阅读说明：本技术 Mir-92a或mir-145在治疗安格曼综合征中的用途 (Use of MIR-92A or MIR-145 for treating angeman syndrome ) 是由 马修·杜林 于 2019-06-14 设计创作，主要内容包括：负调节SNHG14基因活性的微小RNA的表达可用于治疗安格曼综合征。用于治疗安格曼综合征的此类微小RNA包括例如MIR-92a和/或MIR-145及其类似物和变体。可以使用表达载体诸如例如AAV载体转导细胞以将MIR-92a和/或MIR-145引入到靶组织以治疗安格曼综合征。(The expression of micrornas that negatively regulate the activity of SNHG14 gene may be used to treat angmann syndrome. Such microRNAs useful in the treatment of Angerman syndrome include, for example, MIR-92a and/or MIR-145 and analogs and variants thereof. Cells can be transduced with expression vectors such as, for example, AAV vectors to introduce MIR-92a and/or MIR-145 to target tissues to treat angmann syndrome.)

1. A method for treating angeman syndrome in a patient in need thereof, comprising administering to the patient a vector comprising a nucleic acid encoding microrna-145, primary miR145, precursor miR145, microrna-92 a, primary miR-92a, or precursor miRNA-92a, wherein the nucleic acid is operably linked to a promoter, wherein one or more symptoms of the angeman syndrome are ameliorated.

2. The method of claim 1, wherein following said administering, expression of microrna-145, primary miR145, precursor miR145, microrna-92 a, primary miR-92a, or precursor miRNA-92a in said patient is associated with reduced symptoms of said angeman syndrome.

3. The method of claim 1, wherein the promoter is selected from the group consisting of: CAG promoter, CMV promoter, human synapsin 1 gene promoter (hSyn), dynorphin promoter, encephalomyelin promoter and CaMKII promoter.

4. The method of claim 1, wherein the promoter is a CAG promoter.

5. The method of claim 1, wherein the vector comprises a woodchuck post-transcriptional regulatory element (WPRE).

6. The method of claim 1, wherein said vector comprises a bovine growth hormone polyadenylation sequence (BGHpA).

7. The method of claim 1, wherein the carrier comprises a fluorescent reporter cassette.

8. The method of claim 1, wherein the vector is an adeno-associated virus (AAV).

9. The method of claim 8, wherein the adeno-associated virus is AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, or AAVRec 3.

10. The method of claim 1, wherein the vector is a lentivirus.

11. The method of claim 1, wherein the vector is delivered to a target location in the brain of the patient.

12. The method of claim 1, wherein the vector is administered via a route selected from the group consisting of: oral, buccal, sublingual, rectal, topical, intranasal, vaginal and parenteral.

13. The method of claim 1, wherein the vector is administered directly to the target location.

14. The method of claim 1, wherein the method provides amelioration of at least one symptom selected from the group consisting of: developmental delay, mental disability, speech impairment, gait ataxia and/or limb tremor, seizures, microcephaly, improper happy ending, frequent laughing, hydrophile, smile, and excitability.

15. The method of claim 1, further comprising applying ultrasound to a target location in the brain of the patient to enhance permeability of the blood brain barrier of the patient at the target location, wherein the carrier is delivered to the target location.

16. A method of treating anglman syndrome in a patient in need thereof, comprising administering to the patient an effective amount of a pharmaceutical composition that increases the level of microrna-145 and/or microrna-92 a molecules in the brain of the patient.

17. The method of claim 16, wherein the composition comprises microrna-145, primary miR145, precursor miR145, microrna-92 a, primary miR92a, or precursor miR92 a.

18. The method of claim 16, wherein the composition comprises a vector comprising a nucleic acid encoding microrna-145, primary miR145, precursor miR145, microrna-92 a, primary miR92a, or precursor miR92 a.

19. The method of claim 16, wherein following said administering, expression of microrna-145, primary miR145, precursor miR145, microrna-92 a, primary miR92a, or precursor miR92a in the patient is associated with reduced symptoms of an angeman syndrome disorder.

20. The method of claim 18, wherein the nucleic acid encoding microrna-145, primary miR145, precursor miR145, microrna-92 a, primary miR92a, or precursor miR92a is operably linked to a promoter.

21. The method of claim 20, wherein the promoter is selected from the group consisting of: CAG promoter, CMV promoter, human synapsin 1 gene promoter (hSyn), dynorphin promoter, encephalomyelin promoter and CaMKII promoter.

22. The method of claim 18, wherein the vector comprises a woodchuck post-transcriptional regulatory element (WPRE).

23. The method of claim 18, wherein the carrier comprises a fluorescent reporter cassette.

24. The method of claim 18, wherein the vector is an adeno-associated virus (AAV).

25. The method of claim 24, wherein the adeno-associated virus is AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, or AAVRec 3.

26. The method of claim 18, wherein the vector is a lentivirus.

27. The method of claim 18, wherein the vector is delivered to a target location in the brain of the patient.

28. The method of claim 18, wherein the vector is administered via a route selected from the group consisting of: oral, buccal, sublingual, rectal, topical, intranasal, vaginal and parenteral.

