US20190022251A1

US20190022251A1 - Adeno-Associated Virus Virions for Treatment of Epilepsy

Info

Publication number: US20190022251A1
Application number: US16/069,370
Authority: US
Inventors: Shin-ichi Muramatsu; Keiji Oguro; Kuniko Shimazaki
Original assignee: Gene Therapy Research Institution Co Ltd; Jichi Medical University
Current assignee: Gene Therapy Research Institution Co Ltd; Jichi Medical University
Priority date: 2016-01-15
Filing date: 2017-01-13
Publication date: 2019-01-24
Also published as: JP6924441B2; WO2017122789A1; JPWO2017122789A1; EP3403675A4; EP3403675B1; EP3403675A1; US20220275399A1; ES2871527T3

Abstract

Provided is a novel gene therapy means for neurological diseases including epilepsy. The present invention provides: a recombinant adeno-associated virus vector for use in the treatment of neurological diseases including epilepsy, which comprises a polynucleotide encoding a protein capable of improving the excitation-inhibiting function of an inhibitory synapse in vivo, preferably neuroligin-2 protein; a pharmaceutical composition comprising said recombinant vector; and others. The present invention also provides a method for treating a disease such as epilepsy using the recombinant vector.

Description

TECHNICAL FIELD

The present invention relates to genetically recombinant adeno-associated virus (rAAV) virions for neural diseases. More specifically, the present invention relates to rAAV for treatment of neuropsychiatric diseases such as epilepsy, schizophrenia, autism spectrum disorder, mental retardation, anxiety, manic-depressive psychosis, migraine, phobic and compulsive symptoms, drug addiction, Angelman syndrome, dyskinesia, dystonia, Alzheimer's disease, and developmental disorders (attention deficit hyperactivity disorder and Asperger's syndrome).

BACKGROUND ART

Epilepsy is classified into partial convulsions that are seizures generated partially in body parts as a result of occurrence of the abnormal excitation of neurons in the brain at relatively limited sites, and generalized seizures (e.g., tonic-clonic seizures) that are generalized convulsions generated because the abnormally excited neurons in the brain affect the cortex entirely. A generalized seizure causes the loss of consciousness, and is a disease that causes no convulsion, but may cause impaired consciousness alone or abnormal psychiatric symptoms (e.g, psychomotor seizures). Current general therapeutic approaches are mainly pharmacotherapies using antiepileptic drugs (e.g., phenytoin, carbamazepine, and valproic acid). Surgical treatment is also performed for intractable epilepsy cases.
About 30% of epilepsy cases is accounted for by intractable diseases, the seizures of which are not suppressed by drug treatment. In the case of inner temporal lobe epilepsy, surgical treatment such as temporal lobectomy may be effective. However, the excision of bilateral hippocampi causes a decrease in memory retention, so that if seizure foci are located in bilateral hippocampi, such a case is not a candidate for surgery. Moreover, the number of patients with intractable epilepsy such as childhood epileptic encephalopathy (e.g., West syndrome) having unknown seizure foci is estimated to be about 100,000 in Japan, and no curable therapy exists for such patients.
To treat such intractable epilepsy, a method for treating each disease by a gene therapy targeting nerve cells has also been examined. As a means (vector) for delivering a therapeutic gene to nerve cells, a means of using a recombinant adeno-associated virus (rAAV) is known in the art. Examples of such rAAV include those disclosed in International Publications WO2012/057363, WO2008/124724, WO2003/093479, and the like.
Means for adjusting neural activity targeting synapse-associated proteins have been studied. For example, the document of Kohl, C et al. (Non Patent Literature 1) discloses that the direct administration of an rAAV vector expressing neuroligin 2 (NLGN2) that is a synapse localized protein of nerve cells to hippocampi to overexpress NLGN2, results in altered social behavior and inhibitory synaptic transmission, but does not describe any specific treatment of the disease. The document of Moe et al. (Non Patent Literature 2) discloses that epilepsy symptoms are alleviated by treatment of epilepsy using an rAAV vector expressing neuropeptide Y. Moreover, the document of Fang et al. (Non Patent Literature 3) discloses that epilepsy symptoms are alleviated as a result of direct administration of an rAAV vector expressing the antisense of neuroligin 1 (NLGN1) that is a synapse localized protein of nerve cells to hippocampi.

PRIOR ART DOCUMENTS

Patent Literatures

Patent Literature 1: WO 2012/057363
Patent Literature 2: WO 2008/124724
Patent Literature 3: WO 2003/093479

Non Patent Literatures

Non Patent Literature 1: Kohl, C. et al., PLOS ONE, 2013 February, vol. 8, e56871
Non Patent Literature 2: Moe′, F. M. et al., JASPER'S BASIC MECHANISMS OF THE EPILEPSY, 2012,
Non Patent Literature 3: Kullmann, D. M. et al., Nature Reviews Neurology, 2014, vol. 10, page 300-304
Non Patent Literature 4: Fang et al., Mol. Neurobiol. 2014 Nov. 27 [Epub]

SUMMARY OF INVENTION

Technical Problem

A novel medicine for treating epilepsy by gene transfer is required.
Furthermore, such a medicine is desired to be advantageous in actual medication such that it has fewer side effects, and can be more simply administered, for example.

Solution to Problem

As a result of intensive studies to establish a gene therapy for epilepsy, the inventors of the present application have discovered that through preparation of a recombinant adeno-associated virus vector comprising a polynucleotide encoding neuroligin 2 that is a protein for improving the excitation-inhibiting capability of inhibitory synapses, and administration of the vector to a living subject, epilepsy symptoms are improved, and thus have completed the invention of the present application.
Specifically, the present application provides a recombinant adeno-associated virus (rAAV) vector as described below for treatment of the following nerve cell-related diseases such as epilepsy, and a pharmaceutical composition comprising the vector, for example.
{1} A recombinant adeno-associated virus vector, comprising a polynucleotide encoding a protein for improving an excitation-inhibiting function of inhibitory synapses in a living subject, which is used for treatment of a disease selected from the group consisting of epilepsy, schizophrenia, autism spectrum disorder, mental retardation, anxiety, manic-depressive psychosis, migraine, phobic and compulsive symptoms, drug addiction, Angelman syndrome, dyskinesia, dystonia, Alzheimer's disease, and developmental disorders (attention deficit hyperactivity disorder and Asperger's syndrome).
{2} The recombinant adeno-associated virus vector according to {1}, wherein the polynucleotide comprises a nucleotide sequence encoding a neuroligin 2 protein which comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6, or an amino acid sequence having about 90% or more identity with said amino acid sequence and binding to neurexin.
{3} The recombinant adeno-associated virus vector according to {1} or {2}, wherein the disease is epilepsy.
{4} The adeno-associated virus recombinant vector according to any one of {1} to {3}, wherein the recombinant adeno-associated virus vector comprises:
a protein having a variant amino acid sequence in which tyrosine at position 445 in the amino acid sequence of the wild-type AAV1 capsid protein is substituted with phenylalanine;
a protein having a variant amino acid sequence in which tyrosine at position 445 in the amino acid sequence of the wild-type AAV2 capsid protein is substituted with phenylalanine; or
a protein having a variant amino acid sequence in which tyrosine at position 446 in the amino acid sequence of the wild-type AAV9 capsid protein is substituted with phenylalanine.
{5} The recombinant adeno-associated virus vector according to any one of {1} to {4}, wherein the above polynucleotide comprises a promoter sequence selected from the group consisting of a synapsin I promoter sequence, a myelin basic protein promoter sequence, a neuron specific enolase promoter sequence, a calcium/calmodulin-dependent protein kinase II (CMKII) promoter sequence, a tubulin αI promoter sequence, a platelet-derived growth factor β chain promoter sequence, a glial fibrillary acidic protein (GFAP) promoter sequence, a L7 promoter (cerebellar Purkinje cell specific promoter) sequence, a glial fibrillary acidic protein (hGfa2) promoter sequence, and a glutamate receptor delta 2 promoter (cerebellar Purkinje cell specific promoter) sequence, and a glutamic acid decarboxylase (GAD65/GAD67) promoter sequence.
{6} The recombinant adeno-associated virus vector according to any one of {1} to {5}, wherein the above polynucleotide comprises an inverted terminal repeat (ITR) selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV8, and AAV9.
{7} The recombinant adeno-associated virus vector according to any one of {1} to {6}, wherein the above polynucleotide further comprises a polynucleotide for inhibiting the excitation of excitatory synapses.
{8} A pharmaceutical composition, comprising the recombinant adeno-associated virus vector according to any one of {1} to {7}.
{9} The pharmaceutical composition according to {8}, which is administered intracerebrally.
{10} The pharmaceutical composition according to {8}, which is administered intrathecally.
{11} The pharmaceutical composition according to {8}, which is administered peripherally.
{12} The pharmaceutical composition according to any one of {8} to {11}, which is used in combination with a chemotherapeutic agent for a neuropsychiatric disease.
{13} A method for treatment of a disease selected from the group consisting of epilepsy, schizophrenia, autism spectrum disorder, mental retardation, anxiety, manic-depressive psychosis, migraine, phobic and compulsive symptoms, drug addiction, Angelman syndrome, dyskinesia, dystonia, Alzheimer's disease, and developmental disorders (attention deficit hyperactivity disorder and Asperger's syndrome), comprising administering to the living subject a recombinant adeno-associated virus vector that comprises a polynucleotide having a nucleotide sequence encoding a protein for improving the excitation-inhibiting function of inhibitory synapses in a living subject.
{14} The treatment method according to {13}, which is combined with a chemotherapy.

