阅读说明：本技术 治疗精神分裂症和其他神经精神病症的方法 (Methods of treating schizophrenia and other neuropsychiatric disorders ) 是由 S·A·戈德曼 Z·刘 于 2019-12-11 设计创作，主要内容包括：本公开涉及恢复受试者中神经胶质细胞的K～(+)摄取的方法。这些方法涉及在有效于恢复所述神经胶质细胞的K～(+)摄取的条件下向所述受试者施用SMAD4抑制剂。本公开还涉及治疗或抑制受试者的神经精神病症的发作的方法。这些方法涉及在有效于治疗或抑制有此需要的受试者的所述神经精神病症的发作的条件下,向所述受试者施用SMAD4抑制剂。(The present disclosure relates to restoring K to glial cells in a subject + The method of uptake. These methods involve treating a subject with a K effective to restore the glial cells + Administering to the subject an inhibitor of SMAD4 under conditions of ingestion. The present disclosure also relates to methods of treating or inhibiting the onset of a neuropsychiatric disorder in a subject. These methods involve administering to a subject in need thereof an inhibitor of SMAD4 under conditions effective to treat or inhibit the onset of the neuropsychiatric disorder in the subject.)

1. A method for recovering damaged K⁺Channel-functional glial cell K₊A method of ingestion, the method comprising

Effective in restoring K with damage⁺K of the glial cell of channel function⁺Administering an inhibitor of SMAD4 to the glial cell under conditions of uptake.

2. The method of claim 1, wherein the glial cells are glial progenitor cells.

3. The method of claim 2, wherein said administering is performed under conditions effective to restore astrocyte differentiation of glial progenitor cells.

4. The method of claim 1, wherein the glial cell is an astrocyte.

5. The method of claim 1, wherein the SMAD4 inhibitor is an inhibitory nucleic acid molecule selected from the group consisting of a SMAD4 antisense oligonucleotide, a SMAD4 shRNA, and a SMAD4 siRNA.

6. The method of claim 1, wherein the SMAD4 inhibitor is a small molecule selected from the group consisting of 5-fluorouracil, valproic acid, vorinostat, and PR-629.

7. The method of claim 1, wherein there is a compromised K₊The glial cell that is channel-active is a glial cell of a subject with a neuropsychiatric disorder.

8. The method of claim 8, wherein the neuropsychiatric disorder is schizophrenia.

9. A method for restoring glial cell K in a subject⁺A method of ingestion, the method comprising:

selection of glial cells with damage K⁺A subject of ingestion, and

at a K effective for restoring said glial cell⁺Administering to the selected subject an inhibitor of SMAD4 under conditions of ingestion.

10. The method of claim 9, wherein the glial cells are glial progenitor cells.

11. The method of claim 10, wherein said administering is performed under conditions effective to restore astrocyte differentiation of glial progenitor cells of said subject.

12. The method of claim 9, wherein the glial cell is an astrocyte.

13. The method of claim 12, wherein said administering is effective to restore astrocyte K⁺Conditions of steady stateThe process is carried out as follows.

14. The method of claim 9, wherein the SMAD4 inhibitor is an inhibitory nucleic acid molecule selected from the group consisting of a SMAD4 antisense oligonucleotide, a SMAD4 shRNA, and a SMAD4 siRNA.

15. The method of claim 9, wherein the SMAD4 inhibitor is a small molecule selected from the group consisting of 5-fluorouracil, valproic acid, vorinostat, and PR-629.

16. The method of claim 9, wherein the SMAD4 inhibitor is packaged in a nanoparticle delivery vehicle.

17. The method of claim 15, wherein the delivery vehicle comprises a glial cell targeting moiety.

18. The method of claim 9, wherein the selected subject has or is at risk of having a neuropsychiatric disorder.

19. The method of claim 18, wherein the neuropsychiatric disorder is selected from the group consisting of schizophrenia, autism spectrum disorder, and bipolar disorder.

20. The method of claim 19, wherein the neuropsychiatric disorder is schizophrenia.

21. The method of claim 9, wherein the administering is performed under conditions effective to reduce neuronal excitability in the subject.

22. The method of claim 9, wherein the administering is performed under conditions effective to reduce the incidence of epilepsy in the subject.

23. The method of claim 9, wherein the administering is performed under conditions effective to improve a cognitive disorder in the subject.

24. The method of claim 9, wherein the administering is performed using intracerebral delivery, intrathecal delivery, intranasal delivery, or by direct infusion into the ventricle.

25. The method of claim 9, wherein the subject is a human.

26. A method of treating or inhibiting the onset of a neuropsychiatric disorder in a subject, the method comprising:

selecting a subject having or at risk of having a neuropsychiatric disorder, an

Administering to the selected subject an inhibitor of SMAD4 under conditions effective to treat or inhibit the onset of the neuropsychiatric disorder in the subject.

27. The method of claim 26, wherein the glial cells are glial progenitor cells.

28. The method of claim 26, wherein said administering is performed under conditions effective to restore astrocyte differentiation of glial progenitor cells of said subject.

29. The method of claim 26, wherein the glial cell is an astrocyte.

30. The method of claim 26, wherein the SMAD4 inhibitor is an inhibitory nucleic acid molecule selected from the group consisting of a SMAD4 antisense oligonucleotide, a SMAD4 shRNA, and a SMAD4 siRNA.

31. The method of claim 26, wherein the SMAD4 inhibitor is a small molecule selected from the group consisting of 5-fluorouracil, valproic acid, vorinostat, and PR-629.

32. The method of claim 26, wherein the SMAD4 inhibitor is packaged in a nanoparticle delivery vehicle.

33. The method of claim 31, wherein the delivery vehicle comprises a glial cell targeting moiety.

34. The method of claim 26, wherein the neuropsychiatric disorder is selected from the group consisting of schizophrenia, autism spectrum disorder, and bipolar disorder.

35. The method of claim 34, wherein the neuropsychiatric disorder is schizophrenia.

36. The method of claim 26, wherein the administering is performed under conditions effective to reduce neuronal excitability in the subject.

37. The method of claim 26, wherein the administering is performed under conditions effective to reduce the incidence of epilepsy in the subject.

38. The method of claim 26, wherein the administering is performed under conditions effective to improve a cognitive disorder in the subject.

39. The method of claim 26, wherein the administering is performed using intracerebral delivery, intrathecal delivery, intranasal delivery, or by direct infusion into the ventricle.

40. The method of claim 26, wherein the subject is a human.

Technical Field

The disclosure relates to methods for treating a mammal having a compromised K⁺Recovery of potassium glial cell (K) in glial cells for channel function⁺) The method of uptake. These methods are useful for treating a subject having a neuropsychiatric condition.

Background

Schizophrenia is a psychiatric disorder characterized by delusions, auditory hallucinations, and cognitive impairment that affects about 1% of The population worldwide, but is still poorly understood (Allen et al, "Systematic Meta-Analyses and Field Synopsis of Genetic Association Studies in Schizophrania: The SzGene Database," Nature Genetics 40:827 & 834 (2008); Sawa and Snayder, "Schizophrania: reverse applications to a Complex Disease," Science 296:692 & 695 (2002)). In the past decade, it has become clear that many Schizophrenia-related genes are involved in the development and physiological processes of glial cells (Yin et al, "synthetic dyefunction in schizophrena," adv. exp. med. biol.970:493-516 (2012)). Thus, both astrocytic and oligodendrocyte dysfunction are implicated in the etiology of schizophrenia. Astrocytes play a crucial role, inter alia, in the structural development of the neural network and in the coordination of the activity of the neural circuits, which play an important role by releasing glial transmitters, maintaining synaptic density and regulating synaptic Potassium and neurotransmitter levels (Christopherson et al, "Thromboplandinsare Astrocytes-Secreted Proteins That protein CNS synergy," Cell 120:421 (433) (2005); Chung et al, "ascent media synergy interaction Through MEGF10 and MERKpathwaters," Nature 504:394 (2013); and Thrane et al, "Ammonia Triggers neural differentiation and Seizes by pair pacifying interaction Point interference," Nat.19: 1648 (2013)). However, the role that astrocytic dysfunction plays in the development of neuropsychiatric disorders such as schizophrenia is not clear. The present disclosure is directed to overcoming this and other deficiencies in the art.

Disclosure of Invention

A first aspect of the present disclosure relates to a method of restoring glial cell K⁺Method of uptake, wherein the glial cell has an impaired K⁺The channel functions. The method involves effectively recovering a K with damage⁺Channel-functional glial cell K⁺Administering an inhibitor of SMAD4 to the glial cell under conditions of uptake.

Another aspect of the disclosure relates to a method of restoring glial cell K in a subject⁺The method of uptake. The method involves selecting glial cells K that have an impairment⁺Ingested subject and at a K effective to restore said glial cells⁺Administering to the selected subject an inhibitor of SMAD4 under conditions of ingestion.

Another aspect of the present disclosure relates to a method of treating or inhibiting the onset of a neuropsychiatric disorder in a subject. This method involves selecting a subject having, or at risk of having, a neuropsychiatric disorder, and administering to the selected subject an inhibitor of SMAD4 under conditions effective to treat or inhibit the onset of the neuropsychiatric disorder in the subject.

To investigate the role of glial pathology in neurological and neuropsychiatric disorders such as schizophrenia, a protocol for the generation of Glial Progenitor Cells (GPC) from induced pluripotent Cells (iPSCs) was established (Wang et al, "Human iPSC-Derived oligomeric Progeneitor Cells Can Myelinate and Resue a Mouse Model of genetic hybridization," Cell Stem Cell 12:252-264(2013), which is incorporated herein by reference in its entirety). This model allows the production of GPC and its derived astrocytes and oligodendrocytes from patients with schizophrenia in a manner that preserves their genetic integrity and functional repertoire. This protocol provides a means by which astrocytes derived from patients with Schizophrenia can be assessed for differentiation, gene expression and physiological function in vitro and in vivo following implantation in immunodeficient mice (Windrem et al, "human ipsc global Mouse clinical recent global constraints to Schizophrenia," Cell Stem Cell 21:195-208.e6(2017), which is incorporated herein by reference in its entirety). It was noted that such human glial chimeric mice, which were colonized with iPSC-derived GPC generated from schizophrenic patients, exhibited significant abnormalities in both astrocytic differentiation and mature structure associated with significant physiological and behavioral abnormalities. Importantly, RNA sequence analysis revealed that these developmental defects in schizophrenia GPC were associated with down-regulation of a panel of core differentiation-associated genes whose transcriptional targets included many transporters, channel and synaptic modulators that found similar defects in schizophrenia glial cells.

As described herein, targetable signaling nodes that can mitigate such schizophrenia-associated gliosis are identified. To this end, iPSC GPC was generated from patients with childhood onset schizophrenia or their normal Controls (CTRs), and astrocytes were generated from these cells. The gene expression patterns and astrocyte functional differentiation of GPC from schizophrenia and controls were compared. Excessive TGF signaling has been found to play a critical role in the dysfunctional differentiation of schizophrenic GPC, the role of TGF in this cellular context is signaled by SMAD4, and phenotypically normal aspects can be restored to SCZ glial cells by SMAD4 inhibition.

Drawings

FIGS. 1A-1F show efficient generation of hGPC from SCZ iPSC. Flow cytometry revealed that in SCZ (4 SCZ lines, n.gtoreq.3/line) and CTR (4 CTR lines, n.gtoreq.3/line) derived hipSCs>90% of the undifferentiated hipscs expressed SSEA4 (fig. 1A). The expression of NPC marker CD133 was not different between SCZ and CTR derived lines at the Neural Progenitor (NPC) stage (fig. 1B). The hGPC defined by CD140a was similarly generated from SCZ and CTR derived iPSCs as well, and CD140a⁺The relative proportion of cells did not differ in the SCZ and CTR hpgc cultures (fig. 1C). Expression of CD44 in SCZ-and CTR-derived lines at the astrocyte progenitor stageThere was no difference therebetween (fig. 1D). PDGF. alpha.R after incubation with BMP4⁺The percentage of glial cells was significantly higher in the SCZ lines (4 SCZ lines, n.gtoreq.3 per line) than in the CTR lines (4 CTR lines, n.gtoreq.3 per line) (FIG. 1E). S100 β in addition to GFAP⁺Astrocytes were significantly higher in the CTR line relative to the SCZ line (fig. 1F). FSC, forward scatter. A scale: 50 μm. By two-tailed t-test,. star.p<0.001; and NS: is not significant; mean. + -. SEM.

FIGS. 2A-2J show impaired astrocyte differentiation in SCZ GPC. As shown in FIGS. 2A-2D, hNPC highly expressed both SOX1 and PAX6 in both SCZ and CTR (4 different patients and respective lines, n.gtoreq.3 per line) at the Neural Progenitor (NPC) stage. Similarly, the efficiency of hGPC production as defined by PDGFR α/CD140a did not differ between SCZ and CTR lines (4 different patient-specific lines each, n.gtoreq.3 per line) (FIGS. 2E-2G). In contrast, GFAP, as shown in FIGS. 2H-2J⁺The proportion of astrocytes was in CTR lines (4 CTR lines, n.gtoreq.3/line [ 70.1. + -. 2.4%)]) The comparison was performed on SCZ lines (4 SCZ lines, n.gtoreq.3/line, [ 39.9. + -. 2.0 ]]) Is significantly higher. A scale: 50 μm; by two-tailed t-test,. star.p<0.001; and NS: is not significant; mean. + -. SEM.

FIGS. 3A-3E show that TGF-beta signaling-dependent transcripts are up-regulated in SCZ GPC. FIG. 3A is a schematic of the Ingeneity Pathway Analysis of RNA-seq data, revealing that TGF β -dependent transcription is up-regulated in SCZ hGPC. Up-regulated genes include LTBP1, LTBP2, IGFBP3, TGFB1, PDGFB, GDF3, GDF7, BMP1, and BMP 5. Down-regulated genes include AMH and BMP 3. qPCR confirmed that TGF β pathway-associated and up-regulated genes (including BMP1, BMPR2, RUNX2, SERPINE1, BAMBI, etc.) were significantly up-regulated in SCZ hpgc (4 SCZ lines, 3 replicates per line) relative to CTR cells (4 CTR lines, 3 replicates per line) (fig. 3B). In contrast, as shown in fig. 3C, the expression of these genes was not the same between SCZ and CTR lines at the NPC stage. Principal Component Analysis (PCA) revealed a similar methylation state between CTR and SCZ-derived ipscs (fig. 3D). Fig. 3E is a heatmap showing that variability in iPSC methylation status is primarily due to gender and individual strains (p <0.05), not due to disease state or age. P <0.05, p <0.01 by two-tailed t-test; and NS: is not significant; mean. + -. SEM.

FIGS. 4A-4B show validation of BAMBI overexpression and knockdown. In CTR hpgpc transduced with lentivirus-BAMBI (4 CTR lines, 3 replicates per line), qPCR confirmed significant overexpression of BAMBI (fig. 4A). SCZ hpgc (4 SCZ lines, 3 replicates per line) expressed high levels of BAMBI relative to CTR hpgc, whereas lentiviral-BAMBI-shRNAi transduction of SCZ hpgc suppressed BAMBI expression to the level of CTR hpgc (fig. 4B). For a and B, by one-way ANOVA, # P < 0.001; mean. + -. SEM.

FIGS. 5A-5C show a deficit in glial differentiation that mimics SCZ in the normal hGPC expression phenotype of BAMBI. Fig. 5A-5B show that overexpression of the membrane-bound BMP antagonist BAMBI in CTR hpgc (4 CTR lines, 3 replicates per line) significantly reduced the efficiency of astrocytic transformation. However, BAMBI knockdown in SCZ hpgc (4 SCZ lines, 3 replicates per line) was not sufficient to restore astrocyte differentiation (fig. 5B). In addition to BAMBI, BMP antagonists Follistatin (FST) and gremlin1(GREM1) were also up-regulated in SCZ hpgc relative to controls (fig. 5C). A scale: 50 μm; p <0.001, for B one-way ANOVA; for C, by two-tailed t-test, # P < 0.001; and NS: is not significant; mean. + -. SEM.

Figures 6A-6D show SMAD4 regulating astrocytic differentiation of SCZ GPC. Figure 6A is a schematic of SMAD4 that modulates the expression of the TGF β and BMP pathways by: 1) phosphorylation of both SMAD2/3 and SMAD 1/5/8; 2) SMAD nuclear translocation and target promoter activation, including early induction of the endogenous BMP inhibitors BAMBI, Follistatin (FST) and gremlin1(GREM 1); and 3) subsequent feedback inhibition of BMP signaling. The graph of fig. 6B shows that BAMBI, FST and GREM1 were all significantly overexpressed in SCZ CD140a sorted hdgcs relative to control-derived hdgcs. SMAD4 knockdown in SCZ hpgc (4 SCZ lines, 3 replicates/line) then suppressed expression of BAMBI, FST, and GREM1 to control levels. Figure 6C is a set of immunochemical images showing that SMAD4 knockdown in SCZ hpgc restored astrocytic differentiation to CTR hpgc (4 SCZ lines, 3 replicates per line). DOX (-)/(+) means short/long term culture with DOX. SMAD4 knockdown following astrocyte induction caused GFAP-defined astrocyte loss in the SCZ and CTR groups as mediated by continuous doxycycline exposure, as shown in the graph of fig. 6D. DOX (-)/(+) means short/long term culture with DOX. A scale: 50 μm; p <0.05, p <0.01, p < 0.001; one-way ANOVA; and NS: is not significant; mean. + -. SEM.

Fig. 7A-7C show validation of SMAD4 knockdown. Fig. 7A is a graph showing differences in SMAD4 mRNA levels between SCZ and control hdgpc and astrocytes, as reflected in CD140 a-sorted hdgpc (left panel) and CD 44-sorted astrocytes (right panel). Fig. 7B is a schematic of an experimental plan for assessing the effect of transient doxycycline-regulated SMAD4 knockdown on astrocyte differentiation of SCZ and CTR patient-derived hdgpcs. Fig. 7C is a graph showing SMAD4 expression. SCZ CD140a sorted hdcp (4 SCZ strains, 3 replicates per strain) was transduced with Doxycycline (DOX) -inducible lentivirus-SMAD 4-shRNAi and then induced by DOX to drive expression of SMAD 4-shRNAi. The culture was then switched to astrocyte differentiation conditions and DOX was withdrawn, allowing SMAD4 expression and astrocyte maturation (DOX only in the GPC phase), or persisted, continuing to suppress SMAD4 expression during astrocyte maturation (DOX maintained in the AST phase). Lentiviral SMAD4-shRNAi strongly inhibited SMAD4 expression in DOX, whereas SMAD4 expression was unaffected in the absence of DOX induction. DOX (-)/(+) means short/long term culture with DOX. P <0.01 by one-way ANOVA; and NS: is not significant; mean. + -. SEM.