29. The method of claim 18, wherein the vector is administered directly to the target location.

30. The method of claim 18, wherein the composition comprises a vector that is a non-viral vector.

31. The method of claim 30, wherein the non-viral vector is a liposome-mediated delivery vector.

Technical Field

The present disclosure relates generally to the use of the expression of micrornas that negatively modulate the activity of SNHG14 gene for the treatment of angmann syndrome. Such microRNAs for use in the treatment of Angelman syndrome (Angelman) include, for example, MIR-92a and/or MIR-145 and analogs and variants thereof. More specifically, the disclosure relates to the use of expression vectors such as, for example, AAV vectors, which can be used to transduce cells to introduce MIR-92a and/or MIR-145 to target tissues to treat angmann syndrome.

Background

Angman Syndrome (AS) is a neurodevelopmental disorder characterized by severe developmental delay or mental disability, severe speech impairment, gait ataxia and/or limb tremor, seizures, microcephaly, and unique behaviors with inappropriate happy behavior including frequent laughing, hydrophilic (after for water), smiling, and excitement. Microcephaly and seizures are also common. Developmental delay was first noted at about six months of age; however, the unique clinical features of angeman syndrome do not manifest until one year of age, and may take several years before a correct clinical diagnosis is evident.

Many of the characteristic features of angmann syndrome are caused by the loss of function of the gene called UBE3A, which encodes the ubiquitin protein ligase E3A (UBE3A) gene. (Kishino, T. et al. Nat Genet (1997)12: 385-. One typically inherits one copy of the UBE3A gene from each parent. Two copies of the gene are open (active) in many bodily tissues, however, in certain regions of the brain, only the copy inherited from the human mother (maternal copy) is active. This parent-specific gene activation is caused by a phenomenon known as genomic imprinting. UBE3A is maternally imprinted in the brain, making it almost exclusively expressed by maternal chromosomes, while paternal chromosomes are epigenetic silenced. (Albrecht, U.S. et al, Nat Genet (1997)17: 75-78). If the maternal copy of the UBE3A gene is lost due to chromosomal changes or gene mutations, a human will not have an active copy of the gene in some parts of the brain.

Several different genetic mechanisms can inactivate or delete the maternal copy of the UBE3A gene. Most cases of angman syndrome (about 70 percent) occur when the maternal chromosome 15 segment containing the gene is missing. In other cases (about 11%), angman syndrome is caused by a mutation in the maternal copy of the UBE3A gene.

The small nucleolar RNA host gene 14(SNHG14), alternatively known as UBE3A-ATS, will extend antisense into the UBE3A gene and thus play a potential role in inhibiting paternal UBE3A expression and imprinting. There is still much to be understood how the deficiencies in the protein product of UBE3A lead to the neurodevelopmental defect observed in angmann syndrome. Accordingly, there remains a need for improved and/or additional therapies for treating a subject diagnosed as having angleman syndrome.

SUMMARY

Methods of treating Angmann Syndrome (AS) in a patient in need thereof are provided, the methods comprising delivering to the patient an effective amount of a composition that negatively modulates the activity of SNHG14 gene. Such a method of treatment comprises delivering to the patient an effective amount of a composition that increases the level of microRNA-145 and/or microRNA-92 a molecules in the central nervous system of the patient. The methods disclosed herein are designed to negatively modulate the activity of the SNHG14 gene.

Methods of treating angmann syndrome in a patient in need thereof are provided, the methods comprising delivering to the patient an effective amount of a composition that increases the level of microrna-145 and/or microrna-92 a molecules in cells of the central nervous system of the patient. Methods of treating angmann syndrome in a patient in need thereof are provided, the methods comprising administering to the patient a vector encoding microrna-145, a primary miRNA145, or a precursor miRNA 145. Methods of treating angmann syndrome in a patient in need thereof are provided, the methods comprising administering a vector encoding microrna-92 a, primary miR92a, or precursor miRNA92 a. In embodiments, the increased level of microRNA-145 or microRNA-92 a causes an improvement in one or more symptoms of Angermann syndrome.

In embodiments, a vector encoding microrna-145, primary miR145, or precursor miR145 causes an increase in the level of microrna-145 in patients with angeman syndrome and is associated with a reduction in the symptoms of the disorder. In embodiments, a vector encoding microrna-92 a, primary miR92a, or precursor miR92a causes increased levels of microrna-92 a in patients with angeman syndrome, and is associated with reduced symptoms of the disorder.

In certain aspects, a vector comprising a nucleic acid encoding microRNA-145, primary miR145 or precursor miR145 comprises a promoter operably linked to the nucleic acid encoding microRNA-145, primary miR145 or precursor miR 145. In embodiments, the vector comprises a woodchuck post-transcriptional regulatory element (WPRE). In an embodiment, the vector includes a bovine growth hormone polyadenylation sequence (BGHpA). In embodiments, the vector comprises a fluorescent reporter cassette. In embodiments, the vector is an adeno-associated virus. In embodiments, the vector is a lentivirus. In embodiments, the vector comprising a nucleic acid encoding microrna-92 a, primary miR92a, or precursor miR92a comprises a promoter operably linked to the nucleic acid encoding microrna-92 a, primary miR92a, or precursor miR92 a. In embodiments, the nucleic acid encoding microRNA-92 a is microRNA-92 a-3 p. In embodiments, the vector comprises a woodchuck post-transcriptional regulatory element (WPRE). In an embodiment, the vector includes a bovine growth hormone polyadenylation sequence (BGHpA). In embodiments, the vector comprises a fluorescent reporter cassette. In embodiments, the vector is an adeno-associated virus. In embodiments, the vector is a lentivirus. In embodiments, the vector is an AAV vector.