Advantageous Effects of Invention

Enhancement of the functions of a synaptic inhibitory system by a gene therapy according to the invention of the present application is useful as a method for treatment of epilepsy. The composition of the invention of the present application can be expected to be effective for even patients with intractable epilepsy having unspecified seizure foci, such as childhood epileptic encephalopathy (e.g., West syndrome).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1a shows the result of confirming the expression of the recombinant neuroligin 2 protein in a mouse to which vascular-administration-type rAAV was administered by the FLAG antibody staining of tissue sections. FIG. 1b shows the result of detecting the expression of the recombinant neuroligin 2 in a mouse to which a control was administered.

FIG. 2 shows the results of measuring epileptic seizure frequencies of mice subjected to 3 types of intracardiac administration (intracardiac administration of vascular-administration-type rAAV expressing NLGN2, vascular-administration-type rAAV expressing GFP protein, and physiological saline) at ages in weeks plotted on the horizontal axis.

FIG. 3 shows the results of seizure duration of mice subjected to the above 3 types of intracardiac administration.

FIG. 4 shows the results of seizure intensity of mice subjected to the above 3 types of intracardiac administration.

FIG. 5 shows the results of aggregating the results of seizure duration×seizure intensity in FIG. 3 and FIG. 4.

FIG. 6 shows the results of thresholds when mice subjected to the above 3 types of administration were subjected to electric stimulation at each age in weeks.

FIG. 7a shows the results of measuring the seizure frequencies of epilepsy of mice to which vascular-administration-type rAAV expressing NLGN2, vascular-administration-type rAAV expressing GFP protein, and physiological saline were topically administered to hippocampi at ages in weeks indicated on the horizontal axis (FIG. 7a ), as well as the results of the same of mice to which vascular-administration-type rAAV expressing NLGN2, and vascular-administration-type rAAV expressing GFP protein were intracardially administered at ages in weeks plotted on the horizontal axis (FIG. 7b ).

FIG. 8 shows the results of measuring seizure duration of mice in FIG. 7.

FIG. 9 shows the results of measuring seizure intensity of mice in FIG. 7.

FIG. 10 shows the aggregated results of seizure duration×seizure intensity of mice in FIG. 7.

FIG. 11 shows thresholds of electric stimulation measured at each age in weeks of mice in FIG. 7.

DESCRIPTION OF EMBODIMENTS

In this application, a recombinant adeno-associated virus vector is provided for treatment of a disease selected from the group consisting of epilepsy, schizophrenia, autism spectrum disorder, mental retardation, anxiety, manic-depressive psychosis, migraine, phobic and compulsive symptoms, drug addiction, Angelman syndrome, dyskinesia, dystonia, Alzheimer's disease, developmental disorders (attention deficit hyperactivity disorder, and Asperger's syndrome), which comprises a polynucleotide encoding a protein for improving the excitation-inhibiting function of inhibitory synapses in a living subject.