FIGS. 8A-8B show the expression of potassium channel (KCN) -associated genes in SCZ hGPC. Fig. 8A is a heatmap showing the differentially expressed potassium channel genes in SCZ-derived hdcp lines. Each SCZ-derived hdcp line was compared separately to three pooled CTR-derived hdcp lines (FDR 5%, FC >2.00[ if applicable ]). The indicated genes were found to be differentially expressed in at least three of the four evaluated SCZ-derived hdcp lines. qPCR confirmed that the potassium channel-associated genes (including ATP1a2, SLC12a6, and KCNJ9) were all significantly down-regulated in SCZ hpgc (4 SCZ lines, 3 replicates per line) relative to CTR cells (4 CTR lines, 3 replicates per line (fig. 8B)). By two-tailed t-test,. p < 0.01; mean. + -. SEM.

FIGS. 9A-9E show a decrease in potassium uptake by SCZ astrocytes. FIG. 9A is Na +/K + -ATPase pump, NKCC1 Na⁺/K⁺/2Cl^-Schematic representation of the involvement of cotransporters and inward rectifying K + channels in the regulation of potassium uptake by astrocytes. qPCR confirmed that several K + channel-associated genes were down-regulated relative to CTR cells in SCZ CD44+ astrocyte biased GPC, as shown in fig. 9B. SCZ and CTR CD44+ GPC were cultured in FBS with BMP4 to produce mature GFAP + astrocytes, which were then assessed for K⁺Taking; results were normalized to total protein and cell number. FIG. 9C shows K binding to CTR astrocytes⁺Uptake (4 CTR lines, 5 replicates per line) of K compared to SCZ astrocytes⁺Uptake was significantly reduced (4 SCZ lines, 5 replicates per line). Astrocytes were treated with ouabain, bumetanide and tolytin to assess which potassium transporter classes were functionally impaired in SCZ astrocytes relative to controls (4 lines per control, 4 replicates/line). Both ouabain and bumetanide were effective in reducing K of CTR astrocytes⁺Uptake (fig. 9D, grey bar), while neither affected K + uptake by SCZ astrocytes (fig. 9E, purple bar). For B and C, by two-tailed t-test,. P<0.05，**P<0.01，***P<0.001; for D, by one-way ANOVA<0.001; and NS: is not significant; mean. + -. SEM.

FIGS. 10A-10C show the generation of astrocytes from SCZ CD44+ astrocyte-biased progenitor cells. Inducing differentiation of SCZ-derived and CTR-derived CD44+ astrocyte precursors into astrocytes. Immunostaining with GFAP showed that there was no significant difference in astrocyte production efficiency between SCZ-derived lines (fig. 10A, right panel; 4 SCZ lines, 5 replicates per line) and CTR-derived lines (fig. 10A, left panel; 4 CTR lines, 5 replicates per line) (see also the graph of fig. 10B). qPCR revealed that GFAPmRNA expression was not different between SCZ and CTR derived CD44+ astrocyte precursors, as shown in fig. 10C. A scale: 50 μm. For B and C, two-tailed t-test; and NS: is not significant; mean. + -. SEM.

Detailed Description

A first aspect of the present disclosure relates to a method of restoring K + uptake by glial cells, wherein the glial cells have an impaired K⁺The channel functions. The method involves effectively recovering a K with damage⁺Channel-functional glial cell K⁺Administering an inhibitor of SMAD4 to the glial cell under conditions of uptake.

Another aspect of the disclosure relates to a method of restoring glial cell K in a subject⁺The method of uptake. The method involves selecting glial cells K that have an impairment⁺Ingested subject and at a K effective to restore said glial cells⁺Administering to the selected subject an inhibitor of SMAD4 under conditions of ingestion.

As used herein, "glial cells" include glial progenitor cells, oligodendrocyte-biased progenitor cells, astrocyte-biased progenitor cells, oligodendrocytes, and astrocytes. Glial progenitor cells are bipotent progenitor cells in the brain that are capable of differentiating into oligodendrocytes and astrocytes. Glial progenitor cells may be identified by their expression of certain stage-specific surface antigens, such as gangliosides recognized by the A2B5 antibody and PDGFR α (CD140a), as well as stage-specific transcription factors, such as OLIG2, NKX2.2, and SOX 10. Oligodendrocyte-biased progenitors and astrocyte-biased progenitors are identified by their acquired expression of stage-selective surface antigens, including, for example, CD9 and lipid sulfatides recognized by the O4 antibody for oligodendrocyte-biased progenitors and CD44 for astrocyte-biased progenitors. Mature oligodendrocytes are identified by their expression of myelin basic protein, and mature astrocytes are most commonly identified by their expression of Glial Fibrillary Acidic Protein (GFAP). In one embodiment of the methods described herein, K + uptake is restored in the glial progenitor cells. In another embodiment, in astrocyte biasRecovery of K into progenitor cells⁺And (4) taking. In another embodiment, K is restored in astrocytes⁺And (4) taking.

According to these aspects of the disclosure, there is a compromised K⁺The ingested cells have a reduced K compared to normal healthy glial cells⁺The ingested glial cells, in particular glial progenitor cells, astrocyte-biased progenitor cells and/or astrocytes. In one embodiment, with reduced K⁺An ingested glial cell is one in which one or more potassium channel-encoding genes are down-regulated, resulting in a glial cell with reduced expression of the corresponding potassium channel protein. In particular, down-regulation of the expression of one or more potassium channel coding genes selected from the group consisting of⁺The intake is reduced: KCNJ9, KCNH8, KCNA3, KCNK9, KCNC1, KCNC3, KCNB1, KCNF1, KCNA6, SCN3A, SCN2A, SCNN1D, SCN8A, SCN3B, SLC12A6, SLC6a1, SLC8A3, ATP1a2, ATP1A3, ATP2B 2.

Thus, in one embodiment, glial cells K are selected for having damage⁺The subject taking involves assessing potassium uptake by glial cells of the subject, comparing the potassium uptake level of the glial cells to the potassium uptake level of a population of control healthy glial cells, and selecting glial cell K⁺A subject with reduced intake. In another embodiment, glial cells K are selected for having damage⁺The subject of uptake involves assessing the glial cell expression level of one or more potassium channel-encoding genes selected from the group consisting of: KCNJ9, KCNH8, KCNA3, KCNK9, KCNC1, KCNC3, KCNB1, KCNF1, KCNA6, SCN3A, SCN2A, SCNN1D, SCN8A, SCN3B, SLC12A6, SLC6a1, SLC8A3, ATP1a2, ATP1A3, ATP2B 2. In another embodiment, glial cells K are selected for having damage⁺The ingested subjects are involved in assessing glial protein expression of one or more potassium channels, including GIRK-3 (encoded by KCNJ9), potassium voltage gated channel subfamiliesFamily H member 8 (encoded by KCNH 8), potassium voltage gated channel subfamily A member 3 (encoded by KCNA 3), potassium channel subfamily K member 9 (encoded by KCNK 9), potassium voltage gated channel subfamily C member 1 (encoded by KCNC 1), potassium voltage gated channel subfamily C member 3 (encoded by KCNC 3), potassium voltage gated channel subfamily B member 1 (encoded by KCNB 1), potassium voltage gated channel subfamily F member 1 (encoded by KCNF 1), potassium voltage gated channel subfamily a member 6 (encoded by KCNA 6), type 3 sodium channel protein subunit α (encoded by SCN 3A), type 2 sodium channel protein subunit α (encoded by SCN 2A), amiloride sensitive sodium channel subunit δ (encoded by SCNN 1D), type 8 sodium channel protein subunit α (encoded by SCN 8A), sodium channel subunit β -3 (encoded by SCN 3B), solute carrier family 12 member 6 (i.e., K).⁺/Cl^-Cotransporter 3) (encoded by SLC12A 6), sodium and chloride dependent GABA transporter 1 (i.e., GAT-1) (encoded by SLC6A 1), Na⁺/Ca⁺²Swap 3 (encoded by SLC8A 3), Na⁺/K⁺Transport ATPase subunit alpha-2 (encoded by ATP1A 2), Na⁺/K⁺Transport atpase subunit α -2 (encoded by ATP1a 3), plasma membrane calcium transport atpase 2 (i.e., PMCA2) (encoded by ATP2B 2). Selecting the subject for treatment using the methods described herein if the level of one or more potassium channel proteins is reduced.

Potassium uptake, potassium channel gene expression, potassium channel protein expression, and SMAD4 gene expression can all be assessed using the methods described herein and methods well known to those skilled in the art. These parameters can be assessed in a glial cell sample taken from the subject. Alternatively, one or more of these parameters may be assessed in a subject-derived sample of induced pluripotent stem cells (ipscs) derived glial cells. ipscs can be obtained from virtually any somatic cell of a subject including, for example, but not limited to, fibroblasts, dermal fibroblasts obtained, for example, by skin sample or biopsy, synoviocytes from synovial tissue, keratinocytes, mature B cells, mature T cells, pancreatic beta cells, melanocytes, hepatocytes, foreskin cells, cheek cells, lung fibroblasts, peripheral blood cells, bone marrow cells, and the like. ipscs can be obtained by methods known in the art, including the use of integrating viral vectors (e.g., lentiviral vectors, inducible lentiviral vectors, and retroviral vectors), vectors such as transposons and lentiviral vectors labeled with loxP sites (floxed) and non-integrating vectors such as adenovirus and plasmid vectors, to deliver the above genes that promote Cell reprogramming (see, e.g., Takahashi and Yamanaka, Cell 126:663-676 (2006); Okita et al, Nature 448:313-317 (2007); Nakagawa et al, Nat. Biotechnol.26:101-106 (2007); Takahashi et al, Cell 131:1-12 (2007); Meissner et al Nat. Biotech.25:1177-1181 (2007); Yu et al Science 318:1917-1920 (2007); Park et al Nature 451:141-146 (2008); and U.S. patent application No. 2008/0233610, which are incorporated herein by reference in their entirety). Other methods for producing IPS cells include those disclosed in the following documents: WO2007/069666, WO2009/006930, WO2009/006997, WO2009/007852, WO2008/118820, Ikeda et al, U.S. patent application publication No. 2011/0200568 to Egusa et al, U.S. patent application publication No. 2010/0156778 to Musick, U.S. patent application publication No. 2012/0276070 to Musick, and U.S. patent application publication No. 2012/0276636 to Nakagawa; shi et al, Cell Stem Cell 3(5):568-574 (2008); kim et al, Nature 454:646-650 (2008); kim et al, Cell 136(3), 411-419 (2009); huangfu et al, Nature Biotechnology 26: 1269-; ZHao et al, Cell Stem Cell 3: 475-; feng et al, Nature Cell Biology 11:197-203 (2009); and Hanna et al, Cell 133(2): 250-. Methods of driving ipscs to Glial Progenitor Cell (GPC) fate and astrocyte fate are described herein and known in the art, see, e.g., Wang et al, "Human iPSC-Derived oligomeric promoter Cells can molecule and research a motor Model of genetic hybridization," Cell Stem Cell 12: 252-.

In one embodiment, with compromised K⁺The ingested glial cells are glial cells from subjects with neuropsychiatric disorders. As referred to herein, a "neuropsychiatric disorder" includesAny brain disease with psychotic symptoms including, but not limited to, dementia, amnestic syndrome and personality behavior changes. K known to be involved in damage in glial cells⁺Neuropsychiatric disorders that function and are suitable for treatment using the methods described herein include, but are not limited to, schizophrenia, autism spectrum disorders, and bipolar disorder.

Accordingly, another aspect of the present disclosure relates to a method of treating or inhibiting the onset of a neuropsychiatric disorder in a subject. This method involves selecting a subject having, or at risk of having, a neuropsychiatric disorder, and administering to the selected subject an inhibitor of SMAD4 under conditions effective to treat or inhibit the onset of the neuropsychiatric disorder in the subject.

In one embodiment, the subject treated according to the present disclosure is a subject suffering from, or at risk of suffering from, schizophrenia. Schizophrenia is a chronic and serious psychiatric disorder that affects how an individual thinks, feels, and behaves. Several Staging models of The disease state have been proposed so far (Agius et al, "The Staging Model in Schizophrania, and Clinical applications," Psychiator. Danub.22(2): 211; 220 (2010); "McGorry et al," Clinical Staging: a ecological Model and Practical Strategy for New Research and Better Health and Social issues for pathological and Related Disorders, "Can.J.Psychiatry 55(8): 486-. However, in general, schizophrenia progresses in at least three stages: prodromal phase, first onset and chronic phase. There is also heterogeneity of individuals at all stages of The condition, some of which are considered to be at an ultra-High Risk, clinically High Risk, or at Risk for The onset of Psychosis (Fusar-Poli et al, "The psychology High-rise State: a Comprehensive State-of-The-Art Review," JAMA psychology 70:107-120(2013), which is incorporated herein by reference in its entirety).

The methods described herein are applicable to treatment ofSubjects treated for any stage of schizophrenia and any level of psychosis at risk, as all stages will involve impaired glial cell K⁺And (4) taking. For example, in one embodiment, a subject treated according to the methods described herein is a subject at risk of developing schizophrenia. Such a subject may have one or more genetic mutations in one or more genes associated with the development of schizophrenia selected from the group consisting of: ABCA13, ATK1, C4A, COMT, DGCR2, DGCR8, DRD2, MIR137, NOS1AP, NRXN1, OLIG2, RTN4R, SYN2, TOP3B YWHAE, ZDHHC8 or chromosome 22(22q 11). In another embodiment, the subject may be in the prodromal phase of the disease and exhibit one or more early symptoms of schizophrenia, such as anxiety, depression, sleep disorders, and/or transient intermittent psychotic syndrome. In another embodiment, a subject treated according to the methods described herein is experiencing psychotic symptoms of schizophrenia, e.g., hallucinations, delusions.

In another embodiment, the methods described herein are used to treat a subject having autism or a related disorder. Related Disorders include, but are not limited to, Asperger's Disorder, pervasive developmental Disorder not otherwise specified, childhood disintegrative Disorder, and Rett's Disorder, which differ in the severity of symptoms, including difficulties in social interactions, communication, and abnormal behavior (McPartland et al, "austism and Related Disorders," handbb Clin neuron 106: 407-. The methods described herein are applicable to the treatment of each of these conditions and any stage of the condition. In one embodiment, a subject treated according to the methods described herein does not exhibit symptoms of any autism or related condition. In another embodiment, the subject treated exhibits one or more early symptoms of autism or related condition. In yet another embodiment, a subject treated according to the methods described herein exhibits multiple symptoms of autism or a related condition.

In another embodiment, the methods described herein are used to treat a subject having bipolar disorder. Bipolar disorder is a group of symptoms characterized by long-term emotional instability, disorders of circadian rhythms, and fluctuations in energy levels, mood, sleep, and self and other opinion. Bipolar disorders include, but are not limited to, bipolar disorder type I, bipolar disorder type II, cyclothymic disorder, and bipolar disorder not otherwise specified.

Generally, bipolar disorder is a progressive condition that progresses into at least three stages: prodromal, symptomatic, and residual phases (Kapczinski et al, "Clinical indications of a Staging Model for Bipolar Disorders," Expert Rev Neurother 9: 957-. The methods described herein are suitable for treating subjects suffering from any of the above-described bipolar disorders and subjects at any stage of a particular bipolar disorder. For example, in one embodiment, a subject treated according to the methods described herein is a subject in an early prodromal phase that exhibits symptoms of mood instability/swing, depression, rapid thinking, anger, irritability, physical agitation, and anxiety. In another embodiment, the subject treated according to the methods described herein is a subject in the symptom phase or residual phase.

As used herein, the terms "subject" and "patient" specifically include human and non-human mammalian subjects. As used herein, the term "non-human mammal" extends to, but is not limited to, domestic pets and domestic animals. Non-limiting examples of such animals include primates, cattle, sheep, ferrets, mice, rats, pigs, camels, horses, rabbits, goats, dogs, and cats.

According to the present disclosure, to have a compromised K⁺Glial cells that function as channels are administered an inhibitor of SMAD 4. In another embodiment, the subject has damaged glial cells K⁺Ingested subjects were administered SMAD4 inhibitors. In another embodiment, K is administered to a subject with or without the involvement of damaged glial cells⁺An ingested neuropsychiatric disorder or a subject at risk of having the neuropsychiatric disorder is administered an inhibitor of SMAD 4. Smad4 (also known as maternal confrontation to paralytic Homolog 4 (Heat Against Decapentaplegic Homolog 4, MADH4) and DPC4) represents the most unique member of the Smad family. This protein serves as a shared hetero-oligomerization partner in complex with pathway-restricted Smad (Lagna et al, "Partnership beta DPC4 and SMAD Proteins in TGF-beta Signalling Pathways," Nature 383:832-836 (1996); Zhang et al, "The Tumor supressor Smad4/DPC 4as a Central Mediator of Smad Function," Current.biol.7: 270-276(1997), which is incorporated herein by reference in its entirety). Smad4 has been shown to exhibit two distinct functions within the Smad signaling cascade, although it does not interact with TGF- β receptors. Smad4 facilitates binding of Smad Complexes to DNA by its N-terminus, and provides, by its C-terminus, the activation signal required for Smad Complexes to stimulate transcription (Liu et al, "Dual Role of the Smad4/DPC4 Tumor supressor in TGFbeta-index transcription Complexes," Genes Dev.11:3157-3167(1997), which is incorporated herein by reference in its entirety).

The SMAD4 amino acid sequence is provided as SEQ ID NO 1 below.