In embodiments, the vector is delivered to a target location in the central nervous system of the patient. In embodiments, the target location is the brain of the patient. In embodiments, the route of administration of the carrier is oral, buccal, sublingual, rectal, topical, intranasal, vaginal, or parenteral. In embodiments, the vector is administered directly to the target site.

In embodiments, ultrasound is applied to a target location in the brain of the patient to enhance permeability of the blood brain barrier of the patient at the target location, wherein microRNA-145 or microRNA-92 a is delivered to the target location.

Detailed Description

Described herein are methods and compositions for treating angmann syndrome comprising administering a composition that negatively modulates the activity of the SNGH14 gene. For patients with angmann syndrome, such compositions include, for example, microrna-145, primary miR145, or precursor miR 145. Also described herein are methods and compositions for treating angmann syndrome, the methods comprising administering microrna-92 a, primary miR92a, or precursor miR92a to a patient having angmann syndrome.

In embodiments, vectors encoding microRNA-145, primary miR145 or precursor miR145 are provided. In embodiments, a vector encoding microrna-145, primary miR145, or precursor miR145 is administered to a patient having angeman syndrome, wherein the patient exhibits amelioration of one or more symptoms of the disorder. In embodiments, vectors encoding microrna-92 a, primary miR92a, or precursor miR92a are provided. In embodiments, a vector encoding microrna-92 a, primary miR92a, or precursor miR92a is administered to a patient having angmann syndrome, wherein the patient exhibits amelioration of one or more symptoms of the disorder.

microRNA-145, primary miR145, precursor miR145, microRNA-92 a, primary miR92a and/or precursor miR92a are collectively referred to herein as microRNAs (microRNAs or microRNAs). Administration of microRNA-145, primary miR145, precursor miR145, microRNA-92 a, primary miR92a and/or precursor miR92a to a patient is collectively referred to herein as microRNA therapy. Microrna treatment increases the level of the corresponding active microrna molecule in the cell. The increase may be produced by directly providing microrna to the cell, or may be produced by indirectly providing microrna to the cell, such as via a vector. The microrna can include RNA or DNA molecules that also include additional sequences. An increase in the level of a corresponding active microrna molecule in a cell of the CNS, e.g., the brain of a patient, is associated with an improvement in one or more symptoms of angeman syndrome.

One or more primary mirnas may be used in the compositions and methods described herein. Any suitable form of primary mRNA may be used. The primary mRNA may be processed and acted upon within the cell to obtain the function of the miRNA, e.g., may be converted to a precursor miRNA and then to a mature form. Alternatively, the miRNA may initially be a miRNA precursor. In embodiments, the compositions and methods include precursor mirnas that undergo cleavage by a double-stranded endonuclease, type III rnase, known as Dicer, resulting in incomplete miRNA-miRNA duplexes of about 20-25 nucleotides in size. This duplex comprises the mature miRNA strand and its opposite complementary miRNA strand. One or more precursor mirnas can be used in the compositions and methods described herein. The precursor miRNA may act to obtain the function of the miRNA. Any suitable form of precursor miRNA may be used. It is also contemplated that the miRNA of the compositions and methods described herein can be a mature miRNA.

The micrornas can be delivered to the cells in a non-expression vector manner or an expression vector manner. Expression vectors and vectors are used interchangeably herein. In embodiments, the microrna may be isolated or purified prior to use in subsequent steps. The microrna can be isolated or purified prior to introduction into the cell. "introduction" into a cell includes known methods of transfection, transduction, infection, and other methods for introducing an expression vector or heterologous nucleic acid into a cell. The template nucleic acid or amplification primer may be isolated or purified before it is transcribed or amplified. Isolation or purification can be carried out by a number of methods known to those skilled in the art for nucleic acids. Delivery of micrornas can be carried out in several formats, such as by encapsulating chemically modified RNA portions or by unmodified RNA portions in viral or non-viral delivery containers (vessel). Non-expression vector delivery means include nanoparticles, microparticles, liposomes, polymers, microspheres, etc. that can be targeted to brain cells. The microrna can also be delivered as a plasmid or microcarrier based expression system, where it can then be expressed and processed by the RNAi machinery in the cell to form mature micrornas.

The nucleic acid construct for miRNA expression may be produced recombinantly. Such expression vectors are provided herein. Expression vectors are nucleic acid vehicles (carrier nucleic acids) into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. Expression vectors include plasmids, cosmids, recombinant viruses such as adeno-associated virus (AAV), adenovirus, retrovirus, poxvirus, and other viruses known in the art (phage, animal, and plant viruses), and artificial chromosomes (e.g., YAC). One of ordinary skill in the art is well equipped with techniques for constructing expression vectors by standard recombinant techniques. In embodiments, the expression vector with the microrna is delivered to a cell of the patient. The nucleic acid molecules are delivered to the cells of the patient in a form where they can be taken up and advantageously expressed, so that therapeutically effective levels can be achieved.

Nucleic acid molecules for use in the vectors disclosed herein include nucleic acid molecules encoding mammalian microRNA-145, primary miR145, precursor miR145, microRNA-92 a, primary miR92a or precursor miR92 a. Such nucleic acids are well known in the art and are publicly available. In an embodiment of the invention, human microRNA-145, primary miR145, precursor miR145, microRNA-92 a, primary miR92a or precursor miR92a sequences are used in a vector. The mature sequence of human microRNA-92 a, also known as microRNA-92 a-3p, is:

(SEQ ID NO:1)5'-UAUUGCACUUGUCCCGGCCUGU-3’，

the mature sequence of human microRNA-145, also known as microRNA-145-5 p, is: (SEQ ID NO:2)5 'GUCCAGUUUUCCCCAGGAAUCCCU-3'.

