STXBP1 AS PSYCHIATRIC BIOMARKER IN MURINE MODEL SYSTEM AND THEIR USES

• US 20110268747A1
• Filed: 08/18/2009
• Published: 11/03/2011
• Est. Priority Date: 08/20/2008
• Status: Active Grant

• Current Assignee: Brainco Biopharma, S.L., Universidad Del Pais Vasco - Euskal Herriko Unibertsitatea
• Original Assignee: Universidad Del Pais Vasco - Euskal Herriko Unibertsitatea
• Inventors: Martinez Martinez, Antonio, Callado, Luis Felipe, Ferrer-Alcon, Marcel, Uriguen, Leyre, Guerrero Martinez, Maria Jose, Simon Buela, Laureano, Meana, Jose Javier
• Granted Patent: US 9,267,151 B2
• Time in Patent Office: Days
• US Class Current: 424/172.1
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 67/027 New or modified breeds of vertebrates
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

A non-human transgenic animal having a polynucleotide encoding an STXBP1 polypeptide, which polynucleotide is operably linked to a promoter, wherein said transgenic animal has greater than wild-type expression of the STXBP1 polypeptide in at least one brain region, as well as related vectors, methods of producing transgenic animals, in vitro and in vivo screening methods for potential therapeutic agents, and methods for treating and diagnosing neuropsychiatric illness are disclosed.

Claims

1. A transgenic rodent having a polynucleotide encoding an STXBP1 polypeptide, which polynucleotide is operably linked to a brain-specific promoter, wherein said transgenic rodent has greater than wild-type expression of the STXBP1 polypeptide in at least one brain region selected from the group consisting of cortex, striatum, hippocampus, and cerebellum.

2. The transgenic rodent according to claim 1, wherein said polynucleotide is present in a higher than wild-type copy number.

3. The transgenic rodent according to claim 1, wherein said polynucleotide encodes:

(i) an STXBP1 polypeptide having an amino acid sequence having at least 80% amino acid sequence identity to the sequence of SEQ ID NO;

2;

(ii) an STXBP1 polypeptide having the amino acid sequence of SEQ ID NO;

2

(iii) an STXBP1 polypeptide having an amino acid sequence having at least 80% amino acid sequence identity to the sequence of SEQ ID NO;

4;

(iv) an STXBP1 polypeptide having the amino acid sequence of SEQ ID NO;

4;

or

(v) an active fragment of any one of (i)-(iv) having at least 200 amino acids,

and wherein said STXBP1 polypeptide of any one of (i)-(iv) or said active fragment of (v) is capable of binding to a syntaxin polypeptide.

6. The transgenic rodent according to claim 1, wherein the brain-specific promoter is an EAAT3 promoter.

7. The transgenic rodent according to claim 1, wherein the brain-specific promoter comprises a polynucleotide having at least 80% nucleic acid sequence identity to the sequence of SEQ ID NO:

6 or a polynucleotide having the sequence of SEQ ID NO;

6.

9. The transgenic rodent according to claim 1, having at least 10% greater expression of the STXBP1 polypeptide in said at least one brain region, as measured by Western blot, immunofluorescence or qPCR of an STXBP1 mRNA.

11. The transgenic rodent according to claim 1, wherein the rodent is a mouse.

17. A method of producing the transgenic rodent of claim 1, comprising:

introducing a vector of into one or more cells of the rodent at an embryonic stage, wherein said vector comprises a polynucleotide encoding an STXBP1 polypeptide operably linked to a brain-specific promoter and optionally further regulatory sequences.