The nucleic acid sequence encoding SMAD4 is provided as SEQ ID NO 2

According to the present disclosure, a suitable SMAD4 inhibitor is any agent or compound capable of reducing the level of SMAD4 expression and/or SMAD4 signaling activity in a subject's glial cells relative to the level of SMAD4 expression and/or signaling activity that occurs in the absence of the agent. Suitable inhibitors may inhibit SMAD mRNA expression or protein expression, may block SMAD4 post-translational processing, may inhibit the interaction of SMAD4 with other signaling proteins, or may block SMAD4 nuclear translocation.

In one embodiment, the SMAD4 inhibitor is a small molecule inhibitor. An exemplary SMAD4 Inhibitor suitable for use in the methods disclosed herein is the Deubiquitinase Inhibitor PR-619 (i.e., 2, 6-diamino-3, 5-dithiocyanopyridine; CAS number 2645-32-1) that Reduces the Expression level of SMAD4, as described in Soji et al, "Deubiquitinase Inhibitor PR-619 reduction Smad4 Expression and depression reduction in lumen with unified aqueous oxygen evolution," PLoS 13(8): e0202409(2008), which is incorporated herein by reference in its entirety. Another exemplary small molecule inhibitor of SMAD4, which is Valproic acid (see, e.g., Mao et al, "Valproic acid inhibition temporal delivery in renal cell by discovery SMAD4 expression," mol.Med.Rep.16(5):6190-6199(2017) and Lan et al, "Valproic acid (VPA) inhibition the temporal delivery in renal cell view the dual delivery of SMAD4," J Cancer Res Clin Oncol.142(1):177-85(2016), which is incorporated herein by reference in its entirety), also functions by decreasing SMAD4 expression and is suitable for use in the methods described herein. Another exemplary small molecule inhibitor of SMAD4 suitable for use in the methods described herein is 5-fluorouracil (5-FU) that reduces the levels of SMAD4 protein, as taught by Okada et al, "Regulation of transformation growth factor is involved in the efficiency of the efficacy of combined 5-fluorootic and interferon alpha-2b therapy of advanced hepatocellular Carcinoma," Cell Death Discov.4:42(2018), which is incorporated herein by reference in its entirety. Another exemplary SMAD4 Inhibitor suitable for use in the methods described herein is the HDAC Inhibitor vorinostat, which inhibits SMAD4 nuclear translocation, as described by Sakamoto et al, "A Histone deacylase Inhibitor precursors superior Epithelial-metabolic transfer and inhibitors chemical in Biliary Transcer," PLoS One 11(1): e0145985(2016), which is incorporated herein by reference in its entirety. Mitogen-activated protein kinase (MAPK) -specific inhibitors also block SMAD4 nuclear translocation as disclosed in Jiang et al, "MAPK inhibitors modulated Smad2/3/4complex Cell-nuclear translocation in myofibrasts via Imp7/8 differentiation," Mol Cell biochem.406(1-2):255-62(2015), which is incorporated herein by reference in its entirety). Thus, MAPK-specific inhibitors (particularly ERK, JNK, and p 38-specific inhibitors) serve as another class of small molecule inhibitors useful in the methods disclosed herein. Suitable inhibitors in this Class are known in the art and include, for example, but are not limited to Ulitetinib (Ulixertinib) (ERK Inhibitor) (BVD523) (Sullivan et al, "First-in-Class ERK1/2Inhibitor Ulixertinib (BVD-523) in Package with MAPK Mutant Advanced Solid solutions: solutions of a Phase I Dose-estimation and Expansion Study," Cancer Discov.8(2):1-12 (2017)), which is incorporated herein by reference in its entirety; CC-401, SP600125, AS601245, AS602801, D-JNK-1, and BI-78D (JNK Inhibitors) (Cicenas et al, "JNK, p38, ERK, and SGK1 Inhibitors in Cancer," Cancers 10:1(2018), which are incorporated herein by reference in their entirety); SCIO-469 (Tapidimod (Talmapimod)), BIRB-796 (Durmamod (Doramapimod)), LY2228820 (Ramietinib), VX-745 and PH-797804 (selective p38 Inhibitors) (Cicenas et al, "JNK, p38, ERK, and SGK1 Inhibitors in Cancer," Cancers 10:1(2018), which are incorporated herein by reference in their entirety).

Another class of SMAD4 inhibitors suitable for use in the methods disclosed herein includes inhibitory peptides. One suitable peptide inhibitor of SMAD4 is the SBD peptide capable of blocking the interaction of the SMAD4 protein (Urata et al, "A peptide present blocks the interaction of NF-. kappa. B p65 subbunnit with Smad4 enzymes BMP2-induced osteopenias," J Cell physiology.233 (9):7356-7366(2018), which is incorporated herein by reference in its entirety). The SBD peptide corresponds to the amino-terminal region within the transactivation of p65, which interacts with the MH1 domain of SMAD4 (referred to as Smad4 binding domain (SBD)) (see Urata et al, "A peptide blocks the Interaction with Smad4 enhanced BMP2-Induced osteogenesis," J Cell physiology.233 (9):7356-7366(2018) and Hirata-Tsuchiya et al, Inhibition of BMP2-Induced Bone Formation by the p65 bunit of NK-kB video Interaction with SMAD4, "mol. Binding of SBD peptides to SMAD4 blocked SMAD4 interaction with other proteins such as p 65. An exemplary SBD peptide has amino acid sequence APGLPNGLLSGDEDFSSIADMDFSALLSQISS (SEQ ID NO: 35).

Another suitable peptide inhibitor of SMAD4 is a pilin-like protein (CLP or Cotl 1; UniProtKB accession number Q14019), which is an F-actin binding protein. This protein inhibits SMAD4 by causing post-translational downregulation of SMAD4(Xia et al, "Cooperatin-like protein CLP/Cotl1 supresses cleavage growth of high activity of IL-24/PERP and inhibition of non-canonical TGF β signaling," Oncogene 37(3):323-331(2018), which is incorporated herein by reference in its entirety). Thus, a recombinant form of CLP/Cotl1 having the amino acid sequence SEQ ID NO:8 (shown below) or an active fragment thereof is suitable for use in the methods disclosed herein.

In another embodiment, the SMAD4 inhibitor is an inhibitory nucleic acid molecule selected from the group consisting of a SMAD4 antisense oligonucleotide, a SMAD4 shRNA, a SMAD4 siRNA, and a SMAD4 RNA aptamer.

The use of antisense approaches to inhibit in vivo translation of genes and subsequent protein expression is well known in the art (e.g., U.S. Pat. No. 7,425,544 to Dobie et al; U.S. Pat. No. 7,307,069 to Karras et al; U.S. Pat. No. 7,288,530 to Bennett et al; U.S. Pat. No. 7,179,796 to Cowsert et al, which is incorporated herein by reference in its entirety). Suitable Antisense nucleic acids, according to the present disclosure, are nucleic acid molecules (e.g., molecules containing DNA nucleotides, RNA nucleotides, or modifications (e.g., modifications that increase the stability of the molecule, such as 2' -O-alkyl (e.g., methyl) substituted nucleotides) or combinations thereof) that are complementary to or hybridize to at least a portion of a particular nucleic acid molecule encoding SMAD4 (see, e.g., Weintraub, h.m., "Antisense DNA and RNA," Scientific am.262:40-46(1990), which is incorporated herein by reference in its entirety). SEQ ID NO 2 above is an exemplary nucleic acid molecule encoding SMAD 4. Suitable antisense oligonucleotides for use in the methods described herein are at or up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length and comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobases relative to a target SMAD4 nucleic acid or a specified portion thereof. The antisense nucleic acid molecule hybridizes to its corresponding target SMAD4 nucleic acid molecule to form a double-stranded molecule that interferes with translation of the mRNA, as the cell will not translate the double-stranded mRNA.

The SMAD4 antisense nucleic acid can be introduced into a cell as an antisense oligonucleotide, or can be produced, for example, using gene therapy methods, in a cell into which a nucleic acid encoding the antisense nucleic acid has been introduced. anti-SMAD 4 antisense oligonucleotides suitable for use in accordance with the methods described herein are disclosed in Monia et al, U.S. Pat. No. 6,013,787 and Kretschmer et al, "Differential Regulation of TGF- β Signaling Through Smad2, Smad3, and Smad4," Oncogene 22:6748-6763(2003), which are incorporated herein by reference in their entirety.

The SMAD4 siRNA is a double-stranded synthetic RNA molecule of about 20-25 nucleotides in length with short 2-3 nucleotide 3' overhangs at both ends. A double stranded siRNA molecule refers to both the sense and antisense strands of a portion of a target mRNA molecule, in this case a portion of the nucleotide sequence of SMAD4, i.e., SEQ ID No. 2 encoding SMAD 4. siRNA molecules are typically designed to target a region of about 50-100 nucleotides from the start codon of the SMAD4 mRNA target. Upon introduction into the cell, the siRNA complex initiates an endogenous RNA interference (RNAi) pathway, resulting in cleavage and degradation of the target SMAD4 mRNA molecule. siRNA molecules targeting SMAD4 and other members of the SMAD4 transcription complex useful in the methods disclosed herein are disclosed in U.S. patent No. 9,035,039 to Dhillon et al and pupampu-Dove et al, "protected Tumor Immunity Using Aptamer-Targeted RNAi to Render CD8+ T Cells resist to TGF β Inhibition," j. Various modifications of siRNA compositions have been described, such as the incorporation of modified nucleosides or motifs into one or both strands of the siRNA molecule to enhance stability, specificity and efficacy, and are suitable for use in accordance with this aspect of the disclosure (see, e.g., WO2004/015107 to Giese et al; WO2003/070918 to McSwiggen et al; WO1998/39352 to Imanishi et al; U.S. patent application publication No. 2002/0068708 to Jesper et al; U.S. patent application publication No. 2002/0147332 to Kaneko et al; U.S. patent application publication No. 2008/0119427 to Bhat et al, which are incorporated herein by reference in their entirety).

Short or small hairpin RNA molecules are functionally similar to siRNA molecules, but contain longer RNA sequences that produce tight hairpin turns. shRNA is cleaved into siRNA by cellular mechanisms, and gene expression is silenced by cellular RNA interference pathways. Described herein are shRNA molecules that effectively interfere with SMAD4 expression, and which comprise the following nucleic acid sequences: 5 'GUAAGUAGCUGGCUGACCA-3' (SEQ ID NO:3) targeting SMAD4 nucleotide sequence 5'-TGGTCAGCCAGCTACTTAC-3' (SEQ ID NO:4) and 5'-AGAAGUGAGUCAUAUUCAU-3' (SEQ ID NO:6) targeting SMAD4 nucleotide sequence 5'-ATGAATATGACTCACTTCT-3' (SEQ ID NO: 7). Other shRNA molecules that inhibit SMAD4 expression and that are suitable for use in accordance with the methods described herein are art-inhibited, see, e.g., WO2016115558 to Doiron, which is incorporated herein by reference in its entirety.

Nucleic acid aptamers that specifically bind to SMAD4 are also suitable for use in the methods as described herein. Nucleic acid aptamers are single-stranded, partially double-stranded or double-stranded nucleotide sequences that are capable of specifically recognizing a selected target molecule, i.e., the SMAD4 protein having the amino acid sequence SEQ ID No. 1 or the SMAD4 nucleic acid molecule having the nucleotide sequence SEQ ID No. 2, by mechanisms other than Watson-Crick base pairing or triplex formation. Aptamers include, but are not limited to, defined sequence segments and sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides, and nucleotides comprising a backbone modification, a branch point, and a non-nucleotide residue, group, or bridge.

Modifications to the inhibitory nucleic acid molecules described herein (i.e., SMAD4 antisense oligonucleotides, sirnas, shrnas, PNAs, aptamers) include substitutions or alterations to internucleoside linkages, sugar moieties, or nucleobases. Modified inhibitory nucleic acid molecules are generally preferred over native forms because they have desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. For example, chemically modified nucleosides can be used to increase the binding affinity of a shortened or truncated antisense oligonucleotide to its target nucleic acid. Thus, comparable results can often be obtained with shorter antisense compounds having such chemically modified nucleosides.

The inhibitory nucleic acid molecule targeted to SMAD4 may optionally contain one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides can confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the nucleic acid molecule. In certain embodiments, the nucleoside comprises a chemically modified ribofuranosyl ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents including 5 'and 2' substituents, bridging non-geminal ring atoms to form Bicyclic Nucleic Acids (BNAs), replacement of ribosyl epoxy atoms with S, N (R) or C (R1) (R)2 (where R ═ H, C1-C12 alkyl or protecting groups), and combinations thereof. Examples of chemically modified sugars include 2 ' -F-5 ' -methyl substituted nucleosides, replacement of the ribosyl epoxy atom with S and further substitution at the 2 ' -position.

In certain embodiments, the nucleoside is modified by replacement of the ribose ring with a sugar substitute (sometimes referred to as a DNA analog), such as a morpholino ring, cyclohexenyl ring, cyclohexyl ring, or tetrahydropyranyl ring.

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, but functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both the natural nucleobase and the modified nucleobase are capable of participating in hydrogen bonding. Such nucleobase modifications may confer nuclease stability, binding affinity, or some other beneficial biological property to the SMAD4 inhibitor nucleic acid molecule. Modified nucleobases include synthetic and natural nucleobases, such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of a nucleic acid molecule to its target nucleic acid. Other modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-amino adenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C.ident.C-CH 3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine bases, 5-halo (specifically 5-bromo), 5-trifluoromethyl, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

The naturally occurring internucleoside linkages of RNA and DNA are 3 'to 5' phosphodiester linkages. Inhibitory nucleic acid molecules having modified internucleoside linkages comprise internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methyl phosphates, phosphoramidates, and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free bonds are well known. In certain embodiments, an inhibitory nucleic acid molecule targeted to a SMAD4 nucleic acid comprises one or more modified internucleoside linkages.

The inhibitory nucleic acid molecules described herein can be covalently linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the resulting inhibitory nucleic acid molecule. Typical conjugate groups include a cholesterol moiety and a lipid moiety. Additional conjugate groups include carbohydrates, polymers, peptides, inorganic nanostructured materials, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

The inhibitory nucleic acid molecules described herein can also be modified to have one or more stabilizing groups, such as cap structures, typically attached to one or both ends of the inhibitory nucleic acid molecule to enhance properties (e.g., nuclease stability). These end modifications protect the inhibitory nucleic acid molecule from exonuclease degradation and aid in intracellular delivery and/or localization. The cap structure may be present at the 5 'end (5' -cap) or the 3 'end (3' -cap), or may be present at both ends. Cap structures are well known in the art and include, for example, reverse-deoxy alkali-free caps. Other 3 'and 5' -stabilizing groups that can be used to cap one or both ends of an inhibitory nucleic acid molecule to confer nuclease stability include those disclosed in WO 03/004602 to Manoharan, which is incorporated herein by reference in its entirety.

In another embodiment, a suitable SMAD4 inhibitor is any agent or small molecule capable of reducing, blocking, or preventing the level of interaction in a glial cell relative to the interaction of SMAD4 with SMAD2 and 3 and/or the interaction of SMAD4 with SMAD1, 5, and 8 that occurs in the absence of the agent.

In another embodiment, a suitable SMAD4 inhibitor is any agent or small molecule capable of antagonizing or reducing SMAD4 activity in a glial cell relative to the level of SMAD4 activity that occurs in the absence of the agent.

In one embodiment, the SMAD4 inhibitor for use according to the methods described herein is packaged into a nanoparticle delivery vehicle (delivery vehicle) to effect delivery of the inhibitor to the subject's glial cells. Suitable nanoparticle delivery vehicles for delivering SMAD4 inhibitors across the blood-brain barrier and/or to glial cells include, but are not limited to, liposomes, protein nanoparticles, polymer nanoparticles, metal nanoparticles, and dendrimers.

Liposomes are spherical vesicles of about 80-300nm in size, composed of a phospholipid and steroid (e.g., cholesterol) bilayer. Liposomes are biodegradable and have low immunogenicity. The SMAD4 inhibitors as described herein may be incorporated into liposomes using an encapsulation method. Liposomes are taken up by target cells by adsorption, fusion, endocytosis or lipid transfer. Release of SMAD4 inhibitor from liposomes depends on liposome composition, pH, osmotic gradient, and surrounding environment. Liposomes can be designed to release SMAD4 inhibitors in an organelle-specific manner to achieve nuclear delivery of, for example, SMAD4 inhibitors.

Methods and types of liposomes useful for delivering the SMAD4 inhibitors described herein to glial cells are known in the art, see, e.g., Liu et al, "Paclitaxel loaded liposomes purified with a multi functional peptide for the purposes of glioma targeting," Biomaterials 35: 4835-4847 (2014); gao et al, "Glioma targeting and blood-blue barrier specificity by dual-targeting doxorubicin ligands," Biomaterials 34: 5628-; zong et al, "Synergistic dual-ligand and doxorubicin ligands immunogenic targeting and therapeutic efficacy of broad glioma in analytes," Mol pharm.11: 2346-; yermisci et al, "systematic incorporated particulate pigments acids of the Blood-particulate barrier and product neuroprotection," J Cerebr Blood F Met.35: 469-475 (2015), which is incorporated herein by reference in its entirety.

In another embodiment, the SMAD4 inhibitor described herein is packaged in a polymeric delivery vehicle. Polymeric delivery vehicles are structures that are typically about 10 to 100nm in diameter. Suitable polymeric nanoparticles for encapsulating SMAD4 inhibitors as described herein may be made from synthetic polymers (e.g., poly-epsilon-caprolactone, polyacrylamide, and polyacrylate) or natural polymers (e.g., albumin, gelatin, or chitosan). The polymeric nanoparticles used herein may be biodegradable, such as poly (L-lactide) (PLA), Polyglycolide (PGA), poly (lactic-co-glycolic acid) (PLGA), or non-biodegradable, such as polyurethane. The polymeric nanoparticles used herein may also contain one or more delivery-enhancing surface modifications. For example, in one embodiment, the polymeric nanoparticles are coated with a nonionic surfactant to reduce immunological interactions as well as intermolecular interactions. The surface of the polymeric nanoparticle may also be functionalized to attach or immobilize one or more targeting moieties as described below, such as an antibody or other binding polypeptide or ligand that directs the nanoparticle across the blood-brain barrier and/or to glial cells for glial cell uptake (i.e., glial progenitor cell or astrocyte uptake).