In addition to nucleic acids encoding wild-type microRNA-145 and microRNA-92 a, variants of such nucleic acids may also be used in the methods of the invention. For example, such variants can affect the localization of micrornas within a cell. Such variants may result in decreased nuclear localization of micrornas as cytoplasmic localization increases. In aspects of the invention, microRNA-145 and microRNA-92 a mimetics may also be used to treat Angermann syndrome. Such mimics are commercially available (see e.g., Sigma Aldrich).

Any suitable expression vector known to those of skill in the art can be used to deliver the micrornas herein to a target location in a cell of the central nervous system. Following such delivery, cells in the target site are transfected with micrornas, thereby increasing the levels of these micrornas in the brain of the patient. Transduction viral (e.g., retroviral, adenoviral, lentiviral, and adeno-associated viral) vectors can be used for somatic gene therapy, particularly due to their high infection efficiency and stable integration and expression.

In embodiments, the expression vector may be a stable integrating vector or a stable non-integrating vector. Examples of suitable vectors are lentiviruses and adeno-associated viruses (AAV). Lentiviruses are a subset of retroviruses. Lentiviruses can integrate into the genome of non-dividing cells such as neurons. Lentiviruses are characterized by high infection efficiency, long-term stable expression of the transgene, and low immunogenicity. In embodiments, lentiviral vectors can be used to deliver micrornas to the brain.

AAV is a defective parvovirus known to infect many cell types and is not pathogenic to humans. AAV can infect both dividing and non-dividing cells. In embodiments, AAV vectors may be used herein to deliver micrornas to the brain. Any known adeno-associated virus (AAV) may be used herein, for example, AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, and AAVRec3 may be used in conjunction with neurons. AAV vectors for use in the methods disclosed herein include the AAV vectors described in U.S. provisional patent application No. 62/550,458, which is hereby incorporated by reference in its entirety. Additional suitable AAV serotypes have been developed by pseudotyping, i.e. mixing the capsid and genome from different viral serotypes. Thus, for example, AAV2/7 indicates a virus containing the genome of serotype 2 packaged in a capsid from serotype 7. Other examples are AAV2/5, AAV2/8, AAV2/9, and the like. Hybrid AAV capsid serotypes rec1, rec2, rec3 and rec4 are produced by rearranging fragments of capsid sequences that match AAV8 in all three non-human primate AAV serotypes cy5, rh20 and rh 39. See, Charbel et al, PLoS one.2013apr 9; e60361 (8) (4). The terms rec3AAV and AAVRec3 may be used interchangeably herein. Self-complementary adeno-associated viruses (scAAV) can also be used as vectors. Although AAV packages single-stranded DNA and requires a process for second strand synthesis, scAAV packages anneal together to form the two strands of double-stranded DNA. scAAV allows rapid expression in cells by skipping second strand synthesis.

One of ordinary skill in the art can construct suitable vectors using known techniques. Suitable vectors may be selected or constructed that contain, in addition to the microRNAs, appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, marker genes and other appropriate sequences. Those skilled in the art are familiar with appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, marker genes, and other suitable sequences.

The expression vectors herein include appropriate sequences operably linked to a coding sequence or ORF to facilitate its expression in the targeted host cell. "operably linked" sequences include both expression control sequences, such as a promoter adjacent to the coding sequence, and expression control sequences that act in trans or distally to control the expression of the desired product.

Typically, the vector includes a promoter that facilitates expression of the microRNA in the target cell. The promoter may be selected from a number of constitutive or inducible promoters that can drive expression of the selected transgene in the brain. Examples of constitutive promoters include the CMV immediate early enhancer/chicken β -actin (CBA) promoter-exon 1-intron 1 element, the RSV LTR promoter/enhancer, the SV40 promoter, the CMV promoter, the dihydrofolate reductase (DHFR) promoter, and the phosphoglycerate kinase (PGK) promoter.

Specificity can be achieved exclusively by regional and cell type specific expression of the receptor, for example using tissue or region specific promoters. Viral gene promoter elements may help determine the type of cell expressing the microrna. Some promoters are non-specific (e.g., CAG, a synthetic promoter), while others are neuron-specific. CAG promoters are strong synthetic promoters that can be used to drive high levels of expression. The CAG promoter consists of: 1) cytomegalovirus (CMV) early enhancer element, 2) the promoter, first exon, and first intron of the chicken β -actin gene, and 3) the splice acceptor of the rabbit β -globin gene. In embodiments, the promoter is a CAG promoter. Neuronal specific promoters include (e.g., synaptoprotein; hSyn) or promoters that favor a particular neuronal type, such as the dynorphin promoter, the enkephalin promoter, the GFAP (glial fibrillary acidic protein) promoter that favors astrocytes, or the CaMKIIa promoter that favors corticoglutamic cells but may also target gabaergic cells under the cortex. In embodiments, the promoter is the camkhia (α CaM kinase II gene) promoter, which can drive expression in the forebrain. Other neuronal cell type specific promoters include the NSE promoter, the tyrosine hydroxylase promoter, the myelin basic protein promoter, the glial fibrillary acidic protein promoter, and the neurofilament gene (heavy, medium, light) promoter.