1. Excitation Control in Excitatory Synapse and Inhibitory Synapse

In this application, the term “synapse(s)” refers to junctional complexes between a synaptic knob formed of each swollen axonal terminal of nerve cells, and its target neuron or myocyte. In a living subject, excitatory synapses for transmitting excitation and inhibitory synapses for inhibiting the excitation transmission are present. Moreover, most synapses are chemical synapses (slow signal transduction) that are mediated by transmission of chemical substances. Another type of synapses includes electric synapses exhibiting quick response in terms of time, but are uncommonly observed in the central nervous system of a mature mammal.
In excitatory synapses, amino acids such as glutamic acid, aspartic acid, cysteic acid, and homocysteic acid function as transmitters, excitatory postsynaptic potential (EPSP) is generated, and then when the electric potential exceeds a threshold, excitation (impulse) transmission is performed. On the other hand, in inhibitory synapses, amino acids such as γ-aminobutyric acid (GABA), glycine, taurine, alanine, cystathionine, and serine function as transmitters, and then inhibitory postsynaptic potential (IPSP) is generated, which is considered to suppress the impulse of postsynaptic neurons or makes the generation thereof difficult. EPSP and IPSP include fast EPSP or fast IPSP exhibiting a rapid time course (the entire time course is within 100 milliseconds) and slow EPSP or slow IPSP exhibiting extremely slow time course lasting for tens of seconds to tens of minutes. Here, in rapid IPSP, GABA associated with a GABA_Areceptor (Cl⁻) channel and glycine associated with a glycine receptor (Cl⁻) channel are known to act. As slow IPSP transmitters, GABA_Bacting through a GABA_Breceptor as well as acetylcholine and catecholamine are known to act.
Examples of a protein for improving the excitation-inhibiting function of inhibitory synapses include neuroligin 2 and neurexin involved in synapse stabilization, GABA receptor, glutaminedecarboxylase (GAD) involved in GABA biosynthesis, Na⁺ channel protein and Cl⁻ channel protein involved in glycine transport, neuropeptide Y, gephyrin that is a scaffold protein, SLITRK3 that is a transmembrane protein and involved in inhibitory synapse formation, and PTPRD that is receptor-type tyrosine phosphatase binding to SLITRK3. A polynucleotide contained in the vector of the present invention comprises preferably a nucleotide sequence encoding a neuroligin 2 protein (SEQ ID NO: 2, 4 or 6) as a protein for improving the excitation-inhibiting function of inhibitory synapses.
The term “neuroligin (NLGN)” refers to a membrane protein family existing in a postsynaptic membrane, and is generally classified into neuroligins 1 to 4. Each of these neuroligins specifically binds to a cell adhesion molecule neurexin (Neurexin: NRXN) protein of a presynaptic membrane in order to connect a synapse preterminal and a postsynaptic site. Neuroligin 1 is located in excitatory synapses, and is considered to mediate excitatory synaptic transmission. On the other hand, neuroligin 2 is located in inhibitory synapses, and is considered to mediate inhibitory synaptic transmission. Moreover, neuroligin 3 is expressed in both excitatory synapses and inhibitory synapses, heart, pancreas, and the like, and neuroligin 4 is expressed in heart, liver and the like.
Neurexin proteins that are binding partners of neuroligins are generally classified into neurexin 1α to 3α and 1β to 3β. Herein, α neurexins and β neurexins are long-chain proteins and short-chain proteins, respectively, which are generated from the same gene by the action of different promoters. Neuroligin 2 to be used in the invention of the present application functionally binds to neurexin 1α.
A known amino acid sequence can be used as the amino acid sequence of the neuroligin 2 protein to be used in the invention of the present application. Examples of such amino acid sequence include Genbank Accession No. AAM46111 (human), EDL12455 (mouse), and EDM04903 (rat). Examples of such proteins of other animal species that can be used herein include proteins derived from mammals such as monkey, dog, pig, cattle and horse. The amino acid sequences of human, mouse and rat neuroligin 2 proteins are represented by SEQ ID NO: 2, 4 and 6, respectively.
Furthermore, examples of the neuroligin 2 protein to be used in the invention of the present application include a protein that has an amino acid sequence having about 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6, and is capable of binding to a neurexin 1α protein under physiological conditions. The larger numerical values are generally more preferred. In addition, the amino acid sequence of human neuroligin 2 and that of mouse neuroligin 2 share 98% or more identity, and the amino acid sequence of human neuroligin 2 and that of rat neuroligin 2 share 91% or more identity. In the present invention, the phrase “a variant protein functions to a degree equivalent to that of the original protein” (for example, a protein exhibits binding ability equivalent to that of the original protein) means that, for example, the specific activity ranges from about 0.01 to 100, preferably ranges from about 0.5 to 20, and more preferably ranges from about 0.5 to 2, but examples thereof are not limited thereto.
Furthermore, examples of the neuroligin 2 protein to be used in the invention of the present application include a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6 or an amino acid sequence that has the above identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6, in which one or more amino acids are deleted, substituted, inserted and/or added, and is capable of binding to the neurexin 1α protein under physiological conditions. Among the above amino acid deletion, substitution, insertion and addition, two or more types thereof may take place simultaneously. An example of such a protein is a protein comprising an amino acid sequence that is prepared from the amino acid sequence of SEQ ID NO: 2, 4 or 6 by deletion, substitution, insertion and/or addition of, for example, 1 to 50, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36, 1 to 35, 1 to 34, 1 to 33, 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9 (1 to several), 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or one amino acid residue, and is capable of binding to a neurexin a protein under physiological conditions. The smaller number of the above amino acid residues to be deleted, substituted, inserted and/or added are generally more preferred.
Examples of amino acid residues in the protein (polypeptide) of the present invention, which can be substituted with each other, are as described below. Amino acid residues included in the same group can be substituted with each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, and cyclohexylalanine;
Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, and 2-aminosuberic acid;
Group C: asparagine, and glutamine;
Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, and 2,3-diaminopropionic acid;
Group E: proline, 3-hydroxyproline, and 4-hydroxyproline;
Group F: serine, threonine, and homoserine; and
Group G: phenylalanine, and tyrosine.
A neuroligin protein in which an amino acid residue(s) is substituted can be prepared according to a method known by persons skilled in the art, such as a general genetic engineering technique. Such genetic engineering procedures can be referred to, for example, Molecular Cloning 3rd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press. 2001, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997.
Furthermore, examples of a polynucleotide that is preferably used in the invention of the present application include a polynucleotide having the polynucleotide sequence of SEQ ID NO: 1, 3 or 5, in which 1 or more (for example, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9 (1 to several), 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and 1) nucleotides are deleted, substituted, inserted and/or added, and encoding the protein that comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6, or a protein that comprises an amino acid sequence prepared from the amino acid sequence of SEQ ID NO: 2, 4 or 6 by deletion, substitution, insertion and/or addition of one or more amino acids as described above, and is capable of binding to a neurexin 1α. Among these deletion, substitution, insertion and addition, two or more types thereof may be contained in combination simultaneously. A smaller number of the above nucleotides to be deleted, substituted, inserted and/or added are generally more preferred. Moreover, examples of a preferable polynucleotide in the invention of the present application include a polynucleotide which is hybridizable under stringent hybridization conditions to SEQ ID NO: 7, 9 or 11 or its complementary sequence and encodes the amino acid sequence of SEQ ID NO: 2, 4 or 6, and a polynucleotide encoding a protein which comprises an amino acid sequence prepared from the amino acid sequence of SEQ ID NO: 2, 4 or 6 by deletion, substitution, insertion and/or addition of one or more amino acids as described above, and is capable of binding to a neurexin.
Hybridization can be performed by well-known methods or methods modified therefrom, for example, methods described in Molecular Cloning (3rd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press. 2001), etc. When commercially-available libraries are used, hybridization may be performed in accordance with the methods described in instructions provided by manufacturers, etc. As used herein, the term “stringent conditions” may be any of low stringent conditions, moderate stringent conditions and high stringent conditions. The term “low stringent conditions” refers to conditions of, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, and 50% formamide at 32° C. The term “moderate stringent conditions” refers to conditions of, for example, 5×SSC, 5× Denhardt's solution, 0.5% SDS, and 50% formamide at 42° C. The term “high stringent conditions” refers to conditions of, for example, 5×SSC, 5× Denhardt's solution, 0.5% SDS, and 50% formamide at 50° C. Under these conditions, it can be expected that DNA with higher homology is obtained efficiently at higher temperatures. Multiple factors are involved in hybridization stringency including temperature, probe concentration, probe length, ionic strength, time, salt concentration and others, but persons skilled in the art can appropriately select these factors to achieve similar stringency.
Examples of such a hybridizable polynucleotide include polynucleotides having, e.g., 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more identity with the nucleotide sequence of SEQ ID NO: 7, 9 or 11, as calculated by using default parameters under a homology search software, such as FASTA and BLAST. In general, the larger numerical value of the above homology is more preferred.
The identity or homology between amino acid sequences or polynucleotide sequences can be determined using the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990; Proc. Natl Acad. Sci. U.S.A., 90: 5873, 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul S. F. et al., J. Mol. Biol. 215: 403, 1990). When a nucleotide sequence is analyzed using BLASTN, the parameters are, for example, score=100 and word length=12. When an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score=50 and word length=3. When BLAST and Gapped BLAST programs are used, default parameters for each of the programs are employed.