Methods and types of polymeric Nanoparticles useful for the delivery of SMAD4 inhibitors as described herein to glial cells are known in the art, see, e.g., koffee et al, "Nanoparticles engineering antibody delivery of blood-antibody barrier-affinity probes for in vivo optical and magnetic resistance imaging," Proc Natl Acad Sci S a.108: 18837-; ZHao et al, "The perfect of The polyurethane loaded poly (butyl acrylate) coated with The polystyrene 80on The block-resist barrier and its protective effect against cellulose emulsion/hydrolysis in front," Biol phase wall.36: 1263-1270 (2013); yermisci et al, "systematic incorporated particulate pigments acids of the Blood-particulate barrier and product neuroprotection," J Cerebr Blood F Met.35: 469-475 (2015), which is incorporated herein by reference in its entirety.

In another embodiment, the compositions of the present disclosure are packaged in a dendrimer nanocarrier delivery vehicle. Dendrimers are unique polymers with well-defined size and structure. Exemplary nanomolecules having a dendritic structure suitable for use as delivery vehicles for SMAD4 inhibitors as described herein include, but are not limited to, glycogen, amylopectin, and proteoglycans. Methods of encapsulating therapeutic compositions (e.g., compositions described herein) in the internal structure of dendrimers are known in the art, see, e.g., D' emeruele et al, "dendromer-Drug interactions," Adv Drug delivery Rev 57: 2147-2162 (2005), which is incorporated herein by reference in its entirety. The surface of the dendrimer is suitable for attachment of one or more targeting moieties, such as antibodies or other binding proteins and/or ligands capable of targeting the dendrimer across the blood-brain barrier and/or to glial cells as described herein.

An exemplary dendrimer for encapsulating the SMAD4 inhibitor for administration and delivery to a subject in need thereof is poly (amidoamide) (PAMAM). PAMAM has been used to deliver protein and nucleic acid therapeutics to target cells of interest. Methods of encapsulating therapeutic agents in PAMAMs and methods of delivering therapeutic agents to the central nervous system using PAMAMs are also known in the art and may be used herein, see, e.g., Cerqueira et al, "Multifunctionalized CMCht/PAMAM dendrimer nanoparticles modulators the cellular uptake by y assays and oligonucleotides in primary cells of cells," Macromol biosci.12: 591-597 (2012); nance et al, "systematic timer-drug flow of ischemia-induced neural bottom matrix in flow," J Control Release 214: 112-; natali et al, "Dendriers as drivers: dynamics of PEGylated and method-loaded Dendrimers in aqueous solution," Macromolecules 43: 3011-3017 (2010); han et al, "Peptide conjugated PAMAM for targeted doxorubicin delivery to transporter expressed regulators," Mol Pharm 7: 2156-2165 (2010); kannan et al, "Dendrimer-based Postnat Therapy for neuro-fluidic and Cerebral Palsy in a Rabbit Model," Sci. Transl. Med.4:130 (2012); and Singh et al, "form and form-PEG-PAMAM dendrimers: synthesis, characterization, and targeted anti-drug delivery in molecular bearing," Bioconjugate Chem 19: 2239-.

In another embodiment, the SMAD4 inhibitor as disclosed herein is packaged in silver nanoparticles or iron oxide nanoparticles. Methods and preparations of silver and iron oxide nanoparticles useful for the delivery of SMAD4 inhibitors described herein to glial cells are known in the art, see, e.g., Hohnholt et al, "Handling of iron oxide and silver nanoparticles by astrocytes," neurohem res.38: 227-239 (2013), which is incorporated herein by reference in its entirety.

In another embodiment, the SMAD4 inhibitor described herein is packaged in gold nanoparticles. Gold nanoparticles are small particles (<50nm) that enter cells via endocytic pathways. In one embodiment, Gold Nanoparticles are coated with Glucose to facilitate transfer of the Nanoparticles across the blood Brain barrier and uptake of the Nanoparticles by Astrocytes via GLUT-1 receptors, as described by Gromnicova et al, "Glucose-coated Gold Nanoparticles across the enzyme assays and Enter assays In vitro," PLoSONE 8(12): e81043(2013), which is incorporated herein by reference In its entirety.

In another embodiment, the composition of the present disclosure is packaged in silica nanoparticles. Silica nanoparticles are biocompatible, highly porous and easily functionalized. The silica nanoparticles are amorphous in shape and range in size from 10 to 300 nm. Silica Nanoparticles suitable for Delivery of therapeutic compositions such as SMAD4 inhibitors to the CNS for glial uptake are known In the art, see, e.g., Song et al, "In vitro Study of Receptor-mediated Silica Nanoparticles Delivery Across Blood Brain Barrier," ACS application.mater.interface 9(24): 20410-; tamba et al, "Tailored Surface silicon Nanoparticles for Blood-Brain Barrier networking: Preparation and In vivo Investigation," Arabian J.chem.doi.org/10.1016/j.arabjc.2018.03.019(2018), which is incorporated herein by reference In its entirety.

In another embodiment, the SMAD4 inhibitor is packaged into a protein nanoparticle delivery vehicle. Protein nanoparticles are biodegradable, metabolizable and easily modified to allow capture of therapeutic molecules or compositions and attachment of targeting molecules as needed. Suitable Protein Nanoparticle Delivery vehicles known in the art and used to deliver therapeutic compositions to the central nervous system include, but are not limited to, Albumin particles (see, e.g., Lin et al, "Blood-broad Barrier peptide Nanoparticles for biomedical Drug Delivery system for anticoagulant Protein Therapy," ACS Nano 10(11):9999-10012(2016), and Ruan et al, "substtate P-modified Human Serum Nanoparticles with bound Protein for Targeted Therapy B8 (2016)," 85-96 (2018): which is incorporated herein by reference in its entirety), Gelatin Nanoparticles (see, e.g., Zo et al, "Gelatin Nanoparticle Delivery system for polypeptide Nanoparticles for" antibiotic Drug Delivery system B8 (2016) ", which is incorporated herein by reference in its entirety) and Lactoferrin Nanoparticles (see, e.g., Kumari et al, "overlying Blood Brain Barrier with Dual Purpose plasma Temozolamide Loaded Lactoferrin Nanoparticles for coating Glioma (SERP-17-12433)," Scientific Reports 7:6602(2017), which is incorporated herein by reference in its entirety).

Nanoparticle-mediated delivery of therapeutic compositions can be achieved passively (i.e., based on the normal distribution pattern of liposomes or nanoparticles in the body) or by active targeted delivery. Active targeted delivery involves altering the natural distribution pattern of the delivery vehicle by attaching a targeting moiety to the outer surface of the liposome. In one embodiment, a delivery vehicle as described herein is modified to include one or more targeting moieties, i.e., targeting moieties that facilitate delivery of the liposome or nanoparticle across the blood-brain barrier and/or targeting moieties that facilitate glial uptake (i.e., glial progenitor uptake and/or astrocytic uptake). In one embodiment, a delivery vehicle as described herein is surface modified to express a targeting moiety suitable for achieving blood brain barrier penetration. In another embodiment, a delivery vector as described herein is surface modified to express a targeting moiety suitable for glial uptake. In another embodiment, the delivery vector described herein is surface modified to express a dual targeting moiety.

Targeting moieties that facilitate liposome or nanoparticle delivery across the blood brain barrier utilize receptor-mediated, transporter-mediated, or adsorption-mediated transport across the barrier. Suitable targeting moieties for achieving blood-brain barrier passage include antibodies and ligands that bind to endothelial cell surface proteins and receptors. Exemplary targeting moieties include, but are not limited to, cyclic RGD peptides (Liu et al, "Paclitaxel loaded peptides purified with a multifunctionality and targeting for a glioma," Biomaterials 35: 4835-4847 (2014), which is incorporated herein by reference in its entirety); a cyclic A7R Peptide that binds VEGFR2 and neuropilin-1 (Ying et al, "A Stabilized Peptide Ligand for Multi functional gliomas Targeted Drug Delivery," J.Contr.Rel.243:86-98(2016), which is incorporated herein by reference in its entirety); transferrin, peptides or antibodies capable of binding to Transferrin Receptor (Zong et al, "synthetic dual-ligand binding and Therapeutic affinity of peptide in a chemicals," Mol phase.11: 2346. sub.235773 (2014); Yiemi et al, "systematic incorporated into peptide bound peptide transfer a crystals of peptide in a protein complex. sub.394-peptide binding and protein transcription," J center Blood F.35: 469. sub.2015); and Wei et al, "Brain bound peptide bound system Delivery of peptide in a protein of peptide in a protein binding protein of peptide in a protein binding protein of peptide in a protein of peptide binding protein in a protein of peptide; folate proteins or peptides that bind folate receptors (Gao et al, "Glioma targeting and blood-blue barrier specificity by dual-targeting doxorubin lipids," Biomaterials 34: 5628-; lactoferrin albumin or a peptide binding to the lactoferrin Receptor (Song et al, "In vitro Study of Receptor-mediated Silica Nanoparticles Delivery Across Blood Brain Barrier," ACS application. Mater. Interfaces 9(24): 20410-; low density lipoprotein receptor ligands, such as ApoB and ApoE (Wagner et al, "upper Mechanisms of ApoE-modified nanoparticules on Brain Capillary intrinsic Cells as a Blood-bridge Barrier Model," PLoS One 7: e32568(2012), which is incorporated herein by reference in its entirety); substance P peptides (Ruan et al, "Substance P-modified Human Serum Albumin Nanoparticles Loaded with Paclitaxel for Targeted Therapy of Glioma," Acta pharmaceutical Sinica B8 (1):85-96(2018), which is incorporated herein by reference in its entirety); and angiopep-2(An2) peptides (Demeule et al, "Conjugation of a broad-pendant peptide with neuroleptin proteins polypeptides inhibitory properties," J.Clin.invest.124: 1199-1213 (2014), which is incorporated herein by reference in its entirety). Other suitable targeting moieties include ligands for amino acid transporters, such as Glutathione for transport through Glutathione transporters (Rip et al, "Glutathione PEGylated Liposomes: Pharmacokinetics and Delivery of Cargo Across the Blood-Brain Barrier in rates," J.drug Target 22:460-67(2014), which is incorporated herein by reference in its entirety), and Choline derivatives for Delivery through Choline transporters (Li et al, "Choline-derivative-modified Nanoparticles for Brain-targeting Gene Delivery," adv.Mater.23:4516-20(2011), which is incorporated herein by reference in its entirety).

The second targeting moiety is a moiety that facilitates glial cell delivery and uptake. Suitable targeting moieties for achieving astrocytic uptake include, but are not limited to, Low Density Lipoprotein (LDL) receptor ligands or peptides thereof capable of binding to LDL Receptors and Oxidized LDL Receptors on astrocytes (Lucarelli et al, "The Expression of Native and Oxidized LDL Receptors in proteins Microvessels specificity Enhanced by assay-derived solvent Factor(s)," FEBS Letters 522(1-3):19-23(2002), incorporated herein by reference in its entirety), glucose or other glycans capable of binding to GLUT-1 receptors on Astrocytes (Gromnicova et al, "Glucose-coated Gold Nanoparticles Transfer across Human Brain protein endothelial and inner assays In vitro," PLoS ONE 8(12): e81043(2013), which is incorporated herein by reference In its entirety) and platelet-derived growth factors or peptides thereof capable of binding to PDGFR α of glial cells.

Glial cell delivery of inhibitory nucleic acid molecules described herein (e.g., SMAD4 antisense oligonucleotides, SMAD4 siRNA, SMAD4 shRNA) may also be achieved by packaging such nucleic acid molecules in viral vectors. Several viral vectors are known to inherently target Astrocytes In vivo, such as Lentiviral vectors (Colin et al, "Engineered Lentiviral Vector Targeting assays In vivo," Glia57:667-679(2009), and Cannon et al, "Pseudotype-dependent Lentiviral Transduction of assaying or neurones In the Rat substentia Nigra," exp.Neurol.228:41-52(2011), which are incorporated herein by reference In their entirety, and adeno-associated viral vectors (Furman et al, "Targeting assays analytes neuroreagents In a. Mouse Model of Alzheimer's Disease," J.Neurosis.32: 16129-40), which are incorporated herein by reference In their entirety, and are suitable for use In the inhibition of nucleic acid delivery according to the methods described herein, and for the delivery of nucleic acids according to SMAD molecules described herein.

In one embodiment, the vector is an adeno-associated virus (AAV) vector. A number of therapeutic AAV vectors suitable for delivering a nucleic acid SMAD4 inhibitor or a polynucleotide encoding an inhibitor of the SMAD4 protein described herein to the central nervous system are known in the art. See, e.g., Deverman et al, "Gene Therapy for Neurological Disorders: Progress and Prospectra," Nature Rev.17:641-659(2018), which is incorporated herein by reference in its entirety. Suitable AAV vectors include serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 in native form or engineered for enhanced tropism. AAV vectors known to have tropism for the CNS and particularly suitable for therapeutic expression of the SMAD4 nucleic acid molecules described herein include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9 in native form or engineered for enhanced tropism. In one embodiment, the AAV vector is an AAV2 vector. In another embodiment, the AAV vector is an AAV5 vector as described in Vital et al, "Anti-Tau structural scFv MC1 Antibody functional Tau specifices in Adult JNPL3 Mice," Acta Neuropathin. Commun.6:82(2018), optionally containing a GFAP or CAG promoter and a post-woodchuck hepatitis virus (WPRE) translational regulatory element. In another embodiment, the AAV vector is an AAV9 vector as described by Haiyan et al, "Targeting Root Cause by systematic scAAV9-hIDS Gene Delivery: Functional Correction and recovery of Server MPSII in Mice," mol.ther.methods Clin.Dev.10:327-340(2018), which is incorporated herein by reference in its entirety. In another embodiment, the AAV vector is an AAVrh10 vector as described by Liu et al, "Vectored Intra particulate Immunations with the Anti-Tau Monoclonal Antibody PHF1 Markedly reduce Tau Pathology in variant transduction Mice," J.Neurosci.36(49):12425-35(2016), which is incorporated herein by reference in its entirety.

In another embodiment, the AAV vector is a hybrid vector comprising the genome of one serotype (e.g., AAV2) and a capsid protein of another serotype controlling tropism (e.g., AAV1 or AAV 3-9). See, for example, Broekman et al, "Adeno-associated Virus Vectors segmented with AAV8 Capsule move Efficient and this AAV-1 or-2 segmented for Wireless Gene Delivery to the New Motor Mouse," Neuroscience 138:501-510(2006), which is incorporated herein by reference in its entirety. In one embodiment, the AAV vector is an AAV2/8 hybrid vector as described in "AAV-mediated Expression of Anti-Tau ScFv primers in a Mouse Model of Tauopathy," J.Exp.Med.214(5):1227(2017), which is incorporated herein by reference in its entirety, by Ising et al. In another embodiment, the AAV vector is an AAV2/9 hybrid vector as described by Simon et al, "A Rapid Gene Delivery-Based Mouse Model for Early-Stage Alzheimer Disease-Type Taupathiy," J.Neuropath.Exp.Neurol.72(11):1062-71(2013), which is incorporated herein by reference in its entirety.

In another embodiment, the AAV vector is the following: have been engineered or selected so that CNS transduction is enhanced following intraparenchymal administration, e.g., AAV-DJ (Grimm et al, J.Viol.82:5887-5911(2008), which is herein incorporated by reference in its entirety); increased transduction of stem or progenitor cells, such as SCH9 and AAV4.18(Murlidharan et al, J.Virol.89: 3976-; enhanced retrograde transduction, such as rAAV2-retro (Muller et al, nat. Biotechnol.21:1040-1046(2003), which is incorporated herein by reference in its entirety); or enhanced transduction of the adult CNS following IV administration, e.g., AAV-PHP.B and AAVPHP.eB (Deverman et al, nat. Biotechnol.34: 204-.

As used herein, "treatment" or "treatment" includes the administration of an SMAD4 inhibitor to partially or fully restore or derepress potassium channel gene expression in glial cells, partially or fully restore potassium channel uptake activity in glial cells, and partially or fully restore potassium homeostasis in glial cells and surrounding tissues. With respect to treating a subject having a neuropsychiatric condition, "treating" includes any indication of success in ameliorating the condition, including any objective or subjective parameter, such as a reduction, alleviation, reduction of symptoms (e.g., reduction of neuronal excitability), or making a patient more tolerant to the condition (e.g., reduction of epileptic events); slowing the progression of the condition; the disease state is not worsened; or improving the physical and mental health of the subject. Treatment or amelioration of symptoms can be based on objective or subjective parameters; including results of physical examination, neurological examination, and/or psychiatric evaluation.

As referred to herein, "under effective conditions" refers to an effective dose, route of administration, frequency of administration, formulation of SMAD4 inhibitor, and the like, that plays a role in achieving the desired therapeutic benefit for the subject. An effective amount of an inhibitor of SMAD4 for treating a subject according to the methods described herein is a dose of an inhibitor of SMAD4 that partially or fully derepresses potassium channel gene expression, thereby restoring potassium channel uptake function (partially or fully) to allow restoration of brain potassium homeostasis. In the case of administering an inhibitor of SMAD4 to a subject suffering from a neuropsychiatric disorder, such as schizophrenia, the effective dose is a dose that induces differentiation of glial progenitor cells into astrocytes. In another embodiment, an effective dose is a dose required to restore brain potassium homeostasis to a level sufficient to reduce extracellular levels of potassium, reduce neuronal excitability, and reduce the level of an epileptic event. In another embodiment, an effective dose to treat a subject having a neuropsychiatric disorder is a dose effective to improve a cognitive disorder in the subject. The effective dosage and administration conditions for a particular subject will vary, for example, depending on the health and physical condition of the individual to be treated, the mental and emotional abilities of the individual, the stage of the disorder, the type of SMAD4 inhibitor, the route of administration, the formulation, the attending physician's assessment of the medical condition, and other relevant factors.