Expression control sequences may also include appropriate transcription initiation, termination, and enhancer sequences; highly efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance the stability of a nucleic acid or protein; and, when desired, sequences that enhance product processing and/or secretion. Many diverse expression control sequences, including natural and non-natural, constitutive, inducible and/or tissue-specific expression control sequences, are known in the art and may be used herein depending on the type of expression desired.

In addition to promoters, expression control sequences for eukaryotic cells typically include enhancers such as those derived from immunoglobulin genes, SV40, CMV, and the like, and polyadenylation sequences that may include splice donor and splice acceptor sites. Polyadenylation sequences are typically inserted 3' to the coding sequence and 5 ' to the 3' ITR sequence. Illustrative examples of poly a signals that can be used in the vectors herein include a poly a sequence (e.g., AATAAA, ATTAAA, or AGTAAA), a bovine growth hormone poly a sequence (BGHpA), a rabbit β -globin poly a sequence (r β gpA), or another suitable heterologous or endogenous poly a sequence known in the art.

Regulatory sequences useful herein may also include introns, such as an intron located between the promoter/enhancer sequence and the coding sequence. One useful intron sequence is derived from SV40 and is referred to as the SV 40T intron sequence. Another includes woodchuck hepatitis virus post-transcriptional element (WPRE). WPRE is a DNA sequence that when transcribed yields a tertiary structure that enhances expression.

The vectors herein may comprise reporter genes, such as those encoding fluorophores. Fluorophores are fluorescent compounds that can re-emit light, typically after excitation at a particular frequency. They may be used as tags or markers that can be attached to, for example, proteins to allow the proteins to be localized. Many suitable fluorophores are known in the art. The fluorophores can be classified by the color they emit, e.g., blue, cyan, green, yellow, orange, red, and other colors. For example, mCherry, mRasberry, mTomato and mRuby are red fluorophore proteins; citrine, venus and EYFP are yellow fluorophore proteins. Green Fluorescent Protein (GFP) is a commonly used fluorophore.

The micrornas described herein (whether delivered by expression vectors or by non-expression vector forms) are used to treat angmann syndrome. Symptoms of angman syndrome may include, but are not limited to, intellectual disability, speech deficits, seizures, and characteristic behavioral characteristics. Behavioral characteristics of angman's syndrome include happy ending, laughter easily, short attention, hyperkinetic behavior, masticating of objects, sleep disturbance, and hydrophilicity. The methods of treatment herein may include providing an improvement in one or more of the foregoing symptoms.

In certain aspects, patients suspected of carrying a genetic defect leading to angmann's syndrome may be tested prior to treatment to detect and confirm the presence of such defects. In one example, a patient may be tested to detect a defect in the UBE3A gene. Molecular genetic testing (methylation testing and UBE3A sequence analysis) can identify changes in approximately 90% of individuals.

Targeted therapy according to the present disclosure may be implemented after the location or suspected location of abnormal activity associated with angman syndrome in a patient has been determined. Methods of determining the location of abnormal activity in the brain or otherwise affected neuronal tissue are well known in the art. In embodiments, a region determined to be the origin of aberrant activity may be targeted.

In some embodiments, the vectors disclosed herein are administered directly to the central nervous system, e.g., the brain or spinal cord. Any method known in the art for administering a vector directly to the central nervous system can be used. Vectors can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, superior thalamus, pituitary, substantia nigra, pineal), cerebellum, telencephalon (striatum, cerebrum including occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, striatum, cerebrum and hypothalamus. The vector may be delivered into the cerebrospinal fluid by, for example, lumbar puncture. In addition, when administration is performed intravenously, ultrasound can be applied to a target location in the brain of the patient to enhance permeability of the blood-brain barrier of the patient at the target location to facilitate uptake of the carrier. The application of ultrasound to enhance the permeability of the blood brain barrier of patients is disclosed in serial No. 62/471,635, the contents of which are incorporated herein in their entirety.

Methods for directly administering materials to a target site, such as the brain, are well known. For example, a Burr hole, such as a Burr hole, may be drilled into the skull and a needle of appropriate size may be used to deliver the carrier or non-carrier vehicle to the target location. In embodiments, a portion of the skull can be removed to expose dural matter at or near the target location (craniotomy), and a carrier or non-carrier vehicle can be administered directly to the target location. In embodiments, the carrier or non-carrier vehicle is injected into the cranium using stereotactic (stereotaxic cordinate), micropipettes, and automated pumps to deliver the carrier or non-carrier vehicle precisely to the desired area with minimal damage to surrounding tissues.

In certain aspects, the micropump can be used to deliver a pharmaceutical composition comprising a carrier or non-carrier vehicle containing a microrna to a target region in the brain. The composition may be delivered immediately or over an extended period of time, e.g., over 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, or more. After delivery of the vector to the target location in the brain, a sufficient amount of time may be allowed to pass to allow expression of the microrna at the target location.

In certain aspects, the carrier or non-carrier delivery vehicle herein can be administered systemically. Systemic delivery includes oral, buccal, sublingual, rectal, topical, intranasal, vaginal and parenteral modes of administration. Examples of parenteral administration modes include intravenous administration mode, intraperitoneal administration mode, intramuscular administration mode, and subcutaneous administration mode. In embodiments, the carrier or non-carrier delivery vehicle will circulate until they contact a target location in the CNS, including the brain, where they deliver or cause micrornas to be expressed and function, e.g., to aid network formation and/or to modulate neuronal signaling networks.