2. Target Disease in the Present Invention

The invention of the present application provides an rAAV vector useful for treatment of a disease selected from the group consisting of epilepsy, schizophrenia, autism spectrum disorder, mental retardation, anxiety, manic-depressive psychosis, migraine, phobic and compulsive symptoms, drug addiction, Angelman syndrome, dyskinesia, dystonia, Alzheimer's disease, developmental disorders (attention deficit hyperactivity disorder, and Asperger's syndrome), and particularly epilepsy.
Epilepsy refers to pathological conditions in which excessive, synchronous discharging of cerebral nerve cells results in repeated clinical seizures falling in the identical type in one individual (e.g., generalized tonic-clonic seizure, absence seizure, seizure with auditory hallucination, and tonic seizure of a part of extremities). According to the classification of International League Against Epilepsy (ILAE) in 1981, clinical seizures are divided into partial seizures (simple partial seizure and complex partial seizure), generalized seizures (absence seizure, myoclonus seizure, tonic-clonic seizure, atonic seizure), and common variable seizures. Furthermore, according to the “Classification of epilepsy, epilepsy syndrome, and related seizure disorders” of ILAE in 1989, epilepsy is classified into localization-related epilepsy (sub-classified into age-related, symptomatic, cryptogenic epilepsy), generalized epilepsy (sub-classified into idiopathic, cryptogenic or symptomatic epilepsy), cases that cannot be determined to be focal or generalized epilepsy, and special syndrome (e.g., febrile convulsion).
An example of epilepsy with a brief seizure in flexion, which begins at infancy (around 1 year old) as a cardinal sign is West syndrome (or spasmus nutans). This disease forms a series of momentary tonic seizures by which the patient bends his/her upper part of the body and head part forward continuously. There are various causes of West syndrome, and the causes including congenital brain malformation, neurocutaneous syndrome such as tuberous sclerosis, and inborn errors of metabolism such as vitamin B6 deficiency are known. West syndrome is often accompanied by mental retardation, and generally evolves as the patient grows into generalized epilepsy mainly associated with generalized tonic-clonic seizure (grand mal epilepsy), or other types of epilepsy, such as Lennox syndrome, temporal lobe epilepsy, and the like. The rAAV vector of the present invention can have a therapeutic effect against West syndrome.
3. Recombinant Adeno-Associated Virus (rAAV) Vector of the Present Invention
In the invention of the present application, as a vector for delivering a gene to be used for controlling synaptic functions to nervous system cells, a recombinant adeno-associated virus vector (herein also referred to as “vascular-administration-type vector”) described in WO 2012/057363, which is capable of efficiently delivering genes to nerve cells also through peripheral administration, or a vector described in WO 2008/124724 etc., can be used, for example. The rAAV vector of the present invention can pass through the blood-brain barrier of a living subject, and thus is capable of introducing a therapeutic gene of interest to nervous system cells of the brain, the spinal cord or the like of a patient by an administration means for delivery to the brain through the blood-brain barrier, such as by peripheral administration to the patient. Moreover, the vector can also be administered intrathecally or directly to a target site in the brain.
The rAAV vector of the present invention can be prepared from preferably natural adeno-associated virus type 1 (AAV1), type 2 (AAV2), type 3 (AAV3), type 4 (AAV4), type 5 (AAV5), type 6 (AAV6), type 7 (AAV7), type 8 (AAV8), type 9 (AAV9) or the like, but examples thereof are not limited thereto. The nucleotide sequences of these adeno-associated viral genomes are known and can be referred to the nucleotide sequences of GenBank accession numbers: AF063497.1 (AAV1), AF043303 (AAV2), NC_001729 (AAV3), NC_001829.1 (AAV4), NC_006152.1 (AAV5), AF028704.1 (AAV6), NC_006260.1 (AAV7), NC_006261.1 (AAV8), and AY530579 (AAV9), respectively. Among them, the types 2, 3, 5 and 9 are human-derived. According to the present invention, it is particularly preferred to use the capsid protein (VP1, VP2, VP3 or the like) derived from AAV1, AAV2 or AAV9. Among human-derived AAVs, AAV1 and AAV9 were reported to have comparatively high multiplicity of infection on nerve cells (Taymans, et al., Hum Gene Ther 18:195-206, 2007, etc.).
A capsid protein to be contained in the rAAV vector used in the present invention is preferably a variant protein, as described in WO2012/057363, WO2008/124724 or the like, which has an amino acid sequence in which at least one tyrosine is substituted with another amino acid such as phenylalanine as compared with the wild-type amino acid sequence. Examples thereof include a variant protein having the amino acid sequence (SEQ ID NO: 9) formed by substitution of tyrosine at position 445 with phenylalanine from the amino acid sequence of a wild-type AAV1 capsid protein, a variant protein having an amino acid sequence (SEQ ID NO: 10) in which the tyrosine residue at position 444 in the amino acid sequence of a wild-type AAV2 capsid protein is substituted with the phenylalanine residue, and a variant protein having the amino acid sequence (SEQ ID NO: 11) in which the tyrosine residue at position 446 in the amino acid sequence of a wild-type AAV9 capsid protein is substituted with the phenylalanine residue (WO2012/057363 and WO 2008/124724). Such a capsid protein has a function of forming a capsomere solely or in combination with the other capsid protein members (for example, VP2 and VP3). Moreover, a polynucleotide comprising a therapeutic gene of interest to be delivered to nervous system cells is packaged in the capsomere.
When the rAAV vector of the present invention is administered into the blood stream, the rAAV vector can pass through the blood-brain barrier of a living subject including an adult and a fetus. In the present invention, examples of nervous system cells as targets of gene transfer include, at least nerve cells contained in the central nervous system such as the brain and the spinal cord, and examples of the cells may further include neuroglial cells, microglial cells, astrocytes, oligodendrocytes, ependymocytes, and cerebrovascular endothelial cells. The percentage of nerve cells in nervous system cells to which a gene is transferred is preferably, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100%.
The Rep protein used in the present invention may have the same amino acid sequence identity described above, and may contain deletion, substitution, insertion and/or addition of the same number of amino acid residues described above, as long as it has known functions to the same degree such as a function of recognizing an ITR sequence and replicating the genome depending on the sequence, a function of recruiting and packaging a wild-type AAV genome (or rAAV genome) into a viral vector, and a function of forming the rAAV vector of the present invention. Examples of the range of functionally equivalent degrees include ranges described in description concerning the above specific activity. In the present invention, preferably, a Rep protein derived from known AAV3 is used.
A polynucleotide encoding the Rep protein used in the present invention may have the same number of identity described above or may contain deletion, substitution, insertion and/or addition of nucleotides in the same number described above, as long as it encodes a Rep protein having known functions to the same degree, such as a function of recognizing an ITR sequence and replicating the genome depending on the sequence, a function of recruiting and packaging a wild-type AAV genome (or rAAV genome) into a viral vector, and a function of forming the rAAV vector of the present invention. Examples of the range of functionally equivalent degrees include ranges described in description concerning the above specific activity. In the present invention, preferably, a rep protein derived from AAV3 or AAV2 is used.
In an embodiment of the present invention, capsid protein VP1's (VP1, VP2 and/or VP3) encoded by an internal region of the above wild-type AAV genome, and the Rep protein are used through incorporation of a polynucleotide encoding them into an AAV helper plasmid. The capsid proteins (VP1, VP2 and/or VP3) and the Rep protein used in the present invention may be incorporated into one, two, three or more types of plasmid, if necessary. In certain cases, one or more types of these capsid proteins and Rep protein may be contained in the AAV genome. In the present invention, preferably, the capsid proteins (VP1, VP2 and/or VP3) and the Rep protein are all encoded by one type of polynucleotide and provided in the form of an AAV helper plasmid.
A polynucleotide to be packaged in the rAAV vector of the present invention (referred to as the polynucleotide) can be prepared by substituting a polynucleotide of the internal region (specifically, one of or both the rep gene and the cap gene) located between ITR on the 5′ side and that on the 3′ side of the wild-type genome with a gene cassette containing a polynucleotide encoding a protein of interest (therapeutic gene), a promoter sequence for transcription of the polynucleotide, and the like. Preferably, ITR on the 5′ side and that on the 3′ side are located at the 5′ end and the 3′ end of the AAV genome, respectively. Preferably, the rAAV genome of the present invention includes 5′-ITR and 3′-ITR contained in AAV1, AAV2, AAV3, AAV4, AAV8 or AAV9 genome. In general, since an ITR portion easily takes a sequence wherein the complementary sequence is replaced (flip and flop structure), and the 5′ to 3′ direction may be reversed in the ITR contained in the rAAV genome of the present invention. In the rAAV genome of the present invention, the length of the polynucleotide which is replaced by the internal region (i.e., therapeutic gene) is preferably similar to the length of the original polynucleotide from a practical viewpoint. Specifically, it is preferred that the rAAV genome of the present invention has almost the same size as 5 kb, which is the full length of the wild type genome, for example, about 2 kb to 6 kb, preferably about 4 kb to 6 kb. Except for the length of a transcription regulatory region including a promoter, polyadenylation, etc. (assuming that the length is e.g., about 1 kb to 1.5 kb), the size of a therapeutic gene to be incorporated into the rAAV genome of the present invention preferably ranges from about 0.01 kb to 3.7 kb, more preferably, about 0.01 kb to 2.5 kb, and further preferably, about 0.01 kb to 2 kb, in length, but not limited thereto.
In general, a polynucleotide to be packaged in a recombinant adeno-associated virus vector may take times (several days) until the therapeutic protein of interest is expressed, when the genome is single-stranded. In such a case, a therapeutic gene to be introduced may be designed to be an sc (self-complementary) type in order to exhibit an effect within a shorter time period. Details about this procedure is described in Foust K D, et al. (Nat Biotechnol. 2009 January; 27(1):59-65), for example. The polynucleotide packaged in the rAAV vector of the present invention may be a non-sc type or a sc type.
In an embodiment, the rAAV vector of the present invention comprises a polynucleotide (i.e., such a polynucleotide is packaged) comprising, preferably, a nerve cell-specific promoter sequence and a therapeutic gene operably linked to the promoter sequence. As the promoter sequence to be used in the present invention, a nerve cell-specific promoter sequence is derived from nerve cells, neuroglial cells, oligodendrocytes, cerebrovascular endothelial cells, microglial cells, or ventricular epithelial cells, for example, but the examples thereof are not limited thereto. Specific examples of such promoter sequence include, but are not limited to, a synapsin I promoter sequence, a myelin basic protein promoter sequence, a neuron specific enolase promoter sequence, a glial fibrillary acidic protein promoter sequence, a L7 promoter (cerebellar Purkinje cell specific promoter) sequence, a glutamate receptor delta 2 promoter (cerebellar Purkinje cell specific promoter) sequence, a glial fibrillary acidic protein (hGfa2) promoter sequence, and a glutamic acid decarboxylase (GAD65/GAD67) promoter sequence. Moreover, in the rAAV vector of the present invention, promoter sequences such as a calcium/calmodulin-dependent protein kinase II (CMKII) promoter sequence, a tubulin αI promoter sequence, a platelet-derived growth factor β chain promoter sequence, and the like can also be used. The above promoter sequences may be used independently or in optional combination of two or more thereof. In addition, the above promoter sequences may be strong promoter sequences that are generally used, such as a CMV promoter and a CAG promoter. Examples of particularly preferable promoter sequences in the present invention include a synapsin I promoter sequence, a myelin basic protein promoter sequence, a L7 promoter (cerebellar Purkinje cell specific promoter) sequence, and a glutamate receptor delta 2 promoter (cerebellar Purkinje cell specific promoter). Furthermore, known sequences such as an enhancer sequence which assists in transcription of mRNA, translation into a protein, etc., a Kozak sequence, an appropriate polyadenylation signal sequence, etc may also be contained.
A therapeutic gene of interest to be incorporated into the rAAV genome of the present invention is delivered with high efficiency to nerve cells and then integrated into the genome of the cells. When the rAAV vector of the present invention is used, the therapeutic gene can be transferred to about 10 times more, about 20 times or more, about 30 times or more, about 40 times or more or about 50 times or more of the nerve cell as compared with a conventional rAAV vector. The number of nerve cells carrying the gene transferred thereto can be determined, e.g., by preparing an rAAV vector for packaging the rAAV vector genome with any marker gene incorporated therein, administering the rAAV vector to an animal to be tested, and then measuring the number of nervous system cells expressing the marker gene (or marker protein) incorporated in the rAAV vector genome. The marker gene to be used herein is selected from known genes. Examples of such marker gene include a LacZ gene, a green fluorescence protein (GFP) gene, and a light emitting protein gene (e.g., firefly luciferase).

