) OB cells were differentiate into osteoblastic lineage with b-glycerophosphate and Ascorbic acid for 7 days and alkaline phosphatase activity was measure and normalized to cell viability. It is herewith demonstrated that CRMP4 deficiency promotes osteoblast differentiation in vitro.

Referring to Figure 12 there is shown the effect of different signalling molecules on osteoblast differentiation of CRMP^^'O versus WT OB. A&B) WT, CRMP4^+/~ OB and CRMP4^' OB were induced to osteoblast differentiation in the presence of different signalling molecules that are known to be involved in osteogenesis including BMP-2,-4,- 7, TGFb1 &2, Inhibitors for GSK3b kinase, Wnt3a conditioned media (Wnt CM) and Sema3A. It is herewith demonstrated that BMPs signalling pathway is involved in mediating the regulatory function of CRMP4 on osteoblast differentiation. CRMP4 deficiency increases the responsiveness of osteoblast to BMPs-induced ALP activity.

Referring to Figure 13 WT and CRMP4-/- OB were induced to osteoblast differentiation in the presence of different BMPs for 12 days and matrix mineralization in cultured cells was stained with Alizarin Red and eluted for quantification. Based thereon it may be concluded that CRMP4 deficiency increases the responsiveness of osteoblast to BMPs- induced matrix mineralization. Referring to Figure 14 WT and CRMP4 OB were induced with different concentration of BMP-2 for 24h and qPCR analysis was performed for the indicated genes. Based thereon it may be concluded that several of BMP2-responsive genes were shown to be up-regulated in CRMP4^' OB.

Referring to Figure 15 WT and CRMP4-/- MSC cells were isolated from the bone marrow of 2 months old mice and cultured in osteogenic media. A) CRMP4-/- MSC cells displayed increased number of CFU-F stained for ALP activity as compared to WT MSC control cells. B) MSC from CRMP4-/- and WT mice were induced with BMP2 for 1 week (ALP activity) and for 12 days matrix mineralization. Based thereon it may be concluded that loss function of CRMP4 significantly stimulates the BMP2-induced osteoblast differentiation in MSC cells.

Referring to Figure 16 WT and CRMP4-/- OB were induced with BMP-2 (100 ng/ml) and western blot was performed for the indicated proteins of canonical and non-canonical BMP2 signals. Based thereon it may be concluded that CRMP4 deficiency enhances the activation of both canonical and non-canonical BMP2 signalling pathways in osteoblasts.

Referring to Figure 17 there is shown the effect of specific BMP-type I receptor inhibitor LDN-193189 on BMP2 -induced osteoblast differentiation. (A) WT and CRMP4-/- OB were treated with BMP2 in the presence of different concentrations of LDN-193189 for 6 days during osteoblast differentiation. (B) Cells were incubated with different concentration of LDN for 30 min and then induced with BMP2 for 10 min. Western blot was performed for the indicated proteins. Based thereon it may be concluded that BMP- type I receptor is partially involved in mediating the activation of BMP2 signalling in CRMP4-/- OB cells.

Referring to Figure 18 there is shown the BMP2-induced migration of CRMP4-/- OB in response to BMP2. A) Oris™ Cell Migration Assay was used to study the dose- dependent effect of BMP2 on cellular migration of CRMP4-/- OB vs WT OB. Imaging analysis and quantification of cell layer area of migrated cells was analyzed by high content Operetta imaging system. B) Western blot analysis of BMP2-induced FAK phosphorylation. Based thereon it may be concluded that BMP2 enhances the migration of CRMP4-/- OB via stimulating the FAK phosphorylation. Reference List

1. Charrier, E., Reibel, S., Rogemond, V., Aguera, M., Thomasset, N., and Honnorat, J. (2003) Mol. Neurobiol. 28, 51 -64

2. Goshima, Y., Nakamura, F., Strittmatter, P., and Strittmatter, S. M. (1995) Nature 376, 509-514

3. Niisato, E., Nagai, J., Yamashita, N., Abe, T., Kiyonari, H., Goshima, Y., and Ohshima, T. (2012) Dev. Neurobiol.

4. Kowara, R., Menard, M., Brown, L., and Chakravarthy, B. (2007) Biochem. Biophys. Res. Commun. 363, 190-193

5. Rosslenbroich, V., Dai, L., Baader, S. L., Noegel, A. A., Gieselmann, V., and Kappler, J. (2005) Exp. Cell Res. 310, 434-444