The microrna is used in an amount effective against angmann syndrome in a patient. The dosage of the active ingredient depends on the age, weight and individual condition of the patient, individual pharmacokinetic data and the mode of administration. In the case of human subjects having a body weight of about 70kg, the daily dose of microRNA administration may be from 0.01mg/kg body weight to 100mg/kg body weight, for example from 0.1mg/kg body weight to 50mg/kg body weight, from 1mg/kg to 20mg/kg body weight, administered in a single dose or in several doses.

In embodiments, ultrasound therapy is used to enhance delivery of micrornas to target locations in the brain by disrupting the blood brain barrier. The use of focused ultrasound energy herein disrupts the BBB without adversely affecting the carrier, non-carrier delivery vehicle, microrna, and/or the brain tissue itself. The use of ultrasonic energy herein can increase the rate of delivery of the carrier, non-carrier delivery vehicle and/or microrna to a target location in the brain, reduce side effects that may be associated with delivery of the carrier, non-carrier delivery vehicle and/or microrna to a target location in the brain, reduce the amount of dose, while concentrating the carrier, non-carrier delivery vehicle and/or microrna at the target location, and can allow for controlled release of the amount of the carrier, non-carrier delivery vehicle and/or microrna at the target location. Methods for delivering ultrasound energy through the skull are known in the art. See, e.g., U.S. patent No. 5,752,515 and U.S. publication No. 2009/0005711, both of which are hereby incorporated by reference in their respective entireties. See also Hynynen et al, NeuroImage 24(2005) 12-120.

In accordance with the present disclosure, microrna treatment provides an improvement in one or more symptoms of angeman syndrome that persists for more than 1 hour following administration to a patient. In embodiments, microrna treatment provides an improvement in one or more symptoms of the disorder that persist for more than 2 hours after administration to a patient. In embodiments, microrna treatment provides an improvement in one or more symptoms of the disorder that persist for more than 3 hours after administration to a patient. In embodiments, microrna treatment provides an improvement in one or more symptoms of the disorder that persist for more than 4 hours after administration to a patient. In embodiments, microrna treatment provides an improvement in one or more symptoms of the disorder that persist for more than 6 hours after administration to a patient. In embodiments, the microrna treatment provides an improvement in one or more symptoms of the disorder that persist for more than 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after administration to a patient. In embodiments, according to the present disclosure, there is provided an improvement in at least one symptom lasting 12 hours after administration to a patient. In embodiments, the microrna treatment provides an improvement in the next day function of the patient. For example, the microrna can provide an improvement in one or more symptoms of the disorder that persist for more than about, e.g., 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after administration and waking from overnight sleep.

In embodiments, the methods described herein are effective to reduce, delay, or prevent one or more other clinical symptoms of angeman syndrome. For example, the effect of microRNA therapy on target sites in the central nervous system (including the brain) of a patient can be compared to that of an untreated patient or a patient prior to treatment. In embodiments, the patient's symptoms, pharmacological and/or physiological indicators (indicators) prior to treatment are measured and measured one or more times again after treatment has begun. In embodiments, the control is a reference level or an average value determined based on a symptom, pharmacological, or physiological indicator in one or more patients (e.g., healthy patients) that are measured not to have the disease or disorder to be treated. In embodiments, the amount of miR-145 and/or miR-92a in brain tissue prior to treatment is compared to the amount of miR-145 and/or miR-92a in brain tissue after treatment. In embodiments, the effect of the treatment is compared to conventional treatments within the purview of one skilled in the art (purview).

An effective treatment for angeman syndrome as disclosed herein can be established by showing a reduction in the frequency or severity of symptoms (e.g., more than 10%, 20%, 30%, 40%, or 50%) after a period of time compared to baseline. For example, patients with microrna treatment may be randomly assigned a placebo as an adjunct to standard therapy during a 2-month double-blind period after a 1-month baseline period. The primary outcome measure may include the percentage of responders to microrna and to placebo, defined as having experienced at least a 10% to 50% reduction in symptoms from baseline during the second month of the double-blind period.

In embodiments, a pharmaceutical composition comprising a carrier, a non-carrier delivery vehicle, and/or a microrna can provide a conventional release profile or a modified release profile. Pharmaceutical compositions may be prepared using pharmaceutically acceptable "carriers" comprising materials that are considered safe and effective. "vehicle" includes all components present in a pharmaceutical formulation except for the active substance or ingredient. The term "carrier" includes, but is not limited to, diluents, binders, lubricants, disintegrants, fillers and coating compositions. Those skilled in the art are familiar with such pharmaceutical carriers and with methods of formulating (compounding) pharmaceutical compositions using such carriers.