4. Other Therapeutic Genes

As other means or additional means for improving the excitation-inhibiting function of inhibitory synapses, for example, a means of enhancing the expression of neurexin 1α that is a binding partner of neuroligin 2, and a means of improving the intracellular signal transduction of neuroligin 2 can be expected. Alternatively, as such other means or additional means, a means of lowering the functions of excitatory synapses, such as suppressing the expression of a protein involving the operation of excitatory synapses, which is specifically a means of reducing the number of neuroligin 1 by using the antisense of neuroligin 1 (Non Patent Literature 4: Fang et al., Mol. Neurobiol. 2014 November) can also be useful.
The rAAV vector of the present invention may express different proteins for controlling synaptic functions. Examples of such different proteins include neutralizing antibodies against proteins and receptors existing on synaptic membranes (including antigen-binding sites, Fab, Fab2, single-chain antibody (scFv), etc.). Examples of the classes of these antibodies include IgG, IgM, IgA, IgD, and IgE.
For example, for inhibition of the functions of excitatory synapses, a therapeutic gene to be incorporated into the rAAV genome of the present invention may be a polynucleotide for modifying (for example, disrupting or lowering) a function of a target endogenous gene, or a polynucleotide for changing (for example, lowering) an expression level of an endogenous protein, such as an antisense molecule, a ribozyme, interfering RNA (iRNA), and micro RNA (miRNA). For example, in order to effectively inhibit the expression of a target gene by using an antisense sequence, preferably, the length of an antisense nucleic acid is 10 or more nucleotides, 15 or more nucleotides, 20 or more nucleotides, or 100 or more nucleotides, or even more preferably 500 or more nucleotides. In general, the length of an antisense nucleic acid to be used is shorter than 5 kb, and is preferably shorter than 2.5 kb.
By using a ribozyme, the mRNA encoding a protein of interest can be specifically cleaved to decrease the expression of the protein. For the design of such a ribozyme, reference may be made to various known publications (see e.g., FEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285, 1988; Nucl. Acids. Res. 17: 7059, 1989; Nature 323: 349, 1986, etc.).
The term “RNAi” refers to a phenomenon that, when a double-stranded RNA with a sequence identical or similar to a target gene sequence is introduced into cells, expression of both a target foreign gene introduced and the target endogenous gene is decreased. Examples of RNA used herein include double-stranded RNA of 21 to 25 nucleotides in length that triggers RNA interference, such as dsRNA (double strand RNA), siRNA (small interfering RNA), shRNA (short hairpin RNA) or miRNA (microRNA). These RNAs can be locally delivered to a desired site by a delivery system using liposomes, or a vector that generates the double-stranded RNA described above can be used for local expression thereof. Methods for preparing or using such double-stranded RNA (dsRNA, siRNA, shRNA or miRNA) are known from many publications (see, e.g., National Publication of International Patent Application No. 2002-516062, U.S. Patent No. 2002/086356A, Nature Genetics, 24(2), 180-183, 2000 February).
To use these other therapeutic genes, for example, a known internal ribosome entry site (IRES) sequence is allowed to intervene in a polynucleotide contained in the vector of the present invention. When the rAAV genome of the present invention is a non-sc type, it is possible to select promoters with more varied lengths and genes of interest, and also a plurality of genes of interest. A polynucleotide to be packaged in the rAAV vector of the present invention has a full length of preferably about 5 kb or less (about 4.7 kb or less when an ITR region is excluded).
5. Preparation of the rAAV Vector of the Present Invention
A general method can be employed as a method for preparing the rAAV vector of the present invention. For example, the method may comprise a step of transfecting a cultured cell with: (a) a first polynucleotide encoding a capsid protein (generally referred to as an AAV helper plasmid), and (b) a second polynucleotide (carrying a therapeutic gene of interest) to be packaged in the rAAV vector of the present invention; and may further comprise a step of transfecting the cultured cell with (c) a plasmid encoding an adenovirus-derived factor, also referred to as an adenovirus (AdV) helper plasmid, or a step of infecting cultured cells with an adenovirus. The method can also comprise a step of culturing the transfected cultured cell and a step of collecting the recombinant adeno-associated virus vector from the culture supernatant. Furthermore, (d) an example of a method for preparing the rAAV vector of the present invention includes a method for producing an rAAV in a large scale by preparing baculoviruses containing the above polynucleotides (a) and (b), respectively, and then infecting insect cells, Sf9 or the like with the viruses. This method is already known and also used in Examples of the Description.
A nucleotide encoding the capsid protein of the present invention in the first polynucleotide (a) is preferably operably bound to a known promoter sequence that is operable in cultured cells. As such a promoter sequence, for example, a cytomegalovirus (CMV) promoter, an EF-1α promoter, an SV40 promoter, and the like can be appropriately used. Furthermore, the first polynucleotide can comprise a known enhancer sequence, a Kozak sequence, a polyA addition signal sequence and the like, as appropriate.
The second polynucleotide (b) comprises a therapeutic gene at a position where it is operable with a nervous system cell-specific promoter. Furthermore, the second polynucleotide can comprise a known enhancer sequence, a Kozak sequence, a polyA addition signal sequence, and the like as appropriate. The first polynucleotide can further comprise a cloning site, which can be cleaved by various known restriction enzymes, and is located downstream from the nervous system cell-specific promoter sequence. A multicloning site containing a plurality of restriction enzyme recognition sites is more preferred. Persons skilled in the art may incorporate a therapeutic gene of interest downstream of the nervous system cell-specific promoter, in accordance with known genetic engineering procedures. For such genetic engineering procedures, see, e.g., Molecular Cloning 3rd Edition, J. Sambrook et al., Cold Spring Harbor Lab: Press. 2001, etc.
In preparation of the rAAV vector of the present invention, a helper virus plasmid (e.g., adenovirus, herpes virus or vaccinia) is used and can be introduced into cultured cells simultaneously with the above first and second polynucleotides. Preferably, the preparation method of the present invention further comprises a step of introducing an adenovirus (AdV) helper plasmid. In the present invention, preferably, AdV helper is derived from a virus of the same species as that of cultured cells. For example, when human cultured cells 293T are used, a human AdV-derived helper virus vector can be used. As such an AdV helper vector, a commercially available AAV Helper-Free System (Agilent Technologies, catalog No. 240071) can be used, for example.
In preparation of the rAAV vector of the present invention, examples of a method for transfecting cultured cells with the above one or more types of plasmid, which can be used herein, include various known methods such as the calcium phosphate method, lipofection method, and electroporation method, etc. Such methods are described in, e.g., Molecular Cloning 3rd Ed., Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997, etc.
6. Pharmaceutical Composition Containing the rAAV Vector of the Present Invention