In one embodiment, with compromised K⁺The glial cells that function as channels are glial progenitor cells. As shown in the examples herein, upregulation of SMAD4 in glial progenitor cells inhibits K⁺Channel gene expression and subsequent inhibition of K of glial progenitor cells⁺And (4) taking. K⁺The reduction in uptake inhibits terminal glial progenitor cell differentiation. Thus, in one embodiment, an effective dose of an inhibitor of SMAD4 is a dose that enhances astrocytic maturation of glial progenitor cells, thereby reducing, eliminating or inhibiting the onset of neuropsychiatric disease, symptoms of neuropsychiatric disease, or side effects of the disease.

In another embodiment, with compromised K⁺The glial cells that function as channels are astrocytes. Inhibition of SMAD4 in astrocytes restores K in affected astrocytes⁺Uptake and subsequent K⁺And (4) steady state. Inhibition of SMAD4 in astrocytes (in which potassium channel expression and function is altered) of subjects with neuropsychiatric disorders reduces neuronal excitability, reduces incidence of epilepsy (seizure epilepsy), and improves cognitive disorders. Thus, treatment with an effective dose of an inhibitor of SMAD4 reduces, alleviates, arrests, or inhibits the development of symptoms or conditions associated with schizophrenia, autism spectrum disorder, bipolar disorder, or any other neuropsychiatric disorder. Treatment may be prophylactic, to prevent or delay the onset of a disease, condition, or disorderMaking or worsening, or preventing the manifestation of clinical or subclinical symptoms thereof. Alternatively, treatment can be therapeutic to inhibit and/or alleviate symptoms after the disease, condition, or disorder manifests itself.

Can be used for restoring glial cells K in a subject (e.g., a subject with a neuropsychiatric condition)⁺The ingested inhibitor of SMAD4 can be administered parenterally, topically, orally, or intranasally for therapeutic treatment. Intramuscular injection (e.g., into arm or leg muscles) and intravenous infusion are suitable methods of administering the SMAD4 inhibitors disclosed herein. In some methods, such molecules are administered as sustained release compositions or devices such as medipadds^TMDevices (Elan pharm technologies, Dublin, Ireland). Alternatively, the SMAD4 inhibitors disclosed herein are administered parenterally by intracerebral delivery, intrathecal delivery, intranasal delivery, or by direct infusion into the ventricles of the brain.

In one embodiment, parenteral administration is by infusion. The infused SMAD4 inhibitor may be delivered by a pump. In certain embodiments, widespread distribution of infused SMAD4 inhibitors is achieved by delivery to the cerebrospinal fluid by intracranial administration, intrathecal administration, or intracerebroventricular administration.

In certain embodiments, the infused SMAD4 inhibitor is delivered directly to the tissue. Examples of such tissues include striatal tissue, intracerebroventricular tissue, and caudate nucleus tissue. Specific localization of SMAD4 inhibitors can be achieved by direct infusion into the target tissue.

In certain embodiments, parenteral administration is by injection. The injection may be delivered with a syringe or pump. In certain embodiments, the injection is a bolus administered directly to the tissue. Examples of such tissues include striatal tissue, intracerebroventricular tissue, and caudate nucleus tissue. Specific localization of agents including antisense oligonucleotides can be achieved by injection into the target tissue.

In certain embodiments, the specific localization of a SMAD4 inhibitor, such as a SMAD4 antisense oligonucleotide, to a target tissue improves the pharmacokinetic properties of the inhibitor compared to the broad spread of the SMAD4 inhibitor. The specific localization of the SMAD4 inhibitor improves potency compared to the widespread of the inhibitor, thus less inhibitor needs to be administered to achieve a similar pharmacology. By "similar pharmacology" is meant the amount of time (e.g., duration of action) that the target SMAD4 mRNA and/or the target SMAD4 protein is down-regulated/inhibited. In certain embodiments, the method of specifically localizing the SMAD4 inhibitor (e.g., by bolus injection) reduces the median effective concentration of the inhibitor (EC50) by about 20-fold.

In another embodiment, an inhibitor of SMAD 4as described herein is co-administered with one or more other agents. According to this embodiment of the present disclosure, such one or more additional agents are designed to treat the same disease, disorder, or condition as the SMAD4 inhibitors described herein, or one or more symptoms associated therewith. In one embodiment, the one or more additional pharmaceutical agents are designed to treat undesirable side effects of one or more pharmaceutical compositions of the present disclosure. In one embodiment, an inhibitor of SMAD 4as described herein is co-administered with another agent to treat an undesired effect. In another embodiment, an inhibitor of SMAD 4as described herein is co-administered with another agent to produce a combined effect. In another embodiment, an inhibitor of SMAD 4as described herein is co-administered with another agent to produce a synergistic effect.

In one embodiment, the SMAD4 inhibitor and another agent as described herein are administered simultaneously. In another embodiment, the SMAD4 inhibitor and another agent as described herein are administered at different times. In another embodiment, the SMAD4 inhibitor and another agent as described herein are prepared together as a single formulation. In another embodiment, the SMAD4 inhibitor and the other agent as described herein are prepared separately.

In certain embodiments, agents that may be co-administered with a SMAD4 inhibitor as described herein include antipsychotics, such as haloperidol, chlorpromazine, clozapine, quetiapine, and olanzapine; antidepressants such as fluoxetine, sertraline hydrochloride, venlafaxine, and nortriptyline; tranquilizers, e.g. benzodiazepinesClonazepam, paroxetine, venlafaxine, and a beta-blocker; and mood stabilizers such as lithium, valproate, lamotrigine, and carbamazepine.

Examples

Materials and methods

Patient identification, protection and sampling. Patients from which these lines were derived were diagnosed with a disability level of schizophrenia that occurs early in adolescents; all patients and their guardians had obtained consent/approval from children and adolescent psychiatrists under the supervision of one of us (RLF) and were blinded to subsequent line assignments according to the approval protocol of the University hospital Medical Center Institutional Review Board (University hospital Case Medical institute Institutional Review Board). No study investigator has access to the patient identifier.

Cell origin and strain. The iPSC line of schizophrenia origin was generated from patients with childhood onset schizophrenia and the control line was generated from age and gender appropriate control subjects; all iPSC lines were obtained as previously reported (Windrem et al, "Human iPSC Global Mouse clinical scientific recent controls to Schizophrania," Cell Stem Cell 21:195-208.e6(2017), which is incorporated herein by reference in its entirety). Another control line (C27; Wang et al, "Human iPSC-derived oligomeric Progenetor Cells Can Myelinate and research a Mouse Model of genetic hybridization," Cell Stem Cell 12:252-264(2013), which is incorporated herein by reference in its entirety) is enthusiastically supplied by the Lorenz student Phd (Memoral Sloan-Kettering). Lines of control origin include: CWRU-22(26 year old male), CWRU-37(32 year old female), CWRU-208(25 year old male), and C27; SCZ-derived lines include CWRU-8(10 year old female), CWRU-51(16 year old male), CWRU-52(16 year old male), CWRU-193(15 year old female), CWRU-164(14 year old female), CWRU-29(12 year old male), CWRU-30(12 year old male), and CWRU-31(12 year old male) (Windrem et al, "Human iPSC Global Mouse Chimeras derived geographic contacts to Schizophrania," Cell Stem Cell 21:195-208.e6(2017), which is incorporated herein by reference in its entirety; see Table 1). CWRU-51/52 and CWRU-29/30/31 included different lines from the same patient and were assessed to estimate inter-line variability from a single patient. All iPSCs were produced from Fibroblasts by retroviral expression of Cre-cleavable mountain Factors (Yamanaka Factors) (Oct4, Sox2, Klf4, c-Myc) (Takahashi et al, "Induction of Pluripent Stem Cells from Human Fibroblasts by Defined factories," Cell 131:861 872(2007), which is incorporated herein by reference in its entirety), and pluripotency and nuclear stability were verified as described (Windrem et al, "Human iPSC Global Mouse Cells derived Global contacts to Schizophra," Cell Stem Cell 21:195-208.e6(2017), which is incorporated herein by reference in its entirety).

TABLE 1 patient derived iPSC lines for this study

The lines used in this study have been previously described and published in Windrem et al, "Human iPSC Global Mouse clinical scientific derived Global connections to Schizophrania," Cell Stem Cell 21:195-208.e6(2017), which is incorporated herein by reference in its entirety. Additional manipulations added to this study (astrocyte differentiation and resulting differentiated astrocyte versus K)⁺Assessment of uptake) is indicated in the middle column, while karyotype normal and available CGH array data are indicated in the two rightmost columns. All of these lines had normal karyotypes. CGH arrays showed that several lines had sporadic indels, but no line had previously been associated with schizophrenia or autism spectrum disorders.

hiPSC culture and passaging. Hipscs were cultured on irradiated Mouse Embryo Fibroblasts (MEFs) in hESC medium (see below) supplemented with 10ng/ml bFGF (Invitrogen, 13256-. Media changes were performed daily and after 4-7 days of culture, cells were passaged at 80% confluence. For hiPSC passage, cells were first incubated with 1ml of collagenase (Invitrogen, 17104-. The pellet was resuspended in bFGF-containing ES medium and plated onto fresh irradiated MEF at 1:3-1: 4.

GPC and astrocytes were generated from hipscs. When the hipscs reached 80% confluence, they were incubated with 1ml of dispase (Invitrogen, 17105-; they were cultured in bFGF-free ES medium for 5 days. Eb was plated onto polyornithine (Sigma, P4957) and laminin (VWR, 47743) coated dishes at DIV6 and cultured for 10 days in nerve induction medium (NIM; see below) supplemented with 20ng/ml bFGF, 2. mu.g/ml heparin and 10. mu.g/ml laminin ("Wang et al," Human iPSC-derived oligomeric Progenetor Cells Can Myleinate and research a Mouse Model of genetic hybridization, "Cell Stem Cell 12:252-264(2013), which is incorporated herein by reference in its entirety).

At DIV 25, Eb was gently scraped with a 2ml glass pipette and then cultured in NIM plus 1. mu.M of chiorphinamide (Calbiochem, 80603-730) and 0.1. mu.M of RA (Sigma, R2625). At DIV33, NPC appeared and was continuously switched to NIM with 1. mu.M of purple morphinamine and 10ng/ml bFGF for 7 days, then to Glial Induction Medium (GIM) with 1. mu.M of purple morphinamine (Wang et al, "Human iPSC-Derived oligomeric Progenetor Cells Can Myelinate and research a Motor Model of genetic hybridization," Cell Stem Cell 12:252-264(2013), which is incorporated herein by reference in its entirety), for 15 days. At DIV 56, the resulting glia spheres were mechanically dissected under a dissecting microscope with a microsurgical blade and switched to GIM with 10ng/ml PDGF, 10ng/ml IGF and 10ng/ml NT3, with medium changes every 2 days. At DIV 80-100, CTR GPC was incubated with 10ng/ml BMP4(PeproTech, AF-120-05ET) and 0.5. mu.M DMH1(Sigma, D8946-5MG) for 2 weeks, and SCZ GPC was transduced with lentivirus-SMAD 4-shRNAi for 2 weeks, both to verify K⁺Expression of the transporter gene. In DIV 150-180, GPC was combined with mouse anti-CD 44 microbeads (1:50)Incubated, and then incubated with rabbit anti-mouse IgG2a + b microbeads (1:100) and further sorted by magnetic cell sorting (MACS) with magnetic posts. Then make CD44⁺Cells were matured into astrocytes in M41 supplemented with 10% FBS (VWR, 16777-one 014) and 20ng/mL BMP4 for 4 weeks.

The media formulations are listed in table 2(hESC medium and neural medium) and table 3 (glial cell medium and astrocyte induction medium).

TABLE 2 culture Medium formula basal Medium, hESC Medium and neural Medium

TABLE 3 Medium formulation glial cell culture Medium and astrocyte Induction Medium

FACS/MACS sorting. Cells were incubated with Accutase (Fisher Scientific, SCR005) at 37 ℃ for 5 minutes to obtain a single cell suspension, and then centrifuged at 200RCF for 10 minutes. These GPCs were resuspended in cold Miltenyi wash buffer with primary antibody (phycoerythrin (PE) conjugated mouse anti-CD 140a, 1:50 for FACS; mouse anti-CD 140a, 1:100 for MACS) and incubated on ice for 30 minutes with gentle rotation every 10 minutes. After primary antibody incubation, these cells were then washed and incubated with secondary antibodies (rabbit anti-mouse IgG2a + b microbeads, 1:100) and then sorted by MACS on magnetic columns or directly by FACS on FACSAria IIIu (Becton-Dickinson). Sorted cells were counted and plated on polyornithine and laminin coated 24-well plates for further experiments. Antibodies and dilutions are listed in table 4.

TABLE 4 antibodies for FACS/MACS sorting

RT-PCR. Total RNA was extracted from the cell lines using miRNeasy mini kit (Qiagen, 217004) and then reverse transcribed to cDNA using Taqman reverse transcription kit (Fisher Scientific, N808080234). The relative expression of mRNA was measured by Bio-RAD S6048, which was further normalized to the expression of 18S mRNA.

The primer sequences are listed in table 5.

TABLE 5 RT-PCR primers

In vitro immunocytochemistry. Cells were first fixed with 4% paraformaldehyde for 5 minutes at room temperature. After 3 washes with D-PBS (Invitrogen, 14190-250) containing thimerosal (Sigma, T5125), the cells were permeabilized with 0.1% saponin (Fluka Analytical, 47036) plus 1% goat or donkey serum for 15 min at room temperature. Cells were further blocked with 5% goat or donkey serum plus 0.05% saponin for 15 minutes at room temperature. After incubation with the primary antibody overnight at 4 ℃, cells were incubated with the secondary antibody for 30 minutes at room temperature. Counts of immunofluorescent cells were obtained from 10 random fields per replicate, and each sample had three replicates. See table 4 for antibodies and dilutions used.

And (4) methylation. DNA was extracted from iPSC strains using QIAamp DNA mini kit (Qiagen, 56304) and then whole genome Methylation analysis was performed using Illumina Methylation Epic array; this analysis was performed at UCLA neurosciences Genomics Core. Raw data (IDAT) files from intensity data are imported into R and normalized using the preprcessQuantille function from the minifi Package (Arye et al, "Minfi: aFlexible and Comprehensive Bioconductor Package for the Analysis of Infinium DNA Methylation microarray," biologics 30:136301369(2014), which is incorporated herein by reference in its entirety). Probes with poor quality signals were excluded based on a set threshold value (>0.01) for detection p-value. Probes are also excluded if they map to multiple genomic locations of the sex chromosome or if they contain a single nucleotide polymorphism at a CpG site. After pretreatment, the samples were evaluated by principal component analysis based on the characteristics of methylation intensity (M value). To determine whether covariates (gender, age, cell line, etc.) can account for changes in methylation status of the samples, a linear regression model was fitted to the covariates and each principal component. Covariates with significant p-values (<0.05) are highlighted, indicating a meaningful relationship between changes in covariates (predictor variables) and changes in principal component values (reaction variables).

Molecular cloning and virus construction. The Human cDNA encoding SMAD4 (GE Healthcare, MHS6278) was cloned downstream of the EF1 alpha promoter in pTANK-EF1 a-IRES-mChery-WPRE (Benraiss et al, "Human Gla Can Both inductor and research assays of Disease Photopype in Huntington Disease," Nature Communications 7:11758(2016), which is incorporated herein by reference in its entirety). Lentiviral vectors allow tandem expression of SMAD4 with the reporter gene mCherry. The doxycycline-inducible shRNA (gene target sequence: TGGTCAGCCAGCTACTTAC (SEQ ID NO:4) or ATGAATATGACTCACTTCT (SEQ ID NO:7)) of human SMAD4 in SMAD4 pSMART-TRE3G-EGFP-Puro-WPRE was ordered from GE Healthcare (V3SH 11252). BAMBI previously produced Human shRNA and cDNA of BAMBI (Sim et al, "Complementary Patterns of Gene Expression by Human Oligodendronate Progenerants and the same Environment prediction definitions of Progenerants Maintenance and Difference," Ann. neuron.59: 763-779(2006), which is incorporated herein by reference in its entirety). Correct insertion in the final construct was verified by sequencing. The plasmid was then co-transfected with pLP-VSV (Invitrogen, K497500) and psPAX2(Didier Trono gift, Addgene 12260) by X-treemeGENE (Roche, 06366236001) into 293FT cells (Fisher Scientific, R70007) for lentivirus production. The supernatant of 293T cells was then collected and centrifuged at 76000RCF for 3 hours to concentrate the virus (Beckman, L8-70, Ultratrifuge). Serial 10-fold dilutions of the virus were then prepared and transduced into 293T cells and fluorescent colonies were counted to assess virus titer.

Transduction of the cells. CD140a isolation by MACS⁺hGPC and then transduced with lentivirus-TRE 3G-SMAD4-shRNAi or lentivirus-EF 1 alpha-BAMBI-shRNAi or their corresponding scrambled control viruses. Lentiviral-EF 1a-BAMBI-shRNAi effectively inhibited the expression of the target gene (FIG. 4B). Cells infected with lentivirus-TRE 3G-SMAD4-shRNAi were treated with doxycline (Fisher, CN19895510) doxycycline at 0.5. mu.g/ml 4 days after viral infection; this treatment was maintained for 1 week, after which the experiment was started; during this period, the cells were maintained in glial cell induction medium. SMAD4 mRNA expression decreased to control under doxycycline<30 percent; no inhibition was noted in the absence of doxycycline (FIGS. 7A-7C).

Potassium uptake. Astrocytes were plated at 30,000 cells/well on polyornithine and laminin coated 24-well plates. For potassium uptake assays, astrocytes were contacted with⁸⁶Rb (1.0-3.3. mu. Ci/well) were incubated together for 15 minutes and then washed three times with ice-cold artificial cerebrospinal fluid (aCSF, 500. mu.L/well). 0.5N NaOH (200. mu.L/well) was loaded into each well for cell lysis, the wells were loaded into 5ml of a mixture (Ultima Gold, Fisher Scientific, 509050575), and counted by scintillationThe measurement was performed by a machine (Beckman Coulter, LS6500) and the results were normalized to total protein (BCA protein assay kit, Fisher Scientific, 23227) and cell number (hemocytometer, Fisher Scientific, 02-671-54). The aCSF solution contains (in mM): 124NaCl, 2.5KCl, 1.75NaH₂PO₄、2MgCl₂、2CaCl₂0.04 vitamin C, 10 glucose and 26NaHCO₃，pH 7.4。

Quantitative and statistical analysis. Statistical parameters including precision n, center, dispersion, precision measure (mean ± SEM), and statistical significance are reported in the figures and legend. All analyses were performed using GraphPad PRISM 6 using one-way ANOVA and two-tailed t-test. Statistical significance was considered to be a P-value of less than 0.05. Significance was expressed as p <0.05, p <0.01 and p < 0.001. The graphs and figures were plotted and assembled using Prism 6.