6. Cole, A. R., Causeret, F. , Yadirgi, G., Hastie, C. J., McLauchlan, H., McManus, E. J., Hernandez, F., Eickholt, B. J., Nikolic, M., and Sutherland, C. (2006) J. Biol. Chem. 281 , 16591 -16598

7. Uchida, Y., Ohshima, T., Sasaki, Y., Suzuki, H., Yanai, S., Yamashita, N., Nakamura, F., Takei, K., Ihara, Y., Mikoshiba, K., Kolattukudy, P., Honnorat, J., and Goshima, Y. (2005) Genes Cells 10, 165-179

8. Post, S., Abdallah, B. M., Bentzon, J. F., and Kassem, M. (2008) Bone 43, 32-39

9. DE JONG, D.S. et al.; BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 320, No. 1 , 2004, pages 100-107

10. DE JONG, D.S. et al.; JOURNAL OF BONE AND MINERAL RESEARCH, Vol. 19, No. 6, 2004, pages 947-958

SEQUENCE LISTING

<110> Syddansk Universitet

<120> INHIBITOR OF DPYSL3/CRMP4 FOR TREATMENT OF BONE LOSS <130> P1283DK00 <160> 2

<170> Patentln version 3.5

<210> 1

<21 1 > 49

<212> PRT

<213> Homo sapiens

<400> 1

Val Phe Asp Leu Thr Thr Thr Pro Lys Gly Gly Thr Pro Ala Gly Ser Ala Arg Gly Ser Pro Thr Arg Pro Asn Pro Pro Val Arg Asn Leu His Ser Gly Phe Ser Leu Ser Gly Thr Gin Val Asp Glu Gly Val Arg Ser Ala

<210> 2

<21 1 > 19

<212> DNA

<213> Homo sapiens

<400> 2

gtgttgatga cgtacgtta

<210> 3

<21 1 > 19

<212> RNA <213> Artificial Sequence <220>

<223> siRNA <400> 3

guguugauga cguacguua

<210> 4

<21 1 > 21

<212> RNA

<213> Artificial Sequence <220>

<223> siRNA <400> 4

ccaaacgguu gugaucuaaa g

<210> 5

<21 1 > 21

<212> RNA

<213> Artificial Sequence <220>

<223> siRNA

<400> 5

uaacuccuuc augguuuaua u

<210> 6

<21 1 > 21 <212> RNA

<213> Artificial Sequence <220>

<223> siRNA

<400> 6

cggcauagau ggaacccauu a

Claims

1. An antibody, fragments hereof, or derivatives hereof, which specifically blocks CRMP4 phosphorylation sites at the c-terminal end by targeting the epitope having the sequence VFDLTTTPKGGTPAGSARGSPTRPNPPVRNLHSGFSLSGTQVDEGVRSA (SEQ ID 1) for use in treating or preventing bone loss in a patient.

2. The antibody according to claim 1 , wherein the antibody is selected from the group consisting of an isolated polyclonal antiserum, a preparation of purified polyclonal antibodies, and a preparation containing one or more monoclonal antibodies.

3. The antibody according to claim 1 or 2, wherein the bone loss is associated with ankylosing spondylitis, renal osteodystrophy, osteoporosis, glucocorticoid-induced osteoporosis, Paget's disease, abnormally increased bone turnover, periodontitis, bone fractures, rheumatoid arthritis, osteoarthritis, peri prosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, multiple myeloma, bone loss associated with microgravity, Langerhan's Cell Histiocytosis (LHC), or bone loss associated with renal tubular disorders, or bone loss associated with bed-ridden conditions.

4. The antibody according to any one of the claims 1-3, wherein the bone loss is associated with osteoporosis.

5. siRNA binding to the CRMP4 gene sequence GTGTTGATGACGTACGTTA (SEQ ID 2) for use in treating or preventing bone loss in a patient.

6. siRNA for use according to claim 5, wherein the siRNA is selected from the group consisting of GUGUUGAUGACGUACGUUA (SEQ ID NO 3), CCAAACGGUUGUGAUCUAAAG (SEQ ID NO 4), UAACUCCUUCAUGGUUUAUAU (SEQ ID NO 5), and CGGCAUAGAUGGAACCCAUUA (SEQ ID NO 6)