The rAAV vector of the present invention can comprise genes useful for treatment of neurological disorders, particularly, diseases relating to protein dysfunction in synapses (e.g., schizophrenia and autism spectrum disorder). The rAAV vector comprising these genes can be administered intravascularly to pass through the blood-brain barrier, and thus can be incorporated into nerve cells of the brain, the spinal cord, and the retina. The rAAV vector comprising such a therapeutic gene can be contained in the pharmaceutical composition of the present invention. As such therapeutic genes, for example, polynucleotides encoding the above-mentioned antibodies, neurotrophic factor (NGF), growth factor (HGF), acidic fibroblast growth factor (aFGF), miRNA and the like can be selected. It can be expected to treat neurological disorder through peripheral administration of such rAAV vector to a test subject.
The active ingredient of the pharmaceutical composition of the present invention may be formulated solely or in combination therein, and can also be provided as a pharmaceutical preparation by formulation with a pharmaceutically acceptable carrier or an additive for a pharmaceutical preparation. In this case, the active ingredient of the present invention may be contained in an amount e.g., 0.1 to 99.9 wt % in the preparation.
Examples of the pharmaceutically acceptable carriers or additives that can be used include excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, dissolution agents, dissolution aids, isotonic agents, pH regulators, stabilizers, etc. For oral administration, excipients that are generally used in the art, such as microcrystalline cellulose, sodium citrate, calcium carbonate, disintegrants such as starch and alginic acid, granulation binders such as polyvinylpyrrolidone, and lubricants can be used in combination. When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient may be used in combination with various sweeteners or corrigents, coloring agents or dyes, and, if necessary, emulsifying and/or suspending agents as well, together with diluents such as water, ethanol, propylene glycol, glycerin, etc. and combinations thereof.
Examples of the pharmaceutical preparations suitable for oral administration can include powders, tablets, capsules, fine granules, granules, liquids or syrups, etc. Examples of the pharmaceutical preparations suitable for parenteral administration can include injections, intrathecal injections, suppositories, etc. For parenteral administration, solutions of the active ingredient of the present invention dissolved in either sesame or peanut oil or in aqueous propylene glycol solution may be employed. The aqueous solutions should be appropriately buffered (preferably pH 8 or higher) as necessary; it is first necessary to render the liquid diluent isotonic. As such a liquid diluent, physiological saline can be used. The thus prepared aqueous solutions are suitable for intravenous injection. On the other hand, the oily solutions are suitable for intra-articular injection, intra-muscular injection and subcutaneous injection. The preparation of all these solutions under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. Furthermore, the active ingredient of the present invention can also be administered topically to the skin, etc. In this case, topical administration is desirably performed by way of creams, jellies, pastes, ointments and the like, in accordance with standard pharmaceutical practice.
The dose of the pharmaceutical composition of the present invention is not particularly limited, and an appropriate dose can be chosen depending on various conditions such as type of disease, age and symptoms of the patient, administration route, therapeutic goal, presence or absence of concurrent drugs, etc. The dose of the pharmaceutical composition of the present invention is, but not limited to, for example, 1 to 5,000 mg, and preferably 10 to 1,000 mg per day for an adult (e.g., body weight of 60 kg). Such daily dose may be administered in 2 to 4 divided doses per day. When vg (vector genome) is used as a dosage unit, the dose can be selected from, but not limited to, e.g., the range from 10⁹to 10¹⁴vg, preferably, 10¹⁰to 10¹³vg, and more preferably, 10¹⁰to 10¹²vg per kg body weight.
7. Administration of the rAAV Vector of the Present Invention
The rAAV of the present invention is capable of passing through the blood-brain barrier of a living subject (including incomplete fetal and newborn blood-brain barriers, and established adult blood-brain barriers) and thus capable of delivering genes in the rAAV to nervous system cells of the brain, the spinal cord, and the like through peripheral administration to a living subject (including adults and fetuses or newborns). Furthermore, the rAAV vector to be used in the present invention can target nerve cells contained in an adult's brain, spinal cord, and the like through peripheral administration. As used herein, the term “peripheral administration” refers to administration routes which those skilled in the art usually understand as peripheral administration, including intravenous administration, intraarterial administration, intraperitoneal administration, intracardiac administration, intramuscular administration, and umbilical intravascular administration (e.g., the target is a fetus), and so on. Furthermore, an administration method, which involves using a fluid other than blood that is fluidly communicated with the brain, such as intrathecal administration, can also be used for the rAAV vector of the present invention. In another embodiment, the rAAV vector of the present invention can also be locally administered to a target site within the brain, such as hippocampi. For example, when the rAAV of the present invention is administered via intrathecal administration into a spinal fluid, or via peripheral administration into blood, a means for administration simpler than intraparenchymal administration can be provided.
8. Kit for Preparation of the rAAV Vector of the Present Invention
In another embodiment, the present invention provides a kit for preparing the rAAV of the present invention. Such a kit can contain, for example, (a) a first polynucleotide for expression of capsid protein VP1 or the like, and (b) a second polynucleotide to be packaged in the rAAV vector. For example, the first polynucleotide comprises a polynucleotide encoding the amino acids of SEQ ID NO. For example, the second polynucleotide may or may not comprise a therapeutic gene of interest, but can preferably comprise various restriction enzyme cleavage sites for incorporation of such a therapeutic gene of interest.
The kit for preparing the rAAV vector of the present invention can further contain any component described herein (e.g., an AdV helper.). The kit of the present invention may further include instructions describing the protocols for preparation of the rAAV vector using the kit of the present invention.
9. Chemotherapeutic Agent to be Used in Combination with the rAAV of the Present Invention
The rAAV vector according to the invention of the present application can also be used in combination with an existing chemotherapeutic agent. Examples of such a chemotherapeutic agent include phenytoin, carbamazepine, valproic acid, topiramate, lamotrigine, rufinamide, phenobarbital, diazepam, clonazepam, ethosuximide, zonisamide, gabapentin, levetiracetam, midazolam, clobazam, and propofol. For example, after administration of the rAAV of the invention of the present application, a significant reduction in the dose of the above chemotherapeutic agent can be expected.

10. Determination of Therapeutic Effects

The therapeutic effects of the rAAV vector of the present invention can be determined using a known means for determining if excitation can be inhibited by the therapeutic effects. Examples of such a known means include, but are not limited to, analysis of behavior levels, analysis of the pharmacodynamics of labeled transmitters (e.g., GABA), measurement of excitatory postsynaptic potential and inhibitory postsynaptic potential, measurement of changes in threshold of epilepsy induced by medicines or electric stimulation, brain wave, optical topography, and positron emission tomography (PET).