Example 1 impairment of astrocyte differentiation in SCZ GPC

ipscs were generated from skin samples obtained from patients with childhood onset Schizophrenia as well as healthy young adult controls without known psychiatric disease, as previously described (winnrem et al, "Human iPSC global Mouse childras regenerative global constraints to Schizophrenia," Cell Stem Cell 21:195-208.e6(2017), which is incorporated herein by reference in its entirety). Although age, sex, race, diagnosis and medication history are accompanied by cell line identifiers, researchers cannot obtain patient identifiers except the attending psychiatrist. Briefly, fibroblasts were isolated from each sample; from these cells, 8 hiPSC lines were obtained from patient samples and normal controls (5 juvenile onset schizophrenia patients and 3 healthy gender matched and age similar controls) (table 1). The Steward reflecting MCCA lentivirus (mixture et al, "Generation of Transmission free-specific Experimental Cell culture Cell slow virus 1728.," Differentiation of molecular library derived from Induced tissue culture Cell 2010-free, "transformation of Transmission free-specific Experimental Cell baffled" 2010-free, "Cell 131:861 872 (2007); Welstead et al," Generation iPS Cell from MEFS Through expressed Expression of Sox-2, Oct-4, c-Myc, and Klf4, "J.Vis. Exp.14:734(2008), which is incorporated herein by reference in its entirety) was used to encode Oct4, Sox2, Klf4 and c-Myc (Takahashi et al," introduction of transcription free-free discrete testing Cell Defined by Defined genes Cell filters) (mixture et al, "propagation free-dispersed tissue sample Cell baffled" 12. upright sample Cell, "Stem Cell Res Ther 3:43(2012), which is incorporated herein by reference in its entirety), produces ipscs. A fourth hipSC control line C27(Chambers et al, "high effective Conversion of Human ES and iPS Cells by Dual Inhibition of SMAD signalling," Nature Biotechnol.27:275-280(2009), which is incorporated herein by reference in its entirety) was also used to ensure that all genomic and phenotypic data are consistent with previous work (Wang et al, "Human iPSC-derived Oligodendronate Progeneitor Cells Can Myleination and research Mouse Model of genetic Hyponel," Cell Stem Cell 12:252-264(2013), which is incorporated herein by reference in its entirety). RNA sequencing and immunolabeling were used to assess pluripotent gene expression to confirm that all lines were pluripotent. Using Short Tandem Repeat (STR) based DNA fingerprinting, the identity of each iPSC line was confirmed to match the parental donor fibroblasts and karyotyping and comparative genomic hybridization was performed on each line to confirm genomic integrity. In addition, whole genome methylation of these iPSC lines was aligned to compare their methylation status.

Glial differentiation efficiency of Cells obtained from SCZ patients and control subjects (n ═ 4 lines, from 4 different patients, each with ≧ 3 replicates per patient, controls per line versus pair) was first compared by directing these iPSC Cells towards GPC fates (Wang et al, "Human iPSC-derived oligomer Progeneitor Cells Can Myleinate and research a Mouse Model of genetic hybridization," Cell Stem Cell 12:252-264(2013), which is incorporated herein by reference in its entirety) as described previously and assessing expression of stage-specific markers of maturation over time. By flow cytometry, all ipscs tested were found to exhibit typical colonies and express pluripotency markers, including SSEA4 (fig. 1A). At the Neural Progenitor Cell (NPC) stage, both ICC and flow cytometry revealed no difference in the expression levels of the stage-selective marker, paired box protein PAX-6(PAX6), sex-determining region Y-box1(SOX1) and cell surface marker prominin-1/CD133 between the CTR and SCZ-derived lines (FIGS. 2A-2D; FIG. 1B). In the GPC phase, expression of GPC-selective platelet-derived growth factor receptor alpha (PDGFR α/CD140a) was then assessed (Sim et al, "CD140a identities a position of high hly Myelogenic, Migration-compatibility and efficient engineering Human Oligordonity promoter Cells," Nature Biotechnol.29: 934-. At the astrocyte progenitor stage, flow cytometry confirmed that the expression level of the cell surface marker CD44 was not different between CTR and SCZ-derived lines (fig. 1D). Thus, no difference in differentiation of SCZ and CTR ipscs was found throughout the GPC stage and the astrocyte progenitor stage.

At this point, SCZ and CTR derived GPC were further differentiated into astrocytes by incubation in M41 medium supplemented with 20ng/ml BMP4 for 4 weeks. The immunolabeling revealed GFAP in the control lines (4 CTR lines, n.gtoreq.3/line, average of 4 CTR lines 70.1. + -. 2.4%)⁺The proportion of astrocytes was significantly higher than SCZ lines (4 SCZ lines, n.gtoreq.3/line, mean value 39.9 ± 2.0% for 4 SCZ lines, P <0.001 by two-tailed t-test) (fig. 2H-2J). S100 β in addition to GFAP⁺The percentage of astrocytes was significantly higher in CTR lines relative to SCZ lines (fig. 1F). In contrast, PDGF α R⁺The ratio of GPC was significantly higher in BMP4 treated SCZ glial cells (4 SCZ lines, n.gtoreq.3/line) relative to BMP4 treated CTR glial cells (4 CTR lines, n.gtoreq.3/line) (FIG. 1E). This defect in astrocytic differentiation was consistently observed in all SCZ GPCs relative to CTR cells, and was associated in vitro with the previously described defect in astrocytic differentiation in vivo (Windrem et al, "Human iPSC Global Mouse cells derived Global boundary Contibu)Ion to Schizophrania, "Cell Stem Cell 21:195-208.e6(2017), which is incorporated herein by reference in its entirety).

Example 2 SCZ GPC upregulates the expression of the BMP signalling inhibitor BAMBI

To identify molecular chaperones for SCZ GPC defective astrocyte differentiation, FACS-sorted CD140a from 3 different CTR-derived and 4 SCZ-derived lines at time points 154 to 242 days in vitro were used early⁺GPC performed on RNA-seq (Windrem et al, "Human iPSC Global Mouse cells derived Global contacts to Schizophrania," Cell Stem Cell 21:195-208.e6(2017), which is incorporated herein by reference in its entirety). mRNA was isolated from these cells using polyA-selection for RNA sequencing on the Illumina HiSeq 2500 platform with approximately 4500 ten thousand 1 × 100 bp reads per sample. Raw counts were analyzed for fold change at 5% FDR and log2>Disease-disorder genes were identified under 1. By this approach, 118 mrnas consistently and significantly differentially expressed from CD140a sorted SCZ hpcs relative to their control iPSC hpcs have been identified (winnrem et al, "Human iPSC global Mouse chiceras derived global constraints to Schizophrenia," Cell Stem Cell 21:195-208.e6(2017), which is incorporated by reference herein in its entirety). In these, many genes involved in glial lineage progression in SCZ hpgc are down-regulated relative to their normal controls, suggesting that astrocytic differentiation in SCZ is impaired in a cell-autonomous manner due to the intrinsic defect of SCZ-derived glial progenitor cells.

Using these early data, in this study, the approach that is regulated significantly differently in SCZ hpgc was first identified using the Induction Pathway Analysis (IPA). It was found that in these, the BMP signaling-related transcripts were up-regulated in SCZ hppc compared to CTR hppc (fig. 3A). Then, qPCR confirmed that the expression of many TGF β pathway modulators, including BAMBI, was indeed significantly elevated in SCZ GPC (fig. 3B). In contrast, these BMP signaling-related transcripts differed in the NPC stage between SCZ and CTR lines (fig. 3C). Methylation status of CTR-derived and SCZ-derived ipscs were similar (fig. 3D); the less variability marked between lines in the iPSC methylation state appears to be due to gender and line, not disease state or subject age (fig. 3E). Thus, the upregulation of BAMBI and other TGF β and BMP pathway modulators noted in SCZ hpgc is not due to any systemic disease-dependent differences in methylation patterns between CTR and SCZ cells at the pluripotent stem cell stage.

BMP4 is a strong stimulator of astrocyte Differentiation by Human GPC, and BAMBI is a strong antagonist of BMP 4-induced astrocyte induction, acting as a pseudoreceptor and thus a dominant inhibitory inhibitor of BMP signaling (Sim et al, "comparative Patterns of Gene Expression by Human Oligodendrocyte precursors and therapy Environment prediction inhibitors of Progeneitor Maintance and Differentiation," Ann neuron 59: 763-. BAMBI expression is also activated by TGF-beta and BMP receptor dependent Signaling as a compensatory negative feedback reaction (Onictchouk et al, "silence of TGF-beta Signaling by the pseudoprocessor BAMBI," Nature 40:480-485(1999), which is incorporated herein by reference in its entirety). Thus, the RNA-seq qPCR data revealed that both BMP signaling dependent transcripts and BAMBI were up-regulated in SCZ hGPC, but not in SCZ hNPC (FIGS. 3B-3C). These data indicate that up-regulation of BMP signaling is specific for SCZ glial cells and occurs first at the glial progenitor stage, and that this process is associated with up-regulation of BAMBI expression, which in turn inhibits astrocytic differentiation of SCZ hpgc.

Based on this, determination of BAMBI overexpression in normal control subject-derived hdpcs by inhibiting differentiation of these hdpcs can mimic or regenerate SCZ GPC phenotype. For this purpose, the expression of BAMBI was genetically regulated in hpgpc, i.e. both SCZ and CTR derived GPC (fig. 4A-4B). It was found that overexpression of BAMBI in CTR GPC significantly reduced the efficiency of astrocyte conversion (4 CTR lines, 3 repeats per line, mean of 4 CTR lines/36.4% ± 4.3%), yielding cells similar to SCZ hpgpc in their refractoriness to final astrocyte maturation (4 SCZ lines, 3 repeats per line, mean of 4 SCZ lines/45.5% ± 3.6%; p ═ 0.12 by two-tailed t-test) (fig. 5A-5B). However, BAMBI knockdown in SCZ GPC did not rescue astrocytic differentiation in the cells, suggesting that BAMBI overexpression caused resistance of SCZ hpgpc to maturation, but was not sufficient in this regard (fig. 5A-5B). Thus, when qPCR was used to assess the expression of alternative inhibitors of BMP signaling, the mrnas encoding both BMP and BMP-dependent signalling potent antagonists Follistatin (FST) and gremlin1(GREM1) were found to be significantly up-regulated by SCZ GPC (SCZ vs CTR; 4 SCZ and 4 CTR strains, 3 replicates per strain; ddCt of FST 2.45 ± 0.39, p < 0.05; GREM1 ═ 3.38 ± 0.53, p < 0.01; two-tailed t-test) (fig. 5C).

Example 3-astrocytic differentiation by SCZ GPC can be rescued by SMAD4 knockdown.

SMAD4 is essential for typical BMP signaling because it serves as a co-effector of multiple upstream signals in response to which it translocates to The nucleus where BMP and TGFB regulatory genes are activated (Herhaus and Sapkota, "The empirical coils of deubiquitting Enzymes (DUBs) in The TGFbeta and BMP Pathways," Cell Signal 26: 2186-. These include BAMBI as well as FST and GREM1, all of which function in concert with negative feedback modulators of glial-promoting BMP Signaling (Brazil et al, "BMP Signaling: Agony and Antagony in the Family," Trends Cell Biol 25:249-264 (2015); Onchichouk et al, "sizing of TGF-beta Signaling by the pseudo-selector BAMBI," Nature 40:480-485(1999), which is incorporated herein by reference in its entirety) (FIG. 6A). Based on this, it was hypothesized that SMAD4 knockdown in hdcp by inhibiting early expression of BAMBI, FST, and GREM1 could enhance astrocyte differentiation from hdcp. Furthermore, to the extent that the block of differentiation in SCZ hpgc is due to SMAD 4-mediated overexpression of the endogenous BMP inhibitor, it was assumed that SMAD4 knockdown thus differentially enhanced astrocytic differentiation by SCZ hpgc. To test this possibility, Doxycycline (DOX) induction of SMAD4 shRNAi was used to conditionally knock down SMAD4 expression in SCZ and CTR hpgpc, and then its BMP regulatory gene expression was assessed by qPCR (fig. 7A-7C). It was found that SMAD4 knockdown did inhibit expression of BMP signaling dependent genes, including BAMBI, FST and GREM1 (SCZ-LV-scrambled vs SCZ-LV-SMAD 4-shRNA; 4 different patient iPSC lines/group, 3 replicates/line; ddCt of BAMBI: 2.56 + -0.35, p < 0.05; FST: 2.38 + -0.24, p < 0.01; GREM 1: 3.04 + -0.45, p < 0.05; all comparisons were by ANOVA and post-hoc t-test) (FIG. 6B). Importantly, transient DOX-induced SMAD4 knockdown, in which shRNAi expression was restricted to the progenitor state, strongly promoted astrocytic differentiation of SCZ GPC, overcoming its relative block in astrocytic differentiation to effectively rescue the astrocytic phenotype (fig. 6C-6D). Specifically, SMAD4 Knockdown (KD) in SCZ GPC restored GFAP-defined astrocyte differentiation efficiency to the level of CTR GPC (SCZ-SMAD 4-shRNA: 56.8% + -3.8% at the GPC stage; CTR line: 62.2% + -4.0%; p >0.05, one-way ANOVA; mean + -SE of 4 different patient lines/group, n ≧ 3 repeats/line) (FIGS. 6C-6D). In contrast, continuous SMAD4 knockdown following astrocyte induction caused a GFAP-defined astrocyte depletion in both the SCZ and CTR groups, as mediated by continuous DOX exposure (as outlined in fig. 7B) (fig. 6C-6D). Thus, maintaining the mature astrocytic phenotype appears to require ongoing SMAD4 signaling equally in SCZ and CTR astrocytes.

Taken together, these data indicate that aberrant BMP signaling produced by over-expression of the driver BMP signaling inhibitor inhibited astrocyte differentiation in SCZ GPC, and this differentiation defect could be rescued by SMAD4 knockdown. Nevertheless, once SCZ GPC proceeded to astrocyte differentiation, SMAD4 expression was then required to maintain astrocyte phenotype identically in CTR and SCZ astrocytes, consistent with its previously described function as an effector of BMP-mediated astrocyte maturation (Kohyama et al, "BMP-induced REST regulations the Establishment and Maintenance of phenotypic Identity," J.cell biol.189:159-170(2010), which is incorporated herein by reference in its entirety). These data indicate that pathological BMP-dependent signaling in SCZ GP can define its astrocytic maturation and that this cytopathology can result in part from SMAD 4-dependent overexpression by GPC of endogenous inhibitors of neuroglia-promoting BMP signaling.

Example 4 SCZ astrocytes exhibit reduced potassium uptake

Along with the impaired astrocytic differentiation of SCZ GPC, RNA-seq data indicate that those successfully differentiated astrocytes may still be functionally impaired. In particular, RNA-seq revealed transcriptional downregulation of a number of potassium channel (KCN) encoding genes, including Na, in SCZ GPC⁺-K⁺ATP enzyme, Na⁺-K⁺/2Cl^-Cotransporter (NKCC) and inward rectifying potassium channels of the Kir family (FIG. 8A) (Windrem et al, "Human iPSC Global Mouse reactors recent Global control to Schizophrania," Cell Stem Cell 21:195-208.e6(2017), incorporated herein by reference in its entirety), all of which play an important role in the uptake of potassium by astrocytes (Larsen et al, "constraints of the Na (+/K (+) -ATPase, NKCC1, and Kir4.1 to Hippocampus K (+) Clearance and Volume Responses," Gla 62: 608-. In these deregulated KCN genes, Na was encoded in each of the 4 SCZ lines assessed, compared to the 4 control lines⁺/K⁺-ATPase Pump, NKCC1 Na⁺/K⁺/2Cl^-Cotransporter and Kir3.3 Voltage gated K⁺ATP1A2, SLC12A6 and KCNJ9(Bottger et al, "glutamic-System Defects Bei and Psychiatic Manifection in a A facial tissue Type 2Disease-Mutation motion Model," Sci.Rep.6:22047 (2016); Gamba and Friedman, "thumb casting library: The Na (+) (K (+):2Cl (-) transport, NKCC2, and The Calcium-Sensoreptor, CaSR," Pcluger Arch Arch.667: 61-76 (2009); Lesage et al, "Molecular Properties of neural G-Protein-Activated chemistry J.1995, and Biotech et al, 28660. Biotech. J.28270. Repent et al, incorporated by reference in their entirety)And (6) adjusting. These findings indicate that K + uptake by SCZ glial cells is extensively impaired.