In embodiments, pharmaceutical compositions comprising a carrier, a non-carrier delivery vehicle, and/or a microrna are suitable for parenteral administration, including, for example, intramuscular (i.m.), intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), or intrathecal (i.t). Parenteral compositions must be sterile for administration by injection, infusion or implantation into the body, and may be packaged in single-dose or multi-dose containers. In embodiments, the liquid pharmaceutical composition for parenteral administration to a patient comprises any of the respective amounts of active substance described above, e.g., carrier, non-carrier delivery vehicle and/or microrna. In embodiments, the pharmaceutical composition for parenteral administration is formulated to a total volume of about, e.g., 0.1ml, 0.25ml, 0.5ml, 0.75ml, 1ml, 1.25ml, 1.5ml, 1.75ml, 2ml, 2.25ml, 2.5ml, 2.75ml, 3ml, 3.25ml, 3.5ml, 3.75ml, 4ml, 4.25ml, 4.5ml, 4.75ml, 5ml, 10ml, 20ml, 25ml, 50ml, 100ml, 200ml, 250ml, or 500 ml. In embodiments, the volume of the pharmaceutical composition comprising the expression vector is a microliter amount. For example, 0.1 microliter to 10 microliter or more may be injected. For example, 0.1 microliters, 0.2 microliters, 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, 1.0 microliters, 1.25 microliters, 1.5 microliters, 1.75 microliters, 2.0 microliters, 2.25 microliters, 2.5 microliters, 2.75 microliters, 3.0 microliters, 3.25 microliters, 3.5 microliters, 3.75 microliters, 4.0 microliters, 4.25 microliters, 4.5 microliters, 4.75 microliters, 5.0 microliters, 5.25 microliters, 5.5 microliters, 5.75 microliters, 6.0 microliters, 6.25 microliters, 6.5 microliters, 6.75 microliters, 7.0 microliters, 8.25 microliters, 8.5 microliters, 8.75, 9.0 microliters, 9.25 microliters, 9.5, 9.75 microliters, or 10 microliters. In embodiments, the composition is contained in a micropipette, bag, glass vial (visual), plastic vial, or bottle (bottle).

In embodiments, the pharmaceutical composition for parenteral administration comprises the respective amounts described above. In embodiments, a pharmaceutical composition for parenteral administration comprises from about 0.0001mg to about 500mg of active agent, e.g., a carrier, a non-carrier delivery vehicle, and/or a microrna. In embodiments, a pharmaceutical composition for parenteral administration to a patient comprises an active substance, e.g., a carrier, a non-carrier delivery vehicle, and/or a microrna, at a corresponding concentration of about 0.001mg/ml to about 500 mg/ml. In embodiments, pharmaceutical compositions for parenteral administration comprise the active agent at a corresponding concentration of, for example, about 0.005mg/ml to about 50mg/ml, about 0.01mg/ml to about 50mg/ml, about 0.1mg/ml to about 10mg/ml, about 0.05mg/ml to about 25mg/ml, about 0.05mg/ml to about 10mg/ml, about 0.05mg/ml to about 5mg/ml, or about 0.05mg/ml to about 1 mg/ml. In embodiments, the pharmaceutical composition for parenteral administration comprises the active substance at a corresponding concentration of, for example, about 0.05mg/ml to about 15mg/ml, about 0.5mg/ml to about 10mg/ml, about 0.25mg/ml to about 5mg/ml, about 0.5mg/ml to about 7mg/ml, about 1mg/ml to about 10mg/ml, about 5mg/ml to about 10mg/ml, or about 5mg/ml to about 15 mg/ml.

In an embodiment, a pharmaceutical composition for parenteral administration is provided, wherein the pharmaceutical composition is stable for at least six months. In embodiments, a pharmaceutical composition for parenteral administration exhibits no more than about a 5% reduction in active agent for, e.g., at least 3 months or 6 months. In embodiments, the amount of carrier or non-carrier vehicle degrades by no more than about, e.g., 2.5%, 1%, 0.5%, or 0.1%. In embodiments, the degradation is less than about, e.g., 5%, 2.5%, 1%, 0.5%, 0.25%, 0.1% for at least six months.

In an embodiment, a pharmaceutical composition for parenteral administration is provided, wherein the pharmaceutical composition remains soluble. In embodiments, pharmaceutical compositions for parenteral administration are provided that are stable, soluble, locally-compatible, and/or ready-to-use. In embodiments, the pharmaceutical compositions herein are ready-to-use for direct administration to a patient in need thereof.

Pharmaceutical compositions provided herein for parenteral administration may comprise one or more excipients, such as solvents, solubility enhancers, suspending agents, buffers, isotonic agents, stabilizers, or antimicrobial preservatives. When used, the excipients of the parenteral composition will not adversely affect the stability, bioavailability, safety and/or efficacy of the carrier, non-carrier delivery vehicle and/or microrna used in the composition. Accordingly, parenteral compositions are provided in which there is no incompatibility between any of the components of the dosage form.

In embodiments, the parenteral composition comprising a carrier, a non-carrier delivery vehicle, and/or a microrna comprises a stabilizing amount of at least one excipient. For example, the excipient may be selected from the group consisting of: buffers, solubilizers, tonicity agents, antioxidants, chelating agents, antimicrobial agents and preservatives. One skilled in the art will appreciate that excipients may have more than one function and be classified into one or more specific groups.

In embodiments, the parenteral composition comprises a carrier, a non-carrier delivery vehicle and/or a microrna, and an excipient, and wherein the excipient is present at a weight percentage (w/v) of less than about, e.g., 10%, 5%, 2.5%, 1%, or 0.5%. In embodiments, the excipient is present at a weight percentage of between about, e.g., 1.0% to 10%, 10% to 25%, 15% to 35%, 0.5% to 5%, 0.001% to 1%, 0.01% to 1%, 0.1% to 1%, or 0.5% to 1%. In embodiments, the excipient is present at a weight percentage of between about, e.g., 0.001% to 1%, 0.01% to 1%, 1.0% to 5%, 10% to 15%, or 1% to 15%.

In embodiments, the parenteral composition can be administered as needed, e.g., once daily, twice daily, three times daily, four times daily, five times daily, six or more times daily, or continuously depending on the needs of the patient.