25. An in vivo method for identifying an agent for use in the treatment of neuropsychiatric illness, comprising:

(i) administering a test agent to the transgenic rodent of claim 1 and subsequently measuring, directly or indirectly, the expression of an STXBP1 polypeptide in at least one brain region relative to the expression of the STXBP1 polypeptide in at least one brain region of a control transgenic rodent of claim 1, which has not been exposed to the test compound;

or

(ii) administering a test agent to the transgenic rodent of claim 1 and subsequently assessing the presence or severity of one or more schizophrenia-related behaviours in the transgenic animal relative to the one or more schizophrenia-related behaviours in a control transgenic rodent of claim 1, which has not been exposed to the test agent,

wherein a reduction in said expression in (i), said one or more schizophrenia-related behaviours in (ii), or a combination thereof, due to the test agent indicates that the test agent is potentially useful in the treatment of neuropsychiatric illness.

26. The method according to claim 25, wherein said one or more schizophrenia-related behaviours are selected from:

reduced motor activity in an open-field test;

reduced time spent in open arms of an elevated plus maze;

reduced social interaction;

increased recognition index in a novel object recognition task; and

decreased prepulse inhibition of startle response.

27. The method according to claim 25, wherein the test agent is found to reduce said expression in (i), said one or more schizophrenia-related behaviours in (ii), or a combination thereof.

40. The method of claim 17, wherein the brain-specific promoter is an EAAT3 promoter and optionally further regulatory sequences.

41. The method of claim 17, wherein the brain-specific promoter comprises a polynucleotide having at least 80% nucleic acid sequence identity to the sequence of SEQ ID NO:

6 or a polynucleotide having the sequence of SEQ ID NO;

6.

43. The method according to claim 17, wherein said polynucleotide encodes:

(i) an STXBP1 polypeptide having an amino acid sequence having at least 80% amino acid sequence identity to the sequence of SEQ ID NO;

2;

(ii) an STXBP1 polypeptide having the amino acid sequence of SEQ ID NO;

2

(iii) an STXBP1 polypeptide having an amino acid sequence having at least 80% amino acid sequence identity to the sequence of SEQ ID NO;

4;

(iv) an STXBP1 polypeptide having the amino acid sequence of SEQ ID NO;

4;

or

(v) an active fragment of anyone of (i)-(iv) having at least 200 amino acids,

and wherein said STXBP1 polypeptide of anyone of (i)-(iv) or said active fragment of (v) is capable of binding to a syntaxin polypeptide.

44. The method of claim 17, further comprising extracting DNA from the rodent to confirm the incorporation of the polynucleotide into the genome of the rodent.

45. The method of claim 25, wherein the neuropsychiatric illness comprises schizophrenia, bipolar disorder, or a combination thereof.

46. A method for treating a neuropsychiatric illness in a subject, comprising administering to the subject a therapeutically effective amount of an agent identified or identifiable for use in the treatment of the neuropsychiatric illness, by any one or any combination of:

(a) an in vitro method comprising;

(i) contacting a cell that expresses an STXBP1 polypeptide with a test agent and measuring, directly or indirectly, expression of the STXBP1 polypeptide in a control cell which has not been exposed to the test agent;

or

(ii) contacting an STXBP1 polypeptide with a test agent and measuring, directly or indirectly, binding of the STXBP1 polypeptide to a syntaxin polypeptide relative to the binding of a control STXBP1 polypeptide which has not been exposed to the test agent to a syntaxin polypeptide,

wherein the test agent is found to reduce said expression in (i) or said binding in (ii);

(b) an in vitro screening method comprising;

contacting at least one cell that expresses an STXBP1 polypeptide with a test agent; and

detecting whether said test agent alters an STXBP1-related activity as compared with said activity in the absence of the test agent, wherein the test agent is found to inhibit said STXBP1-related activity;

or

(c) an in vivo method according to claim 25.

47. A method according to claim 46, wherein said agent comprises:

an antibody molecule or binding fragment thereof capable of binding to an STXBP1 polypeptide;

or

an antisense nucleic acid, ribozyme, triple helix molecule, siRNA or other nucleic acid capable of inhibiting STXBP1 gene expression.

48. The method of claim 46, wherein the neuropsychiatric illness is schizophrenia, bipolar disorder, or a combination thereof.

4-5. -5. (canceled)

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42. (canceled)