11. Terms Used in the Description

The meaning indicated by each term as used herein is as described below. Terms not particularly described herein are intended to refer to meanings that are normally understood by persons skilled in the art.
As used herein, the terms “virus or viral vector”, “virus virion,” and “virus or viral particles” are interchangeably used, unless otherwise indicated.
As used herein, the term “nervous system” refers to an organ system made up of nerve tissues. As used herein, the term “nervous system cells” include at least nerve cells included in the central nervous system including brains, spinal cords, etc. and may further include neuroglial cells, microglial cells, astrocytes, oligodendrocytes, ependymocytes, cerebrovascular endothelial cells, etc.
As used herein, the term “polynucleotide” is interchangeably used with “nucleic acid,” “gene” or “nucleic acid molecule,” which is intended to mean a nucleotide polymer. As used herein, the term “nucleotide sequence” is used interchangeably with “nucleic acid sequence” or “base sequence,” which is represented by a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T). For example, the “polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof” is intended to mean a polynucleotide comprising a sequence shown by the respective deoxynucleotides A, G, C and/or T of SEQ ID NO: 1, or a fragment thereof.
Each of “virus or viral genome” and “polynucleotide” according to the present invention may exist in the form of a DNA (e.g., cDNA or genomic DNA), respectively, and may also be in the form of an RNA (e.g., mRNA). Each of the viral genome and the polynucleotide as used herein may be a double-stranded or single-stranded DNA. Single-stranded DNA or RNA may be a coding strand (also known as a sense strand) or a non-coding strand (also known as an anti-sense strand). Regarding the explanation herein for placing a promoter, a gene of interest, polyadenylation signal, etc. in the gene, which are encoded by the rAAV genome, if the rAAV genome is a sense strand, the strand itself is described and if it is an antisense strand, its complementary strand is described, unless otherwise specified.
As used herein, the terms “protein” and “polypeptide” are interchangeably used and intended to mean a polymer of amino acids. The polypeptide as used herein is represented in accordance with conventional peptide designation, in which the N-terminus (amino terminus) is on the left hand and the C-terminus (carboxyl terminus) on the right hand. The partial peptide in the polypeptide of the present invention (as used herein, may briefly be referred to as the partial peptide of the present invention) includes a partial peptide of the polypeptide of the present invention described above, preferably having the same properties as those of the above polypeptide of the present invention.
As used herein, the term “plasmid” refers to various known gene elements, for example, a plasmid, a phage, a transposon, a cosmid, a chromosome, etc. The plasmid can be replicated in a particular host and transport gene sequences between cells. As used herein, the plasmid contains various known nucleotides (DNA, RNA, PNA and a mixture thereof) and may be a single strand or a double strand, and preferably a double strand. As used herein, the term “rAAV vector plasmid” is intended to include a double strand formed by rAAV vector genome and its complementary strand, unless otherwise specified. The plasmid used in the present invention may be linear or circular.
As use herein, the term “packaging” refers to the events including preparation of single-stranded viral genomes, assembly of coat (capsid) proteins, enclosure of viral genome within a capsid (encapsidation), and the like. When an appropriate plasmid vector (normally, a plurality of plasmids) is introduced into a cell line that allows packaging under appropriate conditions, recombinant viral particles (i.e., virus virions, viral vectors) are constructed and secreted into the culture.

EXAMPLES

The present invention is described below in more detail by referring to Examples, but the scope of the invention should not be limited to the following Examples.

Experimental Outline

Intracardiac administration of rAAV-Neuroligin2 (rAAV-NL2) at 6 weeks of age For:-Seizure frequency-Duration-Intensity-Duration×Intensity-Changes in threshold for electric stimulation
Intracerebral Expression of Neuroligin2-Carrying Intravascular-Administration-Type rAAV
Reports concerning the gene therapy for epilepsy using model animals are found here and there, but they all involve performing topical administration stereotactically. For application to a human patient, administration methods without invasive procedures are desired. This time, the inventors of the present application prepared an intravascular-administration-type adeno-associated virus (rAAV) vector, administered the vector to EL mice naturally developing epilepsy (Suzuki, Proc. Jpn. Acad., Ser.B89 (2013)), and then observed the condition of intracerebral expression and the presence or the absence of an effect of suppressing seizure.

Experimental Materials and Methods

Recombinant Adeno-Associated Virus (rAAV) Vector
The vector to be used in the Examples is previously disclosed AAV9/3 (having tyrosine mutation (Y446→F) introduced into AAV9 capsid, and ITR of AAV3) carrying a Synapsin I promoter (WO 2012/057363). To differentiate from endogenous Neuroligin 2 (NL2), an rAAV vector expressing neuroligin2 with the N-terminus, to which a FLAG tag (DDDDK) sequence had been bound, was prepared (AAV9/3-Syn1-FLAG (DDDDK)-NL2), and then administered to subject animals.
Administration to Animal
EL mice (6 weeks of age, male, body weight: 22-32 g) were used.
Under 2-4% sevoflurane anesthesia, inracardiac injection of AAV9/3-Syn1-FLAG (DDDDK)-NL2 was performed at 4.1×10¹³vector genome/ml×0.1 ml/mouse (NL2 inracardiac injection group n=10). A group to which AAV9/3-Syn1-AcGFP-WPRE was administered via intracardiac injection at 2.3×10¹³vg/ml×0.1 ml/mouse (n=17), and a group to which only physiological saline was administered at 0.1 ml/mouse (n=14) were designated as control groups. Also, for comparison with topical administration, a group of mice to which AAV9/3-Syn1-FLAG (DDDDK)-NL 2 was injected to the bilateral hippocampal CA3 region (0.5 mm anterior to and 3.0 mm lateral to bregma, and 2.0 mm from the brain surface) at 4.1×10¹³vg/ml×0.005 ml (n=3) was designated as the topical administration group.

TABLE 1

		Number of
Administration site	Titer vg/mouse	mice

rAAV-NL2	Intracardiac	4.1 × 10¹²	10
rAAV-GFP	Intracardiac	2.3 × 10¹²	17
Physiological saline	Intracardiac	—	14
rAAV-NL2	Bilateral hippocampi	2.0 × 10¹¹	3
rAAV-GFP	Bilateral hippocampi	1.1 × 10¹¹	3
Physiological saline	Bilateral hippocampi	—	3

Evaluation by Angular Acceleratory Stimulation
After administration of the vector and the like, each of the mice was rotated with the tail held for predetermined times (8 rotations) every week until 22 weeks of age, and the behavior of each of the mice was video-recorded. Thereafter, the presence or the absence of seizures, duration when a mouse had developed a seizure, and the intensity thereof were observed on video.
Seizure intensity was scored as follows:
1 point: no seizure;
2 points: only raised the tail or only shook the body;
3 points: developed a clear seizure, but kept the posture without falling down; and
4 points: developed a severe seizure and could not keep the posture and fell down sideways.
Seizure duration was scored as follows:
1 point: no seizure;
2 points: 1-10 seconds;
3 points: 11-20 seconds;
4 points: 21-30 seconds;
5 points: 31-60 seconds; and
6 points: 61 seconds or longer.
The seizure incidence, the mean seizure duration, the mean seizure intensity, and the mean seizure duration×intensity of each group were evaluated every week.
Evaluation by Electric Stimulation
At 5 (before administration of the vector), 12, 18, 22 weeks of age, electrodes were placed on both ears of a mouse, electric stimulation (the following parameters were used in Neuropack S1 (NIHON KOHDEN): duration: 1 ms, interval: 50 ms, 10 train, strength: max 50 mA every 0 mA to 5 mA) was applied to induce an epileptic seizure, and then the seizure threshold was measured. If no seizure was induced by 50 mA, the seizure threshold was determined to be 60 mA for evaluation. For only the topical administration group of mice, this procedure was performed at 5, 12, and 22 weeks of age.
Statistical Processing was Evaluated by the Following Tests.
Seizure incidence: Fisher's exact test
Other: Welch's t test
Histological Analysis
Preparation of brain specimen: Each mouse was deeply anesthetized with pentobarbial, and then 4% paraformaldehyde-containing 0.1M phosphate buffer (pH7.4) was injected through the left ventricle for perfusion fixation. After fixation, the brain was dissected out, immersion fixed in a fixative for a half day, transferred into 15% sucrose-containing 0.1M phosphate buffer (pH 7.4), and then stored in a refrigerator until a histochemical experiment.
rAAV9-GFP Expression and Cell Identification
40-μm sagittal sections were prepared using a cryo-microtome and then GFP expression was identified at each brain site using a fluorescence microscope. GFP-expressing cells were identified by double staining with the following markers.
Nerve cells: NeuN or MAP2; glial cells: GFAP; and inhibitory interneurons: Parvalbumin
rAAV9-NL2 Expression and Cell Identification
In a manner similar to that in identification of GFP, 40-μm sagittal sections were prepared, and the transgene NL2 expression was identified by FLAG (DDDDK) antibody staining. FLAG antibody was purchased from Abcam plc., and the expression was identified under a fluorescence microscope by image processing using a Alexafluor 488 conjugated secondary antibody.