Based on these genomic data, K in SCZ astrocytes was assessed⁺Whether the uptake is indeed impaired. To address this hypothesis, qPCR was used to confirm whether these K + channel-associated genes are dysregulated in SCZ glial cells. They were indeed significantly down-regulated, thus validating the RNA-seq analysis (fig. 8B and fig. 9B). Next, functional K was assessed directly in cultured SCZ-and CTR-derived astrocytes⁺And (4) taking. To obtain mature SCZ and CTR astrocyte cultures, CD 44-sorted glial progenitor cells were cultured for 4 weeks in basal medium supplemented with 10% Fetal Bovine Serum (FBS) and 20ng/ml BMP4 to enhance differentiation of mature fibrillar astrocytes expressing Glial Fibrillary Acidic Protein (GFAP) (fig. 10A-10C). Astrocyte maturation was achieved by SCZ-derived as well as CTR-derived progenitor cells under these conditions of high astrocyte development and using cells that had been sorted for the early astrocyte marker CD44 (fig. 10A-10C). Astrocytes from 4 different SCZ and 4 different CTR lines were then used with the conjugate for K⁺Uptake of alternative monovalent cations 86Rb (Larsen et al, "controls of the Na (+)/K (+) -ATPase, NKCC1, and Kir4.1 to Hippocpal K (+) Clearance and Volume Responses," Glia 62:608-622(2014), incorporated herein by reference in its entirety) was incubated together and rubidium uptake measured as a function of cell number and total protein. K + uptake in SCZ glia cells (4 SCZ cell lines, 5 replicates per cell line) was dramatically reduced relative to CTR glia cells (4 CTR cell lines, 5 replicates per cell line), normalized by cell number and total protein (FIG. 9C; by two-tailed t-test, P + uptake in SCZ glia cells (4 CTR cell lines, 5 replicates per cell line) and normalized by cell number and total protein<0.001)。

Because of different potassium Na codes in SCZ glial cells⁺/K⁺The genes for the ATPase pump and the inward rectifying channel are deregulated, so that the three potassium uptake mechanisms are blocked separately using the drugs ouabain, bumetanide and tolypeptide. The effect of these drugs on astrocytes has not been previously assessed, so different concentrations of each drug were first testedTo determine the modulation of human astrocyte K⁺Optimal dosage range for ingestion. Targeting Na separately⁺/K⁺ATP-ase Pump and NKCC1 encoded Na⁺/K⁺/2Cl^-Ouabain and bumetanide of cotransporter obviously inhibit K in CTR neuroglia cell⁺Uptake, whereas the tropipeptide products targeting the Kir channel did not (FIGS. 9D-9E, left panels). In sharp contrast, neither ouabain nor bumetanide affected the SCZ astrocyte pair K⁺Uptake of (FIG. 9D-9E, right panel). This indicates that K of SCZ-derived astrocytes⁺The reduced function of uptake may be attributed primarily to Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^-The co-transporter function is down-regulated, rendering these cells ineffective for treatment with ouabain and bumetanide.

Discussion of examples 1 to 4

These data indicate that astrocytic differentiation in GPC derived from childhood onset schizophrenia is impaired, and that this maturation defect can be rescued by inhibition of BMP signaling via SMAD4 knockdown. Importantly, Astrocyte depletion has recently been noted in both Cortical and subcortical areas of patients with Schizophrenia, and this may be particularly pronounced in the white matter (Rajkowska et al, "Layer-specific Reductions in GFAP-reactive Expression in the Dorsolaral Prefrontal Cortex in Schizophrania," Schizophrase Res 57: 127-. Astrocytes play a key role in Neural Circuit formation and stability (Christopherson et al, "Thrombospondins are advanced-secreted Proteins that is CNS synergy," Cell 120: 421-. Thus, any such developmental defect in astrocytic differentiation in SCZ GPC may result in a severe defect in the initial formation or stability of the neural circuit, which is one of the hallmarks of schizophrenia (Penzes et al, "Dendritic Spine pathology in Neuropsychiatric Disorders," Nat Neurosci 14:285-293(2011), which is incorporated herein by reference in its entirety). In this regard, RNA-seq data indicate that TGFBR and BMP Signaling are up-regulated in SCZ GPC, which is associated with activation of downstream BMP regulatory genes including the competitive inhibitor BAMBI that promotes glial BMP Signaling (Onchichouk et al, "silence of TGF-beta Signaling by the PseuD promoter BAMBI," Nature 40:480-485(1999), which is incorporated herein by reference in its entirety). It has been previously noted that high Expression of BAMBI in adult GPC significantly inhibited astrocytic Differentiation, as induced by BMP4(Sim et al, "comparative Patterns of Gene Expression by Human Oligodendrocyte precursors and theory Environment precursors of promoter Expression and Differentiation," Ann Neurol 59:763-779(2006), which is incorporated herein by reference in its entirety), suggesting that pathological elevation of BMP signaling-induced BAMBI Expression in SCZ hdcp relative to normal control hdcp may be sufficient to inhibit Differentiation into mature astrocytes. In addition to BAMBI, several other inhibitors of TGF β/BMP signaling, including FST and GREM1 (Brazil et al, "BMP Signalling: Agony and Antagony in the Family," Trends Cell Biol 25:249 264(2015), which is incorporated herein by reference in its entirety) are also upregulated by SCZ GPC; these may allow SCZ hpgc to avoid astrocyte fate, even after BAMBI knockdown.

Notably, activation of typical TGF signaling is dependent on activation of SMAD2/3 via the TGF pathway or SMAD1/5/8 via BMP receptor dependent signaling; each of these effectors requires nuclear translocation in combination with SMAD4 followed by activation of its downstream gene target (Hata and Chen, "TGF-beta Signaling from candidates to Smads," Cold Spring Harb Perspect Biol 8 (2016); Herhaus and Sapkota, "The emulsifying rounds of Deubilizing Enzymes (DUBs) in The TGFbeta and BMP Pathways," Cell Signal 26:2186-2192(2014), which is incorporated herein by reference in its entirety). Thus, SMAD4 knockdown was found to effectively inhibit BMP signaling-induced expression of endogenous BMP inhibitors, and as such significantly promoted astrocytic differentiation of other differentiation-resistant SCZ GPC. Importantly, this differentiation response of hdcp to SMAD4 inhibition is only indicated at the hdcp stage and only in SCZ hdcp; control patient-derived hdgpc did not show this enhanced differentiation in response to SMAD4 inhibition. Thus, modulation of SMAD4 may represent a strategy suitable for alleviating the deficit in glial cell differentiation in schizophrenia.

Glial cell maturation is precisely regulated in human brain Development (Goldman and Kuypers, "How to Make an oligomerization," Development 142: 3983-. Astrocytes have multiple roles in the CNS, including energy support for neurons and oligodendrocytes, potassium buffering, neurotransmitter circulation, and synapse formation and maturation; likewise, Astrocytes play a critical Role in the formation and maintenance of Neural circuits (Blanco-Suarez et al, "Role of intersection in CNS Disorders," J.Physiol.595: 1903-. Astrocytes also contribute to the lymphatic system by regulating cerebrospinal fluid flow through the Brain interstitium (Xie et al, "Sleep drive metabolism from the administration Brain," Science 342:373-377(2013), which is incorporated herein by reference in its entirety). Thus, delayed differentiation of SCZ astrocytes may have a significant impact on neural network formation, organization and maturation functions, etc.

It has been found that many potassium transporters are down-regulated in SCZ glial cells. Interestingly, previous global genomic association studies (GWAS) have identified associations of potassium pump, transport and channel genes with schizophrenia. For example, the Chromosome 1q21-q22 Locus containing KCNN3 has a significant association with Familial Schizophrenia (Brzustowicz et al, "Location of a Major Suscientific Locus for family Schizophrania on Chromosome 1q21-q22," Science 288:678-682(2000), which is incorporated herein by reference in its entirety). KCNN3 is widely expressed in the human brain and selectively modulates neuronal excitability and neurotransmitter release in monoaminergic neurons (O' Donovan and Owen, "Candidate-gene Association students of Schizophrenia," am.J.hum.Genet.65:587-592(1999), which is incorporated herein by reference in its entirety). In addition to KCNN3, many other potassium channel genes are also associated with Schizophrenia, including KCNQ2 and KCNAB1(Lee et al, "Pathway Analysis of Genome-wide Association Study in Schizophrania," Gene 525:107-115(2013), which is incorporated herein by reference in its entirety). Recently, a new De Novo Mutation in ATP1A3, a subunit of the sodium potassium pump, was particularly associated with Childhood Onset Schizophrenia (Smedemarrk-Margulies et al, "ANovel De Novo Mutation in ATP1A3 and Childhood-Onset Schizophrenia," Cold Spring Harb Mol Case Stud 2, a001008(2016), which is incorporated herein by reference in its entirety).

Down-regulation or dysfunction of these potassium transporters in GPC and its derived astrocytes may contribute significantly to the disease phenotype of schizophrenia. Potassium channels, pumps and transport genes are described in GPC (Coppi et al, "UDP-glucose enhance Outward K (+) Current New for Cell Differentiation and Stuctions Cell Differentiation by Activating the GPR17 Receptor in oligomer concentrates," Glaa 61:1155-⁺in Material Gray Matter, "J.neurosci.33: 2432-; zhang and Barres, "Astrocyte Heterogeneity: anExpressed broadly in the comprehensive of "Current Opinion in Neurobiology 20:588-594(2010), which is incorporated herein by reference in its entirety), where they regulate not only proliferation, Migration and Differentiation, but also glial Cell-to-neuron relationships (Coppi et al," UDP-glucose Enhances Outward K (+) Current neurological for Cell Differentiation and stimulation by activation of the GPR17 recept in Oligodendron predictors, "Glaa 61:1155-1171 (2013); maldonado et al, "Oligodendrocyte precrosor Cells are Accurate records of Local K⁺in Mature Gray Matter, "J.Neurosci.33: 2432-2442(2013), which is incorporated herein by reference in its entirety). In relation to the latter, astrocytes also pass all three major K' s⁺Transport mechanisms (including Na +/K + -ATPase, NKCC1 cotransporter, and inward-rectifying Kir channels) modulate synaptic K⁺Uptake (Larsen et al, "constraints of the Na (+)/K (+) -ATPase, NKCCl, and Kir4.1 to Hippocamppal K (+) Clearance and Volume Responses," Glia 62: 608-.

Thus, deregulated K + transport and potassium channel gene expression have been associated with a variety of neurological and psychiatric disorders. Several Kir genes, including Kir4.1, are involved in astrocytic potassium buffering and glutamate uptake, and deletions of these genes were noted in both Huntington's Disease and multiple sclerosis (Seifert et al, "assay Dysfunction in Neurological Disorders: a Molecular Perfect," Nat Rev Neurosis 7: 194:. 206 (2006); Tong et al, "assay Kir4.1 Ion Channel deficiencies to neural Dysfunction in Huntington's Disease Model: 703(2014), which is incorporated herein in its entirety by reference). In addition, mutations in astrocyte ATP1A2 (the sodium potassium pump α 2 isoform) may be causally related to familial hemiplegic migraine (Bottger et al, "glutamic-system Defects Beam and Psychiaatri)c Manifests in a family Hemipelegic Migraine Type 2 Disease-stimulation Mouse Model, "Sci Rep 6:22047 (2016); swarts et al, "family Hemipple Migraine Mutations effects Na, K-ATPase Domain Interactions," Biochim Biophys Acta 1832:2173-2179(2013), which is incorporated herein by reference in its entirety. In all of these examples, glial cell K⁺Uptake was compromised as in SCZ glial cells, and all of these lesions were associated with phenotypic hyperexcitability. Indeed, in a mouse model of schizophrenia, extracellular K⁺Elevations have been shown to alter Neuronal Excitability and neural circuit stability (Crabtree et al, "Alteration of neural activity and Short-term synthetic Plasticity in the preceding Cortex of a Mouse Model of Mental ilness," J.Neurosci. (2017), which is incorporated herein by reference in its entirety). Thus, the SCZ glial cell pair K⁺The reduction in uptake may be an important contributor to the pathogenesis of schizophrenia, particularly with respect to those schizophrenia phenotypes associated with hyperexcitability and epilepsy, which are enhanced in the context of disrupted potassium homeostasis.

Thus, these data reveal defective differentiation of astrocytes by SCZ GPC, potential reversibility of the defect by SMAD4 knockdown, and K by SCZ glial cells⁺Defective uptake of (4). The resulting deficiency in synaptic potassium homeostasis may be expected to significantly reduce neuronal firing thresholds, while enhancing network desynchronization (Benraiss et al, "Human Glaa Can Board approach and research applications of Disease photosype in Huntington Disease," Nature Communications 7:11758(2016), which is incorporated herein by reference in its entirety). Likewise, it is contemplated that glial cell K⁺The positive regulator of uptake may have practical value for the treatment of Schizophrenia (Calcaterra et al, "Schizophrania-associated hERG Channel Kv11.1-3.1 bits a Unit Trafficking Definitions for High Through put Screening," Sci Rep 6:19976 (2016); He et al, "Current pharmaceutical students on hERG Potas Channels," Trends Mol Med 19: 227-; rahmanzadeh et al, "rock of the Effect of Bumetanide, a Selective NKCC1 Inhibitor, in Patients with Schizophrania: A Double-bed-Randomized Trial," Psychiatry Clin Neurosci 71:72-73(2017), which is incorporated herein by reference in its entirety). Taken together, these findings identify the causal role of astrocytopathy on neuronal dysfunction of SCZ, and in so doing, suggest a tractable set of molecular targets for their treatment.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the following claims.

Sequence listing

<110> university of Rochester

<120> methods of treating schizophrenia and other neuropsychiatric disorders

<130> 147400.03771 (6-18129)

<150> 62/778,145

<151> 2018-12-11

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<170> PatentIn version 3.5

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Glu Thr Phe Ala Lys Arg Ala Ile Glu Ser Leu Val Lys Lys Leu Lys

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Glu Lys Lys Asp Glu Leu Asp Ser Leu Ile Thr Ala Ile Thr Thr Asn

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Arg Leu Gln Val Ala Gly Arg Lys Gly Phe Pro His Val Ile Tyr Ala

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Arg Leu Trp Arg Trp Pro Asp Leu His Lys Asn Glu Leu Lys His Val

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Lys Tyr Cys Gln Tyr Ala Phe Asp Leu Lys Cys Asp Ser Val Cys Val

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Asn Pro Tyr His Tyr Glu Arg Val Val Ser Pro Gly Ile Asp Leu Ser

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Gly Leu Thr Leu Gln Ser Asn Ala Pro Ser Ser Met Met Val Lys Asp

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Glu Tyr Val His Asp Phe Glu Gly Gln Pro Ser Leu Ser Thr Glu Gly

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His Ser Ile Gln Thr Ile Gln His Pro Pro Ser Asn Arg Ala Ser Thr

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Glu Thr Tyr Ser Thr Pro Ala Leu Leu Ala Pro Ser Glu Ser Asn Ala

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Thr Ser Thr Ala Asn Phe Pro Asn Ile Pro Val Ala Ser Thr Ser Gln

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Pro Ala Ser Ile Leu Gly Gly Ser His Ser Glu Gly Leu Leu Gln Ile

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Ala Ser Gly Pro Gln Pro Gly Gln Gln Gln Asn Gly Phe Thr Gly Gln

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Pro Ala Thr Tyr His His Asn Ser Thr Thr Thr Trp Thr Gly Ser Arg

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Thr Ala Pro Tyr Thr Pro Asn Leu Pro His His Gln Asn Gly His Leu

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Gln His His Pro Pro Met Pro Pro His Pro Gly His Tyr Trp Pro Val

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His Asn Glu Leu Ala Phe Gln Pro Pro Ile Ser Asn His Pro Ala Pro

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Glu Tyr Trp Cys Ser Ile Ala Tyr Phe Glu Met Asp Val Gln Val Gly

325 330 335

Glu Thr Phe Lys Val Pro Ser Ser Cys Pro Ile Val Thr Val Asp Gly

340 345 350

Tyr Val Asp Pro Ser Gly Gly Asp Arg Phe Cys Leu Gly Gln Leu Ser

355 360 365

Asn Val His Arg Thr Glu Ala Ile Glu Arg Ala Arg Leu His Ile Gly

370 375 380

Lys Gly Val Gln Leu Glu Cys Lys Gly Glu Gly Asp Val Trp Val Arg

385 390 395 400

Cys Leu Ser Asp His Ala Val Phe Val Gln Ser Tyr Tyr Leu Asp Arg

405 410 415

Glu Ala Gly Arg Ala Pro Gly Asp Ala Val His Lys Ile Tyr Pro Ser

420 425 430

Ala Tyr Ile Lys Val Phe Asp Leu Arg Gln Cys His Arg Gln Met Gln

435 440 445

Gln Gln Ala Ala Thr Ala Gln Ala Ala Ala Ala Ala Gln Ala Ala Ala

450 455 460

Val Ala Gly Asn Ile Pro Gly Pro Gly Ser Val Gly Gly Ile Ala Pro

465 470 475 480

Ala Ile Ser Leu Ser Ala Ala Ala Gly Ile Gly Val Asp Asp Leu Arg

485 490 495

Arg Leu Cys Ile Leu Arg Met Ser Phe Val Lys Gly Trp Gly Pro Asp

500 505 510

Tyr Pro Arg Gln Ser Ile Lys Glu Thr Pro Cys Trp Ile Glu Ile His

515 520 525

Leu His Arg Ala Leu Gln Leu Leu Asp Glu Val Leu His Thr Met Pro

530 535 540

Ile Ala Asp Pro Gln Pro Leu Asp

545 550

<210> 2

<211> 8789

<212> DNA

<213> Intelligent (Homosapien)