In an embodiment, a parenteral composition of active substances is provided wherein the pH of the composition is between about 4.0 to about 8.0. In embodiments, the pH of the composition is between, for example, about 5.0 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0. In embodiments, the pH of the composition is between, for example, about 6.5 to about 7.5, about 7.0 to about 7.8, about 7.2 to about 7.8, or about 7.3 to about 7.6. In embodiments, the pH of the aqueous solution is, for example, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.7, about 7.8, about 8.0, about 8.2, about 8.4, or about 8.6.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The terms "about" or "approximately" as used herein mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, as practiced in the art. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, and/or up to 1% of a given value. Alternatively, in particular with respect to biological systems or processes, the term may mean within an order of magnitude of the value, preferably within 5 times the value, and more preferably within 2 times the value.

"ameliorating" refers to the treatment of angeman syndrome.

By "improvement in next day function" or "wherein there is improvement in next day function" is meant improvement upon waking from an overnight sleep period, wherein the beneficial effects of administering microrna therapy to the patient are tailored to at least one symptom of the syndrome or disorder herein, and are objectively discernible by the patient or by an observer at a time period, e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, etc., upon waking.

"treating", "treatment" or "treatment" may refer to the following: alleviating or delaying the onset of clinical symptoms of a disease or disorder in a patient who may be suffering from or susceptible to the disease or disorder but who has not yet experienced or exhibited clinical or subclinical symptoms of the disease or disorder. In certain embodiments, "treating" or "treatment" may refer to preventing the appearance of clinical symptoms of a disease or disorder in a patient who may be suffering from or susceptible to the disease or disorder but does not yet experience or display clinical or subclinical symptoms of the disease or disorder. "treating", "treating" or "treatment" also refers to inhibiting a disease or disorder, e.g., arresting or reducing its development or at least one clinical or subclinical symptom thereof. "treating", "treating" or "treatment" also refers to alleviating the disease or disorder, e.g., causing regression of the disease or disorder or at least one clinical or subclinical symptom thereof. The benefit to the patient to be treated may be statistically significant, mathematically significant, or at least perceptible to the patient and/or physician. Nevertheless, prophylactic (preventative) and therapeutic (curative) treatments are two separate embodiments of the disclosure herein.

By "pharmaceutically acceptable" is meant "molecular entities and compositions that are generally considered safe", e.g., that are physiologically tolerable and do not typically produce allergic or similar untoward reactions such as gastric upset and the like when administered to a human. In embodiments, the term refers to molecular entities and compositions approved by a regulatory agency of the Federal or a state government as a GRAS list (approved by the FDA under sections 204(s) and 409 of the Federal Food, Drug and Cosmetic Act) or similar list (the U.S. pharmacopeia or another generally recognized pharmacopeia for use in animals, and more particularly in humans).

An "effective amount" or "therapeutically effective amount" can mean a dose sufficient to alleviate one or more symptoms of a syndrome, disorder, disease or condition being treated or to otherwise provide a desired pharmacological and/or physiological effect. An "effective amount" or a "therapeutically effective amount" may be used interchangeably herein.

A combination of (a combination of) "or" administered with (an administered along) may be used interchangeably and means that two or more agents are administered during the course of therapy. These agents may be administered together at the same time or separately at spaced intervals. These agents may be administered in a single dosage form or in separate dosage forms.

A "patient in need thereof" may include an individual, e.g., a mammal such as a human, canine, feline, porcine, rodent, etc., that has been diagnosed as having angman syndrome, which method may be provided to any individual, including, for example, where the patient is a neonate, an infant, a pediatric patient (6 months to 12 years of age), an adolescent patient (12-18 years of age), or an adult (over 18 years of age). The patient includes a mammal. Such patients include, for example, patients diagnosed as having a genetic defect in the Ube3a gene.

"prodrug" refers to a pharmacological substance (drug) that is administered to a subject in an inactive (or significantly less active) form. Once administered, the prodrug is metabolized in the body (in vivo) to a compound having the desired pharmacological activity.

"analog" and "derivative" are used interchangeably and refer to a compound that has the same core as the parent compound but can differ from the parent compound by bond order, the absence or presence of one or more atoms and/or groups of atoms, and combinations thereof. Enantiomers are examples of derivatives. Derivatives may differ from the parent compound, for example, in one or more substituents present on the nucleus, which may include one or more atoms, functional groups or substructures. In general, it is contemplated that derivatives may be formed from the parent compound, at least in theory, via chemical and/or physical processes.

The term "pharmaceutically acceptable salt" as used herein refers to derivatives of a compound as defined herein, wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, non-toxic base addition salts with inorganic bases. Suitable inorganic bases such as alkali metal bases and alkaline earth metal bases include metal cations such as sodium, potassium, magnesium, calcium, and the like. Pharmaceutically acceptable salts can be synthesized from the parent compound by conventional chemical methods.

It is understood that the examples and embodiments provided herein are exemplary embodiments. Those of skill in the art will envision many modifications to the examples and embodiments consistent with the scope of the disclosure herein. Such modifications are intended to be covered by the claims.

Sequence listing

<110> Olympic medical Co

Matthew Dulling

<120> use of MIR-92A or MIR-145 for the treatment of Angerman syndrome

<130> 2262-63 PCT

<150> US 62/684,774

<151> 2018-06-14

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic microRNA-92 a-3p

<400> 1

uauugcacuu gucccggccu gu 22

<210> 2

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic microRNA-145-5 p

<400> 2

guccaguuuu cccaggaauc ccu 23