Experimental Results and Discussion

Hippocampal slices (specimens) were subjected to measurement of changes in intracellular Ca²⁺ influx upon ischaemic loading based on changes in fluorescence of rhod 2-AM (DOJINDO catalog No.: R002). As compared with DDY mice, EL mice exhibited, in the CA3 region, significant increases in intracellular Ca²⁺ influx induced in hippocampi by ischaemiac loading, suggesting the vulnerability of the inhibitory system in the CA3 region. Next, the expression of intervening cells of an excitation inhibition system in EL hippocampi was histologically examined by immunostaining with the parvalbumin antibody (catalog No.: LS-C39101). No significant difference was found between EL mice and DDY mice in terms of the number of parvalbumin-positive cells in each hippocampal region, suggesting possible changes at the synaptic level. Based on the above results, an rAAV vector expressing a gene of an inhibitory system synapse-related molecule was prepared, the vector was administered to EL via stereotactic hippocampal injection and intravascular injection, and then intracerebral distribution was histologically observed by staining using a FLAG antibody (FIG. 1a , and FIG. 1b ). As shown in FIG. 1a , FLAG-tagged NLGN2 was broadly expressed in hippocampal and cerebral cortical nerve cells as a result of intravascular administration of the vector the present invention, so that successful gene delivery by rAAV could be confirmed.
Subsequently, the intravascular injection group was observed for the presence or the absence of the effect of suppressing epileptic seizure (FIG. 2 to FIG. 6). A group to which a green fluorescent protein EGFP expression AAV vector was administered, and a group to which physiological saline was administered were designated as control groups. The effect of suppressing epilepsy was evaluated for each attribute; that is, frequency of seizure development, intensity, duration, and seizure intensity×duration of a mouse at each age in weeks. In addition, significant differences were observed at positions marked with “*” and “**” in the figures. The group to which NLGN2 had been administered suppressed seizures more significantly than the control groups, and exhibited no change in threshold for electric stimulation (FIG. 2 to FIG. 6). No change in threshold for electric stimulation suggested that the initiation of the operation of inhibitory synapses remained unchanged from the time before introduction and suggested the low side effect.
Expression of the gene of interest was observed in hippocampal neurons of the hippocampal injection group, and in whole brain neurons including hippocampal neurons of the intravascular administration group (the results not shown). Compared with the control groups, the hippocampal injection group, specifically, the group to which NLGN2 had been administered exhibited a significant difference in seizure frequency in some cases, but exhibited overall no significant difference in the effect (FIG. 7 to FIG. 11). In the figures, significant differences were observed for those indicated with “*” and “**”. Moreover, compared with the intracardiac administration groups, the intracardiac administration groups were generally observed to tend to exhibit the higher effects on suppressing epilepsy than the other groups in any attribute of seizure frequency, seizure duration and seizure intensity.
The target molecules were supplied to whole brain neurons by the intravascular-administration-type AAV vector. The excitation inhibitory effect of said molecules is capable of suppressing epileptic seizures without changing the threshold for electric stimulation, suggesting a possibility of a non-invasive epilepsy gene therapy as a more advantageous therapeutic method.

INDUSTRIAL APPLICABILITY

The use of the rAAV vector of the present invention can be expected to treat (e.g., alleviation, improvement, and repair) genetic malfunctions in nervous system cells (including congenital and acquired malfunctions).

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1: human neuroligin2 nucleotide sequence
SEQ ID NO: 2: human neuroligin2 amino acid sequence
SEQ ID NO: 3: mouse neuroligin2 nucleotide sequence
SEQ ID NO: 4: mouse neuroligin2 amino acid sequence
SEQ ID NO: 5: rat neuroligin2 nucleotide sequence
SEQ ID NO: 6: rat neuroligin2 amino acid sequence
SEQ ID NO: 7: Flag-tagged mouse neuroligin2 nucleotide sequence
SEQ ID NO: 8: Flag-tagged mouse neuroligin2 amino acid sequence
SEQ ID NO: 9: AAV1 capsid protein Y445F variant amino acid sequence
SEQ ID NO: 10: AAV2 capsid protein Y444F variant amino acid sequence
SEQ ID NO: 11: AAV9 capsid protein Y446F variant amino acid sequence

Claims

1. A recombinant adeno-associated virus vector, wherein the virus vector comprises a polynucleotide encoding a protein for improving an excitation-inhibiting function of inhibitory synapses in a living subject, and is used for treatment of a disease selected from the group consisting of epilepsy, schizophrenia, autism spectrum disorder, mental retardation, anxiety, manic-depressive psychosis, migraine, phobic and compulsive symptoms, drug addiction, Angelman syndrome, dyskinesia, dystonia, Alzheimer's disease, and developmental disorders (attention deficit hyperactivity disorder and Asperger's syndrome),

wherein the polynucleotide comprises a nucleotide sequence encoding a neuroligin 2 protein which comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6, or an amino acid sequence having about 90% or more identity with said amino acid sequence and capable of binding to neurexin, and

wherein the recombinant adeno-associated virus vector comprises:

a protein having a variant amino acid sequence in which tyrosine at position 445 in the amino acid sequence of a wild-type AAV1 capsid protein is substituted with phenylalanine;

a protein having a variant amino acid sequence in which tyrosine at position 445 in the amino acid sequence of a wild-type AAV2 capsid protein is substituted with phenylalanine; or

a protein having a variant amino acid sequence in which tyrosine at position 446 in the amino acid sequence of a wild-type AAV9 capsid protein is substituted with phenylalanine.

2. (canceled)

3. The recombinant adeno-associated virus vector according to claim 1, wherein the disease is epilepsy.

4. (canceled)

5. The recombinant adeno-associated virus vector according to claim 1, wherein the polynucleotide comprises a promoter sequence selected from the group consisting of a synapsin I promoter sequence, a myelin basic protein promoter sequence, a neuron specific enolase promoter sequence, a calcium/calmodulin-dependent protein kinase II (CMKII) promoter sequence, a tubulin α1 promoter sequence, a platelet-derived growth factor β chain promoter sequence, a glial fibrillary acidic protein (GFAP) promoter sequence, a L7 promoter (cerebellar Purkinje cell specific promoter) sequence, a glial fibrillary acidic protein (hGfa2) promoter sequence, and a glutamate receptor delta 2 promoter (cerebellar Purkinje cell specific promoter) sequence, and a glutamic acid decarboxylase (GAD65/GAD67) promoter sequence.

6. The recombinant adeno-associated virus vector according to claim 1, wherein the polynucleotide comprises an inverted terminal repeat (ITR) selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV8, and AAV9.

7. The recombinant adeno-associated virus vector according to claim 1, wherein the polynucleotide further comprises a polynucleotide for inhibiting the excitation of excitatory synapses.

8. A pharmaceutical composition, comprising the recombinant adeno-associated virus recombinant vector according to claim 1.

9. The pharmaceutical composition according to claim 8, which is administered intracerebrally.

10. The pharmaceutical composition according to claim 8, which is administered intrathecally.

11. The pharmaceutical composition according to claim 8, which is administered peripherally.

12. The pharmaceutical composition according to claim 8, which is used in combination with a chemotherapeutic agent for a neuropsychiatric disease.