<400> 2

atgctcagtg gcttctcgac aagttggcag caacaacacg gccctggtcg tcgtcgccgc 60

tgcggtaacg gagcggtttg ggtggcggag cctgcgttcg cgccttcccg ctctcctcgg 120

gaggcccttc ctgctctccc ctaggctccg cggccgccca gggggtggga gcgggtgagg 180

ggagccaggc gcccagcgag agaggccccc cgccgcaggg cggcccggga gctcgaggcg 240

gtccggcccg cgcgggcagc ggcgcggcgc tgaggagggg cggcctggcc gggacgcctc 300

ggggcggggg ccgaggagct ctccgggccg ccggggaaag ctacgggccc ggtgcgtccg 360

cggaccagca gcgcgggaga gcggactccc ctcgccaccg cccgagccca ggttatcctg 420

aatacatgtc taacaatttt ccttgcaacg ttagctgttg tttttcactg tttccaaagg 480

atcaaaattg cttcagaaat tggagacata tttgatttaa aaggaaaaac ttgaacaaat 540

ggacaatatg tctattacga atacaccaac aagtaatgat gcctgtctga gcattgtgca 600

tagtttgatg tgccatagac aaggtggaga gagtgaaaca tttgcaaaaa gagcaattga 660

aagtttggta aagaagctga aggagaaaaa agatgaattg gattctttaa taacagctat 720

aactacaaat ggagctcatc ctagtaaatg tgttaccata cagagaacat tggatgggag 780

gcttcaggtg gctggtcgga aaggatttcc tcatgtgatc tatgcccgtc tctggaggtg 840

gcctgatctt cacaaaaatg aactaaaaca tgttaaatat tgtcagtatg cgtttgactt 900

aaaatgtgat agtgtctgtg tgaatccata tcactacgaa cgagttgtat cacctggaat 960

tgatctctca ggattaacac tgcagagtaa tgctccatca agtatgatgg tgaaggatga 1020

atatgtgcat gactttgagg gacagccatc gttgtccact gaaggacatt caattcaaac 1080

catccagcat ccaccaagta atcgtgcatc gacagagaca tacagcaccc cagctctgtt 1140

agccccatct gagtctaatg ctaccagcac tgccaacttt cccaacattc ctgtggcttc 1200

cacaagtcag cctgccagta tactgggggg cagccatagt gaaggactgt tgcagatagc 1260

atcagggcct cagccaggac agcagcagaa tggatttact ggtcagccag ctacttacca 1320

tcataacagc actaccacct ggactggaag taggactgca ccatacacac ctaatttgcc 1380

tcaccaccaa aacggccatc ttcagcacca cccgcctatg ccgccccatc ccggacatta 1440

ctggcctgtt cacaatgagc ttgcattcca gcctcccatt tccaatcatc ctgctcctga 1500

gtattggtgt tccattgctt actttgaaat ggatgttcag gtaggagaga catttaaggt 1560

tccttcaagc tgccctattg ttactgttga tggatacgtg gacccttctg gaggagatcg 1620

cttttgtttg ggtcaactct ccaatgtcca caggacagaa gccattgaga gagcaaggtt 1680

gcacataggc aaaggtgtgc agttggaatg taaaggtgaa ggtgatgttt gggtcaggtg 1740

ccttagtgac cacgcggtct ttgtacagag ttactactta gacagagaag ctgggcgtgc 1800

acctggagat gctgttcata agatctaccc aagtgcatat ataaaggtct ttgatttgcg 1860

tcagtgtcat cgacagatgc agcagcaggc ggctactgca caagctgcag cagctgccca 1920

ggcagcagcc gtggcaggaa acatccctgg cccaggatca gtaggtggaa tagctccagc 1980

tatcagtctg tcagctgctg ctggaattgg tgttgatgac cttcgtcgct tatgcatact 2040

caggatgagt tttgtgaaag gctggggacc ggattaccca agacagagca tcaaagaaac 2100

accttgctgg attgaaattc acttacaccg ggccctccag ctcctagacg aagtacttca 2160

taccatgccg attgcagacc cacaaccttt agactgaggt cttttaccgt tggggccctt 2220

aaccttatca ggatggtgga ctacaaaata caatcctgtt tataatctga agatatattt 2280

cacttttgtt ctgctttatc ttttcataaa gggttgaaaa tgtgtttgct gccttgctcc 2340

tagcagacag aaactggatt aaaacaattt tttttttcct cttcagaact tgtcaggcat 2400

ggctcagagc ttgaagatta ggagaaacac attcttatta attcttcacc tgttatgtat 2460

gaaggaatca ttccagtgct agaaaattta gccctttaaa acgtcttaga gccttttatc 2520

tgcagaacat cgatatgtat atcattctac agaataatcc agtattgctg attttaaagg 2580

cagagaagtt ctcaaagtta attcacctat gttattttgt gtacaagttg ttattgttga 2640

acatacttca aaaataatgt gccatgtggg tgagttaatt ttaccaagag taactttact 2700

ctgtgtttaa aaagtaagtt aataatgtat tgtaatcttt catccaaaat attttttgca 2760

agttatatta gtgaagatgg tttcaattca gattgtcttg caacttcagt tttatttttg 2820

ccaaggcaaa aaactcttaa tctgtgtgta tattgagaat cccttaaaat taccagacaa 2880

aaaaatttaa aattacgttt gttattccta gtggatgact gttgatgaag tatacttttc 2940

ccctgttaaa cagtagttgt attcttctgt atttctaggc acaaggttgg ttgctaagaa 3000

gcctataaga ggaatttctt ttccttcatt catagggaaa ggttttgtat tttttaaaac 3060

actaaaagca gcgtcactct acctaatgtc tcactgttct gcaaaggtgg caatgcttaa 3120

actaaataat gaataaactg aatattttgg aaactgctaa attctatgtt aaatactgtg 3180

cagaataatg gaaacattac agttcataat aggtagtttg gatatttttg tacttgattt 3240

gatgtgactt tttttggtat aatgtttaaa tcatgtatgt tatgatattg tttaaaattc 3300

agtttttgta tcttggggca agactgcaaa cttttttata tcttttggtt attctaagcc 3360

ctttgccatc aatgatcata tcaattggca gtgactttgt atagagaatt taagtagaaa 3420

agttgcagat gtattgactg taccacagac acaatatgta tgctttttac ctagctggta 3480

gcataaataa aactgaatct caacatacaa agttgaattc taggtttgat ttttaagatt 3540

ttttttttct tttgcacttt tgagtccaat ctcagtgatg aggtaccttc tactaaatga 3600

caggcaacag ccagttctat tgggcagctt tgtttttttc cctcacactc taccgggact 3660

tccccatgga cattgtgtat catgtgtaga gttggttttt ttttttttta atttttattt 3720

tactatagca gaaatagacc tgattatcta caagatgata aatagattgt ctacaggata 3780

aatagtatga aataaaatca aggattatct ttcagatgtg tttacttttg cctggagaac 3840

ttttagctat agaaacactt gtgtgatgat agtcctcctt atatcacctg gaatgaacac 3900

agcttctact gccttgctca gaaggtcttt taaatagacc atcctagaaa ccactgagtt 3960

tgcttatttc tgtgatttaa acatagatct tgatccaagc tacatgactt ttgtctttaa 4020

ataacttatc taccacctca tttgtactct tgattactta caaattcttt cagtaaacac 4080

ctaattttct tctgtaaaag tttggtgatt taagttttat tggcagtttt ataaaaagac 4140

atcttctcta gaaattgcta actttaggtc cattttactg tgaatgagga ataggagtga 4200

gttttagaat aacagatttt taaaaatcca gatgatttga ttaaaacctt aatcatacat 4260

tgacataatt cattgcttct tttttttgag atatggagtc ttgctgtgtt gcccaggcag 4320

gagtgcagtg gtatgatctc agctcactgc aacctctgcc tcccgggttc aactgattct 4380

cctgcctcag cctccctggt agctaggatt acaggtgccc gccaccatgc ctggctaact 4440

tttgtagttt tagtagagac ggggttttgc ctgttggcca ggctggtctt gaactcctga 4500

cctcaagtga tccatccacc ttggcctccc aaagtgctgg gattacgggc gtgagccact 4560

gtccctggcc tcattgttcc cttttctact ttaaggaaag ttttcatgtt taatcatctg 4620

gggaaagtat gtgaaaaata tttgttaaga agtatctctt tggagccaag ccacctgtct 4680

tggtttcttt ctactaagag ccataaagta tagaaatact tctagttgtt aagtgcttat 4740

atttgtacct agatttagtc acacgctttt gagaaaacat ctagtatgtt atgatcagct 4800

attcctgaga gcttggttgt taatctatat ttctatttct tagtggtagt catctttgat 4860

gaataagact aaagattctc acaggtttaa aattttatgt ctactttaag ggtaaaatta 4920

tgaggttatg gttctgggtg ggttttctct agctaattca tatctcaaag agtctcaaaa 4980

tgttgaattt cagtgcaagc tgaatgagag atgagccatg tacacccacc gtaagacctc 5040

attccatgtt tgtccagtgc ctttcagtgc attatcaaag ggaatccttc atggtgttgc 5100

ctttattttc cggggagtag atcgtgggat atagtctatc tcatttttaa tagtttaccg 5160

cccctggtat acaaagataa tgacaataaa tcactgccat ataaccttgc tttttccaga 5220

aacatggctg ttttgtattg ctgtaaccac taaataggtt gcctatacca ttcctcctgt 5280

gaacagtgca gatttacagg ttgcatggtc tggcttaagg agagccatac ttgagacatg 5340

tgagtaaact gaactcatat tagctgtgct gcatttcaga cttaaaatcc atttttgtgg 5400

ggcagggtgt ggtgtgtaaa ggggggtgtt tgtaatacaa gttgaaggca aaataaaatg 5460

tcctgtctcc cagatgatat acatcttatt atttttaaag tttattgcta attgtaggaa 5520

ggtgagttgc aggtatcttt gactatggtc atctggggaa ggaaaatttt acattttact 5580

attaatgctc cttaagtgtc tatggaggtt aaagaataaa atggtaaatg tttctgtgcc 5640

tggtttgatg gtaactggtt aatagttact caccatttta tgcagagtca cattagttca 5700

caccctttct gagagccttt tgggagaagc agttttattc tctgagtgga acagagttct 5760

ttttgttgat aatttctagt ttgctccctt cgttattgcc aactttactg gcattttatt 5820

taatgatagc agattgggaa aatggcaaat ttaggttacg gaggtaaatg agtatatgaa 5880

agcaattacc tctaaagcca gttaacaatt attttgtagg tggggtacac tcagcttaaa 5940

gtaatgcatt tttttttccc gtaaaggcag aatccatctt gttgcagata gctatctaaa 6000

taatctcata tcctcttttg caaagactac agagaatagg ctatgacaat cttgttcaag 6060

cctttccatt tttttccctg ataactaagt aatttctttg aacataccaa gaagtatgta 6120

aaaagtccat ggccttattc atccacaaag tggcatccta ggcccagcct tatccctagc 6180

agttgtccca gtgctgctag gttgcttatc ttgtttatct ggaatcactg tggagtgaaa 6240

ttttccacat catccagaat tgccttattt aagaagtaaa acgttttaat ttttagcctt 6300

tttttggtgg agttatttaa tatgtatatc agaggatata ctagatggta acatttcttt 6360

ctgtgcttgg ctatctttgt ggacttcagg ggcttctaaa acagacagga ctgtgttgcc 6420

tttactaaat ggtctgagac agctatggtt ttgaattttt agtttttttt ttttaaccca 6480

cttcccctcc tggtctcttc cctctctgat aattaccatt catatgtgag tgttagtgtg 6540

cctcctttta gcattttctt cttctctttc tgattcttca tttctgactg cctaggcaag 6600

gaaaccagat aaccaaactt actagaacgt tctttaaaac acaagtacaa actctgggac 6660

aggacccaag acactttcct gtgaagtgct gaaaaagacc tcattgtatt ggcatttgat 6720

atcagtttga tgtagcttag agtgcttcct gattcttgct gagtttcagg tagttgagat 6780

agagagaagt gagtcatatt catattttcc cccttagaat aatattttga aaggtttcat 6840

tgcttccact tgaatgctgc tcttacaaaa actggggtta caagggttac taaattagca 6900

tcagtagcca gaggcaatac cgttgtctgg aggacaccag caaacaacac acaacaaagc 6960

aaaacaaacc ttgggaaact aaggccattt gttttgtttt ggtgtcccct ttgaagccct 7020

gccttctggc cttactcctg tacagatatt tttgacctat aggtgccttt atgagaattg 7080

agggtctgac atcctgcccc aaggagtagc taaagtaatt gctagtgttt tcagggattt 7140

taacatcaga ctggaatgaa tgaatgaaac tttttgtcct ttttttttct gttttttttt 7200

ttctaatgta gtaaggacta aggaaaacct ttggtgaaga caatcatttc tctctgttga 7260

tgtggatact tttcacaccg tttatttaaa tgctttctca ataggtccag agccagtgtt 7320

cttgttcaac ctgaaagtaa tggctctggg ttgggccaga cagttgcact ctctagtttg 7380

ccctctgcca caaatttgat gtgtgacctt tgggcaagtc atttatcttc tctgggcctt 7440

agttgcctca tctgtaaaat gagggagttg gagtagatta attattccag ctctgaaatt 7500

ctaagtgacc ttggctacct tgcagcagtt ttggatttct tccttatctt tgttctgctg 7560

tttgaggggg ctttttactt atttccatgt tattcaaagg agactaggct tgatatttta 7620

ttactgttct tttatggaca aaaggttaca tagtatgccc ttaagactta attttaacca 7680

aaggcctagc accaccttag gggctgcaat aaacacttaa cgcgcgtgcg cacgcgcgcg 7740

cgcacacaca cacacacaca cacacacaca cacaggtcag agtttaaggc tttcgagtca 7800

tgacattcta gcttttgaat tgcgtgcaca cacacacgca cgcacacact ctggtcagag 7860

tttattaagg ctttcgagtc atgacattat agcttttgag ttggtgtgtg tgacaccacc 7920

ctcctaagtg gtgtgtgctt gtaatttttt ttttcagtga aaatggattg aaaacctgtt 7980

gttaatgctt agtgatatta tgctcaaaac aaggaaattc ccttgaaccg tgtcaattaa 8040

actggtttat atgactcaag aaaacaatac cagtagatga ttattaactt tattcttggc 8100

tctttttagg tccattttga ttaagtgact tttggctgga tcattcagag ctctcttcta 8160

gcctaccctt ggatgagtac aattaatgaa attcatattt tcaaggacct gggagccttc 8220

cttggggctg ggttgagggt ggggggttgg ggagtcctgg tagaggccag ctttgtggta 8280

gctggagagg aagggatgaa accagctgct gttgcaaagg ctgcttgtca ttgatagaag 8340

gactcacggg cttggattga ttaagactaa acatggagtt ggcaaacttt cttcaagtat 8400

tgagttctgt tcaatgcatt ggacatgtga tttaagggaa aagtgtgaat gcttatagat 8460

gatgaaaacc tggtgggctg cagagcccag tttagaagaa gtgagttggg ggttggggac 8520

agatttggtg gtggtatttc ccaactgttt cctcccctaa attcagagga atgcagctat 8580

gccagaagcc agagaagagc cactcgtagc ttctgctttg gggacaactg gtcagttgaa 8640

agtcccagga gttcctttgt ggctttctgt atacttttgc ctggttaaag tctgtggcta 8700

aaaaatagtc gaacctttct tgagaactct gtaacaaagt atgtttttga ttaaaagaga 8760

aagccaacta aaaaaaaaaa aaaaaaaaa 8789

<210> 3

<211> 19

<212> RNA

<213> Artificial (Artificial)

<220>

<223> SMAD4 shRNA

<400> 3

guaaguagcu ggcugacca 19

<210> 4

<211> 19

<212> DNA

<213> Intelligent (Homosapien)

<400> 4

tggtcagcca gctacttac 19

<210> 5

<211> 21

<212> DNA

<213> Artificial (Artificial)

<220>

<223> disordered shRNA

<400> 5

aagttgcaaa tcgcgtctct a 21

<210> 6

<211> 19

<212> RNA

<213> Artificial (Artificial)

<220>

<223> SMAD4 shRNA

<400> 6

agaagugagu cauauucau 19

<210> 7

<211> 19

<212> DNA

<213> Intelligent (Homosapien)

<400> 7

atgaatatga ctcacttct 19

<210> 8

<211> 142

<212> PRT

<213> Artificial (Artificial)

<220>

<223> recombinant CLP/cotl1

<400> 8

Met Ala Thr Lys Ile Asp Lys Glu Ala Cys Arg Ala Ala Tyr Asn Leu

1 5 10 15

Val Arg Asp Asp Gly Ser Ala Val Ile Trp Val Thr Phe Lys Tyr Asp

20 25 30

Gly Ser Thr Ile Val Pro Gly Glu Gln Gly Ala Glu Tyr Gln His Phe

35 40 45

Ile Gln Gln Cys Thr Asp Asp Val Arg Leu Phe Ala Phe Val Arg Phe

50 55 60

Thr Thr Gly Asp Ala Met Ser Lys Arg Ser Lys Phe Ala Leu Ile Thr

65 70 75 80

Trp Ile Gly Glu Asn Val Ser Gly Leu Gln Arg Ala Lys Thr Gly Thr

85 90 95

Asp Lys Thr Leu Val Lys Glu Val Val Gln Asn Phe Ala Lys Glu Phe

100 105 110

Val Ile Ser Asp Arg Lys Glu Leu Glu Glu Asp Phe Ile Lys Ser Glu

115 120 125

Leu Lys Lys Ala Gly Gly Ala Asn Tyr Asp Ala Gln Thr Glu

130 135 140

<210> 9

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 9

ctggataccg cagctaggaa 20

<210> 10

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 10

ccctcttaat catggcctca 20

<210> 11

<211> 17

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 11

tgcggccgat tgtgaac 17

<210> 12

<211> 21

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 12

cctcttttct ctgcggaacg t 21

<210> 13

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 13

ctaccatgga ccatcctgct 20

<210> 14

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 14

cctatcccaa ggtcttgctg 20

<210> 15

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 15

gtggacgagg caagagtttc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 16

ttcccgaggt ccatctactg 20

<210> 17

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 17

tcaggaacct caccttggac 20

<210> 18

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 18

gcacagtggg gagaagagag 20

<210> 19

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 19

gattgatgac aagggcatgg 20

<210> 20

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 20

cccatagggt gagaaaacca 20

<210> 21

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 21

atcgccactc cagctacatc 20

<210> 22

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 22

ggcagcatca cagtagcatc 20

<210> 23

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 23

ccatttccaa tcatcctgct 20

<210> 24

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 24

acctttgcct atgtgcaacc 20

<210> 25

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 25

ggaggacgtg aatgacaaca 20

<210> 26

<211> 23

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 26

cacgttctca cacgtttctt tac 23

<210> 27

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 27

ggccagtgca actctttcta 20

<210> 28

<211> 21

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 28

ctgtagttca gggcagttga g 21

<210> 29

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 29

gttatcctcg agggcatggt 20

<210> 30

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 30

cgtcctccag agtcagcact 20

<210> 31

<211> 22

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 31

aactgttaga cgacggacat ag 22

<210> 32

<211> 20

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 32

cttcggtctg gtgtccattt 20

<210> 33

<211> 22

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 33

tgaaccatcc aacgacaatc ta 22

<210> 34

<211> 21

<212> DNA

<213> Artificial (Artificial)

<220>

<223> primer

<400> 34

cttgctgagg taccatgttc t 21

<210> 35

<211> 32

<212> PRT

<213> Artificial (Artificial)

<220>

<223> SBD peptides

<400> 35

Ala Pro Gly Leu Pro Asn Gly Leu Leu Ser Gly Asp Glu Asp Phe Ser

1 5 10 15

Ser Ile Ala Asp Met Asp Phe Ser Ala Leu Leu Ser Gln Ile Ser Ser

20 25 30